Relaxin 2008
Fifth International Conference on Relaxin and Related Peptides
May 18–23, 2008 | Sheraton, Maui | Hawai’i, USA
Organizing Committee
Gillian D. Bryant-Greenwood (chair)
Carol A. Bagnell
Daniele Bani
Ross Bathgate
O. David Sherwood
Josh Silvertown
Elaine Wolff (Conference Secretary)
www.conferences.uiuc.edu/relaxin
Foreword
As Chair of the organizing committee, I welcome you to Relaxin 2008 and hope that you will learn a lot and also update
your knowledge about relaxin and its related peptides, meet new colleagues and old friends, forge new collaborations
and at the same time, enjoy the beauty of the Island of Maui.
I am truly grateful for all the assistance, suggestions, and ideas from the organizing committee. Their help with the
program was particularly valuable. I have been very fortunate to have Elaine Wolff as the conference secretary and to
tap her memory of Relaxin 2004. The sponsorship from U.S. government sources, private foundations, commercial
organizations, and private individuals has made it possible to offer some financial support for 20 new investigators to
attend and present their data at this conference. The recipients of these awards are listed in this program. This generosity
has also allowed us to keep the costs as low as possible. I recognize the difficulty of raising funds for travel and
accommodation and particularly thank all the invited speakers for their efforts to come and present their most recent and
exciting data. They have made this all worthwhile.
On behalf of the organizing committee, I have great pleasure in welcoming you to Relaxin 2008 and wish you a very
productive and pleasant time in our beautiful Hawaiian Islands.
Gillian D. Bryant-Greenwood
Chair
We are pleased to announce the following as Sponsors of Relaxin 2008:
Major Sponsors
Corthera, Inc. (formerly BAS Medical, Inc.)
Dr. Peter Bryant-Greenwood gave in memory of his father, Dr. Frederick C. Greenwood
Dr. Bernard and Jane Steinetz gave in memory of their son, Carl Steinetz
University of Guelph, Canada
Department of Obstetrics and Gynecology, University of Hawaii—Sponsor of the Opening Dinner
United States Department of Agriculture (USDA)—New Investigator Registration Grants
Sponsors
March of Dimes—New Investigator Housing Grants
Phoenix Pharmaceuticals, Inc.
Supporters
Pacific Biosciences Research Center, University of Hawaii—Sponsor of the Opening Reception
Johnson & Johnson Pharmaceutical Research and Development, LLC
R&D Systems, Inc.—New Investigator Housing Grant
Relaxin 2008
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New Investigator Award Recipients
iv
Alsadek Bogzil
University of Manchester, United Kingdom
Priya Maseelall
New Jersey Medical School, USA
Gabrielle Callander
Howard Florey Institute, Australia
Barbara McGowan
Imperial College London, United Kingdom
Joseph Chen
Rutgers University, USA
Jonathan McGuane
University of Florida, USA
Dan Debrah
University of Pittsburgh, USA
Fazel Shabanpoor
Howard Florey Institute, Australia
Amy-Lynn Frankshun
Rutgers University, USA
Sudhir Singh
University of Nebraska Medical Center, USA
Michelle Halls
Monash University, Australia
Craig Smith
Howard Florey Institute, Australia
Linda Haugaard-Jonsson
University of Kalmar, Sweden
Yu May Soh
The University of Melbourne, Australia
Andras Kern
University of Hawaii, USA
Emma van der Westhuizen
Monash University, Australia
Zhen Li
Baylor College of Medicine, USA
Lenka Vodstrcil
The University of Melbourne, Australia
Sherie Ma
Howard Florey Institute, Australia
Su Ee Wong
Howard Florey Institute, Australia
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Relaxin 2008
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Social Program
Sunday, May 18, 2008
1:00–6:00 PM
Registration: Kahului Foyer
5:00–6:00 PM
Welcome Reception: Courtyard
Pacific Biosciences Research Center, University of Hawaii—Sponsor
Includes Accompanying Persons: yellow, green, pink name badges
6:00–7:30 PM
Opening Dinner: Courtyard
Department of Obstetrics and Gynecology, University of Hawaii—Sponsor
Includes Accompanying Persons: yellow, green, pink name badges
7:30–8:00 PM
Geology of the Hawaiian Islands: Wailuku Room
Dr. Robert Wright, School of Earth, Ocean Science and Technology, University of Hawaii
Monday, May 19, 2008
8:00–9:00 AM
Continental Breakfast: Courtyard
Includes Accompanying Persons: green, pink name badges
12:30–1:30 PM
Lunch provided: Anuenue Lawn
Includes Accompanying Persons: green name badges
Tuesday, May 20, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room
Includes Accompanying Persons: green, pink name badges
Afternoon Free—lunch and dinner on your own
Wednesday, May 21, 2008
8:00–9:00 AM
Continental Breakfast: Courtyard
Includes Accompanying Persons: green, pink name badges
12:30–1:30 PM
Lunch provided: Anuenue Lawn
Includes Accompanying Persons: green name badges
5:30 PM
Buses leave from hotel lobby for “Ulalena” (dinner not included)
Includes Accompanying Persons: yellow, green, pink name badges
Thursday, May 22, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room
Includes Accompanying Persons: green, pink name badges
Afternoon Free—lunch and dinner on your own
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Friday, May 23, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room
Includes Accompanying Persons: green, pink name badges
12:45 PM
Closing Lunch provided: Anuenue Lawn
Includes Accompanying Persons: green name badges
Key to Accompanying Persons name badge colors:
Accompanying Person Option #1 (yellow name badge): includes Sunday Evening welcome reception and dinner and Wednesday evening
excursion to Ulalena.
Accompanying Person Option #2 (green name badge): includes Sunday Evening welcome reception and dinner; Monday, Wednesday, and
Friday luncheons; Monday through Friday continental breakfasts; Wednesday evening excursion to Ulalena.
Accompanying Person Option #3 (pink name badge): includes Sunday Evening welcome reception and dinner; Monday through Friday
continental breakfasts; Wednesday evening excursion to Ulalena (no luncheons included).
Information for Conference Presenters
Poster Presentations (designated as “P” in the abstracts)
The posters will be divided into three sessions. Due to space limitations, posters will not be displayed for the entire
conference. Poster Session I will be from 7:30–9:00 AM on Tuesday; Poster Session II will be from 7:30–9:00 AM on
Thursday; Poster Session III will be from 7:30–9:00 AM on Friday. The presenting author should be by his or her poster
and available for discussion during the assigned session. Posters should be affixed to the poster boards between 7:00
and 7:30 AM on the day of the session. Numbers will be posted on the display boards. Posters should remain in place
throughout the rest of the day and removed at the end of the program that day.
A space of 8 feet wide by 4 feet high (244 cm wide x 122 cm high) is available for each poster.
There will be no audiovisual capability in the poster presentation area.
All Oral Presentations (designated as “O” in the abstracts)
In order to ensure the seamless progression of talks within each session, our technical staff will need to have all
presentations and laptops ready before the session begins. Presentations must be submitted to our technical staff no later
than 30 minutes before the beginning of each morning, afternoon, or evening session. Persons wishing to project from
their own laptop computer must bring it to the meeting room at least 15 minutes before the beginning of the session in
which they will be speaking.
Presenters may bring their talks to our staff on CD-ROM disks or USB flash drives. In addition, we strongly recommend
that you also upload a copy of your PowerPoint presentation before coming to the conference. Uploaded files will be
tested on the equipment we will use for the sessions. We will let you know if there are any problems. The deadline for
presentation uploads is 5:00 PM (Hawaiian Standard Time) Friday, May 16, 2008. After that time, presentations will only
be accepted at the Conference.
Relaxin 2008
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Relaxin 2008 Program The Fifth International Conference on Relaxin and Related Peptides
Sunday, May 18, 2008
1:00–6:00 PM
Registration: Kahului Foyer
5:00–6:00 PM
Welcome Reception: Courtyard
Pacific Biosciences Research Center, University of Hawaii—Sponsor
6:00–7:30 PM
Opening Dinner: Courtyard
Department of Obstetrics and Gynecology, University of Hawaii—Sponsor
7:30–8:00 PM
Geology of the Hawaiian Islands: Wailuku Room
Dr. Robert Wright, School of Earth, Ocean Science and Technology, University of Hawaii
Posters will be displayed in the Kihei/Wailea rooms. Platform Presentations will be in the Wailuku Room.
* New Investigators
Monday, May 19, 2008
8:00–9:00 AM
Continental Breakfast: Courtyard
9:00–10:30 AM
Platform Session I: Relaxin and Related Peptides Structure and Function #1
Presentations O1–4
Chairs: C. Schwabe and G. Tregear
9:00 AM
30 min
Structure and Activity in the Relaxin Family of Peptides
O-1
Geoffrey Tregear, Howard Florey Institute, Australia
9:30 AM
30 min
The Chemical Synthesis of Relaxin and Related Peptides: From Yesterday to Tomorrow
O-2
John Wade, Howard Florey Institute, Australia
10:00 AM
15 min
Development of High Affinity INSL3 Antagonists
O-3
*Fazel Shabanpoor, Howard Florey Institute, Australia
10:15 AM
15 min
The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of
the Different RXFP Receptors
O-4
Mohammed Hossain, Howard Florey Institute, Australia
10:30–11:00 AM
Morning Break
11:00–12:30 PM
Platform Session 2: Relaxin and Related Peptides Structure and Function #2
Presentations O5–8
Chairs: D. Craik and J. Wade
11:00 AM
30 min
Structural Insights into the Function of Relaxins
O-5
Johan Rosengren, University of Kalmar, Sweden
11:30 AM
30 min
Probing the Functional Domains of Relaxin-3 and the Creation of a Selective Antagonist for
GPCR135 over Relaxin Receptor LGR7
Changlu Liu, Johnson & Johnson Pharmaceuticals, Inc., USA
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-6
12:00 PM (15)
15 min
Structural Characterization of the R3/I5 Chimeric Relaxin Peptide
O-7
*Linda Haugaard-Jonsson, University of Kalmar, Sweden
12:15 PM (15)
15 min
Degradation of Relaxin Family Peptides by Insulin-degrading Enzyme
O-8
Robert Bennett, Veterans Affairs Medical Center, Omaha, Nebraska, USA
12:30–1:30 PM
Lunch provided: Anuenue Lawn
1:30–3:00 PM
Platform Session 3: Reproductive Functions of Relaxin and Related Peptides #1
Presentations O9–12
Chairs: C. Bagnell and G. Bryant-Greenwood
1:30 PM
25 min
Relaxin in Human Pregnancy: Consequences of Too Little or Too Much of A Good Thing!
O-9
Laura Goldsmith, Department of Obstetrics and Gynecology, New Jersey Medical School of
UMDNJ, USA
1:55 PM
25 min
Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and Inhibit
Apoptosis in the Mouse Cervix and Vagina
O-10
David Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at
Urbana-Champaign, USA
2:20 PM
25 min
The Good, The Bad and The Ugly: Lessons in Female Reproductive Tract Physiology from the
Relaxin Gene Knockout (Rln-/-) Mouse
O-11
Laura Parry, Department of Zoology, The University of Melbourne, Australia
2:45 PM
15 min
Decreased Hyaluronic Synthase Expression in the Cervix of Pregnant Relaxin-Deficient (Rln-/-)
Mice
O-12
*Yu May Soh, Department of Zoology,The University of Melbourne, Australia
3:00–3:30 PM
Afternoon Break
3:30–5:15 PM
Platform Session 4: Reproductive Functions of Relaxin and Related Peptides #2
Presentations O13–17
Chairs: L. Goldsmith and L. Parry
3:30 PM
25 min
Milk-Borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of Neonatal Tissues
O-13
Carol Bagnell, Department of Animal Sciences, Rutgers University, USA
3:55 PM
25 min
Relaxin and Maternal Lactocrine Programming of Neonatal Uterine Development
O-14
Frank Bartol, Department of Animal Sciences, Auburn University, USA
4:20 PM
25 min
Relaxin in the Marmoset Monkey: There Are Two Sides to Every Story
O-15
Almuth Einspanier, Institute of Physiological Chemistry, Leipzig, Germany
4:45 PM
15 min
Expression of LGR7 in the Primate Corpus Luteum Implicates the Corpus Luteum as a
Relaxin Target Organ
O-16
*Priya Maseelall, Department of Obstetrics and Gynecology, New Jersey Medical School, USA
5:00 PM
15 min
Characterization and Biological Activity of Prorelaxin in Porcine Milk
O-17
*Amy-Lynn Frankshun, Department of Animal Sciences, Rutgers University, USA
Dinner on your own
7:00–8:00 PM
Meet the Professors for Trainees: Coral Reef Room
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Tuesday, May 20, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room and Poster Session I
Posters P1–20
Regioselective Disulfide Solid Phase Synthesis of Mouse Relaxin
P-01
John D. Wade, Howard Florey Institute, Australia
Trafficking of the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135)
P-02
*Emma T. van der Westhuizen, Monash University, Australia
Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodination and Isolation of
Peptide by One-step Ether Precipitation
P-03
Suode Zhang, Howard Florey Institute, Australia
Chemical Synthesis and Biological Activity Evaluation of β-Ala and γ-Abu Substituted Insulin-like
Peptide 3 Analogs
P-04
Suode Zhang, Howard Florey Institute, Australia
Synthesis, Conformation and Biological Activity of Novel Dicarba Analogs of
Human Relaxin-3/INSL7
P-05
Mohammed Akhter Hossain, Howard Florey Institute, Australia
Human Insulin-like Peptide 5 (INSL5): Synthesis and Activity on RXFP3 and RXFP4 Receptors
P-06
Mohammed Akhter Hossain, Howard Florey Institute, Australia
De novo Design and Synthesis of Cyclic and Linear Peptides to Mimic the B-chain Binding
Elements of Human Relaxin Family Peptides
P-07
Mohammed Akhter Hossain, Howard Florey Institute, Australia
Investigating the Molecular Mechanics of Relaxin Family Peptide Receptor Function Using Time
Correlated Single Photon Counting Confocal Microscopy
P-08
Ross A.D. Bathgate, Howard Florey Institute, Australia
P-09
Withdrawn
Identification of the Relaxin-responsive Cells in the Human Chorion and Decidua
P-10
Jaime S. Horton, University of Hawaii, USA
RXFP1 Is Expressed on Sperm Acrosome and Relaxin Stimulates the Acrosomal Reaction of
Human Spermatozoa
P-11
Lisa Gianesello, University of Padova, Italy
Involvement of Relaxin in Endometriosis
P-12
Sara S. Morelli, New Jersey Medical School, USA
Relaxin and Neopterin as Local Ovarian Granulosa Cell Growth- and Colony-stimulating Factors
P-13
Elissaveta Borissova Zvetkova, IEMAM – BAS, Bulgaria
The Lethal Phenotype in Relaxin-Deficient (Rln-/-) Mice Is Not Due to Impaired Mammary Gland
Development or Milk Protein Synthesis
Laura J. Parry, The University of Melbourne, Australia
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-14
Laser Microdissection of Neonatal Porcine Endometrium for Tissue Specific Evaluation of Relaxin
Receptor (RXFP1) Expression in Response to Perinatal Zearalenone Exposure
P-15
Anne A. Wiley, Auburn University, USA
The Effects of Relaxin-deficiency on Uterine Angiogenesis in Early Pregnancy
P-16
Jane E. Girling, Monash University, Australia
The Effects of Fetal and Placental Numbers on Blood Concentrations of Relaxin in the Rabbit
P-17
Phillip A. Fields, University of South Alabama, USA
Cervical Softening in the Common Marmoset Monkey (Callithrix jacchus)
P-18
Almuth Einspanier, Institute of Physiological Chemistry, Germany
Perinatal Zearalenone Exposure Affects RXFP1, RXFP2 and Morphoregulatory Gene Expression in
the Neonatal Porcine Uterus
P-19
*Joseph C. Chen, Rutgers University, USA
The Effects of Blocking Progesterone Action on Relaxin Receptor Expression in the Myometrium
of Pregnant Rats
P-20
*Lenka A Vodstrcil, The University of Melbourne, Australia
9:00–10:30 AM
Platform Session 5: Relaxin Family Receptors #1
Presentations O18–21
Chairs: A. Agoulnik and J. Rosengren
9:00 AM
30 min
Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function
O-18
Ross Bathgate, Howard Florey Institute, Australia
9:30 AM
30 min
Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function
O-19
*Andras Kern, Pacific Biosciences Research Center, University of Hawaii, USA
10:00 AM
15 min
Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome ProliferatorActivated Receptor γ
O-20
*Sudhir Singh, Department of Biochemistry and Molecular Biology, University of Nebraska, USA
10:15 AM
15 min
Development of a High-throughput Receptor-binding Assay Using Time-Resolved Fluorescent
Europium Lanthanide for Screening Insulin-like Peptide 3 (INSL3) Analogues for their Receptor
Binding Affinity
O-21
*Fazel Shabanpoor, Howard Florey Institute, Australia
10:30–11:00 AM
Morning Break
11:00–12:45 PM
Platform Session 6: Relaxin Family Receptors #2
Presentations O22–27
Chairs: R. Summers and C. Schwabe
11:00 AM
25 min
Structure, Function and Evolution of Ligands and Receptors of the Insulin/Relaxin Peptide Family
O-22
Pierre De Meyts, Hagedorn Research Institute, Denmark
11:25 AM
20 min
Activation of Relaxin Related Receptors by Short, Linear Peptides Derived from a Collagen
Containing Precursor
O-23
Ronen Shemesh, Compugen Ltd., Israel
11:45 AM
15 min
RXFP1 Couples to the Gαi3-Gβγ-PI3K-PKCζ Pathway via the Final 10 Amino Acids of the Receptor
C-terminal Tail
O-24
*Michelle Halls, Department of Pharmacology, Monash University, Australia
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12:00 PM
15 min
Local Regulation of Relaxin Receptors in the Myometrium of Pregnant Rats
O-25
*Lenka Vodstrcil, Department of Zoology, The University of Melbourne, Australia
12:15 PM
15 min
The Role of the N-Terminal Leucine Rich Repeat Cap in Mediating INSL3 Specificity to Relaxin
Family Peptide Receptor 2
O-26
Daniel Scott (Ross Bathgate), Howard Florey Institute, Australia
12:30 PM
15 min
The Relaxin Family Peptide Receptors (RXFPs): A New Subgroup of Dimeric, Cooperative GPCRs
O-27
Angela Manegold Svendsen, Hagedorn Research Institute, Denmark
Afternoon Free—lunch and dinner on your own
7:00–8:30 PM
Platform Session 7: Physiology of Insulin-like Peptide 3 (INSL3)
Presentations O28–30
Chairs: S. Sutton and R. Bathgate
7:00 PM
30 min
INSL3/RXFP2 Signaling in Testicular Descent: Mice and Men
O-28
Alexander Agoulnik, Department of Obstetrics and Gynecology, Baylor College of Medicine, USA
7:30 PM
30 min
New Roles for INSL3 in Adults: Regulation of Bone Metabolism and Association of RXFP2 Gene
Mutations with Osteoporosis
O-29
Alberto Ferlin, Department of Histology, Microbiology and Medical Biotechnologies, University
of Padova, Italy
8:00 PM
30 min
Regulation of INSL3 Transcription in Testicular Leydig Cells
O-30
Jacques Tremblay, Department of Obstetrics and Gynecology, Laval University, Quebec, Canada
Wednesday, May 21, 2008
8:00–9:00 AM
Continental Breakfast: Courtyard
9:00–10:30 AM
Platform Session 8: Relaxin and Related Peptides: Receptor Signaling
Presentations O31–34
Chairs: T. Klonisch and D. Sherwood
9:00 AM
30 min
The "Hot Wires" of the Relaxin-like Factor (INSL3)
O-31
Christian Schwabe, Department of Biochemistry and Molecular Biology, Medical University of South
Carolina, USA
9:30 AM
30 min
Roles of the Receptor, the Ligand and the Cell in the Signal Transduction Pathways Utilised by the
Relaxin Family Peptide Receptors 1-3 (RXFP1-3)
O-32
Roger Summers, Department of Pharmacology, Monash University, Australia
10:00 AM
15 min
Ligand-directed Signalling Pathways at the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135)
Determined Using Reporter Genes
O-33
*Emma van der Westhuizen, Department of Pharmacology, Monash University, Australia
10:15 AM
15 min
Relaxin Activates Multiple cAMP Signalling Pathway Profiles in Different Target Cells
O-34
*Michelle Halls, Department of Pharmacology, Monash University, Australia
10:30–11:00 AM
Morning Break
11:00–12:30 PM
Platform Session 9: Cardiac and Vascular Actions of Relaxin
Presentations O35–38
Chairs: C. Samuel and R. Summers
11:00 AM
25 min
Prominent Role of Relaxin in Improving Post-infarction Heart Remodelling. Clues from in vitro and
in vivo Studies with Genetically Engineered Relaxin-producing Myoblasts
Daniele Bani, Department of Anatomy, Histology and Forensic Medicine, University of Florence, Italy
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-35
11:25 AM
25 min
New Aspects on Cardiovascular Actions of Relaxin: Inotropy and Signaling
O-36
Thomas Dschietzig, Department of Cardiology and Angiology, Charité University Medicine, Germany
11:50 AM
25 min
Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin: Experimental Findings
O-37
Xiao-Jun Du, Baker Heart Institute, The University of Melbourne, Australia
12:15 AM
15 min
Relaxin-Induced Compositional and Geometric Remodeling of Small Renal Arteries
O-38
*Dan Debrah, Department of Bioengineering, University of Pittsburgh, USA
12:30–1:30 PM
Lunch provided: Anuenue Lawn
1:30–3:30 PM
Platform Session 10: Neurobiology of Relaxin and Relaxin-related Peptides
Presentations O39–44
Chairs: R. Bathgate and E. Unemori
1:30 PM
25 min
Relaxin-family Peptide and Receptor Systems in Mammalian Brain: Recent Insights from
Anatomical and Functional Studies
O-39
Andrew Gundlach, Howard Florey Institute, Australia
1:55 PM
25 min
Relaxin-3 and its Role in Neuroendocrine Function
O-40
*Barbara McGowan, Department of Investigative Medicine, Imperial College London, UK
2:20 PM
25 min
Metabolic and Neuroendocrine Responses to RXFP3 Modulation in the CNS
O-41
Steven Sutton, Johnson & Johnson Pharmaceuticals, USA
2:45 PM
15 min
Relaxin-3 Neurons of the Nucleus Incertus Modulate Septohippocampal Theta Rhythm and Spatial
Working Memory in Rats
O-42
*Sheri Ma, Howard Florey Institute, Australia
3:00 PM
15 min
Behavioral Phenotyping of Mixed-background (129SV/B6) Relaxin-3 Knockout
O-43
*Craig Smith, Howard Florey Institute, Australia
3:15 PM
15 min
An in vitro Study of the Protective Effect of Relaxin on Brain Tissue under Ischemic Stress
O-44
Brian Wilson, Department of Biology, Acadia University, Canada
5:30 PM
Buses leave from hotel lobby for “Ulalena” (dinner not included)
Thursday, May 22, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room and Poster Session II
Posters P21–40
Relaxin Activation of Water Drinking in the Mouse: Likely Role of RXFP1 in the Subfornical Organ
P-21
Andrew L. Gundlach, Howard Florey Institute, Australia
Relaxin-3 Neurons in Nucleus Incertus of the Rat: Effect on Activity of Psychological Stressors and
the Light-Dark Cycle
P-22
Pei-Juan Shen, Howard Florey Institute, Australia
Expression and Distribution of the Relaxin Family Peptide Receptors—RXFP1-3—in Primate and
Human Brain
P-23
Pei-Juan Shen, Howard Florey Institute, Australia
Relaxin-3 and RXFP3 Gene Expression and Peptide Distribution in the Brain and Ovary of
Teleost Fish
P-24
Brian C. Wilson, Acadia University, Canada
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xiii
Insulin-like Peptide 7 (Relaxin 3): Distribution and Interaction with Two Functionally Distinct
Binding Sites in the Rat Brain
P-25
Jaw Kang Chang, Phoenix Pharmaceuticals, Inc., USA
Central Administration of Human Relaxin-2 in Adult Male Rats Inhibits Food Intake
P-26
*Barbara M.C. McGowan, Imperial College London, United Kingdom
Verification of a Relaxin-3 Knockout/LacZ Reporter Mouse as a Model of Relaxin-3 Deficiency
P-27
*Craig M. Smith, Howard Florey Institute, Australia
Effects of Intracerebroventricular Injection of RXFP3 Agonist and Antagonist Peptides on
Behaviour of Wildtype and Relaxin-3 Knockout Mice
P-28
*Craig M. Smith, Howard Florey Institute, Australia
Distribution of Relaxin-3 mRNA, Relaxin-3 Immunoreactivity and RXFP3 Binding Sites in the Brain
of the Macaque (Macaca fascicularis)
P-29
*Sherie Ma, Howard Florey Institute, Australia
Relaxin Receptor LGR7 Is Regulated by Estrogen
P-30
*Priya Maseelall, New Jersey Medical School, USA
Prolonged RXFP1 and RXFP2 Signalling Is Due to Weak Internalization and a Lack of
βArrestin Recruitment
P-31
*Gabrielle E. Callander, Howard Florey Institute, Australia
The Regulatory Action of Insulin, IGF1 and Relaxin on Nitric Oxide Synthase Activity and its
Impairment in Rat Skeletal Muscle in Condition of Streptozotocin Diabetes of Type II
P-32
Ludmila A. Kuznetsova, Russian Academy of Sciences, Russia
Synthetic Peptides, Derived from the Third Intracellular Loop of Relaxin Receptor LGR7, as Probes
for Study of Molecular Mechanisms of Relaxin Action on Adenylyl Cyclase Signaling System
P-33
Alexander O. Shpakov, Russian Academy of Sciences, Russia
Regulation of Lgr8 (RXFP2) Expression in the Mouse Fetal Kidney by the Transcription
Factor Pod1
P-34
Laura J. Parry, The University of Melbourne, Australia
Expression Profile of Insulin-like Peptide-5 (INSL5) and GPCR142—A Perspective on Functional
Roles and Future Directions
P-35
Qian Sang, Howard Florey Institute, Australia
Changes in Arterial Function by Chronic Relaxin Infusion Are Mediated by the Leucine Rich Repeat
G Coupled Lgr7/RFXP1 Receptor
P-36
Julianna E. Debrah, Magee-Womens Research Institute, USA
Relaxin Promotes Matrix Metalloproteinase-2 and Decreases Wnt/β-Catenin Expression in the
Neonatal Porcine Heart
P-37
Teh-Yuan Ho, Rutgers University, USA
Boar Testis Acts as a Source and Target Tissue of Relaxin
P-38
Tetsuya Kohsaka, Shizuoka University and Gifu University, Japan
α-Adrenergic Activation Upregulates Expression of Relaxin Receptor RXFP-1 in Cardiomyocytes
XiaoLei Moore, Baker Heart Research Institute, Australia
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-39
Relaxin/RXFP1 Signaling in Prostate Cancer Progression
P-40
Shu Feng, Baylor College of Medicine, USA
9:00–10:30 AM
Platform Session 11: Novel Actions of Relaxin
Presentations O45–49
Chairs: K. Conrad and D. Bani
9:00 AM
25 min
Relaxin’s Induction of Specific MMPs Contributes to Degradation of Cartilage Matrix in Target
Synovial Joints: Receptor Profiles Correlate with Matrix Remodeling Responses
O-45
Sunil Kapila, Department of Orthodontics and Pediatric Dentistry, University of Michigan, USA
9:25 AM
25 min
Relaxin’s Involvement in ECM Homeostasis: Two Diverse Lines of Evidence
O-46
Timothy Cooney, Department of Orthopaedics, Hamot Medical Center, USA
9:50 AM
25 min
Relaxin in the Airway and Lung: Potential as a Novel Treatment for Asthma
O-47
Mimi L.K. Tang, Department of Allergy and Immunology, Royal Children’s Hospital, Melbourne, Australia
10:15 AM
25 min
Scar Reduction and Cosmetic Effects of Relaxin
O-48
Dennis Stewart, Corthera Inc. (BAS Medical Inc.), USA
10:40 AM
15 min
Role of Relaxin during Human Osteoclastogenesis
O-49
Alberto Ferlin, Department of Histology, Microbiology and Medical Biotechnologies, University
of Padova, Italy
10:55–11:15 AM
Morning Break
11:15–1:30 PM
Platform Session12: Renal Actions of Relaxin
Presentations O50–56
Chairs: T. Dschietzig and G. Weiss
11:15 AM
25 min
In Search of the Elusive Vasodepressor Agents of Pregnancy
O-50
Kirk Conrad, University of Florida, USA
11:40 AM
25 min
Relaxin: An Endogenous Renoprotective Factor?
O-51
Tim Hewitson, Department of Nephrology, Royal Melbourne Hospital, Australia
12:05 PM
25 min
Investigations into the Signaling Mechanisms by which Relaxin Inhibits Renal
Myofibroblast Differentiation
O-52
Chrishan Samuel, Howard Florey Institute, Australia
12:30 PM
15 min
Relaxin-induced Changes in Renal Function and RXFP1 Receptor Expression in the Female Rat
O-53
*Alsadek Bogzil, Faculty of Life Sciences, University of Manchester, United Kingdom
12:45 PM
15 min
Vascular Endothelial and Placental Growth Factors: New Players in the Slow Relaxin
Vasodilatory Pathway
O-54
*Jonathan McGuane, Department of Physiology and Functional Genomics, University of Florida, USA
1:00 PM
15 min
Effects of Relaxin on the Development of Mesangial Proliferative Nephritis
O-55
Naoki Ikegaya, Department of Applied Biological Sciences, Shizuoka University, Japan
1:15 PM
15 min
Relaxin Affects Endothelial Progenitor Cell Number and Function
O-56
Mark Segal, Department of Medicine, University of Florida College of Medicine, USA
Afternoon Free—lunch and dinner on your own
Relaxin 2008
xv
7:00–8:30 PM
Platform Session 13: Relaxin and Relaxin-like Peptides in the Blood and Brain
Presentations O57–60
Chairs: A. Einspanier and C. Bagnell
7:00 PM
25 min
Evaluation of Relaxin Blood Profiles as a Means of Assessing Placental Function and
Responsiveness to Therapeutic Strategies in Mares
O-57
Peter Ryan, College of Veterinary Medicine, Mississippi State University, USA
7:25 PM
25 min
Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed-up Species: New
Methods, Uses and Findings
O-58
Bernard Steinetz, Department of Environmental Medicine, New York University School of Medicine, USA
7:50 PM
15 min
Development of Adult Neuronal-specific Relaxin-3 Knockout Rats Using Adeno-associated
Viral-driven miRNA
O-59
*Gabrielle Callander, Howard Florey Institute, Australia
8:05 PM
15 min
Presence and Role of LGR8 (RXFP2) in Thalamo- and Cortico-Striatal Circuits: Effects of
Intrastriatal Injection of INSL3 on Stereotypic Behaviour in the Rat
O-60
Andrew Gundlach, Howard Florey Institute, Australia
Friday, May 23, 2008
7:30–9:00 AM
Continental Breakfast: Kahului Room and Poster Session III
Posters P41–55
Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis
P-41
Robert G. Bennett, Veterans Affairs Medical Center and University of Nebraska
Medical Center, USA
Evaluation of Relaxin’s Anti-fibrotic Actions by SELDI-TOF Mass Spectrometry-based Protein
Profiling of Relaxin KO Mice, a Model of Progressive Fibrosis
P-42
John Wade, Howard Florey Institute, Australia
Novel EGF and IGF1 Adenylyl Cyclase Signaling Mechanisms with Similar
Structural-Functional Organization
P-43
Marianna N. Pertseva, Russian Academy of Sciences, Russia
Investigating the Role of Endogenous Relaxin in Experimental Diabetic Renal Disease
P-44
*Su Ee Wong, Howard Florey Institute, Australia
Determinants of Relaxin-induced Changes in Passive Compliance of Small Renal Arteries:
Geometric vs. Compositional Remodeling
P-45
*Dan O. Debrah, University of Pittsburgh, USA
Relaxin as a Protective Substance in the Preserving Solution for Liver Transplantation:
Spectrophotometric in vivo Imaging of Local Oxygen Supply in an Isolated Perfused Rat
Liver Model
P-46
Markus U. Boehnert, University of Bern, Switzerland
Involvement of Relaxin in Salt-sensitive Hypertension
P-47
Naoki Ikegaya, Shizuoka University, Japan
Volar Oblique Ligament Fibroblasts Express Estrogen, LGR7 Receptors
P-48
Elisa M. Konieczko, Gannon University, USA
Basement Membrane Localization of LGR7 Receptors and TGF-beta in Juvenile Skin Biopsies
Timothy E. Cooney, Hamot Medical Center, USA
xvi
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-49
Relaxin Differentially Regulates Gelatinase Expression and Activity in Cultured Gingival Fibroblasts
P-50
Chrishan S. Samuel, Howard Florey Institute and Department of Biochemistry and Molecular Biology,
The University of Melbourne, Australia
INSL3 Plays a Role in the Balance between Bone Formation and Resorption
P-51
Anastasia Pepe, University of Padova, Italy
Mutations in INSL3-RXFP2 Genes in Cryptorchid Boys
P-52
Alberto Ferlin, University of Padova, Italy
The Mechanisms behind Relaxin on Improving Muscle Healing
P-53
Yong Li, Children’s Hospital of Pittsburgh and University of Pittsburgh, USA
Relaxin Has a Role in the Molecular Dynamics of Cellular Movement
P-54
Thomas Klonisch, University of Manitoba, Canada
In vitro Relaxin H2 Strengthens Fetal Membranes?
P-55
Gillian Bryant-Greenwood, University of Hawaii, USA
9:00–10:20 AM
Platform Session 14: Relaxin and Cancer
Presentations O61–64
Chairs: P. Ryan and A. Einspanier
9:00 AM
25 min
Implications of Leukocyte Responsiveness to Tumor-derived Relaxin
O-61
Michael Cox, The Prostate Center, University of British Columbia, Canada
9:25 AM
25 min
Relaxin and INSL3 Promote Tumor Formation of Human Thyroid Carcinoma Cells
O-62
Thomas Klonisch, Department of Human Anatomy and Cell Science, University of Manitoba, Canada
9:50 AM
15 min
Estrogen and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) Modulate RLN2 Promoter Activity in
Estrogen-receptor Positive T47D Human Breast Cancer Cells
O-63
Sabine Hombach-Klonisch, Department of Human Anatomy and Cell Science, University of
Manitoba, Canada
10:05 AM
15 min
Relaxin Signaling in Human Uterine Fibroids
O-64
*Zhen Li, Department of Obstetrics and Gynecology, Baylor College of Medicine, USA
10:20–11:00 AM
Morning Break
11:00–12:30 PM
Platform Session 15: Relaxin Clinical Trials
Presentations O65–67
Chairs: D. Stewart and M. Cox
11:00 AM
30 min
Scientific Rationale and Design of a Phase I Study of Relaxin in Women with Severe Preeclampsia
O-65
Elaine Unemori, Corthera Inc. (BAS Medical Inc.), USA
11:30 AM
30 min
A Randomized, Double Blind, Placebo Controlled Trial of Relaxin for Cervical Ripening in
Post-date Pregnancies
O-66
Gerson Weiss, Department of Obstetrics and Gynecology, New Jersey Medical School, USA
12:00 PM
30 min
A Pilot Safety and Dose-finding Trial of Intravenous Recombinant Human Relaxin (rhRlx) in
Compensated Congestive Heart Failure
O-67
Thomas Dschietzig, Department of Cardiology and Angiology, Charité University Medicine, Germany
12:30 PM
Closing Remarks
12:45 PM
Closing Lunch provided: Anuenue Lawn
Relaxin 2008
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Abstracts
Monday, May 19, 2008
O-01
Structure and Activity in the Relaxin Family of Peptides
Geoffrey W. Tregear, Ross A.D. Bathgate, Mohammed Akhter Hossain, Feng Lin, Suode Zhang, Fazel Shabanpoor, Daniel
J. Scott, Sherie Ma, Andrew L. Gundlach, and John D. Wade
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and
activation, together with advances in methods for structural characterisation, has enabled the key structural features of
the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarity in
primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the
four known relaxin family peptide receptors (RXFP1-4). Most of our knowledge on structure and function relates to the
relaxin-RXFP1, INSL3-RXFP2 and relaxin 3-RXFP3 systems with information accumulating not only on the critical
ligand structures, but also the domains and residues on the receptor itself that are required for specificity and activation.
These studies provide the framework for the design of small molecule mimetics. While the B-chain cassette R-X-XX-R-X-X-I defined by Büllesbach and Schwabe is essential for binding and activation at RXFP1, it is now recognised
that the A-chain, particularly the N-terminal domain, is also critical for receptor specificity. The inter- and intra-chain
links are clearly required for structural integrity but can be replaced with the more stable dicarba residues. Studies of
the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists.
The relaxin 3 A-chain – INSL5 B-chain chimeric peptide and analogues with C-terminal truncations of the B-chain,
developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are
proving invaluable for in vivo studies of the relaxin 3 RXFP-3 system.
O-02
The Chemical Synthesis of Relaxin and Related Peptides: From Yesterday
to Tomorrow
John D. Wade, Feng Lin, Akhter Hossain, Suode Zhang, and Geoffrey W. Tregear
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Since Sanger’s pioneering discovery that insulin comprises of not one but two peptide chains that were tethered by three
disulfide bonds in a unique disposition, considerable effort has been expended upon developing methods for its efficient
chemical synthesis. Herculean efforts by Chinese, American and German teams led to the successful acquisition in the
mid-1960s of insulin via random combination in solution of the individual S-reduced chains. These findings showed
that all the information required for correct alignment and combination of the two chains are conferred by their primary
structure. However, soon after the subsequent identification and establishment of the insulin superfamily of peptides,
including the relaxins, it became apparent that such methods were not usefully applicable to the chemical assembly of
relaxin as a consequence of the peptide’s less favourable physicochemical properties. However, the refinement of the
solid phase peptide synthesis methodology together with the development of a variety of cysteine S-protecting groups
that were orthogonal to one another soon allowed the successful acquisition of relaxin and related peptides via elegant
regioselective and sequential assembly of the three disulfide bonds. Such methods have been instrumental in allowing
detailed structure-function and biochemical and physiological studies to be carried out. However, there remains a need
for still more efficient means of chemical synthesis of multicystine double-stranded peptides, particularly involving
fewer reactions steps. We describe novel approaches that include the use of single mini-proinsulin-like peptides together
with one-pot sequential disulfide bond formation.
2
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-03
Development of High Affinity INSL3 Antagonists
Fazel Shabanpoora,b, Richard A. Hughesc, Frances Separovicb, and John D. Wadea
a
Howard Florey Institute, bSchool of Chemistry, cDepartment of Pharmacology, The University of Melbourne, Parkville,
Victoria 3010, Australia
Insulin-like peptide 3 (INSL3), also referred to as Leydig-insulin-like (Ley-I-L) and relaxin-like factor (RLF), belongs
to the relaxin-insulin superfamily of peptides. INSL3 plays important roles in the reproductive system, such as the
meiotic progression of arrested oocytes in females and the suppression of male germ cell apoptosis. INSL3 consists
of two chains (A and B) connected via three disulfide bonds. Our previous structure-activity relationship studies on
INSL3 led to identification of a cyclic peptide mimetic of the INSL3 B-chain which inhibits binding of 33P-relaxin to the
receptor RXFP2 in cell culture (pKi=6.65±0.09, n=3). This compound was designed based on the previously identified
INSL3 antagonist (pKi=5.86±0.03, n=3) which we showed to block the spermatogenesis in vivo in rats. However, the
INSL3 analogues that have been developed to date are active within the micromolar range. Therefore, our aim is to
develop specific mimetics of INSL3 with still higher receptor binding affinity for therapeutic applications in the area
of fertility control. To further explore the structure-activity relationships for INSL3 antagonists, we have used SYBYL
molecular modeling software to design three series of mimetics: [1] disulphide-constrained, [2] lactam-constrained, and
[3] those containing helicogenic residues and N-caps. These compounds were synthesized by solid phase methodology
and their secondary structure was determined using circular dichroism (CD) spectroscopy. All mimetics showed a low
level of α-helicity in phosphate buffered saline (PBS, pH 7.5), but dramatically increased helicity upon addition of
20% trifluoroethanol (TFE). Currently, we are performing competition binding assays to determine the affinity of these
analogues for the INSL3 receptor, RXFPR2.
O-04
The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding
and Activation of the Different RXFP Receptors
Mohammed Akhter Hossaina, K. Johan Rosengrenb, Linda M. Haugaard-Jönssonb, Soude Zhanga, Sharon Layfielda, Tania
Ferraroa, Geoffrey W. Tregeara, John D. Wadea, and Ross A. D. Bathgatea
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bDepartment of Chemistry and Biomedical
Sciences, University of Kalmar, Kalmar, Sweden
The role of the A-chain in H2 and H3 relaxin activity is currently unknown. A recent study has shown that INSL3 can be
truncated at the N-terminus of its A-chain by up to 9 residues without affecting RXFP2 binding affinity while becoming
a high affinity antagonist. This suggests that the N-terminus of the INSL3 A-chain contains residues essential for RXFP2
activation. In this study we have synthesized A-chain truncated H2 and H3 relaxin peptides, characterized their structure
by CD spectroscopy and NMR and tested their binding and cAMP activities on RXFP1, RXFP2 and RXFP3. In stark
contrast to INSL3, A-chain truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently
cAMP stimulatory activity with truncation past 8 amino acids. Replacement of these residues at the N-terminus with
4 or 5 alanine residues resulted in the partial rescue of binding activity and potency at RXFP1 but not at RXFP2. H3
relaxin A-chain truncated peptides displayed similar properties on RXFP1 highlighting that H3 relaxin binds to RXFP1
via the same mechanism as H2 relaxin. In contrast, A-chain truncated H3 relaxin peptides showed identical activity on
RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. CD and NMR studies
demonstrated that removal of the N-terminal A-chain residues resulted in a significant decrease in overall structure of
both H2 and H3 relaxin. This study demonstrates the subtle differences in the role of the A-chain in the interactions
between relaxin family peptides and their receptors.
Relaxin 2008
3
O-05
Structural Insights into the Function of Relaxins
K. Johan Rosengrena, Ross A.D. Bathgateb, David J. Craikc, Norelle L. Dalyc, Linda M. Haugaard-Jönssona, Mohammed
Akhter Hossainb, and John D. Wadeb
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden; bHoward Florey Institute, The University
of Melbourne, Parkville, Victoria 3010, Australia; cInstitute for Molecular Bioscience, University of Queensland, Brisbane,
Queensland 4072, Australia
a
The relaxin family of peptide hormones share a structural fold that is characterised by two peptide chains that are crossbraced by three disulfide bonds. Onto this framework, which besides the disulfide array has limited conserved structural
features, various amino acid side chains are presented for interactions with the relaxin receptors. Relaxins activate
two unrelated classes of G-protein coupled receptors. The relaxin and INSL3 receptors (LGR7 and LGR8) belong to
the Leucine-rich repeat containing GPCRs, which have a large N-terminal ligand-binding domain, while the smaller
relaxin-3 and INSL5 receptors (GPCR135 and GPCR142) are classic peptide GPCRs.
Interestingly relaxin-3 has the ability to activate its own receptor GPCR135, as well as the GPCR142 and LGR7
receptors. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to
interact with the various receptors we have studied these peptides using NMR. Full 3D structures of both relaxin-3 and
INSL3 have been determined and revealed a number of interesting features. Both peptides show a significant amount
of line-broadening in certain regions, in particular around the intra A-chain disulfide bond, suggesting that despite the
disulfide bonds the fold is rather dynamic. While the peptides share a common structural core there are significant
differences, particularly around the termini. For example in relaxin-3 the A-chain N-terminus is fully structured into a
helix while in INSL3 the first five residues are disordered. The structural data in combination with mutational studies
provide valuable insights into the structure-activity relationships of relaxins.
O-06
Probing the Functional Domains of Relaxin-3 and the Creation of a Selective
Antagonist for GPCR135 over Relaxin Receptor LGR7
Changlu Liua, Chester Kueia, Steven Suttona, Jonathan Sheltona, Jessica Zhua, Diane Nepomucenoa, Mohammed Akhter
Hossainb, John D. Wadeb, Ross A.D.Bathgateb, Pascal Bonaventurea, and Timothy Lovenberga
a
Johnson & Johnson Pharmaceutical Research & Development, LLC, San Diego, California 92121, USA; bHoward Florey Institute, The
University of Melbourne, Parkville, Victoria 3010, Australia
Both relaxin-3 and its receptor (GPCR135) are predominantly expressed in brain regions known to play important roles
in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding
behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and
other potential functions remain to be studied. Since relaxin-3 also activates the relaxin receptor (LGR7), which is also
expressed in the brain, selective GPCR135 agonists and antagonists are crucial to study the physiological functions
of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric
relaxin-3/INSL5 peptide, designated R3/I5). In this report, we describe the creation of a high affinity antagonist
for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BD23-27)R/I5, consists of a modified
relaxin-3 B-chain (a replacement of Gly23 to Arg and removal of Gly24-Trp27) and the A-chain of INSL5. In vitro
pharmacological studies show that R3(BD23-27)R/I5 binds to human GPCR135 and GPCR142 with high affinity and is
a potent functional GPCR135 antagonist (pA2 = 9.15), but is not a human LGR7 ligand. Furthermore, R3(BD23-27)R/
I5 is also a potent selective rat GPCR135 antagonist. It had a similar binding profile at the rat GPCR135 receptor (pA2 =
9.6) and lacked affinity for the rat LGR7 receptor completely.
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-07
Structural Characterization of the R3/I5 Chimeric Relaxin Peptide
Linda M. Haugaard-Jönssona, Mohammed Akhter Hossainb, Norelle L. Dalyc, Ross A.D. Bathgateb, David J. Craikc, John D.
Wadeb, and K. Johan Rosengrena
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden; bHoward Florey Institute, The University
of Melbourne, Parkville, Victoria 3010, Australia; cInstitute for Molecular Bioscience, University of Queensland, Brisbane,
Queensland 4072, Australia
a
Selective agonist and antagonist for each individual relaxin receptor, would be highly desirable for in vivo studies, in
order to fully understand the biological significance of the different relaxin ligands. Recent reports demonstrated that
such relaxin analogues can be achieved by developing chimera peptides, where the A-chain from one relaxin ligand
is combined with the B-chain from another relaxin ligand. Using this strategy a selective peptide for GPCR135 and
GPCR142 over LGR7 was generated by the combination of relaxin-3 B-chain with the A-chain from INSL5 (R3/I5).
Here we have used NMR spectroscopy to determine the 3D structure of this R3/I5 chimera to gain structural insight into
the consequences of combining chains from two relaxins. In particular it was of interest to see if the relaxin-3 B-chain
has undergone conformational changes or if additional LGR7 binding sites are located in the A-chain and thereby lost
when generating the R3/I5 chimera peptide.
The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B-chain compared to native relaxin-3 and
the INSL5 A-chain displays a relaxin/insulin like fold with two parallel helices. These findings indicate that binding
and activation of the smaller relaxin receptors GPCR135 and GPCR142 mainly requires the B-chain and that the
A-chain function as bulk and support to the structure. In contrast, the larger LGR7 demonstrate a more complex binding
mechanism involving both the A-chain as well as the B-chain.
O-08
Degradation of Relaxin Family Peptides by Insulin-degrading Enzyme
Robert G. Bennetta,b,c, Dean G. Heimanna, and Frederick G. Hamela,b
a
Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA; bDepartments of Internal Medicine, Pharmacology and Experimental
Neuroscience, and cDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha,
Nebraska 68198, USA
Insulin-degrading enzyme (IDE) is a ubiquitously expressed metalloproteinase responsible for the intracellular
degradation of insulin. IDE also degrades other members of the insulin superfamily, including relaxin. No studies have
been reported regarding the interaction of other relaxin-like peptides with IDE. Purified rat liver IDE was utilized to
examine the binding and degradation properties of porcine relaxin, or human InsL3, and relaxin-3. First, we assessed the
ability of each peptide to competitively inhibit the degradation of radiolabeled insulin. As observed previously, relaxin
inhibited insulin degradation less potently than insulin (IC50 182.0nM vs. 36.3nM respectively). In contrast, both InsL3
and relaxin-3 inhibited IDE with potencies approaching that of insulin (IC50 44.4nM and 53.7nM, respectively). Next,
to determine the ability to compete with insulin for binding to IDE, covalent cross-linking of radiolabeled insulin to
IDE was performed in the presence of excess (1µM) relaxin family peptides. All peptides reduced insulin binding to
IDE. Finally, nonreducing SDS-PAGE was performed to assess actual degradation of the peptides. All peptides were
degraded but with varying rates (insulin>relaxin>InsL3=relaxin-3). The degradation of all peptides was reduced by the
IDE inhibitor 1,10-phenanthroline. In summary, the data suggest that relaxin, InsL3 and relaxin-3 all interact with IDE,
and are able to interfere with the binding and degradation of insulin by IDE. The three relaxin family peptides are all
substrates for the proteolytic activity of IDE. Therefore, it is possible that in addition to insulin, IDE may be important
for the cellular proteolysis of relaxin, InsL3 and relaxin-3.
Relaxin 2008
5
O-09
Relaxin in Human Pregnancy: Consequences of Too Little or Too Much of a
Good Thing!
Laura T. Goldsmith
Department of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School of UMDNJ, Newark, New Jersey 07103, USA
With any bona fide hormone or growth factor, the concentrations in circulation can be critical to their effects. Low
concentrations can cause one effect, whereas high concentrations of the same factor can be responsible for an opposite
action. Unlike certain other aspects of life, in endocrinology, more is not necessarily better. This frequently complicates
our understanding of the actions of a hormone. It appears to be the case with relaxin, for which elucidation of its role in
human pregnancy has been very difficult.
Relaxin has beneficial effects upon the endometrium which are responsible for establishment of pregnancy. Relaxin
stimulates endometrial decidualization, the structural and biochemical changes in endometrial parenchymal cells, and
the accompanying angiogenesis, modulation of matrix metalloproteinase activity and increased concentration in local
immune cells required for implantation.
However, our recent data also demonstrate that either too much or too little relaxin can be detrimental. Elevated
circulating maternal relaxin concentrations are associated with premature birth. This is likely due to the effects of relaxin
at the level of the cervix, via upsetting the balance in the maintenance of cervical connective tissue architecture. In
addition, the absence of circulating relaxin during pregnancy or overabundance of circulating relaxin (hyperrelaxinemia)
during pregnancy in women may have negative consequences upon glucose metabolism. Furthermore, decreased
expression of relaxin mRNA and relaxin receptor LGR7 mRNA are associated with endometriosis, which may
impede fertility.
O-10
Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and
Inhibit Apoptosis in the Mouse Cervix and Vagina
LiJuan Yaoa, O. David Sherwooda,b, Paul Cookec, Daryl Melingc, and Alexander Agoulnikd
Department of Molecular and Integrative Physiology, bCollege of Medicine, and cDepartment of Veterinary Biosciences, University of
Illinois at Urbana-Campaign, Illinois 61801, USA; dDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston,
Texas 77030, USA
a
Relaxin promotes growth of the lower reproductive tract in rodents. The objective of this study was to determine whether
stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis
of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (S) and
epithelium (E) from wild-type (wt) and LGR7 knockout (ko) mice: wt-S+wt-E, wt-S+ko-E, ko-S+wt-E and ko-S+ko-E.
Tissue recombinants were grown for 3 weeks as subrenal capsule grafts in intact syngeneic female mice. Then, on day 1
of treatment, the hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17β.
Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on day 8 through 0600 h on
day 10 of treatment. To evaluate cell proliferation, 5-bromo-2-deoxyuridine (BrdU) was injected sc 10 h before cervices
and vaginas were collected at 1000 h on day 10. TUNEL was used to quantify apoptosis. Relaxin markedly increased
proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P<
0.01), but had no effect on tissue recombinants lacking wt stroma, regardless of whether epithelium was derived from wt
or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient
for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in the cervix and vagina,
while epithelial LGR7 does not have any effect on these processes.
6
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-11
The Good, The Bad and The Ugly: Lessons in Female Reproductive Tract
Physiology from the Relaxin Gene Knockout (Rln-/-) Mouse
Laura J. Parry
Department of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia
Targeted gene deletion in embryonic stem cells to develop a null relaxin (Rln-/-) mouse has proved an effective tool to
study the functions of relaxin in female reproductive tract physiology. However, there are a number of discrepancies
in the phenotypes reported between different studies. It is worth considering the variable effects of modifier genes
among inbred strains, phenotypic robustness and redundancy or compensation caused by genes performing similar
functions. We suggest that age is another factor that should be taken into account. The other main limitation of in
vivo mouse models of reproductive tract physiology is the extent to which they can increase our understanding of
pregnancy in humans. Despite these challenges, the pregnant Rln-/- mouse has provided useful information on the
mechanisms of relaxin action in vivo. We have shown that relaxin decreases collagen synthesis but does not increase
matrix metalloproteinase (MMP) expression in the cervix at the end of gestation. Relaxin treatment in Rln-/- mice also
affects synthesis of hyaluronan, cervical hydration and collagen dispersal. In addition, we have identified an interaction
between relaxin and estrogen receptors, and a ligand-specific down-regulation of Lgr7. Our recent studies demonstrate
novel phenotypes in the uterus and placenta of Rln-/- mice related to relaxin’s actions as a vascular hormone. There is a
reduction in endometrial angiogenesis in early gestation and abnormalities in placental development. The consequence
of these phenotypes is that pups born to Rln-/- dams are significantly smaller than their wildtype counterparts, which is
indicative of intrauterine growth restriction.
O-12
Decreased Hyaluronic Synthase Expression in the Cervix of Pregnant RelaxinDeficient (Rln-/-) Mice
Yu May Soha, Lenka A. Vodstrcila,b, and Laura J. Parrya
Department of Zoology and bDepartment of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia
a
The extracellular matrix (ECM) of the cervix changes extensively in a process called cervical ripening in late pregnancy.
This occurs through a complex series of events leading to collagen fibre degradation and dispersal. We tested the
hypothesis that relaxin increases cervical water content by stimulating the hyaluronic synthase (HAS) enzymes that
regulate hyaluronic acid (HA) synthesis. HA is thought to recruit water molecules into the spaces between collagen
fibrils and causes them to disperse. In the first study, cervices were collected from Rln+/+ and Rln-/- mice on days 14.5,
16.5, 18.5 and 19 gestation, and day 1 postpartum. HAS expression was compared between the genotypes by qPCR
analysis. In the second study, Rln-/- mice were implanted with osmotic minipumps on day 12.5 gestation to infuse rhH2
relaxin; cervices were dissected on days 16.5 and 18.5 gestation. has1 and has2 gene expression increased significantly at
term and decreased immediately postpartum in the Rln+/+ mice. Although has1 and has2 were expressed in Rln-/- mice,
there was no increase in expression on day 18.5 gestation compared with Rln+/+ mice. Similarly, cervix wet weight
did not increase in late pregnant Rln-/- mice. Relaxin infusion in pregnant Rln-/- mice significantly increased cervical
has2 but decreased has1 expression compared with saline-controls. Relaxin treatment also caused a 6-fold increase in
cervix wet weight and a 4-fold decrease in Lgr7 expression. These data suggest that relaxin promotes cervical hydration
through an has2-mediated increase in HA, and facilitates cervical ripening by dispersing collagen fibrils in the ECM.
Relaxin 2008
7
O-13
Milk-borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of
Neonatal Tissues
Carol A. Bagnella, Bernard G. Steinetzb, and Frank F. Bartolc
a
Department of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers University, New Brunswick, New Jersey 08901,
USA; bNelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA; cDepartments
of Animal Sciences and Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn,
Alabama 36849, USA
The fact that all newborn mammals drink milk extends the timeframe of maternal influence on development into
neonatal life. While nutritional and immunological benefits of milk are clear, the role of milk as a conduit for bioactive
factors with the potential to affect neonatal development is less well defined. Porcine and canine milk contain
immunoreactive relaxin (RLX) that is transmitted into the circulation of nursing offspring. In the pig, a window of
opportunity for transmission of milk-borne RLX from mother to neonate is open at birth and remains so for the first
three days of neonatal life. Recent in vitro studies showed that RLX in porcine milk is bioactive. Moreover, RLX
receptor (RXFP1) expression is detectable in the porcine female reproductive tract and other somatic tissues at birth. The
lactocrine hypothesis for maternal programming of neonatal development was proposed as a mechanism whereby RLX,
a prototypical milk-borne growth factor in the pig, is delivered to nursing offspring where it affects development of
RXFP1-positive target tissues. Data indicating that treatment of newborn gilts with RLX increased estrogen receptor-α
expression in the uterus and cervix by postnatal day 2 supports a role for RLX in lactocrine programming of the female
reproductive tract. Effects of RLX on Wnt/β-catenin expression in porcine cardiac tissue during this period support a
role for RLX in developmental programming of non-reproductive target tissues as well. Ongoing studies will test the
lactocrine hypothesis for maternal programming of development by RLX and related milk-borne factors. (Support:
USDA-NRI 2003-35203-1357 and 2007-35203-18098)
O-14
Relaxin and Maternal Lactocrine Programming of Neonatal Uterine Development
Frank F. Bartola,b, Anne A. Wileya, and Carol A. Bagnellc
Departments of aAnimal Sciences and bAnatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn
University, Auburn, Alabama 36849, USA; cDepartment of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers
University, New Brunswick, New Jersey 08901, USA
In mammals, including the pig (Sus scrofa domesticus), structural patterning and functional programming of female
reproductive tract tissues involve events that occur shortly after birth. Porcine endometrial development between birth
(postnatal day = PND 0) and PND 15 is estrogen receptor- (ER) dependent and estrogen-sensitive. The endometrium is
relaxin (RLX) receptor- (RXFP1) positive and ERα-negative at birth. Uterine expression of RXFP1 and ERα, detectable
by PND 2, increases with age from PND 0-14. Estrogen exposure during this period that is sufficient to affect uterine
developmental trajectory and adult uterine phenotype alters uterine RXFP1 and morphoregulatory gene expression
patterns, including those for Hoxa10, Wnt7a and MMP-9, by PND 14. Data implicate RXFP1 as an element of the
uterine developmental program. Uterotrophic effects documented for both estrogen and RLX in the neonatal pig are
age-specific and most pronounced after onset of ERα expression when RLX effects can be inhibited by pretreatment
with the ER antagonist ICI 182,780. Thus, crosstalk between RLX and estrogen signaling systems evolves with age
in the porcine uterus. Given that RLX administered from birth stimulates uterine ERα expression and that estrogen
administration during this period stimulates RXFP1 expression by PND 2, a developmentally relevant functional
relationship between these signaling systems is envisioned. Evidence that RLX is present in porcine milk and in the
circulation of nursing offspring supports the lactocrine hypothesis for maternal programming of uterine tissues in which
milk-borne RLX, acting via RXFP1, facilitates ERα expression and porcine endometrial development in the neonate.
(Support: USDA-NRI 2003-35203-1357, USDA-NRI 2007-35203-18098 and NSF EPS-0447675)
8
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-15
Relaxin in the Marmoset Monkey: There Are Two Sides to Every Story
A. Einspaniera, K. Eberta, K. Liedera, M. Eberta, S. Lierb, O. Lampa, R. Einspanierb, N. Beindorffc, C. Schlumbohmc, M. Kempera,
J. Jacoba, and M. Sohra
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, 04103 Leipzig, Germany; bInstitute of Veterinary Biochemistry, Freie
Universität Berlin, Germany, CGerman Primate Center, Göttingen, Germany
a
Many disorders occurring in adult life date back to influences of the intra-uterine environment. Therefore in the first
part of this paper, different factors involved in early pregnancy are analysed while in the second part, the effect of stress
throughout pregnancy on the endocrine situation and physiological parameter are examined in the marmoset monkey.
The results of part I showed a massive increase in gene and protein expression from the RLX system (RLX, RXFP1)
as well as enzymes of estrogen biosynthesis solely throughout the implantation process (day 10/11), which declined
immediately around day 14/15 of pregnancy. Steroid receptor expression remained unchanged expressed throughout
early pregnancy.
In part II, a short-term application of stress hormone during early gestation induced a significant rise of serum RLX and
E2, whereas serum P4 declined throughout the application period. No significant differences compared to uterine growth
rate, gestation length, fetal biparietal diameter, or maternal and newborn body weights were observed in all groups.
However, the early prenatal stressed females showed a significant increase in body weight (456±86g) compared to
controls (344±51g) at 20 months of age, differences in their blood fatty acids levels, and an earlier onset of cycle activity
compared to the control group.
In summary, the RLX-system is involved in the process of implantation and with the enzymes of estrogen biosynthesis
it orchestrates the remodelling of the uterine tissue. However, a stress-induced significant serum increase in RLX and
estrogens during early pregnancy seems to have an epigenetic effect on the female offspring.
Project was kindly supported by DFG (German Research Council) grant EI 333/11-1 and EU (EUPEAH QLR1-CT
2002-02758)
Some of these results are taken from the thesis by K. Ebert, S. Lier, and M. Kemper.
O-16
Expression of LGR7 in the Primate Corpus Luteum Implicates the Corpus Luteum
as a Relaxin Target Organ
Priya Maseelall, Aimee Seungdamrong, Gerson Weiss, Andrea Wojtczuk, and Laura T. Goldsmith
New Jersey Medical School, Department of Obstetrics, Gynecology, and Women’s Health, Newark, New Jersey 07103, USA
In primates, the corpus luteum is the source of circulating relaxin. No studies have determined whether the primate
corpus luteum is also a relaxin target organ. We had the opportunity to study LGR7 expression in human term
pregnancy corpora lutea and non-human primate corpora lutea obtained during the menstrual cycle. Real-time RT-PCR
demonstrated LGR7 mRNA expression in human term pregnancy corpora lutea and in rhesus monkey corpora lutea
taken at early, mid, and late luteal phases. The relative expression of LRG7 mRNA in rhesus monkey corpora lutea
during the mid and late luteal phases of the menstrual cycle was 4 fold higher than in the early luteal phase. Also, since
the primate corpus luteum is LH dependent, we assessed LGR7 mRNA expression using corpora lutea from GnRH
antagonist treated rhesus monkeys. GnRH antagonist treatment, which decreased circulating LH to undetectable levels,
resulted in a five fold increase in luteal LGR7 mRNA expression. Increased luteal expression of LGR7 mRNA in the
mid and late luteal phases of the menstrual cycle may suggest a role for relaxin in the demise of the non-pregnant corpus
luteum. In addition, these data suggest that luteal LGR7 mRNA expression may be regulated by relaxin and/or LH, and
that the primate corpus luteum is a target organ for relaxin.
Relaxin 2008
9
O-17
Characterization and Biological Activity of Prorelaxin in Porcine Milk
Amy-Lynn Frankshuna, Teh Yuan Hoa, Bernard G. Steinetzb, Frank F. Bartolc, and Carol A. Bagnella
Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA; bNelson Institute of Environmental Medicine,
New York University School of Medicine, Tuxedo, New York 10987, USA; cDepartments of Animal Sciences and Anatomy, Physiology and
Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA
a
A lactocrine mechanism for delivery of milk-borne relaxin for maternal programming of neonatal development has
been proposed. In support of this hypothesis porcine relaxin (pRLX) was detected in colostrum and in the circulation
of nursing pigs. While immunoreactive pRLX concentrations are highest in milk during early lactation (9-19 ng/ml),
declining to < 2 ng/ml by postnatal day 14, whether milk-borne pRLX is bioactive is unknown. Therefore, objectives
were to characterize the molecular size and determine the bioactivity of pRLX in milk using a bioassay validated for
use with milk. Sow milk samples collected on lactation days 0 (day of parturition) and 14 were diluted 1:1 with EDTA
to solubilize caseins and centrifuged to remove fat. Samples and native pRLX were resolved under non-reducing/
denaturing conditions by SDS-PAGE. To monitor pRLX bioactivity RXFP1-transfected HEK293T cells were employed
as targets and total cAMP was measured. Immunoblotting revealed a 19 kDa band corresponding to proRLX in day 0
milk. Bioactivity of proRLX in milk was confirmed by an increase in cAMP in response to increasing volumes of day
0 milk. Dilution curves of pRLX standards and day 0 lactation milk were parallel. Incubation with day 14 milk did not
increase cAMP. Addition of porcine insulin or human insulin-like growth factor-I (1 µg/ml) to HEK293T-RXFP1 cells or
treatment of non-transfected cells with pRLX failed to elicit a cAMP response. These data show that proRLX in porcine
milk is bioactive, supporting the lactocrine hypothesis. The extent to which milk-borne pRLX affects development
remains to be determined. (Support: USDA-NRI 2003-35203-1357 and 2007-35203-18098)
10
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Abstracts
Tuesday, May 20, 2008
P-01
Regioselective Disulfide Solid Phase Synthesis of Mouse Relaxin
Feng Lin, Eleni Giannakis, Nicola F. Hanson, Geoffrey W. Tregear, and John D. Wade
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Fibrosis is a leading cause of organ failure where normal tissue is replaced by excess connective tissue. Several organs
are prone to this process regardless of etiology. The hormone, relaxin, is emerging as a novel antifibrotic therapy. It
limits collagen production and reorganization, while stimulating increased collagen degradation. It not only prevents
firbogenesis, but also reduces established scarring. Recent studies on relaxin-deficient mice have established relaxin as
an important, naturally occurring regulator of collagen turnover and provide new insights into the therapeutic potential
of relaxin. Mouse relaxin has an insulin-like structure that consists of 25 residues A-chain and 35 residues B-chain with
three disulfide bonds, it differs from other mammalian relaxins is that the A-chain contains an additional residue within
its sequence. Mouse relaxin could not be successfully prepared by simple combination of the individual chains due to
the very poor solubility of the B-chain. This necessitated recourse to use of a regioselective disulfide bond formation
strategy. Solid phase synthesis of the separate, selectively S-protected A- and B-chains followed by their purification and
subsequent stepwise formation of each of the three disulfides via aeration, thioloysis and iodolysis led to the successful
acquisition of mouse relaxin in good overall yield. Comprehensive chemical characterization confirmed both the correct
chain orientation and the integrity of the synthetic product.
P-02
Trafficking of the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135)
Emma T. van der Westhuizena, John D. Wadeb, Patrick M. Sextona, and Roger J. Summersa
a
Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia
The aim of this study was to examine the influence of relaxin family peptides on the trafficking of the H3 relaxin
receptor, RXFP3. In CHO-K1 and HEK293 cells expressing RXFP3 (also known as SALPR or GPCR135), H3 relaxin
but not H2 relaxin, porcine relaxin or INSL3, competed for [125I]-INSL5/H3 relaxin chimaera binding. RXFP3 was
tagged at the carboxy-terminal tail with a variant of the green fluorescent protein, GFP2, and transiently expressed in
CHO-K1 and HEK293 cells. Competition for [125I]-INSL5/H3 relaxin chimaera binding was observed with H3 relaxin
in CHO-RXFP3-GFP2 (pIC50 = 8.61±0.20) and HEK-RXFP3-GFP2 cells (pIC50 = 8.93±0.50) and was consistent
with that observed at wild type RXFP3 (pIC50 = 8.36±0.16 (CHO-K1); 9.04±0.31 (HEK293)). Similarly, potent
H3 relaxin-stimulated ERK1/2 activation was observed downstream of RXFP3 (pEC50 = 9.47±0.32 (CHO-RXFP3GFP2); 8.99±0.40 (HEK-RXFP3-GFP2) 8.81±0.25 (CHO-RXFP3); 9.04±0.31 (HEK-RXFP3)). Together these results
suggested that RXFP3-GFP2 maintained the same phenotype as wild type RXFP3. Internalization of RXFP3 was
observed using radioligand binding assays and confocal microscopy. Treatment of cells expressing RXFP3 with H3
relaxin but not H2 relaxin, porcine relaxin or INSL3 (all 10nM; n=4) reduced the number of cell surface binding sites.
Likewise, loss of cell surface RXFP3-GFP2 binding sites was observed in cells treated with H3 relaxin (10nM) (n=6).
RXFP3-GFP2 internalization visualized by confocal microscopy, showed surface-expressed receptors sequestered
into vesicle-like structures 5-30 min after H3 relaxin (10nM) treatment. This study demonstrates for the first time that
RXFP3 internalizes following treatment with H3 relaxin, but not other related peptides, in CHO-K1 and HEK293 cells
transiently expressing the receptor.
12
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-03
Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodination and
Isolation of Peptide by One-step Ether Precipitation
Suode Zhang, Feng Lin, Mohammed Akhter Hossain, Fazel Shabanpoor, and John D. Wade
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Multiple disulfide bond-containing peptides have long been challenging synthetic targets. The selection of appropriate
sulfhydryl protecting groups is crucial for subsequent correct disulfide bond formation. Among various sulfhydryl
protecting groups, the S-acetamidomethyl (Acm) protecting group is particularly valuable. Treatment of peptides
containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting groups and
disulfide formation. This method is now commonly used. Unfortunately, there are often side reactions associated with
this reaction, particularly with tryptophan and tyrosine residues, and thus excess iodine should be quenched or adsorbed
as quickly as possible after completion of the disulfide bond formation. This is usually achieved by addition of sodium
bisulfate, sodium thiosulfate, ascorbic acid, powdered zinc dust, activated charcoal or by dilution with water followed by
extraction with carbon tetrachloride. However, the quenching reagents themselves can generate additional side reactions
as does lyophilization of the peptide solution. Thus a new method for the rapid quenching of the iodination and isolation
of peptides is urgently required. In the present study, it was established that simple ether precipitation can fulfill these
requirements. Peptides containing pairs of S-Acm protecting groups are dissolved in 95% acetic acid for iodination.
After completion of the disulfide bond formation, ice-cooled ether is simply added to the reaction solution for quenching
of iodination and simultaneous precipitation of peptide that can be stored or applied directly onto an HPLC column for
purification. This method is particularly beneficial for the disulfide synthesis of insulin-like peptides such as INSL3.
P-04
Chemical Synthesis and Biological Activity Evaluation of β-Ala and γ-Abu
Substituted Insulin-like Peptide 3 Analogs
Suode Zhanga, Ross A. Bathgatea, Geoffrey W. Tregeara, Richard A. Hughesb, and John D. Wadea
Howard Florey Institute and bDepartment of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia
a
Insulin-like peptide 3 (INSL3, also known as relaxin-like factor, RLF) is now recognized to be a major circulating
hormone in the male and female with important fertility regulatory roles. The receptor for INSL3, LGR8, is a member
of the leucine-rich repeat containing G-protein coupled receptor family. Solution NMR spectroscopy showed that INSL3
adopts a characteristic relaxin/insulin-like fold but is a highly dynamic molecule. The B-chain contains a long helical
segment which spans residues B12-B22. Detailed structure-activity studies using synthetic Ala-substituted analogues
of INSL3 have shown that its receptor ectodomain binding site is mediated by five key residues within the B-chain,
with four of these HisB12, ArgB16, ArgB20, ValB19 and Trp B27 located on one face of the helix. We designed two INSL3
analogs in which AlaB17 was substituted with the conformationally different β-Ala and γ-Abu which were expected
to alter the position of the side chains of the critical ArgB16 and ValB19 as well as to confer protective effects from
endoproteolysis. The peptides were prepared by optimized regioselective directed disulfide bond solid phase synthesis
and, after purification, testing for their binding affinity for, and cAMP activity on, cells expressing LGR8. It was found
that the β-Ala-substituted INSL3 analog had, slightly higher binding affinity together with increased potency in LGR8
expressing cells. In contrast, the γ-Abu-substituted INSL3 analog exhibited similar binding affinity and activity to native
INSL3. The observations will provide new directions for the design of novel specific agonists and antagonists of the
LGR8 receptor.
Relaxin 2008
13
P-05
Synthesis, Conformation and Biological Activity of Novel Dicarba Analogs of
Human Relaxin-3/INSL7
Mohammed Akhter Hossaina, Ross A.D. Bathgatea, Bianca J. van Lieropb, Andrea J. Robinsonb, K. Johan Rosengrenc,
Geoffrey W. Tregeara, and John D. Wadea
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bSchool of Chemistry, Monash University, Clayton,
Victoria 3168, Australia; cDepartment of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden
Disulfide bonds are common structural elements in many peptides and proteins where they play a crucial role in
maintaining an active conformation. Such bonds can be susceptible to rupture by intracellular enzymes such as
disulfide reductase which, in turn, can decrease the half life of the biomolecule rendering it less effective as therapeutic
compound. The replacement of disulfide bonds with non-reducible isosteres is a useful means of increasing the in vivo
stability of a protein. Consequently, we have replaced the intramolecular disulfide bond of human relaxin-3 (H3 relaxin),
a peptide that has potential for the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid
phase synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular
bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed
to install the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification and B-chain
combination via two intermolecular disulfide bonds, then furnished dicarba-relaxin-3. Two isomers (cis and trans) of the
dicarba H3 relaxin were isolated and characterized by both CD and NMR spectroscopy which confirmed their structural
similarity to the native peptide. Both analogs demonstrated equivalent binding affinity to native H3 relaxin on RXFP1
and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was
the same as the native peptide, the potency on RXFP1 expressing cells was slightly lower. The in vivo stability of the
peptides is currently under investigation.
P-06
Human Insulin-like Peptide 5 (INSL5): Synthesis and Activity on RXFP3 and RXFP4
Receptors
Mohammed Akhter Hossain, Ross A.D. Bathgate, Chze K. Kong, Geoffrey W. Tregear, and John D. Wade
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Insulin-like peptide 5 (INSL5) was first identified by searching EST databases. Primary structure analysis showed it
to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain structure (A and B) containing the
insulin-like disulfide crosslinks. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142)
coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that
INSL5 is an endogenous ligand for RXFP4. Given that the primary function of INSL5/RXFP4 pair remains unknown, an
effective means of producing sufficient quantities of this peptide and its analogues is needed in order to systematically
investigate its structural and biological properties. A combination of solid phase methods together with regioselective
disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to successful synthesis and
required highly optimized conditions for their acquisition. In particular, the use of strong tertiary amidine, DBU, as
Nα-deprotection base was required for the assembly of the B-chain. Following sequential disulfide bond formation
and chain combination, the resulting synthetic INSL5, obtained in good overall yield, was shown to possess similar
secondary structure like other insulin-like peptides. The peptide was able to inhibit cAMP activity in SKN-MC cells
expressing the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast it had no activity on the human
RXFP3 receptor. Synthetic INSL5 therefore demonstrates equivalent activity to the recombinant peptide and will be a
useful tool to determine the biological function of INSL5.
14
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-07
De novo Design and Synthesis of Cyclic and Linear Peptides to Mimic the B-chain
Binding Elements of Human Relaxin Family Peptides
Mohammed Akhter Hossain, Ross A. D. Bathgate, Geoffrey W. Tregear, and John D. Wade
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Human relaxin family peptides contain distinct amino acids in their B-chains that are responsible for the binding
specificity to Relaxin Family Peptide Receptors (RXFP1-4). In particular the trivalent structure (R-X-X-X-R-X-X-I)
within the B-chain of relaxin is responsible for its specificity to RXFP1. In conjunction with some of these residues Trp27 in INSL3 and the equivalent Trp-25 in H3 relaxin are important for RXFP2 and RXFP3 activity respectively. In an
attempt to mimic these motifs, we have designed six cyclic peptides using regioselectively addressable functionalized
template (RAFT) to direct the side chains of Arg, Arg and Ile towards one direction with or without the additional Trp
residue. RAFT, which adopts a solution structure based on an antiparallel beta sheet conformation closed by two beta
turns centered on Pro-Gly dipeptides, is a means of arranging multiple functional groups in an appropriate orientation.
Additionally, two domain minimized linear analogs of the H3 B-chain with internal salt bridges to increase their
a-helicity were also made to compare their binding affinity with the RAFTs. None of the RAFT peptides showed any
binding affinity for RXFP1, RXFP2 or RXFP3. Circular dichroism spectral analysis showed that all six RAFT molecules
exhibited partial β-sheet structure along with two beta turns but no α-helix. This lack of α-helical structure is the likely
reason for their lack of activity. The linear analogs showed similar affinity to RXFP3 as the native linear H3 relaxin
B-chain suggesting that the internal salt bridges were unable to increase the α-helical structure and increase
binding affinity.
P-08
Investigating the Molecular Mechanics of Relaxin Family Peptide Receptor
Function Using Time Correlated Single Photon Counting Confocal Microscopy
Daniel J. Scotta, Brian Jonesb, Geoffrey W. Tregeara, and Ross A.D. Bathgatea
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bOlympus Australia, Mt. Waverley,
Victoria 3149, Australia
G-protein coupled receptors are dynamic proteins and their cellular signaling properties are influenced by sub-cellular
localization, molecular conformation and oligomerization. In order to investigate the molecular mechanics of relaxin
family peptide (RXFP) receptors, one must be able to analyze receptor states at a single cell level. Recently developed
time-correlated single photon counting (TCSPC) systems enable highly accurate measurement of fluorescence lifetime
(FLIM) and fluorescence correlation spectroscopy (FCS), both powerful techniques for investigating protein dynamics
and interactions, in single cells via a confocal microscope. TCSPC based confocal microscopy was used to investigate
the molecular mechanics of RXFP1 and RXFP2 function, particularly focusing on the role of receptor oligomerization.
RXFP1 and RXFP2 were tagged in several positions with various donor and acceptor fluorophores and their cellular
localization and functional ability to increase cAMP in response to ligand was tested. Functional tagged receptor pairs
were then utilized with TCSPC to measure FLIM-FRET by determining any decrease in fluorescence lifetime of donor
fluorophores as a result of energy transfer to acceptor fluorophores. FLIM-FRET analysis in HEK cells expressing both
donor tagged and acceptor tagged RXFP1 indicated a significant population of closely interacting receptors in all cellular
areas of receptor expression. The signal could be removed by increasing expression of untagged RXFP1, indicating
specific oligomerization. Now that the system is optimized, we aim to use FLIM-FRET and FCS to delve further into
the molecular mechanics and conformational changes that occur during RXFP1 and RXFP2 activation, with the ultimate
goal of aiding agonist and antagonist design.
Relaxin 2008
15
P-09
Withdrawn
P-10
Identification of the Relaxin-responsive Cells in the Human Chorion and Decidua
Jaime S. Horton, Sandra Y. Yamamoto, and Gillian D. Bryant-Greenwood
Department of Cell and Molecular Biology, University of Hawaii, Honolulu, Hawaii, 96813 USA
The human fetal membranes lie between the myometrium and amniotic cavity forming the fetal container or sac. This
complex tissue together with the decidua, forms a large maternal-fetal interface and is the site of the molecular crosstalk which results in human parturition. The decidua is a source of intrauterine relaxin and the choriodecidua expresses
its receptor (LGR7). Since these tissues consist of a variety of maternal and fetal cells, we sought to identify the
primary cell(s) responsible for LGR7 expression and relaxin responsiveness. Decidual stromal cells and macrophages
were isolated by scraping the membranes, followed either by enzymatic digestion and Percoll gradient selection or by
FACS cell sorting, based upon the markers CD14 and CD68. Chorionic cytotrophoblasts were isolated by stripping the
amnion, enzymatic digestion and Percoll gradient selection. The stromal cells showed low LGR7 expression, but a 4 day
treatment with decidualization cocktail significantly increased (p=0.03) LGR7 and prolactin expression. However, their
cAMP response to relaxin was reduced. LGR7 expression by decidual macrophages was variable and about 4-fold less
than THP-1 cells, correlating with their cAMP responses. On the other hand, cytotrophoblasts consistently expressed
LGR7 and demonstrated a strong cAMP dose-response to relaxin. Therefore, the fetal cytotrophoblast is being used to
further characterize its response(s) to relaxin. Supported by NIH grant HD24314.
16
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-11
RXFP1 Is Expressed on Sperm Acrosome and Relaxin Stimulates the Acrosomal
Reaction of Human Spermatozoa
Lisa Gianesello, Alberto Ferlin, Massimo Menegazzo, Anastasia Pepe, and Carlo Foresta
University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for
Male Gamete Cryopreservation, 35121 Padova, Italy
The function of relaxin in human reproductive processes remain poorly understood. Nevertheless, relaxin is produced
by the prostate in men and by the corpora lutea of ovaries and endometrium in women. Previous studies with contrasting
results have suggested a possible role of relaxin on fertilization. In this study we analysed the expression of the
relaxin receptor RXFP1 on human sperm and the in vitro effect of relaxin on sperm function. Measurement of relaxin
concentration in the seminal plasma of 10 normozoospermic men showed that it is present at high levels (1253±445pg/
ml). These concentrations are similar to those found in the serum of pregnant women (1000-3500pg/ml), and
significantly higher to those found in the serum of the same male subjects (32±17pg/ml). RXFP1 protein was detected
by immunofluorescence on the acrosomal cap of human spermatozoa. This data was confirmed by the co-localization of
RXFP1 and the specific acrosomal staining Pisum Sativum, and by the disappearance of RXFP1 fluorescence after the
induction of the acrosomal reaction by calcium-ionophore. The analysis of acrosome reaction in vitro under different
doses of relaxin showed that the hormone increases the percentage of sperm reacted after 1 and 3h. Interestingly, the
entity of acrosome reaction induced by relaxin is similar to that obtained with progesterone, the hormone produced by
the corpus luteus and considered the physiological inductor of acrosome reaction. These data suggest a physiological
role for relaxin produced by the prostate and/or the female genital tract after ovulation.
P-12
Involvement of Relaxin in Endometriosis
Sara S. Morellia, Felice Petragliab, Gerson Weissa, Stefano Luisib, Pasquale Floriob, Jeff Gardnerc, Andrea Wojtczuka, and
Laura T. Goldsmitha
Obstetrics, Gynecology and Women’s Health, and cPediatrics, New Jersey Medical School, Newark, New Jersey, 07101, USA; bPediatrics,
Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
a
Endometriosis, a disease characterized by the presence of endometrial stromal and glandular tissue at an extrauterine
(ectopic) site, affects 10-15% of all reproductive-aged women and is an important contributing factor to chronic pelvic
pain and infertility. Matrix metalloproteinases have been implicated in the establishment of endometriotic lesions. Since
relaxin is a potent inhibitor of endometrial matrix metalloproteinase expression, we hypothesized that relaxin plays
a role in endometriosis. Expression of relaxin mRNA and its LGR7 receptor mRNA in normal human endometrium
were compared to expression in samples from patients with endometriosis. Total RNAs, extracted from ectopic (n=8)
and eutopic (uterine) (n=11) endometrium of patients with endometriosis, and from endometrium of normal controls
(n=12), were subjected to real time RT-PCR. Relaxin mRNA was detectable in normal endometrium from 9 of 12 (75%)
control patients and detectable in a lower proportion of samples [9 of 19 (47.4%)] from patients with endometriosis.
LGR7 mRNA was detectable in all samples, with lower expression in endometriosis samples than in endometrium from
controls. Relaxin receptor LGR7 mRNA levels vary with cycle phase, with greater expression in the secretory phase
(SP) than in proliferative phase (PP). In both phases, LGR7 mRNA levels were lower in ectopic samples than in either
eutopic samples (6.2-fold lower in PP and 19.0-fold lower in SP) or endometrium from normal controls (6.5-fold lower
in PP and 15.6-fold lower in SP). Decreased local expression of relaxin and LGR7 mRNA in ectopic endometrium from
patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective
against endometriosis.
Relaxin 2008
17
P-13
Relaxin and Neopterin as Local Ovarian Granulosa Cell Growth- and Colonystimulating Factors
Elissaveta Zvetkovaa, Roumiana Denkovaa, Violetta Bournevaa, Konstantza Balevaa, Yordanka Martinovaa, Boitcho Nikolova,
Tanya Timevab, and Dietmar Fuchsc
IEMAM-BAS, Sofia, Bulgaria; bInstitute of Obstetrics and Gynecology, MU Sofia, Bulgaria; cInnsbruck Medical Universty, Innsbruck, Austria
a
The influence of relaxin (RLX) and neopterin (NeoPt) on the porcine- and human ovarian granulosa cell proliferation,
differentiation and steroidogenesis has been studied in vitro - in conditions of OGCscultures. It was established that
two local biologically active substances stimulate in vitro OGCs proliferation /cell colony-formation, differentiation
and steroidogenesis (Endocr. Regul., 32, 1998, 33 - 43; Acta morphologica et anthropologica, /Sofia/, 7, 2002, 28 31). The similar mitogenic effects of both cell growth factors - relaxin and neopterin, on the in vitro cultured OGCs,
could be mediated through increased expression of inducible nitric oxide synthase and by activation of NOSII nuclear
transcription factor NF-kappa B. The in vitro model indicating a role of relaxin peptide and of pteridine neopterin as
ovarian granulosa cell proliferative and differentiating factors could be applied in further clinical studies on cellular and
molecular mechanisms associated with physiological and/or neoplastic cell growth.
P-14
The Lethal Phenotype in Relaxin-deficient (Rln-/-) Mice Is Not Due to Impaired
Mammary Gland Development or Milk Protein Synthesis
Laura J. Parrya, Lenka A. Vodstrcila,b, Anna Maddena, Stephanie H. Amira, Katrina Baldwina, Mary E. Wlodekb,
and Kevin R. Nicholasa,c
a
Departments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cCRC for Innovative Dairy
Products, Melbourne, Australia
Relaxin is essential for nipple development during late pregnancy in mice and may also stimulate mammary gland
growth. This study tested the hypothesis that relaxin is involved in milk protein synthesis as well as mammary gland
development in pregnant mice. Comparisons between wildtype (Rln+/+) and relaxin-deficient (Rln-/-) mice showed no
obvious differences in either lobuloalveolar structure or ductal branching in the mammary gland. Mammary explants
obtained from Rln-/- mice on day 16.5 gestation expressed β-casein and α-lactalbumin in response to lactogenic
hormones at a similar level to Rln+/+ mice, implying normal milk protein synthesis. Exogenous relaxin treatment
had no significant effect on milk protein expression in mammary tissue in vitro. These data are explained by a lack
of relaxin receptors in mouse mammary tissues. Pregnant Rln-/- mice chronically infused with human relaxin for
6 days gave birth to live pups without difficulty, and 96% pups survived beyond 7 days. However, birth weights of
the relaxin-treated Rln-/- young were significantly lower than Rln+/+ pups. Infusion of relaxin for 3 days was not
sufficient to prevent pup mortality. The lactiferous ducts of relaxin-treated Rln-/- mice were devoid of milk, but there
were no developmental differences in the lobuloalveolar tissue compared with saline-infused controls. In summary,
mammary gland development in Rln-/- mice is not different from Rln+/+ mice and relaxin is not critical for β-casein or
α-lactalbumin synthesis. The lethal phenotype in Rln-/- mice results from insufficient milk delivery caused by impaired
nipple development and inadequate sucking stimulus from the pups.
18
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-15
Laser Microdissection of Neonatal Porcine Endometrium for Tissue Specific
Evaluation of Relaxin Receptor (RXFP1) Expression in Response to Perinatal
Zearalenone Exposure
Anne A. Wileya, Johannes Kauffoldb, Martin Wähnerc, Bethany Crean-Harrisa, Dori J. Millera, Carol A. Bagnelld,
and Frank F. Bartola,e
Departments of aAnimal Sciences and eAnatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn
University, Auburn, Alabama 36849, USA; bFaculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany; cAnhalt
University of Applied Science, 06406 Bernburg, Germany; dDepartment of Animal Sciences, Endocrinology and Animal Biosciences
Program, Rutgers University, New Brunswick, New Jersey 08901, USA
In the pig (Sus scrofa domesticus), relaxin receptor (RXFP1) expression is detectable in the endometrium at birth
(postnatal day = PND 0). Relaxin can stimulate and may support organizationally critical estrogen receptor (ER) -α
expression in the endometrium and endometrial RXFP1 expression is estrogen-sensitive in the neonatal pig. Here,
objectives were to: (1) employ laser microdissection (LMD) to enable tissue-specific quantification of the effects of
perinatal exposure to zearalenone (ZEA), a selective ER modulator, on endometrial RXFP1 expression; and (2) relate
such effects to glandular epithelial cell proliferation patterns as reflected by proliferating cell nuclear antigen (PCNA)
labeling index (LI) in the neonatal porcine uterus. Litters from pregnant gilts fed ZEA (1500μg ZEA/kg feed/day) or
vehicle from 14 days prepartum through postpartum day 21 were used as a source of neonatal tissues. Neonates were
cross-fostered at birth to generate four ZEA exposure groups (n=6 neonates/group), including unexposed controls and
exposures limited to pre-, post-, or pre- and postnatal periods. Uteri, obtained on PND 20-21, were fixed in Xpress
Molecular Fixative (Sakura Finetek, USA) and tissue cross-sections (7μm) were subjected to LMD. Total RNA
extracted from excised endometrium (approximately 800ng from 10-14 cross-sections/neonate) was evaluated for
RXFP1 mRNA by quantitative RT-PCR. Epithelial PCNA LI was determined using immunohistochemistry and image
analysis. Both endometrial RXFP1 expression (P<0.06) and glandular epithelial PCNA LI (P<0.05) were reduced on
postnatal day 20-21 in ZEA-exposed neonates. Thus, perinatal ZEA exposure affects molecular and cellular events,
including RXFP1 expression, associated with endometrial programming in the neonatal pig. (Support: AiF ‘’Otto von
Guericke’’ e.V.; NSF EPS-0447675; USDA 2007-35203-18098)
P-16
The Effects of Relaxin-deficiency on Uterine Angiogenesis in Early Pregnancy
Jane E. Girlinga, Lee Lee Ngb, and Laura J. Parryb
a
Centre for Women’s Health Research, Monash Institute of Medical Research and Monash University Department of Obstetrics and
Gynaecology, Clayton, Victoria 3168, Australia; bDepartment of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia
The human endometrium undergoes regular periods of growth and regression during the menstrual cycle, including
concomitant remodelling of the vasculature. Previous studies have used mouse models to show that oestrogen and
progesterone stimulate angiogenesis (new blood vessel growth) within the endometrium. Endometrial angiogenesis
also occurs in the early stages of mouse pregnancy, which coincides with an increase in circulating progesterone.
Relaxin stimulates VEGF from human endometrial stromal cells in vitro and increases endometrial vascularization
in ovariectomized steroid-primed primates in vivo. To date, no studies have investigated the effects of relaxin on
endometrial angiogenesis in early pregnancy. Our aim was to test the hypothesis that endometrial angiogenesis would
be reduced in relaxin-deficient mice (Rln-/-) in comparison to their wildtype (Rln+/+) counterparts. Uterine tissues
were collected from Rln-/- and Rln+/+ mice on days 1 to 4 of pregnancy, before implantation. All mice were treated
with BrdU prior to dissection to allow the number of blood vessel profiles containing proliferating endothelial cells
(PVPs) to be quantified by double CD31/BrdU immunohistochemistry. Consistent with published studies, PVPs were
first observed on days 3 and 4 of pregnancy. However, the percentage of PVPs was reduced in Rln-/- mice compared to
Rln+/+ mice (Day 3: median = 4.4% versus 19.6%, Day 4: 9.6% versus 22.2%). We subsequently identified relaxin and
relaxin receptors in the mouse endometrium in early pregnancy. Our data suggest that locally synthesized relaxin acts in
synergy with progesterone to initiate endometrial angiogenesis in early pregnancy.
Relaxin 2008
19
P-17
The Effects of Fetal and Placental Numbers on Blood Concentrations of Relaxin in
the Rabbit
Phillip A. Fieldsa and Michael J. Fieldsb
Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA; b Animal
Sciences Department, University of Florida, Gainesville, Florida 32611, USA
a
The following study was conducted in order to determine the effects of conceptus (fetus and placenta) and/or fetal
numbers on the blood concentrations of relaxin in the rabbit. Experiment 1: Day 18 pregnant rabbit conceptuses
were surgically removed through incisions in the uterine horns. Rabbits were left with either 7, 5, 3 or 1 intact
implantation sites. Blood was collected from the ear vein on days 5, 10, 15, 18, 25, 30, 32 and 35 of pregnancy and
relaxin concentrations determined by RIA using porcine relaxin antiserum. There was no statistical difference between
relaxin concentrations in rabbits carrying 1 vs 7 conceptuses. Experiment 2: A total fetectomy was conducted on day
18 pregnant rabbits. However, not all placentas were removed. Rabbits were left with either 7, 5, 3 or 1 placentas
minus fetuses. Blood was collected from the ear vein on days 5, 10, 18, 20, 25, 30, 32 and 35 of pregnancy and
relaxin concentrations determined by RIA using porcine relaxin antiserum. There was no significant difference in the
peak concentrations of relaxin observed at day 25. However, there was a significantly sharper decrease in relaxin
concentrations after day 25 in rabbits with only 1 placenta vs those carrying 7, 5 or 3 placentas. The results indicate that
the number of conceptuses does not influence blood concentrations of relaxin in the rabbit and that there is potentially a
compensatory mechanism with regard to placental size and relaxin secretion.
P-18
Cervical Softening in the Common Marmoset Monkey (Callithrix jacchus)
Christina Simon and Almuth Einspanier
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, An den Tierkliniken 1, 04103 Leipzig, Germany
Tissue remodeling of the cervix is required for the last trimester of pregnancy, which is under endocrine control, e.g.
relaxin (RLX) and estradiol (E2). The purpose of the present study was to examine the influence of RLX and E2 on
the cervical tissue of the marmoset monkey in two experimental designs. Firstly, an in vivo experiment was set up to
determine the intracervical diameter under topically applied RLX. Secondly, an in vitro experiment was carried out to
investigate cervical tissue of ovariectomized marmosets treated systemically with E2, RLX and a combination thereof.
In vivo locally applied RLX induced a distinct significant widening of the cervix (0.84±0.4 mm in diameter). Under in
vitro investigation the wet weights of the cervical tissue were significantly increased after all three hormonal treatments
(E2: 0.27±0.07g, RLX: 0.25±0.04g, E2+RLX: 0.30±0.11g vs. control: 0.10±0.04g). Furthermore, loosening of the
connective tissue structure and an increase in the number of eosinophile cells as well as higher expression of matrix
metalloproteinases and the relaxin receptor RXFP1 were detected in the cervical tissue after all hormone treatments
compared to controls. However, strongest signals of all parameters were present under combined treatment with E2 and
RLX. To sum up, our results demonstrated for both hormones a widening and a remodeling effect on the primate cervix
with the most prominent effects under combined application of E2 and RLX, suggesting mutual influences in their effect
on cervical tissue.
This project was supported by the DFG (German Research Council) grant Ei 333/11-1.
20
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-19
Perinatal Zearalenone Exposure Affects RXFP1, RXFP2 and Morphoregulatory
Gene Expression in the Neonatal Porcine Uterus
Joseph C. Chena, Anne A. Wileyb, Johannes Kauffoldc, Martin Wähnerd, Frank F. Bartolb,e, and Carol A. Bagnella
Department of Animal Science, Rutgers University, New Brunswick, New Jersey 08901, USA; Departments of bAnimal Sciences and
Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA;
c
Faculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany; dAnhalt University of Applied Science, 06406 Bernburg,
Germany
a
e
The mycotoxin zearalenone (ZEA) is a selective estrogen receptor modulator (SERM) that can contaminate cereal feeds
leading to reproductive disorders. ZEA consumption by periparturient females may result in exposure of offspring to
SERMs during pre- and postnatal life, the latter as a consequence of consumption of xenoestrogen-contaminated milk.
Exposure of gilts to estrogen within 14 days from birth disrupts the uterine developmental program and has lasting
effects on adult uterine capacity. Such effects are marked by alterations in normal patterns of uterine relaxin/insulin-like
factor 3 receptors (RXFP1/RXFP2) and morphoregulatory gene expression (i.e. Wnt/Hoxa axis). To determine effects
of perinatal ZEA exposure on uterine expression of genes associated with endometrial development in the neonate,
pregnant sows were fed ZEA (1500 µg ZEA/kg feed/day) or vehicle from 14 days before farrowing through postnatal
day (PND) 20-21, when neonatal uterine tissues were collected. At birth, gilts were cross-fostered to generate four ZEA
exposure groups (n=5-6/group): unexposed controls or exposures limited to prenatal, postnatal, or pre and postnatal
(continuous) periods. Transcripts were measured by quantitative RT-PCR using cyclophilin as a housekeeping gene.
At PND 20-21, uterine Wnt7a, Hoxa10 and RXFP2 mRNA levels were decreased in neonates exposed continuously to
ZEA (p<0.05). Uterine RXFP1 mRNA levels were decreased in postnatal and continuously exposed groups (p<0.05).
Uterine Wnt4 mRNA levels were unchanged. Results indicate that perinatal exposure to milk-borne ZEA can affect
gene expression associated with neonatal uterine programming by altering expression of patterning and relaxin family
receptor genes. (Support: AiF "Otto von Guericke" e.V; USDA 2007-35203-18098)
P-20
The Effects of Blocking Progesterone Action on Relaxin Receptor Expression in the
Myometrium of Pregnant Rats
Lenka A Vodstrcila,b, Laura J Parrya, Stephen J Lyec, Oksana Shynlovac, and Mary E Wlodekb
a
Departments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cSamuel Lunenfeld Research
Institute, Mt. Sinai Hospital, Toronto, Ontario M5G1X5, Canada
The actions of relaxin are mediated by a Type C GPCR, Lgr7 (RXFP1). Myometrial Lgr7 expression is highest in earlyto mid- gestation in the rat but decreases in late gestation when serum levels of relaxin are elevated and progesterone
(P4) decreases. Therefore, we hypothesized that functional P4 withdrawal is necessary for the down-regulation in
myometrial Lgr7 at term. Conversely, preventing P4 withdrawal will inhibit the decrease in Lgr7 expression. We
conducted two studies to test these hypotheses. I) Rats were treated with the P4 receptor antagonist, RU486 (10mg/kg),
or vehicle on day 16 (n=6) or 19 (n=4) of gestation and myometrial tissue was collected 24 hours later. II) Pregnant rats
received 4 daily subcutaneous injections of P4 (16mg/kg) starting on day 20 gestation to prevent P4 withdrawal (n=4/
stage; day 20, 21, 22 and 23). Myometrial Lgr7 expression was compared with vehicle treated controls. Administration
of RU486 to block the actions of P4 caused a significant decrease in myometrial Lgr7 gene expression in treatment
groups at both stages of gestation, compared with controls. In contrast, daily injections of P4 had no significant effect on
Lgr7 expression after 1-4 days of treatment. These data demonstrate that functional progesterone withdrawal contributes
to the down-regulation in myometrial Lgr7 at the end of pregnancy. However, prolonging the length of gestation with
P4 did not compensate for the inhibitory effects of the fetal-placental unit and/or high circulating relaxin at the
stages examined.
Relaxin 2008
21
O-18
Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2
Receptor Function
Ross A.D. Bathgatea,b, Daniel J. Scotta, Gabrielle E. Callandera, Tracey N. Wilkinsona, Emma J. Hopkinsa, Despina E.
Ganellaa, Brigham J. Hartleya, Sharon Layfielda, Tania Ferraroa, Paul Gooleyb, Marco Mudac, John D. Wadea,
and Geoffrey W. Tregeara,b
Howard Florey Institute and bDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia;
Serono Research Institute, Rockland, Massachusetts 02370, USA
a
c
The receptors for relaxin and INSL3 are now well characterized as Relaxin Family Peptide (RXFP) receptors, RXFP1
and RXFP2, respectively. They are G-protein coupled receptors (GPCRs) with closest similarity to the glycoprotein
hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs). Additionally RXFP1 and RXFP2
are unique in the LGR family in that they contain an LDL class A (LDLa) module at their N-terminus. Studies from
our lab and others have demonstrated that activation of RXFP1 and RXFP2 involves a three stage process. Primary
ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the leucine
rich repeats (LRRs) of the ectodomain. There is a secondary binding site in the transmembrane (TM) exoloops which
may be mediated by A-chain interactions. Receptor signalling through cAMP then requires the unique LDLa module
as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation
for a GPCR. Furthermore, we have identified truncated RXFP1 splice variants that are secreted proteins containing the
LDLa module. These variants act as antagonists of RXFP1 function and may be endogenous regulators of RXFP1 action.
Moreover, we have discovered numerous additional human RXFP1 and RXFP2 splice variants that are co-expressed in
numerous human tissues and may have important modulatory roles. Current studies are focussed on resolving the sites
of secondary binding interaction, the role of the LDLa module in activation and the influence of receptor oligomerization
on receptor function.
O-19
Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function
Andras Kern and Gillian D. Bryant-Greenwood
Pacific Biosciences Research Center, University of Hawaii, Honolulu, Hawaii 96822, USA
The LGR7/RXFP1 and LGR8/RXFP2 receptors are unique receptors among the GPCRs in having an LDL-A module.
Their complex gene organization, among the intron richest of the GPCRs, suggests that alternative splicing is a common
occurrence. We have therefore investigated the role of the LDL-A module and showed the identity, expression and
functions of three LGR7 splice variants in the human decidua. Point mutations of conserved residues or complete
deletion of this module resulted in loss of the cAMP response to relaxin. Its glycosylation also impacted LGR7
cell surface delivery and therefore receptor activation. The wild-type (WT) LGR7 was expressed as both precursor
and mature forms, but deletion of the LDL-A module resulted in expression of only the mature form. Three new
alternatively spliced variants of LGR7 were identified, all containing a truncated extracellular region. Their functional
characterization showed them exerting dominant negative effects on the WT-LGR7, by preventing its homodimerization,
maturation and subsequent trafficking to the cell surface, resulting in loss of function. The treatment of cells with relaxin
induced desensitization and internalization of LGR7 into endosomal vesicles. Internalization was associated more with
β-arrestin 2 than β-arrestin 1. In summary, different mechanisms have been identified for controlling the cell surface
expression and function of the LGR7 protein, which are likely to be significant for the role of relaxin in
human parturition.
Supported by NIH grant HD24314.
22
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-20
Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome
Proliferator-activated Receptor Gamma
Sudhir Singha and Robert G. Bennetta,b,c
a
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA; bDepartments
of Internal Medicine and Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198,
USA; cVA Medical Center, Omaha, Nebraska 68105, USA
Relaxin has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by activators of
peroxisome proliferator-activated receptorγ (PPARγ), raising the possibility that a mechanism for relaxin’s antifibrotic
effects may involve activation of the PPARγ pathway. To test this possibility, PPAR activity was assessed by transient
transfection of 293T cells with RXFP1 and a reporter vector with a PPAR response element upstream of luciferase
(PPRE-luc). Relaxin increased PPRE activation in a concentration-dependent manner, maximally at 1nM, with an
EC50 of 24 pM. In contrast, InsL3 did not activate PPRE, demonstrating that the relaxin effect was RXFP1 specific.
The PPARγ agonists rosiglitazone (rosi) and 15-deoxy-δ12,14-prostaglandin J2 (15dPGJ2) increased PPRE activity,
but PPARα or PPARδ agonists had little effect. The combined effect of relaxin+rosi and relaxin+15dPGJ2 was more
than additive (Relaxin (Rlx) 169.2±9.1%, Rosi 158.7±15.0%, Rlx+Rosi 270±25.9%, PGJ 135.5±22.1%, Rlx+PGJ
248±41.6%). A PPARγ ligand blocker (GW9662) reduced PPRE activation in response to rosi but not relaxin, suggesting
that relaxin stimulates ligand-independent PPAR activation. Relaxin’s effect was mimicked by forskolin, but not
blocked by pertussis toxin, consistent with a Gαs coupled mechanism. Finally, both relaxin and rosi increased the levels
of CD36, a PPARγ target gene, in THP-1 cells, suggesting that relaxin stimulates PPARγ activity. In summary, relaxin
activates PPARγ and the combined effect of relaxin and PPARγ agonists was superadditive, suggesting that both agents
might be used together for an increased antifibrotic effect. The better understanding of this pathway might help in
amelioration of fibrotic diseases.
O-21
Development of a High-throughput Receptor-binding Assay using Time-Resolved
Fluorescent Europium Lanthanide for Screening Insulin-like Peptide 3 (INSL3)
Analogues for their Receptor Binding Affinity
Fazel Shabanpoora,b, Richard A. Hughesc, Ross A.D. Bathgatea, Frances Separovicb, and John D.Wadea
a
Howard Florey Institute, bSchool of Chemistry, cDepartment of Pharmacology, The University of Melbourne, Parkville,
Victoria 3010, Australia
The assessment of the receptor binding affinity of analogues of INSL3 typically requires the use of 125I-labelled INSL3
which has a number of disadvantages including its short half-life, high cost, the need to be prepared prior to use and
radioactivity safety issues. To overcome these problems, we have developed a high throughput receptor binding assay
using a europium-based time-resolved fluorescence (TRF) label. The emitted fluorescent light from this probe has unique
characteristics of long fluorescence halftime, a large Stoke’s shift and a sharp emission peak, which allow measurement
of signal to be made after the decay of short-lived background fluorescence. Initially, INSL3 was labeled post-synthesis
with a europium chelate at free amino groups. Competition binding assays were undertaken to determine the affinity
(pIC50) of labeled INSL3 for RXFP2 in HEK 293 cell line expressing RXFP2. The pIC50 of the INSL3 for RXFP2 using
Eu-INSL3 was similar to that of 125I-labelled INSL3, 9.45 and 9.35 respectively. The europium TRF receptor-binding
assay is quicker than 125I-radioactive assay as it is possible to directly measure the level of binding without the need to
transfer the cells. Moreover, the Eu-INSL3 had a low nonspecific binding and high signal to noise ratio. However, postsynthesis labeling of INSL3 has the disadvantage of not knowing where the Eu-chelate binds and the number of chelates
per INSL3 molecule. Consequently we have developed efficient solid phase synthesis technique to prepare specifically
mono-labelled peptide, which we have shown possesses optimum biophysical properties for receptor binding studies.
Relaxin 2008
23
O-22
Structure, Function and Evolution of Ligands and Receptors of the Insulin/Relaxin
Peptide Family
Pierre De Meytsa, Angela Manegold Svendsena, Waseem Sajida, Milka Vreclb, Anders Hedingc, John D. Waded, Ross A.D.
Bathgated, and Jane Nøhra
a
Receptor Systems Biology Laboratory, Hagedorn Research Institute, DK-2820 Gentofte, Denmark; bInstitute of Anatomy, Histology and
Embryology, Veterinary Faculty, University of Ljubljana, Sl-1000 Ljubljana, Slovenia; c7TM Pharma A/S, DK-2970 Hørsholm, Denmark;
d
Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia
The insulin/IGF/relaxin peptide family in humans comprises three ligands (insulin, IGF-I and IGF-II) that bind to RTKs,
and seven peptides related to relaxin that bind to GPCRs. The recent crystallographic structure of the insulin receptor
(IR) extracellular domain has ended decades of speculation regarding the mechanism of IR binding and negative
cooperativity. It supports a model whereby two sites on insulin crosslink alternatively two partial binding sites on each
IR half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of RTKs as well as
cytokine receptors. Surprizingly, recent studies indicate that constitutive dimerization and negative cooperativity is also
an ubiquitous property of GPCRs, showing allosteric mechanisms and negative cooperativity similar to those described
for the IR. I will describe the similarities and differences in the insulin and IGF-I binding kinetics and mechanisms of
activation of both IR and IGF-IRs and in the binding of H2 relaxin, INSL3, and a chimeric INSL3/relaxin ligand to
RXFP1, RXFP2, and RXFP3 respectively.
O-23
Activation of Relaxin Related Receptors by Short, Linear Peptides Derived from a
Collagen Containing Precursor
Ronen Shemesha, Chen Hermesha, Amir Toporika, Zurit Levinea, Amit Novika, Assaf Woola, Yossef Kligera, Avi Rosenberga,
Ross A.D. Bathgateb, and Yossi Cohena
Compugen Ltd., Tel-Aviv, Israel; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
a
In a screening effort, based on algorithmic prediction for novel GPCR peptide activators, we were able to identify
and examine two novel peptides, short, linear and derived from a natural, previously unidentified precursor protein,
containing a collagen-like repeat. Both peptides seemed to show a significant cAMP stimulatory and inhibitory effect
on CHO-K1 cells transiently transfected with both LGR7 (RXFP1) and LGR8 (RXFP2), after treatment with cAMP
generating Forskolin, as compared to the same cells treated with Forskolin+Relaxin. This activation was not found
for the Relaxin 3 receptor (RXFP3). In a set of follow-up experiments, both peptides were found to stimulate cAMP
production, mostly upon initial stimulation of cAMP by 5uM of Forskolin in cells transfected with either LGR7 or
LGR8. In dye-free cell impedance GPCR activation assay analysis, we were able to show that these peptides were also
able to activate a cellular responses mediated by these receptors. Although untransfected CHO-K1 cells showed some
cellular activation by both Relaxin and at least by one of our newly discovered peptides, LGR7 and LGR8 transfected
cells showed a stronger activation indicating a strong activation of a cellular response through activation of
these receptors.
In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of
the Relaxin/Insulin like family, are potential ligands for the Relaxin related family of receptors, and as such might serve
as novel candidates for Relaxin related therapeutic indications. Both peptides are linear and were found to be active after
being chemically synthesized.
24
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-24
RXFP1 Couples to the Gαi3-Gβγ-PI3K-PKCζ Pathway via the Final 10 Amino Acids
of the Receptor C-terminal Tail
Michelle L. Hallsa, Maria Papaioannoua, John D. Wade b, Bronwyn A. Evansa, Ross A.D. Bathgate b, and Roger J. Summersa
a
Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia
The relaxin family peptide receptors, RXFP1 and RXFP2, are highly similar receptors that share approximately 80%
amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, important
for relaxin-mediated decidualisation and myometrial inhibition. Despite the high homology the receptors couple to
different G-proteins to affect cAMP accumulation. Both RXFP1 and RXFP2 couple to Gαs to increase, and GαoB
to negatively modulate cAMP accumulation. However, only RXFP1 can couple to Gαi3 with time to activate a GβγPI3K-PKCζ pathway to further increase cAMP accumulation. This study aimed to determine the region of RXFP1 that
directs coupling to the delayed Gαi3 pathway using receptor mutagenesis. Receptor chimeras were initially examined:
RXFP1/2 (ectodomain, ED, of RXFP1; membrane-anchored domain, MAD, of RXFP2) lacked signalling via Gαi3
whereas RXFP2/1 (ED of RXFP2; MAD of RXFP1) could activate the delayed pathway in response to both H2 relaxin
and INSL3. This not only suggests that activation of the Gαi3 pathway is dependent upon the MAD of RXFP1, but that
this activation is not unique to H2 relaxin. A truncated RXFP1 receptor (lacking the C-terminal tail) was also unable to
activate the Gαi3 pathway, as was a receptor lacking the final 10 amino acids of the RXFP1 C-terminus. Additionally, a
receptor construct in which the final 10 amino acids of RXFP1 was added to the C-terminal tail of RXFP2 demonstrated
gain-of-function signalling via the Gαi3 pathway. Thus activation of the delayed Gαi3-Gβγ-PI3K-PKCζ cAMP pathway
by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor.
O-25
Local Regulation of Relaxin Receptors in the Myometrium of Pregnant Rats
Lenka A. Vodstrcila,b, Oksana Shynlovac, Stephen J. Lyec, Mary E. Wlodekb, and Laura J. Parrya
a
Departments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cSamuel Lunenfeld Research
Institute, Mt. Sinai Hospital, Toronto, Ontario M5G1X5, Canada
Relaxin’s actions are mediated by a Type C GPCR, Lgr7 (RXFP1). In pregnant mice, there is a significant downregulation in myometrial Lgr7 expression at term. The regulatory mechanisms are unknown but could involve local,
fetal-placental factors. To address this question, we investigated Lgr7 expression in two models of unilateral pregnancy
by qPCR. The gestational profile of myometrial Lgr7 expression was first established in non-pregnant (NP), pregnant
and postpartum (PP) rats. A second group of rats had one ovarian tube ligated before mating and tissues were collected
from gravid and non-gravid horns at various gestational stages. The tammar wallaby has separate gravid and nongravid uteri, so myometrium was collected from paired uteri at different stages of gestation. Lgr7 was expressed in the
rat myometrium at similar levels to NP in early- to mid-gestation, with a significant reduction (70%) in late gestation.
Postpartum receptor expression returned to NP levels. A similar pattern of Lgr7 gene expression was seen in the
cervix without the increase postpartum. A comparison of Lgr7 expression in different rat tissues on day 20 gestation
demonstrated highest expression in the cervix > myometrium = endometrium = vagina >> placenta = fetal membranes.
In the two unilateral pregnancy studies, Lgr7 expression was lower in the gravid uterus compared to the non-gravid
uterus, demonstrating a local influence of the fetal-placental unit on myometrial Lgr7 expression. Our data suggest that
the fetal-placental unit contributes to the down-regulation in myometrial Lgr7 at term, thereby removing the inhibitory
effects of relaxin on uterine contractility.
Relaxin 2008
25
O-26
The Role of the N-Terminal Leucine Rich Repeat Cap in Mediating INSL3 Specificity
to Relaxin Family Peptide Receptor 2
Daniel J. Scott, Tracey Wilkinson, Geoffrey W. Tregear, and Ross A.D. Bathgate
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
We recently defined the leucine-rich repeat (LRR) residues involved in primary INSL3 binding to relaxin family peptide
receptor 2 (RXFP2). Many of these residues are conserved in RXFP1 and have been previously implicated in relaxin
binding. The ability of RXFP2 to accommodate relaxin and INSL3 binding suggests conservation of hormone binding
conformations in these two receptors. However, the inability of INSL3 to bind RXFP1 indicates a degree of specificity in
the INSL3/RXFP2 interaction. We postulated that RXFP2 unique residues are responsible for mediating such specificity,
and inserting such residues into RXFP1 may rescue INSL3 binding. RXFP2 unique residues were identified using
multiple sequence alignments and inserted into RXFP1 to generate RXFP1/2 chimeric receptors. Each RXFP1/2 chimera
was expressed in HEK cells and their ability to bind INSL3 tested. Only one RXFP1/2 chimera exhibited significant
INSL3 binding, RXFP1/2 βN2, which contained 3 RXFP2 specific residues from the N-terminal LRR cap. Interestingly,
replacement of any of these residues with alanine in RXFP2 resulted in no significant loss of INSL3 binding, indicating
that these residues do not bind INSL3. Instead, competition binding assays utilizing mutant INSL3 peptides suggested
that the residues inserted into RXFP1/2 βN2 induced structural changes in the LRRs of RXFP1, allowing the basic
residues from the B-chain of INSL3 to interact with conserved acidic residues in LRR IV and LRR VI. Overall this
demonstrated that there are crucial differences between the relaxin/RXFP1 and the INSL3/RXFP2 interactions, which
will be important to consider when designing relaxin and INSL3 mimetics.
O-27
The Relaxin Family Peptide Receptors (RXFPs): A New Subgroup of Dimeric,
Cooperative GPCRs
Angela Manegold Svendsena, Julie Køniga, Line Eggebrecht Beka, Milka Vreclb, Anders Hedingc, Jesper Bøggild
Kristensend, John D. Wadee, Ross A.D. Bathgatee, Pierre De Meytsa, and Jane Nøhra
a
Receptor Systems Biology Laboratory, Hagedorn Research Institute, DK-2820 Gentofte, Denmark; bInstitute of Anatomy, Histology and
Embryology, Veterinary Faculty, University of Ljubljana, Sl-1000 Ljubljana, Slovenia; c7TM Pharma A/S, DK-2970 Hørsholm, Denmark;
d
Chemical API Supply Isotopes, Novo Nordisk, Novo Nordisk Park, DK-2760 Måløv, Denmark; eHoward Florey Institute of Experimental
Physiology and Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia
Following the demonstration of negative cooperativity for insulin (and later IGF-I) receptors (which are dimeric RTKs),
it was shown over 30 years ago that β2-adrenergic and TSH receptors (now known to be GPCRs) also exhibit negative
cooperativity and therefore might function as dimers or oligomers, but the concept of dimerization in GPCRs was
only recently experimentally validated. A connection between negative cooperativity and dimerization was recently
demonstrated for the glycoprotein hormone receptors, including the TSH and FSH receptors, members of the LGR
subgroup of GPCRs.
To study the dimerization and cooperativity of the GPCRs for relaxin-like peptides, RXFP1, RXFP2 (also LGRs),
and RXFP3, we investigated the kinetics of ligand binding to these receptors. To further evaluate the dimerization, we
performed Bioluminescence Resonance Energy Transfer experiments (BRET).
We showed that dissociation of prebound H2 relaxin, INSL3, and a chimeric INSL5/relaxin ligand from RXFP1,
RXFP2, and RXFP3 respectively was accelerated by unlabeled ligand in an ‘’infinite dilution’’ protocol, indicating
the presence of negative cooperativity. This suggested that these receptors function as dimers or oligomers. The dose
response curve for negative cooperativity for RXFP2 was bell-shaped (like the curve for insulin receptors), whereas it
was monophasic in the case of RXFP1 and RXFP3 (like the curve for IGF-I receptors).
BRET experiments for RXFP1 and RXFP2 confirmed that constitutive dimerization occurs through the 7TM domain.
The ectodomain stabilized the dimerization in both cases. We furthermore demonstrated the heterodimerization of
RXFP1 and RXFP2. BRET experiments are in progress to study the possible homodimerization of RXFP3.
26
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-28
INSL3/RXFP2 Signaling in Testicular Descent: Mice and Men
Shu Fenga, Alberto Ferlinb, Natalia Bogatchevaa, Anne Truonga, Sean Corbettc, Shuo Hanc, Dolores Lambc, Carlo Forestab,
and Alexander I. Agoulnika
Department of Obstetrics and Gynecology, cScott Department of Urology, Baylor College of Medicine, Houston, Texas 77030, USA;
Department of Histology, Microbiology and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova,
Padova, Italy
a
b
The mutations of insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. We
performed mutation screening in human patients with cryptorchidism from different populations in Europe and USA.
Several missense mutations were identified both in INSL3 and RXFP2 genes. The RXFP2 mutation T222P is the
only one strongly associated with the mutant phenotype found in more than 1000 patients and controls analyzed.
The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely
compromised receptor cell membrane expression. We have also detected a number of mutations in INSL3 gene including
a novel mutation in a family with a history of undescended testis. In mice the Rxfp2 gene expression was markedly
increased after birth in male reproductive tissues and was readily detectable in the epididymis, Leydig cells, and germ
cells of the testis. The strongest expression was detected in adult cremaster muscle. INSL3 treatment increased cell
proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine
signaling in these cells. We generated RXFP2-iCre transgenic mice expressing improved Cre recombinase (iCre)
under the control of a 2.4-kb mouse RXFP2 promoter. The iCre was expressed in the gubernacular ligament at E14.5,
indicating that this promoter is able to drive RXFP2 gene expression during transabdominal testis descent. Finally, we
have produced mice with cre/loxP activated shRNA transgene targeting RXFP2. The analysis of the receptor knockdown
in different organs and at different developmental stages is currently underway.
O-29
New Roles for INSL3 in Adults: Regulation of Bone Metabolism and Association of
RXFP2 Gene Mutations with Osteoporosis
Alberto Ferlina, Anastasia Pepea, Lisa Gianeselloa, Andrea Garollaa, Shu Fengb, Arianna Facciollia, Roy Morelloc, Alexander I.
Agoulnikb, and Carlo Forestaa
a
University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for
Male Gamete Cryopreservation, 35121 Padova, Italy; bDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston,
Texas 77030, USA; cDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells and acts by binding to its specific G-protein
coupled receptor RXFP2 (Relaxin family peptide 2). INSL3 is involved in testicular descent and mutations in INSL3
and RXFP2 genes cause cryptorchidism. The physiological role of INSL3 in adults unknown, although substantial
INSL3 circulating levels are present. After extensive clinical, biochemical and hormonal investigation, including bone
densitometry by DEXA, on 25 young men (age 27-41) with the well characterized T222P mutation in the RXFP2 gene,
we found that 16 of them (64%) have significantly reduced bone density. No other cause of osteoporosis was evident
in these subjects, whose testosterone and gonadal function were normal. Expression analysis of INSL3 and RXFP2 on
human bone biopsy and human and mouse osteoblast cell cultures performed by RT-PCR and immunohistochemistry
showed the presence of RXFP2 in these cells. Real-time cAMP imaging analysis and proliferation assay of osteoblasts
under the stimulus of INSL3 showed a dose- and time-dependent increase in cAMP and cell proliferation, and specific
osteoblast gene activation was observed by real-time PCR after INSL3 stimulation. Lumbar spine and femoral bone of
Rxfp2-deficient mice were studied by static and dynamic histomorphometry and micro CT respectively, and showed
decreased bone mass, mineralizing surface, bone formation and osteoclast surface compared to wild-type littermates,
compatible with a functional osteoblast impairment. This study identified for the first time a role for INSL3 in adults,
demonstrating a modulating effect on bone metabolism and linking RXFP2 gene mutations with human osteoporosis.
Relaxin 2008
27
O-30
Regulation of INSL3 Transcription in Testicular Leydig Cells
Jacques J. Tremblay
Division of Reproduction, Perinatal and Child Health, CHUQ Research Centre, Quebec City, Quebec, G1V 4G2, Canada; Centre for
Research in Biology of Reproduction, Department of Obstetrics and Gynecology, Faculty of Medicine, Laval University, Quebec City,
Quebec, G1V 0A6, Canada
Insulin-like 3 (INSL3) is almost exclusively expressed in the gonads. During fetal life, INSL3 is produced by testicular
Leydig cells and it regulates testicular descent. In adults, INSL3 is expressed in the testis and the ovary where it acts
as a survival factor for germ and follicular cells. In contrast to its well defined roles in reproductive development and
function, very little is known about the molecular mechanisms regulating INSL3 transcription in gonadal cells. In several
mammalian species, the entire INSL3 locus is located within the last intron of the JAK3 gene which suggests that INSL3
regulatory elements must be located within a relatively short 5’ flanking region. So far, two transcription factors have
been shown to activate INSL3 transcription in Leydig cells: the nuclear receptors SF1 and NUR77. Because they are
also found in tissues that do not express INSL3, additional factors must be involved in directing INSL3 expression in
Leydig cells. Through 5’ deletions and site-directed mutagenesis, we have mapped a short region that confers about 70%
of activity to the human INSL3 promoter. Within this region, we identified a consensus binding site for the Kruppellike factor KLF6 which we found to be expressed in Leydig cells. KLF6 can activate the INSL3 promoter and this
transactivation is blunted when the KLF element is mutated. Furthermore, we found that KLF6 cooperates with SF1
and NUR77 to further enhance INSL3 transcription. Altogether, these data provide new insights into the molecular
mechanisms regulating INSL3 expression in Leydig cells. (Supported by CIHR)
28
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Abstracts
Wednesday, May 21, 2008
O-31
The "Hot Wires" of the Relaxin-like Factor (INSL3)
Christian Schwabe and Erika E. Büllesbach
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA
Peptide hormones regulate important functions in about 10 power 14 cells in a human as they bind to their specific cell
surface receptors, yet for decades it has remained unknown what structures within the receptor-bound hormone are
initiating a specific response in the target cells.
Structure/function analysis of RLF suggested that binding and signaling are associated with different regions of the
surface. Whereas the B chain segment between cysteine 11 and tryptophan 27 mediates binding, signaling is a function
of the N-terminal region and involves both chains. Elimination of the N-terminal residues up to B9 or alternately,
removal of residues 1 to 9 of the A-chain, do not reduce binding avidity of RLF whereas signaling ceased in either case.
This discovery made it possible to explore the structure of the signal initiation site of RLF.
Our work showed that the function of the "hot wires" of RLF is independent of the amino acid side-chains in the
signaling region and that trans-membrane signal induction may be a function of peptide bonds of the ligand, forced
into the signaling position by the specific interaction of RLF with the leucine-rich repeat G-protein coupled receptor 8
(LGR8). In addition, our observations offer an explanation for ligand crosstalk as well as for the to date inexplicable
ability of some anti-receptor antibodies to elicit a specific biological response.
O-32
Roles of the Receptor, the Ligand and the Cell in the Signal Transduction Pathways
Utilised by the Relaxin Family Peptide Receptors 1-3 (RXFP1-3)
Roger J. Summersa, Ross A.D. Bathgateb, John D. Wadeb, Emma T. van der Westhuizena, and Michelle L. Hallsa
a
Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia
The relaxin family peptides act on four G protein-coupled receptors now termed relaxin family peptide receptors
(RXFP1-4). The leucine-rich repeat containing RXFP1 and RXFP2 and the small peptide-like RXFP3 and RXFP4
are the physiological targets for relaxin, insulin-like (INSL) peptide 3, relaxin-3 and INSL5, respectively. Activation
of RXFP1 or RXFP2 causes increased cAMP that results from Gs-mediated activation and GoB-mediated inhibition of
adenylate cyclase. With RXFP1, an additional later increase in cAMP involves βγ subunits released from Gi3 and the
final 10-amino acids in the C-terminus. The overall cAMP response to activation of RXFP1 is also highly dependent
on the cell type expressing the receptor. In contrast, RXFP3 and RXFP4 inhibit adenylate cyclase and RXFP3 causes
ERK1/2 phosphorylation. RXFP3 receptors characterised using receptor binding and inhibition of adenylate cyclase
show a competition profile of H3 relaxin> H3 relaxin B-chain>> H2 relaxin=porcine relaxin=INSL3. However,
examination of signalling using reporter genes in CHO-K1 RXFP3 cells revealed activator protein - 1 (AP-1) activation
in response to both H3 and H2 relaxin but not INSL-3. This suggests that RXFP3 activates the c-Jun N-terminal kinase
(JNK) pathway but also that H2 relaxin interacts with RXFP3 at a site distinct from that identified by H3 relaxin. These
examples from the RXFP receptors demonstrate the importance of the C-terminal tail of GPCRs in signal transduction
and also that the ligand and the cell type have important roles in the pathways activated.
30
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-33
Ligand-directed Signalling Pathways at the Relaxin Family Peptide Receptor 3
(RXFP3; GPCR135) Determined Using Reporter Genes
Emma T. van der Westhuizena, Brigham J. Hartleyb, John D. Wadeb, Patrick M. Sextona, and Roger J. Summersa
a
Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia
The H3 relaxin receptor, RXFP3, has been identified and characterized as a Gi/o-coupled receptor that inhibits forskolinstimulated cAMP accumulation. This study characterizes signalling pathways activated by RXFP3 in CHO-K1 cells
stably expressing RXFP3, using reporter genes. Time course studies using 8 reporter gene constructs were carried out in
CHO-K1 RXFP3 cells with H3 relaxin, H2 relaxin and INSL3. Robust activator protein (AP)-1 activation was observed
in cells treated with both H3 relaxin and H2 relaxin, but not with INSL3 (all 0.1µM), suggesting that RXFP3 may
activate the stress-activated protein kinase (c-Jun N-terminal kinase; JNK) signal transduction pathways. H3 relaxin also
strongly activated nuclear factor (NF)-κB signalling pathways, that were not activated by either H2 relaxin or INSL3 (all
0.1µM). No reporter gene activation was observed in cells transfected with heat shock element (HSE), cAMP response
element (CRE; cAMP/PKA and p38 MAPK/JNK signalling pathways), serum response element (SRE; ERK and JNK
signalling pathways), glucocorticoid response element (GRE; cell cycle related pathways), E-box DNA binding element
(Myc; cell proliferation related pathways) or nuclear factor of activated T-cells (NFAT; PKC/Ca2+ signalling pathways)
when stimulated with H3 relaxin, H2 relaxin or INSL3 (all 0.1µM). This study demonstrated that RXFP3 activates
signalling pathways upstream of AP-1 elements, when stimulated with H3 relaxin or H2 relaxin and that RXFP3 can also
activate NF-κB signalling pathways when stimulated with H3 relaxin.
O-34
Relaxin Activates Multiple cAMP Signalling Pathway Profiles in Different
Target Cells
Michelle L. Hallsa, Ross A.D. Bathgateb, and Roger J. Summersa
a
Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia
The receptor for H2 relaxin, the relaxin family peptide receptor 1 (RXFP1), activates multiple pathways leading to
cAMP accumulation when expressed in human embryonic kidney (HEK) 293T cells. In this cell system, RXFP1 initially
couples to both Gαs (to increase cAMP) and GαoB (to negatively modulate cAMP), but with time recruits coupling to
a Gαi3-Gβγ-PI3K-PKCζ pathway to further increase cAMP. Although cAMP signalling in this system is relatively well
defined, the signalling pathways activated by relaxin in its target cells and tissues are still unclear. This study aimed
to examine the cAMP signalling of RXFP1 in cells that endogenously express the receptor. Seven cell types derived
from various backgrounds were screened for receptor expression. RXFP1 expression was identified in THP-1 (human
monocyte cell line), T-47D (human breast cell line), Colo16 (human lung cell line), rat cardiac fibroblasts and rat renal
fibroblasts from obstructed kidneys. Although of small magnitude in comparison to THP-1 cells, relaxin-induced cAMP
responses were observed. Only in THP-1 cells and rat cardiac fibroblasts was there activation of the Gαi3-Gβγ-PI3KPKCζ pathway leading to cAMP accumulation. In all other cells there was activation of a combination of the initial
pathways to affect cAMP. T-47D cells could activate only Gαs, whereas Colo16 and rat renal fibroblasts from obstructed
kidney could activate both Gαs and GαoB pathways. Thus the signalling pathways activated by relaxin are highly
dependent upon the cell line under investigation, and this may help to explain the varied physiological responses exerted
by relaxin in its different target tissues.
Relaxin 2008
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O-35
Prominent Role of Relaxin in Improving Post-infarction Heart Remodelling: Clues
from in vitro and in vivo Studies with Genetically Engineered Relaxin-producing
Myoblasts
Daniele Bania, Silvia Nistria, Lucia Formiglia, Elisabetta Meaccib, Fabio Francinic, and Sandra Zecchi-Orlandinia
Departments of aAnatomy, Histology and Forensic Medicine, bBiochemical Sciences, cPhysiological Sciences, University of Florence, I-50139
Florence, Italy
Stem cell transplantation and gene therapy are viewed as promising approaches for treatment of the post-infarcted heart
and prevention of deleterious cardiac remodelling and heart failure. We explored this issue by transplanting mouse
C2C12 myoblasts, transfected to express eGFP or eGFP/RLX, to swine with chronic myocardial infarction. One month
later, C2C12 myoblasts selectively settled in the ischemic scar around blood vessels showing an activated endothelium
(ICAM-1-,VCAM-positive). They did not trans-differentiate towards a cardiac phenotype, but did induce extracellular
matrix (ECM) remodeling by the secretion of matrix metalloproteases (MMP) and increase microvessel density by
VEGF expression. C2C12/RLX myoblasts displayed greater efficacy to engraft the post-ischemic scar and to induce
ECM remodelling and angiogenesis than C2C12/GFP. By echocardiography, C2C12-engrafted swine, especially those
which received C2C12/RLX, showed better heart contractility than the untreated controls. Hence, the advantage afforded
by the grafted myoblasts on cardiac function is primarily dependent on their paracrine effects on ECM remodelling
and vascularization.
Another intriguing possibility is that local cardiomyoblasts could be re-activated during adult life to differentiate and
regenerate cardiac tissue. Therefore, we studied whether C2C12/GFP and C2C12/RLX myoblasts could influence the
differentiation of mouse neonatal cardiac cells in co-culture. We observed that C2C12 cells established close contacts
with cardiomyocytes, mediated by the formation of functional gap junctions, and tended to form a sort of structural
support guiding the newly formed cardiac cells during their growth and differentiation into a network of spontaneously
beating cardiomyocytes. Compared with cardiomyocytes alone, those grown together with C2C12/GFP and, even
more, C2C12/RLX showed increased morphological and biochemical signs of differentiation (troponin-T, ANP, cardiac
myosin). These findings indicate that C2C12 myoblasts and RLX may favor differentiation of cardiomyocytes and
suggest that cell grafting and RLX gene therapy could reduce cardiac remodelling and help myocardial regeneration.
O-36
New Aspects on Cardiovascular Actions of Relaxin: Inotropy and Signaling
Thomas Dschietziga, Cornelia Bartscha, Hans-Tilman Kinkela, Franz Paul Armbrusterb, Christoph Richtera, Gert Baumanna,
and Karl Stangla
a
Department of Cardiology and Angiology, Charité University Medicine Berlin, Campus Mitte, 10117 Berlin, Germany; bImmundiagnostik
AG, Bensheim, Germany
We have shown that relaxin acts as compensatory mediator in human heart failure and that the peptide - apart from
its signaling via LGR7 -activates the glucocorticoid receptor (GR). Here, we summarize our latest investigations into
relaxin’s inotropic effects in human myocardium and into relaxin-GR signaling. To explore inotropy we used human
right and left atrial as well as right and left ventricular myocardial samples obtained from donor hearts and from
explanted, terminally failing hearts. In right and left atrial preparations from both donor and failing hearts, human
relaxin-2 evoked comparable positive inotropic effects which were mediated by protein kinase A and ATP-sensitive
potassium channels. On the other hand, a positive inotropic effect of relaxin-2 could be found neither in donor nor in
failing right and left ventricular myocardium. The finding of the preserved atrial inotropic effect of relaxin is of potential
relevance to the ongoing therapeutical use of the peptide in human heart failure.
We have also investigated the auto-regulation of relaxin in different cell models. Our experiments, which included
silencing of GR gene expression, CAT promoter assays,chromatin immuno-precipitation, and immunofluorescence,
show that a positive self-regulatory loop of human relaxin-2 gene expression exists and that it critically involves GR
and relaxin/GR binding to the relaxin promoter. This finding deepens our understanding of the complex relaxin signal
transduction and may also be relevant to the emerging clinical use of the peptide.
32
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-37
Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin:
Experimental Findings
Xiao-Jun Dua, Qi Xua, Edna Lekgabea,b, Xiao-Ming Gaoa, Geoffrey W. Tregearb, Ross A.D. Bathgateb, and Chrishan S. Samuelb
Baker Heart Research Institute, and bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
a
Cardiac fibrosis is a hallmark of heart disease, contributes to development of heart failure and arrhythmias, and forms a
key therapeutic target. There is a major unmet need for more selective, effective and safe anti-fibrotic drugs. Following
earlier findings of cardiac fibrosis phenotype in relaxin-1 deficient (Rln-/-) mice and anti-fibrotic property of relaxin
in other organs, we studied anti-fibrotic action of endogenous or exogenous relaxin in diseased hearts. In Rln-/- and
wildtype mice subjected to aorta banding (8-weeks), the extent of cardiac hypertrophy, fibrosis and ventricular
dysfunction were comparable between both genotypes, suggesting lack of protection in this model by endogenous
relaxin albeit its cardiac expression was elevated. Relaxin therapy (via osmotic minipump for 2-weeks) reversed
cardiac fibrosis in mice with cardiac-restricted overexpression of β2-adrenergic receptors (β2-TG) or spontaneously
hypertensive rats (SHRs, 9-10 mo). Molecular measures suggest inhibited fibroblast activation/proliferation, reduced
collagen synthesis and elevated matrix metalloproteinase-2. Enhanced oxidative stress, inflammation and significant
matrix remodelling characterize the β2-TG cardiomyopathy model, which responds well to relaxin administration or
virally-mediated gene transfection. However, relaxin failed in reversing cardiac fibrosis in 22-mo-old SHRs implying
that the efficacy is disease-stage dependent. In mice with surgically induced ischemic heart disease (transmural
infarction,ischemia/reperfusion), relaxin therapy inhibited scar formation, increased angiogenesis and alleviated
cardiomyocyte apoptosis without adverse effect. In these studies, however, we detected limited functional benefit by
the treatment.
Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct
mechanisms. Future research needs to explore functional improvement following fibrosis reversal, cellular and molecular
mechanisms involved, and changes in relaxin receptor/signalling in diseased myocardium.
O-38
Relaxin-induced Compositional and Geometric Remodeling of Small Renal Arteries
Dan O. Debraha, Julianna E. Debrahb, Sanjeev G. Shroffa, and Kirk P. Conradc
a
Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA; bMagee-Womens Research Institute,
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA; cPhysiology and Functional Genomics and Obstetrics and
Gynecology, University of Florida, Gainesville, Florida 32610, USA
We have recently shown that administration of recombinant human relaxin (rhRlx) to nonpregnant female rats increases
global arterial compliance. Additionally, we found that relaxin alters the passive mechanical properties of small renal
arteries (SRA) in mice and rats and that these arteries exhibit an increase in wall area. The goals of the present study
were to (1) examine the effects of relaxin on SRA wall composition and (2) characterize the determinants of the relaxininduced increase in SRA wall area.
Nonpregnant female mice were administered rhRlx for 5 days after which SRA were isolated. We measured arterial
collagen, elastin, and total protein using the Sircol collagen assay, the Fastin elastin assay, and the Pierce BCA
protein assay, respectively. Additionally, we quantified arterial smooth muscle cell (SMC) size and density using
immunofluorescent imaging techniques.
Compared to control mice, SRA isolated from rhRlx-treated mice were characterized by a significant reduction in
collagen to total protein ratio (0.13±0.01 vs 0.19±0.01 µg collagen/µg protein; mean±SEM; P<0.02), as well as a
significant increase in SMC density (6.1±0.1 vs 5.0±0.1 cells/1000 µm2; P<0.001). In contrast, there were no significant
changes in elastin content (104±4 vs 97±14 µg elastin/mg dry weight) or SMC size (111.3±4.3 vs 105.0±3.2 µm2)
between the two groups.
We conclude that: (1) relaxin reduces relative collagen content of SRA and (2) the relaxin-induced increase in SRA wall
area is primarily due to SMC hyperplasia and not hypertrophy.
Relaxin 2008
33
O-39
Relaxin-family Peptide and Receptor Systems in Mammalian Brain: Recent Insights
from Anatomical and Functional Studies
Andrew L. Gundlach
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Significant advances have been made since the Relaxin 2004 conference in our knowledge of the distribution and
function of the three major relaxin-family peptide/receptor systems present in the brain, i.e. the relaxin/RXFP1, INSL3/
RXFP2 and relaxin-3/RXFP3 pairings. Studies in our laboratory have attempted to construct detailed comparative maps
of these systems in major experimental species (rat/mouse) and higher species (primate/human), in order to determine
whether subsequent functional experiments in rat and/or mouse will be reflective of human physiology (see Ma et al.,
Sedaghat et al., Shen et al., this meeting). These studies have so far revealed a degree of variation in the distribution of
RXFP1 and RXFP2 mRNA/protein in rat and mouse brain, in contrast to a conserved distribution of RXFP3 seen in rat,
mouse, primate and human. In line with this latter data, the distribution of relaxin-3 neurons and projections is similar in
rat, mouse and primate. We have also assessed the regulation of expression of the different relaxin-family peptides and
receptors - examining the effect of pregnancy and lactation on RXFP1 levels in rat and mouse brain (see Piccenna et al.)
and the effect of psychological stressors on relaxin-3 expression (see Banerjee et al.). Important collaborative studies
using new, selective agonist and antagonist peptides have revealed the functional importance of RXFP2 in rat basal
ganglia (Sedaghat et al.) and endogenous relaxin-3 signaling in the septohippocampal system and associated behaviours
(Ma et al., Smith et al.). These and other studies (McGowan et al., Sutton et al.) are now rapidly revealing the precise
details of relaxin peptide biology within the brain.
O-40
Relaxin-3 and its Role in Neuroendocrine Function
Barbara M.C. McGowan, Sarah A. Stanley, Mohammad A. Ghatei, and Stephen R. Bloom
Department of Investigative Medicine, Division of Investigative Science, Imperial College London, Hammersmith Campus, London
W12 ONN, United Kingdom
The hypothalamus plays a key role in the regulation of energy homeostasis and endocrine function. Relaxin-3 is a
hypothalamic neuropeptide belonging to the insulin superfamily of peptides. It is expressed in the nucleus incertus of the
brainstem which has projections to the hypothalamus and is thought to act in the brain via the RXFP3 receptor, though
the RXFP1 receptor may also play a role. RXFP3 and RXFP1 are present in the hypothalamic paraventricular nucleus
(PVN), an area with a well characterised role in the regulation of energy balance and stress. The PVN also modulates
reproductive function by providing inputs to hypothalamic GnRH neurons. The physiological roles for relaxin-3 remain
to be established. Evidence for a role of relaxin-3 as a hypothalamic orexigenic peptide will be reviewed, including
its effects on the hypothalamo-pituitary-thyroid axis and energy expenditure. Studies pointing towards a putative role
of relaxin-3 in the hypothalamic-pituitary-gonadal axis will be discussed together with recent work on the effects of
relaxin-3 on the stress axis. Central endocrine effects of relaxin-3 will be compared to ‘classical’ relaxin. We conclude
that relaxin-3 may act as a hypothalamic signal to co-ordinate appetite, stress and reproductive status. Further studies
will be required to determine whether these are physiological roles for relaxin-3 and to determine the receptors involved.
34
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-41
Metabolic and Neuroendocrine Responses to RXFP3 Modulation in the CNS
Steven Suttona, Jonathan Sheltona, Craig Smithb, John Williamsb, Sujin Yuna, Tim Motleya, Chester Kueia, Andrew Gundlachb,
Changlu Liua, and Timothy Lovenberga
a
Johnson & Johnson Pharmaceutical Research and Development, LLC, San Diego, California 92121, USA; bHoward Florey Institute, The
University of Melbourne, Parkville, Victoria 3010, Australia
Neuroanatomical studies have shown relaxin-3 neurons, primarily found in the rodent nucleus incertus (NI), project
widely to a large number of areas expressing the relaxin-3 receptor (RXFP3) and this data suggests relaxin-3/RXFP3
signaling modulates sensory, emotional and neuroendocrine processing. The similar distribution of this receptor-ligand
pair in the brain of rat, mouse and monkey suggests that experimental findings obtained in lower species will translate
to higher species. A role for relaxin-3 and RXFP3 in modulating stress responses is strongly suggested by the expression
of CRF-R1 by NI cells, increased relaxin-3 expression in NI after stress or CRF injection, and hormonal responses to
icv relaxin-3 injection. Recent data (Ma et al., this meeting) is consistent with a further role for this ligand-receptor in
modulating memory. In addition, relaxin-3 has been reported to modulate feeding and body weight control. Acute or
chronic central (icv or iPVN) injections of relaxin-3 have shown a consistent stimulatory effect on food consumption,
while relaxin was inactive suggesting the phagic effect of relaxin-3 was mediated by RXFP3. We have confirmed
the role of RXFP3 in modulating feeding and body weight using a selective RXFP3 agonist (IR3/I5) and antagonist
{R3(D23-27R)/I5}, collecting feeding, body weight, hormone and body composition data. In addition, we have
preliminary body weight and fMRI data from relaxin-3 KO mice, which on a 129:B6 background are smaller and leaner
than congenic controls. These data suggest relaxin-3, acting through RXFP3, is involved in coordinating stress, learning/
memory and feeding responses as predicted on the basis of neuroanatomy.
O-42
Relaxin-3 Neurons of the Nucleus Incertus Modulate Septohippocampal Theta
Rhythm and Spatial Working Memory in Rats
Sherie Maa, Angel Nuñezb, Francisco E. Olucha-Bordonauc, Feng Lina, Chester Kueid, Steven W. Suttond, John D. Wadea,
Changlu Liud, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bDepartamento de Anatomia, Histología y
Neurociencia Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain; cDepartamento de Anatomía y Embriología
Humana, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain; dJohnson & Johnson Pharmaceutical Research and
Development, LLC, San Diego, California 92121, USA
The neuropeptide relaxin-3 (RLN3) is enriched in GABA neurons of the nucleus incertus (NI), which are highly
responsive to psychological stressors and corticotropin-releasing hormone. These neurons innervate many forebrain
areas containing high densities of the RLN3 receptor, "GPCR135" or "RXFP3". The septum and hippocampus are two
regions that are particularly enriched in RLN3 fibres/terminals and RXFP3 in the rat and mouse brain. High-frequency,
rhythmic neuronal activation or ‘theta rhythm’, occurs in the hippocampus during memory processing, particularly
during spatial navigation; and GABAergic and cholinergic neurons projecting from the septum to the hippocampus are
capable of generating and pacing theta activity. We have shown in the anaesthetized rat that the NI acts as a key relay
for the induction of hippocampal theta rhythm activity by pontine and somatosensory activation. In the current study, we
therefore determined if endogenous and/or exogenous activation of septal RXFP3 was involved in this process, using
anaesthetized and conscious rats. In anaesthetized rats, acute activation of RXFP3 in the medial septum by a specific
RXFP3 agonist (R3/I5) increased the level of hippocampal theta rhythm; an effect blocked by prior septal injection of
an RXFP3 antagonist (R3(B"delta"23-27)R/I5). In conscious rats, injection of the RXFP3 antagonist into the medial
septum inhibited spatial learning performance in the spontaneous alternation test, an effect that was reversible by coadministration of an equimolar amount of the agonist, R3/I5. Our observations reveal an important action of RLN3/
RXFP3 in the ‘septohippocampal system’ that potentially links higher cognitive functions with the control of stress and
metabolic and neuroendocrine balance.
Relaxin 2008
35
O-43
Behavioral Phenotyping of Mixed-background (129SV/B6) Relaxin-3
Knockout Mice
Craig M. Smitha, Andrew J. Lawrencea, Steve W. Suttonb, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and
Development LLC, San Diego, California 92121, USA
In order to assess the behavioral phenotype of mice lacking relaxin-3, two independent cohorts of adult male and female
relaxin-3 knockout (KO) mice and their wildtype (WT) littermates (n = 8-12) were subjected to a battery of standard
behavioral tests. There were no genotype-related deficits in motor coordination (assessed on the accelerating rotarod)
or spatial memory (Y-maze), and levels of anxiety were equivalent (in large open-field, elevated plus-maze, and light/
dark box tests). Sex-based differences were observed, however. Female KOs displayed a "hypoactive" phenotype in
locomotor cells that was also detected in the large-open field and Y-maze. Combined with results from further testing
of females in social-interaction and object-recognition paradigms, this data suggests that relaxin-3 deficiency in female
mice may result in reduced exploratory drive, possibly due to the hypothesized role for relaxin-3 in hippocampal theta
rhythm generation (Ma et al., this meeting). Male KO mice did not display this hypoactivity, although they did display
signs of a "depressive-like" phenotype, with a strong trend to spend more time in the Porsolt posture during the forced
swim test, after either an acute or chronic stress (15-min restraint, daily for 1 or 7 days). This is consistent with findings
that relaxin-3 neuron activity is strongly influenced by CRF and psychological stressors (Banerjee et al., this meeting).
Preliminary results from measures of pre-pulse inhibition of acoustic startle in these mice also indicate that relaxin-3
may be involved in sensorimotor gating.
O-44
An in vitro Study of the Protective Effect of Relaxin on Brain Tissue
under Ischemic Stress
Brian C. Wilsona, Peter Milnea, Barry O’Connellb, and Tarek Salehb
a
Department of Biology, Acadia University, Wolfville, NS, B4P 2R6, Canada; bDepartment of Biomedical Sciences, Atlantic Veterinary
College, Charlottetown, PE, C1A 4P3, Canada
Relaxin pretreatment reduces the size of the infarct that develops following middle cerebral artery occlusion in
anesthetized rats. A nitric oxide synthase III inhibitor completely negates this effect, which suggests that an endothelinnitric oxide cascade is involved that might improve collateral perfusion of brain parenchyma in the affected zone.
We evaluated an organotypic brain slice culture technique to study the direct actions of relaxin on neural cells under
ischemic stress. Four coronal brain slices (400µm) at the level of somatosensory cortex were obtained from 21 neonatal
rats. Slices were cultured under standard conditions for 14 days. Experimental slices were placed in a deoxygenated,
glucose-free balanced salt solution (gfBSS) in 95% N2 (balance CO2) for one hour whereas control slices were
transferred to oxygenated BSS with glucose. An additional group of slices were incubated in gfBSS containing 100
nM H2 relaxin for the same period. Subsequently, all three groups of brain slices were transferred back into normal,
oxygenated media. Slices were analysed at 0, 4, 8 and 24 hours following ischemic challenge using propidium iodide
(PI) fluorescence to highlight cellular damage or death. The percentage of dead and dying cells in a slice served as a
measure of the severity of ischemic damage and was compared between groups. Relaxin treatment attenuated PI
staining at 4, 8 and 24 hours post-ischemia; this suggests that the progression of cellular death was attenuated. Relaxin
may have direct actions on cells improving their chance of survival during an ischemic challenge. Funded by NSERC
and Pfizer Canada.
36
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Abstracts
Thursday, May 22, 2008
P-21
Relaxin Activation of Water Drinking in the Mouse: Likely Role of RXFP1 in the
Subfornical Organ
Loretta Piccenna, Michael J. McKinley, Lesley L. Walker, Andrew J. Lawrence, and Andrew L. Gundlach
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
The subfornical organ (SFO) is one of a group of nuclei known as the circumventricular organs and is strongly
associated with osmoregulatory activity. Neurons in the SFO of adult rat brain express high levels of receptors for
the peptide hormone, relaxin; and intracerebroventricular (icv) administration of human relaxin to pregnant and
non-pregnant rats produces increased vasopressin secretion, blood pressure and water drinking. Intravenous (iv)
administration of relaxin also increases water drinking via activation of SFO neurons. Finally, SFO ablation in adult rats
diminishes relaxin-induced drinking, further substantiating the fundamental involvement of the SFO in this behaviour.
Recent studies indicate that the native receptor for relaxin is LGR7 or "RXFP1". However, human relaxin has high
affinity for both RXFP1 and RXFP2 and both receptors are present in rat and mouse brain. In studies to establish
whether relaxin acts through RXFP1 and/or RXFP2 to induce water consumption, we have conducted studies in wildtype
(WT) and LGR7-KO/LacZ reporter mice. Following icv injection of human relaxin (100 & 200 ng/µl), LGR7-KO mice
drank no water, or drank less than WT mice over a 5-120 min period, suggesting that relaxin acts primarily via RXFP1 to
induce drinking. Furthermore, mice display high levels of RXFP1 expression, but not RXFP2, in the SFO. These studies
did reveal an apparent behavioural activation of LGR7-KO mice by central injections of human relaxin, including
increased grooming, which is thought to reflect activation of RXFP2 (Sedaghat et al., this meeting). This possibility can
be tested using an RXFP2 antagonist, and mouse relaxin, which is more selective than the human peptide for mouse
RXFP1 cf. RXFP2.
P-22
Relaxin-3 Neurons in Nucleus Incertus of the Rat: Effect on Activity of
Psychological Stressors and the Light-Dark Cycle
Avantika Banerjee, Pei-Juan Shen, Ross A.D. Bathgate, and Andrew L. Gundlach
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
Relaxin-3 (RLN3) is distinguished by its enriched expression in the pontine nucleus incertus (NI) and its broad
projections, postulated to participate in ascending neural circuits involved in behavioural activation, biorhythms and
stress responses. In studies to investigate this hypothesis, we are examining the regulation of RLN3 expression by
different experimental stimuli. Initially, we studied the effect of swim stress on RLN3 expression in NI neurons. Rats
(3-5/group) were subjected to two 10-min forced-swim sessions 24-h apart and killed over a time-course of 0.5-24 h.
Expression of RLN3 in NI neurons was assessed by ISHH and a rapid and significant increase in both RLN3 hnRNA
and mRNA was observed (70-135% at 1-2 h, p ≤ 0.05). Swim stress induced increases in RLN3 expression were blunted
by pre-treatment with a CRF-R1 antagonist. We also studied the pattern of RLN3 expression across the light-dark cycle.
Rats were housed under 12:12h light/dark conditions for 7 days (lights on at 07:00 - designated as Zeitgeber time [ZT]
0) and were killed at ZT0, 4, 8, 12, 16, 20. RLN3 mRNA and hnRNA levels displayed an upward trend during the dark
phase with a peak at ZT 20 that was significantly above the lowest levels of expression during the light phase at ZT8,
suggesting a possible interaction between RLN3 neurons and circuits regulating circadian rhythm that warrants further
investigation. Studies are now in progress to explore the effect of ‘stressors’ such as foot-shock and restricted food
access on RLN3 neuron activity.
38
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-23
Expression and Distribution of the Relaxin Family Peptide Receptors - RXFP1-3 - in
Primate and Human Brain
Pei-Juan Shena, Sherie Maa, Ross A.D. Bathgatea, Jose L. Lanciegob, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bCenter for Applied Medical Research, University
of Navarra, 31008 Pamplona, Spain
Anatomical studies have established the presence and widespread distribution of native receptors for relaxin, INSL3
and relaxin-3 in rat and mouse brain, suggesting important roles for these systems in sensorimotor, autonomic and
neuroendocrine function. However, the precise profile of expression and distribution of these neuropeptides and their
receptors in higher species including human and their significance for neurological and psychiatric disease is not
established. In order to establish which experimental species best models these systems in human brain and in light of
some distinctive differences in their relative distributions in rat and mouse brain; this study examined the localization
of LGR7 (or RXFP1), LGR8 (or RXFP2) and GPCR135 (or RXFP3) in macaque and postmortem human brain using
radioligand binding and autoradiography. Fresh-frozen sections from the brain of macaque (Macaca fascicularis)
and sections of cerebral cortex and hippocampus from a neurologically-normal 40 year-old male were incubated with
[125I]-h-relaxin to detect RXFP1, [125I]-h-INSL3 to detect RXFP2 and [125I]-R3/I5 to detect RXFP3. In results so far,
specific [125I]-h-relaxin binding sites were detected in human cerebral cortex, not hippocampus; [125I]-h-INSL3 sites
were detected in macaque cortex; and specific [125I]-R3/I5 binding was detected in the macaque septum, hippocampus
and thalamus, areas that express RXFP3 in rat and mouse brain. Studies are continuing to confirm and extend these
initial findings using brain sections from additional primate brains and samples from other human brain areas. This
comparative mapping will provide an anatomical framework for the interpretation of research in animals to identify the
biological roles of relaxin family peptides in normal physiology and in human disease.
P-24
Relaxin-3 and RXFP3 Gene Expression and Peptide Distribution in the Brain and
Ovary of Teleost Fish
Brian C. Wilsona, Emily K. Fletchera, Rebecca Rappaporta, Sara Good-Avilaa, and Laura Parryb
a
Department of Biology, Acadia University, Wolfville, NS, B4P 2R6, Canada; bDepartment of Zoology, The University of Melbourne,
Parkville, Victoria 3010, Australia
Bioinformatic and phylogenetic analyses suggest that teleost fish possess 5 or 6 relaxin-3 genes with three forms, -3a,
3b, 3c strongly related to mammalian relaxin-3. Teleost genomes also contain predicted sequence for the relaxin-3
receptor RXFP3. RT-PCR was used to determine if relaxin-3a, -3b and -3c and RXFP3 are expressed in brain and gonad
of zebrafish, Danio rerio, and immunohistochemistry was used to determine the distribution of relaxin-3 and its receptor
in the ovary and through the rostro-caudal extent of the brains of killifish, Fundulus heteroclitus. In addition, the effect
of exogenous human relaxin-3 on steroidogenesis was studied using enzyme immunoassays and cultures of killifish
ovarian follicles. Relaxin-3a, -3b and -3c genes, and the RXFP3 gene, were expressed in the brain and gonads of male
and female fish regardless of reproductive condition. Immunostaining for relaxin-3 and GPCR135 was observed in key
brain areas mediating visual perception and visually-guided behaviours. In addition, structures involved with integrating
information from the lateral line system displayed relaxin-3 staining. This supports data suggesting that relaxin-3 is a
neuropeptide involved in sensory-motor integration and in fish, may play a role in prey capture or predator avoidance. In
the ovary, relaxin-3-immunoreactivity was observed in vitellogenic and post-vitellogenic follicles as well as the corpus
luteum. Treatment with human relaxin-3 stimulated estradiol production in vitellogenic and post-vitellogenic follicles,
but had no effect on the release of the primary progestin, maturation-inducing steroid. This suggests that ovarian
relaxin-3 may be involved with yolk deposition and processes of oocyte maturation. Funded by NSERC Canada.
Relaxin 2008
39
P-25
Insulin-like Peptide 7 (Relaxin 3): Distribution and Interaction with Two Functionally
Distinct Binding Sites in the Rat Brain
Jaw Kang Changa, Jun Yanga, Eugen Brailoiub, Siok L Dunb, Jason Grieshoberb, Jin J. Luob,c, G. Cristina Brailoiub,
and Nae J. Dunb
Phoenix Pharmaceuticals, Inc., Burlingame, California 94010, USA; Departments of bPharmacology and cNeurology, Temple University
School of Medicine, Philadelphia, Pennsylvania 19140, USA
a
Immunohistochemical studies by means of a rabbit polyclonal antiserum against the C-connecting peptide of the human
insulin-like peptide 7 (INSL7) revealed INSL7-immunoreactive (irINSL7) cell bodies in the pontine nucleus incertus,
with few cells scattered in the lateral or ventrolateral periaquaductal gray area. The mean number of irINSL7 neurons
in the nucleus incertus from three rats was about 1200. Numerous labeled cell processes were noted in the forebrain
areas; with a high density in the septum, hypothalamus, and thalamus. Optical imaging with a voltage-sensitive dye
DiSBAC4(3) or a calcium-sensitive dye Fura-2AM was used to assess the effect of INSL7 on dissociated, cultured
rat hypothalamic neurons. INSL7 (100 nM) applied by superfusion depolarized or hyperpolarized a population of
hypothalamic neurons. INSL7 (100 nM) increased intracellular calcium concentrations [Ca2+]i in a population of
hypothalamic neurons with two distinct patterns: 1) a sustained elevation with a plateau phase lasting for minutes; and
2) a fast, transitory rise followed by oscillations. In a Ca2+-free HBSS, the two types of response to INSL7 were not
substantially modified, suggesting that the rise in [Ca2+]i is mainly derived from intracellular stores. Our result shows
that INSL7 is expressed in neurons of the nucleus incertus, which provide dense innervation to the forebrain including
the hypothalamus. Functional studies suggest that INSL7 causes a membrane depolarization or hyperpolarization in a
population of rat cultured hypothalamic neurons or two patterns of calcium mobilization, raising the possibility that the
peptide may interact with two functionally distinct binding sites.
P-26
Central Administration of Human Relaxin-2 in Adult Male Rats Inhibits Food Intake
Barbara M.C. McGowan, Nicholas E. White, Debabrata Roy, Sarah A. Stanley, Waljit Dhillo, James V. Gardiner, Mohammad
A. Ghatei, and Stephen R. Bloom
Department of Investigative Medicine, Division of Investigative Science, Imperial College London, Hammersmith Campus,
London W12 ONN, United Kingdom
Relaxin belongs to the insulin superfamily, which includes “classical” relaxin, (relaxin-1 in most species, relaxin-2 in
humans) and the recently identified orexigenic hypothalamic neuropeptide relaxin-3. Central administration of relaxin-3
increases food intake in male Wistar rats, an effect thought to be mediated via the RXFP3 receptor expressed in the
hypothalamic paraventricular nucleus (PVN). Relaxin is expressed in the rat PVN, arcuate nucleus, and supraoptic
nucleus, hypothalamic regions known to play an important role in appetite. These areas express the relaxin receptor
RXFP1, also localised to the circumventricular organs where relaxin is known to affect water intake. We hypothesized
that central injections of human relaxin-2 in adult male rats may exert an effect on appetite.
Intracerebroventricular (ICV) injections of human H2 (18-540 pmol) to satiated rats significantly decreased food intake
1h post-administration in the early dark phase [2.51±0.24g (vehicle) vs 1.59±0.37g (18pmol H2), 1.59±0.23g (180pmol
H2), 1.51±0.26g (540pmol H2), p<0.05 for all doses H2]. IntraPVN administration of H2 (18-540 pmol) significantly
decreased 1h food intake in satiated rats in the early dark phase [3.13±0.35g (vehicle) vs 1.35±0.33g (18pmol H2),
p<0.001, 1.61±0.31g (180pmol H2), p<0.01, 1.23 ± 0.32g (540pmol H2), p<0.001]. Cumulative food intake was
suppressed up to 8h post-injection of H2 (180-540 pmol) compared to vehicle. ICV administration of H2 (180 pmol)
decreased feeding behaviour, and increased grooming and head-down behaviour. These results suggest that H2 may be
exerting an effect on appetite through RXFP1 in the PVN.
40
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-27
Verification of a Relaxin-3 Knockout/LacZ Reporter Mouse as a Model of Relaxin-3
Deficiency
Craig M. Smitha, Pei-Juan Shena, Sherie Maa, Steve W. Suttonb, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and
Development LLC, San Diego, California 92121, USA
A relaxin-3 knockout (KO) mouse strain was generated by deletion of exon 2 of the relaxin-3 gene and replacement
with a LacZ reporter gene cassette downstream and in-frame with the endogenous relaxin-3 promoter (Lexicon Genetics
Inc.). In this study, biochemical and anatomical studies were completed to determine the effectiveness of the gene
deletion, levels of reporter gene activity, and the effect of the gene deletion on relaxin-3 receptor (RXFP3) levels. The
genotype of mice was routinely determined from tail samples using RT-PCR and gel electrophoresis separation of
different-sized DNA fragments corresponding to the WT and/or KO allele (using primers unique for each allele). In
immunohistochemical studies using a polyclonal relaxin-3 antibody, KO mice were shown to lack relaxin-3 staining
in coronal sections through the medial septum, while strong relaxin-3 immunoreactivity was observed in equivalent
sections from WT mice. The activity of the LacZ reporter gene as a marker of relaxin-3 gene expression was confirmed,
with strong X-Gal staining detected in sections through the nucleus incertus from KO and heterozygous mice, consistent
with this region containing a high density of relaxin-3 positive cells in WT mice. In radioligand binding studies using
[125I]-R3/I5 (a chimeric peptide selective for RXFP3) and brain sections from 3 WT and 3 KO mice, we observed
equivalent densities of RXFP3 binding sites in a number of brain areas, indicating that the expression of the receptor
is not highly dependent upon activation by the ligand and demonstrating the feasibility of future studies to replace the
native ligand in relaxin-3 KO mice by central RXFP3 agonist injections or viral-based methods.
P-28
Effects of Intracerebroventricular Injection of RXFP3 Agonist and Antagonist
Peptides on Behaviour of Wildtype and Relaxin-3 Knockout Mice
Craig M. Smitha, Natalie L. Downera, Sherie Maa, Akhter Hossaina, Andrew J. Lawrencea, John D. Wadea, Steve W. Suttonb,
and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and
Development LLC, San Diego, California 92121, USA
Neuroanatomical studies in rat and mouse have revealed that a discrete population of relaxin-3 neurons in the nucleus
incertus (NI) projects to a wide range of forebrain areas expressing the relaxin-3 receptor, RXFP3 - predicting a role for
relaxin-3 signaling in sensory, limbic and neuroendocrine processing. In this regard, expression of CRF-R1 by NI cells,
increased relaxin-3 in NI after stress or icv CRF injection, and hormonal responses to icv relaxin-3 injection, suggests
a role for relaxin-3 in modulating stress responses in rats. In addition, relaxin-3 has been reported to modulate feeding
and body weight in rats, with central injections of relaxin-3 producing a consistent stimulation of food consumption and
body weight gain. More recent studies have confirmed the role of RXFP3 in feeding, using a selective RXFP3 agonist
(R3/I5) and antagonist (R3(Bδ23-27)R/I5) (Sutton et al., this meeting). The availability of specific RXFP3 agonist
and antagonist peptides and relaxin-3 KO mice (Smith et al., this meeting) prompted the present study to examine
the behavioral consequences of icv injections of R3/I5, (R3(Bδ23-27)R/I5) or both (cf vehicle) in wildtype (WT)
and relaxin-3 KO mice. Mice are anesthetized, and a guide cannula is stereotaxically inserted 1 mm above the lateral
ventricle. After recovery for 7 days, guide cannula location is checked by an injection of angiotensin II and a predicted
drinking response. In subsequent testing, mice are being assessed for levels of feeding, exploratory behaviour, and stress
and anxiety responses. Relaxin-3 KO mice will be assessed for differences in response sensitivity to the RXFP3 agonist
and for an absence of responses to the antagonist seen in WT mice.
Relaxin 2008
41
P-29
Distribution of Relaxin-3 mRNA, Relaxin-3 Immunoreactivity and RXFP3 Binding
Sites in the Brain of the Macaque (Macaca fascicularis)
Sherie Maa, Pei-Juan Shena, Qian Sanga, Jose L. Lanciegob, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bCentre for Applied Medical Research, University
of Navarra, 31008 Pamplona, Spain
Relaxin-3 (RLN3) is the ancestral relaxin peptide that preferentially binds and activates ‘GPCR135’ or ‘RXFP3’. In
the rat, RLN3 mRNA and immunoreactivity (IR) are abundant in pontine nucleus incertus (NI) neurons that project
to numerous forebrain regions, including cingulate cortex, medial septum, lateral preoptic area, lateral/dorsomedial
hypothalamus and ventral hippocampus. Functional studies suggest that NI/RLN3 neurons are involved in stress
responses, feeding and somatosensory processing. In order to confirm the relevance of this conserved neuropeptide to
higher species, this study initially examined the presence and distribution of RLN3-IR elements throughout the brain of
Macaca fascicularis, using a RLN3 C-peptide-directed antibody. In perfusion-fixed sections from four brains, RLN3IR was observed in nerve fibers in various regions, including the central gray, thalamus, and lateral and dorsomedial
hypothalamus. Staining was highly enriched in the septum, hippocampus, ventral tegmental area, and interpeduncular
and supramammillary nuclei. RLN3-IR was readily observed in nerve terminals and boutons in these areas, indicating
that RLN3 is appropriately positioned for synaptic release. Immunostained cell bodies were observed in the pontine
central gray. In situ hybridization with an antisense RLN3-riboprobe revealed RLN3 mRNA-containing cells in the
pontine central gray. The distribution of RXFP3 protein was determined by labeling fresh-frozen brain sections with
[125I]-R3/I5. The distribution of binding sites correlated with the distribution of RLN3-IR. The broad distribution of
RLN3-positive axons and terminals and RXFP3 in the forebrain of Macaca fascicularis is consistent with observations
in the rat and mouse and predicts an important role for RLN3 signaling in primate and human brain.
P-30
Relaxin Receptor LGR7 Is Regulated by Estrogen
Priya Maseelall, Gerson Weiss, Andrea Wojtczuk, and Laura T. Goldsmith
Department of Obstetrics, Gynecology, and Women’s Health, New Jersey Medical School, Newark, New Jersey 07103, USA
Relaxin has pronounced effects upon the mammalian reproductive tract and is involved in the maintenance of pregnancy.
Relaxin is an important remodeller of female reproductive tract connective tissue. Unfortunately, despite its significance
in relaxin action, understanding of LGR7 regulation is limited. Since the actions of relaxin in certain target tissues appear
to require estrogen exposure, we hypothesized that relaxin action in the cervix is modulated by estrogen. We tested this
hypothesis by determining expression of LGR7 and whether estrogen regulates its expression in a well established,
relaxin responsive, in vitro model of human term pregnancy cervix, lower uterine segment fibroblasts (LUSF). LGR7
mRNA was detected in LUSF using quantitative real-time RT-PCR. This was verified by the expected amplicon size
of 192 base pairs and a single peak at 83.3°C seen on melt analysis. LGR7 mRNA levels in LUSF were significantly
increased by estradiol to mean levels of 146% ± 6.5 (M ± SE, n=2 experiments, each performed using multiple RT-PCR
reactions) above those of control, untreated cells (p = 0.04). These data are the first demonstration of estrogen regulation
of relaxin receptor LGR7 expression. That estrogen positively regulates relaxin receptor expression in LUSF, suggests
estrogen amplification of relaxin action in the cervix.
42
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-31
Prolonged RXFP1 and RXFP2 Signalling Is Due to Weak Internalization and a Lack
of βArrestin Recruitment
Gabrielle E. Callandera, Walter G. Thomasb, and Ross A.D. Bathgatea
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bBaker Heart Research Institute, Melbourne,
Victoria 3004, Australia
A number of studies have observed that relaxin can induce prolonged physiological responses. This raises the question
as to whether RXFP1 is regulated by the same mechanisms described for most G protein-coupled receptors (GPCR). To
address this, we investigated the mechanisms that normally contribute to receptor regulation, including internalization,
trafficking of βarrestins and receptor phosphorylation of both RXFP1 and RXFP2. In HEK293 cells expressing
RXFP1 or RXFP2, and in the absence of IBMX, both receptors still elicit cAMP responses up to 6 hours after initial
stimulation. In accord with this result, RXFP1 and RXFP2 did not demonstrate any significant internalization in
response to relaxin or INSL3, as compared to the AT1 receptor, which undergoes rapid and robust internalization in
response to AngII stimulation. Additionally co-expression of GPCR kinases was found to have no effect on the rate
of internalization for either RXFP1 or RXFP2. Confocal microscopy was utilised to follow the trafficking of GFPlabelled βarrestins after receptor activation. Neither RXFP1 nor RXFP2 activation resulted in recruitment of βarrestins
to the cell surface, whereas the AT1 receptor rapidly recruits both βarrestins 1 and 2. Receptors immunoprecipitated
from [32P] metabolically labelled cells were used to investigate the agonist-specific phosphorylation. Weak receptor
phosphorylation was observed only at 30 mins, perhaps explaining the lack of internalization, but indicating the presence
of second messenger-dependent phosphorylation. These results suggest that the prolonged actions of relaxin and related
peptides reflect the absence of desensitization of RXFP1 and RXFP2 by the classically described mechanisms.
P-32
The Regulatory Action of Insulin, IGF1 and Relaxin on Nitric Oxide Synthase
Activity and its Impairment in Rat Skeletal Muscle in Condition of Streptozotocin
Diabetes of Type II
Ludmila A. Kuznetsova, Oksana V. Chistyakova, and Marianna N. Pertseva
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223, St. Petersburg, Russia
We investigated the influence of peptides of insulin superfamily on the nitric oxide synthase (NOS) activity in rat
skeletal muscle of control and diabetic groups. It was shown that endothelial NOS (eNOS) and neuronal NOS (nNOS)
activity is present in the membrane-bound form in the muscles. The stimulation of the NOS activity by insulin, insulinlike growth factor 1 (IGF-1) and relaxin was revealed. The treatment of muscles by antibodies against the nNOS led to
disappearance of stimulatory effects of insulin superfamily peptides on nNOS activity. The specific antibodies against
the eNOS have no effect on the insulin and IGF-1 NOS-activating action, but blocked the effect of relaxin. This give
evidence that the target for stimulating action of the insulin and IGF-1 is nNOS, meanwhile relaxin activates only eNOS.
It was revealed that in rat skeletal muscles with experimental diabetes of type II (80 and 180 days) the activity of NOS is
decreased and the activating action of the peptides on the membrane-bound forms of NOS is weakened. Reduced NOS
activity is associated with a decrease in the regulation of the nNOS and eNOS activities by the peptides. The conclusion
was made that in the condition of type II diabetes in the rat muscle tissues the NOS signaling pathway which mediates
the action of hormones and growth factor of insulin nature is impaired. The investigation was supported by grants:
Russian Foundation for Basic Research 06-04-48809; Basic Research for Medicine, 2007.
Relaxin 2008
43
P-33
Synthetic Peptides, Derived from the Third Intracellular Loop of Relaxin Receptor
LGR7, as Probes for Study of Molecular Mechanisms of Relaxin Action on Adenylyl
Cyclase Signaling System
Alexander O. Shpakova, Ivan A. Guryanovb, Ludmila A. Kuznetsovaa, Svetlana A. Plesnevaa, Gennady P. Vlasovb, and
Marianna N. Pertsevaa
I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, 194223, St. Petersburg, Russia;
Institute of Macromolecular Compounds of Russian Academy of Sciences, 199004, St. Petersburg, Russia
a
b
The synthetic peptides 619-629, 619-629-Lys(Palm) and 615-629, corresponding to the C-terminal region of the third
intracellular loop (C-ICL3) of the relaxin receptor of type 1 (LGR7), were used for identification of the types of relaxin
receptor participating in the realization of relaxin stimulating effect on adenylyl cyclase (AC) activity in the rat tissues
(brain, myocardium, skeletal muscles) and the muscle tissues of invertebrates - the earthworm Lumbricus terrestris,
the mollusks Anodonta cygnea and Lymnaea stagnalis. The peptides 619-629-Lys(Palm) and 615-629 in competition
manner inhibit the AC stimulating effect of relaxin in the brain and myocardium but don’t change this effect in the
other tissues. The data obtained show that receptor LGR7 participates in realization of AC stimulating effect of relaxin
in brain and myocardium, meanwhile give evidence in favor of the another AC signaling mechanisms of relaxin action
(in particular six-components, discovered by authors) in skeletal muscles and muscles of invertebrates, which did not
involve the receptor of LGR7 type. The AC stimulating effect of relaxin in brain and myocardium was decreased in the
presence of C-terminal peptide 385-394 of mammalian Gαs-subunit and was blocked by treatment with cholera toxin.
Thus, in brain and myocardium the relaxin stimulates AC via receptor LGR7 and Gs-protein. The coupling between
them is mediated by the interaction of C-ICL3 of the receptor and C-terminal segment of Gαs. The investigation was
supported by grants: Russian Foundation for Basic Research 06-04-48809, Basic Research for Medicine, 2007.
P-34
Regulation of Lgr8 (RXFP2) Expression in the Mouse Fetal Kidney by the
Transcription Factor Pod1
Mary Familari, Duc Vu, and Laura J. Parry
Department of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia
Lgr8 (or RXFP2) is expressed in the developing and adult rat kidney. At embryonic stage E18.5, Lgr8 mRNA
was localised to putative mesangial cells in the developing glomerulus, suggesting that Insl3-Lgr8 is involved in
glomerulogenesis. This process begins just after the renal tubule undergoes segmentation to form a nephron, with the
glomerulus at the proximal end, around E12-13 in mouse. One of the main regulatory factors in glomerulogenesis is
the basic helix-loop-helix (bHLH) transcription factor Pod1, which is expressed in the stroma. Null Pod1 mice have
a marked reduction in the number of nephrons and delayed glomerulogenesis. The promoter region of the Lgr8 gene
contains an E-box consensus sequence that can bind bHLH factors, including Pod1. Therefore, we examined the
expression of Lgr8 relative to Pod1 in the fetal and neonate kidney and tested the hypothesis that Lgr8 is a downstream
target of Pod1. Fetal and neonate kidneys were collected at E14.5, E16.5 and E18.5, and day 1, 4 and 7 postpartum. Lgr8
and Pod1 expression were compared between these stages using qPCR. In the second study, fetal kidneys were dissected
from Pod1-/- mice on E18.5 to assess the effects on Lgr8 expression. Lgr8 gene expression was highest on E14.5 and
E16.5 but decreased significantly on E18.5 and in the neonate kidney. This was not correlated with increased Pod1
expression. However, there was a significant increase in Lgr8 expression in the E18.5 kidneys of Pod1-/- mice. These
data suggest that Pod1 may negatively regulate Lgr8 expression in the developing glomerulus.
44
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-35
Expression Profile of Insulin-like Peptide-5 (INSL5) and GPCR142—A Perspective
on Functional Roles and Future Directions
Qian Sanga, Steven W. Suttonb, Ross A.D. Bathgatea, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and
Development, San Diego, California 92121 USA
Insulin-like peptide-5 (INSL5) was first characterized as a member of the insulin gene superfamily by Conklin et al. in
1991. Human INSL5 cDNA was isolated from a library constructed from sigmoid colon tissues from a Crohn’s disease
patient and mouse cDNA was obtained from libraries constructed from irradiated colon and thymus. Quantitative RTPCR analysis of human and mouse tissues revealed that INSL5 was highly expressed in the colon and thymus, but
the physiological role of INSL5 is unknown and yet to be explored. In the present study, we examined the presence
and distribution of INSL5 mRNA in the adult mouse using in situ hybridization histochemistry. Using a DIG-labeled
antisense riboprobe, we observed apparent INSL5 mRNA expression in the large intestine and in the thymus, where
specific labeling was present in the cortex and not the medulla. INSL5 expression in the thymus cortex suggest that
INSL5 may be involved in cellular immunity processes; and the thymus cortex contains T-cell precursors and is where
early events of T-cell development and positive selection occur. Recent bioinformatics and molecular studies have
established that the native receptor for INSL5 is GPCR142 or "RXFP4". Unlike other relaxin peptide-receptor pairings,
INSL5/ RXFP4 do not appear to be important for brain function, but high levels of RXFP4 mRNA have been detected
in human intestine and colon, suggesting that INSL5-RXFP4 signaling may play a role in these tissues. Further studies
are planned to localize the expression of INSL5 and RXFP4 in mouse GI-tract, as a basis for functional studies in normal
and gene-knockout mice.
P-36
Changes in Arterial Function by Chronic Relaxin Infusion Are Mediated by the
Leucine Rich Repeat G Coupled Lgr7/RFXP1 Receptor
Julianna E. Debraha, Alexander Agoulnikb, and Kirk P. Conradc
a
Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA; bDeparment of Obstetrics and Gynecology, Baylor College of
Medicine, Houston, Texas 77030, USA; Department of Physiology and Functional Genomics, University of Florida College of Medicine,
Gainesville, Florida 32610, USA
Circulating relaxin contributes to renal circulatory adaptations in pregnancy. Chronic administration of recombinant
human relaxin (rhRLX) to nonpregnant rodents inhibits myogenic reactivity and increases passive compliance of small
renal arteries (SRA), thereby mimicking pregnancy. We hypothesize that these arterial responses to rhRLX are mediated
by the Lgr7/RFXP1, and not the Lgr8/RFXP2 receptor. Lgr7 and Lgr8 receptor-deficient, and wild-type virgin mice
were investigated. rhRLX or vehicle (VEH) was infused for 5 days. SRA were then isolated and mounted in a pressure
arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a 20 mmHg step
increase in intraluminal pressure). SRA from rhRLX-infused Lgr7 wild-type mice showed inhibited myogenic reactivity
with a 6.1 ± 0.5% increase in diameter whereas the arteries from rhRLX-infused Lgr7 knock-out mice exhibited robust
myogenic reactivity with only a 1.2 ± 0.4% change in diameter (p=0.001). In contrast, myogenic reactivity of SRA was
comparably inhibited in both the rhRLX-infused Lgr8 knock-out and wild-type mice (p=NS). SRA from VEH-treated
mice, regardless of genotype, all exhibited robust myogenic reactivity. rhRLX infusion increased arterial compliance of
SRA from Lgr7 wild-type, but not from Lgr7 knock-out mice (p=0.01). In contrast, arteries from rhRLX-infused Lgr8
knock-out mice showed increased arterial compliance relative to VEH-infused knock-out animals (p=0.001). Thus,
relaxin-induced inhibition of myogenic reactivity and increase in passive compliance of small renal arteries is mediated
by the Lgr7, and not the Lgr8 receptor.
Relaxin 2008
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P-37
Relaxin Promotes Matrix Metalloproteinase-2 and Decreases Wnt/β-Catenin
Expression in the Neonatal Porcine Heart
Teh-Yuan Hoa, Megan A. Diltsa, Frank F. Bartolb, and Carol A. Bagnella
Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA; bDepartments of Animal Sciences and
Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA
a
Relaxin (RLX), reported to play an important role in cardiovascular remodeling, is linked to activation of matrix
metalloproteinases (MMPs). RLX is found in the circulation of nursing pigs from birth and RLX receptor (RXFP1)
transcripts have been identified in the neonatal heart. The neonatal pig heart undergoes remodeling in the first few
days of life with left ventricular (LV) hypertrophy and collagen accumulation due to volume overload. Wnt/β-catenin
signaling has also been implicated in cardiac remodeling. Therefore, objectives of this study were to determine the
effects of RLX on the myocardial Wnt/β-catenin signaling system and MMP expression at postnatal day (PND) 2. The
LV myocardium was obtained from gilts on PND 2 after administration of vehicle (PBS) or porcine RLX (20 ug/kg BW/
in PBS every 6h for 48h; n=4/group). In other studies, LV myocardial explants from PND 2 gilts (n = 3) were incubated
with vehicle or porcine RLX (1-100 ng/ml) for 24 hr when both medium and tissue were collected. Results showed that
myocardial Wnt7a and Wnt4 gene expression decreased following RLX administration in vivo, while there was no effect
on Wnt5a expression. Immunoreactive myocardial β-catenin protein was reduced in RLX-treated animals. Zymographic
analysis of medium from RLX-treated heart explants showed an increase in proMMP-2, but not proMMP-9 activity.
Western blot analysis indicated that RLX increased myocardial MMP-2 protein as compared to controls. Data suggest
that the RLX-induced decline in Wnt/β-catenin expression at PND 2, together with increased MMP-2 activity, may be
important for neonatal cardiac remodeling. (Support USDA-NRI 2003-35203-1357 and USDA-2007-35203-18098)
P-38
Boar Testis Acts as a Source and Target Tissue of Relaxin
Tetsuya Kohsakaa,b, Shinichi Katoa, Siqinb, Itaru Minagawaa, Keiichiro Yogoa,b, Tatsuo Kawarasakic, and Hiroshi Sasadad
a
Laboratory of Animal Reproduction, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan; bDivision of Animal Resource
Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan; cShizuoka Swine and Poultry
Experimental Station, Kikugawa 439-0037, Japan; dLaboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku
University, Sendai 981-8555, Japan
Testicular function is regulated by the complex interplay of many different molecules that includes both endocrine and
paracrine signaling, and proper regulation is critical for optimum reproductive capacity. Among molecules, relaxin has
also been suggested to be implicated in regulating testicular function in rodents. Also in boars, there is limited evidence
that the testis might be a candidate source and target of relaxin, although it is presently unknown whether relaxin actually
has any effect on the testis. To identify that the boar testis is a source and target tissue of relaxin, we characterized
the expression and cellular localization of both relaxin and its own receptor LGR7 by molecular and immunological
approaches. Relaxin gene expression increased through puberty onwards, while LGR7 gene expression changed little
during pubertal development. Cellular expression of relaxin gene and protein was restricted to the Leydig cells, whereas
LGR7 gene and protein were expressed by both Leydig cells and seminiferous epithelial cells. Interestingly, relaxin
protein was only detected in the testis as 18 kDa form (the expected size of prorelaxin), but not as the 6 kDa mature
form, during pubertal development because of lacking the processing enzyme for prorelaxin. Because recombinant and
native prorelaxin is known to have a high bioactivity, it is possible that the 18 kDa protein expressed in the boar testis
also has substantial bioactivity. Thus, we conclude that the testis is both a source and target tissue of relaxin in boars,
suggesting that a functional relaxin/LGR7 hormone-receptor network operates within the boar testis.
46
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-39
α-Adrenergic Activation Upregulates Expression of Relaxin Receptor RXFP-1 in
Cardiomyocytes
XiaoLei Moore, Alice Hong, and Xiao-Jun Du
Experimental Cardiology Lab, Baker Heart Research Institute, Melbourne, Australia
Relaxin family peptide receptor-1 (RXFP-1) mediates multiple effects of relaxin. However, factors regulating RXFP-1
expression remain largely undefined. We have explored whether activation of α- and β-adrenergic receptors (AR), a
class of GPCRs, may regulate expression RXFP-1 in cardioyocytes. Neonate rat cardiomyocytes (NRCM) in culture
were treated with α1- or β-AR agonists for 24 hr, in the presence or absence of specific inhibitors. RXFP-1 mRNA level
was analysed by real-time qPCR with results normalized by reference genes. Compared with untreated NRCMs, α1-AR
agonists (100 μM methoxamine, 5-50 μM phenylephrine or 10 μM norepinephrine) increased RXFP-1 expression by
approximately 3 folds (P<0.0001). In contrast, β1-AR agonists (10 μM dobutamine) reduced RXFP-1 expression by
50% (P=0.001) while β2-AR agonist zinterol (1 μM) showed no effect. The upregulation of RXFP-1 mediated by α1-AR
agonists was blocked by selective inhibitors, respectively, for protein kinase-C (PKC, 1 μM BIM or 10 μM Ro320432),
protein kinase-A (PKA, 2 μM KT5720 or 1 μM H89) or ERK (30 μM PD98059 or 10 μM U0126). In mice with
cardiomyocyte-restricted overexpression of α1A- and α1B-AR, RXFP-1 expression, relative to non-transgenic littermates,
increased by 3.2-fold (P<0.0001) and 7.6-fold (P<0.02), respectively, in keeping with the in vitro finding. RXFP-1
expression was not altered in hearts of β2-AR transgenic mice.
Conclusion: α1-AR activation up-regulates RXFP-1 gene expression with signal pathways involving PKA, PKC and
ERK, while activation of β1-AR, but not β2-AR, down-regulates RXFP-1 expression. This is the first demonstration
on distinct regulation by adrenergic signalling of RXFP-1 expression, which links effects of relaxin with
sympathoadrenergic activities.
P-40
Relaxin/RXFP1 Signaling in Prostate Cancer Progression
Shu Fenga, Irina U. Agoulnikb, Zhen Lia, Hee Dong Hanc, Gabriel Lopez-Beresteinc, Anil Soodc, Michael M. Ittmannd,
and Alexander I. Agoulnika
a
Departments of Obstetrics and Gynecology, bMolecular and Cellular Biology, dPathology, Baylor College of Medicine, Houston, Texas
77030, USA; cDepartment of Gynecologic Oncology, MD Anderson Cancer Center, University of Texas, Houston, Texas 77030, USA
We investigated the role of relaxin/RXFP1 signaling in prostate cancer progression. Quantitative RT-PCR analysis
showed that while the mRNA expression of relaxin receptor RXFP1 was maintained at the same level in human prostate
cancer and normal samples, relaxin mRNA expression was significantly higher in prostate cancer samples compared
with the normal prostate tissues. Relaxin immunohistochemistry using tissue microarrays confirmed higher relaxin
protein expression in prostate cancer samples. Stimulation with relaxin increased LNCaP and PC3 cells proliferation,
invasiveness, and adhesion in vitro. Alternatively, the suppression of relaxin/RXFP1 expression via short interfering
RNAs decreased prostate carcinoma cells invasiveness by 90-95%, cell growth by 10- 25% and increased cell apoptosis
about 3 times. The RXFP1 gene suppression in PC3 cells resulted in decreased phosphorylation of protein kinase B
(AKT). Illumina microarray analysis of changes in gene expression in PC-3 cells caused by RXFP1 siRNA knock down
revealed several potential targets of relaxin signaling in prostate cancer. The effect of activation of the relaxin signaling
on prostate cancer progression in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) was
analyzed in mice with transgenic relaxin overexpression and Rxfp1-deficient mice. The Tg(Rln1), TRAMP males had
shorter median survival time, associated with the decreased apoptosis of tumor cells, whereas Rxfp1-/-, TRAMP mice
on average lived longer than the wild type TRAMP mice. Suppression of RXFP1 expression in PC3 xenografts in nude
mice using siRNA chitosan nanoparticles resulted in decrease of tumor size. Thus, relaxin/RXFP1 might be a potential
therapeutic target in the treatment of prostate cancer.
Relaxin 2008
47
O-45
Relaxin’s Induction of Specific MMPs Contributes to Degradation of Cartilage
Matrix in Target Synovial Joints: Receptor Profiles Correlate with Matrix
Remodeling Responses
Sunil Kapila and Wei Wang
Department of Orthodontics and Pediatric Dentistry, The University of Michigan, Ann Arbor, Michigan 48109, USA
The long-term goal of our studies is to understand the mechanisms by which relaxin and estrogen potentially contribute
to joint diseases particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we
showed that relaxin produces a dose-dependent induction of tissue degrading enzymes of the matrix metalloproteinase
(MMP) family, specifically MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase), and MMP13 (collagenase-3) in cell isolates and tissue explants from the rabbit and mice TMJ disc fibrocartilage. Further in vitro
tissue explant studies demonstrated that the induction of these MMPs is accompanied by loss of tissue collagen and
glycosaminoglycans (GAGs), which was blocked by adding a pan-MMP inhibitor to the cultures. We also found the
targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with beta-estradiol or relaxin
or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The
modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic
symphysis, and differed from that of the knee meniscus, which did not show any changes in its matrix composition.
Finally, we found that the two target tissues showing the greatest modulation of MMPs and matrix loss, namely the
TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors (ER)-alpha and -beta, relaxin-1
(LGR7) receptor, and INSL3 (LGR8) receptor that differed substantially from those in cells from the knee meniscus.
Together, these findings suggest a novel model for targeted tissue turnover of cartilages of specific joints through
hormone-mediated induction of select MMPs. (Supported by NIH R29 DE11993, KO2 DE00458 and RO1 DE018455)
O-46
Relaxin’s Involvement in ECM Homeostasis: Two Diverse Lines of Evidence
Timothy E. Cooneyb, John D. Lubahnb, Justine M. Schoberb, and Elisa Konieczkoa
a
Department of Biology, Gannon University, Erie, Pennsylvania 16541, USA; bDepartments of Orthopaedics, Orthopaedic Research, and
Urology, Hamot Medical Center, Erie, Pennsylvania 16550, USA
Introduction: Burgeoning evidence suggests that the hormone, relaxin, modulates collagen in the ECM of diverse tissues
(Garber et al., 2001; Williams et al, 2001; Samuel et al., 2002; Masterson et al., 2004; Samuel et al., 2004). In separate
lines of study, we provide further substantiation of this notion.
Methods: LGR7 Expression in Ligament Fibroblasts: Immunofluorescence (IF) was used to probe isolated fibroblasts
derived from explant culture for vimentin, actin, LGR7, and estrogen receptor beta. Ligaments were obtained as surgical
waste from thumb reconstruction patients. Four specimens have been examined to-date.
LGR7 Expression in Pediatric Skin Biopsies: Immunohistochemistry (IHC) was used on frozen sections from 24
biopsies from children undergoing genitoplasty. A subset of samples were also probed for TGF-B isoforms. Appropriate
controls were used. Finally, a subset of patient blood was assayed for relaxin using an ELISA-based method.
Results: IF data showed that cells expressed vimentin and actin, consistent with fibroblast morphology. Cells derived
from two of three female patients expressed LGR7 receptors; the solitary male was negative. Given the small sample,
however, the data are preliminary.
IHC data demonstrated LGR7 receptors in the basement membrane of the epidermis, regardless of gender. Most tissue
expressed TGF-B. Finally, serology suggested that relaxin was detectable in these children.
Discussion: Our two lines of research provide additional evidence for diverse tissue tropism for relaxin. In particular,
connective tissue as diverse as ligaments and basal lamina keratinocytes express LGR7. These data lend support to our
contention that relaxin affects ligament integrity and wound healing.
48
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-47
Relaxin in the Airway and Lung: Potential as a Novel Treatment for Asthma
Mimi L.K. Tanga, Chrishan S. Samuelb, and Simon R. Roycea
a
Department of Allergy and Immunology, Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria 3050
Australia; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
We have identified an important role for relaxin in the regulation of collagen deposition and remodeling in the airway/
lung, and demonstrate a potential role for relaxin as a novel treatment for asthma, targeting airway fibrosis and airway
hyperresponsiveness (AHR) in an animal model of allergic airway disease (asthma). We have previously reported that
mice deficient in relaxin develop age-related changes similar to airway remodeling in asthma, including subepithelial
fibrosis, epithelial remodeling, and AHR; and that mice deficient in relaxin have markedly increased airway fibrosis in
an animal model of allergic airway disease (AAD). It is now demonstrated that relaxin and the relaxin receptor LGR7
are co-localised to cells involved in the remodeling process in asthma – airway epithelium, fibroblasts, smooth muscle
cells, and that relaxin expression by these cells is significantly reduced in human asthma and in a murine model of AAD.
Together, these findings suggest a role for relaxin as an inhibitor of airway fibrosis and AHR in asthma. The effect of
exogenous relaxin treatment on airway remodeling and airway hyperresponsiveness was examined in a chronic model of
AAD. Recombinant relaxin reversed collagen deposition in the airways and significantly reduced AHR when compared
to vehicle treated control animals. Matrix metalloprotease levels were increased, suggesting a possible mechanism for
the antifibrotic effect.
Conclusion: Relaxin plays an important role in the negative regulation of airway fibrosis during homeostasis and in
asthma. Relaxin treatment can reverse remodeling changes of airway fibrosis and significantly inhibit AHR in an animal
model of AAD (asthma).
O-48
Scar Reduction and Cosmetic Effects of Relaxin
Dennis R. Stewart
Corthera, Inc. (formerly BAS Medical, Inc.), San Mateo, California 94402, USA
Relaxin has previously been tested in rodent wound healing models and been shown to promote angiogenesis and speed
healing. However, pigs have been shown to be a better model for human skin in dermatology studies so juvenile pigs
were selected for a study of scar reduction and cosmetic appearance. Twelve 20 x 6 mm excisional wounds were created
per animal and relaxin was administered systemically and/or by topical application for 6 weeks. Systemic relaxin was
administered via infusion pumps at a rate of 125 ug/kg/day. Some wounds were irrigated with relaxin solution during the
first week. Topical formulations of relaxin with 0, 0.5 or 5 mg/ml and applied twice daily for weeks 2-3 and then daily
for weeks 3-6. Visual assessments of wound closure and scab removal were made on day 7 following wound creation.
Assessments of healing and cosmetic appearance were made by a dermatologist at weeks 2, 4 and 6. Wound sites were
collected at 6 weeks and evaluated histologically for granulation tissue, inflammation and collagen organization. Wounds
in animals receiving systemic relaxin had improved appearance with less redness, reduced granulation tissue and lower
amounts of inflammation. They showed a more well knit collagen structure compared to controls. Wounds treated with
topical formulations did not show improvement over controls. The topical formulation used was found to have a short
residence time which likely limited penetration of relaxin. Reformulated relaxin preparations with improved penetration
might be useful as a topical treatment for wounds to prevent or reduce scarring.
Relaxin 2008
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O-49
Role of Relaxin during Human Osteoclastogenesis
Arianna Facciolli, Anastasia Pepe, Lisa Gianesello, Carlo Foresta, and Alberto Ferlin
University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for
Male Gamete Cryopreservation, 35121 Padova, Italy
The aim of this study is to characterize the possible role of relaxin of human osteoclasts. In fact, we are currently
investigating the effects of relaxin and INSL3 on bone metabolism, and previous studies showed en effect of relaxin
on peripheral blood mononuclear cells (PBMCs), the precursors of osteoclasts. Analysis of RXFP1 and RLN-2 mRNA
expression in primary cell culture of human osteoclasts obtained from PBMCs by RT-PCR showed only the presence
of RXFP1 transcripts. Immunofluorescence analysis confirmed the expression in osteoclasts also of the corresponding
RXFP1 protein. We then analysed the in vitro effect of relaxin on osteoclastogenesis (from PBMCs to mature osteoclasts,
about 14 days) and activated mature osteoclasts. We added relaxin 50 pM during osteoclastogenesis and collected the
cells every 2-3 days. RNA from these cells was used to perform quantitative RT-PCR for the analysis of expression of
4 osteoclast specific genes (TRAP, CTSK, RANK, NFATc1). Relaxin produced a significant increase in CTSK, RANK
and NFATc1 gene expression but not on TRAP. However, acute (minutes, hours) and chronic (days) stimulation of the
activated mature cells with the same dose of relaxin did not modify gene expression. This study shows, for the first time,
a role for the hormone relaxin on human bone metabolism regulating osteoclastogenesis.
O-50
In Search of the Elusive Vasodepressor Agents of Pregnancy
Kirk P. Conrada, Lee A. Danielsonb, Sanjeev G. Schroffc, Dan O. Debrahc, Arundhathi Jeyabalanc, Jacqueline Novakd, John M.
Davisone, and Marie Smithe
Universities of Florida, bNew Mexico, cPittsburgh, and dWalsh University, USA; eUniversity of Newcastle upon Tyne, United Kingdom
a
Understanding the mechanisms underlying arterial adaptations to normal pregnancy may lead to novel therapies for
the prevention and treatment of preeclampsia, as well as certain renal and cardiovascular diseases in the nonpregnant
population. Our early work challenged the prevailing hypothesis that vasodilatory prostaglandins mediate maternal renal
and systemic hemodynamic adaptations to pregnancy. Rather, we provided evidence for a major role of NO and its upand down-stream mediators, the endothelial ETB receptor and cGMP, respectively. In search of reproductive endocrine
signals, we postulated a role for the ovarian hormone, relaxin. We subsequently showed that relaxin administration to
conscious female and male rats increases renal blood flow and glomerular filtration rate (GFR), and inhibits myogenic
reactivity of small renal arteries ex vivo. Identical renal circulatory changes are observed during pregnancy, and they
are prevented by relaxin neutralizing antibodies or ovariectomy. In nonpregnant women and men, relaxin is also a
potent renal vasodilator, and women lacking circulating relaxin show markedly subdued increases in GFR during
early pregnancy. Relaxin administration to conscious male and female rats increases cardiac output and global arterial
compliance, and reduces systemic vascular resistance. Identical changes are observed during pregnancy, and they
are completely or partly abrogated by relaxin neutralizing antibodies during mid and late gestation, respectively.
Investigations of another candidate vasodilator, hCG, suggests that it may play a role in some organ circulations during
pregnancy, but not the kidney. Finally, additional players in the relaxin vasodilatory pathway have been identified,
including Lgr7/RXFP1, vascular gelatinases and angiogenic growth factors.
50
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-51
Relaxin: An Endogenous Renoprotective Factor?
Tim D. Hewitsona,b and Chrishan Samuelc,d
a
Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria 3050 Australia; bDepartment of Medicine, The University of
Melbourne, cHoward Florey Institute and dDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne,
Victoria 3010, Australia
Fibrosis, so called scarring, is a key cause of pathology and dysfunction in a variety of organs. The biology of healing
and scarring are similar, albeit with very different outcomes. While the kidney has some capacity for repair after acute
injury, renal fibrosis represents a failure of the wound-healing process after prolonged injury. Typical of this process
in general, renal fibrosis is the result of a disproportionate accumulation of extracellular matrix that occurs in ongoing
kidney disease. Renal scarring is both progressive and multifactorial, ultimately leading to end-stage renal failure and the
requirement for dialysis or kidney transplantation.
Regardless of the underlying etiology, renal mesenchymal cells such as the fibroblast are the cellular basis of this
process. Activity and differentiation of these cells are regulated by a number of profibrotic cytokines, growth factors,
vasoactive mediators and other signals, with transforming growth factor-beta being amongst the most important.
However, we now also recognise that there are a number of endogenous antifibrotic factors that are renoprotective by
counteracting these signals to limit matrix synthesis and organisation, and reduce net accumulation. Relaxin is one
such factor. The absence of endogenous relaxin results in spontaneous fibrosis with aging and also accelerates the
pathogenesis of progressive fibrosis after injury. Both effects are reversed by replenishment with recombinant H2relaxin.
This talk summarises recent developments in the understanding of the cellular and molecular mechanisms in fibrosis, and
how these are influenced by relaxin.
O-52
Investigations into the Signaling Mechanisms by which Relaxin Inhibits Renal
Myofibroblast Differentiation
Chrishan S. Samuela,b, Ishanee Mookerjeea,b, Michelle L. Hallsc, Roger J. Summersc, Geoffrey W. Tregeara,b,
and Tim D. Hewitsond,e
a
Howard Florey Institute and Departments of bBiochemistry and Molecular Biology and dMedicine, The University of Melbourne, Parkville,
Victoria 3010, Australia; cDepartment of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; eDepartment of Nephrology,
Royal Melbourne Hospital, Parkville, Victoria 3050 Australia
Derived from fibroblasts, myofibroblasts are the principal cells that are responsible for the synthesis and reorganization
of excess matrix in renal interstitial fibrosis. Recognized from their de novo expression of α-smooth muscle actin
(α-SMA), myofibroblast differentiation and activity can be influenced by several factors including a combination
of growth factors and other soluble mediators, extracellular matrix components and mechanical stress. Relaxin has
previously been shown to inhibit renal myofibroblast differentiation in vitro, an effect partly mediated through its
ability to interfere with the TGF-β pathway via inhibition of Smad2 phosphorylation and translocation. Furthermore,
endogenous relaxin has been shown to protect the kidney from a myofibroblast-mediated model of injury in vivo.
However, the pathways involved in the interaction between relaxin and TGF-β remain unknown. In this study, we
therefore sought to further investigate the signaling mechanisms by which human gene-2 (H2) relaxin regulates (myo)
fibroblast differentiation in vitro by examining the effect of relaxin on mixed populations of fibroblasts/myofibroblasts
propagated from injured rat kidneys. Cultures containing ~70% myofibroblasts were assessed for relaxin receptor mRNA
expression and used to determine which i) relaxin receptors (RXFP1, RXFP2), ii) G-proteins and iii) signaling pathways
(PI3 kinase, nitric oxide, Smad2) were involved in the regulation of α-SMA by H2 relaxin. Our findings suggest that
relaxin protects the injured kidney by signaling through RXFP1 in addition to the nitric oxide and Smad2 pathways to
inhibit renal myofibroblast differentiation.
Relaxin 2008
51
O-53
Relaxin-induced Changes in Renal Function and RXFP1 Receptor Expression in the
Female Rat
Alsadek H. Bogzil and Nick Ashton
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Pregnancy is associated with profound changes in renal structure and function. The aim of this study was to determine
whether relaxin induces pregnancy-like changes in renal function in female rats. Female Sprague Dawley rats received
human recombinant relaxin (0.4 μg/h or 4 μg/h) or vehicle (20 mM sodium acetate) via an osmotic minipump for 7 days
prior to the measurement of renal function using a servo-controlled fluid replacement system to maintain euvolaemic
body fluid status. Extracellular fluid volume was determined by calculating the dilution of injected 3H inulin. Kidneys
were harvested from relaxin-treated and pregnant rats (day 11, corresponding to maximal plasma [relaxin]) to quantify
RXFP1 receptor expression. Seven-day administration of relaxin at 0.4 μg/h resulted in a decrease in plasma osmolality
(control 292.6±2.1 vs relaxin 236.9±6.0 mOsm/kg), sodium and chloride concentrations (P<0.05); extracellular fluid
volume was increased (control 19.5±0.4 vs relaxin 21.6±0.7 ml/100g bwt, P<0.05). Urine flow rate was unaffected,
but sodium (control 4.9±0.5 vs relaxin 2.8±0.4 μmol min-1 100g bwt-1), chloride and osmolar excretion rates were
decreased significantly (P<0.01). RXFP1 expression (protein and mRNA) was increased in the kidneys of rats treated
with relaxin at 0.4 μg/h and in pregnant rats. Immunohistochemistry showed that RXFP1 was present on the proximal
tubules and inner medullary collecting ducts. In contrast, relaxin-treatment at 4 μg/h for 7 days had no effect on renal
function in female rats. These data suggest that relaxin may alter renal electrolyte handling in a manner similar to that
seen in pregnancy.
O-54
Vascular Endothelial and Placental Growth Factors: New Players in the Slow
Relaxin Vasodilatory Pathway
Julianna Debrahb, Jonathan T. McGuanea, Jacqueline Novakd, J. Peter Rubinc, and Kirk P. Conrada
Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610, USA; bMageeWomens Research Institute, Pittsburgh, Pennsylvania 15213, USA; cUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
15213, USA; dWalsh University, North Canton, Ohio 44720, USA
a
The mechanism of relaxin’s "slow" vasodilatory effect in rats involves MMP2- or MMP9-mediated conversion of
big endothelin (ET) to ET1-32, which activates the endothelial ETB receptor/NO pathway. Because LGR7/RXFP1 is
predominantly expressed in the vascular smooth muscle (VSM), we reasoned that this vasodilatory response to relaxin,
which mediates renal vasodilation in vivo and reduced myogenic reactivity of small renal arteries ex vivo, is transduced
from the VSM to the endothelium, and hypothesized that vascular endothelial and/or placental growth factors (VEGF
and PlGF) could fulfill this function.
Small rat and mouse renal and human subcutaneous arteries were studied in a pressure arteriograph. In arteries pretreated with control antibodies, myogenic reactivity (measured by the percent change in diameter in response to a
20mmHg step increase in intraluminal pressure) was inhibited by relaxin treatment (30ng/ml) for 3 hours. In contrast,
pre-treatment with VEGF-neutralizing antibodies blocked the response to relaxin and restored myogenic reactivity in
rat and mouse small renal (1.4±0.8 and 0.3±0.9% [VEGF Abs] vs 8.7±1.1 and 5.4±0.4% [control Abs], respectively;
p<0.001) as well as human subcutaneous arteries (0.9±0.7% [VEGF Ab] vs 6.4±1.0% [control Ab]; p=0.003).
Furthermore, pre-treatment with PlGF-neutralizing antibodies similarly negated the effect of relaxin in rat and mouse
small renal (-0.7±0.8 and 1.2±1.1% [PlGF Abs] vs 7.1±0.9 and 5.4±0.4% [control Abs], respectively; p#less than or
equal to#0.005) as well as human subcutaneous arteries (-2.7% [PlGF Ab] vs 5.9% [control Ab]).
These data indicate that VEGF and PlGF are essential players in the relaxin vasodilatory pathway, potentially involved in
cross-talk between VSM and endothelium.
52
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-55
Effects of Relaxin on the Development of Mesangial Proliferative Nephritis
Naoki Ikegayaa, Tetsuya Kohsakab, Takuya Yoshidac, Hiromichi Kumagaic, Tatsuo Yamamotod, and Akira Hishidad
a
Medical Care Center, bDepartment of Applied Biological Sciences, Shizuoka 422-8529, Shizuoka University, cDepartment of Clinical
Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, d1st Department of Medicine, Hamamatsu
University, School of Medicine 431-3192, Hamamatsu, Japan
Relaxin (RLX), belonging to the insulin family, has been known as a hormone of pregnancy. Although potent antifibrotic
and vasodilatory properties of RLX are recently reported, the involvement in nephritis has not been fully elucidated. In
this study, we examined the expression of RLX receptors and effects of RLX administration in experimental nephritis
induced by anti-thymocyte serum (ATS). When examined immunohistochemically the expression of RLX receptors in
normal and nephritic kidneys, nephritic glomeruli showed an increased immunostaining compared to normal glomeruli.
The administration of RLX by osmotic minipump for 7 days to rats with ATS nephritis did not cause a significant
alteration in blood pressure or body weight. On the 7th day, however, a significant reduction in urinary protein (66.6
vs. 35.8 mg/day, p<0.05) was recognized in rats treated with RLX. Histologic studies revealed the amelioration of
glomerular accumulation of PAS positive matrix in rats received RLX. A significant reduction of phosphorylated
SMAD2 as a marker of TGF-β activation was observed in rats treated with RLX compared to rats without RLX
treatment. These results indicate that RLX may play a significant role in the development of mesangial proliferative
glomerulonephritis in rats independently of blood pressure, suggesting that RLX can be a potential therapeutic tool for
nephritic diseases.
O-56
Relaxin Affects Endothelial Progenitor Cell Number and Function
Mark Segala, Laura Sautinaa, Elaine Beema, and Kirk Conradb
a
Departments of Medicine, bPhysiology and Functional Genomics, and Ob/Gyn, University of Florida College of Medicine, Gainesville,
Florida 32610, USA
Relaxin is known to induce vasodilation that is endothelial and nitric oxide (NO)-dependent. Endothelial progenitor
cells (EPC) are derived from the bone marrow and can repair vascular endothelium. Since previous studies have shown
a profound effect of intracellular NO on the number and function of EPC. We hypothesized that relaxin would increase
the number of circulating cells in vivo and stimulate EPC migration in vitro via an NO dependent mechanism. Male mice
were implanted with osmotic minipumps containing recombinant human relaxin (rhRLX) or vehicle (VEH). rhRLX was
infused at 1µg/hour. After 5 days, the number of EPC colonies and serum levels of rhRLX were determined. Relaxintreated mice had more EPC colonies derived from blood than VEH-treated mice (3 +/- 0.8 vs 0.7 +/- 0.5, p <0.05), and
there was a significant correlation between the number of EPC colonies and serum rhRLX concentrations (R2 = 0.42). In
addition, we demonstrated that human EPC migrate equally well towards rhRLX and stromal-derived factor (SDF)-1, the
major chemokine for EPC. Interestingly, EPC chemotaxis was inhibited by L-NAME, an inhibitor of NO synthase. Last
we demonstrated that addition of rhRLX to human EPC leads to an increase in NO. These results suggest that relaxin can
lead to an increase in NO in EPC and modify EPC number in vivo and behavior in vitro. These findings have profound
implications for the therapeutic potential of relaxin. On a cautionary note, relaxin may contribute to progression of
diabetic retinopathy during pregnancy.
Relaxin 2008
53
O-57
Evaluation of Relaxin Blood Profiles as a Means of Assessing Placental Function
and Responsiveness to Therapeutic Strategies in Mares
Peter L. Ryana, David L. Christiansena, Richard M. Hoppera, Carol A. Bagnellb, Wendy E. Vallac, and Michelle M. LeBlancd
Mississippi State University, College of Veterinary Medicine, Mississippi State, Mississippi 39762, USA; bRutgers University, New Brunswick,
New Jersey 08901, USA; Intervet Incorporated, Millsboro, Delaware 19966, USA; cRood and Riddle Equine Hospital, Lexington,
Kentucky 40511, USA
a
Placental insufficiency is regarded as the primary factor contributing to late-term abortion and perinatal death of
foals. Often when problems associated with late-term pregnancy in the horse are manifested the condition is well
advanced and therapeutic intervention may be ineffective in rescuing the pregnancy. If a compromised pregnancy due
to placental insufficiency could be identified early, the pregnancy might be sustained through medical intervention.
Since the placenta is the sole source of circulating relaxin in mares we hypothesized that systemic relaxin may serve
as a biomarker of placental function, fetal well-being and predictor of pregnancy outcome at delivery. To test this
hypothesis we monitored plasma relaxin in mares (light breeds) with normal and problematic pregnancies from clinical
cases presented to the veterinary hospital and pregnant mares experimentally inoculated with Streptococcus equi
zooeidemicus to induce uterine infection. Upon establishment of placentitis, mares were assigned to different therapeutic
strategies and responsiveness monitored. Blood was collected during the third trimester of pregnancy to determine
relaxin concentrations using a homologous equine relaxin radioimmunoassay. The results showed a positive relationship
between lower circulating relaxin and poor pregnancy outcome in mares with compromised placental function in
addition to improved profiles in response to drug therapy. Data support the hypothesis that relaxin has the potential to
serve as a useful means of monitoring placental function and fetal-well being in horses. Relaxin, as a diagnostic assay,
could assist veterinarians identify mares with at-risk pregnancies in a more timely fashion, evaluate therapeutic efficacy,
placental function and fetal well-being.
O-58
Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed-up
Species: New Methods, Uses and Findings
Bernard Steinetza, Salamia Lasanoa, Florine de Haas van Dorsserb, Stephen Glickmanc, and William Swansond
a
Department of Environmental Medicine, NYU School of Medicine, Tuxedo, New York 10987, USA; bDepartment of Veterinary Medicine,
University of Cambridge, United Kingdom; cDepartment of Psychology, University of California, Berkeley, California 94720, USA; dCenter
for Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA
The human population explosion has pushed many mammalian wildlife species to the brink of extinction.
Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. Serum
immunoactive relaxin provided an early and reliable indicator of true pregnancy in Asian and African elephants and
Sumatran rhinoceroses, and is thus an important aid for conservationists studying reproduction in subjects with very long
pregnancies. Unfortunately, the immunoassay was not useful in the giant panda, because of its variable implantation time
and gestation length. These studies required judicious selection of relaxin antisera and standards developed in domestic
species: equine relaxin RIAs for elephants; porcine relaxin RIAs for Sumatran rhinos and pandas.
Our canine relaxin RIA has now been adapted and validated to measure relaxin in the serum and urine of felids,
including domestic, Pallas’ and sand cats, Arabian leopards and cheetahs. Moreover, a commercially available canine
serum relaxin kit (Witness Relaxin Kit, Synbiotics), has been adapted for reliable detection of relaxin in felid urine.
The porcine relaxin RIA has also been utilized to investigate the role of relaxin in the reproductive processes of the
spotted hyena, a species in which the female fetuses are severely masculinized in utero. Indeed, this species might well
now be extinct were it not for the timely secretion of relaxin to enable both copulation and birth of young through
the clitoris.
Given appropriate immunoassay reagents, relaxin determination thus provides a powerful tool for conservationists and
biologists investigating reproduction in endangered and exotic species.
54
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-59
Development of Adult Neuronal-specific Relaxin-3 Knockout Rats Using Adenoassociated Viral-driven miRNA
Gabrielle E. Callandera, Walter G. Thomasb, Andrew L. Gundlacha, and Ross A.D. Bathgatea
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bBaker Heart Research Institute, Melbourne,
Victoria 3004, Australia
Recent in vivo peptide administration studies suggest a role for relaxin-3 in feeding and stress, however the function
of relaxin-3 remains elusive due to the significant challenges of the technique, including limited permeability of the
blood-brain barrier and problems with peptide stability, absorption and metabolism. Additionally, results from knockout
(KO) mice must be interpreted with caution as KOs often develop functional compensation. In studies designed to
avoid such obstacles, we are employing RNA interference and viral gene transfer, to modulate neuronal relaxin-3
expression in the adult rat brain. We have designed, constructed and characterized adeno-associated viral particles
containing relaxin-3 (pAAV-Rln3) or microRNA (pAAV-miRNA) targeting relaxin-3. Media collected from HEK293T
cells transiently expressing pAAV-Rln3, stimulated RXFP1-mediated cAMP accumulation in a dose-dependent fashion,
similar to H3 relaxin, whereas media from cells transfected with a control viral vector had no effect. Three constructs,
containing miRNA directed against different targets within the relaxin-3 transcript, were then tested for their ability to
reduce the expression of relaxin-3 in pAAV-Rln3 transfected cells. All miRNA constructs significantly reduced relaxin3-induced cAMP responses, however miRNA499 was most effective. To investigate the role of relaxin-3 in feeding,
rAAV-miRNA499 and a control virus rAAV-miRC, were produced and purified for stereotaxic injection into the nucleus
incertus of male rats. Post-infection body weight and food consumption are currently being monitored. These viral
tools can be readily utilised in vivo to investigate the role of relaxin-3 in other behaviours including arousal/motivation,
biorhythms and responses to stress.
O-60
Presence and Role of LGR8 (RXFP2) in Thalamo- and Cortico-Striatal Circuits:
Effects of Intrastriatal Injection of INSL3 on Stereotypic Behaviour in the Rat
Katayoun Sedaghata, Suode Zhanga, Ross A.D. Bathgatea, John D. Wadea, David I. Finkelsteinb, and Andrew L. Gundlacha
a
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bMental Health Research Institute, The University
of Melbourne, Parkville, Victoria 3052, Australia
Leucine-rich repeat-containing G-protein-coupled Receptor 8 (LGR8 or "RXFP2") is the native receptor for the peptide
hormone, insulin-like peptide-3 (INSL3), and the aim of these studies was to determine the anatomical distribution of
LGR8 (mRNA and protein) and its functional role in rat brain. In situ hybridization of [35S]-labeled oligonucleotides
revealed high densities of LGR8 mRNA in the intralaminar thalamus (especially the parafascicular nucleus), distinct
layers of frontal cortex and the medial habenula. Autoradiography of [125I]-INSL3 binding sites revealed high densities
of LGR8 protein in distinct bands of frontal and motor cortex, in the thalamus and corpus striatum, and in medial
habenula-fasciculus retroflexus-interpeduncular nucleus pathway. In conscious rats, bilateral injection of INSL3 (~1040 pmol/side in 0.5 µl) into the LGR8-rich ventrolateral striatum, via indwelling guide cannulae, produced a rapid and
strong behavioural activation, with a distinct upward-sniffing displayed for between 2-40 min post-injection. Injection of
the LGR8 antagonist, Ades(1-8)-INSL3 (~40 pmol), did not produce sniffing, but co-injection with an equal amount of
INSL3, blunted the effect of the agonist peptide. Co-injection of amphetamine (20 µg) with INSL3 (40 pmol) produced
a significant increase in sniffing behaviour cf. INSL3 alone. In light of the anatomical data suggesting the presynaptic
location of LGR8 on excitatory thalamo- and cortico- striatal projections, and the behavioural effects observed, we
suggest that INSL3-induced sniffing behavior is produced by LGR8-induced activation of glutamate release in the
striatum. This in turn may alter dopamine release from nigrostriatal inputs and alter the activity of target medium spiny
neurons - ideas that can be tested experimentally.
Relaxin 2008
55
Notes
56
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Abstracts
Friday, May 23, 2008
P-41
Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis
Robert G. Bennetta,b,c, Dean G. Heimanna, and Dean J. Tumaa,b
a
Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA; bDepartments of Internal Medicine, Pharmacology and Experimental
Neuroscience, and cDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha,
Nebraska 68198, USA
Relaxin has antifibrotic effects in a number of tissues, including the liver. Previous studies have focused on the effects
of relaxin in the prevention of hepatic fibrosis, but not after establishment of the disease. To address this issue, we
compared the effects of relaxin treatment either before or after the establishment of hepatic fibrosis. In the progressive
fibrosis study, mice were treated intraperitoneally with carbon tetrachloride (CCl4) for 4 weeks, concomitantly with
porcine relaxin administered by osmotic pumps at 0.5mg/kg/day. In these animals, relaxin caused a reduction in
collagen and smooth muscle actin content, indicating reduced hepatic stellate cell activation. Serum levels of the liver
enzymes ALT and AST were significantly reduced in the relaxin-treated animals, suggesting improved liver function.
In the second experiment, CCl4 was administered for 4 weeks, at which time mice were treated with both CCl4 and
relaxin at 0.5mg/kg/day for 1 week further. In relaxin-treated mice, both liver collagen and smooth muscle actin content
were reduced compared to untreated mice. However, in contrast to the experiment above, relaxin treatment did not
significantly reduce the ALT/AST levels. In conclusion, the data suggest that relaxin treatment concomitant with liver
injury results in reduced collagen deposition and hepatic stellate cell activation, and improved liver function. Relaxin
treatment after fibrosis establishment also reduced collagen and hepatic stellate cell activation, but did not improve
overall liver function within the 1 week treatment time examined. Studies are underway to determine the effect of
extended relaxin treatment on established hepatic fibrosis.
P-42
Evaluation of Relaxin’s Anti-fibrotic Actions by SELDI-TOF Mass Spectrometrybased Protein Profiling of Relaxin KO Mice, a Model of Progressive Fibrosis
Eleni Giannakisa, Mary Macrisa, Geoffrey W. Tregeara,b, Chrishan S. Samuela,b, and John D. Wadea,b,c
a
Howard Florey Institute and Departments of bBiochemistry and Molecular Biology and cChemistry, The University of Melbourne, Parkville,
Victoria 3010, Australia
Fibrosis (organ scarring) is a leading cause of morbidity and mortality, and is characterized by an overproduction of
collagen and causes a hardening of vital tissues leading to a progressive loss of their function. The peptide hormone,
relaxin (RLX) has known anti-fibrotic properties and is currently under investigation as a possible therapeutic agent.
However, to be most effective, RLX needs to be administered in the early stages of disease progression. The primary
aim of this study was to develop diagnostics for the onset of fibrosis to maximize the therapeutic potential of RLX and
provide a means to measure the efficacy of therapeutic intervention strategies. The RLX-1 knockout (-/-) mouse, which
exhibits age-related fibrosis progression, was used as the model for this study. RLX-/- mice were administered with
recombinant human (H2) RLX in citrate buffer (vehicle), which reverses the diseased phenotype, or, as a control, with
vehicle alone each day. Sera from healthy and diseased animals were collected at different time points: 9-months (early
onset of fibrosis) and 12-months (established fibrosis). These were analysed by Surface Enhanced Laser Desorption
Ionisation- Time of Flight- Mass Spectrometry (SELDI-TOF MS). Bioinformatics analysis using Biomarker Wizard©
software revealed the presence of a potential biomarker for early onset fibrosis and which is upregulated in the diseased
animals. Additional studies will involve assessing tissue from healthy and diseased animals and examining earlier time
points before the diseased phenotype has been detected.
58
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-43
Novel EGF and IGF1 Adenylyl Cyclase Signaling Mechanisms with Similar
Structural-functional Organization
Marianna N. Pertseva, Ludmila A. Kuznetsova, Alexander O. Shpakov, and Svetlana A. Plesneva
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223, St. Petersburg, Russia
As we have shown for the first time the hormones of insulin superfamily (insulin, relaxin) are capable to activate
adenylyl cyclase in muscle tissues of vertebrates and invertebrates via six-component signaling cascade: receptortyrosine kinase -> Gi-protein (βγ dimer) -> phosphatidylinositol 3-kinase -> protein kinase C (ζ) -> Gs-protein ->
adenylyl cyclase (AC). The aim of the present study is to prove if this AC signaling mechanism (ACSM) is involved
in the action of IGF1 and EGF - two unrelated growth factors. The deciphering of ACSM was performed with
pharmacological and immunological approach (specific inhibitors and antibodies). It was found that AC activating effect
of both growth factors are realized via: tyrosine kinase (tyrphostin 47 and genistein inhibition), Gi protein (pertussis
toxin ADP ribosylation); phosphatidylinositol 3-kinase (wortmannin inhibition), protein kinase C (ζ) (specific antibody
inhibition), Gs protein (cholera toxin ADP ribosylation). The data obtained and our foregoing results lead to conclusion
that IGF-1 and EGF ACSM have a similar architecture, involving six-component signaling which coincides with that of
insulin and relaxin. It is supposed that this similarity in ACSM of insulin superfamily peptides and EGF is based on their
common growth promoting activity connected with cAMP-system. According to our hypothesis the mitogenic effect of
hormones and growth factors of insulin nature and EGF is realized via ACSM.
The investigation was supported by grants: Russian Foundation for Basic Research 06-04-48809; Basic Research for
Medicine, 2007.
P-44
Investigating the Role of Endogenous Relaxin in Experimental Diabetic
Renal Disease
Su Ee Wonga,b,c, Tim D. Hewitsonc,d, James Beckerd, Geoffrey W. Tregeara,b, Gavin J. Beckerc,d, and Chrishan S. Samuela,b
a
Howard Florey Institute and Departments of bBiochemistry and Molecular Biology and dMedicine, The University of Melbourne, Parkville,
Victoria 3010; Australia; cDepartment of Nephrology, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Pathological complications of diabetes include the accumulation of excess matrix in several organs, including the kidney.
Streptozotocin (STZ)-induced diabetic relaxin wild-type and knock-out mice were used to investigate the ability of
endogenous relaxin to regulate renal collagen turnover. Renal pathology in untreated control animals was compared
to age-matched diabetic animals with a mean blood glucose >14mmol/L after 2 STZ injections (n=6 per genotype
and group). Histochemical and biochemical techniques were used to assess collagen content and sub-types, matrix
metalloproteinase (MMP) expression and activity, tissue inhibitor of metalloproteinase (TIMP) expression and TGF-β1
activity. At 12 weeks post-STZ administration, diabetic animals had higher levels of glycated haemoglobin (by 1.3-fold;
p<0.05), 1.4-fold (p<0.01) higher total renal collagen concentration (hydroxyproline analysis) and >2-fold (p<0.01)
more collagen IV (immunohistochemistry; IHC), with no change in collagen I (IHC), III (SDS-PAGE) or tissue TGFβ1 activity (ELISA). For equivalent levels of hyperglycaemia, no differences in collagen concentration, sub-types or
distribution were observed between genotypes. Activity of the type IV collagenase, MMP-2 (gelatin zymography) and its
inhibitor, TIMP-2 (reverse zymography) were both up-regulated in diabetic mice (p<0.05), with no difference between
genotypes. There was no increased expression of MMP-9, while its inhibitor TIMP-1 was only increased in diabetic
knock-out mice (p<0.05). Findings in this model of early diabetic renal disease suggest that endogenous relaxin does
not alter the pathology of the diabetic mouse kidney. This may be due in part to the normal levels of TGF-β1 activity in
this model.
Relaxin 2008
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P-45
Determinants of Relaxin-induced Changes in Passive Compliance of Small Renal
Arteries: Geometric vs. Compositional Remodeling
Dan O. Debraha, Julianna E. Debrahb, Michael S. Sacksa, Kirk P. Conradc, and Sanjeev G. Shroffa
a
Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA; bMagee-Womens Research Institute,
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA; cDepartments of Physiology and Functional Genomics and
Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32610, USA
We have recently demonstrated that small renal arteries (SRA) from relaxin-treated mice are characterized by both
geometric (increased wall area) and compositional (decreased collagen content) remodeling. Additionally, we found
that these arteries also exhibit increased passive compliance following relaxin treatment. The goal of this work was to
determine the relative contributions of these two forms of remodeling to the increase in
passive compliance.
Nonpregnant female mice were administered recombinant human relaxin (rhRlx) for 5 days after which SRA were
isolated. Ex vivo pressure-diameter data, collected at various longitudinal stretches, were analyzed using a biaxial
constitutive relationship (7-parameter mixed polynomial-exponential model of strain energy function). Vascular wall
mechanical properties at various levels of circumferential and longitudinal strains were quantified in terms of the
corresponding strain energy.
There were no significant differences in strain energy between control and rhRLX-treated groups at low strains
(longitudinal stretch ≤ 1.5, circumferential stretch ≤1.8). At higher strains, however, arteries from the rhRLX-treated
mice exhibited increasingly lower magnitudes of strain energy such that the greatest reduction was observed at the
highest strains.
Our data suggest that under low longitudinal and circumferential loads, the relaxin-induced increase in passive
compliance of SRA is primarily a result of geometric and not compositional remodeling because vascular wall
mechanical properties (strain energy) are unaltered under these conditions. The relaxin-induced compositional
remodeling of SRA becomes functionally relevant only under conditions of high longitudinal and
circumferential loads.
P-46
Relaxin as a Protective Substance in the Preserving Solution for Liver
Transplantation: Spectrophotometric in vivo Imaging of Local Oxygen Supply in an
Isolated Perfused Rat Liver Model
Markus U. Boehnerta, Franz Paul Armbrusterb, and Heidegard Hilbigc
a
Department of Visceral and Transplantation Surgery, University of Bern, Inselspital, 3010 Bern, Switzerland; bImmundiagnostik AG, 64625
Bensheim, Germany; cInstitute of Anatomy, University of Leipzig, 04103 Leipzig, Germany
Reperfusion injury is a well-known problem in organ transplantation. Liver injury caused by ischemia and reperfusion
results from multiple pathologic mechanisms including endothelial damage, neutrophil extravasation, platelet and mast
cell activation as well as peroxidation of cell membrane lipids. Relaxin is known to cause inhibition of platelet and mast
cell activation and vessel dilation. Firstly, the local oxygen supply in liver tissue was investigated by spectrophotometric
in vivo imaging. Secondly, the present study aims to assess the protective effect of relaxin in preservation solution
on liver tissue against cell damage during organ preservation and reperfusion. We used a model of isolated perfused
rat liver, simulating liver transplantation. Organ preservation was performed identically to human transplantations
in 30 male Wistar rats. 64ng of Relaxin per ml solution were applied during preservation. Controls (n=15) were
performed identically without Relaxin. Spectrophotometric imaging of oxygen supply was done using the Erlangen
Micro-Lightguide Spectrophotometer. The oxygen supply was correlated with markers of oxidative cell damage
using immunohistochemistry. Determinants of oxidative stress were malonyldialdehyde (MDA), a metabolite of lipid
peroxidation and myeloperoxidase activity (MPO), a marker for accumulation of neutrophil granulocytes. Compared to
the controls, spectrophotometry qualitatively revealed a better oxygen supply in Relaxin treated liver tissue. Accordingly,
administration of Relaxin in the preservation solution decreased MPO and MDA activity in liver tissue. Our data suggest
that Relaxin has an influence on oxygen distribution in liver tissue and is a promising agent to reduce cell damage in the
liver caused by ischemia and reperfusion injury.
60
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-47
Involvement of Relaxin in Salt-sensitive Hypertension
Naoki Ikegayaa, Tetsuya Kohsakab, Takuya Yoshidac, and Hiromichi Kumagaic
a
Medical Care Center, bDepartment of Applied Biological Sciences, Shizuoka 422-8529, Shizuoka University, cDepartment of Clinical
Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
Although potent anti-fibrotic and vasodilatory properties of relaxin (RLX) are recently reported, the involvement of RLX
in salt-sensitive hypertension has not been elucidated. We investigated effects of RLX on salt-sensitive hypertension.
First, we examined RLX and RLX-receptor (LGR7) in the kidney of male Dahl salt-sensitive (DS) and Dahl saltresistant (DR) rats placed on 8.0% salt diet for 8 weeks. RLX and LGR-7 mRNA was observed in both cortex and
medulla of DS and DR rats. Specific binding of RLX in kidneys was observed and the binding sites of RLX were
similar to LGR-7 immunostaining. By western blotting, renal LGR7 expression increased after 8% NaCl diet. The
increase of LGR7 protein was greater in DS rats than that in DR rats. Next, we examined effects of RLX treatment on
blood pressure, renal histology, apoptosis and TGF-β activation in DS and DR rats placed on an 8% NaCl diet. The
administration of RLX (4 microg/h) to DS rats for 8 weeks significantly reduced systolic blood pressure. Histologic
studies revealed the amelioration of tubulointerstitial fibrosis (-21.8%) and glomerular hypertrophy in rats received
RLX. RLX-treated kidneys showed significantly decreased apoptosis (-40.8%), and phosphorylated Smad2 (-71.0%)
expression in tubules compared to saline control.
These results indicate that RLX-LGR7 axis exists within kidneys and not only extrarenal but also intrarenal regulation of
RLX may work in kidneys. These findings provide background to future clinical trials with RLX as anti-fibrotic therapy
in salt-sensitive hypertension.
P-48
Volar Oblique Ligament Fibroblasts Express Estrogen, LGR7 Receptors
Elisa M. Konieczkoa, John D. Lubahnb, Timothy E. Cooneyb, and Michelle Heida
a
Department of Biology, Gannon University, Erie, Pennsylvania 16541, USA; bDepartments of Orthopaedics and Orthopaedic Research,
Hamot Medical Center, Erie, Pennsylvania 16550, USA
Introduction: Basal joint osteoarthritis (BJA) is predominant in females (Newport, 2000). Deterioration of the volar
oblique ligament (VOL) precedes BJA (Pelligrini et al., 1992). Thus, sex hormones such as estrogen and relaxin may
play a role in ligament integrity. Immunohistochemistry suggests VOL tissue contains estrogen and relaxin receptors
(Krushinski et al., 2002; Lubahn et al, 2006); little is known about isolated VOL fibroblasts. We probed explant
fibroblasts to determine whether LGR7, the putative relaxin receptor, and estrogen receptor beta are expressed.
Methods: With consent, four VOLs were obtained from surgery patients (3 female, 1 male, ages 51-65 YO). Tissue was
minced and cultured in supplemented medium. After seven days, adherent cells were trypsinized, and passaged. Cells
from Passage 3-7 were placed on coverslips, fixed, permeabolized, blocked with serum/PBS, and probed with primary
antibodies for vimentin, actin, estrogen receptor beta, or LGR7. Fluorescent-labeled secondary antibodies were used for
detection. For negative controls, the primary antibody was omitted.
Results: Cells evidenced abundant staining for vimentin and actin, cytoskeletal elements consistent with fibroblast
morphology. Cells obtained from two females stained positively for both LGR7 and estrogen receptor beta; remaining
specimens were negative. In all cases, control slides were negative.
Discussion/Conclusions: These limited data show that 67% of female donors had fibroblasts that expressed sex
hormone receptors; cells from the solitary male donor did not. This provides evidence that some ligament fibroblasts are
competent to respond to relaxin and estradiol. Given our small sample, additional research is warranted to confirm or
refute our findings.
Relaxin 2008
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P-49
Basement Membrane Localization of LGR7 Receptors and TGF-beta in Juvenile
Skin Biopsies
Justine M. Schoberb and Timothy E. Cooneya
Department of Orthopaedic Research; bDepartment of Urology, Hamot Medical Center, Erie, Pennsylvania 16550, USA
a
Introduction: Pediatric genitoplasty produces dermal scarring, compromising dermal sensation and sexual function in
adulthood. Given potential antifibrotic activities, the polypeptide hormone, relaxin, might benefit wound healing. As a
first step, we screened skin biopsies for the relaxin receptor LGR7, as well as TGF-beta, a mediator of scarring.
Methods: Twenty-nine pediatric patients, ages 1-12 YO, provided specimens with consent. Fourteen had labial
deformities, 15 had circumcisions/reconstructions. Specimens were snap-frozen, fixed with cold acetone, quenched of
peroxidase, and blocked with serum. Standard, two-antibody immunohistochemistry localized antigens of interest. Of
the 29 specimens, 24 were screened for LGR7, 17 for TGF-B1, and 16 for TGF-B3. Naïve serum was used as a negative
control; uterine tissue served as a positive control for LGR7 staining. Additionally, blood samples from 16 female
patients were analyzed for relaxin content.
Results: LGR7 was localized to cells lining the basement membrane in most specimens (23/24). As well, TGF-B1 (7/17)
and TGF-B3 (15/16) were co-expressed in this area. Positive and negative controls showed the predicted response.
Eighty-eight percent (14/16) of blood samples had detectable levels of relaxin, averaging 51.5 pg/ml (range: 0-223).
Discussion/Conclusions: Skin biopsies from children evidenced LGR7 and TGF-B coexpression in the cells that
populate the basement membrane of the epidermis, presumably keratinocytes. Systemic relaxin was also present. That
basement membranes in other anatomic areas interact with relaxin has been previously demonstrated (Stemmermann et
al., 1994; McDonald et al., 2003; Mookerjee et al., 2006). Taken together, these data infer a role for relaxin in wound
healing and epithelializaton.
P-50
Relaxin Differentially Regulates Gelatinase Expression and Activity in Cultured
Gingival Fibroblasts
Chrishan S. Samuela,b, Ross Bathgatea,b, Chongxin Zhaoa, Sharon L. Layfielda, Geoffrey W. Tregeara,b, and Dennis R. Stewartc
Howard Florey Institute and bDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010,
Australia; cCorthera Inc., San Mateo, California 94402, USA
a
Many of relaxin’s diverse actions, including its growth- promoting, matrix remodeling and vasodilatory effects are
mediated via regulation of the gelatinases, members of the matrix metalloproteinase (MMP) family. In recent preclinical studies conducted by Corthera Inc. (formerly BAS Medical), the administration of recombinant H2 relaxin to
Beagle dogs was able to prevent orthodontic relapse in a similar manner to that induced by surgical fiberotomy. It was
concluded however, that the relaxin-induced effect may have been potentially enhanced with improved dosing schedules.
We therefore investigated various treatment-regimes involving relaxin on dog gingival fibroblast-stimulated gelatinase
expression and activity under i) basal and TGF-β-stimulated conditions, ii) conditions simulating orthodontic force,
and iii) continuous vs intermittent treatment. Relaxin-treatment of normal gingival fibroblasts significantly increased
MMP-2 (gelatinase-A) expression and activity and MMP-9 (gelatinase-B) expression (all p<0.05 vs control cell values)
at concentrations of 1-100ng/ml, as assessed by gelatin zymography and MMP-2 activity-assay kits. These effects were
not specific to gender, but were enhanced when fibroblasts were stimulated by TGF-β1 or centrifugal force. Maximal
increases in MMP-2 and MMP-9 expression (both p<0.01) were observed when H2 relaxin was continuously applied
to force-activated cells, while intermittent H2 relaxin application only promoted MMP-2 expression and activity.
Furthermore, the H2 relaxin-induced increase in gelatinase expression and activity correlated to decreased gingival
collagen deposition, only when H2 relaxin was administered to TGF-β1-activated fibroblasts. These findings suggest that
relaxin differentially regulates gelatinase expression which may have consequences on matrix remodeling in general.
62
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-51
INSL3 Plays a Role in the Balance between Bone Formation and Resorption
Anastasia Pepea, Lisa Gianeselloa, Arianna Facciollia, Alexander I. Agoulnikb, Alberto Ferlina, and Carlo Forestaa
a
University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for
Male Gamete Cryopreservation, 35121 Padova, Italy; bDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston,
Texas 77030, USA
We have recently demonstrated that the disruption of the INSL3 hormonal system, as observed in humans with
mutations in RXFP2 gene and Rxfp2-KO mice, might have a pathogenic role in osteoporosis. In particular, the low
bone mass phenotype in the Rxfp2-deficient mice is not due to a hyper-resorptive state, but to functional alterations in
the osteoblast compartment causing little bone formation and ultimately a negative balance between bone formation
and bone resorption. In the present study we better characterized the in vitro effect of INSL3 on human osteoblasts
and its interaction with testosterone. Stimulation of human primary osteoblasts with INSL3 at serial concentrations,
from 0.01nM to 10nM, induced a significant and dose-dependent increase in proliferation. We then investigated the
expression of 4 typical osteoblast genes (Osteonectin, M-CSF, Osterix and NFATc1) on cells stimulated with INSL3 and/
or Testosterone at different doses (1pM, 1nM, 1uM). Quantitative RT-PCR analyses show that expression of M-CSF,
Osterix and NFATc1 genes are positively affected by the synergic effect of INSL3 and Testosterone at the highest
concentration used. A significant increase in Osteonectin expression was observed when the osteoblasts were stimulated
with INSL3 or Testosterone alone. This data suggest for the first time that INSL3 could act in synergy with the other
testicular hormone, Testosterone, on osteoblast physiology and could also affect the osteoblast-osteoclast equilibrium
necessary for the development and maintenance of bone tissue.
P-52
Mutations in INSL3-RXFP2 Genes in Cryptorchid Boys
Alberto Ferlina, Daniela Zuccarelloa, Andrea Garollaa, Riccardo Selicea, Cinzia Vinanzia, Francesco Ganza, Carla Cazzadorea,
Biagio Zuccarellob, and Carlo Forestaa
a
University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre
for Male Gamete Cryopreservation, 35121 Padova, Italy; bPaediatric Surgery, Department of Medical and Surgical Paediatric Sciences,
University of Messina, Italy
Cryptorchidism is the most frequent malformation of the urogenital tract in males, with a incidence of 3% at birth, and
it is associated with important consequences in adulthood, such as infertility and testicular cancer. Mutations in the
INSL3 and RXFP2 genes have been associated with human cryptorchidism, but genetic screening performed in adults
with history of cryptorchidism led to contrasting results. In the present study we analysed the frequency of mutations in
these genes (and in the androgen receptor gene, AR) in a large cohort of newborn with cryptorchidism. We study 285
boys (age 6 months-3 years) who underwent unilateral (171 subject) or bilateral (114 subjects) orchidopexy. Mutation
analysis was performed by PCR, DHPLC and sequencing of the two exons of INSL3, all 8 exons of AR and exon 8 of
RXFP2 (where the T222P mutation map). We found 5 RXFP2 mutations in 5 unrelated bilateral cryptorchid boys, 1
INSL3 mutation in a unilateral cryptorchid boy and 3 AR mutation in three unrelated unilateral cryptorchid boy. Overall,
the frequency of INSL3-RXFP2 mutations is therefore 6/285 (2.1%), and 5/114 (4.4%) in bilateral cryptorchidism.
These data well fit with the observation that the incidence of INSL3-RXFP2 gene mutations in infertile adults with
history of cryptorchidism is 4-5%, considering that about 50% of cryptorchid boys will be infertile in adulthood. This
study confirmed the association between INSL3-RXFP2 gene mutations and human cryptorchidism and suggests such a
genetic screening in cryptorchid boys, given the potential association with additional phenotypes in adulthood, such
as osteoporosis.
Relaxin 2008
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P-53
The Mechanisms behind Relaxin on Improving Muscle Healing
Yong Lia,b,c, Xiaodong Mua,b, Haiying Pana, Mia Jeffersona, and Johnny Huarda,b,d
Stem Cell Research Center, Children’s Hospital of Pittsburgh; bDepartment of Orthopaedic Surgery; cDepartment of Pathology and
Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
a
d
Injuries to the muscle represent a large portion of all the injuries encountered professional and recreational sports. These
injuries occur through a variety of mechanisms including direct trauma, such as muscle lacerations and contusions, and
indirect injuries which include strains, ischemia, and neurological dysfunctions. Injured muscle is capable of healing
through the regeneration of the damaged myofibers, however, the process is slow and often results in an incomplete
recovery. The optimal treatments for these types of injuries are not clearly defined, varying widely and dependent on
the type and severity of the injury. Treatments that utilize various growth factors have demonstrated increased muscle
regeneration; however, these approaches have been fraught with the deposition of fibrous scar tissue that ultimately
infers with the complete functional recovery of the muscle. Our previous studies have determined that relaxin, a
polypeptide cytokine/growth factor within the insulin-like growth factor (IGF) family, could improve muscle healing
through the prevention of fibrous scar tissue formation after laceration injury. In the current experiment, we able to
demonstrate that relaxin was able to promote muscle cell differentiation and fusion in vitro and in vivo. We observed that
relaxin, mostly like its’ other family member IGF-1, can stimulate myogenic gene activation in skeletal muscle cells.
Our results also implicated relaxin’s receptor (LGR7), which can be found on the cell surface of skeletal muscle cells, in
it’s ability to positively affect skeletal regeneration. Further research will explore if relaxin can share some of signaling
pathways with IGF-1 which promote myogenic gene expression in muscle cells and improve muscle healing after strain,
an extremely common injury encountered in sports medicine.
P-54
Relaxin Has a Role in the Molecular Dynamics of Cellular Movement
Yvonne Radestockb, Sabine Hombach-Klonischa, Joanna Bialekb, Cuong Hoang-Vub, and Thomas Klonischa
Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada; bClinic of Surgery,
Faculty of Medicine, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
a
GTPases are upstream regulators of cofilin and various other pathways, including Arp3/4 (nucleation of actin at the
plasma membrane), catenins/ E-cadherin (stabilization of adherens junctions), protein kinase C (PKC) isoforms (cell
signaling/ polarization), and adducin (cortical actin network). Relaxin-mediated signaling may involve the action
of small GTPases as suggested by the relaxin-induced cytosolic to plasma membrane translocation of PKC zeta.
Furthermore, the cytoskeletal dynamics associated with the transition from an adherent polarized cell to a motile/
invasive phenotype with ECM-degrading capabilities involves beta-actin polymerization at the leading edge of cellular
movement which is governed by the cofilin capping protein and ARP2/3 pathways. Rho family small GTPases, Cdc42,
Rac1, and RhoA, are monomeric G-proteins that act as key transducers of extracellular signals to the actin cytoskeleton
and are upstream regulators of this process. Stable transfectants of the human thyroid carcinoma cell line FTC-133 secret
bioactive relaxin and display increased motility. Here we show that FTC-133-relaxin clones display an up-regulation
of total and phosphorylated cofilin and the small GTPase proteins Cdc42, Rac and RhoA. Cofilin has F-actin severing/
depolymerising activities and is phosphorylated by LIM kinase which was also increased in RLN and INSL3 clones.
These data provide a first molecular rationale for a novel role of the relaxin-RXFP1 ligand-receptor system as a
morpho-functional regulator of cytoskeletal dynamics. The enhanced expression of Rho family GTPases is likely to
contribute to an enhanced beta-actin polymerization and changes in vesicular trafficking which would manifest in
enhanced motility, altered vesicular secretion profiles, and enhanced ECM-degrading capabilities observed in the
FTC-133-relaxin transfectants.
This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research
Council of Canada (NSERC), Manitoba Medical Foundation (MMSF), and Manitoba Health Research Council (MHRC).
64
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
P-55
In vitro Relaxin H2 Strengthens Fetal Membranes?
Samer Yousfia, James Novaka, Robert Moorea, Deepak Kumara, Maria Pereza, Brian Mercerb, John Moorea,b, and
Gillian Bryant-Greenwoodc
a
Pediatrics, bReproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA; cDevelopmental and Reproductive
Biology, University of Hawaii, Honolulu, Hawaii 96813 USA
Relaxin has been shown to increase extracellular matrix (ECM) remodeling and specifically MMP activity in target
tissues. One study of the effect of relaxin on fetal membrane biophysical properties showed a biphasic pattern with low
doses causing mild weakening and higher doses showing no effect on the tissue. Towards the end of gestation, the fetal
membranes undergo a programmed, biochemically-mediated weakening process with concomitant ECM remodeling and
cellular apoptosis. In addition, we have reported that in vitro incubation of fetal membrane with TNFα and IL-1β can
reproduce the entire weakening and remodeling process. Evidence is less clear for other potential biochemical mediators
and we hypothesized that relaxin would weaken human fetal membrane. Therefore, full-thickness membrane fragments
were incubated with increasing doses of relaxin H2 (0-100 ng/ml) for 1-3 days. The fragments were then strength
tested using our previously published ball-burst methodology and also tested for biochemical markers of remodeling:
gelatinase activity and MMP proteins. Results showed that relaxin induced a dose dependent increase in break strength.
At the highest dose, a consistent 20% increase was observed (P<0.01) with concomitant decreases in MMP9 gelatinase
activity and protein abundance. Therefore, contrary to our expectations, relaxin paradoxically increased fetal membrane
strength at high doses. The mechanism(s) for this relaxin effect are unknown. Supported by NIH grants HD048476 (JM)
and HD24314 (GBG).
Relaxin 2008
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O-61
Implications of Leukocyte Responsiveness to Tumor-derived Relaxin
Kevin A. Figueiredoa, Jodie B. Palmerb, and Michael E. Coxa,c
The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia V6H 3Z6, Canada; bLudwig Institute for Cancer Research,
Austin Health, Heidelberg, Victoria 3084, Australia; cDepartment of Surgery, University of British Columbia, Vancouver, British Columbia
V6H 3Z6, Canada
a
Leukocytes are critical effectors of inflammatory and cancer biology. Cancers are thought to produce chemokine-like
factors that promote leukocyte recruitment and tumor infiltration and suppress immune surveillance. Relaxin is an
endocrine hormone previously implicated in breast and prostate cancer progression. We have shown that relaxin is a
cancer-produced factor with properties important for leukocyte recruitment and activation. Our studies demonstrate that
relaxin expression is up-regulated during neuroendocrine differentiation of the human prostate cancer model, LNCaP. To
examine the impact of relaxin on host cells associated with adenocarcinomas, we generated recombinant 6 His-tagged
relaxin (RLXH) in a mammalian expression system. This immunoreactive and biologically active relaxin preparation
was used to screen a variety of cell types for cAMP responsiveness. Of the cell types screened, none were more
responsive to RLXH than the monocyte/macrophage cell line THP-1 and peripheral blood mononuclear cells (PBMC).
Our studies indicate that relaxin promotes cell-cell clustering, substrate adhesion, and migratory capacity of mononuclear
leukocytes in a relaxin dose-dependent manner proportional to cAMP accumulation. We demonstrate that these
biological responses occur through a relaxin receptor LGR7-dependent mechanism likely involving cAMP-dependent
regulation of the small GTPase, Rap1. Relaxin stimulation of monocytes enhanced chemotaxis of THP-1 and PBMCs to
monocyte chemoattractant protein-1 and induced cytokine expression profiles indicative of type II, immunosuppressive,
macrophage differentiation. Classical roles for relaxin can now be expanded to include a role in targeting mononuclear
leukocytes and that its ability to mediate macrophage activation states have important implications in the context of
tumor-induced inflammation and immunosuppression.
O-62
Relaxin and INSL3 Promote Tumor Formation of Human Thyroid Carcinoma Cells
Thomas Klonischa, Joanna Bialekb, Yvonne Radestockb, Cuong Hoang-Vub, and Sabine Hombach-Klonischa
Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada; bClinic of Surgery,
Faculty of Medicine, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
a
The relaxin/RXFP1 and INSL3/RXFP2 have been established as ligand-receptor systems associated with tumor growth
and metastasis. Both relaxin-like ligands, relaxin and INSL3, and their cognate receptors are present in a variety of tumor
types, including breast, prostate, endometrial, hematopoietic, and thyroid cancer tissues and cell lines. Here we provide
evidence from human thyroid cancer cell lines and nude mouse studies that both relaxin and INSL3 act as autocrine/
paracrine oncogenic factors that can modulate the local tumor environment to facilitate tumor expansion. When injected
into nude mice, thyroid carcinoma transfectants expressing H2 relaxin and human INSL3 developed rapidly growing,
cell-dense tumor masses rich in vascularization but with a poor interstitial matrix. Cultured transfectants displayed
enhanced production of matrix-metalloproteinase 2 (MMP2; gelatinase A) and MT1-MMP. Activation of proMMP2 in
part depends on the formation of a trimolecular complex between MT1-MMP, tissue inhibitor of MMP2 (TIMP2) and
proMMP2. Relaxin and INSL3 clones displayed increased MMP2 activity as determined by processing of a fluorescentlabelled MMP2 substrate and this coincided with enhanced production of MT1-MMP and the presence of TIMP2. A
similar increase in MMP2 and MT1-MMP production was detected when the follicular thyroid carcinoma cell line FTC133 was incubated with recombinant human relaxin and INSL3. In conclusion, we have identified MMP2 and MT1MMP as novel target molecules of relaxin and INSL3 in thyroid carcinoma cells and provide a molecular mechanism for
rapid tumor growth and angiogenesis of these cell clones in nude mice.
This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research
Council of Canada (NSERC) and Manitoba Health Research Council (MHRC).
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Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-63
Estrogen and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) Modulate RLN2
Promoter Activity in Estrogen-receptor Positive T47D Human Breast Cancer Cells
Silke Kietza, Thomas Klonischb, and Sabine Hombach-Klonischb
University Medical Center Goettingen, Pediatric Clinic I, 37075 Goettingen, Germany; bDepartment of Human Anatomy and Cell Science,
University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba R3E 0W3, Canada
a
RLN1 and RLN2 transcripts are increased in human breast cancer specimens. We employed the estrogen-responsive
human breast cancer cell line T47D to determine the ability of estrogen and TCDD to increase RLN1 and RLN2
transcriptional gene activity. T47D cells expressed ERalpha protein and the 17beta-estradiol (E2) target gene pS2
was induced following exposure to 10nM E2. Arylhydrocarbon receptor (AhR) was present in T47D and exposure
to 10nM 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) resulted in induction of the AhR target gene Cyp1A1. RLN1
and RLN2 promoter luciferase constructs were used to test for RLN promoter activation. RLN1 and RLN2 transcripts
were quantified using real-time PCR. When exposed to E2 for 12h and 24h, RLN2 promoter activity was enhanced
and this effect was not observed in the absence of E2. The estrogen receptor (ER) blocker ICI-182780 completely
prevented the E2-induced increase in RLN2 promoter activity indicating that the RLN2 promoter was activated by an
ER-dependent mechanism. After 24h exposure 10 nM TCDD alone did not induce RLN2 promoter activity. However,
when applied simultaneously with E2, TCDD reduced the E2-mediated increase in RLN2 promoter activity suggesting
an anti-estrogenic action in RLN2 transcriptional regulation. Acting alone, TCDD caused an up-regulation of RLN2
transcripts peaking at 8h exposure and preceding the E2-induced RLN2 up-regulation at 12 h. These results were specific
for the RLN2 promoter. In conclusion, our results show that TCDD and E2 stimulate RLN2 transcription by different
mechanisms and that TCDD acts anti-estrogenic in the presence of E2.
This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research
Council of Canada (NSERC), Manitoba Medical Foundation (MMSF) and Manitoba Health Research Council (MHRC).
O-64
Relaxin Signaling in Human Uterine Fibroids
Zhen Lia,b, Jennifer K. Burzawaa, Anne Tronga, Shu Fenga, Xiaowen Tongb, Ertug Kovancia, Aleks Rajkovica,
and Alexander I. Agoulnika
a
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77025, USA; bDepartment of Obstetrics and
Gynecology, Tongji University, 200065 Shanghai, People’s Republic of China
Uterine fibroid tumors (leiomyomata) are the most common gynecological neoplasms clinically affecting up to 30%
of all women during reproductive age. The primary characteristics of uterine fibroids are the increased proliferation of
myometrial cell, production of the abundant extracellular matrix (ECM), and the downregulation of ECM-degrading
enzymes expression. Recent progress in cDNA microarray analysis revealed a distinct similarity of leiomyomata with
other fibrotic processes. The crucial role of several cytokines and growth factors is well-recognized in this disease.
Relaxin, a member of insulin-relaxin peptide family of hormones is strongly expressed in myometrium. Relaxin signals
through its G protein-coupled receptor RXFP1. It regulates ECM remodeling through alterations of collagen deposition,
cell migration and proliferation, and overall tissue homeostasis. Relaxin strongly suppresses the effects of TGF-ß
induced myofibroblast differentiation, the major cytokine involved in the pathogenesis of uterine fibroids. Examination
of public cDNA array gene expression databases revealed a consistent pattern of down-regulation of RXFP1 and several
other genes involved in relaxin signaling, suggesting an involvement of relaxin in fibroid etiology. We used 25 matched
pairs of leiomyomata and normal myometrium samples to analyze expression of RXFP1, RXFP2, caveolin 1, steroid
receptors and their co-regulators at RNA and protein levels using qRT-PCR, immunohistochemistry and Western blot
hybridization. The results of this analysis will be presented.
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O-65
Scientific Rationale and Design of a Phase I Study of Relaxin in Women with
Severe Preeclampsia
Elaine Unemoria, Baha Sibaib, Susy Wooda, and Sam Teichmana
a
Corthera, Inc. (formerly BAS Medical, Inc.), San Mateo, California 94402, USA; bDepartment of Obstetrics and Gynecology, University of
Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA
The pathophysiology of preeclampsia involves profound systemic vasoconstriction. Its etiology may be related to
reduced blood flow to the placenta, leading to the elaboration of soluble factors which increase maternal systemic
vascular resistance and cause renal dysfunction. Reduced bioactivity of vascular endothelial growth factor (VEGF) may
play a central role in this pathophysiology. Previous clinical studies have strongly suggested that relaxin is a systemic
and renal vasodilator. In nonclinical studies, relaxin administration to monkeys has been associated with increased
vessel density in the endometrium and in previous human trials, relaxin administration has been highly correlated
with increased menstrual bleeding in women. VEGF has been proposed as a mechanism for these endometrial effects.
Together, these data suggest that relaxin may be able to relieve systemic and renal vasoconstriction and improve
placental perfusion in women with preeclampsia. As a first step in the development program for relaxin in this indication,
a multi-center, randomized, double blind, placebo-controlled Phase I safety study in women with severe preeclampsia
has been launched in the US. Three doses of relaxin, 3, 10, and 30 μg/kg/day, or placebo will be administered for up to
72 hours in women admitted to the hospital for management of their disease. Although the trial is primarily focused on
safety, signs of efficacy, such as changes in blood pressure and renal markers, will be assessed.
O-66
A Randomized, Double Blind, Placebo Controlled Trial of Relaxin for Cervical
Ripening in Post-date Pregnancies
Gerson Weissa, Sam Teichmanb, Dennis Stewartb, David Naderb, Susy Woodb, and Elaine Unemorib
Department of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School, UMDNJ, Newark, New Jersey 07103, USA,
Corthera, Inc. (formerly BAS Medical), San Mateo, California 94402, USA
a
b
It is standard obstetrical practice to deliver all pregnancies by the 42nd week since longer pregnancy is attendant to
increased fetal demise. This study had two objectives, to determine if 24 hr infusion of pharmacological concentrations
of serum relaxin was safe in pregnant women and to determine whether the elevated relaxin could ripen the cervix
or induce labor. After a small dose escalation phase of the study determined that relaxin could safely be elevated to
approximately 30x over baseline (normal pregnancy) concentrations, the effects of 24 hr of infusion of relaxin vs
placebo on Bishop score, time to dilation, uterine contractions and time to delivery were determined in 25 subjects/
group. The study was multi-centered, randomized, double-blind and placebo controlled, in 72 women > 40 weeks of
pregnancy. Elevation of serum relaxin to approximately 30x over baseline for 24 hr was safe to mother and fetus. Safety
results for treatment and placebo groups were comparable. Infusion of relaxin for 24 hr did not advance cervical ripening
as measured by Bishop score and did not alter secondary end points, including time to delivery. Relaxin infusion did
result in trends of lower blood pressure and improvements in renal markers. Thus, while short term human relaxin
infusion does not advance cervical ripening or labor onset, it was safe in pregnant women with potentially positive
affects on blood pressure and renal function. This suggests use in hypertensive diseases of pregnancy.
68
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
O-67
A Pilot Safety and Dose-finding Trial of Intravenous Recombinant Human Relaxin
(rhRlx) in Compensated Congestive Heart Failure
Thomas Dschietziga, Elaine Unemorib, Susy Woodb, Christoph Richtera, Julia Böhmera, Sam Teichmanb, Gert Baumanna,
and Karl Stangla
a
Department of Cardiology and Angiology, Charité University Medicine Berlin, Campus Mitte, 10117 Berlin, Germany; bCorthera, Inc.
(formerly BAS Medical), San Mateo, California 94402, USA
We have recently established that relaxin is constitutively expressed in human cardiovascular tissues and that the
hormone -upregulated in myocardium - plays a compensatory role in human heart failure (CHF). In this open-label
study, 16 patients with compensated CHF were treated with intravenous rhRlx in one of 3 dose cohorts. Patients
were monitored for 24 hours in the intensive care unit during the infusion period and for 24 hours post-infusion.
Hemodynamic measurements were performed using a Swan-Ganz and arterial catheter. The safety and tolerability
demonstrated by rhRlx in the lowest dose group (A; sequential treatment for 8 hours each with dosages equivalent to
10, 30, and 100 µg/kg/day) allowed escalation to Group B (240, 480, and 960 µg/kg/day), and the highest safe dose, 960
µg/kg/day, was then selected for 24 hours of dosing in Group C. In all subjects and at all dosages, rhRlx was shown to
be safe and well tolerated. In these well-medicated patients, rhRlx produced relevant improvements in hemodynamic
parameters, including pulmonary wedge pressure, cardiac index, and systemic vascular resistance. Markers of renal
function showed improvement in all subjects. Transient increases compared to baseline in some of these renal markers
were also observed in some subjects post-dosing. Rebound hypertension or changes in heart rate were not observed.
No incidences of hypotension were recorded during or post-dosing. Overall, the safety and efficacy profile of rhRlx in
patients with compensated CHF indicates that this drug may be of value in the treatment of patients with acute heart
failure syndrome and worsening of renal function.
Relaxin 2008
69
Participants
Agoulnik, Alexander
Baylor College of Medicine
agoulnik@bcm.edu
Craik, David
University of Queensland
d.craik@imb.uq.edu.au
Gundlach, Andrew
Howard Florey Institute
andrew.gundlach@florey.edu.au
Amento, Edward
Molecular Medicine Research Institute
eamento@mmrx.org
De Aizpurua, Henry
Howard Florey Institute
henry.deaizpurua@florey.edu.au
Halls, Michelle
Monash University
halls.michelle@gmail.com
Bagnell, Carol
Rutgers, the State University of New Jersey
bagnell@aesop.rutgers.edu
De Meyts, Pierre
Receptor Systems Biology Laboratory
pdm@novonordisk.com
Haugaard-Jonsson, Linda
University of Kalmar
linda.jonsson@hik.se
Bani, Daniele
University of Florence
daniele.bani@unifi.it
Debrah, Dan
University of Pittsburgh
debrahdo@upmc.edu
Hewitson, Tim
Royal Melbourne Hospital
tim.hewitson@mh.org.au
Bartol, Frank F.
Auburn University
bartoff@auburn.edu
Debrah, Julianna
Magee Womens Research Institute
debrahje@upmc.edu
Ho, Teh-Yuan
Rutgers, the State University of New Jersey
tho@aesop.rutgers.edu
Bathgate, Ross
Howard Florey Institute
bathgate@florey.edu.au
Dschietzig, Thomas
Charité Hospital
thomas.dschietzig@t-online.de
Hombach-Klonisch, Sabine
University of Manitoba
hombach@cc.umanitoba.ca
Bennett, Robert
VA Medical Center/University of Nebraska
rgbennet@unmc.edu
Du, Xiao-Jun
Baker Heart Research Institute
xiaojun.du@baker.edu.au
Horton, Jaime
University of Hawaii
jshorton@hawaii.edu
Boehnert, Markus
University of Bern
markusboehnert@yahoo.de
Einspanier, Almuth
Institute of Physiological Chemistry
einspanier@vetmed.uni-leipzig.de
Hossain, Mohammed
Howard Florey Institute
akhter.hossain@florey.edu.au
Bogerd, Jan
Utrecht University
j.bogerd@uu.nl
Feng, Shu
Baylor College of Medicine
shuf@bcm.edu
Ikegaya, Naoki
Shizuoka University
nike11@d9.dion.ne.jp
Bogzil, Alsadek
Manchester University
bogzil@hotmail.com
Ferlin, Alberto
University of Padova
alberto.ferlin@unipd.it
Kapila, Sunil
University of Michigan
skapila@umich.edu
Bryant-Greenwood, Gillian
University of Hawaii
gbg@pbrc.hawaii.edu
Fernandes Gonçalves, Roseli
São Paulo University
ona@usp.br
Kern, Andras
University of Hawaii
kerna@pbrc.hawaii.edu
Callander, Gabrielle
Howard Florey Institute
gab.c@florey.edu.au
Fields, Mike
University of Florida
fields@ufl.edu
Klonisch, Thomas
University of Manitoba
klonisch@cc.umanitoba.ca
Chang, Jaw-Kang
Phoenix Pharmaceuticals Inc.
jawkangchang@yahoo.com
Fields, Phillip
University of South Alabama
College of Medicine
pfields@usouthal.edu
Koester, Anja
Eli Lilly and Company
Anja_Koester@lilly.com
Charles, Chris
University of Guelph
charlesc@uoguelph.ca
Chen, Joseph
Rutgers, the State University of New Jersey
jchen@aesop.rutgers.edu
Conrad, Kirk
University of Florida
kpconrad@ufl.edu
Cooney, Timothy
Hamot Medical Center
tim.cooney@hamot.org
Cox, Michael E.
The Prostate Centre at VGH
mcox@interchange.ubc.ca
70
Foresta, Carlo
University of Padova
carlo.foresta@unipd.it
Frankshun, Amy-Lynn
Rutgers, the State University of New Jersey
FRANKSHUN@AESOP.Rutgers.edu
Girling, Jane
Monash Institute of Medical Research
jane.girling@med.monash.edu.au
Goldsmith, Laura
New Jersey Medical School
goldsmit@umdnj.edu
Good-Avila, Sara
Acadia University
sara.good-avila@acadiau.ca
Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008
Kohsaka, Tetsuya
Shizuoka University
t-kohsaka@agr.shizuoka.ac.jp
Konieczko, Elisa
Gannon University
konieczk001@gannon.edu
Layfield, Sharon
Howard Florey Institute
shaz@florey.edu.au
Li, Zhen
Baylor College of Medicine
zl1@bcm.edu
Liu, Changlu
Johnson & Johnson Pharmaceutical Research &
Development
cliu9@prdus.jnj.com
Lobb, Derek
McMaster University
Lobbd@McMaster.ca
Samuel, Chrishan
Howard Florey Institute
chrishan.samuel@florey.edu.au
Tang, Mimi
Murdoch Childrens Research Institute
mimi.tang@mcri.edu.au
Ma, Sherie
Howard Florey Institute
sherie.ma@florey.edu.au
Sanborn, Barbara
Colorado State University
Barbara.Sanborn@colostate.edu
Tregear, Geoffrey
Howard Florey Institute
geoffrey.tregear@florey.edu.au
Makineni, Rao
makinenir@aol.com
Scarpa, Antonio Luigi
IBSA Lugano CH
slotan@tiscali.it
Tremblay, Jacques
CHUQ Research Centre - Laval University
jacques-j.tremblay@crchul.ulaval.ca
Schwabe, Christian
Medical University of South Carolina
schwabec@musc.edu
Unemori, Elaine
Corthera, Inc.
eunemori@corthera.com
Segal, Mark
University of Florida
segalms@medicine.ufl.edu
van der Westhuizen, Emma
Monash University
emma.zumpe@med.monash.edu.au
Shabanpoor, Fazel
Howard Florey Institute
fazel.shabanpoor@florey.edu.au
Vodstrcil, Lenka
University of Melbourne
L.vodstrcil@pgrad.unimelb.edu.au
Shemesh, Ronen
Compugen Ltd.
ronen.shemesh@cgen.com
Wade, John
Howard Florey Institute
john.wade@florey.edu.au
Shen, Pei-Juan
Howard Florey Institute
pei-juan.shen@florey.edu.au
Weiss, Gerson
New Jersey Medical School
weissge@umdnj.edu
Sherwood, O. David
University of Illinois
od-sherw@uiuc.edu
Wiley, Anne A.
Auburn University
wileyaa@auburn.edu
Singh, Sudhir
University of Nebraska Medical Center
ssing1@unmc.edu
Willcox, Jordan
University of Guelph
jwillcox@uoguelph.ca
Slieker, Lawrence
Lilly Research Laboratories
slieker_lawrence_j@lilly.com
Wilson, Brian
Acadia University
brian.wilson@acadiau.ca
Smith, Craig
Howard Florey Institute
craig.smith@florey.edu.au
Wlodek, Mary
The University of Melbourne
m.wlodek@unimelb.edu.au
Soh, Yu May
The University of Melbourne
y.soh2@pgrad.unimelb.edu.au
Wong, Su Ee
Howard Florey Institute
suee.wong@mh.org.au
Steinetz, Bernard
NYU School of Medicine
steinetz@env.med.nyu.edu
Yamamoto, Sandra
University of Hawaii
sandy@pbrc.hawaii.edu
Stewart, Dennis
Corthera, Inc.
drstewart@corthera.com
Yegorov, Sergey
Acadia University
087491y@acadiau.ca
Summers, Roger
Monash University
roger.summers@med.monash.edu.au
Zhang, Suode
Howard Florey Institute
suode.zhang@florey.edu.au
Manegold Svendsen, Angela
Hagedorn Research Institute
agms@hagedorn.dk
Maseelall, Priya
New Jersey Medical School
pmaseelall@aol.com
McGowan, Barbara
Imperial College
b.mcgowan@imperial.ac.uk
McGuane, Jonathan
University of Florida
jtmcguane@ufl.edu
Moore, Xiao
Baker Heart Research Institute
shirley.moore@baker.edu.au
Morelli, Sara
New Jersey Medical School
sara.s.morelli@gmail.com
Nøhr Larsen, Jane
Hagedorn Research Institute
jnql@hagedorn.dk
O’Byrne, Elizabeth
Rutgers University
lizobyrne@yahoo.com
Parry, Laura
University of Melbourne
ljparry@unimelb.edu.au
Rajpert-De Meyts, Ewa
Copenhagen University Hospital
erm@rh.hosp.dk
Rinderknecht, Ernst
Corthera, Inc.
erinderknecht@corthera.com
Rosengran, K. Johan
University of Kalmar
johan.rosengren@hik.se
Ryan, Peter
Mississippi State University
ryan@cvm.msstate.edu
Sacher, Frank
Bayer Schering Pharma AG
frank.sacher@bayerhealthcare.com
Salimova, Ekaterina
EMBL
eks@embl.it
Sutton, Steven
Johnson & Johnson
ssutton@prdus.jnj.com
Tan, Ronald
rap3tan@netvigator.com
Relaxin 2008
71
Notes
Notes
Notes
Notes
Notes
Notes