Molecular and
Experimental Medicine
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Joe Krueger, Ph.D., Research Associate, and
Brunehilde Felding-Habermann, Ph.D., Associate Professor,
Department of Molecular and Experimental Medicine
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The Scripps Research Institute. All rights reserved.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005 241
Roger C. Cornell, M.D.
Adjunct Professor
Carlo Ferrari, M.D.
Adjunct Associate Professor
Alfredo A. Jalowayski, Ph.D.
Adjunct Assistant Professor
Laura M. Crisa, M.D.
Assistant Professor
Mitchell H. Friedlaender, M.D.
Adjunct Professor
John T. Curnutte, M.D.,
Ph.D.
Adjunct Associate Professor
Jeffrey S. Friedman, M.D.,
Ph.D.
Assistant Professor
Eric F. Johnson, Ph.D.
Professor
Acting Head, Division of
Biochemistry
Ernest Beutler, M.D.*
Chairman and Professor
Head, Division of Hematology
George L. Dale, Ph.D.
Adjunct Associate Professor
Theodore Friedmann, M.D.
Adjunct Professor
Masahiro Aoki, M.D., Ph.D.
Adjunct Assistant Professor
Arthur D. Dawson, M.D.
Adjunct Professor
Andrew J. Gale, Ph.D.
Assistant Professor
Hiroshi Asahara, M.D., Ph.D.
Assistant Professor
Albert B. Deisseroth, M.D.,
Ph.D.
Adjunct Professor
Roberta A. Gottlieb, M.D.
Associate Professor
DEPAR TMENT OF
MOLECULAR AND
E X P E R I M E N TA L
MEDICINE
S TA F F
Bonno N. Bouma, Ph.D.
Adjunct Professor
Mathew E. Brunson, M.D.
Adjunct Assistant Professor
Joel N. Buxbaum, M.D.
Professor
Head, Division of Research
Rheumatology
Carlos J. Carrera, M.D.
Adjunct Assistant Professor
Dennis A. Carson, M.D.
Adjunct Professor
Sergio D. Catz, Ph.D.
Assistant Professor
Francis V. Chisari, M.D.
Professor
Head, Division of
Experimental Pathology
Sandra Christiansen, M.D.
Adjunct Associate Professor
Gregory J. del Zoppo,
M.D.**
Associate Professor
Giacomo A. DeLaria, M.D.
Adjunct Associate Professor
Thomas F. Deuel, M.D.
Professor
Head, Division of Molecular
Oncology
Kazuhiro Kakimi, M.D., Ph.D.
Adjunct Assistant Professor
Thomas J. Kipps, M.D., Ph.D.
Adjunct Professor
Lawrence E. Kline, D.O.
Adjunct Associate Professor
John H. Griffin, Ph.D.**
Professor
James A. Koziol, Ph.D.
Professor
Head, Division of
Biomathematics
Andras Gruber, M.D.
Adjunct Assistant Professor
Daniel F. Kripke, M.D.
Adjunct Professor
Luca G. Guidotti, D.V.M.,
Ph.D.
Associate Professor
Thomas J. Kunicki, Ph.D.**
Associate Professor
Anne M. Hanneken, M.D.
Associate Professor
Stuart A. Lipton, M.D., Ph.D.
Adjunct Professor
Fu-Tong Liu, M.D., Ph.D.
Adjunct Professor
Ralph B. Dilley, M.D.
Adjunct Professor
Mary J. Heeb, Ph.D.**
Associate Professor
Darryl D’Lima, M.D.
Adjunct Assistant Professor
Paul G. Heyworth, Ph.D.
Adjunct Associate Professor
Martin Lotz, M.D.
Professor
Head, Division of Arthritis
Research
Darlene J. Elias, M.D.
Adjunct Assistant Professor
James A. Hoch, Ph.D.
Professor
Head, Division of Cellular
Biology
Christopher Lee Marsh, M.D.
Adjunct Associate Professor
Brunehilde FeldingHabermann, Ph.D.
Associate Professor
Frank M. Huennekens, Ph.D.
Professor Emeritus
James R. Mason, M.D.
Adjunct Assistant Professor
Jack Clausen, M.D.
Adjunct Professor
S E C T I O N C O V E R F O R T H E D E PA R T M E N T O F M O L E C U L A R A N D E X P E R I M E N TA L
Clifford W. Colwell, Jr., M.D.
Adjunct Professor
M E D I C I N E : Confocal immunoflourescence photomicrograph (400X) of a Vero cell infected with
porcine endogenous retrovirus and stained to localize the expression of the viral Gag protein. During
the viral life cycle, the production of viral proteins and their traffic to the cell membrane are critical
Brian R. Copeland, M.D.
Adjunct Assistant Professor
steps in the assembly of new viral particles. Understanding the molecular mechanisms of intracellular
retroviral protein traffic is important for devising strategies to treat or prevent these infections. Work
done in the laboratory of Daniel R. Salomon, M.D., by Katherine Marcucci and David Valente.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
242 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
David A. Mathison, M.D.
Adjunct Professor
Alessandro Sette, Ph.D.
Adjunct Professor
Robert McMillan, M.D.
Professor Emeritus
Farhad F. Shadan, M.D.,
Ph.D.
Adjunct Assistant Professor
William E. Miller, M.D.
Adjunct Assistant Professor
Jorge J. Nieva, M.D.
Assistant Professor
Zhixing K. Pan, M.D., Ph.D.
Adjunct Assistant Professor
Sanford J. Shattil, M.D.
Adjunct Professor
Alexander R. Shikhman, M.D.,
Ph.D.
Assistant Professor
Anthony J. Pelletier, Ph.D.
Adjunct Assistant Professor
Inmaculada Silos-Santiago,
M.D., Ph.D.
Adjunct Associate Professor
Marta Perego, Ph.D.
Associate Professor
Gregg J. Silverman, M.D.
Adjunct Associate Professor
Paul J. Pockros, M.D.
Adjunct Assistant Professor
Ronald A. Simon, M.D.
Adjunct Professor
K. Michael Pollard, Ph.D.
Associate Professor
Peter J. Sims, M.D., Ph.D.
Professor
Giuseppe Remuzzi, M.D.
Adjunct Professor
Jack C. Sipe, M.D.
Associate Professor
Michael W. Robertson,
Ph.D.
Associate Professor
Joseph A. Sorge, M.D.
Adjunct Professor
John S. Romine, M.D.
Adjunct Associate Professor
Zaverio M. Ruggeri, M.D.**
Professor
Head, Division of Experimental
Hemostasis and Thrombosis
Peter K. Vogt, Ph.D.
Professor
Head, Division of Oncovirology
Matthias G. von Herrath,
M.D.
Adjunct Associate Professor
Jerry L. Ware, Ph.D.***
Associate Professor
University of Arkansas
Little Rock, Arkansas
Therese Wiedmer, Ph.D.
Associate Professor
Xiaohua Wu, Ph.D.
Assistant Professor
Takao Yagi, Ph.D.
Associate Professor
Williamson B. Strum, M.D.
Adjunct Assistant Professor
Eng M. Tan, M.D.
Professor
Bruce E. Torbett, Ph.D.
Associate Professor
Enrique Saldivar, M.D., Ph.D.
Adjunct Assistant Professor
Constantine Tsoukas, Ph.D.
Adjunct Associate Professor
Daniel R. Salomon, M.D.
Associate Professor
Susan L. Uprichard, Ph.D.
Assistant Professor
Alan Saven, M.D.
Adjunct Professor
Kottayil I. Varughese, Ph.D.**
Associate Professor
Nicholas J. Schork, Ph.D.
Adjunct Professor
John H. Vaughan, M.D.
Adjunct Professor
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
SENIOR RESEARCH
A S S O C I AT E S
Hiroshi Deguchi, M.D., Ph.D.
Jane Eddleston, Ph.D.***
University of California
San Diego, California
Asa B. Gustafsson, Ph.D.
Sanshiro Hashimoto, M.D.***
Yamada Medical Center
Tokyo, Japan
Yuichi Kamikubo, Ph.D.
Richard D. Milner, M.D.,
Ph.D.
Dong-Er Zhang, Ph.D.
Associate Professor
Laurent O. Mosnier, Ph.D.
Bruce L. Zuraw, M.D.
Adjunct Associate Professor
Deirdre M. O’Sullivan, Ph.D.
Keith Stephenson, Ph.D.
Mario P. Tschan, Ph.D.***
Universität Bern
Bern, Switzerland
Reha Celikel, Ph.D.
Jill M. Waalen, M.D.
Mei-Hui Hsu, Ph.D.
Chengqun Huang, M.D.,
Ph.D.
Jennifer L. Johnson, Ph.D.
Robert J. Russo, M.D., Ph.D.
Adjunct Assistant Professor
Quansheng Zhou, Ph.D.
John C. Yu, M.D., Ph.D.
Adjunct Associate Professor
S TA F F S C I E N T I S T S
Donald D. Stevenson, M.D.
Adjunct Professor
Ji Zhao, Ph.D.
Subramanian Yegneswaran,
Ph.D.
R E S E A R C H A S S O C I AT E S
Klaus Kuhn, Ph.D.
Eun-Young Ahn, Ph.D.
Pauline L. Lee, Ph.D.
Cinzia Ambrosi, Ph.D.
Patrizia Marchese, Ph.D.
Shinichi Asabe, Ph.D.
Tsaiwei Olee, Ph.D.
Andreas Bader, Ph.D.
Brian Savage, Ph.D.
Dong Bai, Ph.D.
Stefan Wieland, Ph.D.
Jennifer L. Barber-Singh,
Ph.D.
Akemi Yagi, Ph.D.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005 243
Cristina Bongiorni, Ph.D.
David W.H. Girdwood, Ph.D.
Yumi Kurokawa, M.D., Ph.D.
Kristen E. Bower, Ph.D.
Natalya Greyz, M.D.
Anita Y. Boyapati, Ph.D.
Shawn Patrick Grogan, Ph.D.
Victoria Kutilek, Ph.D.***
Abbott Laboratories
Abbott Park, Illinois
Nathan R. Brady, Ph.D.
Marco Gymnopoulos, Ph.D.
Pablo G. Landart, Ph.D.
Katia Maria Cabral, Ph.D.
Lynn E. Hancock, Ph.D.***
Kansas State University
Manhattan, Kansas
Estelle Leclerc, Ph.D.
Wolf-Achim Hassenpflug, M.D.
Enbo Liu, Ph.D.
Koji Hiraoka, M.D., Ph.D.***
Kurume University
Kurume, Japan
M. Isabel Velazquez Lopez,
M.D., Ph.D.
Richard M. Reisdorph,
Ph.D.***
University of Colorado
Denver, Colorado
Natalia Reixach, Ph.D.
Leila Reynald, Ph.D.
Maria del Carmen Carles,
Ph.D.***
Gwathmey Inc.
Cambridge, Massachusetts
Anna E. Cartier, Ph.D.
Alan Yueh-Luen Lee, Ph.D.
Bruno Sainz, Jr., Ph.D.
David M. Cauvi, Ph.D.
Jiann-Kae Luo, Ph.D.
Jaiweon Hwang, Ph.D.
Yunchao Chang, Ph.D.
Takuma Mabuchi, M.D., Ph.D.
Matteo Iannacone, M.D.
Emily I. Chen, Ph.D.
Uzen Savas, Ph.D.
Guillaume A. Schoch,
Ph.D.***
University of Oxford
Oxford, England
Holly N. Maier, Ph.D.
Dennis A. Sheeter, Ph.D.
Masanori Isogawa, M.D.
Guofeng Cheng, Ph.D.
Mathieu Marella, Ph.D.
Jin Shi, Ph.D.
Tatsuo Ito, M.D.
Stephanie Cherqui, Ph.D.
Florent M. Martin, Ph.D.
Misako Shibakura, Ph.D.
Hao Jiang, Ph.D.
Takuya Chiba, Ph.D.***
Nagasaki University
Nagasaki, Japan
Michael D. Robek, Ph.D.***
Yale University
New Haven, Connecticut
Yuri Martina, Ph.D.
Margaret L. Just, Ph.D.***
eBioscience, Inc.
San Diego, California
Usha Srinivasan, Ph.D.
Sohye Kang, Ph.D.
Constantino Martinez,
Ph.D.***
University of Arkansas
Little Rock, Arkansas
Mou-Chieh Kao, Ph.D.
Eiko Nakamaru-Ogiso, Ph.D.
Jaroslav Truksa, Ph.D.
Sharookh B. Kapadia, Ph.D.
Akiko Okumura, Ph.D.
Ngoc Thanh Truong, Ph.D.
Kazunobu Kato, M.D.,
Ph.D.***
University of California
Davis, California
Fumihiko Okumura, Ph.D.
Masanao Tsuda, Ph.D.
Erin N. Olson, Ph.D.
Billyana C. Tsvetanova, Ph.D.
Sandrine Pacquelet, Ph.D.
Arsen Volkov, Ph.D.
Natalie M. Pecheniuk, Ph.D.
Ji Wang, Ph.D.
Luke F. Peterson, Ph.D.
Yang Wang, Ph.D.
Jung Hwan Kim, Ph.D.
Pablo Perez Pinera, M.D.
Zhuangzhi Wang, Ph.D.
Keun Il Kim, Ph.D.***
Sookmyung Women’s
University
Seoul, Korea
Gian Marco Podda, M.D.
Martin R. Weber, Ph.D.
A. Venkateshwar Reddy,
Ph.D.***
Novartis Institute for
Functional Genomics
San Diego, California
Andrea K. White, Ph.D.
Maria Cortes-Mateos, Ph.D.
Hendrik Szurmant, Ph.D.
Noboru Taniguchi, M.D.
Ian D. Dang, Ph.D.
Chinh T. Dao, Ph.D.
Maria F. Del Papa, Ph.D.
Adam Denley, Ph.D.
Gabriela D.C. Denning,
Ph.D.***
Emory University School of
Medicine
Atlanta, Georgia
Alexandra K. Kiemer,
Ph.D.***
Saarland University
Saarbrücken, Germany
Alejandra R. Diaz, Ph.D.
Jonathon M. Flanagan, Ph.D.
Tatsuya Fukushima, Ph.D.
Takayuki Furumatsu, M.D.,
Ph.D.***
Okayama Medical Center
Okayama, Japan
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The Scripps Research Institute. All rights reserved.
Sunil M. Kurian, Ph.D.
Adam C. Wilson, Ph.D.
244 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Tetsuo Yamashita, Ph.D.
* Joint appointment in The Skaggs
Institute for Chemical Biology
Ming Yan, Ph.D.
** Joint appointment in the
Department of Cell Biology
Xia Yang, Ph.D.
*** Appointment completed; new location shown
Jason K. Yano, Ph.D.
Zhengyi Ye, Ph.D.
Jinseong Yi, Ph.D.
Antonella Zampolli, Ph.D.
Wei Zhang, Ph.D.
Haiyan Zhao, Ph.D.
Li Zhao, Ph.D.
Jin Zhong, Ph.D.
Weiguo Zou, Ph.D.
Masahiko Zuka, M.D., Ph.D.
S C I E N T I F I C A S S O C I AT E S
Fanny E. Almus, Ph.D.
Joseph R. Biggs, Ph.D.
Jose A. Fernandez, Ph.D.
Gabriele E. Foos, Ph.D.
Terri P. Gelbart, B.S., M.T.
Julie M. Ruedi, B.A.
Byoung Boo Seo, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Ernest Beutler, M.D.
Chairman’s Overview
he ancestor of the Department of Molecular and
Experimental Medicine was the Department of
Clinical Research. After merging with the Department of Biochemistry and the Department of Cellular Biology, the Department of Clinical Research was renamed,
but the words experimental medicine in our present
name still have sometimes erroneously been interpreted
as signifying that most or all of the members of the
department are physicians involved in carrying out clinical studies. Such is not the case, of course. Very few
of our faculty members have an M.D. degree, and even
fewer function as physicians. Neither is the clinical application of research findings a major motivating force when
our faculty members choose their research agenda.
Rather, it is, as it should be, intellectual curiosity and,
from a practical point of view, fundability that are the
driving forces. Although our research programs are not
set primarily by the hope that the findings will be implemented in the care of patients, we are, of course, pleased
when something we have learned can benefit humans.
And because at least some of our faculty have a strong
medical background, the connection between our studies and application of the study findings to medicine may
T
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245
be more apparent. In this year ’s overview, I will try to
highlight some of the discoveries that are being made in
the Department of Molecular and Experimental Medicine
that may eventually have an impact on human health.
For many years Peter Vogt, head of our Division of
Oncovirology, has been studying genes that can induce
cancer in chickens. In a seminal paper published in
Science in 1997, Dr. Vogt and colleagues reported that
overproduction of the catalytic subunit of phosphotidylinositol-3′-kinase produced cancer in chickens. Recently,
researchers in several laboratories have followed up on
this finding and have shown that in human tumors,
mutations of the gene for the kinase are often present.
Because upregulation causes cancer, inhibition of this
enzyme might be an effective treatment in tumors in
which this gene has mutated. The cell-based assays for
oncogenic phosphotidylinositol-3′-kinase developed in
the Vogt laboratory could be readily modified to provide
the basis of a high-throughput screen for mutant-specific inhibitors of the kinase, possibly leading to a new
class of drugs effective in cancer.
Another approach to the treatment of cancer is being
pursued in the laboratory of Brunhilde Felding-Habermann. Her group began its work by identifying the
ligands involved in the adherence of platelets to tumor
cells. The results led to the discovery that the tumor
cells displayed an activated conformer of integrin αvβ3.
With the collaboration of Kim Janda and his group, an
antibody to this tumor antigen was found by probing
one the combinatorial antibody libraries developed in
Richard Lerner’s laboratory. When given to mice, the
antibody has pronounced antitumor activities and is a
promising candidate for a new treatment for patients
whose cells display the cognate antigen.
Sometimes a gene that is discovered by a scientist
turns out to have a function quite different from that
originally postulated. This has been the case with the
gene for phospholipid scramblase 1 discovered by Peter
Sims and Theresa Wiedmer and with the gene for pleiotrophin discovered by Thomas Deuel. In each instance,
the gene has regulatory functions that involve it in cancer growth. Both genes are thus attractive targets for
the treatment of cancer.
Of the different types of hepatitis viruses, hepatitis C
virus has been the most difficult to study because there
has been no way to grow the virus in vitro. Now Frank
Chisari and his group have overcome this problem. In a
recent issue of Proceedings of the National Academy
of Sciences, Chisari and his coauthors describe this
246 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
advance and point out that it provides a powerful tool
for the analysis of host-virus interactions that should
facilitate the discovery of antiviral drugs and vaccines
for the treatment and prevention of hepatitis C. A possible new treatment approach to hepatitis has emerged
from the unexpected discovery that blood platelets play
a role in furthering hepatitis virus infections. A collaboration between Luca Guidotti and Frank Chisari working
with hepatitis and Zaverio Ruggeri’s group working with
platelets could lead to an entirely new approach to
hepatitis therapy.
The work being performed in Joel Buxbaum’s laboratory in collaboration with members of the laboratory
of Jeffrey Kelly has major implications with respect to
heart disease, particularly among African Americans.
Ten percent of African Americans more than 60 years
old who have severe congestive heart failure carry the
TTR V122I allele of transthyretin as compared with only
2.1% of control subjects. The basic studies being performed on the mechanism by which this protein forms
abnormal deposits in the heart are leading to the identification of drugs that may stabilize the mutant enzyme
in such a way that the formation of the abnormal deposits
will be prevented. Preliminary clinical trials are already
under way.
Sadly, one of our most gifted faculty members, who
had made major contributions to our understanding of
human disease, passed away last year. Bernie Babior
had been at Scripps Research, serving as head of the
Division of Biochemistry, for 18 years. He was a great
scientist, an extraordinary mentor for the young, and a
valued colleague and friend. The first annual lecture
honoring his memory was given by Seymour Klebanoff
of the University of Washington in June 2005.
In my 2002 overview, I pointed out the difficulties
that prevent rapid translation of laboratory results into
clinical benefits. I regret to write that this situation has
not improved one iota since that time; perhaps it is worse.
Elias Zerhouni, the director of the National Institutes of
Health, has recognized this difficulty, but his “road map”
approach misses the principal source of difficulty. The
problem is not in the number of trained scientists, it is
not in the lack of chemical libraries, and it is not the
lack of a network connecting various clinical research
facilities. The problem is in the web of conflicting and/or
unreasonable rules that an investigator must negotiate
to start an investigation. Once everything else has been
accomplished, including preclinical toxicity studies and
production of the new remedy via the required good manPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ufacturing practices, delays of 1 or 2 years before a
study can be approved and initiated are the rule, not
the exception. Until a proper balance is reached between
the rights of individuals and the needs of society, many
years will pass before the discoveries enumerated in this
overview reach the pharmacy.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
INVESTIGATORS’ R EPORTS
D IVISION
OF
ARTHRITIS RESEARCH
247
Mechanisms of
Chondrocyte Aging
K. Kühn, M. Lotz
Martin Lotz, M.D., Division Head
Novel Approaches to the
Treatment of Osteoarthritis
D. D’Lima, C.W. Colwell, Jr., M. Lotz
steoarthritis is the most prevalent joint disease.
Effective therapies for the treatment of osteoarthritis are urgently needed but currently are
lacking. A large number of therapeutic targets for osteoarthritis have been identified, and many drugs that
might be useful in treatment of this disease have been
effective in animal models. However, in clinical trials in
humans, orally administered drugs directed at diverse
targets were not effective or were associated with systemic adverse reactions. Most of these clinical trials
were performed with diverse populations of patients
with osteoarthritis who differed in the duration and
severity of disease and in risk factors. This heterogeneity is a potential variable that may confound clinical trials on disease-modifying drugs for treatment of
osteoarthritis.
Joint injury is a known risk factor for the development of posttraumatic osteoarthritis. Compared with
patients who already have arthritis, patients with joint
injuries are a more promising and homogenous population for clinical trials. In a clinical study, we are using
radiography, magnetic resonance imaging, and assays
of biochemical markers of osteoarthritis to monitor the
progression of cartilage lesions in patients with meniscus injury.
Because osteoarthritis is limited to a single joint
in this study, the patients are also suitable for local
intra-articular therapy. Local therapy has advantages:
it results in high concentrations of drugs at the relevant site, and it is associated with a reduced risk for
systemic adverse reactions. We completed studies in
animal models of osteoarthritis and showed that injection of injured joints with inhibitors of cell death was
effective in reducing the severity of posttraumatic osteoarthritis. The long-term goal of this project is to develop
local therapy with cell death inhibitors for the prevention of posttraumatic osteoarthritis in humans.
O
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The Scripps Research Institute. All rights reserved.
artilage aging is associated with changes in chondrocyte biosynthetic functions and cell proliferation
and survival. These changes may contribute to the
pathogenesis of osteoarthritis. Mechanisms responsible for
aging-associated changes in chondrocytes, such as the
compromised response to growth factors, remain to be
identified. We analyzed protein expression profiles in chondrocytes to determine mechanisms of chondrocyte aging.
Articular cartilage from the knee was obtained from
young and old human tissue donors. Total cellular protein extracts were analyzed by using 2-dimensional gel
electrophoresis. Proteins with differential expression in
young vs old donors were isolated and identified by
using liquid chromatography and mass spectrometry.
β-Actin was among the proteins with the largest agingrelated differences. Reduced levels of β-actin in chondrocytes from old donors were confirmed by Western blot
analysis of additional cell preparations. Levels of β-actin
mRNA did not differ between old and young donors.
Previous studies indicated aging-associated changes
in the chondrocyte response to growth factors such as
transforming growth factor β. Stimulation of cultured
chondrocytes with transforming growth factor β increased
expression of β-actin in chondrocytes from both young and
old donors, but protein levels remained lower in cells from
older donors. Expression of F-actin was increased in cartilage from old donors, and the ratio of F-actin to G-actin
was higher in cultured chondrocytes from old donors.
When plated on collagen type II, chondrocytes from
young donors spread faster than did chondrocytes from
old donors. Treatment with transforming growth factor β
enhanced spreading to a greater extent in cells from
young donors than in cells from old donors. These differences in the organization of chondrocyte microfilaments can contribute to aging-dependent changes in
chondrocyte function and survival.
C
Aminosugars and Osteoarthritis
A.R. Shikhman, D.C. Brinson, M. Lotz
G
lucosamine and its derivatives are commonly
used remedies for treatment of the symptoms
of osteoarthritis. Mechanisms of aminosugar-
248 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
mediated antiarthritic activities are still poorly understood. Aminosugar screening in vitro indicated that
N-acetylglucosamine has beneficial anti-inflammatory
and anabolic effects on human articular chondrocytes.
A potential mechanism of action of aminosugars is their
modification of facilitated glucose transport. Although
N-acetylglucosamine is not imported by chondrocytes,
it interacts with the glucose transporter GLUT1 expressed
on the plasma membrane and enhances facilitated
glucose transport. This facilitated transport results in
several beneficial metabolic events, including activation of protein kinase C and subsequent acceleration
of glucose flux through the hexosamine pathway and
stimulation of the synthesis of sulfated glycosaminoglycans and hyaluronic acid. A key consequence of
N-acetylglucosamine interactions with chondrocytes in
vitro is better maintenance of homeostasis of cartilage
extracellular matrix; the degree of homeostasis is predictive of chondroprotective activity in vivo.
The therapeutic efficacy of N-acetylglucosamine was
also shown in rabbits with experimental osteoarthritis.
N-acetylglucosamine injected intramuscularly did not
have chondroprotective effects but was mildly anti-inflammatory. In contrast, N-acetylglucosamine given intra-articularly reduced cartilage degradation and also suppressed
synovitis. The chondroprotective effect of N-acetylglucosamine was better than that of hyaluronan.
As a prerequisite to studying the chondroprotective
activity of N-acetylglucosamine in humans, we analyzed
the safety of intra-articularly administered N-acetylglucosamine in patients with osteoarthritis in the knee.
Results of the phase 1 clinical trial indicated the safe
range of N-acetylglucosamine concentrations suitable
for further use in trials to determine the efficacy of
treatment with this aminosugar.
IL-1 Signaling in Chondrocytes
T. Olee, J. Valbracht, M. Lotz
he cytokine IL-1 is a key mediator of joint inflammation and cartilage destruction in arthritis. Determination of the signaling pathways involved in
chondrocyte activation by IL-1 may lead to the detection of new therapeutic targets for arthritis. We determined the role of tyrosine kinases in the IL-1 response
of chondrocytes.
Differential expression of tyrosine kinases in normal and osteoarthrithic cartilage was analyzed by using
T
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The Scripps Research Institute. All rights reserved.
DNA arrays and reverse transcriptase–polymerase chain
reaction. The effects of inhibitors of tyrosine kinases,
mutants of tyrosine kinases, and small interfering RNA
on IL-1–induced protein expression in cultured chondrocytes were analyzed by using an enzyme-linked immunosorbent assay. Activation of tyrosine kinases and MAP
kinases was analyzed by using Western blotting with
phospho-specific antibodies.
We found that expression of proline-rich tyrosine
kinase 2 (PYK2) mRNA was increased in osteoarthritic
cartilage. Analysis of articular cartilage by immunohistochemistry indicated that the majority of chondrocytes
were positive for PYK2 protein. Phosphorylated PYK2
was detectable in cultured chondrocytes and increased
in response to stimulation with IL-1. The PYK2-specific
kinase inhibitor AG-17 completely inhibited production
of IL-1–induced nitric oxide and IL-6 in chondrocyte
monolayers and in cartilage explant cultures. Pharmacologic inhibitors of other tyrosine kinases such as focal
adhesion kinase and Src did not significantly affect
these IL-1 responses. Mutants of PYK2 in which the
kinase was inactive or autophosphorylation was defective and PYK2-specific small interfering RNA inhibited
IL-1–induced production of IL-6 in chondrocytes in vitro.
In a subset of chondrocyte preparations, AG-17 and
PYK2 small interfering RNA prevented IL-1–induced
MAP kinase activation. These results indicate that PYK2
is a new component of IL-1 signaling in chondrocytes
and a potential therapeutic target in arthritis.
Cartilage Stem Cells
S. Grogan, M. Lotz
rticular cartilage contains mature chondrocytes
and a population of immature cells with characteristics of mesenchymal stem cells (MSCs).
Transplantation of these cells or activation by extracellular stimuli that promote their differentiation into chondrocytes may be useful in tissue repair. In addition, aberrant
activation and differentiation of MSCs may contribute
to the pathogenesis of arthritis. Most of the current
knowledge of MSC biology is based on studies with
bone marrow–derived cells. Bone marrow–derived MSCs
have multilineage capacity and can form cartilage, bone,
adipose tissue, tendon, and muscle.
Identification of markers that define MSCs in bone
marrow and other tissues would facilitate isolation of
these cells for research purposes and therapeutic appli-
A
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
cations. The Notch-1 receptor may be a marker for MSCs.
The receptor is expressed on most bone marrow–derived
MSCs and on a subpopulation of cartilage cells. Notch
signaling is required during the early phases of chondrocytic differentiation of MSCs. We suggest that Notch is a
regulator of chondrogenesis and a marker for identification
and isolation of MSCs for applications in tissue repair.
PUBLICATIONS
Daouti, S., Latario, B., Nagulapalli, S., Buxton, F., Uziel-Fusi, S., Chirn, G.W.,
Bodian, D., Song, C., Labow, M., Lotz, M., Quintavalla, J., Kumar, C. Development of comprehensive functional genomic screens to identify novel mediators of
osteoarthritis. Osteoarthritis Cartilage 13:508, 2005.
Kinney, R.C., Schwartz, Z., Week, K., Lotz, M.K., Boyan, B.D. Human articular
chondrocytes exhibit sexual dimorphism in their responses to 17β-estradiol. Osteoarthritis Cartilage 13:330, 2005.
Liu, J., Numa, M.M., Liu, H., Huang, S.J., Sears, P., Shikhman, A.R,, Wong, C.-H.
Synthesis and high-throughput screening of N-acetyl-β−hexosaminidase inhibitor
libraries targeting osteoarthritis. J. Org. Chem. 69:6273, 2004.
Shikhman, A.R., Amiel, D., D’Lima, D., Hwang, S.B., Hu, C., Xu, A., Hashimoto, S.,
Kobayashi, K., Sasho, T., Lotz, M.K. Chondroprotective activity of N-acetylglucosamine in rabbits with experimental osteoarthritis. Ann. Rheum. Dis. 64:89, 2005.
Shikhman, A.R., Brinson, D.C., Valbracht, J., Lotz, M. Facilitated transport and
selective metabolic effects of glucosamine and N-acetylglucosamine in human
articular chondrocytes. Am. J. Physiol., in press.
Transcriptional Regulation of
Skeletal Framework and
Joint Diseases
M. Tsuda, N. Taniguchi, T. Ito, T. Furumatsu, K. Yoshida,
H. Asahara
hondrogenesis is a multistep pathway in which
multipotential mesenchymal stem cells (MSCs)
differentiate into chondrocytes. The transcription
factor Sox9 regulates chondrocyte differentiation and
cartilage-specific expression of genes such as COL2A1,
which encodes collagen type II α1. However, Sox9 is
expressed not only in chondrogenic tissue but also in
nonchondrogenic tissues, suggesting that Sox9 requires
a molecular partner or partners to control chondrogenesis and expression of chondrogenic genes.
We found that peroxisome proliferator-activated
receptor γ coactivator-1α (PGC-1α) is a coactivator for
Sox9 during chondrogenesis. Expression of PGC-1α is
induced at sites of chondrogenesis during limb development in mouse embryos and during chondrogenesis in
human MSC cultures. PGC-1α directly interacts with
Sox9 and promotes Sox9-dependent transcriptional
activity, suggesting that PGC-1α acts as a transcriptional
C
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
249
coactivator for Sox9. Consistent with this finding, in
MSCs, disruption of PGC-1α by small interfering RNA
inhibits COL2A1 expression during chondrogenesis.
Furthermore, overexpression of both PGC-1α and Sox9
induced expression of chondrogenic genes, including
COL2A1, with subsequent chondrogenesis in the MSCs
and in developing chick limbs. Together, our results
suggest a transcriptional mechanism for chondrogenesis that is coordinated by PGC-1α.
Signaling by transforming growth factor β (TGF-β)
is necessary for chondrogenic differentiation. Previously,
we showed that CREB-binding protein (CBP/p300) acts
as an important Sox9 coactivator during chondrogenesis. This past year, we investigated the relationship
between TGF-β–dependent Smad2/3 signaling pathways and the Sox9-CBP/p300 transcriptional complex
at the early stage of chondrogenesis.
Overexpression of Smad3 strongly induced the primary chondrogenesis of human MSCs. In addition,
Smad3 enhanced the transcriptional activity of Sox9
and the expression of COL2A1, and treatment of Smad3
with small interfering RNA inhibited these activities. We
found that Smad2/3 associated with Sox9 in a TGF-β–
dependent manner and formed transcriptional complexes
with Sox9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was
increased by Smad3 overexpression and was suppressed
by small interfering RNA that affected Smad3.
Our findings indicate that Smad3 has a stronger
potential than Smad2 for stimulating the Sox9-dependent
transcriptional activity by modulating the interaction
between Sox9 and CBP/p300. These results suggest that
the Smad3 pathway plays a key role in Sox9-dependent
transcriptional activation in primary chondrogenesis.
PUBLICATIONS
Furumatsu, T., Tsuda, M., Taniguchi, N., Tajima, Y., Asahara, H. Smad3 induces
chondrogenesis through the activation of SOX9 via CREB-binding protein/p300
recruitment. J. Biol. Chem. 280:8843, 2005.
Kato, T., Asahara, H., Kurokawa, M.S., Fujisawa, K., Hasunuma, T., Inoue, H., Tsuda, M.,
Takahashi, S., Motokawa, S., Sumida, T., Nishioka, K. HTLV-I env protein acts as a major
antigen in patients with HTLV-I-associated arthropathy. Clin. Rheumatol. 23:400, 2004.
Kawakami, Y., Tsuda, M., Takahashi, S., Taniguchi, N., Esteban, C.R., Zemmyo, M.,
Furumatsu, T., Lotz, M., Belmonte, J.C., Asahara, H. Transcriptional coactivator
PGC-1α regulates chondrogenesis via association with Sox9. Proc. Natl. Acad. Sci.
U. S. A. 102:2414, 2005.
Nishida, K., Komiyama, T., Miyazawa, S., Shen, Z.N., Furumatsu, T., Doi, H.,
Yoshida, A., Yamana, J., Yamamura, M., Ninomiya, Y., Inoue, H., Asahara, H.
Histone deacetylase inhibitor suppression of autoantibody-mediated arthritis in
mice via regulation of p16INK4a and p21(WAF1/Cip1) expression. Arthritis
Rheum. 50:3365, 2004.
Omoto, S., Nishida, K., Yamaai, Y., Shibahara, M., Nishida, T., Doi, T., Asahara, H.,
Nakanishi, T., Inoue, H., Takigawa, M. Expression and localization of connective
tissue growth factor (CTGF/Hcs24/CCN2) in osteoarthritic cartilage. Osteoarthritis
Cartilage 12:771, 2004.
250 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
D IVISION
OF
B IOCHEMISTRY
Eric F. Johnson, Ph.D., Acting Division Head
Cytochrome P450: Regulation,
Structure, and Function
E.F. Johnson, K.J. Griffin, M.-H. Hsu, Ü. Savas,
G.A. Schoch, J.K. Yano
ammalian genomes generally contain 50–100
genes for the P450 monooxgenases. Some
P450s have specialized roles in autocrine,
paracrine, and endocrine signal transduction pathways,
and most P450s play defensive roles by converting
toxic compounds to less toxic forms that are more soluble and more easily excreted than are the parent compounds. Each xenobiotic-metabolizing P450 generally
oxidizes structurally diverse substrates, leading to a
wide-ranging protective capacity for elimination of toxic
chemicals. We wish to understand how the structural
diversity and regulation of the P450s that metabolize
xenobiotics contribute to a person’s ability to avoid the
adverse effects of environmental chemicals and alter
the clearance and bioavailability of therapeutic drugs.
Although extensive information on the conditional
expression of P450 genes in experimental species is
available, in humans, the transcriptional responses of
P450 genes to environmental stimuli and to physiologic
changes are poorly understood. To address this problem,
we used both cell lines and transgenic mice to study
expression of human family 4 P450 genes. These genes
encode enzymes that are involved in both signal transduction and xenobiotic metabolism. Studies with cell
lines are providing new information about endocrine
and autocrine signal transduction pathways that govern
the conditional expression of these genes in response to
nutritional, hormonal, and xenobiotic signals. Research
is in progress to determine whether more complex
physiologic conditions such as pregnancy or energy
restriction alter the expression of the human enzymes
in transgenic mice.
We also pioneered using x-ray crystallography to
determine the atomic structures of P450s that contribute
extensively to drug metabolism in humans. In collaboration with C.D. Stout, Department of Molecular Biology,
we are defining the structural features of individual
human P450s that contribute to the unique catalytic
selectivities of the enzymes. Our goal is to better under-
M
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
stand the adverse affects of the oxidation of drugs and
toxins and the potential for metabolic drug-drug interactions that can arise from inhibition of P450s if multidrug therapies are used.
Mammalian P450s are tethered to the endoplasmic
reticulum by a transmembrane segment at the amino
terminus and by additional interactions of the catalytic
domain with the cytoplasmic side of the membrane.
Although membrane proteins are difficult to crystallize,
we developed methods to express, purify, and crystallize genetically modified mammalian P450s. Through
these studies, we discovered how the flexibility of the
P450s and the diversity of their amino acid sequences
shape catalytic specificity.
Specific P450s, such as 3A4 and 2C8, oxidize relatively large compounds such as the immunosuppressant
cyclosporin and the antitumor drug paclitaxel. Structures determined for 3A4 and 2C8 indicate how the
architecture of these enzymes has adapted to accommodate these large substrates.
P450s can also oxidize relatively small substrates
such as chloroform, ethanol, and nicotine. Recently,
we solved the structure of the P450 2A6, the principal
nicotine-oxidizing enzyme. Although 2A6 plays a prominent role in detoxification of nicotine, it also can activate
the tobacco smoke–specific carcinogen nitrosamine
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the
carcinogenic form. Several reports indicate that because
of the increased side effects of nicotine, persons who
are genetically deficient in 2A6 activity are less likely
to smoke than are persons not genetically deficient in
this activity, and those who are deficient in 2A6 activity are less likely to have lung cancer if they do smoke.
The structure of 2A6 will contribute to the design and
development of inhibitors that could alter smoking
behavior and diminish the likelihood of tobacco-related
lung cancers.
PUBLICATIONS
Mellet, A., Marques-Soares, C., Schoch, G., Macherey, A.-C., Jaouen, M.,
Dansette, P.M., Sari, M.-A., Johnson, E.F., Mansuy, D. Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and sitedirected mutants. Biochemistry 43:15379, 2004.
Poulos, T.L., Johnson, E.F. Structures of cytochrome P450 enzymes. In: Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed. Ortiz de Montellano, P.R. (Ed.). Plenum Publishing, New York, 2005, p. 87.
Savas, Ü., Hsu, M.-H., Griffin, K.J., Bell, D.R., Johnson, E.F. Conditional regulation of
the human CYP4X1 and CYP4Z1 genes. Arch. Biochem. Biophys. 436:377, 2005.
Wester, M.R., Yano, J.K., Schoch, G.A., Yang, C., Griffin, K.J., Stout, C.D., Johnson, E.F. The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-Å resolution. J. Biol. Chem. 279:35630, 2004.
Yano, J.K., Wester, M.R., Schoch, G.A., Griffin, K.J., Stout, C.D., Johnson, E.F.
The structure of human microsomal cytochrome P450 3A4 determined by x-ray
crystallography to 2.05-Å resolution. J. Biol. Chem. 279:38091, 2004.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Mechanisms of
Regulated Secretion
S.D. Catz, J.L. Johnson, B.A. Ellis, J.M. Ruedi, D. Noack, S.
Pacquelet, B. Young
A GENERAL ROLE FOR RAB27 AND JFC1 IN
SECRETORY CELLS
he traffic of secretory vesicles to the plasma membrane in eukaryotic cells is essential for normal
cell function. The specificity of vesicular transport
relies on Rab GTPases that act as membrane organizers and on their specific effectors that transduce many
steps in cell trafficking. Rab27a, the only Rab known
to associate directly with a disease in humans (Griscelli
syndrome), plays a central role in regulated secretion.
We are studying the mechanisms involved in vesicle
exocytosis, in particular, the function of Rab27a effectors in this process.
Recently, we identified JFC1/Slp1, a member of the
synaptotagmin-like protein family of effectors characterized by the presence of a Rab27-binding motif in its
amino terminus and by C2 domains capable of binding
phosphatidylinositol-3,4,5-trisphosphate in its carboxy
terminus. Using a combination of functional analysis
and confocal microscopy, we discovered that JFC1 and
Rab27a regulate the androgen-dependent secretion of
prostatic-specific acid phosphatase and prostatespecific antigen in human prostate carcinoma cells
in a process that involves the phosphatidylinositol-3′kinase pathway.
In other studies, we have made progress in characterizing the secretory machinery used by the many
secretory organelles present in human neutrophils.
Neutrophils have at least 4 distinct secretory organelles.
During activation and phagocytosis, neutrophils can
differentially secret the contents of the organelles into
the acceptor compartment and/or integrate vesicular
proteins (e.g., cytochrome b 558) into acceptor membranes. The secretion of these organelles is thought to
be hierarchical. We aim to identify the secretory machineries that control the differential secretory behavior of
these organelles.
Both the prostate and the neutrophil secretion studies
will be complemented by using the ashen (Rab27aash)
mouse model.
T
I L - 1 R E C E P T O R – A S S O C I AT E D K I N A S E - 4 A N D
NADPH OXIDASE
For more than 20 years, it has been known that
lipopolysaccharide can elicit neutrophil priming for the
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
251
production of superoxide anion in response to formylated peptides. This phenomenon has been explained
in part by the ability of lipopolysaccharide to upregulate the NADPH oxidase assembly. However, it is still
unclear how the signaling downstream activation of
Toll-like receptor 4 is involved in the upregulation of
the NADPH oxidase. Recently, we discovered that IL-1
receptor–associated kinase-4, a kinase involved in the
signaling downstream activation of Toll-like receptors,
phosphorylates the NADPH oxidase cytosolic factor
p47phox. Using mass spectrometric analysis, we identified a threonine-rich domain in p47phox that is the
target of phosphorylation by the kinase. We are analyzing the physiologic importance of these findings.
PUBLICATIONS
Johnson, J.L., Pacquelet, S., Lane, W.S., Eam, B., Catz, S.D. Akt regulates the
subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.
Traffic 6:667, 2005.
Preserving Vision in Glaucoma
and Macular Degeneration:
Neuroprotective Effects of
the Flavonoids
A. Hanneken, J. Johnson, F.-F. Lin
erve cell damage is the primary cause of visual
loss in patients with glaucoma, retinal detachments, and macular degeneration. Among people more than 75 years old, 30% have one of these
conditions, and the incidence is expected to increase
as the population ages. Restoring the function of injured
nerve cells has been particularly difficult both in the
CNS and in ocular diseases.
We formed an interdisciplinary research team with
P. Maher and her colleagues at the Salk Institute, La
Jolla, California, to explore the ability of flavonoids to
prevent oxidative stress, the type of nerve injury thought
to occur in the eye. This collaboration was started and
the approach was validated after a large, 10-year clinical trial sponsored by the National Eye Institute indicated that antioxidants could reduce the progression
of macular degeneration.
Flavonoids have 4 properties that make them promising candidates for the treatment of eye diseases. The
compounds are potent antioxidants and free-radical
scavengers, they induce neuronal recovery after injury,
they are antiangiogenic (i.e., inhibit growth of blood
N
252 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
vessels that occurs in macular degeneration and diabetic retinopathy), and they are well tolerated orally.
Specific flavonoids can enhance the production of glutathione, block the production of reactive oxygen species, and prevent the late influx of calcium, all of which
are activities that prevent specific events in the nerve
cell death pathway. In addition, the flavonoids can activate the antioxidant response element, which induces
the expression of genes that increase cells’ resistance
to oxidative stress. The ability of flavonoids to restore
the health of injured neuronal cells and induce the outgrowth of neurites gives these compounds a unique
set of advantages compared with other antioxidants.
The goal of our research is to determine the specific
classes and the chemical structures of dietary flavonoids
that are the most effective at preventing oxidative stress–
induced cell death in retinal ganglion nerve cells and
retinal pigment epithelial cells. We are also investigating the mechanisms behind this protective activity. We
screened multiple different flavonoids for their ability
to protect these cell types from oxidative stress induced
by hydrogen peroxide and tert-butyl hydroperoxide
(Fig. 1). To date, we have identified several flavonoids
that are both potent and effective in preventing cell
death in each of these cell types (Table 1). We found
other flavonoids that have limited or no effect.
As illustrated in Figure 2, luteolin, a flavonoid present in spinach, prevents oxidative stress–induced cell
death in retinal pigment epithelial cells, the cells that
die in patients with macular degeneration. Luteolin has
no cellular toxic effects over a wide range of concentrations. Other flavonoids, including quercetin, fisetin,
F i g . 1 . Chemical structures of the dietary flavonoids. EGCG =
(–)-epigallocatechin gallate.
T a b l e 1 . The potency of various flavonoids in protecting primary human retinal pigment epithelium cells from oxidative stress–induced cell death*
Free hydroxyl
Flavonoid
Flavone
Flavonol
Isoflavone
Flavanone
Flavanol
Anthocyanidin
positions
5,6,7
3′,4′,5,7
3,6
3,7
3,5,7
3,3′,4′,7
3,4′,5,7
3,3′,4′,5,7
3,3′methoxy,4′,5,7
3,3′,4′,5,5′,7
4′,5,7
4′,5,7
3′,4′,5,7
3,3′,4′,5,7
3,3′,4′,5,7
3,3′,4′,5,7
3,3′,4,4′,5,7
EC50, µM
Common name
Baicalein
Luteolin
t-BOOH
8±1
2±1
7±1
9±1
26 ± 1
Galangin
Fisetin
3±1
Kaempferol
~50
Quercetin
6±1
Isorhamnetin
>>50
Myricetin
>50
No
Genistein
Naringenin
No
Eriodictyol
7±1
>50
Taxifolin
Catechin
No
Epicatechin
No
Epigallocatechin-3-gallate 22 ± 1
Cyanidin
No
H2O2
21 ± 1
3±1
11 ± 1
8±1
61 ± 1
5±1
No
11 ± 2
No
No
No
No
11 ± 1
No
No
No
30 ± 2
No
LD50, µM
>>100
>50
>>50
27
70
>50
~50
>50
>50
>>50
>50
>>50
>100
>>50
>>50
>>50
>100
>>50
* Half maximal effective concentrations (EC50) were determined by exposing retinal pigment epithelium cells to tert-butyl hydroperoxide (t-BOOH) or hydrogen peroxide (H2O2) in the presence
of flavonoids. The doses of the compounds that caused 50% cell death (LD50) were measured by using exposure to flavonoids alone. Values represent the average of 2–3 independent experiments. Each study was conducted with 2–4 replicates. No = little or no efficacy at doses up to 50 µM.
Published by TSRI Press®. © Copyright 2005,
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MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
253
will be directed toward validating and expanding
these results.
This research is the result of a partnership formed
between the Scripps Mericos Eye Institute and Scripps
Research aimed at bringing together the promise of
biomedical research and the practice of medicine.
PUBLICATIONS
Lin, F.-F., Maher, P., Hanneken, A. Flavonoids protect human retinal pigment epithelial
cells from oxidative stress-induced death. Invest. Ophthalmol. Vis. Sci., in press.
Maher, P., Hanneken A. Flavonoids protect retinal ganglion cells from oxidative
stress-induced death. Invest. Ophthalmol. Vis. Sci., in press.
Maher, P., Hanneken, A. The molecular basis of oxidative stress-induced cell
death in an immortalized retinal ganglion cell line. Invest. Ophthalmol. Vis. Sci.
46:749, 2005.
NADH Dehydrogenases
T. Yagi, A. Matsuno-Yagi, B.B. Seo, E. Nakamaru-Ogiso,
M.-C. Kao, T. Yamashita, M. Marella, J. Barber-Singh
STRUCTURE AND FUNCTION OF PROTONT R A N S L O C AT I N G N A D H - Q U I N O N E O X I D O R E D U C TA S E
F i g . 2 . Luteolin protects retinal pigment epithelial cells from oxidative stress–induced cell death. H2O 2 = hydrogen peroxide;
t-BOOH = tert-butyl hydroperoxide.
and eriodictyol, are also effective; genistein and myricetin
are ineffective.
On the basis of these early findings, we compiled
a list (Table 2) of the fruits and vegetables that contain
the highest concentrations of the flavonoids that are
the most effective at preventing oxidative stress–induced
cell death in cells derived from the eye. Future studies
T a b l e 2 . Dietary flavonoids that protect retinal cells from injury
and death in macular degeneration
Flavonoid
Dietary source
Luteolin
Spinach, wild greens, hot peppers,
celery, thyme, parsley, mint
Quercetin
Onions (especially yellow), cranberries,
cocoa, wild greens, capers, fennel,
spinach, chives, celery, cherries,
blueberries, apples, kale, red wine
Eriodictyol
Peppermint, citrus juices (lemon, lime,
sour orange)
Fisetin
Strawberries, tomatoes, onion, oranges,
apples, peaches, grapes, kiwifruit,
persimmons
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
he proton-translocating NADH dehydrogenase of
mitochondria (complex I) is responsible for energy
coupling in the respiratory chain. Complex I is
composed of 46 unlike subunits and contains 1 FMN
and 8 iron-sulfur clusters as cofactors. The protontranslocating NADH dehydrogenase of bacteria, NDH-1,
is similar to complex I in terms of electron carriers
and inhibitor specificity. However, in contrast to complex I, NDH-1 is composed of 14 unlike subunits
(NuoA–NuoN). Both NDH-1 and complex I consist of
2 major domains: the peripheral segment and the membrane segment. The peripheral domain of NDH-1 contains 7 subunits, NuoB–NuoG and NuoI. Of these, NuoB
and NuoI act as connectors between the 2 domains.
The membrane domain of NDH-1 appears to be composed of 7 subunits (NuoA, NuoH, and NuoJ–NuoN),
which are homologs of mitochondrial DNA-encoded
subunits of complex I.
In one of our current projects, we are clarifying the
functional roles of conserved residues in the membrane
subunits. To explore these roles, we use site-directed
mutagenesis with chromosomal DNA manipulation. As
shown in Figure 1, we found that D79 and E81 in
NuoA and V65 in NuoJ play important roles in energy
coupling of NDH-1.
In another project, we are characterizing cofactors
of NDH-1. In certain bacteria, NDH-1 contains an addi-
T
254 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
F i g . 1 . Essential amino acid residues of membrane domain subunits NuoA and NuoJ of the NADH-quinone oxidoreductase.
tional iron-sulfur cluster binding motif in the subunit
NuoG. Researchers have proposed that this additional
unique cysteine sequence motif is responsible for the
electron paramagnetic resonance signals from the
binuclear cluster N1c of the Escherichia coli NDH-1.
To characterize the N1c cluster at the subunit level and
distinguish it from 3 other iron-sulfur clusters (N1b, N4,
and N5) accommodated in the NuoG subunit, we individually inactivated 4 iron-sulfur cluster binding sites
by substituting alanine for all 4 conserved cysteine
(or histidine) residues. We overexpressed wild-type and
individual iron-sulfur cluster mutant NuoG subunits as
maltose-binding protein fusion protein in E coli. We
compared electron paramagnetic resonance spectra of
the individually mutated NuoG subunits and identified
the [4Fe-4S] electron paramagnetic resonance signals.
On the basis of current analyses and the fact that
the cysteine sequence motif coordinates only [4Fe-4S]
clusters in other known enzymes such as periplasmic
nitrate reductase in Paracoccus denitrificans, we concluded that the N1c binding motif undoubtedly ligates
a [4Fe-4S]. Therefore, we propose to change its misleading name, N1c, to N7.
We attempted expression of NDI1 in vivo in skeletal muscles and brains (substantia nigra and striatum)
in rodents. In all tissues tested, immunohistochemical
staining showed the presence of Ndi1 in the injected
area at 1–2 weeks after injection of the gene (Fig. 2)
Expression persisted for at least 7 months. Furthermore,
the expressed Ndi1 protein stimulated NADH dehydrogenase activity, suggesting that the expressed Ndi1 is
functionally active. We also confirmed that the expression of Ndi1 induced no inflammatory response in the
tissue examined. The data indicate that the gene NDI1
will be a promising therapeutic tool in the treatment
of diseases caused by impairments in complex I.
F i g . 2 . Visualization of the NADH dehydrogenase activity of
Ndi1 expressed in the nigral area of the brain of a rat. Images on
the left (noninjected side) and right (NDI1-injected side) show the
neurons (arrows). Bars = 20 µm.
PUBLICATIONS
Kao, M.-C., Di Bernardo, S., Nakamaru-Ogiso, E., Miyoshi, H., Matsuno-Yagi, A.,
Yagi, T. Characterization of the membrane domain subunit NuoJ (ND6) of the
NADH-quinone oxidoreductase from Escherichia coli by chromosomal DNA manipulation. Biochemistry 44:3562, 2005.
Kao, M.-C., Di Bernardo, S., Perego, M., Nakamaru-Ogiso, E., Matsuno-Yagi, A.,
Yagi, T. Functional roles of four conserved charged residues in the membrane
domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase
from Escherichia coli. J. Biol. Chem. 279:32360, 2004.
Nakamaru-Ogiso, E., Yano, T., Yagi, T., Ohnishi, T. Characterization of the iron-sulfur cluster N7 (N1c) in the subunit NuoG of the proton-translocating NADHquinone oxidoreductase from Escherichia coli. J. Biol. Chem. 280:301, 2005.
MOLECULAR REMEDY OF COMPLEX I DEFECTS
Studies suggest that defects in mitochondrial complex I are involved in many human diseases, such as
encephalomyopathies and some cases of Parkinson’s
disease. However, no effective remedies for complex I
deficiencies have been established. We have adopted
a gene therapy approach in which we use the gene
NDI1, which encodes Ndi1, the single subunit NADH
dehydrogenase of Saccharomyces cerevisiae. Our earlier results indicated that Ndi1 can replace or supplement the functionality of complex I in various cultured
cells. For this approach to be useful, in vivo studies
must indicate that the mature protein is correctly
placed in mitochondria.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Seo, B.B., Nakamaru-Ogiso, E., Cruz, P., Flotte, T.R., Yagi, T., Matsuno-Yagi, A.
Functional expression of the single subunit NADH dehydrogenase in mitochondria in
vivo: a potential therapy for complex I deficiencies. Hum. Gene Ther. 15:887, 2004.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
D IVISION
OF
B IOMATHEMATICS
James A. Koziol, Ph.D., Division Head
255
We estimate the total number (N) of protein-coding genes by using a classical technique, maximum
likelihood. The relevant data are shown in Figure 1.
Application of CaptureRecapture Models to Estimation
of Protein-Coding Gene Count in
the Human Genome
J.A. Koziol, A.C. Feng
he role of our division is to apply mathematical
and statistical principles and techniques to outstanding problems of biological interest. The following is an example of the research we do.
Two groups, the International Human Genome
Sequencing Consortium (IHGSC) and Celera Genomics,
have made independent efforts to sequence the human
genome with annotation of putative genes. Draft
sequences were first reported by the 2 groups in 2001,
and updates were published subsequently. Scientists
at Scripps Research compared early Ensembl annotations
of Celera and IHGSC and found some discrepancies in
the predicted transcript sets. Later, other researchers
suggested that many of these discrepancies could be
explained by the fundamental differences between the
2 genomic assemblies. More recently, reexamination of
the updated annotations from Celera and IHGSC again
revealed nonoverlapping gene sets in the 2 transcripts.
Using these experimental findings, we used a mathematical model to estimate the total number of proteincoding genes in the human genome.
Our mathematical model is a capture-recapture
model for closed populations with time-varying capture
probabilities. This model is not new; it has been widely
used to estimate population sizes in biometry and
ecology, where it is commonly referred to as the M t
model. The relevant assumptions of the M t model in
our setting are as follows: (1) a closed population, that
is, the number of genes in the human genome remains
the same during the human genome project; (2) in a
given genome assembly, all sequences have equal
chance of being annotated regardless of their annotation history, but the probabilities of a sequence being
annotated may vary among these genome assemblies;
and (3) identical sequences always predict the same
gene, and there is no prediction error.
T
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Fig. 1. Total numbers of unique protein-coding genes in the RefSeq,
Ensembl, and Celera gene sets as resolved with BLAST analysis
and use of data releases from June 2002. The total unique gene
count is 28,475 (the sum of counts in all of the subsets depicted
in the Venn diagram).
We may invoke the Mt model with 3 sampling (capture)
occasions, in which we have successively observed n1 =
25,856 genes (Celera, the sum of 8245 + 4945 +
11,917 + 749), n2 = 19,177 genes (Ensembl), and
n 3 = 13,105 genes (RefSeq), and a total of M 4 =
28,475 unique genes. Recapture frequencies are also
immediately available from the figure. When these val^
ues are used, the maximum likelihood estimate N1 of
N is 29,077, with an associated 95% confidence interval of 29,021–29,133. Note that any “reasonable”
statistical estimate of N must exceed the observed
number of genes (28,475); the maximum likelihood
estimate is quite close to this number and suggests that
gene identification is almost complete.
However, the maximum likelihood estimate is substantially larger than the consensus figure of around
25,000 genes in the human genome. How can we
explain these discrepancies? If the assumptions underlying the Mt model are tenable, then invocation of the estimation procedures is straightforward and appropriate.
The closure assumption of the M t model, that the size
of the population (the number of protein-coding genes
in the human genome) remains constant during the study
period, seems acceptable. Furthermore, that the ascertainment probabilities for the 3 sampling occasions
(Ensembl, Celera, and RefSeq) differ seems obvious.
However, others can question whether interpretation
or definition of the key characteristic of the target
256 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
population, namely, protein coding, is consistent in all
3 data sources and whether ascertainment is errorless
in each data set. Fragmented or fused genes, paralogs
(sequences in the same species that share direct common ancestors with the current sequences), and pseudogenes can all lead to possibly inflated gene counts in
Figure 1 (particularly, the 8245 genes unique to Celera).
Subsequent sequencing endeavors could perhaps profit
from this exercise, and researchers should pay particular
attention to the untoward effects of high false-positive
rates of ascertainment.
D IVISION
OF
the signal-sensing domains. Deletion studies indicated
that sporulation in B anthracis is a consequence of the
concerted activities of several of these sensor kinases.
The sporulation pathway regulates the production of
the genes for anthrax toxin through at least one Spo0Acontrolled regulator. Studies on the transcription of the
gene that encodes the protective antigen component of
the toxin revealed the extent of the promoter region
responsible for gene activation and the relationship of the
region to the positive transcription activator AtxA. The
goal of these studies is to understand the specific and
global controls that regulate virulence in B anthracis.
C ELLULAR B IOLOGY
James A. Hoch, Ph.D., Division Head
Sensor Kinases That Regulate
Sporulation and the Synthesis
of Toxins
J.A. Hoch, M. Perego, B. Tsvetanova, T. Fukushima,
A. White, H. Szurmant, A. Volkov, N.T. Truong
ormation of endospores in Bacillus subtilis is a
model for understanding the mechanism of developmentally programmed gene expression. Several
dozen genetically dispersed sporulation operons are
regulated coordinately as temporal classes during the
time required to complete the formation of spores. This
complex developmental program is under the control
of the spo0 genes, which control entry of the cell into
sporulation and the production of toxins and virulence
factors in pathogens such as Bacillus anthracis.
The transcription factor Spo0A is the key master
regulator of the initiation of developmental transcription.
The activity of the protein is controlled by a reversible
phosphorylation-dephosphorylation mechanism. The
pathway to Spo0A activation is a sequential series of
phosphorylation reactions involving, sequentially, the
Spo0F and Spo0B proteins, for which we coined the
term phosphorelay. The probability that sporulation
will be initiated depends on the competition between
kinases and phosphatases.
Previously, we identified 5 sensor kinases involved
in signaling the initiation of sporulation in B subtilis.
Our studies in B anthracis revealed 9 sensor kinases
for sporulation that differ from those of B subtilis in
F
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Computational Analysis of
Molecular Specificity in
2-Component Signaling
J.A. Hoch, T. Hwa*
* University of California, San Diego, California
n both prokaryotes and eukaryotes, a large number
of structurally similar pathways are used to propagate vastly different signals specific to the respective targets of the pathways. Elucidating the myriads
of these pathways is a key challenge of postgenome
biology. A central question in understanding signal
transduction is how a signaling protein distinguishes
its true partner from the much larger number of similar
partners present in the cell. Without this specificity,
unintended cross talk among the pathways will greatly
reduce the fidelity of signal transduction. On the other
hand, designed cross talk at specific stages between
specific pathways provides the means of combinatorial
signal integration that may greatly increase the signal
processing capability of the cell. In collaboration with
T. Hwa, University of California, San Diego, my colleagues and I are examining the molecular interactions
that underlie partner recognition; the focus of these
studies is the 2-component system, the prevalent signaling system in bacteria.
Two-component signaling involves the autophosphorylation of a sensor kinase (SK), usually in response to
the presence of an external signal, and the subsequent
transfer of the phosphate group to a response regulator (RR). A total of 30–40 similar SK-RR pairs have
been identified in bacteria such as Escherichia coli and
Bacillus subtilis, and more than 100 occur in Nostoc
punctiformis. These signaling proteins clearly evolved
I
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
from a progenitor pair by extensive gene duplication
and evolved individual recognition specificity while
maintaining structural homology.
To detect the molecular components that determine
the specificity of protein-protein interactions, we created a software program to examine covariation between
signaling partners relative to the background covariation between noninteracting proteins belonging to the
same family. For each SK-RR pair, a sequence profile
of the interaction domains was generated by using the
hidden Markov model (HMM). The dozen known examples of signaling partners in B subtilis were used as a
training set and were iteratively refined by using genomic sequence data. Applying the HMM profiles to the
approximately 200 sequenced bacterial genomes, we
identified about 600 SK-RR pairs that are adjacently
located in the genome.
For each adjacent SK-RR pair, we computed the
“mutual information” for each pair of residue positions.
We found high mutual information content among a
dozen or so residues from SKs and RRs. The form of
the mutual information was used to construct a scoring function, which was used to rank the degree of
specific interaction for each possible pairing of SK and
RR in an organism (regardless of the proximity of the
genes for the pair in the genome) for each organism.
The interaction scores that were obtained clearly
separated into 2 classes. The adjacent pairs all had
high scores. Most of the nonadjacent pairs had low
scores. A few nonadjacent pairs had high scores. Upon
closer examination, a number of these high-scoring nonadjacent pairs could be identified with known interacting pairs.
Currently, we are developing a more rigorous statistical measure to characterize the significance of the
high-scoring pairs. We are also developing a systematic
way of scoring and evaluating interactions that involve
orphaned SKs or RRs (i.e., those that do not have a
partner located in the immediate vicinity in the genome).
Molecular Dynamics of
Response Regulators
J.A. Hoch, J. Cavanagh*
* North Carolina State University, Raleigh, North Carolina
O
n the basis of studies of the backbone dynamics
of the response regulator Spo0F, we proposed a
model in which communication of information
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257
through the core of the Spo0F protein, between buried
and surface-bound residues, is responsible for the dissociation of sensor kinases from response regulators
after phosphorylation. We defined a region on Spo0F
that moves in a dynamically concerted fashion, driven
by the motion of the imidazole ring of histidine at
position 101.
The imidazole ring moves in response to a conformational change in the aspartic acid binding pocket
upon phosphorylation. Movement of the ring disrupts
packing interactions, a condition that alters the topology of the kinase recognition site, thereby causing the
kinase to dissociate. On the basis of nuclear magnetic
resonance, mass spectrometry, and homology modeling studies, we are developing new models for the specificity of response regulators.
Tetratricopeptide Proteins in
Bacterial Signal Transduction
M. Perego, C. Bongiorni, C. Ambrosi, A. Diaz
etratricopeptide repeats (TPRs) are structural
domains found in proteins in a wide variety of
organisms, from eukaryotes to prokaryotes. Each
repeat consists of 34 amino acids that fold into a pair
of antiparallel α-helixes of equal lengths. TPR motifs
are found in tandem arrays of 3–16 motifs per protein. The repeats organize themselves as a scaffold
with a right-handed superhelical structure with a central groove. TPR domains are thought to be an ancient
module that promotes protein-protein interactions.
Each of the 11 members of the Bacillus subtilis Rap
family of proteins contains 6 TPR motifs. The RapA,
RapB, and RapE members of this family act as negative
regulators of the initiation of sporulation by binding to
the phosphorylated Spo0F response regulator and promoting its dephosphorylation. Activities of RapA and
RapE are inhibited by the pentapeptides PhrA and PhrE,
respectively. The pentapeptides are encoded within
precursor proteins that follow an export-import processing pathway that results in the active inhibitor.
We found that RapC and RapF regulate competence
development by binding to the DNA-binding domain of
the ComA response regulator and inhibiting the ability
of ComA to bind DNA. The activities of RapC and RapF
on ComA are in turn inhibited by the PhrC and PhrF
peptides, respectively. These findings are new exam-
T
258 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
ples of how TPR motifs and their structural organization have been adapted for different specific functions
within the B subtilis Rap family.
Rap and Phr proteins are commonly found in the
genome of sporulating bacilli. We analyzed the genome
of Bacillus anthracis and found 6 genes that encode
Rap proteins; each gene is followed by a gene that
encodes a Phr peptide. Five rap-phr genes are chromosomally located; a sixth gene is present on the pX01
plasmid, which also carries the genes for components
of the toxin protein. Through genetic and biochemical
analysis, we determined that one chromosomally encoded
system and the plasmid-encoded Rap-Phr system regulate the initiation of sporulation in B anthracis and
thus may influence toxin production and virulence.
activate transcription of the gene that encodes gelatinase, a zinc-metallo protease. Gelatinase is required
for the formation of biofilms by E faecalis. Because
growth of bacteria as a biofilm is strongly associated
with the development of human infections, such as
infective endocarditis and urinary tract infections, our
findings suggest that gelatinase may present a unique
target for therapeutic intervention against biofilm-based
enterococcal infection.
Phosphorelay Proteins: Structure,
Molecular Recognition, and
Phosphosignaling
K.I. Varughese, H. Zhao, J.A. Hoch
Signal Transduction in
Enterococcus faecalis
M. Perego, F. Del Papa, J. Hwang
nterococci are commensal bacteria within the
intestinal tract in mammals but also can cause
disease in compromised hosts. The acquisition
of resistance to multiple antibiotics by enterococci makes
infections caused by these microorganisms clinically
challenging. The ability of the bacteria to adapt and
respond to different environmental stimuli, including
the host environment, led us to investigate the role of
2-component signal transduction in the physiology and
pathogenesis of Enterococcus faecalis.
Using a bioinformatic approach, we identified 17
2-component systems consisting of a sensory histidine
kinase and a cognate response regulator and an additional orphan response regulator. We inactivated each
response regulator with the exception of the ortholog
of the YycF essential protein of gram-positive organisms.
We tested the effect of the deletions on a number of
physiologic conditions and detected defects in growth,
antibiotic resistance, stress response, and formation of
biofilms. We are using these mutant strains to analyze
the role of signal transduction in pathogenesis in vivo
and to determine the extent of the regulon controlled
by each 2-component system.
Analysis of the 2-component system encoded by
the gene fsr revealed that this system is the only one
that affects growth of enterococci as a biofilm on solid
surfaces. The role of the fsr system in biofilms is to
E
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ransfer of phosphoryl groups from one protein to
another is the basis for propagating information
in phosphorylation-activated signal transduction.
Bacteria, many lower eukaryotes, and plants have
adapted the phosphorylation-dependent phosphorelay
model to couple signal recognition, signal transduction,
and signal response. The initiation of developmental
gene expression in the sporulation of Bacillus subtilis
is directly controlled by a phosphorelay. In a phosphorelay, the histidine kinases are dephosphorylated by a
common response regulator, Spo0F. Phosphorylated
Spo0F is the substrate for the Spo0B phosphotransferase, which mediates transfer of a phosphoryl group
from Spo0F to Spo0A, the ultimate transcription factor.
In recent years, we determined the crystal structures
of Spo0F, Spo0B, and the molecular complex of these
2 proteins. The 2 molecules associate, bringing the
active aspartates and histidines in proximity for catalysis. The core of the contact surface between the 2
proteins is formed by hydrophobic regions from both
proteins. The Spo0F residues that make up this core are
similar in all response regulators, suggesting that the
binding most likely is initiated through the same residues in all interacting response regulator–kinase pairs.
The transcription factor Spo0A is a 2-domain protein
with an N-terminal receiver domain and a C-terminal
effector domain. The molecule is activated on phosphorylation of the receiver domain; the effector domain then
binds to a specific DNA sequence known as the 0A
box. We determined the crystal structure of the effector domain in complex with a DNA duplex containing
the 0A box.
T
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
The effector domain consists mostly of 7 helices.
The third and fourth helices form a helix-turn-helix
motif for DNA binding, and the recognition helix interacts with the 0A box in the major groove of the DNA.
From the protein-DNA interactions, we established the
origin of specificity. In the crystal lattice, 2 protein
molecules bind to DNA and interact in a head-to-tail
fashion to form a dimer.
Spo0A controls the transcription of several hundred
genes in all spore-forming bacilli, including genes for
sporulation and toxin regulation in pathogens such as
Bacillus anthracis. The protein-protein and protein-DNA
interfaces revealed in the crystal structure of Spo0A
provide a basis for interpreting activation of transcription and for designing drugs to counter infections by
these bacteria.
259
D IVISION OF EXPERIMENTAL
H EMOSTASIS AND THROMBOSIS
Zaverio M. Ruggeri, M.D., Division Head
The Vessel Wall at the Interface
of Hemostasis, Thrombosis,
and Inflammation
Z.M. Ruggeri, F. Almus-Jacobs, R. Celikel, N. Greyz,
R. Habermann, Y. Kamikubo, M. Machin, P. Marchese,
R. McClintock, G.L. Mendolicchio, J. Orje, G.M. Podda,
J. Roberts, B. Savage, M. Shibakura, A. Zampolli
PUBLICATIONS
Bongiorni, C., Ishikawa, S., Stephenson, K., Ogasawara, N., Perego, M. Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems. J. Bacteriol. 187:4353, 2005.
Hancock, L.E., Perego, M. The Enterococcus faecalis fsr two-component system
controls biofilm development through production of gelatinase. J. Bacteriol.
186:5629, 2004.
Hancock, L.E., Perego, M. Systematic inactivation and phenotypic characterization
of two-component signal transduction systems of Enterococcus faecalis V583. J.
Bacteriol. 186:7951, 2004.
Kao, M.C., Di Bernardo, S., Perego. M. Nakamaru-Ogiso, E., Matsuno-Yagi, A.,
Yagi, T. Functional roles of four conserved charged residues in the membrane
domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase
from Escherichia coli. J. Biol. Chem. 279:32360, 2004.
Kojetin, D.J., Thompson, R.J., Benson, L.M., Naylor, S., Waterman, J., Davies,
K.G., Opperman, C.H., Stephenson, K., Hoch, J.A., Cavanagh, J. Structural analysis of metal binding to the Bacillus subtilis response regulator Spo0F: a possible
role for metalloregulation in the initiation of sporulation. Biometals, in press.
Mukhopadhyay, D., Varughese, K.I. A computational analysis on the specificity of
interactions between histidine kinases and response regulators. J. Biomol. Struct.
Dyn. 22:555, 2005.
Szurmant, H., Nelson, K., Kim, E.-J., Perego, M., Hoch, J.A. YycH regulates the
activity of the essential YycFG two-component system in Bacillus subtilis. J. Bacteriol. 187:5419, 2005.
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lood circulates within conduits (arteries, veins,
and connecting capillary beds) that are lined
by a continuous layer of endothelial cells attached
to underlying components of the vascular wall. Oxygen,
nutrients, and metabolic products are exchanged between
circulating blood and tissues, and continuous monitoring goes on to detect perturbations within the vasculature. When a deviation from normal is detected, for
example, a wound causing bleeding or invading pathogens such as bacteria or viruses, numerous interactions
occur between circulating cells, soluble components of
blood, and the vessel wall to correct the abnormality.
Thus, platelets and the coagulation system form clots
that control blood loss from a wound, and white cells
kill and remove invading foreign organisms. It is becoming increasingly evident that all these functions are well
integrated into synergistic defense mechanisms, which
in turn may become a cause of disease if local conditions trigger an excessive and/or protracted response.
Atherosclerosis, or the formation of lipid-rich plaques
in arterial walls, is a situation in which inflammatory
processes mediated mainly by leukocytes cause chronic
alterations of the vascular surface, to which platelets
may react, precipitating the formation of occlusive
thrombi that curtail blood flow and result in organ
damage. Traditionally, our research has been directed
toward understanding the role of platelets in arresting
hemorrhage and their participation in the pathologic
events that lead to the acute occlusion of atherosclerotic arteries, causing myocardial infarction and stroke.
More recently, we have started to study how inflammatory mediators influence platelet function and the
B
260 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
ability of these cells to form clots and conversely how
platelets may support the defense roles of leukocytes
during infectious diseases and immune-mediated reactions. The common themes underlying these efforts
are the definition of the specific role of different components of the vascular wall, cellular and noncellular,
in initiating platelet responses and the evaluation of
the role of hemodynamic forces caused by the flow of
blood in modulating these functions.
Thrombus formation and platelet interactions with
other vascular cells begin when a transition occurs with
respect to the constituents of the vessel wall with which
platelets are in contact, changing from a healthy endothelial cell monolayer to a reactive cell surface or an
exposed extracellular matrix normally shielded from
directly contacting blood elements. A superficial disruption of endothelial integrity exposes basement membrane components such as proteoglycans, collagen
type IV, nidogen, laminin, and fibulin. Deeper lesions
expose other vascular wall collagens, notably types VI,
III, and I.
For many years, efforts have been devoted to elucidating what components of the extracellular matrix
activate platelet responses leading to the formation of
stable clots. The current concept is that collagens play
a major role in this regard and a synergistic role with
von Willebrand factor in areas of the circulation characterized by rapid blood flow. Two platelet membrane
glycoproteins, the integrin α2β1 (glycoprotein Ia-IIa)
and glycoprotein VI, interact directly with collagen. A
congenital deficiency in these glycoproteins causes a
mild bleeding tendency. To dissect the complex pathways
of collagen-induced platelet activation and aggregation, we generated mice with single or combined deficiencies in glycoprotein VI and α2β1. This research is
being done in collaboration with J. Ware, University of
Arkansas, Little Rock, Arkansas, and T.J. Kunicki,
Department of Molecular and Experimental Medicine.
Initial results indicate that the response of platelets
deficient in collagen receptors to a challenge with purified collagens follows the model known today, namely
collagen-induced platelet activation depends on glycoprotein VI and is further modulated by α2β1. Nevertheless, we also found that challenge with complex
extracellular matrices elicits a response from platelets
that lack either receptor, suggesting that constituents
other than collagens in the vessel wall may play a relevant thrombogenic role. Moreover, the membrane of
cells within the vascular wall may contribute to the
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induction of platelet adhesion and aggregation and of
fibrin deposition when exposed to flowing blood at
sites of vascular injury.
In view of this complexity, a clear challenge for
future studies is to obtain more definitive information
on the thrombogenic properties of cellular surfaces
and extracellular matrices deposited by relevant cells
of the vessel wall, including endothelial cells, fibroblasts,
and smooth muscle cells. As emerging evidence indicates
that multiple vascular and blood elements, all subjected to complex regulatory mechanisms, most likely
are involved in the response of platelets and leukocytes
to vascular lesions, we think that studies in whole
organisms are required for the definitive verification of
mechanistic concepts developed to explain these phenomena. One of our immediate goals, therefore, is to
couple ex vivo experiments with cells and cell products
with in vivo models of vascular injury to understand
precisely the mechanisms that govern platelet interactions with components of the vessel wall and other
blood elements. We think that these studies will provide novel mechanistic information on processes that
are central to normal hemostasis and pathologic arterial thrombosis, thus allowing a better definition and
characterization of potential targets for antithrombotic
intervention that may yield new treatments for patients
at risk for cardiovascular and cerebrovascular events.
In addition, we may discover new pathogenetic mechanisms relevant for the progression of infectious and
inflammatory diseases.
PUBLICATIONS
Baglia, F.A., Shrimpton, C.N., Emsley, J., Kitagawa, K., Ruggeri, Z.M., López,
J.A., Walsh, P.M. Factor XI interacts with the leucine-rich repeats of glycoprotein
Ibα on the activated platelet. J. Biol. Chem. 279:49223, 2004.
Federici, A.B., Canciani, M.T., Forza, I., Mannucci, P.M., Marchese, P., Ware, J.,
Ruggeri, Z.M. A sensitive ristocetin co-factor activity assay with recombinant glycoprotein Ibα for the diagnosis of patients with low von Willebrand factor levels.
Haematologica 89:77, 2004.
Kasirer-Friede, A., Cozzi, M.R., Mazzucato, M., De Marco, L., Ruggeri, Z.M.,
Shattil, S.J. Signaling through GP Ib-IX-V activates αIIbβ3 independently of other
receptors. Blood 103:3403, 2004.
Mazzucato, M., Cozzi, M.R., Pradella, P., Ruggeri, Z.M., De Marco, L. Distinct
roles of ADP receptors in von Willebrand factor-mediated platelet signaling and
activation under flow. Blood 104:3221, 2004.
Mendolicchio, G.L., Ruggeri, Z.M. New perspectives on von Willebrand factor
functions in hemostasis and thrombosis. Semin. Hematol. 42:5, 2005.
Ruggeri, Z.M. Platelet and von Willebrand factor interactions at the vessel wall.
Hämostaseologie 24:1, 2004.
Ruggeri, Z.M. Type IIB von Willebrand disease: a paradox explains how von Willebrand factor works. J. Thromb. Haemost. 2:2, 2004.
Varughese, K.I., Ruggeri, Z.M., Celikel, R. Platinum-induced space-group transformation in crystals of the platelet glycoprotein Ibα N-terminal domain. Acta Crystallogr. D Biol. Crystallogr. 60:405, 2004.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Vasudevan, S., Celikel, R., Ruggeri, Z.M., Varughese, K.I., Kunicki, T.J. A single
amino acid change in the binding pocket alters specificity of an anti-integrin antibody
AP7.4 as revealed by its crystal structure. Blood Cells Mol. Dis. 32:176, 2004.
Role of HLA-G in the Regulation
of Allogeneic Immune Responses
L. Crisa, R. Prinsen, V. Cirulli,* B.E. Torbett
* University of California, San Diego, California
LA-G is a nonpolymorphic class Ib HLA molecule that may mediate immunologic tolerance
at sites of immune privilege, such as the anterior chamber of the eye, the testis, the thymus, and the
cytotrophoblast. Several explanations of the immunoregulatory functions of HLA-G have been considered. The
limited polymorphism of HLA-G in humans may allow
the recognition of tissues expressing high levels of this
molecule as “self,” thereby preventing the activation of
autoreactive or alloreactive T cells and natural killer
cells. Alternatively, HLA-G may foster the development
of specific immunoregulatory lymphocytes capable of
downregulating alloreactivity. Our previous finding that
HLA-G is expressed in the thymic medullary epithelium
in humans strongly supports both of these possibilities.
Thus, the purpose of HLA-G expression in the thymic
medulla may be both to educate developing T cells to
recognize HLA-G as self and to induce the selection of
HLA-G–specific immunoregulatory T-cell populations.
We focus on the immune responses elicited by
HLA-G in human thymocytes and peripheral T cells. Our
goals are to dissect the molecular mechanisms of HLA-G
immune functions and then use this information to
bioengineer HLA-G expression in tissues suitable for
transplantation. Particular emphasis is given to models of
pancreatic islet transplantation for the treatment of
diabetes. For this purpose, we have generated lines of
human pancreatic cells that express either low or high levels of membrane-bound or soluble recombinant HLA-G.
These HLA-Glow and HLA-Ghigh human pancreatic cell
lines are useful tools for studies of HLA-G functions both
in vitro and in vivo in models of cell transplantation.
Another promising line of research for the bioengineering of cells for transplantation was provided by our
work on the identification of endothelial cell progenitors in human cord blood. While studying human thymopoiesis in a chimeric mouse model in which mice
are reconstituted with human cord blood, we discovered that cord blood hemopoietic stem cells engrafted
H
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261
in these mice not only reconstituted the bone marrow
and repopulated the human thymic grafts but also
contributed to the formation of new blood vessels at
sites of wound healing.
We are characterizing this population of putative
endothelial progenitors to be used as another target
cell type for transplantation. Specifically, we have defined
some of the growth and differentiation signals required
for the expansion ex vivo of human bone marrow–derived
endothelial progenitors. Currently, using a mouse model
of bone marrow–derived vasculogenesis, we are characterizing the immunologic and angiogenic properties
of bone marrow–derived endothelium. Ultimately, cotransplantation of HLA-G–transduced allogeneic tissue with
endothelial cell progenitors to express HLA-G and/or
enhancing recruitment of bone marrow–derived endothelium with intrinsic immunomodulatory properties
may endow tissue grafts with an additional level of
immunoprotection. This approach may be useful in the
development of novel strategies for the induction of
immunologic tolerance and/or avoidance of rejection
after transplantation.
Cerebral Microvessel-Neuron
Responses to Ischemia
G.J. del Zoppo, S. Fukuda, R. Milner, J. Hallenbeck,* E. Lo**
* National Institutes of Health, Bethesda, Maryland
** Massachusetts General Hospital, Boston, Massachusetts
troke is a vascular disorder with neurologic consequences and an enormous social impact.
Understanding the interactions between neurons
and their supply microvessels can provide insight into
communication and control of neuronal activation and
coordinate responses to local injury (e.g., ischemia).
Continuing studies of the relationships between microvessels and neurons (the neurovascular unit) during focal
brain ischemia indicate that alterations in the vasculature are distinctly related to neuronal injury within the
early moments of the ischemic injury and that preservation of the normal relationships is time dependent.
We hypothesized that alterations in the intercellular
matrix of cerebral microvessels by active proteases are
reflected by injury to neighboring neurons and the extracellular matrix of the neurons. The results of our studies
have supported the concept of the neurovascular unit.
In vivo observations extended our original findings
that heparan sulfate proteoglycans (perlecan) in the
S
262 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
lamina in the cerebral vasculature are more sensitive
than are laminins, collagen IV, and cellular fibronectin
after focal ischemia. In vitro studies confirmed that
heparan sulfate proteoglycans are the most sensitive to
the protease activities generated during focal ischemia.
In studies on the contributions of cathepsin L to
perlecan degradation, we found that focal degradation
of the matrix ligand in cerebral microvessels in the
ischemic core and generation of cathepsin L by nearby
neurons with signs of injury occur within 2 hours of
the onset of ischemia. Heparanase, which can cleave
heparan sulfate side chains, is generated in the same
early time frame and is expressed by neurons. A detailed
quantitative assessment of the relationship between
cathepsin L–immunoreactive microvessels and neurons
indicated that the microvessel-neuron expression of
cathepsin L is scattered among normal pairs within the
ischemic core. The mechanism of cathepsin L upregulation is unknown. Nonetheless, these findings support
the premise that injury of microvessels and injury of
neurons are linked through rapid degradation of vascular and extravascular matrix.
The effect of matrix proteolysis on the intrinsic permeability of the cerebral microvessels has not been
explored. Two mechanisms are possible, both of which
involve cellular matrix receptors, either certain integrins or dystroglycans: (1) loss of the receptor attachment to complementary matrix ligands, and/or (2) loss
of matrix components. With either mechanism, alterations of endothelial cells in the matrix and the proximity of astrocytes to their endothelium are disturbed,
potentially disrupting endothelial cell-cell lateral contacts and increasing permeability.
To model this condition in vitro, we used different
matrices to prepare primary murine cultures of brain
endothelial cells and astrocytes. Both endothelial cells
and astrocytes express functional matrix adhesion receptors. Endothelial cells also express the tight junction
proteins claudin-5, occludin, and zonula occludens protein-1 at cell-cell borders, whereas astrocytes express
occludin and zonula occludens protein-1, but not
claudin-5. Because focal ischemia in vivo leads to loss
of barrier function and rapid disappearance of matrix
and matrix receptors, an in vitro model of ischemic injury
to endothelial cells and glia was established. The results
revealed that hypoxia and glucose deprivation induced
marked loss of certain integrins and dystroglycan from
astrocytes, but not from endothelial cells. These changes
resemble the changes that occur after ischemia in vivo,
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
thereby establishing a system that can be used to
study the molecular events responsible for these
changes in vivo.
With the availability of both organ and cellular systems, we can now investigate the vascular part of the
neurovascular unit, the integrity of the matrix of endothelial cells and astrocytes, and the signaling mechanisms within the neurovascular unit. These studies may
offer novel therapeutic avenues to reduce the pathologic
changes that occur after ischemic injury in the brain.
PUBLICATIONS
del Zoppo, G.J. Lessons from stroke trials using anti-inflammatory approaches that
have failed. Ernst Schering Res. Found. Workshop 47, 2004, p. 155.
del Zoppo, G.J. Thrombin: maybe not so spellbinding. Neurology 14:768, 2004.
del Zoppo, G.J., Kalafut, M. Mechanisms of thrombosis and thrombolysis. In:
Stroke: Pathophysiology, Diagnosis, and Management, 4th ed. Mohr, J.P., et al.
(Eds.). Churchill Livingstone, New York, 2004, p. 785.
Fukuda, S., Fini, C.A., Mabuchi, T., Koziol, J.A., Eggleston, L.L, Jr., del Zoppo,
G.J. Focal cerebral ischemia induces active proteases that degrade neurovascular
matrix. Stroke 35:998, 2004.
Hamann, G.F., del Zoppo, G.J. Vascular biology of cerebral arteries and microvessels. In: Stroke: Pathophysiology, Diagnosis, and Management, 4th ed. Mohr, J.P.,
et al. (Eds.). Churchill Livingstone, New York, 2004, p. 775.
Smith, C.S., Emsley, H.C.A., Gavin, C.M., Georgiou, R.F., Vail, A., Barberan,
E.M., del Zoppo, G.J., Hallenbeck, J.M., Rothwell, N.J., Tyrrell, P.J., Hopkins,
S.J. Tyrell, P.J. Peak plasma interleukin-6 and other peripheral markers of inflammation in the first week of ischaemic stroke correlate with brain infarct volume,
stroke severity and long-term outcome. BMC Neurol. 4:2, 2004.
Wardlaw, J., Berge, E., del Zoppo, G.J., Yamaguchi, T. Thrombolysis for acute
ischemic stroke. Stroke 35:2914, 2004.
Targeting Metastasis of Human
Solid Tumors
B.F. Felding-Habermann, M.R. Weber, E.I. Chen,
D. O’Sullivan, W. Hassenpflug, J.R. Yates, A. Saven,
K.D. Janda, R.A. Lerner
CONVERGENT EVOLUTION OF ANTIBODIES AND
LIGANDS FOR TUMOR CELL RECEPTORS
otentially, a combinatorial antibody library can
contain an individual’s entire immunologic record.
Such libraries could be used to detect and recover
any antibody ever made by the individual, irrespective
of whether the antibody is currently being produced.
To determine if such “fossil records” can be used to
detect antibodies with disease-fighting potential, we
screened combinatorial antibody libraries from patients
with cancer for immunoglobulins that react with metastatic tumor cells. We detected antibodies specific for
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MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
the activated form of integrin αvβ3, a tumor cell adhesion receptor associated with a metastatic phenotype.
In a remarkable example of convergent evolution,
2 of these antibodies contained an integrin recognition
motif of the natural ligand for the adhesion receptor
within the third complementarity-determining region.
In a mouse model of breast cancer, both of the antibodies interfered with lung colonization by cancer cells
and inhibited existing metastatic disease (Fig. 1). The
results suggest that at some time these antibodies were
part of a patient’s surveillance system against metastatic
cells, targeting αvβ3 and disrupting the functions of
the receptor.
263
from the primary tumor, entry into the circulation, arrest
within the vascular bed of distant organs, and invasion
and proliferation in these new environments. We found
that the high-affinity form of integrin αvβ3 supports specific arrest of circulating tumor cells in blood vessels of
distant target organs and promotes tumor cell extravasation and proliferation within the new environment.
These steps lead to successful establishment of clinically relevant metastatic disease. Strikingly, the activated form of αvβ3 as a key molecule is also a specific
target for host immune defense, which leads to the
production of antibodies against tumor metastases.
C O N V E R G E N T E V O L U T I O N O F M E TA B O L I C
PROPERTIES IN BRAIN CELLS AND TUMOR CELLS
T H AT S P R E A D T O T H E B R A I N
F i g . 1 . Interference with established breast cancer metastases
by the antibody Bc-15, isolated from the combinatorial antibody
library of a patient with cancer. Bc-15 is specific for the activated
form of αvβ3 and mimics natural ligands of the integrin. Mutating
the ligand-receptor recognition motif within the antibody creates a
nonfunctional antibody, Mut-15, that does not interfere with metastasis. Immunodeficient mice were injected with human breast cancer
cells, which established metastases in the lungs. At this advanced
stage, the mice were treated with Bc-15 or Mut-15, and the lungs
were analyzed for metastases 18 days later. Left, Lung metastases
after treatment with Bc-15 or Mut-15. Right, Number of metastatic
foci in the lung tissue of the mice.
The ligand-mimetic nature of these antibodies and
their specificity for a single receptor are unique characteristics. Thus, the convergent evolution of critical
sequences in antibodies and other ligands that bind to
the same target means that the immune response can
find a best chemical solution for optimizing binding
energy and exclusive specificity, even though antibodies evolve in real time whereas evolution of the natural
ligand requires billions of years. This powerful property
of antibodies can be exploited for early diagnosis and
treatment of metastatic disease.
R O L E O F αvβ3 I N M E TA S TAT I C P R O G R E S S I O N
Metastasis depends on specific adhesive, migratory,
and invasive properties of the tumor cells for escape
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
We discovered another striking example of convergent evolution when we analyzed protein expression
profiles of tumor cells that had successfully established
metastases in distinct target organs. Comparing tumor
cells isolated from a patient with breast cancer with
variants of the cells derived from metastases to the brain
or bone in our mouse model, we found that tumor cells
that colonized the brain had adapted to the unique energy
demand for glucose in the brain. These tumor cells
required high concentrations of glucose and processed
glucose in a way similar to that used by brain cells. The
energy metabolism of these tumor cells clearly differed
from that of the circulating tumor cells and from that
of cancer cells that had settled in the bone environment.
These distinct properties of metastatic tumor cells and
their different sensitivities to antitumor drugs associated
with metabolic adaptation to high- vs low-glucose environments may be useful in developing therapeutic regimens that target tumor metastases in the brain.
PUBLICATIONS
Chen, E.I., Florens, L., Axelrod, F.T., Monosov, E., Barbas, C.F. III, Yates, J.R. III,
Felding-Habermann, B., Smith, J.W. Maspin alters the carcinoma proteome.
FASEB J. 19:1123, 2005.
Felding-Habermann, B., Lerner, R.A., Lillo, A., Zhuang, S., Weber, M.R., Arrues, S.,
Gao, C., Mao, S., Saven, A., Janda, K.D. Combinatorial antibody libraries from cancer
patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit
breast cancer metastasis. Proc. Natl. Acad. Sci. U. S. A. 101:17210, 2004.
Lillo, A.M., Sun, C., Gao, C., Ditzel, H., Parrish, J., Gauss, C.M., Moss, J., Felding-Habermann, B., Wirshing, P., Boger, D.L., Janda, K.D. A human single-chain
antibody specific for integrin α3β1 capable of cell internalization and delivery of
antitumor agents. Chem. Biol. 11:897, 2004.
264 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
The Antithrombotic,
Anti-inflammatory, and
Antiapoptotic Protein C Pathway
J.H. Griffin, B.N. Bouma, J. Contreras,* H. Deguchi,
D. Elias, F. Espana,** J.A. Fernández, A.J. Gale, L. Mosnier,
N. Pecheniuk, X. Xu, X. Yang, S. Yegneswaran,
B.V. Zlokovic***
* University of Alabama Medical Center, Birmingham, Alabama
** Universitat de València, València, Spain
*** University of Rochester, Rochester, New York
arious host defense systems act in concert in
normal physiology. Coagulation pathways, fibrinolysis pathways, and anticoagulant mechanisms
prevent bleeding while avoiding harmful blood clots.
The protein C pathway provides antithrombotic, antiinflammatory and antiapoptotic activities and is a focus
of our research.
V
ANTIAPOPTOTIC AND CYTOPROTECTIVE EFFECTS
O F A C T I VAT E D P R O T E I N C
The antiapoptotic activity of activated protein C
(APC), first described in 2001, may provide cytoprotective activity that reduces cell death after a variety
of cellular injuries. Recombinant APC, a well-defined
anticoagulant enzyme, reduced mortality in patients
with severe sepsis in a phase 3 trial. However, 2 potent
anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, did not, suggesting the
physiologic relevance of APC’s less well-defined antiinflammatory and antiapoptotic activities.
Therapy with recombinant APC is associated with
an increased risk of serious bleeding complications
because of the anticoagulant activity of the enzyme. To
generate recombinant APC variants that have reduced
anticoagulant activity and thus are less likely to cause
bleeding, we dissected APC’s anticoagulant activity
from its cytoprotective activity by using site-directed
mutagenesis. Using staurosporine-induced endothelial
cell apoptosis assays, we found that mutations to alanine in 2 APC surface loops that severely reduced
anticoagulant activity resulted in 2 APC variants that
retained normal antiapoptotic activity that requires
protease activated receptor-1 and endothelial cell protein C receptor.
Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
such APC variants may reduce serious risks for bleeding while providing the beneficial effects of APC acting
directly on cells.
Remarkably, new studies show that treatment with
APC may improve transplantation of pancreatic islets
in patients with diabetes. Clinical investigations indicate that significant loss of functional islet mass occurs
in the peritransplant period. Islets are injured as a
result of detrimental effects of brain death, pancreas
preservation, islet isolation, hypoxia, hyperglycemia,
and immune-mediated events. In addition, islets are
injured by exposure to blood and by activation of intrahepatic endothelial and Kupffer cells, resulting in inflammation and thrombosis.
In collaborations with J. Contreras and colleagues
at the University of Alabama Medical Center, we found
that administration of recombinant murine APC significantly reduced loss of functional islet mass after intraportal transplantation of islets in diabetic mice. Compared
with control animals, animals given APC had better
glucose control, higher glucose disposal rates, and higher
arginine-stimulated acute release of insulin. These
effects were associated with reduced levels of plasma
proinsulin, intrahepatic fibrin deposition, and islet
apoptosis early after the transplant. In vitro and in
vivo data indicated that APC treatment was associated
with a significant reduction in the release of proinflammatory cytokines after exposure of hepatic endothelial
cells to islets. APC treatment also prevented activation
and dysfunction of endothelial cells elicited by intrahepatic embolization of isolated islets associated with
transplantation of pancreatic islets. These results show
multiple remarkable beneficial effects of APC in transplantation of pancreatic islets and suggest that APC
therapy may enhance the therapeutic efficacy of such
transplantation in patients with diabetes.
NEUROPROTECTIVE ACTIVITIES OF APC
Stroke is a major cause of morbidity and mortality.
In collaboration with B. Zlokovic and colleagues, University of Rochester, we used human brain endothelium
in vitro and murine in vivo stroke models to study the
neuroprotective activities of the protein C pathway.
Previously, we showed that intravenous infusions of
recombinant APC reduced the size of brain infarctions
and brain edema induced by ischemia. Although the
thrombolytic effects of tissue plasminogen activator
(tPA) are beneficial, its neurotoxic effects are a problem. In recent studies, we found that tPA potentiates
apoptosis in ischemic human brain endothelium and
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
in mouse cortical neurons treated with N-methyl- D aspartate by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3
without amplification through the Bid-mediated mitochondrial pathway. APC promoted activation of antiapoptotic mechanisms in brain cells by acting directly
on endothelium and neurons and blocked the vascular
and neuronal toxic effects of tPA in vitro and in vivo.
APC inhibited tPA-induced caspase-8 activation of
caspase-3 in endothelium and caspase-3-dependent
nuclear translocation of apoptosis-inducing factor in
neurons treated with N-methyl-D-aspartate and reduced
tPA-mediated cerebral ischemic injury in mice. Data
suggest that tPA shifts the apoptotic signal in stressed
brain cells from the intrinsic to the extrinsic pathway,
which requires caspase-8, and that APC blocks the
neurovascular toxic effects of tPA. Thus, we speculate
that APC may add substantially to the effectiveness of
tPA therapy for stroke in humans.
I N F L U E N C E O F L I P I D S O N B L O O D C O A G U L AT I O N
Lipid-containing surfaces, including cell membranes
and lipoproteins, provide sites where procoagulant and
anticoagulant enzymes, cofactors, and substrates can
be assembled to express their activities. Although dyslipoproteinemia is associated with arterial atherothrombosis, little is known about plasma lipoproteins
in patients with venous thrombosis. We used nuclear
magnetic resonance spectroscopy and antigenic levels
of apolipoproteins AI and B to determine the concentrations of subclasses of lipoproteins in blood samples
from 49 men less than 55 years old who had venous
thrombosis and from matched control subjects. Patients
with venous thrombosis had significantly lower levels
of high-density lipoprotein (HDL) particles, large HDL
particles, HDL-cholesterol, and apolipoprotein AI and
significantly higher levels of low-density lipoprotein
(LDL) particles and small LDL particles. The quartilebased odds ratio for decreased levels of HDL particles
and apolipoprotein AI was 6.5 for the patients and 6.0
for the control subjects. When polymorphisms in genes
for hepatic lipase, endothelial lipase, and cholesteryl
ester transfer protein were analyzed, patients differed
significantly from control subjects in the allelic frequency for polymorphisms in cholesteryl ester transfer
protein, consistent with the observed pattern of lower
HDL and higher LDL.
Thus, for the first time, we showed that venous
thrombosis in men is associated with dyslipoproteinemia involving lower levels of HDL particles, elevated
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
265
levels of small LDL particles, and an elevated ratio of
apolipoprotein B to apolipoprotein AI. Moreover, this
dyslipoproteinemia is associated with related differences
in the genotype for cholesteryl ester transfer protein.
Overall, we speculate that procoagulant and anticoagulant lipids and lipoproteins may contribute to a yin-yang
balance that influences the upregulation and downregulation of thrombin generation and that alterations
of this lipid balance may alter the hemostatic balance.
Antithrombotic Mechanisms
M.J. Heeb, B.N. Bouma, K.M.S. Cabral, L. Tonnu
lasma proteins that regulate blood coagulation
can prevent thrombosis, a factor in half of deaths
in the United States. One such protein, the serpin protein Z–dependent protease inhibitor (ZPI), requires
protein Z, negatively charged phospholipids, and calcium
to inhibit procoagulant factor Xa. Possible explanations
for the unusual phospholipid requirement for inhibition
of factor Xa by ZPI are (1) a phospholipid surface is
necessary to colocalize ZPI, protein Z, and factor Xa
and (2) phospholipid is necessary to promote a conformational change in at least one of these molecules.
We found that ZPI also inhibited factor IXa, independently of protein Z. We tested the 2 possible explanations for ZPI inhibition of factor Xa, and we determined
whether phospholipids and calcium are required for ZPI
inhibition of factor IXa. We compared the ability of
phospholipid vesicles and soluble phospholipids to
promote inhibition of factor Xa by ZPI and to promote
the formation of complexes consisting of factor IXa
and ZPI. We used surface plasmon resonance and
activity measurements to determine the phospholipid
and calcium requirements for interactions between
factor IXa and ZPI.
ZPI and protein Z inhibited factor Xa with equal
efficiency in the presence of either soluble phospholipids
or phospholipid vesicles. Formation of complexes consisting of factor Xa and ZPI was promoted by either
phospholipid species. In either case, 2 types of complexes were detected by using antibodies to factor Xa,
but only 1 type was detected by using antibodies to
denatured ZPI. Thus, some factor Xa–ZPI complexes
may be noncovalent, whereas some are covalent. Accordingly, only low levels of stable covalent complexes were
detected. Calcium, but not protein Z or phospholipids,
was required for inhibition of factor IXa by ZPI and for
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266 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
association of factor IXa with ZPI. Thus, phospholipid
most likely is required for inhibition of factor Xa by
ZPI because the phospholipid promotes a favorable
conformation of factor Xa, protein Z, and/or ZPI. In
contrast, ZPI and factor IXa interact independently of
phospholipids and protein Z.
Another anticoagulant protein, protein S, is an essential antithrombotic known as a cofactor for activated
protein C in the inactivation of the procoagulants factor Va and factor VIIIa. We showed that protein S has
direct anticoagulant activity independent of activated
protein C: it directly binds to and inhibits factors Va,
VIIIa, and Xa and competes with procoagulant proteins
for negatively charged phospholipids.
Purified protein S has multimeric forms, and reports
indicated that only multimeric purified protein S had a
direct anticoagulant effect and that plasma contains
only monomeric protein S. Because the protein S in
plasma does have a direct anticoagulant effect, we independently examined whether multimers of protein S exist
in plasma and whether monomers and multimers of
protein S can have similar direct anticoagulant effects.
Immunoblotting revealed the presence of protein S
multimers in citrated plasma that had a direct anticoagulant effect and in fractions of gel-filtered citrated or
hirudinated plasma. Biotin-labeled factor Xa reacted with
the plasma protein S monomers and dimers. Multimers
in plasma were confirmed by using an enzyme-linked
immunosorbent assay with the same monoclonal antibody as coating and detecting antibody. Affinity-purified
protein S was separated into monomers and multimers.
The monomers and several multimers of protein S had
similar specific direct anticoagulant effects and a similar ability to compete with procoagulant factors for
limiting phospholipids. Contrary to one report, purified
protein S monomers, dimers, trimers, and higher forms
were clearly separated by using analytic ultracentrifugation, and multimers remained intact in the presence
or absence of calcium.
Interestingly, recombinant protein S in conditioned
medium was more than 95% monomeric but had direct
anticogulant effects similar to those of both protein S
in plasma and multimeric purified protein S. Multimers
of recombinant protein S were not induced by treatment
with EDTA or pH 2.5 but were induced by chromatography in the presence of EDTA. Thus, formation of protein S multimers may be concentration dependent.
It appears that plasma contains both monomers
and multimers of protein S and that the direct anticoPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
agulant effect of protein S in plasma is similar to that
of affinity-purified monomeric and multimeric protein S
and similar to that of unpurified monomeric recombinant protein S.
Structure and Function of
Coagulation Cofactors
A.J. Gale, D. Rozenshteyn, J.-L. Pellequer,* X. Xu
*CEA/DSV/DIEP, Bagnols ser Cèze, France
oagulation factors Va and VIIIa are highly homologous cofactors of the serine proteases factor Xa
and factor IXa, respectively. These cofactors are
activated by thrombin as positive feedback in the coagulation pathway, and they are the primary targets of activated
protein C (APC) in its downregulation of the procoagulant
pathway. In collaboration with J.-L. Pellequer in France,
we used homology modeling techniques to model the
complex 3-dimensional structures of these multidomain proteins. These models were used to engineer
disulfide bonds between domains in both factor Va
and factor VIIIa. In factor Va, the disulfide bond facilitated investigation of the mechanisms of inactivation of
factor Va by APC cleavage. APC cleavages in factor Va
result in loss of factor Va’s affinity for factor Xa and
loss of catalytic efficiency of the resulting prothrombinase complex.
Factor VIIIa is inactivated by 2 mechanisms. Thrombin activation of factor VIII results in a heterotrimer
that consists of the A1 subunit, the A2 subunit, and
the light chain. Spontaneous dissociation of the A2
subunit and proteolytic cleavage of factor VIIIa by APC
both effectively inactivate the factor. Hemophilia A, a
deficiency of factor VIII, is treated by infusions of purified recombinant factor VIII. But the usefulness of factor VIII is limited; after activation by thrombin, it is
unstable as a result of spontaneous dissociation of the
A2 subunit.
We generated 2 factor VIII mutants in which 2 newly
introduced cysteine residues form a de novo disulfide
bridge that cross-links the A2 and A3 domains. One
mutant cross-links the domain between residues 662
(A2) and 1828 (A3); the other between residues 664
(A2) and 1826 (A3). These interdomain disulfides
were designed to prevent the spontaneous dissociation
of the A2 subunit, thereby inhibiting one of the inactivation pathways for factor VIIIa. Both mutants were
C
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
biologically active, and Western blots showed the band
patterns predicted for the introduction of a new disulfide bridge. Moreover, both mutants were highly stable
after thrombin activation. The results of surface plasmon resonance confirmed that dissociation of the A2
subunit was prevented in the mutants. However, the
engineered disulfide bonds did not prevent the inactivation of factor VIIIa by proteolysis by APC or other
proteases. Currently, we are investigating the function
of these stabilized variants in vivo in mice.
Disulfide–cross-linked factor V provides a unique
tool for studies of the cofactor activity of factor V, and
disulfide–cross-linked mutants of factor VIII provide
unique tools for studies of the cofactor activity of factor VIII and potential new therapies for hemophilia A.
PUBLICATIONS
Bouma, B.N., Mosnier, L.O. Thrombin activatable fibrinolysis inhibitor (TAFI) at
the interface between coagulation and fibrinolysis. Pathophysiol. Haemost.
Thromb. 33:375, 2004.
Contreras, J.L., Eckstein, C., Smyth, C.A., Bilbao, G., Vilatoba, M., Ringland,
S.E., Young, C., Thompson, J.A., Fernández, J.A., Griffin, J.H., Eckhoff, D.E.
Activated protein C preserves functional islet mass after intraportal transplantation:
a novel link between endothelial cell activation, thrombosis, inflammation, and islet
cell death. Diabetes 53:2804, 2004.
Daniels, T., Zhang, J., Gutierrez, I., Elliot, M., Yamada, B., Heeb, M.J., Sheets,
S.M., Wu, X., Casiano, C.A. Antinuclear autoantibodies in prostate cancer: immunity to LEDGF/p75, a survival protein highly expressed in prostate tumors and
cleaved during apoptosis. Prostate 62:14, 2005.
Deguchi, H., Bouma, B.N., Middeldorp, S., Lee, Y.M., Griffin, J.H. Decreased
plasma sensitivity to activated protein C by oral contraceptives is associated with
decreases in plasma glucosylceramide. J. Thromb. Haemost. 3:935, 2005.
Deguchi, H., Pecheniuk, N.M., Elias, D.J., Averell, P.M., Griffin, J.H. High-density
lipoprotein deficiency and dyslipoproteinemia associated with venous thrombosis in
men. Circulation 112:893, 2005.
Griffin, J.H. Regulation of coagulation. In: Williams Hematology, 7th ed. Beutler, E.,
et al. (Eds.). McGraw-Hill, New York, in press.
Hall, M.O., Obin, M.S., Heeb, M.J., Burgess, B.L., Abrams, T.A. Both protein S
and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells. Exp. Eye Res., in press.
Heeb, M.J., Cabral, K.M., Ruan, L. Down-regulation of factor IXa in the factor Xase
complex by protein Z-dependent protease inhibitor. J. Biol. Chem. 280:33819, 2005.
Leiba, M., Seligsohn, U., Sidi, Y., Harats, D., Sela, B.A., Griffin, J.H., Livneh, A.,
Rosenberg, N., Gelernter, I., Gur, H., Ehrenfeld, M. Thrombophilic factors are not the
leading cause of thrombosis in Behçet’s disease. Ann. Rheum. Dis. 63:1445, 2004.
Liu, D., Cheng, T., Guo, H., Fernández, J.A., Griffin, J.H., Song, X., Zlokovic, B.V.
Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat. Med. 10:1379, 2004.
Marx, P.F., Havik, S.R., Bouma, B.N., Meijers, J.C.M. Role of isoleucine residues
182 and 183 in thrombin-activatable fibrinolysis inhibitor. J. Thromb. Haemost.
3:1293, 2005.
Mosnier, L.O., Gale, A.J., Yegneswaran, S., Griffin, J.H. Activated protein C variants
with normal cytoprotective but reduced anticoagulant activity. Blood 104:1740, 2004.
Pecheniuk, N.M., Deguchi, H., Griffin, J.H.. Cholesterol enhances phospholipid- dependent activated protein C anticoagulant activity. J. Thromb. Haemost. 3:340, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
267
Seligsohn, U., Griffin, J.H. Hereditary thrombophilia. In: Williams Hematology, 7th ed.
Beutler, E., et al. (Eds.). McGraw-Hill, New York, in press.
Shi, F.-D., Zhang, J.-Y., Liu, D., Rearden, A., Elliot, M., Nachtsheim, D., Daniels, T.,
Casiano, C.A., Heeb, M.J., Chan, E.K.L., Tan, E.M. Preferential humoral response
in prostate cancer to cellular proteins p90 and p62 in a panel of tumor-associated
antigens. Prostate 63:252, 2005.
van Golde, P.H., van der Westelaken, M., Bouma, B.N., van de Wiel, A. Characteristics of piraltin, a polyphenol concentrate, produced by freeze-drying of red
wine. Life Sci. 74:1159, 2004.
Yegneswaran, S., Mesters, R.M., Fernández, J.A., Griffin, J.H. Prothrombin residues 473-487 contribute to factor Va binding in the prothrombinase complex. J.
Biol. Chem. 279:49019, 2004.
Thrombopoietin Initiates
Demethylation-Based
Transcription of GP6 During
Megakaryocyte Differentiation
Reprinted from Blood, Vol. 105, Kanaji, S., Kanaji, T.,
Jacquelin, B., Chang, M., Nugent, D.J., Komatsu, N.,
Moroi, M., Izuhara, K., Kunicki, T.J., pp. 3888-3892.
Copyright 2005, with permission from the American Society
of Hematology.
lycoprotein VI (GPVI) is an essential platelet
receptor for collagens that is exclusively expressed
in the megakaryocytic lineage. Transcription of
the human gene GP6 is driven largely by GATA-binding
protein 1 (GATA-1), specificity protein 1 (Sp1), and
Friend leukemia integration 1 (Fli-1). In this report,
we show that GPVI expression during megakaryocytic
differentiation is dependent on cytosine-phosphate-guanosine (CpG) demethylation that can be initiated by
thrombopoietin (TPO). Sodium bisulfite genomic
sequencing established that a CpG-rich island within
the GP6 promoter region is fully methylated at 10 CpG
sites in GPVI-nonexpressive cell lines, such as UT-7/EPO
and C8161, but completely unmethylated in GPVI-expressive cell lines, including UT-7/TPO and CHRF288-11. To
further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated
human cord blood cells with TPO. The GP6 promoter
is highly methylated in cord blood mononuclear cells
(progenitors) but not in CD41+-enriched cells obtained
after TPO differentiation. Furthermore, when UT-7/EPOMpl cells, which stably express human C-myeloproliferative leukemia virus ligand (c-Mpl), were treated
with TPO, demethylation of the GP6 promoter was
induced. In every case, demethylation of the GP6
G
268 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
promoter correlated with an increase in mRNA level.
Thus, megakaryocyte-specific expression of the GP6
gene is regulated, in part, by CpG demethylation, which
can be directly initiated by TPO.
An Association of Candidate Gene
Haplotypes and Bleeding Severity
in von Willebrand Disease (VWD)
Type 1 Pedigrees
association was observed with 6 other candidate genes,
GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased
plasma VWF:Ag levels were associated with VWF haplotype 1 (–1793G; P = .02). These results establish
that genetic differences in the adhesion receptor subunits α2, αIIb, and GPVI can influence the phenotype
of VWD type 1.
Functional Genomics in Organ
Transplantation and Islet Cell
Xenotransplantation
Reprinted in part from Blood, Vol. 104, Kunicki, T.J.,
D.R. Salomon, S.M. Kurian, S. Cherqui, U. Srinivasan,
Federici, A.B., Salomon, D.R., Koziol, J.A., Head, S.R.,
Y. Martina, K. Marcucci, D. Valente, H. Ospina, F. Jaramillo,
Mondala, T.S., Chismar, J.D., Baronciani, L., Canciani, M.T.,
D. Campbell, T.J. Kunicki, C. Marsh, S. Head,* C. Lanigan,*
Peake, I.R., pp. 2359-2367. Copyright 2004, with
J. Yates.** J. Hewel,** P.Y. Kwok,*** J. Warrington,****
permission from the American Society of Hematology.
S. Horvath,***** J. Papp,***** C.A. Wilson,†
n arterial flow, the initial transient arrest of platelets on collagen requires von Willebrand factor (VWF)
acting as a molecular bridge between collagen and
the glycoprotein Ib (GPIb) complex. Next, the direct
binding of platelet α2β1 and GPVI to collagen anchors
the platelet more firmly to the matrix and facilitates
signal transduction that leads to an activated, procoagulant platelet monolayer. Genetic variation in any of
the responsible receptors might have an effect on platelet function in vivo, particularly in mild forms of VWD
type 1, which accounts for at least 60% of all cases
of VWD.
VWD type 1 is difficult to diagnose because of bleeding variability and low heritability of VWF levels. We
compared a bleeding severity score and bleeding times
to candidate gene haplotypes within pedigrees of 14
index cases, using a covariance components model for
multivariate traits (Mendel: QTL Association). These
pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo)
levels. The bleeding severity score was derived from a
detailed history. Donors were genotyped using a primer
extension method, and 9 candidate genes were selected
for analysis. VWF:RCo levels had the strongest influence
on bleeding severity score and bleeding time. ITGA2
haplotype 2 (807C) and ITGA2B haplotype 1 (Ile843)
were each associated with increased bleeding severity
scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro 219) was also associated with increased
scores (P = .03) after adjustment for donor age. No
I
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
H. Hartounian,†† R. Bartel††
* DNA Microarray Core, Scripps Research
** Department of Cell Biology, Scripps Research
*** University of California, San Francisco, California
**** Affymetrix, Santa Clara, California
***** University of California, Los Angeles, California
†
Food and Drug Administration, Bethesda, Maryland
††
MicroIslets, La Jolla, California
uccessful transplantation requires the orchestration of complex mechanisms set in motion by
surgical implantation of cells or organs into a
patient. Regulation of the immune response with immunosuppressive drugs has received the most attention. But
equally important is the unique cell biology of the
transplanted tissue that evolves under stress after
transplantation and ultimately determines the function
of the transplant.
One challenge, termed functional genomics, is to
understand the expression and function of genes and
proteins after transplantation. How do immunosuppressive drugs work at this fundamental level? What is the
difference between a successful and an unsuccessful
transplant? Another challenge is to develop an unlimited supply of healthy tissue for transplantation, for
example, pancreatic islet cells to cure diabetes. Animals could be used as donors, called xenotransplantation, although the potential risks for infectious disease
inherent in using animal donors need to be better understood so that this method can be used safely. One strategy would be to create technologies to protect cell
S
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
transplants from rejection and optimize the cells’ function. Delivering therapeutic molecules to the transplanted
tissue could enhance the success of engraftment and function. Finally, progenitor cells could be used to enhance
the formation of new blood vessels, called angiogenesis. Revascularization of cell transplants is a critical
step in successful engraftment and function.
FUNCTIONAL GENOMICS IN ORGAN
269
that multiple tissues become infected with pig endogenous retrovirus, identified the human receptors for this
retrovirus, identified functional defects in nonhuman
primate cells for viral entry and assembly (Fig. 1), and
continued to refine our understanding of the viral biology and potential risks. We think these studies are a
necessary complement to our work in safely advancing
clinical islet xenotransplantation.
T R A N S P L A N TAT I O N
We are using high-density gene chip arrays, tandem
mass spectrometry proteomics, and complex trait genetics based on single nucleotide polymorphisms to establish profiles to diagnose acute and chronic transplant
rejection. These studies include patients with both
kidney and liver transplants. A major objective of these
efforts is to identify new pathways that drive the immune
response and cell biology of organ transplants that might
be used as the next generation of targets for therapy.
For example, with all current drug therapies, the target is the patient’s immune response; none target the
transplant itself, even though the function of the transplant is the ultimate determinant of success or failure.
We would like to test the hypothesis that gene expression profiles can be used to create a metric or simple
diagnostic test for adequate immunosuppression. Physicians could then adjust a patient’s drugs on the basis
of an objective measure. Our long-term goal is to identify genes, proteins, and genetic polymorphisms that
determine the outcome of a transplant to create a systems biology–based understanding of clinical transplantation at the molecular level.
F i g . 1 . Multicolor confocal immunomicrographs show expression
and cellular distribution of porcine endogenous retrovirus proteins
(Gag and Env) in human cells that are productively infected (A) and
in nonhuman primate cells that cannot be productively infected (B).
PUBLICATIONS
Flechner, S.M., Kurian, S.M., Head, S.R., Sharp, S.M., Whisenant, T.C., Zhang, J.,
Chismar, J.D., Horvath, S., Mondala, T., Gilmartin, T., Cook, D.J., Kay, S.A., Walker,
J.R., Salomon, D.R. Kidney transplant rejection and tissue injury by gene profiling of
biopsies and peripheral blood lymphocytes. Am. J. Transplant. 4:1475, 2004.
Flechner, S.M., Kurian, S.M., Solez, K., Cook, D.J., Burke, J.T., Rollin, H., Hammond, J.A., Whisenant, T.C., Lanigan, C.M., Head, S.R., Salomon, D.R. De novo
kidney transplantation without use of calcineurin inhibitors preserves renal function
and histology at two years. Am. J. Transplant. 4:1776, 2004.
Hildbrand, P., Cirulli, V., Prinsen, R.C., Smith, K.A., Torbett, B.E., Salomon, D.R.,
Crisa, L. The role of angiopoietins in the development of endothelial cells from cord
blood CD34+ progenitors. Blood 104:2010, 2004.
P I G I S L E T X E N O T R A N S P L A N TAT I O N A N D T H E R I S K
FOR INFECTIOUS DISEASE
We are using novel technology to create a protective
alginate capsule around pig islets to prevent rejection
and modify this capsule with therapeutic molecules to
enhance islet survival and function after transplantation.
Pig insulin works well in humans with diabetes, and
pigs can be genetically engineered and can be available
in great numbers. We are also using our genomics tools
to study how endothelial progenitors, the progenitor cells
for blood vessels, can be included to further advance
the success of cell transplantation, a proof of concept
for composite tissue engineering.
Although xenotransplantation is a logical strategy
to address current shortages of human donor organs,
a critical concern is the potential of moving infections
from the animals to humans. We established a new
mouse model for pig islet xenotransplantation, showed
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Hildbrand, P., Salomon, D.R. Molecular biology of transplantation and xenotransplantation. In: Molecular Basis of Cardiovascular Disease: A Companion to Braunwald’s
Heart Disease, 2nd ed. Chien, K.R. (Ed.). Saunders, Philadelphia, 2004, p. 649.
Kunicki, T.J., Federici, A.B., Salomon, D.R., Koziol, J., Head, S.R., Mondala,
T.S., Chismar, J.D., Baronciani, L., Canciani, M.T., Peake, I.R. An association of
candidate gene haplotypes and bleeding severity in Von Willebrand disease (VWD)
type 1 pedigrees. Blood 104:2359, 2004.
Kunicki, T.J., Head, S., Salomon, D.R. Platelet receptor structures and polymorphisms. Methods Mol. Biol. 273:455, 2004.
Kurian, S.M., Flechner, S.M., Kaouk, J., Modlin, C., Goldfarb, D., Cook, D.J.,
Head, S., Salomon, D.R. Laparoscopic donor nephrectomy gene expression profiling reveals upregulation of stress and ischemia associated genes when compared
to open donor nephrectomies. Transplantation, in press.
Kurian, S.M., Flechner, S.M., Salomon, D.R. Genomics and proteomics in transplantation. Curr. Opin. Organ Transplant. 10:191, 2005.
Martina, Y., Kurian, S., Cherqui, S., Evanoff, G., Wilson, C., Salomon, D.R.
Pseudotyping of infectious porcine endogenous retrovirus species by xenotropic
murine leukemia virus in a pig islet xenotransplantation model. Am. J. Transplant.
5:1837, 2005.
270 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Control of HIV Type 1, Gene
Delivery, and Regulation of
Hematopoietic Development
variants: β and γ. We found that the β variant functions
as a dominant-negative regulator of the originally
reported DMP1 protein. Currently, we are investigating
the molecular and biological roles of the various isoforms
in the development of normal and leukemic cells.
H I V - 1 P R O T E A S E R E S I S TA N C E
B.E. Torbett, K.S. Barnett, M. Borensztein, L. Crisa,
K.M. Fischer, G.E. Foos, P.A. McClintock, R.C. Prinsen,
J.H. Savage, C.H. Swan, M.P. Tschan, J.A. Witkowski,
S. De Rozieres,* J.H. Elder,* Y.-C. Lin,* A.J. Olson*
* Department of Molecular Biology, Scripps Research
ur research interests include normal and abnormal regulation of myeloid development by the
transcription factors PU.1 and cyclin D–interacting Myb-like protein (DMP1), gene delivery strategies
to disrupt cellular entry of HIV type 1 (HIV-1), and the
structural and biochemical evolution of the resistance
of HIV-1 proteases.
O
PU.1 AND MYELOID DEVELOPMENT
PU.1, a member of the ets family of transcription
factors, is expressed solely in hematopoietic cells and
is necessary for directing myeloid development and for
regulating genes required for monocyte/macrophage and
neutrophil function. PU.1 has 3 major domains: the
transactivation, PEST and ets/DNA-binding domains.
PU.1 interacts with transcription factors, and domains
of PU.1 have been implicated in the function of PU.1.
Myeloid development is controlled by temporal gene
expression of PU.1 and interactions among specific transcription factors. We are addressing which PU.1 domains
regulate myeloid lineage–specific commitment, differentiation, and function. To determine which transcription factors interact with PU.1 and direct myeloid development,
we use a mass spectroscopy and proteomics approach
and cells in which the gene for PU.1 is expressed only
under certain conditions. This approach is enabling us to
identify gene programs regulated by PU.1.
D M P 1 , A P O S I T I V E A N D N E G AT I V E R E G U L AT O R O F
THE TUMOR SUPPRESSOR ARF
Cancer often originates from inactivation and/or
deregulation of the control of gene expression. The transcription factor DMP1 positively regulates expression
of human p14ARF and CD13/aminopeptidase N, thus
playing a role in cell-cycle control, differentiation, and
function of hematopoietic and nonhematopoietic cells.
The tumor suppressor ARF is critical for positive regulation of p53, which in turns controls cellular proliferation and modulates apoptosis. We have identified 2
novel and developmentally expressed human DMP1 splice
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
In patients infected with HIV-1, treatment with
inhibitors of HIV reverse transcriptase and protease
suppresses replication of the virus. The current protease inhibitors are competitive substrate inhibitors.
However, HIV-1 variants evolve that escape the approved
drug treatments by developing resistance to the protease inhibitors. A molecular understanding of the
resistance to protease inhibitors is needed so that new
drugs can be developed that inhibit protease-resistant
viruses and are less likely to induce resistant viruses.
In collaboration with J.H. Elder, Department of
Molecular Biology, we showed that a protease inhibitor,
TL-3, inhibits replication of feline immunodeficiency
virus, simian immunodeficiency virus, and HIV-1. Proteases isolated from HIV-1 variants that became resistant
to TL-3 had diminished affinity for TL-3 and were catalytically efficient. Moreover, mutant proteases with changes
in the flap and basal regions, which are regions distinct
from the catalytic site of the enzymes, were broadly
resistant to all current protease inhibitors. Currently, we
are investigating the relationship between structure and
function in our broadly resistant protease mutants and in
wild-type protease to understand how structure contributes to the biochemical basis of resistance.
HIV-1 VECTOR DELIVERY OF CCR5-INTRABODY
G E N E S T O H U M A N H E M AT O P O I E T I C C E L L S
CXCR4 and CCR5 are the main chemokine receptors
for HIV-1 entry into cells, and blocking these receptors
limits entry of the virus. Naturally occurring polymorphisms of the gene for CCR5 indicate that disruption
of the gene provides protection from viruses that use
CCR5 to gain entry. Because polymorphisms are present in healthy persons, the use of genetic intervention
strategies that prevent or limit expression of CCR5 may
provide protection from initial infection and limit spread.
With C.F. Barbas, Department of Molecular Biology, we
showed that intracellular expression of a CCR5-specific
single-chain antibody (intrabody) efficiently disrupted
expression of CCR5 on the T-cell surface and protected
cells from HIV-1 infection.
We have constructed novel HIV-1 vectors to deliver
genes to stem and mature T cells. To provide an additional measure of cellular protection from HIV-1, we
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
also used RNA interference minigenes, which target
chemokine receptors, and ribozymes, which target viral
genes. We found that decreased expression of CCR5
and CXCR4 protected cells against HIV-1 challenge and
imparted a survival advantage in T cells and macrophages derived from stem cells.
PUBLICATIONS
Britschgi, C., Rizzi, M., Grob, T.J., Tschan, M.P., Hugli, B., Reddy, V.A., Andres,
A.-C., Torbett, B.E., Tobler, A., Fey, M.F. Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated
in cancer 1. Oncogene, in press.
de Rozieres, S., Swan, C.H., Sheeter, D.A., Clingerman, K.J., Lin, Y.-C., HuitrónReséndiz, S., Henriksen, S., Torbett, B.E., Elder, J.H. Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3.
Retrovirology 1:38, 2004.
Heaslet, H., Kutilek, V.D., Morris, G., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout,
C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease
NL4-3. J. Mol. Biol., in press.
DIVISION OF
EXPERIMENTAL PATHOLOGY
Francis V. Chisari, M.D., Division Head
Molecular Biology of Hepatitis B
and C Viruses and the Immune
Response to Their Antigens
epatitis B and C viruses are noncytopathic DNA
and RNA viruses that cause acute and chronic
hepatitis and hepatocellular carcinoma. More
than 500 million persons worldwide are chronically
infected with these viruses, and more than 2 million
persons die of these infections every year. In the Division of Experimental Pathology, we focus on discovering the mechanisms responsible for viral clearance and
disease pathogenesis to prevent and cure infections
caused by hepatitis B and C viruses.
H
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
271
Interferon Prevents Formation
of Replication-Competent
Hepatitis B Virus RNAContaining Nucleocapsids
Reprinted from with permission from Proc. Natl. Acad. Sci.
U. S. A., Vol. 102, Wieland S.F., Eustaquio, A., WhittenBauer, C., Boyd, B., Chisari, F.V., pp. 9913-9917. Copyright
2005 National Academy of Sciences, U.S.A.
e have previously shown that IFN-β inhibits
hepatitis B virus (HBV) replication by noncytolytic mechanisms that either destabilize
pregenomic (pg)RNA-containing capsids or prevent
their assembly. Using immortalized murine hepatocyte
cell lines stably transfected with a doxycycline (dox)inducible HBV replication system, we now show that
replication-competent pgRNA-containing capsids are
not produced when the cells are pretreated with IFN-β
before HBV expression is induced with dox. Furthermore, the turnover rate of preformed HBV RNA-containing capsids is not changed in the presence of IFN-β
or IFN-γ under conditions in which further pgRNA synthesis is inhibited by dox removal. In summary, these
results demonstrate that types 1 and 2 IFN activate
hepatocellular mechanism(s) that prevent the formation
of replication-competent HBV capsids and, thereby,
inhibit HBV replication.
W
Dynamics of Hepatitis B Virus
Clearance in Chimpanzees
J.M. Murray, S.F. Wieland, R.H. Purcell,* F.V. Chisari
* National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
e used mathematical modeling to test the
extent to which the effector functions of
cytopathic and noncytopathic T cells contribute to resolution of infection with hepatitis B virus
in 3 acutely infected chimpanzees. Simulations based
exclusively on cytopathic functions indicated a poor fit
to the data and would require the destruction and regeneration of approximately 16 livers for clearance to occur.
In contrast, a scenario based on a combination of cytopathic and noncytopathic functions provided a significantly better fit to the data and would require 8-fold
W
272 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
less destruction to clear the virus from the liver. These
results suggest that noncytopathic mechanisms that
inhibit viral replication and shorten the half-life of covalently closed circular DNA limit the extent to which cytopathic T cells and tissue destruction are required to
terminate acute hepatitis B virus infection.
Oscillating CD8+ T Cell Effector
Functions After Antigen
Recognition in the Liver
Reprinted from Immunity, Vol. 23, Isogawa, M., Furuichi, Y.
Hepatitis C Virus RNA
Replication Is Regulated
by Host Geranylgeranylation
and Fatty Acids
Reprinted with permission from Proc. Natl. Acad. Sci. U. S. A.,
Vol. 102, Kapadia, S.B., Chisari, F.V., pp. 2561-2566.
Copyright 2005 National Academy of Sciences, U.S.A.
epatitis C virus (HCV) infection is a major cause
of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously
demonstrated that high-level HCV replication during
acute infection of chimpanzees is associated with the
modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty
acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we
demonstrate that Huh-7 cells harboring replicating, fulllength HCV RNAs express elevated levels of ATP citrate
lyase and acetyl-CoA synthetase genes, both of which
are involved in cholesterol and fatty acid biosynthesis.
Further, we confirm that the cholesterol-biosynthetic
pathway controls HCV RNA replication by regulating
the cellular levels of geranylgeranyl pyrophosphate, we
demonstrate that the impact of geranylgeranylation
depends on the fatty acid content of the cell, and we
show that fatty acids can either stimulate or inhibit
HCV replication, depending on their degree of saturation.
These results illustrate a complex cellular-regulatory
network that controls HCV RNA replication, presumably by modulating the trafficking and association of
cellular and/or viral proteins with cellular membranes,
suggesting that pharmacologic manipulation of these
pathways may have a therapeutic effect in chronic
HCV infection.
H
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Chisari, F.V., pp. 53-63. Copyright 2005, with permission
from Elsevier.
hen hepatitis B virus (HBV)-specific CD8 +
cytotoxic T lymphocytes (CTLs) are adoptively transferred into HBV transgenic mice,
they enter the liver, recognize antigen, secrete interferon γ (IFNγ), inhibit viral replication, and kill their
target cells, causing hepatitis. In the current study, we
examined the impact of antigen recognition on the
evolution of the activation phenotype, antiviral effector
functions, expansion and contraction kinetics, and
compartmentalization of the transferred CTLs. The
results reveal that noncytolytic and cytolytic effector
functions and expansion-contraction kinetics of the
CTLs are regulated asynchronously and in an oscillatory manner as a consequence of antigen recognition
in the liver and in association with PD-1 upregulation.
We suggest that such oscillations maximize viral clearance and minimize tissue injury during HBV infection
and that poor coordination of these events could lead
to viral persistence and chronic liver disease.
W
Replication of Hepatitis C Virus
Replicons in Mouse Cells
S.L. Uprichard, J. Chung, A. Althage, F.V. Chisari
fforts to understand and combat infection with
hepatitis C virus (HCV) have been hindered by
the lack of good tissue culture replication systems and small-animal models. One major obstacle to
the development of a mouse model of HCV infection is
the restricted host range of the virus, which, so far as
known, replicates solely in humans and primates. To
address this limitation, we are adapting HCV to replicate in mouse cells in vitro.
Recently, we identified an HCV clone that can replicate in mouse cells, and we established mouse cell
lines that constitutively replicate subgenomic and fulllength HCV replicons at high levels. This development
E
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
provides us the opportunity to create an in vivo mouse
model of HCV infection that we can use to assess the
pathologic effects of the virus, to better understand
HCV immunology (e.g., the immunologic and virologic
basis of recovery vs persistence), and test the in vivo
potential of physiologic and pharmacologic agents for
controlling HCV infection.
Role of Platelets in Survival and
Viral Clearance During Acute
Infection With Lymphocytic
Choriomeningitis Virus
M. Iannacone, G, Sitia, M. Isogawa, J.K. Whitmire,*
F.V. Chisari, L.G. Guidotti
* Molecular and Integrative Neurosciences Department, Scripps Research
n earlier studies, we found that platelets have an
unexpected role in the pathogenesis of liver disease
mediated by cytotoxic T lymphocytes (CTLs). To
investigate the contribution of platelets in the control
of an acute viral infection that involves multiple tissues
and organs, we depleted adult immunocompetent mice
of platelets and infected the animals with a noncytopathic
strain of lymphocytic choriomeningitis virus (LCMV).
Surprisingly, a high percentage of platelet-depleted
mice died within 5–7 days of infection, and reconstitution with platelets prevented their death. Decreased
survival also occurred in platelet-depleted mice that
lacked CD8+ cells, suggesting that CTL-dependent
immunopathologic changes did not markedly contribute
to this process. In addition, platelet-depleted mice did
not clear LCMV from peripheral organs (liver, spleen,
and lungs), and death of these animals was associated
with relatively high titers of virus in the brain. Lack of
viral clearance in platelet-depleted mice was also associated with normal CTL priming but reduced accumulation of virus-specific CTLs (whose function was intact)
in the infected organs.
Like our findings on HBV- and adenovirus-specific
CTL responses in the liver, these results are compatible
with the hypothesis that platelets are necessary for
LCMV-specific CTLs to accumulate and control the infection in peripheral organs. This event may help prevent
viral dissemination from the periphery to the brain, a
process that can be fatal in the absence of platelets.
I
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
273
PUBLICATIONS
Bukh, J., Thimme, R., Satterfield, W., Meunier, J.C., Spangenberg, H.C., Forns, X.,
Chang, K.M., Emerson, S.U., Chisari, F.V., Purcell, R.H. Persistence of HCV after
homologous monoclonal rechallenge is associated with emergence of virus variants.
In: Proceedings of the 11th International Symposium on Viral Hepatitis and Liver
Disease. Jilbert, A.R., et al. (Eds.). Australian Centre for Hepatitis Virology, Melbourne, 2004, p. 375.
Chen, M., Sällberg, M., Hughes, J., Jones, J., Guidotti, L.G., Chisari, F.V., Billaud, J.-N., Milich, D.R. Immune tolerance split between hepatitis B virus precore
and core proteins. J. Virol. 79:3016, 2005.
Chen, M.T., Billaud, J.-N., Sällberg, M., Guidotti, L.G., Chisari, F.V., Jones, J.,
Hughes, J. Milich, D.R. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc. Natl. Acad. Sci. U. S. A.
101:14913, 2004.
Chisari, F.V. Unscrambling hepatitis C virus-host interactions. Nature 436:930, 2005.
Chisari, F.V., Thimme, R., Guidotti, L.G., Wieland, S.F., Purcell, R. Mediators of
recovery from HBV infection. In: Proceedings of the 11th International Symposium
on Viral Hepatitis and Liver Disease. Jilbert, A.R., et al. (Eds.). Australian Centre
for Hepatitis Virology, Melbourne, 2004, p. 81.
Guidotti, L.G., Chisari, F.V. Immunobiology and pathogenesis of viral hepatitis. In:
Mechanisms of Disease. Abbas, J.R., Downing, J.R., Kumar, V. (Eds.). Annual
Reviews, Palo Alto, CA, in press. Annual Review of Pathology; Vol. 1.
Iannacone, M., Sitia, G., Isogawa, M., Castor, M., Lowenstein, P., Chisari, F.V.,
Guidotti, L.G. Platelets mediate CTL-induced immunopathology in the liver. Nat.
Med., in press.
Isogawa, M., Kakimi, K., Kamamoto, H., Protzer, U., Chisari, F.V. Differential
dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to
hepatitis B surface antigen. Virology 333:293, 2005.
Kimura, K., Kakimi, K., Wieland, S., Guidotti, L.G., Moriwaki, H., Chisari, F.V.
The role of innate immunity against hepatitis B virus replication in the liver of
transgenic mice. In: Proceedings of the 11th International Symposium on Viral
Hepatitis and Liver disease. Jilbert, A.R., et al. (Eds.). Australian Centre for Hepatitis
Virology, Melbourne, 2004, p. 300.
Murray, J., Wieland, S.F., Chisari, F.V. Dynamics of acute hepatitis B virus infection and clearance in chimpanzees. Proc. Natl. Acad. Sci. U. S. A., in press.
Neumann-Haefelin, C., Blum, H.E., Chisari, F.V., Thimme R. T cell response in
hepatitis C virus infection. J. Clin. Virol. 32:75, 2005.
Robek, M., Boyd, B., Chisari, F.V. Lambda interferon inhibits hepatitis B and C
virus replication. J. Virol. 79:3851, 2005.
Sakai, A., Thimme, R., Spangenberg, H.C., Govindarajan, S., Emerson, S.U.,
Purcell, R.H., Chisari, F.V., Bukh, J. Rapid emergence of virus variants in acute HCV
with vigorous host cellular immune responses and different outcome. In: Proceedings of
the 11th International Symposium on Viral Hepatitis and Liver Disease. Jilbert, A.R., et
al. (Eds.). Australian Centre for Hepatitis Virology, Melbourne, 2004, p. 412.
Sitia, G., Iannacone, M., Chisari, F.V., Guidotti, L.G. Pathogenesis of hepatitis B
virus in transgenic mice. Monogr. Virol. 25:23, 2005.
Thimme, R., Bukh, J., Spangenberg, H., Wieland, S., Blum, H., Purcell, R., Chisari, F. T-cell response to hepatitis B and C virus: lessons learned from the chimpanzee model. Monogr. Virol. 25:66, 2005.
Uprichard, S.L., Boyd, B., Althage, A., Chisari, F.V. Clearance of hepatitis B virus
from the liver of transgenic mice by short hairpin RNAs. Proc. Natl. Acad. Sci.
U. S. A. 102:773, 2005.
Wieland, S.F., Chisari, F.V. Stealth and cunning: hepatitis B and hepatitis C. J.
Virol. 79:9369, 2005.
274 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
D IVISION
OF
H EMATOLOGY
Ernest Beutler, M.D., Division Head
Regulation of Iron Homeostasis
E. Beutler, K. Crain, J. Flanagan, P. Lee, T. Gelbart, C. West,
H. Peng, J. Waalen, L. Wang
he central focus of our research is to understand
human genetic disease. We do this both by studying patients with such diseases and by trying to
understand the underlying physiology in murine systems.
A major part of our effort is directed to the understanding of iron homeostasis. At the clinical level, we
have studied many samples from patients who have a
disease, hereditary hemochromatosis, in which the body
has lost the ability to properly regulate the amount of
iron that is absorbed. The excess iron that accumulates
in macrophages and hepatocytes may produce damage
to the liver, heart, pancreas, and other organs. In the
past year, in patients with hemochromatosis, we identified mutations of the genes that encode ferroportin,
hemojuvelin, HFE, erythroid-specific 5-aminolevulinate
synthase (ALAS2), transferrin receptor-2, and transferrin.
The main regulator of body iron absorption is the
25 amino acid peptide hepcidin. This peptide inhibits
iron absorption and is upregulated in the hepatocytes
of iron-loaded mice and humans. But treating liver cells
with iron in vitro does not upregulate hepcidin. Such
treatment actually diminishes the amount of hepcidin
mRNA in hepatocytes. We are attempting to reconstruct
an in vitro system in which iron stimulates hepcidin
production. Such a system would enable us to dissect
the pathways that sense the level of iron in the body
and send the appropriate regulatory signal to the liver.
Hepcidin is also upregulated by inflammatory stimuli,
and we have shown that this upregulation is mediated
by IL-1 and IL-6 and is independent of HFE and of transferrin receptor 2. We are also attempting to inhibit the
effect of hepcidin on its receptor, ferroportin. Such an
inhibition might be useful both in the treatment of anemia of chronic inflammation, in which hepcidin appears
to play an important role, and in the treatment of iron
storage disease. We performed hydrodynamic transfection
of mice with hepcidin and are attempting to block the
effect of hepcidin with hepcidin analogs or with soluble
fragments of ferroportin, the hepcidin receptor.
T
Published by TSRI Press®. © Copyright 2005,
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The large, well-documented collection of DNA samples that we developed in the course of our study of
the epidemiology of hemochromatosis has continued
to be a valuable resource in studying various aspects
of human biology. It has been known for more than 25
years that people of African ancestry normally have
lower hemoglobin levels in their blood than do whites
or Asians. Africans also have a very high prevalence of
a mild α-thalassemia mutation, consisting of deletion
of 1 of the 2 α-globin loci. We have now been able to
dissect the possible contribution of α-thalassemia to
this racial difference in hemoglobin levels. Our studies
have shown that this mutation does account for a substantial part of the difference, but there are other presumably genetic factors that also play a role.
PUBLICATIONS
Barton, J.C., Felitti, V.J., Lee, P., Beutler, E. Characteristics of HFE C282Y homozygotes younger than age 30 years. Acta Haematol. (Basel) 112:219, 2004.
Beutler, E. The “ascorbate” effect on 2,3-DPG is known to be due to oxalate. Vox
Sang. 86:199, 2004.
Beutler, E. Enzyme replacement in Gaucher disease. PLoS Med. 1:118, 2004.
Beutler, E. Iron absorption in carriers of the C282Y hemochromatosis mutation.
Am. J. Clin. Nutr. 80:799, 2004.
Beutler, E. “Pumping” iron: the proteins. Science 306:2051, 2004.
Beutler, E., Beutler, L., Lee, P.L., Barton, J.C. The mitochondrial NT 16189 polymorphism and hereditary hemochromatosis. Blood Cells Mol. Dis. 33:344, 2004.
Beutler, E., West, C. Hematologic differences between African-Americans and
whites: the roles of iron deficiency and α-thalassemia on hemoglobin levels and
mean corpuscular volume. Blood 106:740, 2005.
Efferth, T., Bächli, E.B., Schwarzl, S.M., Goede, J.S., West, C., Smith, J.C.,
Beutler, E. Glucose-6-phosphate dehydrogenase (G6PD)-deficiency-type Zurich: a
splice site mutation as an uncommon mechanism producing enzyme deficiency.
Blood 104:2608, 2004.
Higgins, T., Beutler, E., Doumas, B.T. Hemoglobin, iron, and bilirubin. In: Tietz
Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. Burtis, C.A.,
Ashwood, E.R., Bruns, D.E. (Eds.). Saunders, Philadelphia, 2005, p. 1165.
Knisely, A.S., Gelbart, T., Beutler, E. Molecular characterization of a third case of
human atransferrinemia. Blood 104:2607, 2004.
Lee, P., Peng, H., Gelbart, T., Wang, L., Beutler, E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6. Proc. Natl. Acad. Sci. U. S. A.
102:1906, 2005.
Sham, R.L., Phatak, P.D., West, C., Lee, P.L., Andrews, C., Beutler, E. Autosomal
dominant hereditary hemochromatosis associated with a novel ferroportin mutation
and unique clinical features. Blood Cells Mol. Dis. 34:157, 2005.
Sipe, J.C., Waalen, J., Gerber, A., Beutler, E. Overweight and obesity associated
with a missense mutation in fatty acid amide hydrolase (FAAH). Int. J. Obes.
Relat. Metab. Disord. 29:755, 2005.
Waalen, J., Beutler, E. No age-related decrease in frequency of heterozygotes for
the HFE C282Y haemochromatosis mutation. J. Hepatol. 40:1044, 2004.
Waalen, J., Nordestgaard, B.G., Beutler, E. The penetrance of hereditary hemochromatosis. Best Pract. Res. Clin. Haematol. 18:203, 2005.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
275
Cell Death in the Heart
R.A. Gottlieb, Å.B. Gustafsson, C. Huang, M.R. Sayen,
M. Jinno, A. Cartier, A. Hamacher-Brady, Y. Tsukada,
T. Cramer, J. Ycaza
yocardial infarctions result in the death of
half a million persons in the United States
each year. We are interested in understanding the molecular events that commit cells to a death
program after ischemia and reperfusion in the heart.
Although ischemia itself is deleterious because of energy
depletion, further damage ensues with reperfusion, when
a burst of reactive oxygen species is produced and when
apoptosis, or programmed cell death, is activated in
vulnerable cells. Because apoptosis is a tightly regulated program, it may be possible to interfere with the
process and salvage cardiac cells. In addition to death
via apoptosis, cells can die via necrosis, which has
generally been regarded as an unregulated process
that can occur after exposure to high levels of oxidants.
However, recent evidence suggests that so-called necrotic
cell death may also be subject to biochemical regulation. We are defining the biochemical events of cell
death in the heart, both apoptosis and necrosis, to
identify potential therapeutic targets to mitigate reperfusion injury.
Using isolated perfused rat hearts subjected to
global ischemia and reperfusion, we found that calpain is activated during reperfusion, leading to cleavage of Bid, a proapoptotic member of the Bcl-2 family
of antiapoptotic proteins. Bid targets the mitochondria,
resulting in energetic failure and release of proapoptotic factors. The protein apoptosis repressor with caspase recruitment domain is expressed at high levels in
cardiac and skeletal muscle and is strongly protective
against cell death mediated by oxidative stress. We have
shown that this protein interacts with Bax, another
proapoptotic protein, to prevent apoptosis through the
mitochondrial pathway.
We are now investigating the effects of Bnip3,
another member of the Bcl-2 family. In addition to promoting apoptosis, Bnip3 promotes autophagy, a cellautonomous mechanism to remove damaged or unwanted
organelles. Using high-resolution fluorescence microscopy
with 3-dimensional deconvolution to image live cells
expressing fluorescent marker proteins, we showed the
presence of mitochondria in autophagosomes in cells
overexpressing Bnip3 (Fig. 1). Autophagy is upregulated
M
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Bnip3-induced mitochondrial dysfunction results in
increased mitochondrial autophagy. HL-1 cardiac myocytes were
transfected with Bnip3, the mitochondria-targeted red fluorescent
protein mito-DsRed, and the fluorescent autophagosome marker LC3GFP. At 48 hours after transfection, Z-stacks of both mito-DsRed and
LC3-GFP fluorescence were acquired and subjected to 3-dimensional
deconvolution algorithms for enhanced resolution. The magnified overlay is the 3-dimensional rendering of a single mitochondrion-containing autophagosome. Image by A. Hamacher-Brady.
Fig. 1.
after ischemia and reperfusion. Research is under way
to determine whether this upregulation is a cytoprotective
response or yet another pathway to cell death.
We made the novel discovery that chloramphenicol
and other inhibitors of cytochrome P450 monooxygenases reduce ischemia-reperfusion injury in the heart.
These drugs are protective even when administered after
ischemia, suggesting that they may have therapeutic
potential in the treatment of myocardial infarction.
Cytochrome P450 monooxygenases in the heart metabolize arachidonic acid to eicosanoids that regulate contractility and vasomotor tone. Some P450 enzymes are
also potent sources of superoxide, which may contribute
to reperfusion injury. We are investigating the basis for
the protective effect of P450 inhibition. We are focusing on the downstream signal transduction events such
as activation of protein kinase C and inhibition of p38
MAP kinase. We are joined in this effort by P. Wentworth,
Department of Chemistry, and M. Yeager, Department
of Cell Biology.
In collaboration with H. Rosen, Department of Immunology, we are studying the effects of sphingosine 1-phosphate and synthetic receptor-selective agonists in the
heart. We have characterized the hemodynamic effects of
these agents on the normally perfused heart and on
the modulation of ischemia-reperfusion injury and reperfusion arrhythmias. In collaboration with J. Molkentin,
Children’s Hospital Medical Center, Cincinnati, Ohio,
276 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
we are examining the mitochondrial alterations mediated
by deletion and overexpression of cyclophilin D. Cyclophilin D, an inner mitochondrial membrane protein,
plays an important role in regulating mitochondrial
integrity after ischemia and reperfusion. In studies with
C. Glembotski, San Diego State University, San Diego,
California, we are examining mitochondrial alterations
in mice deficient in the gene for MAP kinase kinase 6,
which are resistant to ischemia-reperfusion injury.
PUBLICATIONS
Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, N.S., Osinska, H., Hambleton,
M.A., Brunskill, E.W., Sayen, M.R., Gottlieb, R.A., Dorn, G.W., Robbins, J.,
Molkentin, J.D. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434:658, 2005.
Logue, S.E., Gustafsson, Å.B., Samali, A., Gottlieb, R.A. Ischemia/reperfusion
injury at the intersection with cell death. J. Mol. Cell. Cardiol. 38:21, 2005.
Scarabelli, T.M., Gottlieb, R.A. Functional and clinical repercussions of myocyte apoptosis in the multifaceted damage by ischemia/reperfusion injury: old and new concepts
after 10 years of contributions. Cell Death Differ. 11(Suppl 2):S144, 2004.
Genetics of the Brain Endogenous
Cannabinoid System
J.C. Sipe, A. Gerber
e focus on the genetics of the endogenous
cannabinoid system and its potential role in
neurobehavioral disorders such as drug addiction or abuse and neuroimmunologic diseases such as
multiple sclerosis. In 2002, we discovered a naturally
occurring mutation, P129T, in the human gene for fatty
acid amide hydrolase, the main inactivating enzyme
that controls the steady-state function of the brain
endocannabinoid signaling. In collaborative studies
with B.F. Cravatt, Department of Cell Biology, on the
cell biological abnormalities in humans with the homozygous P129T mutation, we found a biochemical link
between reduced cellular activity of the mutant enzyme
and problem drug use. This research confirmed that
this gene variant causes a marked loss of function.
Additional studies on the genetics of reward and
craving disorders led to our discovery of an important
association between variation in the gene for fatty acid
amide hydrolase and overweight and obesity, suggesting that this variation may be a genetic risk factor in
these common abnormalities.
During our investigations on endocannabinoid signaling in the immune system in humans, we identified
W
Published by TSRI Press®. © Copyright 2005,
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and studied a naturally occurring variation in the gene
for cannabinoid receptor 2 that is markedly more prevalent in patients with autoimmune diseases such as myasthenia gravis and rheumatoid arthritis than in healthy
persons. Because this receptor is involved in the normal
control of the level of the immune response to foreign
antigens, we studied the functional abnormalities caused
by this mutation and its association with autoimmunity.
The presence of functionally significant mutations in the
cannabinoid receptor genes may be a predictive risk
factor for vulnerability to certain autoimmune diseases.
PUBLICATIONS
Chiang, K., Gerber, A.L., Sipe, J.C., Cravat, B.F. Reduced cellular expression and
activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a
link between defects in the endocannabinoid system and problem drug use. Hum.
Mol. Genet. 13:2113, 2004.
Sipe, J.C. The brain endogenous cannabinoid system: a role in reward/craving of
addiction? Med. Hypotheses Res. 1:1, 2004.
Sipe, J.C., Arbour, N., Gerber, A., Beutler, E. Reduced endocannabinoid immune
modulation by a common cannabinoid 2 receptor (CB2) gene polymorphism: possible risk factor for autoimmune disorders. J. Leukoc. Biol. 78:231, 2005.
Sipe, J.C., Waalen, J., Gerber, A., Beutler, E. Overweight and obesity associated
with a missense polymorphism in fatty acid amide hydrolase (FAAH). Int. J. Obes.
(London) 29:755, 2005.
DIVISION
OF
MOLECULAR ONCOLOGY
Thomas F. Deuel, M.D., Division Head
Pleiotrophin: A Cytokine With
Critical Roles in Growth and
Development and in the
Progression of Human Neoplasms
T.F. Deuel, L. Ezquerra-Ruiz, G. Herradon, P. Perez-Pinera,
Y. Chang, M. Zuka, W. Zhang
leiotrophin is a recently identified and cloned
cytokine with diverse roles in normal development and differentiation and in responses to
different injuries. Ptn, the gene for pleiotrophin, is
also a proto-oncogene and is constitutively activated
by mutation in many different highly malignant human
cancers. Through use of dominant-negative mutations
in Ptn, we showed in vivo that the different mutations
that constitutively activate Ptn in premalignant cells
are sufficient by themselves to fully account for the
P
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
“switch” of premalignant cells to highly malignant cells.
The tumors derived from the highly malignant cells with
the active Ptn gene are associated with a high degree
of tumor angiogenesis; activation of Ptn in premalignant
cells thus initiates an “angiogenic switch” and denotes
a poor prognosis for host survival. Because mutations
that activate Ptn are among the most frequent in different highly malignant tumors, these mutations most
likely are among the most devastating in the progression of human neoplasms.
We focus on different aspects of the roles of pleiotrophin in normal growth and development and in neoplastic disease. Our long-range goals are to use pleiotrophin
as a therapeutic agent to stimulate angiogenesis and
to target the pleiotrophin signaling pathway as a site
for treating progression of human neoplasms with constitutive expression of Ptn.
SIGNALING
Pleiotrophin signals through a unique mechanism;
it interacts with and inactivates the receptor protein
tyrosine phosphatase (RPTP) β/ζ, thus increasing tyrosine phosphorylation of the substrates of the RPTP β/ζ
by the continued activity of unknown tyrosine kinases.
Substrates of RPTP β/ζ that we have discovered include
β-catenin, β-adducin, and Fyn, all proteins of critical
importance in the regulation of homophilic cell-cell
adhesion. Stimulation with pleiotrophin disrupts adherent
junction complexes and homophilic cell-cell adhesion,
suggesting the importance of pleiotrophin signaling in
the regulation of these vital cell functions. Because
disruption of homophilic cell-cell adhesion is characteristic of highly malignant cells that express Ptn, our
data suggest that one mechanism through which pleiotrophin stimulates a more aggressive phenotype in
malignant cells is disruption of normal cytoskeletal
architecture. Currently, we are investigating the mechanisms of disruption of cytoskeletal protein complexes in
pleiotrophin-stimulated cells.
ANGIOGENESIS
We found that expression of Ptn is upregulated in
developing microvasculature, macrophages, and astrocytes after acute ischemic brain injury and that pleiotrophin directly injected into ischemic myocardium
induces formation of functional neovasculature in vivo,
including stimulating growth of new capillaries and arterioles that functionally interconnect with existent coronary vascular systems. Furthermore, in other studies,
we showed that reversal of endogenous pleiotrophin
signaling in human glioblastoma cells via introduction
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277
of a dominant-negative Ptn gene reverses both the
malignant and the angiogenic phenotypes of these cells
in vivo. These findings indicate that pleiotrophin is an
angiogenic factor in vivo and that constitutive signaling of the endogenous pleiotrophin in cancer cells is
sufficient to initiate tumor angiogenesis and aggressive
tumor growth. We are now studying the mechanisms
and pathways of pleiotrophin signaling that lead to
angiogenesis in both in vitro and in vivo models.
BREAST CANCER
To extend our studies on pleiotrophin in neoplasia,
we used a dominant-negative Ptn and found that it
reversed the malignant phenotype of human breast cancer cells in vitro and in vivo. Currently, we are determining the mechanisms by which pleiotrophin signaling
stimulates a malignant state in human breast cancer
cells. In vitro, we identified reciprocal signaling between
breast cancer cells that express an activated Ptn and
activated stromal fibroblasts. To further test the relevance of pleiotrophin in promoting the growth of malignant breast cancers in vivo, we used doubly transgenic
mice predisposed to breast cancer. We found that constitutive pleiotrophin signaling driven by the mouse
mammary tumor virus promoter, which directs genes
to mammary gland cells for expression, cooperates with
signals driven by polyoma middle T antigen to accelerate the growth of mouse breast cancers and initiate
formation of new blood vessels in the tumors.
In ongoing collaborative studies with Z.-Y. Wang,
Creighton University, Omaha, Nebraska, we have identified and partially characterized a novel form of the
estrogen receptor. The significance of this finding is
under investigation.
D I F F E R E N T I AT I O N A N D D E V E L O P M E N T
We used microarray chips and real-time reverse
transcriptase–polymerase chain reaction to profile gene
transcripts from tissue from different organs in mice
that lacked the gene for pleiotrophin; mice that lacked
the gene for midkine, a pleiotrophin-related cytokine;
and wild-type mice. We discovered that both pleiotrophin and midkine are major regulators of the catecholamine biosynthesis and the renin-angiotensin pathways
in mouse aorta. In addition, midkine signaling is an
important regulator of Ptn expression, but pleiotrophin
signaling has no effect on levels of expression of the
gene for midkine. The results of these ongoing studies
have provided key insights on pathways previously
unsuspected but now shown to be regulated by this
important 2-member cytokine family. Our findings to
278 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
date suggest the importance of these pathways regulated
by pleiotrophin most likely is not in development but
in response to pleiotrophin/midkine upregulation in
response to injury or in different tumors that express
the genes for these 2 proteins.
PUBLICATIONS
Christman, K.L., Fang, Q., Kim, A.J., Sievers, R.E., Fok, H.H., Candia, A.F., Colley, K.J., Ezquerra, L., Herradon, G., Deuel, T.F., Lee, R.J. Pleiotrophin induces
formation of functional neovasculature in vivo. Biochem. Biophys. Res. Commun.
332:1146, 2005.
Ezquerra, L., Herradon, G., Nguyen, T., Silos-Santiago, I., Deuel, T.F. Midkine, a
newly discovered regulator of the renin-angiotensin pathway in mouse aorta: significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling. Biochem. Biophys. Res. Commun. 333:636, 2005.
Ezquerra, L., Herradon, G., Nguyen, T., Vogt, T.F., Bronson, R., Silos-Santiago, I.,
Deuel, T.F. Pleiotrophin is a major regulator of the catecholamine biosynthesis
pathway in mouse aorta. Biochem. Biophys. Res. Commun. 323:512, 2004.
Herradon, G., Ezquerra, L., Nguyen, T., Silos-Santiago, I., Deuel, T.F. Midkine
regulates pleiotrophin organ-specific gene expression: evidence for transcriptional
regulation and functional redundancy within the pleiotrophin/midkine developmental gene family. Biochem. Biophys. Res. Commun. 333:714, 2005.
Herradon, G., Ezquerra, L., Nguyen, T., Vogt, T.F., Bronson, R., Silos-Santiago, I.,
Deuel, T.F. Pleiotrophin is an important regulator of the renin-angiotensin system in
mouse aorta. Biochem. Biophys. Res. Commun. 324:1041, 2004.
Pariser, H., Ezquerra, L., Herradon, G., Perez-Pinera, P., Deuel, T.F. Fyn is a
downstream target of the pleiotrophin/receptor protein tyrosine phosphatase β/ζ
signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin.
Biochem. Biophys. Res. Commun. 332:664, 2005.
Pariser, H., Herradon, G., Ezquerra, L., Perez-Pinera, P., Deuel, T.F. Pleiotrophin
regulates serine phosphorylation and the cellular distribution of β-adducin through
activation of protein kinase C. Proc. Natl. Acad. Sci. U. S. A. 102:12407, 2005.
complex than their putative role in mediating transbilayer lipid movement.
We showed that PLSCR1 plays a role in modulating
the signaling through multiple growth factor receptors
and that PLSCR1 is itself transcriptionally upregulated
through these same receptors. The gene PLSCR1 is also
a major interferon-regulated gene; induced expression
of PLSCR1 is required for full cellular antiviral activity
of interferon. In addition to its usual location at the
plasma membrane, PLSCR1 can also translocate to
the nucleus. During the past year, we further elucidated
the mechanism by which nuclear import of PLSCR1
occurs. Furthermore, we discovered that PLSCR1, once
imported into the nucleus, can act as a transcription
factor, and we found that inositol 1,4,5-trisphosphate
receptor type 1 is one of its target genes.
A major focus during the past year was the biological
function of another member of the PLSCR family,
PLSCR3. Previously, we found that PLSCR1 is required
for normal maturation of granulocytes. Now we have discovered that PLSCR3 appears to play an important role
in adipocyte maturation or function: mice deficient in
PLSCR3 have a marked increase in depot fat, dyslipidemia, and insulin resistance, a phenotype that in many
respects recapitulates elements of adipose-related insulin
resistance in humans known as metabolic syndrome.
N O N C L A S S I C A L N U C L E A R L O C A L I Z AT I O N S I G N A L
Pariser, H., Perez-Pinera, P., Ezquerra, L., Herradon, G., Deuel, T.F. Pleiotrophin
stimulates tyrosine phosphorylation of β-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase β/ζ. Biochem. Biophys. Res.
Commun. 335:232, 2005.
Wang, Z., Zhang, X., Shen, P., Loggie, B.W., Chang, Y., Deuel, T.F. Identification,
cloning, and expression of human estrogen receptor-α36, a novel variant of human
estrogen receptor-α66. Biochem. Biophys. Res. Commun. 336:1023, 2005.
The Diverse Functions of
Phospholipid Scramblases
T. Wiedmer, J. Zhao, Q. Zhou, I. Ben-Efraim, L. Li, P.J. Sims
he phospholipid scramblase (PLSCR) family composed of PLSCR1, PLSCR2, PLSCR3, and PLSCR4
is a structurally unique class of plasma membrane proteins that were originally cloned and characterized in our laboratory. The first PLSCR, PLSCR1,
was identified on the basis of its capacity to promote
rapid transbilayer movement of membrane phospholipids in response to increases in calcium. However,
more recent data suggest that the biology of PLSCR1
and other members of this family is considerably more
T
Published by TSRI Press®. © Copyright 2005,
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OF PLSCR1 WITH A UNIQUE BINDING SITE IN
IMPORTIN
α
Previously, we identified a nuclear localization signal (NLS) in PLSCR1 and mapped it to a sequence in
the C-terminal region. We showed that this sequence
is sufficient for nuclear import through its association
with the importin α/β heterodimer. Furthermore, mutation of positively charged amino acids within the NLS
in the full-length protein completely blocked its entry
into the nucleus. The identified import sequence in
PLSCR1 is a nonclassical NLS; it lacks a contiguous
stretch of positively charged residues and is enriched
in hydrophobic residues.
The 2.2-Å crystal structure of a complex composed
of the PLSCR1 NLS and the armadillo repeat core of
vertebrate importin α has now been determined. Crystallographic analysis revealed that the PLSCR1 NLS
binds to armadillo repeats 1–4 of importin α, but its
interaction partially overlaps the classical NLS-binding
site. Two lysine residues of the PLSCR1 NLS occupy the
canonical positions indicated as P2 and P5. Remarkably, the critical lysine at position P2 that is essential
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
in other known NLS sequences is dispensable in PLSCR1
NLS. Taken together, these data provide insight into a
novel NLS that presents a distinct motif for binding to
importin α.
R O L E O F P L S C R 1 I N T H E D I F F E R E N T I AT I O N O F
LEUKEMIC CELLS
All-trans retinoic acid (ATRA) has been successfully
used for the treatment of acute promyelocytic leukemia
in what is known as “differentiation therapy.” In this
therapy, drugs are used to induce cancer cells to undergo
terminal differentiation, thus preventing further proliferation of the cells. The mechanism by which ATRA
and other agents induce differentiation of leukemic
cells is incompletely understood.
Previously, we showed that proliferation and terminal differentiation of myeloid precursor cells in response
to selective growth factors are impaired in mice deficient in PLSCR1, and in cells of monocytic and granulocytic lineage, expression of PLSCR1 markedly increases
upon terminal differentiation into neutrophils and macrophages. A truncated form of PLSCR1 has been associated with leukemogenic potential, and in patients with
acute myelogenous leukemia, higher levels of PLSCR1
mRNA were associated with significantly longer overall
survival. To gain insight into a potential role of PLSCR1
in the differentiation of leukemic cells, we investigated
the effects of known differentiation-inducing agents on
PLSCR1 expression in leukemic cell lines.
We found that ATRA increased PLSCR1 expression
in ATRA-sensitive acute promyelocytic leukemia cells
NB4 and HL60 but not in maturation-resistant NB4-LR1
cells. Both ATRA- and phorbol 12-myristate 13-acetate
(PMA)–induced monocytic differentiation is accompanied
by increased expression of PLSCR1. Cell differentiation
induced by ATRA and PMA, but not by vitamin D3 or
dimethyl sulfoxide, results in phosphorylation of protein
kinase Cδ, and inhibition of protein kinase Cδ activity
nearly eliminates ATRA- and PMA-induced expression
of PLSCR1. Ectopic expression of a constitutively active
form of the kinase increases PLSCR1 expression. Finally,
decreasing PLSCR1 expression with small interfering
RNA inhibits ATRA- and PMA-induced differentiation.
Our data suggest that as a protein induced upon activation of protein kinase Cδ, PLSCR1 is required for
leukemic cell differentiation induced by these agents.
A D I P O S I T Y, D Y S L I P I D E M I A , A N D I N S U L I N
R E S I S TA N C E I N M I C E W I T H TA R G E T E D D E L E T I O N
OF PLSCR3
To gain insight into the biological function of another
member of the PLSCR family, we investigated mice
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279
with targeted deletions of the gene for PLSCR3. Like
PLSCR1, PLSCR3 is expressed in many blood cells, but
in contrast to PLSCR1, it is also highly expressed in
fat and muscle. Surprisingly, we discovered that at the
age of 2 months, mice deficient in PLSCR3 had an
aberrant accumulation of abdominal fat, which was
accompanied by insulin resistance, glucose intolerance,
and dyslipidemia. Primary adipocytes and cultured bone
marrow–derived macrophages from PLSCR3-deficient
mice were engorged with neutral lipid, and adipocytes
had a defective response to insulin. Plasma of the deficient mice had elevated levels of non–high-density lipoproteins, cholesterol, triglycerides, nonesterified fatty
acids, and leptin, whereas plasma levels of adiponectin
were decreased. These data suggest that expression of
PLSCR3 may be required for normal adipocyte and/or
macrophage maturation or function. Of interest, obesity, in particular, accumulation of abdominal fat with
formation of enlarged, lipid-engorged adipocytes, is a
key acquired risk factor for the onset of type 2 diabetes.
Our data raise the possibility that deletions or mutations affecting the PLSCR3 locus may contribute to
the risk of lipid-related disorders in humans.
PUBLICATIONS
Chen, M.-H., Ben-Efraim, I., Mitrousis, G., Walker-Kopp, N., Sims, P.J., Cingolani, G. Phospholipid scramblase 1 contains a nonclassical nuclear localization
signal with unique binding site in importin α. J. Biol. Chem. 280:10599, 2005.
Giddings, K.S., Zhao, J., Sims, P.J., Tweten, R.K. Human CD59 is a receptor for the
cholesterol-dependent cytolysin intermedilysin. Nat. Struct. Mol. Biol. 11:1173, 2004.
Wiedmer, T., Zhao, J., Li, L., Zhou, Q., Hevener, A., Olefsky, J.M., Curtiss, L.K.,
Sims PJ. Adiposity, dyslipidemia, and insulin resistance in mice with targeted deletion of phospholipid scramblase 3 (PLSCR3). Proc. Natl. Acad. Sci. U. S. A.
101:13296, 2005.
Zhao, K.W, Li, X., Zhao, Q., Huang, Y., Li, D., Peng, Z.G., Shen, W.Z., Zhao, J.,
Zhou, Q., Chen, Z., Sims, P.J., Wiedmer, T., Chen, G.Q. Protein kinase Cδ mediates
retinoic acid and phorbol myristate acetate-induced phospholipid scramblase 1 gene
expression: its role in leukemic cell differentiation. Blood 104:3731, 2004.
DNA Replication and
S-Phase Checkpoint
X. Wu, E. Olson, E. Liu, A. Lee, T. Chiba
enome instability is a hallmark of the malignant
phenotype and a driving force for tumorigenesis.
S phase is genetically the most vulnerable period
of the cell cycle. In this phase, DNA must be replicated
faithfully and the entire genome must be duplicated
exactly once per cell cycle. Errors and lesions that occur
during DNA replication must be repaired to maintain
G
280 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
genome stability. Cell-cycle checkpoints have evolved
to monitor the integrity of the eukaryotic genome and
ensure the completion of DNA replication and the repair
of DNA damage before cell-cycle progression.
In one area of our research, we focus on a diseaselinked protein, Nbs1, that has essential roles in S-phase
checkpoint and DNA double-strand break repair. Mutations in the gene NBS1 lead to Nijmegen breakage
syndrome, which is characterized by radiation sensitivity,
chromosomal instability, and cancer. Nbs1 is phosphorylated by the checkpoint kinase ATM at multiple sites,
and these phosphorylation events are important for the
S-phase checkpoint. However, the molecular mechanism
by which Nbs1 inhibits further DNA replication upon
DNA damage is not clear.
Recently we observed that the simian virus 40
oncoprotein large T antigen interacts with Nbs1. This
association is required for the oncoprotein to drive
chromosomal hyperreplication and multiple rounds of
DNA replication from the simian virus 40 origin. We
also observed that NBS1 deficiency leads to reinitiation of DNA replication close to replication origins. These
observations suggest a novel role of Nbs1 in the prevention of DNA rereplication. They establish a molecular
link between Nbs1 and DNA replication control, which
will help in elucidating the mechanism that underlies
the S-phase checkpoint function of Nbs1.
The second focus of our research is understanding
how DNA replication is controlled so that DNA is replicated once and only once per cell cycle. Rereplication
of the genome, or even a segment of it, could lead to
genome instability. Cdt1 is a replication licensing factor that is required for the establishment of prereplication complexes for initiation of DNA replication. Cdt1
is degraded when cells enter S phase, a situation that
prevents reestablishment of the prereplication complex
in the same cell cycle. We have studied the mechanisms by which Cdt1 is degraded during the cell cycle.
We found that overexpression of Cdt1 in mammalian
cells leads to DNA rereplication, suggesting that cell
cycle–regulated Cdt1 degradation is a critical event to
prevent DNA rereplication. We are also studying how
checkpoints respond to DNA rereplication.
PUBLICATIONS
Liu, E., Li, X., Yan, F., Zhao Q., Wu, X. Cyclin-dependent kinases phosphorylate
human Cdt1 and induce its degradation. J. Biol. Chem. 279:17283, 2004.
Wu, X., Avni, D., Chiba, T., Yan, F., Zhao, Q., Lin, Y., Heng, H.H.Q., Livingston,
D.M. SV40 T antigen interacts with Nbs1 to disrupt DNA replication control.
Genes Dev. 18:1305, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
D IVISION
OF
O NCOVIROLOGY
Peter K. Vogt, Ph.D., Division Head
Molecular Genetics of Cancer
P.K. Vogt, A. Bader, D. Bai, K. Bower, I. Dang, A. Denley,
G. Denning, M. Gymnopoulos, H. Jiang, S. Kang, A. Kurze,
J. Shi, L. Zhao
he focus of our research is molecular mechanisms
of carcinogenesis. We study viral and cellular
oncoproteins and tumor suppressors, defining
their functions in oncogenesis and identifying molecular
targets for therapeutic intervention. In high-throughput
screens, we look for small molecules that can interact
with these targets and inhibit or reverse oncogenic
cellular transformation.
T
O N C O G E N I C T R A N S F O R M AT I O N
Oncogenic transformation of cells requires changes
in gene activities, regulated at the level of transcription,
translation, or posttranslational modification. These
changes result in a gain of function for specific growthpromoting genes and a loss of function for growthattenuating genes.
C A N C E R - S P E C I F I C M U TAT I O N S I N
P H O S P H AT I D Y L I N O S I T O L - 3 ′- K I N A S E
Several studies published during the past year
described cancer-specific mutations in p110α, the
catalytic subunit of phosphatidylinositol-3′-kinase
(PI3K). The mutations are found in about 30% of most
solid human tumors. The mutations do not map randomly over the gene, rather they are concentrated in
distinct hot spots in the gene that encode the kinase
and the helical domains of the enzyme. This nonrandom distribution of the p110α mutations suggests
that they are selected because they provide a growth
advantage for the cell in which they are expressed.
We chose mutations from 3 predominant hot spots
in the gene for p110α and introduced them into the
wild-type protein. When expressed in normal cells, these
mutant proteins induced strong oncogenic transformation.
The transformed cells had constitutive activation of Akt
and its downstream targets, the major effectors of
PI3K signaling. The mutant proteins also had enhanced
enzymatic activity. The oncogenicity of these gain-offunction mutants in cell culture strongly suggests that
they are a growth-promoting force in the human cancers. Because the mutations are cancer-specific and
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
somatic, they are ideal targets for specific inhibitors
that would interfere with the function of the mutant
enzyme but allow activity of the wild-type protein.
P O S T T R A N S L AT I O N A L R E G U L AT I O N O F F O X O
PROTEINS
FOXO proteins are transcription factors that in
response to specific signals control gene expression
programs dealing with (1) the cell cycle, (2) protection against oxidative damage, and (3) apoptosis. The
overall effects of FOXO proteins are an attenuation of
cell growth. FOXO proteins have tumor-suppressing
potential. In cells transformed by PI3K or Akt oncoproteins, FOXO activity is drastically reduced. The Akt
kinase phosphorylates FOXO on 3 sites, and this phosphorylation interferes with the nuclear functions of FOXO
and initiates nuclear export of the FOXO proteins.
We found that the Akt-phosphorylated, cytoplasmic
FOXO protein is rapidly degraded by the proteasomal
pathway in Akt-transformed cells. The elimination of
FOXO and of its growth-suppressive potential is an essential feature of tumor cells that show gain of function in
the PI3K-Akt pathway. The nuclear functions of FOXO
can also be abolished by a dominant-negative mutant of
FOXO. Cells expressing such a mutant become transformed without additional signals from the Akt pathway.
Y B - 1 , A P R O T E I N D I F F E R E N T I A L LY R E G U L AT E D
IN CANCER
Oncogenic transformation is reflected in profound
changes of the transcriptional program: levels of mRNA
are enhanced for some genes and reduced for others.
Among these differentially expressed genes are those
that control the oncogenic phenotype of the cell. Identification of these transformation-relevant genes is still
a challenge, and we and others have designed various
techniques to achieve this difficult goal.
A recent example of cancer-related, differential regulation is the Y box–binding protein YB-1. YB-1 is transcriptionally downregulated in cells transformed by the
oncoproteins PI3K or Akt. Vector-mediated expression
of YB-1 makes cells resistant to oncogenic transformation by PI3K and Akt but does not affect sensitivity to
transformation by other oncoproteins such as Src or Jun.
Induction of cellular resistance by YB-1 is accompanied
by a change of cellular morphology. Compared with normal fibroblasts, cells expressing YB-1 have a more flattened, strongly adherent shape. This change in cell
morphology is correlated with inhibition of cap-dependent
protein synthesis. Genetic and biochemical analyses
suggest that YB-1 does not affect Akt signaling to the
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
281
target of rapamycin kinase but must bind to mRNA to
inhibit oncogenic cellular transformation. Available data
are in accord with the hypothesis that YB-1 inhibits the
translation of specific proteins required for the induction
and maintenance of the transformed cellular phenotype.
SMALL-MOLECULE INH D
D I M E R I Z AT I O N
Myc is a transcriptional regulator that can strongly
stimulate cell proliferation. It is often overexpressed in
human cancer and plays an important role in determining the oncogenic properties of the cancer cells. To function as a transcription factor, Myc must form a dimer
with the smaller, related protein Max. Only the Myc-Max
heterodimer can bind to DNA and can control transcription from specific sites in the genome. Inhibitors of
Myc-Max dimerization may therefore provide a means
for controlling Myc activity.
In collaborative studies with K. Janda, Department
of Chemistry, we identified new small-molecule inhibitors
of Myc-Max dimerization. These molecules come from
a combinatorial library and have a molecular scaffold
that can insert itself between the 2 interacting protein
surfaces. The inhibitors interfere with Myc-Max dimerization and inhibit DNA-binding activity of Myc. Some
of the compounds also prevent Myc-induced oncogenic
transformation in cell culture. These compounds are
active at low micromolar concentrations and are being
further developed for increased potency.
PUBLICATIONS
Aoki, M., Jiang, H., Vogt, P.K. Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins. Proc. Natl.
Acad. Sci. U. S. A. 101:13613, 2004.
Bader, A.G., Vogt, P.K. An essential role for protein synthesis in oncogenic cellular
transformation. Oncogene 23:3145, 2004.
Bader A.G., Vogt P.K. Inhibition of protein synthesis by Y box-binding protein 1
blocks oncogenic cell transformation. Mol. Cell. Biol. 6:2095, 2005.
Bader, A.G., Vogt, P.K. Leucine zipper transcription factors: bZIP proteins. In:
Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Ganten, D., Ruckpaul, K. (Eds.). Springer, New York, in press.
Bader, A.G., Vogt, P.K. Oncogenes and proto-oncogenes: jun oncogenes. In: Encyclopedia of Respiratory Medicine. Laurent, G., Shapiro, S. (Eds.). Elsevier Academic Press, San Diego, in press.
Bader, A.G., Vogt, P.K. Protein synthesis and cancer. In: Nutritional Genomics:
Nutrients, Genes and Genetic Variation in Health and Disease. Brigelius-Flohé, R.,
Joost, H.-G. (Eds.). Wiley-VCH, New York, in press.
Iacovoni, J.S., Cohen, S.B., Berg, T., Vogt, P.K. v-Jun targets showing an expression pattern that correlates with the transformed cellular phenotype. Oncogene
23:5703, 2004.
Kang, S., Bader, A.G., Vogt, P.K. Phosphatidylinositol 3-kinase mutations identified in
human cancer are oncogenic. Proc. Natl. Acad. Sci. U. S. A. 102:802, 2005.
Kang S., Bader, A.G., Zhao, L., Vogt, P.K. Mutated PI 3-kinases: cancer targets on
a silver platter. Cell Cycle 4:578, 2005.
282 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
Lee, L.V., Bower, K.E., Liang, F.-S., Shi, J., Wu, D., Sucheck, S.J., Vogt, P.K.,
Wong, C.-H. Inhibition of the proteolytic activity of anthrax lethal factor by aminoglycosides. J. Am. Chem. Soc. 126:4774, 2004.
Muira, Y., Kainuma, M., Jiang, H., Velasco, H., Vogt, P.K., Hakomori, S. Reversion of
the Jun-induced oncogenic phenotype by enhanced synthesis of sialosyllactosylceramide (GM3 ganglioslide). Proc. Natl. Acad. Sci. U. S. A. 101:16204, 2004.
Vogt, P.K., Jiang H., Aoki M. Triple layer control: phosphorylation, acetylation and
ubiquitination of FOXO proteins. Cell Cycle 4:908, 2005.
Molecular Mechanisms of
Cancer Development and
Protein Modification by a
Ubiquitin-Like Modifier
D.-E. Zhang, O.A. Malakhova, K.J. Ritchie, L.F. Peterson,
M. Yan, K.-I. Kim, R. Hines, E. Kanbe, A. Boyapati, C. Dao,
J.-K. Luo, Y. Wang, M. Shen, W. Zou, D.W. Girdwood,
J.R. Biggs, J.-H. Kim, E.-Y. Ahn, J. Wang, A. Joo,
F. Okumura, F. Beneyt
AML1 AND ITS FUSION PROTEIN AML1-ETO IN
B L O O D C E L L D I F F E R E N T I AT I O N
cute myeloid leukemia is a major hematopoietic
malignant neoplasm characterized by the proliferation of a malignant clone of myeloid progenitor
cells. One of the most common targets of chromosomal
translocations implicated in this neoplasm is the gene
AML1. The gene was isolated through a study of t(8;21)
chromosomal translocation; the results revealed that the
runt homology domain of AML1 is fused to a gene termed
ETO (MTG8) to form a fusion protein called AML1-ETO.
Subsequent studies indicated that the protein AML1 is
crucial for normal hematopoiesis. We previously discovered that AML1 synergistically activates the expression of a critical myeloid gene, the gene for the M-SCF
receptor, with 2 other important transcription factors
C/EBP and PU.1.
To study the effect of AML1-ETO on hematopoiesis,
we produced various mouse models in which wild-type
AML1 was replaced by AML1-ETO. Currently, we are
identifying cofactors involved in the synergy among
various transcription factors and involved in AML1ETO–associated development of leukemia.
A
A NOVEL UBIQUITIN-SPECIFIC ENZYME, UBP43
In studying genes differentially expressed in AML1ETO mice, we isolated the gene for a novel enzyme
UBP43 (USP18), which belongs to a family of ubiquitin-specific proteases. Like phosphorylation and dephosPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
phorylation, ubiquitylation and deubiquitylation are
mechanisms for protein modification. Recently, we
showed that UBP43 is the only currently known enzyme
that removes a ubiquitin-like modifier, ISG15, from ISG15
conjugates. In mice that lacked the gene for UBP43,
UBP43-deficient bone marrow cells were hypersensitive to treatment with type I interferon and died via
apoptosis in the presence of interferon. Most important,
in UBP43-deficient cells, interferon induced a prolonged
Stat1 tyrosine phosphorylation, DNA binding, and
interferon-mediated gene activation. UBP43-deficient
mice are resistant to certain viral and bacterial infections. Currently, we are analyzing molecular pathways
affected by UBP43.
R O L E O F I S G 1 5 C O N J U G AT I O N I N I M M U N E
RESPONSES
The gene for ISG15 was originally cloned as a gene
highly upregulated by interferon and encodes a small
ubiquitin-like protein. Unlike ubiquitin and other ubiquitin-like modifiers, ISG15 is not present in lower eukaryotes, such as yeast, indicating that it may be associated
with specialized functions in higher eukaryotic cells.
Upon viral infection, bacterial infection, or other stress
stimulation, ISG15 can be detected in cells both in free
and in conjugated form (ISGylation). Using high-throughput Western blot analysis, we identified 4 ISGylated
proteins: Stat1, Jak1, Erk1, and PLCγ1. We also found
that Ubc8 is an ISG15-conjugating enzyme. Regulation
of protein ISGylation may provide valuable treatments
to control cell function and survival. We are using techniques such as gene depletion, protein interaction, biochemical purification, and gene regulation to study the
biological function of this interesting protein modification.
PUBLICATIONS
Chan, E.M., Comer, E.M., Brown, F.C., Richkind, K.E., Holmes, M.L., Chong,
B.H., Shiffman, R., Zhang, D.-E., Slovak, M.L., Willman, C.L., Noguchi, C.T., Li, Y.,
Heiber, D.J., Kwan, L., Chan, R.J., Vance, G.H., Ramsey, H.C., Hromas, R.A.
AML1-FOG2 fusion protein in myelodysplasia. Blood 105:4523, 2005.
Dao, C.T., Zhang, D.-E. ISG15: a ubiquitin-like enigma. Front. Biosci. 10:2701, 2005.
Kim, K.I., Giannakopoulos, N.V., Virgin, H.W., Zhang, D.-E. Interferon-inducible
ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation. Mol. Cell. Biol.
24:9592, 2004.
Ritchie, K.J., Hahn, C.S., Kim, K.I., Yan, M., Rosario, D., Li, L., de la Torre, J.C.,
Zhang, D.-E. Role of ISG15 protease UBP43 (USP18) in innate immunity to viral
infection. Nat. Med. 10:1374, 2004.
Tokarz, S., Berset, C., La Rue, J., Friedman, K., Nakayama, K.I., Nakayama, K.,
Zhang, D.-E., Lanker, S. The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFskp2 ubiquitin ligase. J. Biol. Chem. 279:46424, 2004.
Yan, M., Burel, S., Peterson, L.F., Kanbe, E., Iwasaki, H., Boyapati, A., Hines, R.,
Zhang, D.-E. Deletion of an AML1-ETO C-terminal NCoR/SMRT interacting region strongly
induces leukemia development. Proc. Natl. Acad. Sci. U. S. A. 101:17186, 2004.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
D IVISION OF
RHEUMATOLOGY RESEARCH
W.M. Keck Autoimmune Disease Center
Joel N. Buxbaum, M.D., Division Head
Pathogenesis of Late-Onset
Genetic Diseases Related to
Abnormalities of Protein
Conformation
J.N. Buxbaum, N. Reixach, Z. Ye, L. Friske
e are studying the pathogenesis of human
genetic diseases that are age-dependent and
the result of protein misfolding. We use 3
major approaches: animals transgenic for the human
protein transthyretin, a cause of hereditary peripheral
nerve and heart disease; cell cultures to determine
how the misfolded protein injures its cellular targets;
and genetic epidemiology to identify potential disease
carriers and analyze those hereditary factors that affect
the nature of the disease.
We are doing microarray analyses of gene expression in collaboration with J. Walker, Novartis Institute
for Functional Genomics, San Diego, California. In these
studies, we continue to analyze age-related transcriptional changes in the hearts of old animals transgenic
for transthyretin; we compare the pattern of changes
in the old animals with the patterns in young transgenic
animals of the same genetic strain. Our results to date
indicate that specific groups of genes have changes in
transcription with age, independent of the presence of
the transgene. In transgenic animals matched for age
and sex, additional functional groups of genes have
alterations related to the presence of cardiac deposits
of transthyretin. Our current goal is to discover which
changes are enabling, that is, allow deposition to occur,
and which are reactive, that is, represent the effects
of the protein deposit.
In collaboration with researchers in the laboratory
of J. Kelly, Department of Chemistry, we developed a
tissue culture method to determine the ability of small
molecules to inhibit the toxicity of oligomeric forms of
transthyretin, a protein that can produce heart disease
W
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
283
in humans. Dr. Kelly’s group has synthesized a large
number of compounds that can bind in the normal T4
binding site of transthyretin and inhibit aggregation
and formation of fibrils under nonphysiologic conditions.
With our method, compounds that are efficacious under
nonphysiologic conditions can be evaluated under physiologic conditions in a semi–high-throughput mode for
both efficacy and toxicity before further, more expensive studies in experimental animals.
Using the same tissue culture method, we continued our studies to elucidate the mechanism whereby
oligomeric aggregates of transthyretin produce damage
to heart and nerve cells. We have defined the kinetics
of interaction between the toxic molecular species and
the target cell, and we are analyzing the relative roles
of protein-membrane interaction and internalization in
producing toxic effects and cell death.
We recently expanded our collaborative studies with
investigators at the Institute of Molecular Biology in
Oporto, Portugal; Vanderbilt University, Nashville, Tennessee; and the University of California, San Diego, to
include colleagues from Japan and Sweden. In these
studies, we are exploring the role of genes other than
those that encode the most common European mutation
in transthyretin in determining the phenotype of different
individuals who have the same amyloidogenic (transthyretin V122L) mutation. Portuguese and Japanese carriers
of the same mutation have similar patterns of disease,
whereas Swedish carriers have a very different phenotype. We are now analyzing all 3 populations for variations in those genes that we know are associated with
differences in the age of onset of the autosomal dominant
Portuguese form of familial amyloidotic polyneuropathy.
With colleagues in New York, Boston, and North
Carolina, we have extended our studies of African American individuals more than 60 years old who have a
mutation in the gene for transthyretin (TTRval122ile).
The frequency of the allele indicates that it is a significant risk for congestive heart failure for this population.
Our most recent analyses indicated that compared with
controls matched for age, sex, and ethnicity, carriers
of the allele have considerable differences in cardiac
function. This finding reinforces the notion that those
at risk can be recognized by means of a simple genetic
test before they become ill.
In collaboration with Dr. Kelly and investigators in
Boston; Rochester, Minnesota; London; Umea, Sweden;
Portugal; and Japan, we organized a clinical trial to
determine if a treatment with a compound, approved
284 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
by the Food and Drug Administration for another use,
that inhibits formation of amyloid fibrils by binding to
transthyretin is effective in preventing progression of
clinical disease.
PUBLICATIONS
Alexander, A., Subramanian, N., Buxbaum, J.N., Jacobson, D.R. Drop-in, dropout allele-specific PCR: a highly sensitive, single-tube method for PCR-based allelotyping. Mol. Biotechnol. 28:171, 2004.
Soares, M.L., Coelho, T., Sousa, A., Batalov, S., Conceição, I., Sales-Luís, M.L.,
Ritchie, M.D., Williams, S.M., Nievergelt, C.M., Schork, N.J., Saraiva, M.J.,
Buxbaum, J.N. Susceptibility and modifier genes in Portuguese transthyretin V30M
amyloid polyneuropathy: complexity in a single-gene disease. Hum. Mol. Genet.
14:543, 2005.
as foreign antigens by the host immune system. We discovered that loss of SOD2 in mice is associated with
the development of autoantibodies. The specificity of
these autoantibodies overlaps with specificities seen in
patients with the autoimmune diseases systemic lupus
erythematosus and scleroderma. We found that many
mice transplanted with SOD2-deficient cells make antibody against centromeric proteins, whereas mice with
normal cells do not (Fig. 1).
Oxidative Stress and Disease
J.S. Friedman, M. Welsh, F.M. Martin, J. Yi, G. Bydlon
e are investigating how oxidative stress contributes to aging and disease. We use mouse
models with (1) alterations in proteins, such
as superoxide dismutases (SODs) and peroxiredoxins,
and their genes that have a direct role in defense against
oxidant stress or (2) alterations in pathways, such as
p53, p66Shc, and insulin-like growth factor 1, that
transduce oxidant stress signals and play a role in
longevity. Currently, we are focusing on the effect of
loss of SOD2 on blood cells.
In mice, deficiency in SOD2 results in an anemia
that can be used as a model of the human disorder
sideroblastic anemia. During the past year, we used
cDNA microarrays to compare gene expression profiles
of erythroid (red blood cell) progenitors from normal
and SOD2-deficient animals. Preliminary analysis
revealed abnormalities in mitochondrial metabolism in
the SOD2-deficient animals and suggested that oxidant
stress directly affects iron transport into developing red
blood cells. Because protein turnover is slow or absent
in mature red blood cells, SOD2-deficient cells accumulate high levels of oxidatively damaged proteins. Using
SOD2-deficient cells, we developed novel methods for
enriching and identifying oxidized proteins that can be
used in 2-dimensional gel electrophoresis. Use of these
methods will allow more detailed comparison of changes
in oxidized proteins that are hypothesized to accompany aging, inflammatory processes, and neurodegenerative disease.
Oxidative damage to proteins may also be an important factor in the pathogenesis of autoimmune disease,
because oxidized proteins may be mistakenly identified
W
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Enzyme immunosorbent-linked assay shows that many
mice reconstituted with blood cells deficient in SOD2 (SOD2 –/– )
make antibodies against centromeric proteins, whereas recipients of
normal cells (SOD2+/+) do not. For SOD2+/+ vs SOD2–/–, P = .003.
We also study a molecular circuit (Fig. 2) that
includes p66Shc, the tumor suppressor protein p53,
and putative downstream targets, including forkhead
transcription factors. Evidence suggests that this pathway determines cell fate when oxidant stress occurs
and that activity of this pathway may affect life span,
rates of cancer, and progression of neurodegenerative
disease. In cell culture and animal models, we are
attempting to identify components of this pathway
downstream of p66 that link exposure to oxidants with
F i g . 2 . Certain mutations of the protein p53 lead to accelerated
aging, whereas deletion of the protein p66Shc results in increased
longevity. A possible function of p66Shc is transduction of signals
from p53 that accelerate the rate of aging by inhibiting the expression of genes for antioxidant proteins.
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
changes in mitochondrial function. Of particular interest is the apparent dual role of p66Shc as both a sensor of oxidants and a regulator of endogenous oxidant
production by cells.
PUBLICATIONS
Martin, F.M., Bydlon, G., Friedman, J.S. SOD2 deficiency anemia, sideroblastic
anemia, and red cell oxidative stress. Antioxid. Redox Signal., in press.
Martin, F.M., Bydlon, G., Welsh, M.L., Friedman, J.S. A method for rapid mouse
siderocyte enrichment. Exp. Heme, in press.
Autoimmunity Induced by
Xenobiotics
K.M. Pollard, D. Cauvi, P. Hultman,* M.J. Fritzler,**
D.H. Kono***
* Linköping University, Linköping, Sweden
** University of Calgary, Calgary, Alberta
*** Department of Immunology, Scripps Research
e focus on how interactions between the environment and genetics affect induction of
autoimmune diseases. We use murine models
of systemic autoimmunity in which disease is elicited
by exposure to xenobiotics. An important aspect of our
research is a comparison of the similarities and differences between induced systemic autoimmunity and
idiopathic systemic autoimmunity, such as systemic
lupus erythematosus, in mice and humans.
W
EFFECT OF THE PHYTOESTROGEN COUMESTROL IN
THE NZB/W F1 MURINE MODEL OF SYSTEMIC
L U P U S E R Y T H E M AT O S U S
Coumestrol is a naturally occurring plant estrogen.
Because estrogen influences cellular and humoral immunity and has known effects in murine models of systemic
lupus erythematosus, we investigated the effect of
coumestrol on disease expression in NZB/W F 1 mice.
Female NZB/W F1 mice were fed a ‘‘standard’’ rodent
diet including soy proteins, a nonsoy diet, or a nonsoy
diet with 0.01% coumestrol. Outcome measures included
survival, autoantibody expression, immunoglobulin levels, proteinuria, renal histologic and B-cell immunohistochemical findings, and renal mRNA expression.
At 24 weeks, compared with the other 2 groups of
mice, the group fed a diet containing coumestrol had
decreased levels of autoantibodies detected by immunofluorescence and less splenomegaly. At 39 weeks, the
prevalence of autoantibodies was similar among all 3
groups, but the group fed coumestrol had less proteinuria than did the other 2 groups. Treatment with coumesPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
285
trol had little effect on renal mRNA levels as assessed
by gene array analysis, but functional ontology mapping revealed that genes encoding proteins involved in
the immune response were the ones most often affected.
These results suggest that treatment with coumestrol
may ameliorate some aspects of disease progression
in this model of systemic autoimmunity.
IMMUNOSUPPRESSIVE AND AUTOIMMUNE EFFECTS
OF THIMEROSAL IN MICE
The possible health effects of the organic mercury
compound thimerosal (ethylmercurithiosalicylate), which
is rapidly metabolized to ethylmercury, have recently
been debated, and the effect of this compound on the
immune system is unknown. We examined the effect
of thimerosal by treating A.SW (H-2s) mice, which are
susceptible to induction of autoimmunity by heavy metals, with thimerosal in their drinking water for up to 30
days. The lymph node expression of IL-2 and IL-15
mRNA was increased after 2 days of treatment and that
of IL-4 and IFN-γ mRNA after 6 and 14 days. During the
first 14 days of treatment, the number of splenocytes,
including T cells, B cells, and immunoglobulin-secreting
cells, decreased. A strong immunostimulation occurred
after 30 days of treatment, with increases in splenic
weight; numbers of splenocytes, including T cells, B cells,
and immunoglobulin-secreting cells; and the serum level
of immunoglobulins dependent on type 1 and type 2
helper T cells.
Antinucleolar antibodies targeting the 34-kD nucleolar protein fibrillarin and systemic deposition of immune
complexes developed. The H-2 s strains SJL and B10.S
also responded to thimerosal treatment with production
of antinucleolar antibodies. Antinucleolar antibodies did
not develop in the A.TL and B10.TL strains, which share
background genes with the A.SW and B10.S strains,
respectively, but have a different H-2 haplotype, linking
the susceptibility to H-2. Antinucleolar antibodies did not
develop in thimerosal-treated H-2s mice homozygous
for the nu mutation (SJL-nu/nu) or lacking the T-cell
costimulatory molecule CD28 (B10.S-CD28–/–), indicating that the autoimmune response is T-cell dependent.
Using H-2 s strains with targeted mutations, we
found that IFN-γ and IL-6, but not IL-4, are important
for induction of antinucleolar antibodies by thimerosal.
The maximum added renal concentration of thimerosal
(ethylmercury) and inorganic mercury occurred after
14 days of treatment and was 81 µg of mercury per
gram of tissue. Ethylmercury made up 59% and inorganic mercury 41% of the renal mercury.
286 MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
In conclusion, the organic mercury compound
thimerosal has initial immunosuppressive effects similar to those of methylmercury. However, in contrast to
treatment with methylmercury, in genetically susceptible mice, treatment with thimerosal leads to a second
phase, with T cell–dependent and H-2–linked autoimmunity. This immunostimulation may be partly due to
the inorganic mercury derived from the metabolism of
ethylmercury.
PUBLICATIONS
Havarinasab, S., Häggqvist, B., Björn, E., Pollard, K.M., Hultman, P. Immunosuppressive and autoimmune effects of thimerosal in mice. Toxicol. Appl. Pharmacol.
204:109, 2005.
Hultman, P., Taylor, A., Yang, J.M., Pollard, K.M. The effect of xenobiotic exposure
on spontaneous autoimmunity in (SWR x SJL) F1 hybrid mice. J. Toxicol. Environ.
Health, in press.
Lynes, M.A., Fontenot, A.P., Lawrence, D.A., Rosenspire, A.J., Pollard, K.M.
Gene expression influences on metal immunomodulation. Toxicol. Appl. Pharmacol., in press.
Pollard, K.M. (Ed.). Autoantibodies and Autoimmunity: Molecular Mechanisms in
Health and Disease. Wiley-VCH, New York, in press.
α-chain of FcεRI from the endoplasmic reticulum. We
found that the previously defined endoplasmic reticulum retention signal located near the C terminus of
the α-chain is only weakly functional in steady-state
receptor transport. At the same time, we identified a
novel dilysine motif in the middle of the α-chain cytoplasmic domain sequence that is also functional in
controlling FcεRI transport. We have now determined
that the new motif functions synergistically with the
C-terminal retention motif to strongly regulate escape
of the FcεRI α-chain from the endoplasmic reticulum
and eventual transport along the secretory pathway.
In another study, we investigated the structural basis
of assembly of the αγ subunit, a process previously
thought to occur exclusively through interaction of the
subunit transmembrane domains. Our data revealed that
the cytoplasmic domain determinants of each subunit
mediate αγ association, a finding that may have implications in FcεRI signaling.
INHIBITION OF FC
ε R I - M E D I AT E D
CELLULAR
Pollard, K.M. Systemic autoimmunity. In: Encyclopedic Reference of Immunotoxicology. Vohr, H.-W. (Ed.). Springer, New York, 2005, p. 621.
A C T I VAT I O N B Y A M O N O C L O N A L A N T I B O D Y T O
Pollard, K.M., Arnush, M., Hultman, P., Kono, D.H. Costimulation requirements of
induced murine systemic autoimmune disease. J. Immunol. 173:5880, 2004.
Several years ago, we showed that 5H5F8, a monoclonal antibody to the α-chain of FcεRI, inhibits IgEdependent activation of cells in a mast cell line and in
primary human basophils. Our data indicated that
5H5F8-mediated cell inhibition occurs by a unique
mechanism that does not involve perturbation of the
IgE-binding site. The 5H5F8 epitope has been mapped
to the linear membrane proximal region, and in collaboration with F. Kricek, Novartis Pharma, Vienna, Austria, we confirmed the epitope assignment from the
crystallographic structure of a complex formed between
5H5F8 and a synthetic peptide corresponding to the
antibody’s epitope. As a continuation of this collaboration, we hypothesize that the membrane proximal region
of FcεRI may be critically involved in initiating or propagating FcεRI-dependent signaling. We are testing this
hypothesis by exploring the effect of various mutations
in the membrane proximal region on FcεRI-dependent
function in transduced mast cells.
Pollard, K.M., Hultman, P., Kono, D.H. Immunology and genetics of induced systemic autoimmunity. Autoimmun. Rev. 4:282, 2005.
Schoenroth, L.J., Hart, D.A., Pollard, K.M., Fritzler, M.J. The effect of the phytoestrogen coumestrol on the NZB/W F1 murine model of lupus. J. Autoimmun.
23:323, 2004.
Expression and Function of the
High-Affinity Receptor for IgE
M.W. Robertson, Z. Wang, X. Tian
he high-affinity receptor for IgE (FcεRI) is a cellsurface receptor that is critically involved in the
pathology of the allergic reaction. The receptor
is highly expressed by mast cells and basophils. Upon
stimulation by IgE-antigen complexes, these cells secrete
histamine and other mediators of hypersensitivity, leading
to the formation of the clinical signs and symptoms of
allergy. Our goal is to define the molecular basis of FcεRI
cellular expression and the role of FcεRI structure in initiating or propagating IgE-mediated cellular activation.
T
FC
εRI
A S S E M B LY A N D T R A N S P O R T
We continue to explore how FcεRI αγ2 and αβγ2
isoforms assemble and traffic in cells. Recently, we
reassessed the structural basis of transport of the
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
T H E F C ε R I α -C H A I N
MOLECUL AR AND EXPERIMENTAL MEDICINE 2005
The Importance of
Autoantibody-Defined Epitopes
E.M. Tan, D.R. Liu, T. Himoto,* F.D. Shi,** J.-Y. Zhang,***
E.K.L. Chan****
* Kagawa University School of Medicine, Kagawa, Japan
** St. Joseph’s Hospital, Phoenix, Arizona
*** University of Texas, El Paso, Texas
**** University of Florida, Gainesville, Florida
utoimmunity defined as an immune response by
the host to a self-antigen, at the humoral or cellular level or both, is a common phenomenon. Autoimmunity defined as a disease in which host responses
to self-antigens result in pathophysiologic consequences
is a much rarer phenomenon. Often these important differences are not separated in writings on this subject.
We have focused on the humoral immune responses
in autoimmunity, that is, on autoantibodies to self-antigens that can be detected in the serum of patients with
a variety of diseases. These diseases include the rather
large number of systemic rheumatic diseases with autoantibodies, including systemic lupus erythematosus
(often called lupus), rheumatoid arthritis, scleroderma,
Sjögren syndrome, dermatomyositis, and polymyositis.
We have also worked extensively on defining autoantibodies in the sera of patients with different forms of
cancer. In both systemic rheumatic diseases and cancer,
we have shown that each disease category in which
autoantibodies are present can be characterized by
distinct profiles of antigen-antibody specificities, and
we have proposed that antibody profiles in cancer can
be used as diagnostic markers.
In the course of these studies, the uniqueness of
autoantibody-defined epitopes on antigens has been
striking. One feature is the highly conserved nature of
this region in contrast to other potentially immunogenic
regions of an antigen. For example, in studies on autoantibodies to SSB/La, a nuclear protein antigen in patients
with Sjögren syndrome, 5 different monoclonal antibodies derived by experimental immunization with purified
nuclear SSB/La were all highly reactive in a number of
immunologic assays, but all 5 recognized epitopes that
were not identical with the highly conserved epitope
recognized by human autoantibodies.
Another unique feature of autoantibody-defined
epitopes is that the epitopes reside at or are close to
the functional regions or binding sites of the relevant
antigens. For example, we found that human autoanti-
A
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
287
bodies to proliferating cell nuclear antigen can inhibit
the ability of DNA polymerase δ to use template/primers
of single-stranded nucleotides for DNA replication.
Similarly, human autoantibodies to certain tRNA synthetases can inhibit the aminoacylation function of
these synthetases.
These findings led to the further observation that
the epitope defined by an autoantibody is often a conformation-dependent discontinuous sequence on the protein antigen. These observations might be useful clues
for the design of peptides for immunotherapy. Use of
certain regions as immunotherapeutic agents in vaccines
might not be appropriate because these regions might
not be recognized by the host’s immune system. It might
be important to discover how the host has responded to
a self-antigen, because the host’s immune system could
be indicating that it can make an immune response to a
specific region of an autoantigen but cannot or is unwilling to make immune responses to other regions.
PUBLICATIONS
Himoto, T., Kuriyama, S., Zhang, J.-Y., Chan, E.K.L., Nishioka, M., Tan, E.M. Significance of autoantibodies against insulin-like growth factor II mRNA-binding proteins in patients with hepatocellular carcinoma. Int. J. Oncol. 26:311, 2005.
Megliorino, R., Shi, F.-D., Peng, X.-X., Wang, X., Chan, E,K.L., Tan, E.M., Zhang, J.-Y.
Autoimmune response to anti-apoptotic protein survivin and its association with antibodies to p53 and c-myc in cancer detection. Cancer Detect. Prev. 29:241, 2005.
Shi, F.-D., Zhang, J.-Y., Liu, D., Rearden, A., Elliott, M., Nachtsheim, D., Daniels, T.,
Casiano, C.A., Heeb, M.J., Chan, E.K.L., Tan, E.M. Preferential humoral immune
response in prostate cancer to cellular proteins p90 and p62 in a panel of tumorassociated antigens. Prostate 63:252, 2005.
Tan, E.M. The importance of the autoantibody-defined epitope. In: Autoantibodies
and Autoimmunity: Molecular Mechanisms in Health and Disease. Pollard, K.M.
(Ed.). Wiley-VCH, New York, in press.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.