CVR-2012-183R2
SUPPLEMENTAL MATERIALS AND METHODS
17-Estradiol induced interaction of ER with NPPA regulates gene expression in
cardiomyocytes
Running title: Interaction of ER/NPPA in the heart
Shokoufeh Mahmoodzadeh1, Thi Hang Pham1, Arne Kuehne1, Britta Fielitz1, Elke
Dworatzek1, Georgios Kararigas1, George Petrov2, Mercy M. Davidson3, Vera RegitzZagrosek1, 2
1
Institute of Gender in Medicine, Charité–Universitaetsmedizin Berlin, Germany, 2German
Heart Institute Berlin (DHZB), Berlin, Germany,
3
Department of Radiation Oncology,
Columbia University, New York, USA.
Corresponding Author:
Shokoufeh Mahmoodzadeh
Institute of Gender in Medicine
Center for Cardiovascular Research
Hessische Str. 3-4, 10115 Berlin, Germany
Tel.: +49 (0)30 450 525 187
Fax: +49 (0)30 450 525 943,
E-mail: s.mahmoodzadeh@charite.de
Index:
I)
Supplemental materials and methods
II)
Supplemental Figures
III)
Supplemental table 1
IV)
Reference
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CVR-2012-183R2
I) Supplemental materials and methods:
Study approval: Study approval: Atrium tissues from 5 women undergoing aortic valve
replacement for isolated aortic valve stenosis were obtained after written informed consent
had been given. Human LV myocardial samples used in this study were composed of tissue
samples of non-used donor hearts with originally normal systolic cardiac function, no history
of cardiac disease and normal postmortem histology. However, they did not qualify for
transplantation at time of organ harvesting because of functional reasons. All subjects were
Caucasian. The study followed the rules of the Declaration of Helsinki and was approved by
the ethical committee of Charité university medicine. All animal experiments (isolation of
cardiac myocytes from 1 to 3-day-old Spargue-Dawley rat hearts and LV tissue from ERKO
mice) were approved by the State Agency for Health and Social Affairs (LaGeSo, Berlin,
Germany) for the care and use of laboratory animals.
Cells Culture and Treatment: For in-vitro investigations, we used a human adult left
ventricular cardiomyocyte-like cell line, the AC16 cells1, because this cell line contains
cardiac- and muscle-specific markers, which are a good indication for the presence of a
cardiac transcription program. Additionally, our recent study showed that AC16 cells express
ER.2 Therefore, this cell line was proposed to be an appropriate model for cardiomyocytes
and for studying the effects of E2 and ER. AC16 cells were grown in DMEM/F12
(InvitrogenTM), supplemented with 12.5% FBS (PAA Laboratories), penicillin/streptomycin
(100 U/mL, 100 U/mL, PAA) and Amphotericin B (0.25 µg/mL, Invitrogen) at 37°C in 5% CO2.
Prior to E2-treatment, cells were starved with 2.5% charcoal stripped FBS (cs FBS ) in
phenol-red free DMEM/F12 for 24h, subsequently incubated with the physiological
concentration of E2 (10-8 mol/L; Sigma) or ethanol as vehicle for additional 24h at 37°C in 5%
CO2. ICI 182780 (10-5 mol/L, Tocris) treatment was performed 1h before E2 treatment.
Neonatal rat cardiac myocytes (NNRCM) were isolated from 1 to 3-day-old Spargue-Dawley
rat hearts as described elsewhere.3 Briefly, cells were cultured on gelatin-coated plates
(0.2%, Sigma) in DMEM containing 10% FBS, L-glutamin (2mmol/L), and penicillin-
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streptomycin at 37°C in 5% CO2. After overnight plating, cardiomyocytes were cultured in
phenol red-free DMEM 2.5% cs.FBS for 24 hours, followed by a 24h incubation with vehicle
(ethanol) or E2 (10-8 mol/L) at 37°C in 5% CO2, prior to immunostaining.
Plasmid Construction: For the yeast two-hybrid screening, the full-length (FL) coding
region of human ER and ER-EF domain cDNAs were amplified from the pSG-Hego vector
(hER vector; a kind gift of Prof. P. Chambon, France) as template, using primers containing
BamHI/EcoRI sites (for primer sequences see Table 1) by PCR. The PCR products were
cloned into BamHI/EcoRI restriction site of the lexA DNA-BD of the bait vector pBTM116 (BD
Clontech) to generate hER-FL-pBTM116 (ER-FL), and hER-EF-pBTM116 (ER-EF),
respectively. The prey vector NPPA-MUT-pACT2 was constructed by using the QuikChange®
Site-Directed Mutagenesis Kit (Stratagene) with NPPA-WT-pACT2 vector as template that
was isolated as prey from the human heart BD MatchmakerTM cDNA library (for primer see
Table 1). In the NPPA-WT-pACT2 vector, the last two leucine amino acids of the LXXLL
motif of NPPA (amino acids 118 and 119) were exchanged to arginine and proline amino
acids.
For generation of expression vectors containing the full-length human wild type NPPA
and mutated NPPA-LXXLL sequences, respectively, NPPA-WT and NPPA-LXXLL-MUT
cDNA fragments were amplified using appropriate primers (for primer see Table 1) from
NPPA-WT-pACT2 and NPPA-MUT-pACT2 vectors, respectively, as templates. Both
amplified NPPA cDNA fragments were subsequently subcloned in frame into the
BamHI/AgeI-restriction site of the pcDNA™3.1D/V5-HIS/lacZ vector (Invitrogen™) to
generate NPPA-WT-pcDNA3.1 (NPPA-WT) and NPPA-LXXLL-MUT-pcDNA3.1 (NPPA-MUT)
constructs.
To generate the luciferase reporter construct pGL2-1200-prom, the NPPA promoter
sequence -1200bp/+1bp (relative to the transcription start site), was amplified from genomic
DNA isolated from AC16 cells as template by PCR (for primer see table 1), and subcloned
into the SacI/HindIII site of the pGL2-basic vector (Promega).
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Immunofluorescence (IF) and Confocal Microscopy:
AC16 cells or neonatal rat cardiomyocytes (NNRCM) were grown on 8-chamber culture
slides (BD Bioscience) at a density of 2x105 cells/chamber. Additionally, for colocalization
studies, AC16 cells were also co-transfected with pSG5-Hego vector (ER-vector; containing
Fl-ER) or pFLAG-CMV4-ER (a kind gift from Prof. O. Huber, Jena, Germany, containing
Fl-ER with a flag-tag) and NPPA-WT or NPPA-MUT vectors (containing His-tag). Empty
pCDNA3.1 vector was added to maintain a consistent amount of DNA used for transfection.
Cells were cultured and treated as described above. Subsequently, cells were fixed,
permeabilized and blocked as described previously.2 After blocking, the cells were incubated
with the primary antibodies anti-ER (SRA-1010, C-542, monoclonal mouse antibody, Enzo
life science), and anti-NPPA (FL-153, polyclonal rabbit antibody, Santa Cruz), or anti-Flag
(F1804, Monoclonal mouse ANTI-FLAG M2, Sigma), and anti-His (H-15, sc-803, rabbit
polyclonal antibody, Santa Cruz), at 4°C overnight, before they were incubated with
appropriate secondary antibodies conjugated with either FITC or Cy3 (Jackson
ImmunoResearch Laboratories) at RT for 1h. As negative controls, the primary, secondary or
both antibodies were omitted in the procedure. The nuclei were counterstained with
diamidino-phenylindole (DAPI, DAKO). Subsequently, after several washing steps, slides
were mounted with Vectashield mounting medium for fluorescence (H-1000, Vectashield,
Vector Laboratories). Confocal images were acquired using a Leica TCS-SPE spectral laser
scanning microscope, and images were processed by Leica Application Suite AF software
(version 1.8.0).
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II) Supplemental Figures
- Figure S1:
120
100
80
0
ER
ER +
NPPA-WT
ER +
NPPA-MUT
ER
ER +
NPPA-WT
ER +
NPPA-MUT
rel. mRNA expression of GATA4 / HPRT (% of controls)
B
rel. mRNA expression of BNP/ HPRT (% of controls)
A
120
100
80
0
ERa
ERa +
NPPA-WT
ERa +
NPPA-MUT
rel. mRNA expression of TGF/ HPRT (% of controls)
C
120
100
80
0
Figure S1: Effects of E2-induced interaction of NPPA with E2/ER on transcription of BNP,
GATA4 and TGF. AC16 cells were transiently transfected with ER or co-transfected with
ER and NPPA-WT or NPPA-MUT vectors. After 24h, cells were exposed to E2 or vehicle
for 6h (GATA4) or 24h (BNP and TGF). Total RNA was isolated and quantitative PCR were
performed using specific primers for BNP (A), GATA4 (B), and TGF (C). Data represent
mean ± SEM (n=4, each in duplicate).
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- Figure S2:
LXXLL
NPPA
E2
ERα
E2
a
ERα
LXXLL
NPPA
E2
ERα
E2
LXXLL
ERα
NPPA
NPPA
nu
cle
us
LXXLL
b
LXXLL
NPPA
Figure S2: Proposed model of action: Proposed model for E2-dependent interaction of
ER with NPPA in cardiomyocytes. a) E2-activated ER enhances the transcriptional activity
of NPPA promoter through binding to the ERE within the NPPA promoter. b) NPPA interacts
via its LXXLL motif with E2-activated ER. The E2-ER/NPPA-protein complex represses
the E2/ER-induced NPPA gene expression by preventing the binding of ER to the NPPA
promoter.
III) Supplemental table S1:
List of all primers used in this study:
Primers used in the Y2H assay:
- To generate hER-FL-pBTM116 and hER-EF-pBTM116 constructs, we used the human
ER sequence (ESR1, NM_000125, hER) as a reference sequence for primer design.
Enzyme-restriction sites are underlined.
primer
Product
Primer sequence [5’ o 3’]
Enzyme
hER-FW1
hERα-FL
(1808 bp)
TACAGAATTCATGACCATGACCCTCC
EcoRI
AAATGGATCCTCAGACCGTGGCAGGG
BamHI
hER-RV1
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hER-FW2
hERα-EF
(878 bp)
hER-RV2
TAATGAATTCACGGCCGACCAGATGG
EcoRI
AAATGGATCCTCAGACCGTGGCAGGG
BamHI
- The human NPPA sequence (NM_006172) was used as a reference sequence for primer
design.
primer
primer sequence [5’ o 3’]
NPPA-FW
agctgagggcgcggcccactgcccctcg
aa-exchange
Leucin to Arginin
NPPA-RV
tcgactcccgcgccgggtgacggggagc
Leucin to Prolin
Both primers were designed using the QuikChange ® Primer Design Program from
Stratagene company http://www.stratagene.com/qcprimerdesign. The LXXLL motif within the
NPPA was mutated (red color). The prey vector NPPA-WT-pACT2 served as template.
Primers for sequencing:
The following primers were used to verify the recombinant vectors. For the confirmation of
the inserts in the pCR ® 4-TOPO ® vectors, we used commercially available standard primer
pairs M13-FW and M13-RV and gene-specific primers. For the identification of the prey
inserts in pACT2 vectors, we used either GAL4AD-sequencing primers; the 5'LD
Matchmaker insert, or the insert screening amplimer 3'LD.
Primer
primer sequence [5’ o 3’]
M13 FW
GTAAAACGACGGCCAG
M13 RV
CAGGAAACAGCTATGAC
GAL4 AD
ATACCACTACAATGGA
Matchmaker 5´LD-Insert
CTATTCGATGATGAAGATACCCCACCAAACC
Matchmaker 3´LD-Insert
TCGTAGATACTGAAAAACCCCGCAAGTTCAC
hESR1-FL-FW1
ATGACCATGACCCTCCACA
hESR1-FL-RV1
TCAGACCGTGGCAGGGAAAC
hESR1-EF-FW2
TGACGGCCGACCAGATGGTCA
hESR1-EF-RV2
CTTCAGGGTGCTGGACAGA
Primers for generation of expression vectors:
Primer pairs used for generation of NPPA-WT-pcDNA3.1 (NPPA-WT) and NPPA-LXXLLMUT-pcDNA3.1 (NPPA-MUT) vectors:
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primer
product
primer sequence [5´to 3`]
Enzyme
NPPA-FW
NPPA-WT
(453 bp)
AATAGGATCCATGAGCTCCTTCTCCA
BamHI
ATATACCGGTGTACCGGAAGCTGTT
AgeI
NPPA-MUT
(453 bp)
AATAGGATCCATGAGCTCCTTCTCCA
BamHI
ATATCTCGAGTCAGTACCGGAAGCTGTT
XhoI
NPPA-RV
NPPA-MUT-FW
NPPA-MUT-RV
Primers for the synthesis of NPPA promoter fragment:
The following primers were used to generate the NPPA promoter construct: pGL2-1200-prom
(-1200bp/+1bp; relative to the transcription start site):
primer
promNPPA-FW
promNPPA-RV
product
NPPA-1200prom
(1200 bp)
primer sequence [5´to 3`]
Enzyme
GTCCGAGCTCATTGAATGGTAATGGC
SacI
ATACAAGCTTTGCTGGCGTCGTCAAG
HindIII
Primers for quantitative Real-Time polymerase Chain Reaction (qRT-PCR):
The qRT-PCR was performed with gene-specific, intron-spanning primers. The annealing
temperature was 60°C.
primer
h-NPPA
HPRT
hCx43
hNFATc4
hTGF1
hACTN2
hGATA4
primer sequence [5’ o 3’]
FW: TCTGCCCTCCTAAAAAGCAA
RV: TCAGTACCGGAAGCTGTTACAG
FW: CTTTGCTGACCTGCTGGATT
RV: TATGTCCCCTGTTGACTGGT
FW: CTTTGCTGACCTGCTGGATT
RV: TATGTCCCCTGTTGACTGGT
FW: CCGAGACAGTGTCCCTATCC
RV: TCGGCCAATGATCTCACTC
FW: TCTCCGTGGAGCTGAAGCAATAGT
RV: CACACTGCAAGGTGGACATCAACG
FW: GACCCTGGGTATGATCTGGA
RV: GCTGGTGTGGAAGTTCTG
FW: GGAAGCCCAAGAAACCTGAAT
RV: GGGAGGAAGGCTCTCACTG
hBNP
#HS001735910_m1 (Life Technologies)
hCol I
#Hs01076777_m1 (Life Technologies)
hCol III
#Hs00943782_m1 (Life Technologies)
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Primers for ChIP assay:
primer
primer sequence [5’ o 3’]
NPPA-promR2
FW: ggaaggatgtagaaggaattg
NPPA-promR1
FW: acttgctgcctgttatttcc
NPPA-promR5
FW: cggtgagataaccaaggact
c-Myc
TFF1
RV: caggcttagaggacgcagcc
RV. Atgccttctacatccttccc
RV: cagaccctcagctgcaaga
FW: gagcagcagagaaagggaga
RV: cagccgagcactctagctct
FW: ccggccatctctcactatgaa
RV: cctcccgccagggtaaatac
The primer specific to a proximal region of the human GAPDH promoter was provided by
Diagenode (included in the Transcription Factor ChIP Kit).
IV) References:
1.
2.
3.
Davidson MM, Nesti C, Palenzuela L, Walker WF, Hernandez E, Protas L et al. Novel
cell lines derived from adult human ventricular cardiomyocytes. J Mol Cell Cardiol
2005;39:133-147.
Mahmoodzadeh S, Fritschka S, Dworatzek E, Pham TH, Becher E, Kuehne A et al.
Nuclear factor-kappaB regulates estrogen receptor-alpha transcription in the human
heart. J Biol Chem 2009;284:24705-24714.
Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev
Physiol 2003;65:45-79.
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