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Cardiovascular Research 2002 53(3):728-739; doi:10.1016/S0008-6363(01)00525-9
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Copyright © 2002, European Society of Cardiology

Ovarian hormones induce TGF-β3 and fibronectin mRNAs but exhibit a disparate action on cardiac fibroblast proliferation

Isabelle Merciera,b, Federico Colomboa,b, Sylvie Maderc and Angelino Calderonea,b,*

aDepartément de Physiologie, Université de Montréal, Montréal, Québec, Canada
bInstitut de Cardiologie de Montréal, 5000 rue Belanger est, Montréal, Québec, Canada H1T 1C8
cDepartément de Biochimie, Université de Montréal, Montréal, Québec, Canada

* Corresponding author. Tel.: +1-514-376-3330x3710; fax: +1-514-376-1355 calderon{at}icm.umontreal.ca

Received 6 June 2001; accepted 22 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Prior to menopause, women have a lower risk of cardiovascular disease compared to age-matched men. Despite the well-documented beneficial physiological effects of ovarian hormones on vascular reactivity and growth, very little is known with regard to the direct action on cardiac cells. Objective: The following study examined the pattern of ovarian hormone receptor subtype expression in cardiac fibroblasts, the modulator role of 17β-estradiol and progesterone on growth and their respective influence on putative molecular events of extracellular matrix remodeling. Methods and results: Neonatal rat cardiac fibroblasts were isolated from 1- to 3-day-old Sprague–Dawley rats. Immunofluorescence and Western blot analysis revealed the presence of estrogen receptor-{alpha} (ER-{alpha}), and -β (ER-β) subtypes, with the ER-{alpha} subtype localized on the plasma membrane. Likewise, both progesterone receptor-A (PR-A), and -B (PR-B) subtypes were expressed in cardiac fibroblasts, and the PR-B appeared to be the predominant subtype associated with the plasma membrane. Despite the presence of both ER subtypes, the treatment of cardiac fibroblasts with 1 µM 17β-estradiol exerted a modest decrease in DNA synthesis. By contrast, progesterone treatment caused a dose-dependent decrease in [3H]thymidine uptake, without a concomitant induction of apoptosis. The progesterone-mediated decrease in DNA synthesis was associated with the upregulation of the cyclin-dependent kinase inhibitor p27Kip1, whereas p21cip and proliferating cell nuclear antigen protein levels were unchanged. Lastly, despite the modest effect on DNA synthesis, 17β-estradiol increased the steady-state mRNA levels of transforming growth factor-β3 and fibronectin. Likewise, progesterone increased the expression of both transforming growth factor-β3, and fibronectin mRNA. Conclusion: Collectively, these data are the first to highlight the presence of estrogen and progesterone receptor subtypes on the plasma membrane of neonatal rat cardiac fibroblasts, and further underscore the ability of ovarian hormones to directly suppress DNA synthesis, and influence putative molecular events associated with extracellular matrix remodeling.

KEYWORDS Fibrosis; Gender; Hormones; Receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In pre-menopausal women, the incidence of coronary artery disease and hypertension are lower, compared to age-matched men [1]. These findings are consistent with the premise that ovarian hormones, in particular 17β-estradiol, are cardioprotective in nature. Indeed, hormonal replacement therapy (HRT) in post-menopausal women was associated with a 30–50% decrease in the mortality rate associated with cardiovascular disease, compared to untreated post-menopausal subjects [2]. Based on these clinical observations, the use of HRT is gaining widespread consideration as a potentially important therapeutic approach to limit the incidence of cardiovascular disease in post-menopausal women.

A plethora of studies have attempted to elucidate the mechanism(s) by which 17β-estradiol prevents and/or limits the incidence of coronary artery disease. Important physiological actions of 17β-estradiol include ameliorating the HDL/LDL ratio, reducing vascular tone via the synthesis of the vasodilatory substances nitric oxide, and prostacyclin from endothelial cells, and concomitantly inhibiting the generation of the vasoconstrictors angiotensin II and endothelin-1 [3–8]. In addition, in a coronary artery preparation, 17β-estradiol acted as a calcium channel antagonist inhibiting the contractile response of endothelin-1 [9]. Lastly, both ovarian hormones can potentially limit coronary artery disease via the direct inhibition of vascular smooth muscle cell proliferation [10,11].

Very little is known with regard to the direct effect of 17β-estradiol and progesterone on cardiac cell growth and phenotype. In the adult heart, cardiac fibroblasts constitute 90% of the non-myocyte cells, and ovarian hormone receptors have been detected [12]. Previous studies have demonstrated that the exogenous administration of 17β-estradiol either decreased, increased, or had no effect on DNA synthesis in cultured cardiac fibroblasts [13–15]. By contrast, at least one study demonstrated the exogenous administration of progesterone attenuated DNA synthesis in adult rat cardiac fibroblasts [13]. Thus, the following study attempted to better characterize the effect of ovarian hormones on neonatal rat cardiac fibroblast growth, and elucidate the role of cell cycle proteins in this process. Secondly, identify the isoform and subcellular distribution of ovarian hormone receptors in neonatal rat cardiac fibroblasts. Lastly, examine the influence of ovarian hormones on the expression of putative events associated with extracellular matrix remodeling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cultured neonatal rat cardiac fibroblasts
Neonatal rat ventricular fibroblasts were isolated from 1- to 3-day-old Sprague–Dawley rat pups (Charles River Canada; St Constant, Québec), as previously described [16]. Experiments were performed in accordance with the principles of the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the Montreal Heart Institute. The primary culture of neonatal rat cardiac fibroblasts were maintained in phenol red-free DMEM containing 7% heat-inactivated FBS (previously treated with 1% activated carbon Norit SA3, Aldrich, and 0.1% Dextran T70, Pharmacia, in order to remove endogenous steroid hormones) until confluent (5–7 days). Thereafter, confluent cells were passaged with trypsin–EDTA (0.25%, 0.53 nM; GIBCO BRL), and plated for a period of 24–36 h in phenol red-free DMEM containing 7% FBS. Cardiac fibroblasts were subsequently washed and the media was changed to phenol red- and serum-free DMEM containing ITS (insulin (5 µg/ml); transferrin (5 µg/ml); sodium selenite (5 ng/ml) for 48 h prior to the experimental protocol. All experiments were performed on first and second passaged cells.

2.2. Fractionation of cardiac fibroblast lysates into cytosolic and particulate fractions, and the subsequent detection of estrogen and progesterone receptor subtypes
Cardiac fibroblasts were plated at a density of 100–200 cells/mm2 for 48 h in phenol red-free DMEM containing 7% FBS serum, subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h. Cardiac fibroblasts were subsequently washed twice with PBS (pH 7.4), and resuspended in 1 ml of a buffer containing 20 mM Hepes (pH 7.5), 20 mM β-glycerophosphate, 20 mM NaF, 0.2 mM Na3VO4, 5 mM EDTA, 5 mM EGTA, 0.5 mM PMSF, 25 µg/ml leupeptin, and 5 mM DTT (Buffer A). A fraction of the homogenate was treated with 2% Triton X-100 to obtain the whole cell extract and the remainder of the homogenate was centrifuged for 30 min at 100 000xg (4 °C) to yield supernatant (cytosolic) and pellet fractions. The pellet was resuspended in Buffer A plus 1% (v/v) Triton X-100, and centrifuged as before to yield a supernatant containing the particulate fraction [18]. Total, cytosolic and particulate fractions (~300 µg) were subjected to SDS–polyacrylamide gel (10%) electrophoresis, and subsequently transferred to Hybond-C membrane (Amersham Canada Limited). Equal loading of the samples was confirmed with Ponceau S staining. The membrane was pre-incubated in 10 mM Tris pH 7.4, 150 mM NaCl and 0.1% Tween (v/v) (TBS-T buffer) containing 3% skim milk for 1 h at room temperature, and subsequently incubated overnight at 4 °C with either 1 µg/ml of a rabbit-polyclonal antibody directed against estrogen receptor-{alpha} (ER-{alpha}; Santa Cruz; MC-20, SC-542), a goat-polyclonal antibody directed estrogen receptor-β (ER-β; Santa Cruz; L-20, SC-6822), or a rabbit-polyclonal antibody which recognized both progesterone receptor-A (PR-A) and PR-B subtypes (Santa Cruz; C-20, SC-539). Following overnight incubation, the membranes were washed three times with TBS-T containing 3% skim milk, and subsequently incubated for 1 h at room temperature with the appropriate secondary antibody (1:10 000) conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Following incubation, the membranes were washed three times with TBS-T and the bands were detected by autoradiography using the ECL detection kit (Amersham Canada Limited). Films were scanned with a laser densitometer (Chemilmager 4000 v4.04 software; Alphan Innotech Corporation).

2.3. Immunofluorescent staining of estrogen and progesterone receptors
Cardiac fibroblasts were plated on glass coverslips in a six-well plate (100–200 cells/mm2) containing DMEM–7% FBS, and subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h prior to the experimental protocol. Cells were fixed for 10 min in PBS (pH 7.4)–paraformaldehyde (4%), and then incubated for 10 min with 50 mM NH4Cl. Fixed cells were permeabilized with 1% Triton X-100/PBS for 5 min, washed with PBS, and subsequently treated with PBS-2% BSA for 30 min. Cells were incubated overnight at 4 °C with a polyclonal rabbit anti-ER-{alpha} (Santa Cruz; 1/50: MC-20, SC-542) antibody, or a polyclonal rabbit anti-progesterone receptor (Santa Cruz; 1/50: C-20, SC-539) antibody. Cells were subsequently washed with PBS, treated for 30 min with PBS-2%, and incubated for 1 h with anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC; 1:100; Santa Cruz). The coverslips were mounted on DABCO medium (0.2 µM; Sigma), and cells were observed with a 40x-oil objective mounted on a Zeiss Axiovert 100M confocal microscope.

2.4. DNA and protein synthesis
Cardiac fibroblasts were plated on 24-well plates at a density of 100–200 cells/mm2 for 24 h in phenol red-free DMEM containing 7% FBS, and subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h prior to the experimental protocol. Cardiac fibroblasts were subsequently treated for a period of either 24 or 48 h with either 17β-estradiol (1–1000 nM; Sigma), or progesterone (1–250 nM; Sigma). The stock concentration of ovarian hormones was dissolved in DMSO. The final dilution of DMSO at 1 µM was 1:10 000, which had no effect on cell growth. In parallel experiments, cardiac fibroblasts were treated with either TGF-β1 or TGF-β3 (R&D Systems; Minneapolis, MN) for a period of 24 h. DNA synthesis was assessed by the addition of 1 µCi/ml of [3H]thymidine (ICN Biomedicals Inc., Costa Mesa, CA) for a period of 4–6 h prior to the end of the treatment protocol. Protein synthesis was assessed by the addition of 2 µCi/ml of [3H]Leucine (ICN Biomedicals Inc., Costa Mesa, CA) for a period of 24 h. Cells were washed twice with PBS (4 °C), and cold 5% TCA was added for 30 min to precipitate either DNA or protein. The precipitates were washed twice with cold water and resuspended in 0.4 M NaOH. Aliquots were counted in a scintillation counter.

2.5. TUNEL assay
Cardiac fibroblasts were plated on glass coverslips in a six-well plate (100–200 cells/mm2) containing DMEM–7% FBS, and subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h prior to the experimental protocol. Cells were fixed for 10 min in PBS (pH 7.4)–paraformaldehyde (4%), and incubated for 10 min in 50 mM NH4Cl. Fixed cells were permeabilized with saponin solution (0.075% saponin/1 µM EGTA) for 15 min at room temperature and washed with PBS. The cells were subsequently treated with RNase (5 mg/ml; Sigma) to digest all non-DNA material. The TUNEL assay was performed according to the instructions of the manufacturer (Boehringer-Mannheim, Germany). The reaction was terminated following the addition of a stop solution (500 mg of powdered skim milk in 20 ml of sodium chloride (0.15 M)/sodium citrate (0.1 M) (pH 7; SSC 4x). The nuclei were stained following the addition of 4 µl of extrAvidin-FITC (Sigma) per 200 µl of staining solution (500 mg of dry skim milk, 0.1% Triton X-100 in 10 ml of SSC 4x) for 30 min at room temperature. Following the TUNEL assay, the nuclei were co-stained with 0.01 µM of propidium iodide (Sigma) for 15 min at room temperature. The coverslips were mounted on DABCO medium (0.2 µM), and the cells were observed with a 40x-oil objective mounted on a Zeiss Axiovert 100M confocal microscope.

2.6. Western blot analysis of cell cycle proteins
Cardiac fibroblasts were plated in 100-mm plates at a density of 100–200 cells/mm2 in phenol red-free DMEM containing 7% FBS for 48 h, subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h. Following the experimental protocol, cardiac fibroblasts were washed with PBS (pH 7.4, 4 °C), and lysed in a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 20 mM β-glycerophosphate, 0.5 mM phenylmethylsulfonyl fluoride 1 mM sodium vanadate, 1% Triton X-100, 0.5% nonidet P-40, and 1 µg/ml of leupeptin and aprotinin. Cardiac fibroblasts were scraped, the lysate centrifuged for 5 min, and the supernatant was frozen and stored at –80 °C. BioRad assay was used to determine protein content, and 100 µg of cardiac fibroblast lysate was subjected to SDS–polyacrylamide gel (10%) electrophoresis, and subsequently transferred to Hybond-C membrane (Amersham Canada Limited). Equal loading of the samples was confirmed with Ponceau S staining. The membrane was pre-incubated in 10 mM Tris pH 7.4, 150 mM NaCl and 0.1% Tween (v/v) (TBS-T buffer) containing 3% skim milk for 1 h at room temperature, and subsequently incubated overnight at 4 °C with either 1 µg/ml of a goat-polyclonal antibody directed against p21cip1 (Santa Cruz; C-19; SC-397), goat-polyclonal antibody directed against p27Kip1 (Santa Cruz; C-19; SC-528), or a rabbit polyclonal antibody directed against proliferating cell nuclear antigen (PCNA; Santa Cruz; FL-261; SC-7907). Following overnight incubation, the membranes were washed three times with TBS-T buffer containing 3% skim milk, and subsequently incubated for 1 h at room temperature with either a rabbit anti-goat (1:10 000; Santa Cruz Biotechnology) or goat-anti-rabbit antibody (1:10 000; Santa Cruz Biotechnology) conjugated to horseradish peroxidase. Following incubation, the membranes were washed three times with TBS-T, and the bands were detected by autoradiography using the ECL detection kit (Amersham Canada Limited). Films were scanned with a laser densitometer (Chemilmager 4000 v4.04 software; Alphan Innotech Corporation).

2.7. Northern hybridization
Cardiac fibroblasts were plated in 100-mm plates at a density of 100–200 cells/mm2 in phenol red-free DMEM containing 7% FBS until confluent (5–7 days). Cells were subsequently washed and maintained in phenol red- and serum-free DMEM containing ITS for 48 h prior to the experimental protocol. Total mRNA was isolated by a modification of the technique of Chomczynski and Sacchi [17]. A 1.2-kb fragment of mouse TGF-β3 (American Type Culture Collection (ATCC); Rockville, MD), a 2.4-kb fragment of rat TGF-β1(ATCC), a 0.6-kb fragment of rat fibronectin (courtesy of Dr R.O. Hynes) and a 2-kb fragment of rat GAPDH (ATCC) were labeled with [32P]dCTP to a specific activity of 1–2x106 cpm/ng cDNA by the random hexamer (Pharmacia) priming method and hybridized to nylon membranes for 18–24 h at 42 °C as previously described [16]. The filters exposed to the cDNA probes were washed twice (15 min, room temperature) with 300 mmol/l NaCl/30 mmol/l trisodium citrate and 0.1% SDS, and twice (15 min, 45 °C) with 30 mmol/l NaCl/3 mmol/l trisodium citrate and 0.1% SDS. Nylon membranes were subsequently exposed to Kodak XAR film at –70 °C, and films were scanned with a laser densitometer (Chemilmager 4000 v4.04 software; Alphan Innotech Corporation). All levels of mRNA reported in this paper are normalized to the level of GAPDH mRNA.

2.8. Statistics
Data are represented as the mean±S.E.M. and (n) represents an independent preparation of neonatal rat cardiac fibroblasts. Statistical analysis of dose–response curves on growth were assessed by a one-way ANOVA, whereas cell cycle protein and mRNA expression were evaluated by a Student's unpaired t-test. A value of P<0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. The subcellular localization of progesterone and estrogen receptor subtypes
Western blot analysis revealed the expression of both progesterone receptor-A (PR-A) and progesterone receptor-B (PR-B) subtypes in cardiac fibroblasts (n=3). The fractionation of whole cell lysates detected a signal in the cytosolic fraction, and a modest level of expression in the particulate fraction corresponding to the reported ~114 kDa of the PR-B receptor subtype [19,20] (Fig. 1). In addition, a doublet of ~82 and ~86 kDa was identified in the cytosolic fraction, and corresponded to the PR-A receptor subtype (Fig. 1). Interestingly, a similar immunoreactive doublet (~82 and ~86 kDa) was previously reported for the PR-A receptor in breast tumour cells, as well as in a progestin-responsive breast cancer cell line T47D [19,20]. Immunofluorescence experiments revealed an intense perinuclear/nuclear signal with the progesterone receptor antibody, and further highlighted a modest signal on the plasma membrane (n=3) (Fig. 2). This latter observation was consistent with the Western blot data revealing the expression of the PR-B subtype in the particulate fraction. The estrogen receptor-β (ER-β) subtype antibody revealed a single immunoreactive band of ~59 kDa localized predominantly in the cytosolic fraction (n=3) (Fig. 1), a finding consistent with the reported molecular weight of ER-β (~54 kDa) [21]. Lastly, an antibody directed against the estrogen receptor-{alpha} (ER-{alpha}) subtype detected an immunoreactive doublet at ~68 kDa in both the cytosolic and particulate fractions (n=3) (Fig. 1) [22,23]. Likewise, in the rabbit uterus, an immunoreactive doublet for the ER-{alpha} subtype was also observed [23]. Moreover, these latter findings were confirmed by immunofluorescence, as the ER-{alpha} subtype was detected predominantly in the nucleus, and a modest signal was observed associated with the plasma membrane (n=3) (Fig. 2).


Figure 1
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  		Fig. 1 Subcellular localization of progesterone and estrogen receptor subtypes. (Panels A and B) An immunoreactive band of ~114 kDa was detected in both the cytosolic and particulate fractions, whereas a doublet of ~82 and ~86 kDa was detected exclusively in the cytosolic fraction. These immunoreactive bands correspond to the progesterone receptor-B (PR-B), and progesterone receptor-A (PR-A) subtypes, respectively. (Panel C) An immunoreactive band of ~59 kDa, corresponding to estrogen receptor-β (ER-β) subtype was detected exclusively in the cytosolic fraction. (Panel D) An immunoreactive doublet at ~68 kDa was detected in both the cytosolic and particulate fractions, and corresponds to the estrogen receptor-{alpha} subtype (ER-{alpha}). This pattern of ovarian hormone receptor expression was observed in three separate experiments.

 

Figure 2
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  		Fig. 2 Immunofluorescence approach to assess ovarian hormone receptor localization in cardiac fibroblasts. (Panel A) In untreated cardiac fibroblasts, an intense immunoreactive signal of the estrogen receptor-{alpha} subtype was detected in the nucleus, whereas a modest level of expression was observed on the plasma membrane (indicated by arrow) (n=3). (Panel B) An intense immunoreactive signal of the progesterone receptor was detected in the perinuclear/nuclear regions, and a modest level of expression was observed on the plasma membrane (indicated by arrow) (n=3).

 
3.2. Effect of ovarian hormones on DNA synthesis and apoptosis in cardiac fibroblasts
The treatment of cardiac fibroblasts with progesterone for 24 h resulted in a significant dose-dependent decrease in basal [3H]thymidine uptake (n=5; P<0.05), with a maximal effect observed at 250 nM. This antiproliferative effect of progesterone was maintained following a 48 h treatment (n=3; P<0.05) (Fig. 3). In parallel experiments the progesterone antagonist RU486 (100 nM) inhibited the subsequent increase in [3H]thymidine uptake, and progesterone treatment had no effect on protein synthesis (250 nM dose; n=3; 1±1% change vs. basal). Moreover, a 48 h treatment with 250 nM progesterone had no effect on protein synthesis (data not shown). To assess the potential contribution of apoptosis to the growth-suppressing action of progesterone, the TUNEL assay was performed. A 24 h treatment with a pharmacological dose of progesterone (1 µM) did not promote DNA fragmentation (n=2), whereas the exposure to UV (30 min) resulted in a robust staining of the nuclei, indicative of apoptosis (n=4) (Fig. 4). Although, both ER-receptor subtypes were expressed in neonatal rat cardiac fibroblasts, the 24 h treatment with 17β-estradiol caused a non-significant decrease in DNA synthesis following a 24 h treatment (n=4) (Fig. 3). However, the treatment of cardiac fibroblasts with a pharmacological dose (1 µM) of 17β-estradiol exerted a modest significant decrease in [3H]thymidine uptake (36±8% decrease vs. basal; n=4: P<0.05).


Figure 3
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  		Fig. 3 Progesterone rather than 17β-estradiol decreased [3H]thymidine uptake in cardiac fibroblasts. (Panel A) The treatment of cardiac fibroblasts with progesterone for 24 h (n=5 separate experiments) resulted in a significant dose-dependent decrease in basal [3H]thymidine uptake (n=5; P<0.05). Moreover, following a single application of progesterone, the dose-dependent suppression of DNA synthesis was maintained for 48 h (n=3; P<0.05). (Panel B) Despite the expression of both estrogen receptor-{alpha} and -β receptor subtypes in neonatal rat cardiac fibroblasts, the 24 h treatment with 17β-estradiol (n=4) exerted a modest significant decrease only at 1 µM (36±8% decrease vs. basal; n=4: P<0.05). Statistical analysis of each dose–response curve was assessed by a one-way ANOVA.

 

Figure 4
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  		Fig. 4 Apoptosis does not contribute to the growth-suppressing action of progesterone. (Panel A) In 3-day serum-free untreated cardiac fibroblasts (Basal), apoptosis was not evident, as assessed by the TUNEL assay (TdT; n=4). Nuclear staining was observed with propidium iodide (PI) treatment. (Panel B) A 24 h treatment of cardiac fibroblasts with progesterone (1 µM) did not induce DNA fragmentation (n=2), albeit the nucleus was visible with propidium iodide staining (n=2). (Panel C) By contrast, a robust nuclear signal was detected in UV-treated cardiac fibroblasts (30 min), indicative of apoptosis (n=4). Moreover, an overlap of the TUNEL and propidium iodide fluorescence signal was observed in the nucleus, as reflected by the emergence of a yellow fluorescence (TdT+PI).

 
3.3. The regulation of cell cycle proteins by progesterone
Cell cycle progression is regulated via a balance between cyclin-dependent kinases and cyclin-dependent kinase inhibitors. The following series of experiments examined whether the progesterone-mediated decrease in DNA synthesis was associated with the upregulation of the cyclin-dependent kinase inhibitors p27Kip1, and p21cip1. The treatment of cardiac fibroblasts for 4 h with 100 nM progesterone caused a significant increase in p27Kip1 protein levels (60±14% increase vs. basal; n=5; P<0.05) (Fig. 5), whereas by 24 h (n=5), had returned to levels observed in unstimulated cells. By contrast, p21cip1 protein levels were unchanged at either a 4 (7±10% increase vs. basal; n=3) or 24 h (18±40% increase vs. basal; n=3) exposure to progesterone (Fig. 5). Lastly, the cell cycle protein proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor required for DNA synthesis. The treatment of cardiac fibroblasts for either 4 (5±11% increase vs. basal; n=3) or 24 h (13±2% increase vs. basal; n=3) with progesterone (250 nM) did not affect the protein levels of PCNA, compared to unstimulated cells (Fig. 5).


Figure 5
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  		Fig. 5 Progesterone treatment caused a selective increase in the expression of the cyclin-dependent kinase inhibitor p27Kip1. The treatment of cardiac fibroblasts for 4 h with 100 nM progesterone (PRO) increased p27Kip1 protein levels, whereas by 24 h they had returned to levels observed in unstimulated cells. By contrast, the protein levels of either p21Cip1 or proliferating cell nuclear antigen (PCNA) were unchanged following progesterone treatment.

 
3.4. Progesterone and 17β-estradiol treatment of cardiac fibroblasts increased TGF-β3 and fibronectin mRNA expression
Previous studies have demonstrated that the exposure of human osteosarcoma cells to 17β-estradiol increased TGF-β3 mRNA transcription [24]. Although, the treatment of cardiac fibroblasts with 17β-estradiol (100 nM) had no effect on DNA synthesis, a 4 h exposure significantly increased the steady-state mRNA levels of TGF-β3, whereas TGF-β1 mRNA level was unchanged (Fig. 6). Coincident with the increased expression of TGF-β3 mRNA, a consistent induction of the steady-state mRNA level of fibronectin was observed following a 4 h exposure to 17β-estradiol (100 nM), albeit it did not reach statistical significance (P=0.09; n=3) (Fig. 6). Analogous to 17β-estradiol, progesterone (100 nM) significantly increased the steady-state mRNA levels of TGF-β3 following a 4 h treatment, whereas TGF-β1 mRNA level was unaffected. Lastly, the steady-state mRNA level of fibronectin was significantly increased following a 4 h treatment with progesterone (Fig. 6).


Figure 6
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  		Fig. 6 Ovarian hormones promote an increase in the steady-state mRNA level of transforming growth factor-β3 (TGF-β3) and fibronectin. (Panel A) The 4 h treatment with either 17β-estradiol (E2; 100 nM) or progesterone (PRO; 100 nM) significantly increased the steady-state mRNA levels of TGF-β3. Likewise, the 4 h treatment with either 17β-estradiol (E2; 100 nM) or progesterone (PRO; 100 nM) increased steady-state mRNA levels of fibronectin. The mRNA levels of TGF-β3 and fibronectin were normalized to the level of GAPDH mRNA expression. (Panel B) Semi-quantitative analysis revealed that 100 nM progesterone (PROG) and 17β-estradiol (E2; 100 nM) significantly increased TGF-β3 mRNA, whereas TGF-β1 mRNA did not appreciably change. Progesterone and 17β-estradiol increased fibronectin mRNA levels, albeit the latter did not reach statistical significance (n=0.09 vs. basal). Data are expressed as the percent change versus basal, n=3 for each transcript in response to each steroid hormone, mRNA levels were normalized to the level of GAPDH mRNA, and (*) reflects P<0.01 versus basal.

 
3.5. The autocrine role of TGF-β3 in progesterone-mediated inhibition of DNA synthesis
The increase in TGF-β3 mRNA by progesterone may in part contribute via an autocrine mechanism to the decrease in DNA synthesis following the treatment of cardiac fibroblasts with progesterone. Indeed, the 24 h treatment with either TGF-β1 or TGF-β3 resulted in a significant dose-dependent decrease in [3H]thymidine uptake (P<0.05; n=4–5 for each peptide) (Fig. 7). However, in contrast to progesterone, both TGF-β1 and TGF-β3 significantly increased protein synthesis (P<0.05; n=4–5 for each peptide) (Fig. 7). This latter observation supports the premise that progesterone-mediated increase in TGF-β3 mRNA did not result in the synthesis of a biologically active peptide.


Figure 7
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  		Fig. 7 Transforming growth factor-β attenuated DNA but promotes protein synthesis. (Panel A) The treatment of cardiac fibroblasts with either transforming growth factor-β1 or -β3 for 24 h significantly increased protein synthesis in a dose-dependent manner (n=4–5 for each peptide; P<0.05). (Panel B) By contrast, both transforming growth factor-β1 and -β3 significantly decreased DNA synthesis in a dose-dependent manner (P<0.05). Statistical analysis of each dose–response curve was assessed by a one-way ANOVA.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Previous studies have shown that cardiac fibroblasts represent a cellular target of the ovarian hormones progesterone and 17β-estradiol [13], albeit the subtype and subcellular distribution of either progesterone or estrogen receptor subtypes remain equivocal. Previous studies have reported the existence of two progesterone receptor subtypes, designated PR-A, and PR-B, with a molecular weight of ~80 and ~114 kDa, respectively. In the present study, an antibody which recognized both PR-A and PR-B receptor subtypes detected two immunoreactive bands of ~82 and ~86 kDa in the cytosolic fraction; corresponding to the molecular weight of PR-A. This immunoreactive doublet was unexpected, albeit in breast tumour cells, and a progestin-responsive breast cancer cell line T47D, a similar doublet for the PR-A receptor subtype was also identified [19,20]. In addition, a strong immunoreactive band was detected at ~114 kDa in the cytosolic fraction, whereas a concomitant modest signal was observed in the particulate fraction. The latter immunoreactive band of ~114 kDa corresponds to the reported molecular weight of the PR-B receptor subtype [19,20]. Moreover, the detection of the PR-B receptor subtype in the particulate fraction was consistent with a previous study demonstrating the plasma membrane localization of progesterone receptors in the rat hypothalamus [25]. Indeed, consistent with this latter study, and the Western blot data of the present study, an immunofluorescence approach revealed an intense perinuclear/nuclear signal of the progesterone receptor, and a modest consistent level of expression associated with the plasma membrane. Collectively, these data demonstrate that both PR-A and PR-B subtypes are expressed in neonatal rat cardiac fibroblasts, and the PR-B subtype appeared to be the predominant subtype expressed on the plasma membrane. In parallel experiments, an immunoreactive doublet for the estrogen receptor-{alpha} subtype was observed in the cytosolic and particulate fractions, whereas a single band for the estrogen receptor-β subtype was identified predominantly in the cytosolic pool. An immunoreactive doublet for the estrogen receptor-{alpha} subtype has also been identified in rabbit uterus [23]. Moreover, an immunofluorescence approach revealed an intense nuclear signal and a modest signal on the plasma membrane. These data confirm the Western blot analysis, and are consistent with the subcellular pattern of estrogen receptor-{alpha} subtype described in other cell types [22]. Thus, these data are the first to highlight a unique disparate subcellular pattern of estrogen and progesterone receptor subtype in neonatal rat cardiac fibroblasts. Based on these observations, the physiological role of membrane-bound ovarian hormone receptors on cardiac fibroblast phenotype warrants further investigation.

Previous studies performed on neonatal and adult rat cardiac fibroblasts have reported either decreased, increased or no effect in DNA synthesis following 17β-estradiol treatment [13–15]. The decrease in DNA synthesis in the adult fibroblasts was not inhibited by the pretreatment with an anti-estrogen, thereby supporting the involvement of a non-classical estrogen receptor-dependent signaling pathway [13]. In the present study, despite the presence of both estrogen receptor-{alpha} and -β subtypes, a decrease in DNA synthesis was observed following the administration of a pharmacological dose (1 µM) of 17β-estradiol. By contrast, the treatment of adult rat cardiac fibroblasts with progesterone caused a significant dose-dependent decrease in DNA synthesis [13], and as demonstrated in the present study, a similar growth effect was observed in neonatal rat cardiac fibroblasts. Moreover, the growth-suppressing action of progesterone was maintained for 48 h following a single application of the hormone. It is possible that the decrease in DNA synthesis following progesterone treatment may be in part a result of apoptosis. However, the TUNEL assay revealed the absence of DNA fragmentation following a 24 h exposure to progesterone. Collectively, these data support the premise that progesterone, rather than 17-β-estradiol, exerted a significant growth-suppressing action on neonatal rat cardiac fibroblasts without the concomitant induction of apoptosis.

The cell cycle is composed of four phases denoted as G1, S, G2 and M phase. The G1 and G2 phases represent transitions in the cell cycle prior to DNA synthesis in the S phase, and mitosis in the M phase, respectively [26]. Progression through the cell cycle is positively regulated via the formation of cyclin and cyclin-dependent kinase complexes and negatively by cyclin-dependent kinase inhibitors. Previous studies in vascular smooth muscle cells revealed progesterone-mediated attenuation of DNA synthesis was associated with the concomitant suppression of cyclin A and E expression, whereas cyclin B and D1 were unchanged [11]. These data are consistent with a block in G1 to S phase transition. By contrast, the upregulation of cyclin-dependent kinase inhibitors represents a common mechanism employed by a plethora of growth-suppressing factors. The cyclin-dependent kinase inhibitors p21cip1 and p27kip1 have been shown to suppress cell cycle progression predominantly via a block of G1 to S phase transition [27]. In the present study, progesterone-mediated decrease in DNA synthesis was associated with a concomitant increase in p27kip1 protein levels, whereas p21cip1 protein expression remained unchanged. Lastly, the cell cycle protein proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor required for DNA synthesis was unchanged in response to progesterone treatment [28]. By 24 h, the protein level of p27kip1 expression following progesterone treatment returned to that observed in unstimulated cells, despite the continued suppression of DNA synthesis. Collectively, these data support the premise that progesterone-mediated inhibition of DNA synthesis may occur via a block of G1 to S phase transition in part by the upregulation of p27kip1. However, the sustained suppression of DNA synthesis (following a 48 h treatment) supports the premise that the prolonged antiproliferative action of progesterone most likely required the modulation of either the activity and/or expression of alternative cell cycle proteins.

It has been suggested that the ratio of PR-A and PR-B receptor expression dictates in part the physiological action of progesterone. The PR-A receptor subtype represents the truncated form of PR-B, lacking 164 amino acids in the N-terminal region. Studies have demonstrated these two subtypes have distinct functions and operate through disparate signaling pathways [29]. Specifically, the PR-B receptor subtype acts as a positive activator of progesterone-responsive genes, while the PR-A subtype inhibits gene transcription mediated by numerous steroid hormone receptors [30]. In the present study, both the PR-A and PR-B receptors were expressed in neonatal rat cardiac fibroblasts. Moreover, despite the presence of PR-A, progesterone administration stimulated gene transcription, as the treatment of neonatal rat cardiac fibroblasts increased the expression of transforming growth factor-β3 mRNA, whereas transforming growth factor-β1 mRNA remained unchanged. In parallel experiments, progesterone increased the steady-state mRNA level of the extracellular matrix protein fibronectin. Interestingly, despite the absence of a growth-suppressing effect, 17β-estradiol (100 nM) significantly increased the steady-state mRNA level of transforming growth factor-β3, whereas transforming growth factor-β1 mRNA level was unchanged. These latter findings are consistent with previous studies, as 17β-estradiol treatment promoted transforming growth factor-β3 transcription in osteosarcoma cells [24]. Lastly, fibronectin mRNA levels were consistently increased in response to 17β-estradiol treatment, albeit it did not reach statistical significance. A similar observation was made during cutaneous wound healing, as 17β-estradiol increased fibronectin expression [31]. Thus, based on the observation that the transforming growth factor-β family can influence cardiac fibroblast phenotype and suppress proliferation, the induction of transforming growth factor-β3 may represent an autocrine pathway mediating in part the physiological action of ovarian hormones. Indeed, the treatment of cardiac fibroblasts with either transforming growth factor-β1, or -β3 attenuated DNA synthesis, analogous to that observed with progesterone. However, progesterone treatment had no effect on [3H]leucine uptake, whereas both transforming growth factor-β1, and -β3 stimulated protein synthesis. Thus, this latter finding supports the premise that the induction of transforming growth factor-β3 mRNA by progesterone (as well as 17β-estradiol) did not result in the synthesis of a biologically active peptide in neonatal rat cardiac fibroblasts. Alternatively, a transforming growth factor-β3 peptide may have been synthesized in response to progesterone but remained in an inactive latent form and/or the level of expression of a biologically active peptide may have been insufficient to elicit any growth effect.

In summary, these data highlight a unique subcellular distribution of both progesterone and estrogen receptor subtypes in cultured neonatal rat cardiac fibroblasts. The physiological role of membrane-bound PR-B and estrogen receptor-{alpha} in neonatal rat cardiac fibroblasts remains to be explored. Moreover, progesterone, rather than 17β-estradiol exerted a significant suppressing action on DNA synthesis, thereby supporting an antiproliferative effect of this ovarian hormone on cardiac fibroblast growth. Both progesterone, and 17β-estradiol increased the steady-state mRNA level of transforming growth factor-β3 and fibronectin, thereby supporting a potential role in extracellular matrix remodeling. However, the induction of transforming growth factor-β3 mRNA does not appear to represent a biologically active autocrine mechanism influencing the modulation of DNA synthesis by ovarian hormones in cultured neonatal rat cardiac fibroblasts. Collectively, these observations provide the impetus to further examine the effect of hormonal replacement therapy on myocardial remodeling (e.g. fibrosis) and function in both normal and cardiac disease states.

Time for primary review 34 days.


   Acknowledgements
 
This work was supported by the Heart & Stroke Foundation of Canada & Quebec, and Les Fonds de Recherche de l'Institut de Cardiologie de Montréal. A. Calderone is a Chercheur-Boursier Junior II de la Fonds de Recherche de la Sante de Quebec.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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