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, 115 (2), 315-327

Sex-specific Regulation of Collagen I and III Expression by 17β-Estradiol in Cardiac Fibroblasts: Role of Estrogen Receptors

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Sex-specific Regulation of Collagen I and III Expression by 17β-Estradiol in Cardiac Fibroblasts: Role of Estrogen Receptors

Elke Dworatzek et al. Cardiovasc Res.

Abstract

Aims: Sex differences in cardiac fibrosis point to the regulatory role of 17β-Estradiol (E2) in cardiac fibroblasts (CF). We, therefore, asked whether male and female CF in rodent and human models are differentially susceptible to E2, and whether this is related to sex-specific activation of estrogen receptor alpha (ERα) and beta (ERβ).

Methods and results: In female rat CF (rCF), 24 h E2-treatment (10-8 M) led to a significant down-regulation of collagen I and III expression, whereas both collagens were up-regulated in male rCF. E2-induced sex-specific collagen regulation was also detected in human CF, indicating that this regulation is conserved across species. Using specific ERα- and ERβ-agonists (10-7 M) for 24 h, we identified ERα as repressive and ERβ as inducing factor in female and male rCF, respectively. In addition, E2-induced ERα phosphorylation at Ser118 only in female rCF, whereas Ser105 phosphorylation of ERβ was exclusively found in male rCF. Further, in female rCF we found both ER bound to the collagen I and III promoters using chromatin immunoprecipitation assays. In contrast, in male rCF only ERβ bound to both promoters. In engineered connective tissues (ECT) from rCF, collagen I and III mRNA were down-regulated in female ECT and up-regulated in male ECT by E2. This was accompanied by an impaired condensation of female ECT, whereas male ECT showed an increased condensation and stiffness upon E2-treatment, analysed by rheological measurements. Finally, we confirmed the E2-effect on both collagens in an in vivo mouse model with ovariectomy for E2 depletion, E2 substitution, and pressure overload by transverse aortic constriction.

Conclusion: The mechanism underlying the sex-specific regulation of collagen I and III in the heart appears to involve E2-mediated differential ERα and ERβ signaling in CFs.

Figures

Figure 1
Figure 1
Collagen I and III mRNA level are regulated in a sex-specific manner by E2 in human cardiac fibroblasts. (A) CF isolated from women and (B) men with AS after 12 h vehicle or 10−8 M E2-treatment. Collagen I and III mRNA level were quantified by qPCR and normalized to HPRT. Collagen I and III expression of E2-treated cells are given as fold induction of vehicle-treated cells. Mean ± SEM from 10 to 11 independent samples derived from four independently repeated experiments; **P ≤ 0.01, *P ≤ 0.05, comparison was performed using Mann–Whitney U-test.
Figure 2
Figure 2
Collagen I and III protein level are regulated in a sex-specific manner by E2 in primary rat cardiac fibroblasts. (A) Female and (B) male rCF were analysed after 24 h vehicle or 10−8 M E2-treatment by western blot. Representative immunoblots of collagen I, III, and β-actin are shown. Expression of collagen I and III were quantified and normalized to β-actin. Collagen I and III levels of E2-treated cells are given as fold induction of vehicle-treated cells. Mean ± SEM from five to six independent samples derived from three independently repeated experiments; **P ≤ 0.01, *P ≤ 0.05, comparison was performed using Mann–Whitney U-test.
Figure 3
Figure 3
Collagen I and III mRNA level are regulated in a sex-specific manner by sex-dimorphic ERα- and ERβ-activation. (A) Female and (B) male rCF were analysed after 24 h vehicle, 10−8 M E2, 10−7 M ERα- and ERβ-agonist treatment. Collagen I and III mRNA levels were quantified by qPCR and normalized to HPRT. Collagen I and III expression of E2-, ERα-, and ERβ-agonist-treated cells are given as fold induction of vehicle-treated cells (indicated by the horizontal red line). Mean ± SEM from 9 to 12 independent samples derived from four independently repeated experiments; **P ≤ 0.01, *P ≤ 0.05, comparison between treated cells and respective control was performed using Mann–Whitney U-test.
Figure 4
Figure 4
ERα and ERβ protein and phosphorylation of ERα at serine 118 and ERβ at serine 105 differ between cardiac fibroblasts from male and female rats. (A) ERα and ERβ protein were investigated in non-treated female and male rCF. (B) ERα and ERβ protein was analysed in female and male rCF after 24 h of vehicle or 10−8 M E2-treatment. (C) Phosphorylation level of Ser118 of ERα and (D) of Ser105 of ERβ was analysed in female and male after 24 h of vehicle or 10−8 M E2-treatment. Probes were analysed by western blot and representative immunoblots of ERα ERβ, P-Ser118-ERα, P-Ser105-ERβ, and β-actin are shown. The expression of ER and P-ER were quantified and normalized to β-actin. In non-treated cells (A), relative protein levels of ERα and ERβ in female rCF are given as fold induction of male rCF. Protein and phosphorylation levels of ER of E2-treated cells (BD) are given as fold induction of vehicle-treated cells. Mean ± SEM; ER comparison between sexes in non-treated rCF: female: n = 10 and male: n = 12; E2-treatment: from five to six independent samples derived from three independently repeated experiments; **P ≤ 0.01 and *P ≤ 0.05 comparison was performed using Mann–Whitney U-test.
Figure 5
Figure 5
ER bind in a sex-specific manner within the collagen I and III promoter in rat cardiac fibroblasts in the presence of E2. (AF) ERα and ERβ binding to the rat collagen I and III promoter upon 1 h 10−8 M E2-treatment in rCF was analysed using ChIP assays. (A) Upon E2-treatment, ERα and (B) ERβ bind to the collagen I promoter in female rCF (C), whereas only ERβ binds within the collagen I promoter in male rCF. (D) In female rCF, ERα and (E) ERβ bind within the collagen III promoter, and (F) in male rCF only ERβ binds collagen III promoter upon E2-treatment. Mean ± SEM from four to seven independently repeated experiments per sex. Comparisons between all four groups was performed by one-way ANOVA followed by Dunn's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
Figure 6
Figure 6
E2 modulates collagen I and III mRNA and tissue properties of ECT derived from female and male rat cardiac fibroblasts in a sex-specific manner. (A and C) ECT derived from female and (B and D) male rCF were analysed after 6 days of vehicle or 10−8 M E2-treatment. Collagen I and III mRNA level were quantified by qPCR and normalized to HPRT. Collagen I and III expression and ECT volumes of E2-treated ECT are given as fold induction of vehicle-treated ECT. Mean ± SEM from seven to ten individual ECT derived from four independently repeated experiments; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, comparison between treated ECT and related vehicle control was performed using Mann–Whitney U-test. (EH) Rheological destructive tensile strength measurements of ECT derived from female and male rCF after 6 days of vehicle or 10−8 M E2-treatment were performed in an organ bath. Representative stress-strain curves of vehicle and 10−8 M E2-treated (E) female (slope of vehicle treated ECT: 3.27 ± 0.01; E2-treated: 2.65 ± 0.01) and (G) male ECT are shown (slope of vehicle treated ECT: 3.02 ± 0.01; E2-treated ECT: 4.86 ± 0.01). Changes of the Young’s modulus of (F) female and (H) male ECT are shown. Changes in the Young’s modulus of E2-treated ECT are given as fold induction of vehicle-treated ECT. Mean ± SEM from five to seven individual ECT derived from three independently repeated experiments; **P ≤ 0.01, comparison between E2-treated cells and related vehicle control was performed using Mann–Whitney U-test.
Figure 7
Figure 7
E2 attenuates collagen I and III mRNA level in murine hearts subjected to TAC. (A) Collagen I and (B) III mRNA level were determined in hearts from female mice 4 weeks after TAC. Collagen I and III mRNA level were quantified by qPCR and normalized to RPL0. Mean ± SEM from six to nine mice per group. *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA followed by Tukey's multiple comparison test. Sham, sham operated mice; TAC, transverse aortic constriction; OVX, ovariectomy; placebo, phytoestrogen-free chow and E2-supplementation: 17β-estradiolbenzoate (E2B) in phytoestrogen-free chow.
Figure 8
Figure 8
Schematic presentation on how E2 and ER modulate collagen I and III in a sex-specific manner in cardiac fibroblasts. In female CF, E2 phosphorylates ERα at serine 118 (P-Ser118), which binds to ERβ. Both ER translocate into the nucleus and bind to the identified ERE within the collagen I and III promoter. Recruitment of co-repressor(s) (Co-R) results in down-regulation of collagen I and III gene expression in female cells. In male CF, E2-treatment phosphorylates ERβ at serine 105 (P-Ser105), leading to ERβ homodimer formation, which then translocates into the nucleus and binds to the identified ERE within the collagen I and III promoter. This is accompanied by co-activator (Co-A) recruitment leading to up-regulation of collagen gene expression in male cells.

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