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Estradiol promotes the development of a fibrotic phenotype and is increased in the serum of patients with systemic sclerosis.

Aida-Yasuoka K, Peoples C, Yasuoka H, Hershberger P, Thiel K, Cauley JA, Medsger TA, Feghali-Bostwick CA - Arthritis Res. Ther. (2013)

Bottom Line: We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model.Propyl-pyrazole-triol, but not genistein, significantly increased FN expression.The effects of E2 were abrogated by ICI 182,780.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.

Methods: Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2's effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.

Results: E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.

Conclusion: Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.

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Estradiol increases fibronectin production in vitro. (A) Fibronectin (FN) mRNA expression in primary skin fibroblasts from twins discordant for systemic sclerosis (SSc). FN mRNA was analyzed after 24-hour treatment with 10 nM 17β-estradiol (E2), vehicle (V), or no treatment (control, C) using RT-PCR. Assays were done in duplicate. (B) FN protein expression in culture supernatants of primary skin fibroblasts. FN protein expression was analyzed by western blot in untreated, E2-treated or vehicle-treated fibroblasts for 48 hours. SSc, SSc patient; healthy, healthy twin of an SSc patient; control, unrelated healthy donor. (C) FN protein expression in the extracellular matrix (ECM) and culture supernatants (Sup) of normal skin fibroblasts treated with vehicle, transforming growth factor beta (TGFβ), or E2. ECM and culture supernatants were harvested at the indicated time points. (D) FN levels in the ECM and Sup of normal skin fibroblasts stimulated with vehicle and different concentrations of E2 ranging from 0.1 to 10 nM. (E) Graphical summary of FN levels representing data from four independent experiments. *P = 0.0036. (F) Effect of E2 blockade on the expression of FN protein in the ECM and Sup of normal skin fibroblasts stimulated with E2. Cells were treated with E2 for 48 hours in the presence or absence of the ER antagonist ICI 182,780.
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Figure 1: Estradiol increases fibronectin production in vitro. (A) Fibronectin (FN) mRNA expression in primary skin fibroblasts from twins discordant for systemic sclerosis (SSc). FN mRNA was analyzed after 24-hour treatment with 10 nM 17β-estradiol (E2), vehicle (V), or no treatment (control, C) using RT-PCR. Assays were done in duplicate. (B) FN protein expression in culture supernatants of primary skin fibroblasts. FN protein expression was analyzed by western blot in untreated, E2-treated or vehicle-treated fibroblasts for 48 hours. SSc, SSc patient; healthy, healthy twin of an SSc patient; control, unrelated healthy donor. (C) FN protein expression in the extracellular matrix (ECM) and culture supernatants (Sup) of normal skin fibroblasts treated with vehicle, transforming growth factor beta (TGFβ), or E2. ECM and culture supernatants were harvested at the indicated time points. (D) FN levels in the ECM and Sup of normal skin fibroblasts stimulated with vehicle and different concentrations of E2 ranging from 0.1 to 10 nM. (E) Graphical summary of FN levels representing data from four independent experiments. *P = 0.0036. (F) Effect of E2 blockade on the expression of FN protein in the ECM and Sup of normal skin fibroblasts stimulated with E2. Cells were treated with E2 for 48 hours in the presence or absence of the ER antagonist ICI 182,780.

Mentions: The effect of E2 on FN expression was examined using RT-PCR and western blot analysis. In untreated samples, FN mRNA and protein levels in SSc patient fibroblasts were higher than those in their healthy twins. E2 increased FN mRNA and protein levels in healthy twin and SSc fibroblasts (Figure 1A,B). E2 increased FN mRNA and protein levels in a time-dependent and dose-dependent manner in cell supernatants and ECM (Figure 1C,D). E2 induced production of total FN and EDA-domain-containing matrix FN (Figure 1C) and the increase in secreted FN was significant (Figure 1E). The ER antagonist ICI 182,780 blocked the effect of E2 on FN mRNA and protein expression but did not affect transforming growth factor beta-induced FN levels (Figure 1F).


Estradiol promotes the development of a fibrotic phenotype and is increased in the serum of patients with systemic sclerosis.

Aida-Yasuoka K, Peoples C, Yasuoka H, Hershberger P, Thiel K, Cauley JA, Medsger TA, Feghali-Bostwick CA - Arthritis Res. Ther. (2013)

Estradiol increases fibronectin production in vitro. (A) Fibronectin (FN) mRNA expression in primary skin fibroblasts from twins discordant for systemic sclerosis (SSc). FN mRNA was analyzed after 24-hour treatment with 10 nM 17β-estradiol (E2), vehicle (V), or no treatment (control, C) using RT-PCR. Assays were done in duplicate. (B) FN protein expression in culture supernatants of primary skin fibroblasts. FN protein expression was analyzed by western blot in untreated, E2-treated or vehicle-treated fibroblasts for 48 hours. SSc, SSc patient; healthy, healthy twin of an SSc patient; control, unrelated healthy donor. (C) FN protein expression in the extracellular matrix (ECM) and culture supernatants (Sup) of normal skin fibroblasts treated with vehicle, transforming growth factor beta (TGFβ), or E2. ECM and culture supernatants were harvested at the indicated time points. (D) FN levels in the ECM and Sup of normal skin fibroblasts stimulated with vehicle and different concentrations of E2 ranging from 0.1 to 10 nM. (E) Graphical summary of FN levels representing data from four independent experiments. *P = 0.0036. (F) Effect of E2 blockade on the expression of FN protein in the ECM and Sup of normal skin fibroblasts stimulated with E2. Cells were treated with E2 for 48 hours in the presence or absence of the ER antagonist ICI 182,780.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Estradiol increases fibronectin production in vitro. (A) Fibronectin (FN) mRNA expression in primary skin fibroblasts from twins discordant for systemic sclerosis (SSc). FN mRNA was analyzed after 24-hour treatment with 10 nM 17β-estradiol (E2), vehicle (V), or no treatment (control, C) using RT-PCR. Assays were done in duplicate. (B) FN protein expression in culture supernatants of primary skin fibroblasts. FN protein expression was analyzed by western blot in untreated, E2-treated or vehicle-treated fibroblasts for 48 hours. SSc, SSc patient; healthy, healthy twin of an SSc patient; control, unrelated healthy donor. (C) FN protein expression in the extracellular matrix (ECM) and culture supernatants (Sup) of normal skin fibroblasts treated with vehicle, transforming growth factor beta (TGFβ), or E2. ECM and culture supernatants were harvested at the indicated time points. (D) FN levels in the ECM and Sup of normal skin fibroblasts stimulated with vehicle and different concentrations of E2 ranging from 0.1 to 10 nM. (E) Graphical summary of FN levels representing data from four independent experiments. *P = 0.0036. (F) Effect of E2 blockade on the expression of FN protein in the ECM and Sup of normal skin fibroblasts stimulated with E2. Cells were treated with E2 for 48 hours in the presence or absence of the ER antagonist ICI 182,780.
Mentions: The effect of E2 on FN expression was examined using RT-PCR and western blot analysis. In untreated samples, FN mRNA and protein levels in SSc patient fibroblasts were higher than those in their healthy twins. E2 increased FN mRNA and protein levels in healthy twin and SSc fibroblasts (Figure 1A,B). E2 increased FN mRNA and protein levels in a time-dependent and dose-dependent manner in cell supernatants and ECM (Figure 1C,D). E2 induced production of total FN and EDA-domain-containing matrix FN (Figure 1C) and the increase in secreted FN was significant (Figure 1E). The ER antagonist ICI 182,780 blocked the effect of E2 on FN mRNA and protein expression but did not affect transforming growth factor beta-induced FN levels (Figure 1F).

Bottom Line: We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model.Propyl-pyrazole-triol, but not genistein, significantly increased FN expression.The effects of E2 were abrogated by ICI 182,780.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.

Methods: Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2's effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.

Results: E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.

Conclusion: Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.

Show MeSH
Related in: MedlinePlus