Background/Purpose: Systemic sclerosis (SSc) primarily affects postmenopausal women. This sex bias could partly be explained by the action of estrogens on the immune system and/or fibrogenesis. Since little is known about their direct role in fibrogenesis, our aim was to evaluate the effects of estrogens i) on the pathological activation of dermal fibroblasts induced by transforming growth factor-b (TGF-β) and ii) in the development of experimental dermal fibrosis.

Methods: SSc dermal fibroblasts were stimulated with TGF-β and incubated with different concentrations of 17-β-estradiol and/or tamoxifen, a selective estrogen receptor modulator that display anti-estrogenic properties. Collagen release from fibroblasts was evaluated by mRNA levels of col1a1 and col1a2 and by measuring the concentrations of collagen in cell culture supernatants with the SirCol collagen assay. Differentiation of fibroblasts into myofibroblasts was assessed by the expression of alpha smooth muscle actin (α-SMA). Activation of the TGF-β pathway was evaluated by the expression of phospho-smad-2/3. Effects on estrogen inhibition by gene inactivation (knockout mice for the estrogen receptor-α, ERKOa) or targeted molecular strategy (tamoxifen) were evaluated in the mouse model of bleomycin-induced dermal fibrosis and in the tight skin (Tsk-1) mouse model.

Results: Treatment of SSc dermal fibroblasts with 17-β-estradiol or tamoxifen did not change significantly baseline collagen production and fibroblast differentiation. In SSc fibroblasts stimulated by TGF-β, treatment with 17-β-estradiol led to a significant reduction of COL1A1 and COL1A2 mRNA levels, as well as collagen release in cell culture supernatant. In addition, decreased α-SMA mRNA and protein levels were observed upon treatment with 17-β-estradiol in TGF-b stimulated dermal fibroblasts. 17-β-estradiol also led to a significant reduction of TGF-b dependent phosphorylation of phospho-Smad2/3. In fibroblasts stimulated with TGF-b and treated with 17-β-estradiol, the addition of tamoxifen restored TGF-b signaling and its profibrotic effects on collagen synthesis and fibroblast differentiation.

Estrogen inhibition led to more a severe dermal fibrosis induced by bleomycin. Upon bleomycin injections, ERKO-a mice and mice treated with tamoxifen had a significant increase of dermal thickness (17%, p=0.03 and 20%, p=0.04), hydroxyproline content mice (16%, p=0.02 and 36%, p=0.003, respectively) and number of myofibroblasts (22%, p = 0.01 and 20%, p = 0.04 respectively) compared to control mice. In Tsk-1 mice, treatment with tamoxifen led to significantly enhanced skin fibrosis, with a 31±8% increase of hypodermal thickening (p=0.03) and a 17% increase of hydroxyproline content (p=0.01) compared to control mice.

Conclusion: Our results demonstrate a beneficial effect of estrogen in dermal fibrosis. Estrogens reduce TGF-b dependent activation of SSc dermal fibroblasts and estrogen inhibition leads to a more severe experimental dermal fibrosis. These findings may partly explain the occurrence of SSc in postmenopausal women and the greater severity of the disease in men and open avenue to potential hormonal therapies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/estrogens-inhibit-the-profibrotic-effects-of-transforming-growth-factor-beta-and-protect-from-the-development-of-experimental-dermal-fibrosis/