Background 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.

Objectives 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 Effects on estrogen inhibition by gene inactivation (knockout mice for the estrogen receptor-α, ERKOα) or targeted molecular strategy (tamoxifen, a selective estrogen receptor modulator that display anti-estrogenic properties) were evaluated in the mouse model of bleomycin-induced dermal fibrosis and in the tight skin (Tsk-1) mouse model.

SSc dermal fibroblasts were stimulated with TGF-β and incubated with different concentrations of 17-β-estradiol and/or tamoxifen. 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.

Results Estrogen inhibition increased the activation of canonical TGF-β signaling and exacerbated skin fibrosis both in the bleomycin model and in Tsk-1 mice. Upon bleomycin injections, ERKO-α 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.

In SSc dermal fibroblasts, treatment with 17-β-estradiol significantly decreased the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation, decreased activation of canonical TGF-β signaling, and markedly reduced the expression of TGF-β target genes. Tamoxifen reversed the inhibitory effects of estrogens by restoring Smad2/3 phosphorylation and TGF-β-induced collagen synthesis.

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

Disclosure of Interest None declared