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.