Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. Follistatin-like protein 1 (FSTL1) is upregulated and exerts pro-fibrotic effects in various models of organ fibrosis; however, its specific role and underlying molecular mechanism in SSc-related skin fibrosis remain poorly understood. This study aimed to characterize FSTL1 expression in SSc and elucidate its mechanistic role in regulating skin fibrosis.

Peripheral blood and skin tissue samples were collected from 30 patients with SSc and 26 healthy controls. FSTL1 and inflammatory cytokine levels were measured using quantitative real-time PCR and Western blotting, and their correlations with clinical parameters were assessed. Publicly available single-cell RNA sequencing datasets were analyzed to determine the cellular sources and expression patterns of FSTL1 in SSc skin. An in vitro fibrosis model was established by stimulating human skin fibroblasts (BJ cells) with transforming growth factor-β1 (TGF-β1). Following FSTL1 overexpression or knockdown, BJ cells were treated with TGF-β1, the PI3K inhibitor LY294002, or the PI3K agonist 740Y-P. Expression levels of fibrosis-related markers and PI3K/AKT pathway phosphorylation were evaluated. In vivo, a bleomycin (BLM)-induced SSc mouse model was established, and adeno-associated virus-mediated silencing of Fstl1 was performed to assess its effects on dermal fibrosis and PI3K/AKT signaling.

FSTL1 expression was significantly upregulated in both peripheral blood and skin tissues of SSc patients compared to healthy controls. Peripheral blood FSTL1 levels positively correlated with TNF-α and IgM levels and were notably elevated in patients with interstitial lung disease or digital gangrene. Receiver operating characteristic (ROC) curve analysis demonstrated that FSTL1 had high diagnostic value for SSc (area under the curve [AUC] = 0.909). Single-cell transcriptomic analysis revealed a marked increase in the proportion and expression intensity of FSTL1-positive cells in SSc skin, with fibroblasts being the predominant source. FSTL1 expression in fibroblasts co-localized with activation markers, and pathway enrichment analysis suggested that FSTL1-positive cells may promote fibrosis through modulation of PI3K/AKT signaling. In vitro, TGF-β1 (5 ng/mL) successfully induced a fibrotic phenotype in BJ cells and concurrently upregulated FSTL1 expression. Overexpression of FSTL1 enhanced the expression of collagen I and α-smooth muscle actin (α-SMA), increased PI3K/AKT phosphorylation, and potentiated the pro-fibrotic effects of TGF-β1. Conversely, FSTL1 knockdown attenuated these effects. LY294002 partially reversed FSTL1 overexpression-induced fibrosis, while 740Y-P restored the fibrotic phenotype in FSTL1-silenced cells. In the BLM-induced mouse model, Fstl1 was upregulated in fibrotic skin, and its silencing suppressed PI3K/AKT activation and ameliorated dermal fibrosis.

 FSTL1 expression is significantly elevated in the skin of both SSc patients and mouse models of SSc and is closely associated with the extent of skin fibrosis. Mechanistically, FSTL1 promotes fibroblast activation and enhances TGF-β1-mediated pro-fibrotic signaling through the PI3K/AKT pathway, thereby contributing to the pathogenesis of skin fibrosis in SSc. These findings highlight FSTL1 as a potential diagnostic biomarker and therapeutic target in systemic sclerosis.