TGF-β1 promotes collagen synthesis in systemic sclerosis via upregulating P4HA3.

OBJECTIVE

Systemic sclerosis (SSc) is a complex autoimmune disease resulting in fibrosis of skin and internal organs. Despite progress in treatment, SSc carries high mortality due to organ fibrosis. This study aimed to identify a potential antifibrotic therapeutic for the treatment of SSc.

METHODS

SSc bioinformatics data (GSE181549 and GSE138669) were obtained from the public Gene Expression Omnibus (GEO) database. Subsequently, we conducted differential analysis, Protein-protein interaction (PPI) network construction, gene enrichment analysis to identify a key fibrotic gene, followed by verification.

RESULTS

A total of 107 differentially expressed genes were identified through analysis between SSc patients and healthy controls. P4HA3 (α subunit of Collagen prolyl 4-hydroxylases, C-P4Hs) was identified by PPI network analysis. P4HA3 was the sole upregulated gene and positively correlated with the modified Rodnan skin score. PI3K/AKT signaling pathway was enriched by GSEA for single-gene to obtain P4HA3-related pathways. Single-cell analysis detected predominant TGF-β1 upregulation in immune cells, particularly in CD14 + CD16 - monocytes, and P4HA3 showed elevated expression in SFRP2 high fibroblasts. Immunohistochemistry indicated that P4HA3- and TGF-β1-positive cell numbers were elevated in the dermal layers of SSc patients. P4HA3 was also elevated in bleomycin-treated mice and TGF-β1-induced human fibroblasts. Inhibition of C-P4Hs ameliorated experimental fibrosis in bleomycin-treated mice and TGF-β1-induced human fibroblasts. Hydroxyproline levels were reduced following P4HA3 knockdown in human fibroblasts.

CONCLUSION

Our results suggest that P4HA3 is upregulated by TGF-β1 secreted from immune cells to promote collagen synthesis in SSc. Inhibition of C-P4Hs is a potential therapeutic approach for SSc fibrosis.