LEF1 regulated CTHRC1 promotes silica-induced pulmonary fibrosis through CD44-dependent signaling

Silicosis is an incurable fibrotic lung disease caused by crystalline silica exposure. Aberrant activation of lung fibroblasts into pathological fibroblasts is a hallmark of silicosis. CTHRC1 has been considered a novel fibroblast activation biomarker. This study sought to explore CTHRC1's role in silicosis and determine the signaling pathways it modulates. A combination of transcriptomic analysis of clinical samples, LC-MS/MS, Co-IP, and cationic nanoliposome-based techniques was utilized. In vitro models of lung fibroblast activation induced by TGF-β1, PDGF or recombinant CTHRC1 (rCTHRC1), as well as in vivo models of pulmonary fibrosis induced by silica or bleomycin in mice were established. RNA sequencing, Western blot, RT-qPCR, AlphaFold2 analyses and rescue experiments using siCTHRC1, siCD44, CTHRC1 plasmids, and the PI3K activator 740 Y-P were performed to explore the underlying mechanisms. CTHRC1 is a significantly dysregulated gene implicated in myogenesis and ECM-related pathways. Mechanistically, lymphoid enhancer-binding factor 1 (LEF1) exerts its transcriptional regulator role by binding to the CTHRC1 promoter region in TGF-β1-activated fibroblasts. Further, CTHRC1 mediates its pro-fibroblast-to-myofibroblast transition function by directly binding to the CD44 receptor. AlphaFold2 analyses revealed the binding between CTHRC1 and CD44 is accomplished by three critical CD44 residues: Arg41, Asn172, and Trp650. Rescue experiments demonstrated that the CTHRC1-CD44 complex exerts its pro-fibrotic effects through AKT signaling. In vivo, liposomal Cthrc1 siRNA mitigated fibrogenesis in both silica- and bleomycin-induced mouse fibrosis models. Our results uncover a previously unrecognized role of the LEF1-CTHRC1-CD44 axis in silicosis and highlight the therapeutic target potential of CTHRC1 in fibrotic lung diseases.