Single-cell transcriptome analysis revealing mechanotransduction via the Hippo/YAP pathway in promoting fibroblast-to-myofibroblast transition and idiopathic pulmonary fibrosis development.

Objective: Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal interstitial lung disease, characterized by excessive extracellular matrix (ECM) secretion that disrupts normal alveolar structure. This study aims to explore the potential molecular mechanisms underlying the promotion of IPF development.

Methods: Firstly, we compared the transcriptome and single-cell sequencing data from lung tissue samples of patients with IPF and healthy individuals. Subsequently, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on the differentially expressed genes (DEGs). Furthermore, we employed sodium alginate hydrogels with varying degrees of crosslinking to provide differential mechanical stress, mimicking the mechanical microenvironment in vivo during lung fibrosis. On this basis, we examined cytoskeletal remodeling in fibroblasts MRC-5, mRNA expression of multiple related genes, immunofluorescence localization, and cellular proliferation capacity.

Results: Bioinformatics analysis revealed a series of DEGs associated with IPF. Further functional and pathway enrichment analyses indicated that these DEGs were primarily enriched in ECM-related biological processes. Single-cell sequencing data revealed that fibroblasts and myofibroblasts are the main contributors to excessive ECM secretion and suggested activation of mechanotransduction and the Hippo/YAP signaling pathway in myofibroblasts. Cellular experiments demonstrated that sodium alginate hydrogels with different stiffness can simulate different mechanical stress environments, thereby affecting cytoskeletal rearrangement and Hippo/YAP pathway activity in MRC-5 lung fibroblasts. Notably, high levels of mechanical stress promoted YAP nuclear translocation, increased expression of type I collagen and α-SMA, and enhanced proliferative capacity. Additionally, we also found that fibroblasts primarily participate in mechanotransduction through the Rho/ROCK and Integrin/FAK pathways under high mechanical stress conditions, ultimately upregulating the gene expression of CCNE1/2, CTGF, and FGF1.

Conclusion: Our study uncovers the crucial role of cytoskeletal mechanotransduction in myofibroblast transformation and IPF development through activation of the Hippo/YAP pathway, providing new insights into understanding the pathogenesis of IPF.