TGF-β drives pulmonary fibrosis through dual dysregulation of TNF pathway transcription and splicing

It is well known that TGF-β plays a key role in the progression of pulmonary fibrosis. However, how TGF-β/SMAD3 regulates downstream effector genes and its effect on alternative splicing in pulmonary fibrosis is still not fully understood. Here, we established a TGF-β-induced pulmonary fibrosis model in mouse primary fibroblasts to verify the potential role of TGF-β/SMAD signaling in pulmonary fibrosis. In both NIH3T3 and primary fibroblast models of fibrosis, the TNF signaling pathway was significantly enriched among differentially expressed genes, highlighting its core role in driving fibrotic processes. By integrating RNA-seq, CUT&Tag data from fibrotic primary fibroblasts, we found that TGF-β/SMAD3 indirectly regulated the TNF pathway gene Vcam1 through the transcription factor Isl1. Furthermore, using full-length isoform sequencing, we found that fibrosis was associated with a reduction in the number of genome-wide alternative splicing events, particularly in TNF pathway effector genes like Vcam1 and Vegfd. Dual dysregulation of TNF signaling drives fibroblast resistance to apoptosis and aggravates fibrosis. Thus, our study reveals a unique mechanism by which TGF-β/SMAD signaling regulates transcription and coordinates genome-wide alternative splicing to drive pulmonary fibrosis progression.