Abstract Rationale Given that alternative splicing is prominent in aging-related diseases and that idiopathic pulmonary fibrosis (IPF) is an age-associated pulmonary disease, dysregulation of RNA splicing may play a critical role in the pathobiology of IPF. Methods RNA-seq analysis of gene expression and splicing isoforms was performed using lung tissues from IPF patients and age-matched controls, as well as pluripotent stem cells (iPSC) at differentiation days 0, 15, and 35. Basescope assay, RT-PCR, and western blotting were performed on IPF lung tissue, iPSC-derived alveolar type II-like (iAT2) organoids, human lung epithelial cells and fibroblasts, and Isolated mouse AT2 cells and fibroblasts treated with bleomycin. Results Global RNA splicing events were observed in IPF lungs compared with controls. We identified a total of 2,977 genes with significant differential expression (DEG) (FDR ≤ 5%), and 2,641 RNA splicing variants. Most significant splicing events occurred in genes without differential expression changes. Only 258 genes exhibited both differential expression and alternative splicing (AS). Gene ontology analysis revealed that AS-specific genes were enriched in pathways related to fibrogenesis, mRNA processing, regulation of translation, and cell-cell adhesion. Ingenuity Pathway Analysis (IPA) further indicated that AS genes regulate senescence and aging pathways. Among 2,641 alternatively spliced genes, components of the Hippo signaling pathway, including YAP1/TAZ (Yes-associated protein 1) and TEAD (TEA Domain Transcription Factor), were highly spliced in IPF lung tissue. In particular, the TEAD1 isoform retaining exon 6 (TEAD1Rx6) was predominantly expressed in lung fibroblasts, whereas the isoform lacking exon 6 (TEAD1Ex6) was primarily expressed in alveolar epithelial cells, iAT2, and mouse AT2 cells. TEAD1Ex6 expression progressively increased during iPSC differentiation toward alveolar epithelial lineages and was further elevated in IPF lungs, suggesting its association with epithelial reprogramming or maladaptive repair responses. Conclusions Both alternative splicing and differential gene expression drive transcriptional reprogramming in IPF, either independently or cooperatively. The enrichment of splicing alterations in fibrogenic and aging-related pathways underscores a pivotal role for post-transcriptional regulation in disease progression. Cell type-specific expression TEAD1 isoforms suggests distinct functional roles in epithelial regeneration versus fibroblast activation, providing new mechanistic insights and potential targets for future therapeutic development. This abstract is funded by: Tulane University Carol Lavin Bernick Faculty Grant Program and the Tulane University School of Medicine Pilot Fund (Q.Y.). and a generous gift for research from Mr. Howard Kenyon (J.A.L.).
Yin et al. (Fri,) studied this question.