EPB41L3 suppresses epithelial-mesenchymal transition and proliferation by attenuating TGF beta signaling pathways

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive accumulation of extracellular matrix (ECM). Although EPB41L3 has recently been implicated in both epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transition (FMT), its comprehensive role remains largely unknown. A549 cells stably overexpressing EPB41L3 were stimulated with TGF-β1. Signaling pathways, EMT, and ECM markers were analyzed by western blot. Transcriptomic changes were assessed by RNA sequencing, with differential expression analysis performed using edgeR and functional enrichment analyses conducted using WebGestalt. Cell proliferation, division, apoptosis, senescence, and cell cycle regulation were evaluated via MTT, CFSE, SA-β-gal staining, and western blotting for p16/p21. EPB41L3 overexpression markedly attenuated TGF-β-induced phosphorylation of SMAD2/3, ERK1/2, and GSK3β at both 24 and 48 h, with minimal effects on total protein levels, indicating the suppression of both canonical and non-canonical TGF-β signaling. Consistently, EMT markers, including N-cadherin, β-catenin, SNAIL, and SLUG, were significantly reduced. EPB41L3 also robustly suppressed TGF-β-induced collagen production (COL1A1 and COL3A1), indicating the inhibition of fibrotic ECM accumulation. RNA-seq identified 549 differentially expressed genes in EPB41L3-overexpressing cells, which were enriched in ECM organization, collagen crosslinking, and ECM degradation pathways. Functionally, EPB41L3 suppressed proliferation and division without cytotoxicity. This growth inhibition involved increased early apoptotic priming and p21 induction alongside p16 suppression, suggesting reversible cell cycle restraint rather than senescence. EPB41L3 suppresses TGF-β-dependent EMT and fibrotic ECM remodeling while restraining epithelial proliferation, highlighting its potential as a regulator of fibrogenic responses in IPF.