Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a

Abstract

µ-calpain is implicated in pulmonary fibrosis, however its role in the aberrant differentiation of alveolar epithelial type II cells (AT2), a hallmark of pulmonary fibrosis remains unclear, and its targeted transcription factor has not been addressed. Here, examination of the specimen of fibrosis patients revealed excessive proliferation of AT2 cells. In parallel, AT2 cells exhibited substantial calpain 1 (CAPN1), a catalytic subunit of µ-calpain, and phosphorylated FoxO3a (p-FoxO3a), an important transcription factor in lung tissue. Of note, targeted knockdown of CAPN1 in AT2 cells blocked the progression of bleomycin-induced pulmonary fibrosis, manifested as reduced poorly aerated regions in chest CT image, and decreased content of hydroxyproline and α-SMA. Analysis of nuclear fraction displayed an accumulation of CAPN1 and loss of FoxO3a, which was accompanied with activation of Akt. Knockdown of CAPN1 in A549 cells with siRNA antagonized the process of epithelial-mesenchymal transition and blunted FoxO3a phosphorylation and Akt activation. Conversely, overexpression of CAPN1 accelerated mesenchymal transition, enhanced its nuclear accumulation and the translocation of p-FoxO3a out of nucleus. Finally, inhibition of Akt decreased calpain-elicited FoxO3a phosphorylation, meanwhile, transfection of FoxO3a mutant carrying Thr32A and Ser253A mitigated the calpain-stimulated mesenchymal transition. Collectively, we conclude that nuclear accumulation of µ-calpain in AT2 cells is a critical step to aggravate pulmonary fibrosis. we also identify that inactivation of FoxO3a in a Akt-dependent phosphorylation manner confers to calpain-elicited the aberrant differentiation of AT2 cells.