CMLD-2 Attenuates Myofibroblast Activation and Bleomycin-Induced Pulmonary Fibrosis in Mice through Antagonizing ELAVL1-Mediated Osteopontin mRNA Stabilization

Abstract

AbstractBackgroundFibroblast-myofibroblasts transition (FMT) is one of the hallmark cellular processes in pulmonary fibrosis. This study is to investigate the effects of CMLD-2 in FMT and pulmonary fibrosis, which antagonizes HuR, a supposedly key regulatory RNA binding protein (RBP).MethodsHuR or other deferentially expressed RBPs during TGF-β1-induced FMT were analyzed by transcriptomic methods, and further validated in vitro or in fibrotic lung specimens. The effects of HuR overexpression, down-regulation or inhibition by an antagonist CMLD-2 were analyzed in FMT or bleomycin-induced experimental lung fibrosis. HuR-targeting RNA and their interactions were analyzed by CLIP-seq.ResultsHuR, hnRNPA1, hnRNPE1, TIA1 and TFRC were significantly up-regulated, while ESRP1, ESRP2 and TTP were significantly down-regulated. Cytoplasmic expression of HuR was activated in IPF lung tissue and rat lungs of bleomycin-induced fibrosis. HuR overexpression induced α-SMA and collagen I expression, increased the proliferation and migration capacities of fibroblasts with or without the stimulation of TGF-β1. HuR knockdown by shRNA inhibited the proliferation, transition, collagen production and migration properties in fibroblasts or in TGF-β1-stimulated myofibroblasts. Combinative analysis of RNA-seq and CLIP-seq data revealed major HuR binding motifs and several HuR-bound, differentially expressed mRNAs in FMT, specifically SPP1 mRNA encoding osteopontin. HuR-mediated SPP1 mRNA stabilization was further validated by RIP-PCR and half-life analysis using SPP1 mutant transcripts. Inhibiting HuR using CMLD-2 attenuated SPP1/osteopontin expression, TGF-β1-induced FMT in vitro and bleomycin-induced pulmonary fibrosis in mice. Nuclear-cytoplasmic shuttle of HuR was activated in TGF-β1-induced FMT, which was inhibited by p38MAPK (SB203580) or PKC (Go-6976) inhibition, but not associated with phosphorylation of HuR.ConclusionsFibroblast-myofibroblast transition is activated by HuR-SPP1 mRNA interactions, and CMLD-2 is potentiated to be a therapeutic agent targeting HuR for fibroblast-myofibroblast transition and pulmonary fibrosis.