1. CMLD-2 Attenuates Myofibroblast Activation and Bleomycin-Induced Pulmonary Fibrosis in Mice through Antagonizing ELAVL1-Mediated Osteopontin mRNA Stabilization
- Author
-
Guo Qiongya, Ren Hongyan, Li Meng, Liu Lifan, Li Wenting, Zhang Jingjing, Wang Xiaoli, Hu Yiping, Zang Kaixuan, An Yunxia, Li Lin, Wei Li, Xu Zhiwei, Guo Zhiping, Ulrich Costabel, Zhang Xiaoju, and Wang Zheng
- Abstract
BackgroundFibroblast-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.
- Published
- 2022