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

Guo, Q.; Ren, H.; Li, M.; Liu, L.; Li, W.; Zhang, J.; Wang, X.; Hu, Y.; Zang, K.; An, Y.; Li, L.; Wei, L.; Xu, Z.; Guo, Z.; Costabel, U.; Zhang, X.; Zheng, W.

doi:10.1101/2022.03.20.484975

bioRxiv · 10.1101/2022.03.20.484975

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

Guo, Q.·Ren, H.·Li, M.·Liu, L.·Li, W.·Zhang, J.·Wang, X.·Hu, Y.·Zang, K.·An, Y.·Li, L.·Wei, L.·Xu, Z.·Guo, Z.·Costabel, U.·Zhang, X.·Zheng, W.

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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-{beta}1-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 -SMA and collagen I expression, increased the proliferation and migration capacities of fibroblasts with or without the stimulation of TGF-{beta}1. HuR knockdown by shRNA inhibited the proliferation, transition, collagen production and migration properties in fibroblasts or in TGF-{beta}1-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-{beta}1-induced FMT in vitro and bleomycin-induced pulmonary fibrosis in mice. Nuclear-cytoplasmic shuttle of HuR was activated in TGF-{beta}1-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.

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Guo, Q., Ren, H., Li, M., Liu, L., Li, W., Zhang, J., Wang, X., Hu, Y., Zang, K., An, Y., Li, L., Wei, L., Xu, Z., Guo, Z., Costabel, U., Zhang, X., Zheng, W.. 2022-03-21. CMLD-2 Attenuates Myofibroblast Activation and Bleomycin-Induced Pulmonary Fibrosis in Mice through Antagonizing ELAVL1-Mediated Osteopontin mRNA Stabilization. https://doi.org/10.1101/2022.03.20.484975

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