Role and Mechanism of Rho-Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) in Lipopolysaccharide-Evoked...

Because lung fibroblasts play a key role in the pathogenesis of pneumonia, and rho-associated coiled-coil containing protein kinase 1 (ROCK1) is a regulator of lung inflammation, this study studied the action of ROCK1 on lung fibroblast functions under pneumonic conditions.

WI-38 fibroblasts were stimulated with lipopolysaccharide (LPS) in vitro. A mouse model of pneumonia was produced by LPS induction. IP, Co-IP, and protein stability assays were used to confirm the ubiquitin-specific protease 33 (USP33)/ROCK1 relationship. RIP, Me-RIP, and mRNA stability assays were used to validate the methyltransferase-like 3 (METTL3)/ROCK1 relationship. In LPS-inducible WI-38 cells and serum samples of patients with pneumonia, ROCK1, USP33, and METTL3 levels were increased. ROCK1 deficiency attenuated LPS-evoked apoptosis, inflammation, and oxidative stress in WI-38 fibroblasts and BEAS-2B cells, and also diminished macrophage M1 polarization. Mechanistically, USP33 stabilized ROCK1 protein through deubiquitination, and METTL3 stabilized ROCK1 mRNA in an m6A-IGF2BP1-dependent mode. Depletion of USP33 or METTL3 mitigated LPS-evoked WI-38 cell injuries and macrophage M1 polarization by downregulating ROCK1. Moreover, ROCK1 depletion ameliorated LPS-evoked lung injuries in a pneumonia mouse model.

Our findings suggested that ROCK1 upregulation induced by USP33 and METTL3 affected LPS-evoked dysfunction in WI-38 fibroblasts and lung injuries in pneumonic mice, providing promising therapeutic targets for pneumonia.