USP9X deubiquitinates connexin43 to prevent high glucose-induced epithelial-to-mesenchymal transition in NRK-52E cells.

Epithelial-to-mesenchymal transition (EMT) plays an important role in diabetic nephropathy (DN). Ubiquitin-specific protease 9X (USP9X/FAM) is closely linked to TGF-β and fibrosis signaling pathway. However, it remains unknown whether USP9X is involved in the process of EMT in DN. Our previous study has shown that connexin 43 (Cx43) activation attenuated the development of diabetic renal tubulointerstitial fibrosis (RIF). Here, we showed that USP9X is a novel negative regulator of EMT and the potential mechanism is related to the deubiquitination and degradation of Cx43. To explore the potential regulatory mechanism of USP9X, the expression and activity of USP9X were studied by CRISPR/Cas9-based synergistic activation mediator (SAM) system, short hairpin RNAs, and selective inhibitor. The following findings were observed: (1) Expression of USP9X was down-regulated in the kidney tissue of db/db diabetic mice; (2) overexpression of USP9X suppressed high glucose (HG)-induced expressions of EMT markers and extra cellular matrix (ECM) in NRK-52E cells; (3) depletion of USP9X further aggravated EMT process and ECM production in NRK-52E cells; (4) USP9X deubiquitinated Cx43 and suppressed its degradation to regulate EMT process; (5) USP9X deubiquitinated Cx43 by directly binding to the C-terminal Tyr of Cx43. The current study determined the protective role of USP9X in the process of EMT and the molecular mechanism clarified that the protective effects of USP9X on DN were associated with the deubiquitination of Cx43.