O-GlcNAcylation inhibits hepatic stellate cell activation.

Protein O-GlcNAcylation is a critical post-translational modification regulating gene expression and fundamental cell functions. O-GlcNAc transferase (OGT) emerged as a key regulator of liver pathophysiology and disease. In this study, we aimed to evaluate the role of OGT in hepatic stellate cells (HSCs) and its consequent role in liver fibrosis. OGT protein expression and protein O-GlcNAcylation were significantly decreased in CCl4 - or MCD diet-induced liver fibrosis as compared to normal liver in mice. OGT expression and protein O-GlcNAcylation were also decreased in primary HSCs isolated from liver with CCl4 -induced fibrosis compared to those from normal liver. RNA-seq showed that OGT knockdown in HSCs modulated key signaling pathways involved in HSC activation. Promoter sequence analysis of the differentially expressed genes predicted serum response factor (SRF) as a key transcription factor regulated by OGT. Luciferase reporter assay confirmed that OGT repressed activity of SRF to induce α-SMA transcription. Mutations of specific O-GlcNAcylation sites on SRF increased its transcriptional activity, validating negative regulation of SRF by OGT-mediated O-GlcNAcylation. Our results suggest that OGT functions as a negative regulator of HSC activation by promoting SRF O-GlcNAcylation to protect against liver fibrosis.