The Essential Role of O-GlcNAcylation in Hepatic Differentiation

Abstract

Background & Aims. O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase (OGT), which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and hepatocellular carcinoma. Methods. Single-cell RNA-sequencing was used to investigate hepatocyte differentiation in hepatocyte-specific OGT-KO mice with increased hepatic O-GlcNAcylation and in OGA-KO mice with decreased O-GlcNAcylation in hepatocytes. HCC patient samples and the DEN-induced hepatocellular carcinoma (HCC) model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Results. Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation characterized by dysregulation of glycogen storage and glucose production. OGT-KO mice exacerbated DEN-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, OGA-KO mice with increased hepatic O-GlcNAcylation inhibited DEN-induced HCC. A progressive loss of O-GlcNAcylation was observed in HCC patients. Conclusions. Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy

“…Loss of p53 is associated with aneuploidy 20 , and although we did not see a loss in p53 or MDM2 expression, p53 target p21 was up-regulated with OGT-KO, and increased p21 is associated with aneuploidy involving AurB alterations 30 . We also measured changes in HIPPO signaling specifically decreases in YAP phosphorylation leading to more YAP target gene expression 7 . Conversely, YAP O-GlcNAcylation is correlated with higher activity and OGT knockdown reduces this activity 42 .…”

“…Multi-Omics approaches were used to identify pathways regulated by O-GlcNAc while incorporating the pleotropic nature of O-GlcNAc into the analysis. First, we took OGT or OGA-floxed male mice and injected the animals AAV8-TBG Cre to knockout OGT or OGA in hepatocytes 7,8,10 , and we sacrificed the mice and harvested the livers after 1 or 2-weeks post-injection. Alternatively, we injected mice with OGA inhibitor Thiamet-G (TMG) every other day for 1 or 2-weeks and sacrificed the mice.…”

“…The OGT-KO livers showed some polyploidy but no aneuploidy (Figure 7A). Previously, when OGT-KO livers were subjected to partial liver hepatectomy (PHX), the livers showed increased cell growth and hypertrophy after 1-month 7,10 ; therefore, we challenged control and OGT/OGA-KO livers by performing a PHX and collecting the tissue 2-weeks post PHX. After PHX, OGT-KO livers presented with a robust aneuploid phenotype as measured by DNA content (Figure 7B).…”