Activated PKR Inhibits Pancreatic β-cell Proliferation Through Sumoylation-dependent P53 Stability

Objective: Double-stranded RNA-dependent protein kinase PKR is a critical component in the course of cell cycle arrest and growth inhibition induced by different cytokines. PKR has a close contact with type 2 diabetes, due to its role in insulin resistance in peripheral tissues and anti-proliferative effect on pancreatic β-cells. Activated PKR was found to inhibit β-cell proliferation and cell cycle G1 phase arrest, partially through accumulation of P53. But the molecular mechanisms underlying PKR upregulates P53 remain to elucidate. According to our present results, PKR can be specifically activated by low-dosage of BEPP in PKR overexpressing β-cells and then upregulates P53 through sumoylation-dependent stability. Activated PKR can interact with sumo-conjugating enzyme Ubc9. P53 sumoylation relies on PKR-Ubc9 protein-protein interaction. The aim of this study was to investigate the mechanism of PKR/Ubc9/P53 on β-cell proliferation inhibition in the development of type 2 diabetes.Methods: Mice pancreatic β-cell line(MIN-6) was transfected with PSG5 con-plasmid or wt-PKR plasmid(mouse) to construct normal/PKR-overexpressing β cells. MIN-6 was treated with low dosage BEPP(1u M), high glucose(16.7 m M) and high fatty acid(0.1m M palmitic acid) at the same time or inflammatory cytokine TNF-α to construct an in vitro rodent diabetic model. Hoechst 33258 dyeing, Ed U labeling and flow cytometry analysis were used to respectively detect β-cell proliferation and cell cycle distribution of MIN-6 cell line. Western blot analysis was performed to detect protein levels of p-PKR and its downstream signal molecule p-e IF2α in pancreatic β-cells to observe PKR activation. Co-immunoprecipitationAnalysis was used to detect PKR-Ubc9,P53-Ubc9 and P53-SUMO-1 protein-protein interaction to verify P53 sumoylation, coordinating western blot analysis to examine SUMO-1,Ubc9 and P53 protein level. Immunofluorescence assay analysis was applied to detect co-location of PKR and Ubc9. PKR inhibitor 2-AP, ceramide synthesis enzyme inhibitor FUMO B1 and sumoylation inhibitor Spectomycin B1 were pretreated to establish negative control group.Results: In PKR overexpressing MIN-6 cells, low dosage BEPP, glucolipotoxicity and TNFα stimuli can activate PKR and induce its downstream signal molecular e IF2α phosphorylation. PKR activation can induce β cell proliferation arrest and cell cycle G1 phase arrest. The mechanism of G1 phase arrest was PKR-Ubc9 protein-protein interaction. Sumo-conjugating enzyme Ubc9 triggered P53 sumoylation. Co-location of PKR and Ubc9 can be detected in cytoplasm. Spectomycin B1 inhibited P53 sumoylation and protein level upregulation. Similarly, PKR inhibitor and FUMO B1 could inhibit P53 sumoylation through inhibiting PKR activation.Conclusion: Our research revealed that the activation of ceramide/PKR/Ubc9 signaling pathway upregulated P53 protein level and stability though P53 sumoylation, leading to pancreatic β cell proliferation arrest. P53 sumoylation involved in β cell dysfunction and the mechanism of type 2 diabetes.