Cross Interaction Between OGT and mTOR in T Cells and its Consequences in Autoimmunity

Abstract

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that has been studied extensively, yet an understanding of the mechanisms of this disease remains unclear. It is known that epigenetic mechanisms are involved in the disease. We found O‐GlcNAc Transferase (OGT), an X‐linked gene, overexpressed due to hypomethylation in T cells from lupus patients (J. Autoimmunity, 2013). Furthermore, it was reported that the mechanistic target of rapamycin (mTOR) modifies T cell cytokine patterns and contributes to the TReg defect in patients with lupus. (J. Immunology, 2014).PurposeTo investigate the role OGT has in T cell signaling in order to understand the contribution of its overexpression to lupus. In particular, we investigated the relationship between OGT and the mTOR pathway in T cells to understand their participation in the disease.MethodsHealthy female donors were recruited by advertising. The research was reviewed and approved by the University of South Alabama Review Board for Human Subject Research. Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation, then T cells were isolated by magnetic beads. One fraction was transiently transfected for 48 h with 2μg of OGT siRNA using a nucleofector device (Amaxa). Other fractions were subjected to different treatments. Cells were stimulated after treatments with PMA/Ionomycin for 4h and the whole cell extracts were separated by SDS‐PAGE electrophoresis. Protein expression was analyzed by immunoblotting using specific antibodies. ß actin was used as loading control.ResultsIncreasing the levels of glycosylated proteins with 100 mM Pugnac, a competitive inhibitor of O‐GlcNAc‐β‐N‐acetylglucosaminidase (OGA), or with 5mM Glucosamine, increased the phosphorylation of p70S6K, substrate of mTORC1, which indicates an increase in mTOR activity. On the other side, when OGT is knocked down by siRNA, as expected, the protein expression of OGT levels as well as glycosylated protein levels decreased. Interestingly, the activity of mTOR also decreased. Similar results were observed with 10 nM MHY1485, potent activator of mTOR, or with 50 nM rapamycin and 10 nM Torin‐1, both inhibitors of mTOR, which increased or decreased the phosphorylation of its substrate respectively. However, they were unable to modify the levels of glycosylation.ConclusionsThese results strongly suggest that mTOR activation is OGT‐dependent and is located downstream of OGT in T cells. Overexpression of OGT could be responsible for the increase in mTOR activity in T cells from lupus patients.Support or Funding InformationNIH 5R03 AR067518‐02Summer undergraduate research fellowship (SURF) University of South AlabamaDepartment of Biomedical Science, College of Allied Health, University of South Alabama