Yonsei Biology Professor Cho Jin Won Proves Activation Mechanism for NFκB, a Trigger Factor of the Immune System Under Hyperglycemic Conditions The research team of Professor Cho Jin Won of the Yonsei Biology Department and Dr. Yang Won-Ho, a post-doctoral fellow at the University of California, San Diego, discovered that NFkB activation is associated with O-GlcNAcylation state under hyperglycemic condition. It was shown that modification of p65 (a member of NFkB) by O-linked N-acetylglucosamine (GlcNAc) at Thr 352 inhibits interaction to IkB, thereby continuing NFkB activation in nucleus under hyperglycemic condition. The research was published in the October 28 online issue of the Proceedings of the National Academy of Sciences (PNAS). So far, more than 600 nucelocytoplasmic proteins have been identified as O-GlcNAcylated proteins. This modification occurs on hydroxyl group of Serine or Threonine and can compete with phosphorylation. Thereofore, the O-GlcNAc modification may modulate many important cellular functions. O-GlcNAc modification is now widely believed to be involved in developing diabetic complication. The transcription factor nuclear factor-kB (NFkB) plays an important function in immune responses and inflammation. NFkB is composed of a heterodimer of p65 and p50 subunits in most cell types and stays in the cytoplasm by binding to its inhibitor protein, the IkB. It has been reported that NFkB p65 protein is posttranslationally modified by many other chemicals including phosphate, acetyl group and O-GlcNAc. Although it is known that O-GlcNAcylation of NFkB is involved in hyperglycemia-induced NFkB activation, and is required for lymphocyte activation, the specific sites and the function of O-GlcNAcylation on NFkB activation are not well understood. Further, it was reported that NFkB was continuously activated under diabetic condition. In this study, three approaches, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) overexpression, and O-GlcNAcase inhibitor treatment were made to modulate O-GlcNAcylation state in rat vascular smooth muscle cells (VSMCs). When OGA was overexpressed in VSMCs, O-GlcNAcylation was downregulated and hyperglycemia-induced NFkB activation was inhibited. On the other hand, when OGT was overexpressed and OGA inhibitors were treated, NFkB transcriptional activity was increased. Two O-GlcNAcylation sites, Thr 322 and Thr 352 were identified by mutation study and by using mass spectrometry analysis. The data showed that an increment of O-GlcNAcyaltion on NFkB p65 increased the transcriptional activity of NFkB. Using the cell line which does not have NFkB p65, it was found that O-GlcNAcylation on Thr352 interrupted the interaction between NFkB and IkB. This O-GlcNAcylated NFkB was then translocated to the nucleus and stayed there longer than unmodified NFkB because IkB could not interact to O-GlcNAcylated NFkBs to take them out to cytosol. Furthermore, more activated NFkBs were found in nuclei of various organs from STZ induced diabetic mice. This finding may give some clue as to why NFkB is continuously activated under diabetic conditions, and lead to further research regarding diabetes complications and the development of related drugs.