O‐GlcNAcylation and p50/p105 binding of c‐Rel are dynamically regulated by LPS and glucosamine in BV2 microglia cells

Abstract

BACKGROUND AND PURPOSEPreviously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on LPS-induced inducible NOS (iNOS) through the inhibition of NF-κB activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation. EXPERIMENTAL APPROACHBV2 cells were stimulated with LPS with or without GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interactions were examined by co-immunoprecipitation. KEY RESULTSLPS stimulated the activation of c-Rel, increased the O-GlcNAcylation of c-Rel and enhanced the binding of c-Rel to the NF-κB site in the iNOS promoter; GlcN attenuated these effects of LPS. O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel was increased by LPS and reduced by GlcN. LPS increased the interaction of c-Rel with O-GlcNAc transferase (OGT) and p50/p105, and GlcN suppressed these interactions. Knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, but did not affect either the binding of c-Rel to the iNOS promoter or the transcriptional activity of c-Rel. CONCLUSIONS AND IMPLICATIONSIn BV2 microglial cells, the anti-inflammatory effect of GlcN is mediated by prevention of the prolonged activation of transcription factors, c-Rel and NF-κB. Further clarification of the mechanism by which GlcN exerts this effect will facilitate the development of pharmacological strategies for preventing excessive NO formation when targeting inflammatory diseases of the periphery or CNS.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells

“…7A, upper panels). The amount of c-Rel protein in the WGA precipitate was increased by LPS and this increase was inhibited by GlcN at the 25 mM glucose conditions, as previously described (16). However, WGA binding of c-Rel in response to LPS was dramatically increased by GlcN at the 5 mM glucose conditions (Fig.…”

“…O-GlcNAcylation of c-Rel (16). We performed wheat germ agglutinin (WGA) pulldown assays using WGA-conjugated agarose beads that specifically precipitate proteins containing N-acetylglucosamine or N-acetylneuramic acid.…”

“…2 To whom correspondence should be addressed: 100 Inha Ro, Nam-gu, Incheon 22212, Korea. GlcN by regulating c-Rel O-GlcNAcylation and subsequent NF-B activity in both microglia and macrophage cells (16). Activation of macrophages or microglia by LPS stimulates mitogen-activated protein (MAP) kinases and NF-B.…”

Targeted placental deletion of OGT recapitulates the prenatal stress phenotype including hypothalamic mitochondrial dysfunction

“…OGT broadly affects cellular functioning through the unique posttranslational modification, O-GlcNAc, on serine and threonine residues of over 1,000 different intracellular proteins (11). Many of these protein targets (notably RNA polymerase II, histone deacetylase-2, Tet proteins, and core histone H2B) are part of chromatin structure or directly regulate chromatin confirmation (12)(13)(14)(15)(16)(17). Therefore, we hypothesized that reduced placental OGT may be a mechanism through which EPS alters placental function.…”

“…OGT is an important cellular nutrient, stress, and immune sensor (16,22,31). In the placenta, OGT appears to provide signals as to an altered environment (nutritive and/or stressors) that shapes hypothalamic circuits involved in long-term programming of energy homeostasis and stress responsivity in a sexdependent manner.…”