O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Abstract

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice

“…While there are several members of the MAP3K family that can induce p38 activation, we have shown that ASK1 plays a nonredundant and essential role in activating p38 in the diabetic kidney. This is consistent with a role for ASK1 in high glucose-induced p38 activation in cultured glomerular mesangial cells (36) and the various stresses present in diabetes (e.g., oxidative stress, endoplasmic reticular stress, hyperglycemia, and angiotensin II) that are known to activate ASK1/p38 signaling (37). However, activation of p38 MAPK signaling can operate independent of ASK1 in kidney cells as shown by the unaltered lipopolysaccharide and interleukin-1-induced p38 activation and biological responses seen in Ask1 2/2 tubular epithelial cells (4).…”

p38 Mitogen-Activated Protein Kinase Is Required for Glucosamine-Induced Endothelial Nitric Oxide Synthase Uncoupling and Plasminogen-Activator Inhibitor Expression

“…Indeed, activation of p38 mapk by the HBP has been reported in mesangial cells, leading to PAI-1 gene expression. 31 In the present study we detected a transient activation of p38 mapk by GlcN, which was potentiated There are studies showing that p38 mapk activates OGT in the HBP, leading to enhanced O-GlcNAcylation of proteins. 30 It is unlikely, however, that in the present experimental setting, TNF-α enhances GlcN-induced PAI-1 expression through augmentation of increased OGT activity, because our previous study showed no further increase in O-GlcNAcylation of cellular proteins in endothelial cells exposed to TNF-α, 38 although O-GlcNAcylation of certain proteins could not be fully excluded.…”

“…30 Conversely, O-GlcNAc seems also to be able to activate p38 mapk. 16, 31 The aim of the present study was to investigate the critical role of p38 mapk in GlcN-induced PAI-1 expression and eNOS uncoupling in endothelial cells and in isolated mouse aortas.…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…While the above studies were performed in hepatocytes, subsequent studies in a model of myocardial damage demonstrated that catalase mRNA levels were augmented above baseline following inhibition of O-GlcNAcase (PUGNAc), while overexpression of O-GlcNAcase reduced baseline catalase mRNA levels (Ngoh et al 2011). Notably, in contrast to the studies discussed here, O-GlcNAc is thought to promote ROS generation in models of hyperglycemia and glucose toxicity (Goldberg et al 2011), although the molecular basis for this paradox remains to be defined (Lima et al 2012). …”