P38 mitogen-activated protein kinase mediates hexosamine-induced TGFβ1 mRNA expression in human mesangial cells

Aims/hypothesis. The hexosamine pathway has been implicated in the induction of TGFbeta1 expression and in the pathophysiology of diabetic glomerulopathy. Glucose-induced TGFbeta1 expression is mediated by p38 mitogen-activated-protein-kinase (p38-MAPK) and this kinase is activated in the diabetic glomeruli. We examined whether the p38-MAPK is implicated in hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells. Methods. The products of the hexosamine biosynthetic pathway were increased by the addition of glucosamine or by the overexpression of the rate-limiting enzyme of the hexosamine pathway, glutamine: fructose-6-phosphate amidotransferase (GFAT). Results. Glucosamine addition resulted in cell death. UDP-N-Acetylglucosamine, one of the major hexosamine end-products, was increased in normal (7 mmol/l) and high (25 mmol/l) glucose conditions in GFAT-transfected cells compared to control transfected cells by twofold and 1.7-fold respectively (pless than or equal to0.04) and this was accompanied by a 1.6- and 2.3-fold increase (p less than or equal to 0.02) in TGFbeta1 mRNA expression. Addition of the GFAT inhibitor azaserine (10 mumol/l) prevented the induction of TGFbeta1 in GFAT transfected cells. GFAT overexpression induced an increase in p38-MAPK activation after 6 and 12 h incubation in normal glucose, and this was prevented by the GFAT inhibitor azaserine. Furthermore, high glucose enhanced p38-MAPK activation in GFAT tranfected cells (p less than or equal to 0.04). P38-MAPK inhibition using SB202190 (1 mumol/l) reduced hexosamine-induced TGFbeta1 expression in normal and high glucose. The activation of the p38-MAPK was dependent on protein kinase-C. Conclusion/interpretation. Overexpression of GFAT increases hexosamine accumulation which mediates TGFPI expression via a protein kinase-C and p38-MAPK dependent mechanism. Increased glucose concentrations magnify these effects.