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

Abstract

Aims/hypothesis. The hexosamine pathway has been implicated in the induction of TGFβ1 expression and in the pathophysiology of diabetic glomerulopathy. Glucose-induced TGFβ1 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 TGFβ1 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 (p≤0.04) and this was accompanied by a 1.6-and 2.3-fold increase (p≤0.02) in TGFβ1 mRNA expression. Addition of the GFAT inhibitor azaserine (10 µmol/l) prevented the induction of TGFβ1 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≤0.04). P38-MAPK inhibition using SB202190 (1 µmol/l) reduced hexosamine-induced TGFβ1 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 TGFβ1 expression via a protein kinase-C and p38-MAPK dependent mechanism. Increased glucose concentrations magnify these effects. [Diabetologia (2003) 46:531-537]

Renal p38 MAP kinase activity in experimental diabetes

“…Renal p38 in experimental diabetes R Komers et al lation of products of glycation reactions, and oxidative stress, have been shown to induce p38 signaling in mesangial cells, podocytes and in proximal tubular cells in vitro. 3,16,[23][24][25] In agreement with these findings, present studies, conducted in more clinically relevant settings, show that poor metabolic control is the major determinant of p38 activity in the diabetic kidney. Our findings are also in agreement with limited in vivo evidence in the setting of Type 1 diabetes, suggesting increased expression of P-p38 protein expression in wholekidney homogenates harvested from mice early after induction of STZ-diabetes.…”

“…1 In the kidney, such aberrant activation of MAPK cascades may perturb the balance of humoral and cytokine systems responsible for regulating vascular tone, permeability, renal cellular growth, and composition of the extracellular/mesangial matrix (ie, factors associated with the development of diabetic nephropathy). 2,3 p38 mitogen-activated protein kinase (p38), a member of the MAPK family, is activated by a number of stimuli. As a stress-activated kinase, p38 is activated by physical and chemical stress factors, inflammatory cytokines, and vasoactive and growth factors, resulting in growth promotion, apoptosis, oxidative stress, and vasoconstriction.…”

“…10 Convincing in vitro data support a role for p38 in the cell response to a diabetic milieu. The p38 pathway is activated in rat aortic vascular smooth muscle cells (VSMC), 11,12 in renal cells cultured in high-glucose media, 3,[13][14][15][16] in isolated glomeruli of streptozotocin (STZ)-diabetic rats, 2,15 and is involved in mediating signals relevant for development of diabetic nephropathy.…”

Protein O-GlcNAc Modulates Motility-associated Signaling Intermediates in Neutrophils

“…Several studies, all performed under different cellular contexts, have recently demonstrated that increases in HBP flux can lead to increased MAPK activation (16,78,(83)(84)(85). In addition, HBP-driven changes in p44/p42 activation have been suggested to occur through a Rho GTPase-mediated mechanism in cultured rat aortic smooth muscle cells (78).…”

Direct regulation of insulin secretion by angiotensin II in human islets of Langerhans

“…Isolated rat and human islets, HepG2 human liver carcinoma cells, MIN6 beta cells, and small pieces of mouse heart were lysed in 200 μl ice-cold lysis buffer (100 mmol/l TrisHCl, pH 8.0, 500 mmol/l LiCl, 10 mmol/l EDTA, 0.1% [w/v] lithium dodecylsulphate, 5 mmol/l dithiothreitol) and mRNAs were isolated using a Dynabeads oligo (dT) 25 kit (Dynal, Oslo, Norway) according to the manufacturer's instructions. cDNAs were synthesised from each mRNA sample immediately, using Superscript II reverse transcriptase (200 U), oligo(dT) 18 (1 μg) and random 10-mers (1 μg) as previously described [23], and cDNAs were diluted tenfold in tRNA (10 μg/ml) and stored at −85°C.…”