P38 mitogen-activated protein kinase mediates hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells.
OBJECTIVE
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. 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 micro mol/l) reduced hexosamine-induced TGFbeta1 expression in normal and high glucose. The activation of the p38-MAPK was dependent on protein kinase-C.


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 TGFbeta1 mRNA expression. Addition of the GFAT inhibitor azaserine (10 micro mol/l) prevented the induction of TGFbeta1 in GFAT transfected cells.


CONCLUSIONS
Overexpression of GFAT increases hexosamine accumulation which mediates TGFbeta1 expression via a protein kinase-C and p38-MAPK dependent mechanism. Increased glucose concentrations magnify these effects.
Burt
D J
DJ
Department of Diabetes, Endocrinology and Internal Medicine, Guys Hospital, King's College, 5th Floor Thomas Guy House, SE1 9RT, London, UK.
Gruden
G
G
Thomas
S M
SM
Tutt
P
P
Dell'Anna
C
C
Viberti
G C
GC
Gnudi
L
L
D002478:Cells, Cultured; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005944:Glucosamine / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics*; D053773:Transforming Growth Factor beta1; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases
D012333:RNA, Messenger; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014537:Uridine Diphosphate N-Acetylglucosamine; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose; D005944:Glucosamine