Glutamine:fructose-6-phosphate aminotransferase enzyme activity is necessary for the induction of TGF-beta1 and fibronectin expression in mesangial cells.
OBJECTIVE
Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate aminotransferase (GFAT) as a rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine TGF-beta1, a key mediator in the development of diabetic nephropathy and possibly other diabetic angiopathies. In this study we investigated the effect of enhanced expression of wild-type GFAT and two enzymatically inactive GFAT mutants on TGF-beta1 synthesis in mesangial cells.


METHODS
Mutated human GFAT expression vectors were prepared by PCR-site directed mutagenesis. Wild-type and mutated vectors were transfected into human embryonic kidney 293 cells and mesangial cells and GFAT enzyme activity was assessed by formation of glucosamine-6-phosphate. Production of TGF-beta1 and fibronectin protein was examined by ELISA.


RESULTS
Mutation of histidine 577 or lysine 676 to alanine led to a complete loss of GFAT enzyme activity. An increased concentration of wild-type GFAT in mesangial cells enhanced both TGF-beta1 and fibronectin production 1.5-fold, while mesangial cells transfected with the mutated GFAT constructs showed no effect.


CONCLUSIONS
The data indicate that the hexosamine pathway-mediated induction of TGF-beta1 synthesis in mesangial cells is dependent on GFAT enzyme activity. Our results add to previous observations showing that the hexosamine pathway could increase the transcriptional activity of nuclear proteins leading to enhanced cytokine synthesis.
Weigert
C
C
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany.
Friess
U
U
Brodbeck
K
K
Häring
H U
HU
Schleicher
E D
ED
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D005353:Fibronectins / Q000235:genetics*; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000502:physiology*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D042561:Vascular Endothelial Growth Factor B / Q000235:genetics*
D005353:Fibronectins; D011994:Recombinant Proteins; C467485:VEGFB protein, human; D042561:Vascular Endothelial Growth Factor B; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)