GLUCOSE REGULATION OF TRANSFORMING GROWTH-FACTOR-ALPHA EXPRESSION IS MEDIATED BY PRODUCTS OF THE HEXOSAMINE BIOSYNTHESIS PATHWAY

We have recently shown that glucose and glucosamine regulate the trans cription of transforming growth factor-alpha (TGFalpha) in rat aortic smooth muscle (RASM) cells. Based on the increased potency of glucosam ine compared to glucose, we hypothesized that stimulation of TGFalpha transcription by glucose is mediated through the hexosamine biosynthes is pathway. The yeast cDNA for the rate-limiting enzyme of this pathwa y, glutamine:fructose-6-phosphate amidotransferase (GFA), was therefor e expressed in RASM cells. GFA-transfected cells showed an increase in GFA activity, exhibiting a 2.2-fold increase in the synthesis of gluc osamine-6-phosphate, the first product of the hexosamine biosynthetic pathway. To test the effect of GFA overexpression on TGFalpha transcri ptional activity, cells were transiently cotransfected with GFA along with a reporter plasmid containing the firefly luciferase gene under c ontrol of the TGFalpha promoter. GFA-transfected cells exhibited a glu cose-dependent 2-fold increase in TGFalpha activity compared to contro l cells. Maximal stimulation of TGFalpha-luciferase activity by glucos amine, however, was equivalent in GFA- and control-transfected cells, confirming that the stimulation observed by both agents operated throu gh the same pathway. This increase in TGFalpha activity was inhibited (85% at 0.5 mm glucose and 69% at 30 mm glucose) by the glutamine anal og and inhibitor of GFA, 6-diazo-5-oxonorleucine (10 mum). Control stu dies confirmed that the increased TGFalpha-luciferase activity in the GFA-expressing cells was not an artifact of altered growth, survival, or transfection efficiency. Experiments using pharmacological agents t o stimulate or inhibit protein kinase C and cAMP-dependent kinase do n ot support a role for these second messengers in the signaling pathway . Tunicamycin inhibited the ability of glucose to stimulate TGFalpha a ctivity, suggesting that protein glycosylation does play a role. We co nclude that products of the hexosamine biosynthesis pathway mediate th e stimulation by glucose of TGFalpha in aortic smooth muscle cells.