Increased flux through the hexosamine biosynthetic pathway is associated with altered gene expression. To investigate the underlying mechanisms, we treated glomerular mesangial cells with glucosamine and studied the regulation of the plasminogen activator inhibitor (PAI)-1 gene. Incubating mesangial cells with 2 mmol/l glucosamine for 4 days resulted in a 3.1+/-0.4-fold increase in PAI-1 mRNA levels (P < 0.01) and a 33+/-9-fold increase in the activity of a transiently transfected PAI-1 promoter-luciferase reporter gene (P < 0.01). Cotransfection of an expression vector for a dominant-negative type II TGF-beta receptor with the PAI-1 promoter-reporter gene did not interfere with this effect of glucosamine. However, mutation of 2 putative Sp1 sites in the PAI-1 promoter, at -76 to -71 and -44 to -39, markedly reduced induction of PAI-1 luciferase activity by glucosamine, from 8.9+/-1.9-fold to 1.7+/-0.5-fold (P < 0.01). An electrophoretic mobility shift assay demonstrated that glucosamine increased Sp1 DNA binding by 31+/-11% (P < 0.05), implying that the effects of glucosamine were explained, in part, by changes in Sp1 DNA binding. High glucose (20 mmol/l) also activated the transiently transfected PAI-1 promoter (2.5+/-0.4-fold). This effect was diminished by mutation of both the PAI-1 promoter Sp1 sites (1.2+/-0.3-fold, P < 0.05). In addition, 6-diazo-5-oxo-L-norleucine, a glutamine:fructose-6-phosphate-amidotransferase inhibitor, blocked the induction by high glucose (4.7+/-0.8- to 0.9+/-0.1-fold, P < 0.01). These results indicate that stimulation of the PAI-1 promoter by both high glucose and glucosamine involves Sp1 and that the hexosamine pathway may be involved in the regulation of gene expression by high glucose in glomerular mesangial cells.

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