Abstract
Increased flux through the hexosamine biosynthesis pathway (HBP) has been shown to stimulate the expression of a number of genes. We previously demonstrated in glomerular mesangial and endothelial cells that both high glucose concentrations and glucosamine activated the plasminogen activator inhibitor-1 (PAI-1) gene promoter through the transcription factor, Sp1; and that the glutamine:fructose-6-phosphate amidotransferase inhibitor, 6-diazo-5-oxonorleucine, inhibited the effect of high glucose, but not that of glucosamine. Here, we examined the role of protein kinase C (PKC) isoforms in the regulation of the PAI-1 promoter and Sp1 transcriptional activity by the HBP. In transient transfections, exposure to 2 mm glucosamine or 20 mm glucose for 4 days increased the activities of a PAI-1 promoter-luciferase reporter gene as well as the Sp1 transcriptional activation domain fused to the GAL4 DNA-binding domain cotransfected with a GAL4 promoter-luciferase reporter. Cotransfected dominant negative PKC-betaI and -delta completely blocked the induction of PAI-1 promoter transcription by both sugars, whereas only dominant negative PKC-betaI interfered with Sp1-GAL4 activation. Both glucosamine and high glucose stimulated the in vitro kinase activity of immunoprecipitated PKC-betaI and -delta. Furthermore, 6-diazo-5-oxonorleucine suppressed high glucose-induced PKC kinase activity and Sp1-GAL4 transcriptional activation. These findings demonstrate a requirement for the PKC-betaI and -delta signal transduction pathways in HBP-induced transcription.
Highlights
Prolonged exposure to high glucose concentrations promotes the development of microvascular disease associated with diabetes mellitus [1, 2]
Toxic effects of the dominant negative (DN)-protein kinase C (PKC) isoforms in the presence of glucosamine were ruled out by transfecting a luciferase reporter gene driven by a CMV promoter (CMV-LUC)
This conclusion is further supported by findings that high glucose significantly increased the kinase activity of these same PKC isoforms, and that high glucose-induced plasminogen activator inhibitor-1 (PAI-1) promoter activity was inhibited by DN-PKC-I and -␦
Summary
Prolonged exposure to high glucose concentrations (high glucose) promotes the development of microvascular disease associated with diabetes mellitus [1, 2]. We examined the role of protein kinase C (PKC) isoforms in the regulation of the PAI-1 promoter and Sp1 transcriptional activity by the HBP. High glucose and glucosamine strongly stimulated the activity of a Sp1 transcriptional activation domain-GAL4 DNA binding domain fusion protein cotransfected with a GAL4-luciferase reporter and this was blocked by DN-PKC-I.
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