Posttranslational, reversible O-glycosylation is stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells
Posttranslational, reversible O-glycosylation is stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells
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DOI：
10.1210/en.2005-0523
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发表时间：
2006-01-01
期刊：
影响因子：
4.8
通讯作者：
Fantus, IG
中科院分区：
文献类型：
--
作者：
Goldberg, HJ;Whiteside, CI;Fantus, IG
在高糖(HG)存在下，通过己糖胺生物合成途径(HBP)的代谢通量增加，并潜在地刺激与糖尿病肾病发展相关的基因的表达。HBP有许多合成过程，包括酶促细胞内O-糖基化(O-GlcN酰化)，将单一的O-连接的N-乙酰氨基葡萄糖单糖加到丝氨酸或苏氨酸残基上。尽管HBP和基因表达之间存在着大量的数据联系，但O-GlcN酰化对HG刺激的基因表达的确切作用仍不清楚。在肾小球系膜细胞中，HG刺激的纤溶酶原激活物抑制物-1(PAI-1)基因表达需要HBP和转录因子Sp1。在这项研究中，通过用显性负性O-连锁N-乙酰氨基葡萄糖转移酶限制O-GlcN酰化修饰，通过过度表达中性β-N-乙酰氨基葡萄糖苷酶，以及通过RNA干扰抑制O-连锁β-N-乙酰氨基葡萄糖转移酶的表达，来检测O-GlcN酰化在HG诱导的PAI-1表达中的特定作用。通过这些方法降低O-GlcN酰化水平会抑制HG增加内源性PAI-1mRNA和蛋白水平的能力、PAI-1启动子-荧光素酶报告基因的活性以及Sp1的转录激活。相反，β-N-乙酰氨基葡萄糖苷酶抑制剂O-(2-乙酰氨基-2-脱氧-D-吡喃葡萄糖基)氨基-N-苯基氨基甲酸酯在正常葡萄糖存在下处理可增加Sp1O-GlcN酰化和PAI-1mRNA和蛋白水平。这些发现首次证明，在HBP所服务的通路中，O-GlcN酰化是HG诱导的系膜细胞PAI-1基因表达和Sp1转录激活所必需的。
Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG- stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG- induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral beta-N-acetylglucosaminidase, and by knockdown of O-linked beta-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the beta-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG- induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells.