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Identification and characterization of the human Gb3/CD77 synthase gene promoter.
Hemolytic uremic syndrome (HUS) is triggered by verotoxin (VT) produced by the Escherichia coli O157 strain. Several studies have demonstrated that VT induces endothelial cell (EC) death via the VT receptor globotriaosylceramide (Gb3/CD77) leading to this symptom. Inflammatory mediators which are produced as a result of E. coli O157 infection, increase the expression level of Gb3 in EC. Therefore increased expression of Gb3 is considered as a progression step for HUS. The increased expression of Gb3 is due to the transcriptional upregulation of Gb3/CD77 synthase gene (Gb3S, also known as alpha1,4-galactosyltransferase gene), the mechanism of which still remains unknown. To understand the transcriptional machinery and to elucidate the onset mechanism of HUS, we cloned and characterized the human Gb3S promoter. A modified 5'-RACE was used to determine the transcriptional initiation site, which revealed the presence of a TATA-less GC-rich sequence in the proximal region. Promoter activity measured using a luciferase assay demonstrated that the GC-rich sequence is necessary for the basal transcriptional activity, and two silencer elements located 5'-upstream of this GC-rich region regulated the transcriptional level. Furthermore, we found that the GC-rich sequence contained three potential Sp1 binding sites and that all three Sp1 binding elements acted as positive regulators. Since Sp1 is an inducer of several genes in the presence of the inflammatory cytokines in EC, our results suggest that the transcriptional regulation of the Gb3S gene by Sp1 might affect the VT sensitivity of EC and HUS progression.
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