Isolation and characterization of the human gp130 promoter. Regulation by STATS.
Glycoprotein 130 (gp130), a shared component of all the receptors for the interleukin-6 cytokine family, transduces cytokine signals in part by activating latent cytoplasmic signal transducers and activators of transcription (STATs). STATs subsequently translocate into the nucleus and stimulate gene expression. In the studies reported here, the 5'-flanking region of the human gp130 gene was isolated and the transcription initiation sites were mapped. To demonstrate that the isolated DNA fragment contained a functional promoter, a plasmid construct containing 2433 base pairs of the gp130 5'-flanking region, inserted upstream from the firefly luciferase gene, was transiently transfected into HepG2 hepatoma cells. The construct exhibited constitutive promoter activity. In addition, a 5-h treatment with interleukin-6 or oncostatin M stimulated the activity of this promoter severalfold. Localization of the cytokine response element by 5'-deletion analysis and site-directed mutagenesis revealed a cis-acting binding site for activated STAT complexes. Furthermore, DNA binding analysis demonstrated that this element binds activated STAT1 and STAT3 homo- and heterodimers. This STAT-binding element was sufficient to confer cytokine stimulation to a minimal herpesvirus thymidine kinase promoter. These results establish that the DNA fragment we have isolated contains the human gp130 promoter and that interleukin-6 type cytokines may influence the activity of this promoter via activated STATs.
O'Brien
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CA
Division of Endocrinology, University of Arkansas for Medical Sciences Center of Osteoporosis and Metabolic Bone Diseases, University of Little Rock, Arkansas 72205, USA. cobrien@life.uams.edu
Manolagas
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SC
D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000378:metabolism; D050822:Cytokine Receptor gp130; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015698:Genomic Library; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008156:Luciferases / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018121:Receptors, Cytokine / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D004268:DNA-Binding Proteins; C494199:IL6ST protein, human; D008562:Membrane Glycoproteins; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D050822:Cytokine Receptor gp130; D008156:Luciferases