Role of signal transducers and activators of transcription 1 and -3 in inducible regulation of the human angiotensinogen gene by interleukin-6.
The circulating level of angiotensinogen (AGT) is dynamically regulated as an important determinant of blood pressure and electrolyte homeostasis. Because the mechanisms controlling the regulated expression of human angiotensinogen (hAGT) are unknown, we investigated the inducible regulation of the hAGT gene in well differentiated HepG2 cells. Interleukin-6 (IL-6) stimulation produced a 3.2-fold increase in hAGT mRNA peaking at 96 h after stimulation. Deletional mutagenesis of the hAGT promoter in transient transfection assays identified an IL-6 response domain between nucleotides -350 and -122 containing three reiterated motifs, termed human acute phase response elements (hAPREs). Although mutation of each site individually caused a fall in IL-6-inducible luciferase activity, mutation of all three sites was required to block the IL-6 effect. Electrophoretic mobility shift assay (EMSA), supershift, and microaffinity DNA binding assays indicate IL-6-inducible high-affinity binding of signal transducers and activators of transcription 1 and -3 (STAT1 and -3) to hAPRE1 and -3 but only low-affinity binding to hAPRE2. Expression of a dominant-negative form of STAT3, but not STAT1, produced a concentration-dependent reduction in IL-6-induced hAGT transcription and endogenous mRNA expression. These data indicate that STAT3 plays a major role in hAGT gene induction through three functionally distinct hAPREs in its promoter and suggest a mechanism for its up-regulation during the acute-phase response.
Sherman
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CT
Department of Internal Medicine Sealy Center for Molecular Sciences and Human Biological Chemistry and Genetics The University of Texas Medical Branch Galveston, Texas 77555-1060, USA.
Brasier
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AR
D000209:Acute-Phase Proteins / Q000378:metabolism; D000808:Angiotensinogen / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements / Q000235:genetics; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D017384:Sequence Deletion; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000209:Acute-Phase Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D000808:Angiotensinogen; D008156:Luciferases