Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF-alpha-activated NF-kappaB.
hBVR is a Ser/Thr/Tyr kinase/scaffold protein/transcription factor/intracellular transporter of regulators. hBVR is an upstream activator of the insulin/IGF-1/MAPK/PI3K signaling pathway, and of NF-kappaB. As a reductase, it converts biliverdin to the antioxidant, bilirubin. hBVR gene has 8 exons; exon 1 is not translated. We report the characterization of hBVR promoter and its negative and positive regulation, respectively, by TNF-alpha and hypoxia. The 5' end of exon 1 was defined by primer extension analyses; deletion of an inhibitor sequence 350-425 bp upstream of this exon enhanced the promoter activity. One of two NF-kappaB binding sites in the 836-bp promoter was functional; the P65 subunit of NF-kappaB and TNF-alpha acted as inhibitors. On the basis of EMSA and ChIP assays, TNF-alpha treatment increases binding of NF-kappaB to its regulatory element. Overexpression of IkappaB increased hBVR mRNA. Biliverdin, but not bilirubin, was as effective as TNF-alpha in inhibiting hBVR promoter activity. Only one of 4 hypoxia responsive elements (HREs) bound to HIF-1alpha and ARNT expressed in HEK293A cells. An abasic site was introduced at the 3' G of the HRE. This element bound HIF-1 in the gel shift and in in-cell luciferase assays. hBVR was detected in the nucleus at 1, 2, and 4 h after hypoxia (1% O(2)), at which times its kinase and reductase activities were increased. Because hypoxia positively influences hBVR promoter and phosphorylation and TNF-alpha activated NF-kappaB inhibits the promoter, while biliverdin inhibits both NF-kappaB activity and hBVR promoter, we propose a regulatory mechanism for NF-kappaB by hypoxia and TNF-alpha centered on hBVR/biliverdin.
Gibbs
Peter E M
PE
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Miralem
Tihomir
T
Maines
Mahin D
MD
D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D005091:Exons / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000503:physiopathology*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D050436:Regulatory Elements, Transcriptional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase
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