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Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF‐α‐activated NF‐κB
Author(s) -
Gibbs Peter E. M.,
Miralem Tihomir,
Maines Mahin D.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.09-144592
Subject(s) - aryl hydrocarbon receptor nuclear translocator , microbiology and biotechnology , biliverdin , promoter , biology , transcription factor , luciferase , biliverdin reductase , transcription (linguistics) , biochemistry , transfection , gene expression , gene , heme oxygenase , heme , enzyme , aryl hydrocarbon receptor , linguistics , philosophy
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‐κB. 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‐α 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‐κB binding sites in the 836‐bp promoter was functional; the P65 subunit of NF‐κB and TNF‐α acted as inhibitors. On the basis of EMSA and ChIP assays, TNF‐α treatment increases binding of NF‐κB to its regulatory element. Overexpression of IκB increased hBVR mRNA. Biliverdin, but not bilirubin, was as effective as TNF‐α in inhibiting hBVR promoter activity. Only one of 4 hypoxia responsive elements (HREs) bound to HIF‐1α 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‐α activated NF‐κB inhibits the promoter, while biliverdin inhibits both NF‐κB activity and hBVR promoter, we propose a regulatory mechanism for NF‐κB by hypoxia and TNF‐α centered on hBVR/biliverdin.—Gibbs, P. E. M., Miralem, T., Maines, M. D. Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF‐α‐activated NF‐κB. FASEB J . 24, 3239–3254 (2010). www.fasebj.org