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

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-kappa B. 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-kappa B binding sites in the 836-bp promoter was functional; the P65 subunit of NF-kappa B and TNF-alpha acted as inhibitors. On the basis of EMSA and ChIP assays, TNF-alpha treatment increases binding of NF-kappa B to its regulatory element. Overexpression of I kappa B 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-1 alpha 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-kappa B inhibits the promoter, while biliverdin inhibits both NF-kappa B activity and hBVR promoter, we propose a regulatory mechanism for NF-kappa B by hypoxia and TNF-alpha 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-alpha-activated NF-kappa B. FASEB J. 24, 3239-3254 (2010). www.fasebj.org