Abstract
Biliverdin reductase catalyzes the reduction of biliverdin, the product of heme oxygenase (HO) activity, to bilirubin. The reductase is unique among all enzymes characterized to date in being dual pH/cofactor-dependent. Until now the enzyme was assumed to be a noninducible cytosolic protein. This report, for the first time, demonstrates induction and nuclear localization of reductase in rat kidney in response to HO-1 inducers: bacterial lipopolysaccharide (LPS) and bromobenzene. The study also demonstrates that nuclear localization requires an intact nuclear localization signal and is responsive to cGMP. Specifically 16 h after treatment of rats (i.p.) with LPS (5 mg/kg), there was an increase in nuclear biliverdin reductase as determined by immunostaining, Western blotting, and activity analysis. Induction and nuclear localization of the reductase in kidney was also observed in bromobenzene-treated rats (2 mmol/kg, s.c., 24 h). The reductase message levels, however, were not increased in response to either treatment, suggesting post-transcriptional activation of the reductase by LPS and bromobenzene. The mechanism of nuclear transport of the reductase was examined using HeLa cells transfected with the hemagglutinin-tagged reductase construct. When cells were treated with 8-Br-cGMP the protein translocated into the nucleus. Mutation of the putative nuclear localization signal domain of the reductase blocked nuclear transport of the protein. We suggest the significance of nuclear localization of the reductase may relate to: 1) chain-breaking antioxidant activity of bilirubin; 2) inhibition of superoxide formation by bilirubin; and 3) modulation of the signal transduction pathways.
Footnotes
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Send reprint requests to: Dr. Mahin D. Maines, University of Rochester Medical Center, Department of Biochemistry/Biophysics, 601 Elmwood Ave., Rochester, NY 14642. E-mail:mahin_maines{at}urmc.rochester.edu
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↵1 Current address: Arqule, Boston, MA.
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This study was supported by National Institute of Environmental Health Sciences, National Institutes of Health Grants ES04066 and ES04391.
- Abbreviations:
- HO
- heme oxygenase
- LPS
- lipopolysaccharide
- PCR
- polymerase chain reaction
- NLS
- nuclear localization signal
- NO
- nitric oxide
- MAPK
- mitogen-activated protein kinase
- ERK
- extracellular signal-regulated kinase
- Received September 21, 2000.
- Accepted November 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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