Hypoxia-inducible Factor-1 Mediates Transcriptional Activation of the Heme Oxygenase-1 Gene in Response to Hypoxia

doi:10.1074/jbc.272.9.5375

Hypoxia-inducible Factor-1 Mediates Transcriptional Activation of the Heme Oxygenase-1 Gene in Response to Hypoxia*

From the ^‡ Division of Pulmonary and Critical Care and
^¶Center for Medical Genetics, Departments of Medicine and Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and
^∥Department of Molecular Genetics, Alton Ochsner Medical Foundation Medical Center, New Orleans, Louisiana 70121

**Supported by National Institutes of Health Grant DK43135. To whom correspondence should be addressed:
Dept. of Molecular Genetics, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, LA 70121.
Tel.: 504-842-3314; Fax: 504-842-3381.

Abstract

Exposure of rats to hypoxia (7% O₂) markedly increased the level of heme oxygenase-1 (HO-1) mRNA in several tissues. Accumulation of HO-1 transcripts was also observed after exposure of rat aortic vascular smooth muscle (VSM) cells to 1% O₂, and this induction was dependent on gene transcription. Activation of the mouse HO-1 gene by all agents thus far tested is mediated by two 5′-enhancer sequences, SX2 and AB1, but neither fragment was responsive to hypoxia in VSM cells. Hypoxia-dependent induction of the chloramphenicol acetyltransferase (CAT) reporter gene was mediated by a 163-bp fragment located approximately 9.5 kilobases upstream of the transcription start site. This fragment contains two potential binding sites for hypoxia-inducible factor 1 (HIF-1). A role for HIF-1 in HO-1 gene regulation was established by the following observations: 1) HIF-1 specifically bound to an oligonucleotide spanning these sequences, 2) mutation of these sequences abolished HIF-1 binding and hypoxia-dependent gene activation in VSM cells, 3) hypoxia increased HIF-1α and HIF-1β protein levels in VSM cells, and 4) hypoxia-dependent HO-1 mRNA accumulation was not observed in mutant hepatoma cells lacking HIF-1 DNA-binding activity. Taken together, these data demonstrate that hypoxia induces HO-1 expression in animal tissues and cell cultures and implicate HIF-1 in this response.

Footnotes

↵^§ Supported by a National Institutes of Health NHLBI Multidisciplinary Training Grant.
↵^‡‡ An Established Investigator of the American Heart Association. Supported in part by a grant from the American Heart Association and by National Institutes of Health Grant R01-HL55338.
↵^§§ Supported in part by the National Institutes of Health, NIA Physician Scientist Award K11AG00516 and a Research Grant from the American Lung Association.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U70472[GenBank].
¹ The abbreviations used are:

EPO

erythropoietin

HO-1

heme oxygenase-1

AP-1

activator protein-1

CAT

chloramphenicol acetyltransferase

HIF-1

hypoxia-inducible factor-1

HypRE

hypoxia response element

VSM

vascular smooth muscle

CHO

Chinese hamster ovary

bp

base pair(s)

kb

kilobase pair(s).
² J. A. Forsythe, B.-H. Jiang, N. V. Iyer, R. D. Koos, and G. L. Semenza, submitted for publication.
- Received January 18, 1996.
- Revision received October 30, 1996.