Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit

doi:10.1074/jbc.271.50.32253

Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit*

From the ^¶ Hematology-Oncology Division, Brigham & Women's Hospital, Boston, Massachusetts 02115
^″ Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

^‴ To whom correspondence should be addressed:
Hematology-Oncology Division, LMRC-2, Brigham & Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115.
Tel.: 617-732-5841; Fax: 617-739-0748; E-mail: Bunn{at}calvin.bwh.harvard.edu

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1α protein. Both HIF-1α and HIF-1β mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1α protein was barely detectable in normoxic cells, even when HIF-1α was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1β protein levels remained constant, regardless of pO₂. Hypoxia-induced HIF-1 binding as well as the HIF-1α protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1α protein, whereas treatment of hypoxic cell extracts with H₂O₂ had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1α protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1α protein.

Footnotes

↵^∥ Recipient of the National Research Service Award DK09365-01 from the National Institutes of Health.
↵* This work was supported by National Institutes of Health Grant RO1-DK41234 (to H. F. B) and grants from the National Institutes of Health and Dana Farber Cancer Institute/Sandoz Drug Development Program (to D. M. L.). 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.
↵¹ The abbreviations used are:

HIF-1

hypoxia-inducible factor 1

ARNT

aryl hydrocarbon receptor nuclear translocator

luc

luciferase

CMV

cytomegalovirus

EMSA

electrophoretic mobility shift assay

NEM

N-ethylmaleimide

DTT

dithiothreitol.
↵² Because of catalase and peroxidases in intact cells, following treatment with 1 mM H₂O₂, the cell nuclei would have a much lower concentration of peroxide but would have higher levels of activated oxygen species.
- Received August 5, 1996.
- Revision received September 27, 1996.