The Role of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in Hypoxic Induction of Gene Expression

doi:10.1074/jbc.271.25.15117

The Role of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in Hypoxic Induction of Gene Expression

STUDIES IN ARNT-DEFICIENT CELLS*

From the ^‡ Erythropoietin Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom and the
^” Department of Pathology and Laboratory Medicine and Jonsson Comprehensive Cancer Center, Medical School, UCLA, Los Angeles, California 90095

^# To whom correspondence should be addressed:
Rm. 420, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
Tel.: 44-1865-222382; Fax: 44-1865-222500.

Abstract

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1α, and HIF-1β. One partner, HIF-1β, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. In the present work, ARNT-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of ARNT/HIF-1β in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 was absent in the mutant cells, indicating an essential role for ARNT/HIF-1β. Analysis of deleted ARNT/HIF-1β genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, were necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1β in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persisted in these mutant cells, clearly distinguishing ARNT/HIF-1β-dependent and ARNT/HIF-1β-independent mechanisms of gene activation by both these stimuli.

Footnotes

↵^§ Wellcome Clinical Training Fellow.
↵^¶ MRC Training Fellow.
↵^∥ MRC Clinician Scientist.
↵^‴ Supported by Grant CA 28868 from the NCI, National Institutes of Health.
↵* This work was supported in part by grants from The Wellcome Trust and Medical Research Council (MRC). 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

AHR

aryl hydrocarbon receptor

ARNT

aryl hydrocarbon receptor nuclear translocator

ARNT⁻

ARNT-deficient mutant

Epo

erythropoietin

Glut-1

glucose transporter-1

bHLH

basic helix-loop-helix

DFO

desferrioxamine

LDH-A

lactate dehydrogenase-A

PAS

Per-AHR-ARNT-Sim

PDGF-B

platelet-derived growth factor B chain

PGK-1

phosphoglycerate kinase-1

PIPES

piperazine-N,N′-bis(2-ethanesulfonic acid)

VEGF

vascular endothelial growth factor

XRE

xenobiotic response element.
- Received January 22, 1996.
- Revision received March 25, 1996.