Hypoxia-induced Activation of the Retinoic Acid Receptor-related Orphan Receptor α4 Gene by an Interaction between Hypoxia-inducible Factor-1 and Sp1

Naoki Miki,Megumi Ikuta,Takashi Matsui

doi:10.1074/jbc.m313186200

Abstract

Hypoxia plays a key role in the pathophysiology of many disease states, and expression of the retinoic acid receptor-related orphan receptor alpha (RORalpha) gene increases under hypoxia. We investigated the mechanism for this transient hypoxia-induced increase in RORalpha expression. Reverse transcription-coupled PCR analysis revealed that the steady-state level of mRNA for the RORalpha4 isoform, but not the RORalpha1 isoform, increased in HepG2 cells after 3 h of hypoxia. Transient transfection studies showed that the hypoxia-induced increase in RORalpha4 mRNA occurs at the transcriptional level and is dependent on a hypoxia-responsive element (HRE) located downstream of the promoter. A dominant-negative mutant of hypoxia-inducible factor-1alpha (HIF-1alpha) abrogates the transcription activated by hypoxia as well as the transcription activated by exogenously expressed HIF-1alpha, demonstrating the direct involvement of HIF-1alpha in the transcriptional activation. However, HIF-1 alone was not sufficient to activate transcription in hypoxic conditions but, rather, required Sp1/Sp3, which binds to a cluster of GC-rich sequences adjacent to the HRE. Deletion of one or more of these GC boxes reduced or eliminated the HIF-1-dependent transcription. Together, these results suggest that the hypoxia-responsive region of the RORalpha4 promoter is composed of the HRE and GC-rich sequences and that the transcriptional activation under hypoxia is conferred through the cooperation of HIF-1 with Sp1/Sp3.

Highlights

Members of the nuclear receptor superfamily play intrinsic roles in a variety of cellular processes, such as embryogenesis, cell differentiation, cell growth, and cell homeostasis [1, 2]
Deletion of one or more of these GC boxes reduced or eliminated the HIF-1-dependent transcription. These results suggest that the hypoxia-responsive region of the receptor ␣ (ROR␣)4 promoter is composed of the hypoxia-responsive element (HRE) and GC-rich sequences and that the transcriptional activation under hypoxia is conferred through the cooperation of HIF-1 with Sp1/Sp3
Together with the results obtained in the transfection experiments, these observations indicate that the HIF-1 induced by hypoxia binds to the HRE and activates the transcription of the ROR␣4 promoter

Summary

The abbreviations used are

ROR␣, retinoic acid receptor-related orphan receptor ␣; HIF-1, hypoxia-inducible factor-1; HRE, hypoxiaresponsive element; sg, staggerer; Co2ϩ, cobalt chloride; EMSA, electrophoresis mobility shift assay; GRE, glucocorticoid-responsive element; epo, erythropoietin. Cellular adaptation to hypoxia is associated with induction of a number of genes, including vascular endothelial growth factor, erythropoietin, several glycolytic enzymes, and inducible nitric-oxide synthase [23]. HIF-1 binds directly to a hypoxiaresponsive element (HRE) and activates the transcription of genes to adapt to oxygen deprivation. HIF-1␤ is constitutively expressed and is stable under normoxia, expression of the HIF-1␣ protein is regulated by the level of oxygen (26 – 28). In the present study we investigated the mechanism by which the expression of ROR␣ gene is regulated during hypoxia. The hypoxia-induced activation of the ROR␣4 promoter appears to be regulated through a functional interaction between HIF-1 and Sp1/Sp3

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION