1. Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1
- Author
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Naoki Miki, Takashi Matsui, and Megumi Ikuta
- Subjects
Gene isoform ,Transcriptional Activation ,Hypoxia-Inducible Factor 1 ,Time Factors ,Transcription, Genetic ,Sp1 Transcription Factor ,Retinoic acid ,Receptors, Cytoplasmic and Nuclear ,Receptors, Cell Surface ,Biology ,Receptor Tyrosine Kinase-like Orphan Receptors ,Transfection ,Biochemistry ,Retinoic acid-inducible orphan G protein-coupled receptor ,Cell Line ,chemistry.chemical_compound ,Transcription (biology) ,Humans ,Protein Isoforms ,RNA, Messenger ,Hypoxia ,Promoter Regions, Genetic ,Molecular Biology ,Orphan receptor ,Reverse Transcriptase Polymerase Chain Reaction ,Nuclear Proteins ,Receptor Protein-Tyrosine Kinases ,Nuclear Receptor Subfamily 1, Group F, Member 1 ,Cell Biology ,DNA ,Hypoxia-Inducible Factor 1, alpha Subunit ,Molecular biology ,Neuron-derived orphan receptor 1 ,DNA-Binding Proteins ,Retinoic acid receptor ,Sp3 Transcription Factor ,chemistry ,Trans-Activators ,Gene Deletion ,HeLa Cells ,Plasmids ,Transcription Factors - 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.
- Published
- 2004