Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1.
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.
Mesh Terms:
Cell Line, DNA, DNA-Binding Proteins, Gene Deletion, HeLa Cells, Humans, Hypoxia, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Nuclear Proteins, Nuclear Receptor Subfamily 1, Group F, Member 1, Plasmids, Promoter Regions, Genetic, Protein Isoforms, RNA, Messenger, Receptor Protein-Tyrosine Kinases, Receptor Tyrosine Kinase-like Orphan Receptors, Receptors, Cell Surface, Receptors, Cytoplasmic and Nuclear, Reverse Transcriptase Polymerase Chain Reaction, Sp1 Transcription Factor, Sp3 Transcription Factor, Time Factors, Trans-Activators, Transcription Factors, Transcription, Genetic, Transcriptional Activation, Transfection
Cell Line, DNA, DNA-Binding Proteins, Gene Deletion, HeLa Cells, Humans, Hypoxia, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Nuclear Proteins, Nuclear Receptor Subfamily 1, Group F, Member 1, Plasmids, Promoter Regions, Genetic, Protein Isoforms, RNA, Messenger, Receptor Protein-Tyrosine Kinases, Receptor Tyrosine Kinase-like Orphan Receptors, Receptors, Cell Surface, Receptors, Cytoplasmic and Nuclear, Reverse Transcriptase Polymerase Chain Reaction, Sp1 Transcription Factor, Sp3 Transcription Factor, Time Factors, Trans-Activators, Transcription Factors, Transcription, Genetic, Transcriptional Activation, Transfection
J. Biol. Chem.
Date: Apr. 09, 2004
PubMed ID: 14742449
View in: Pubmed Google Scholar
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