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.
Miki
Naoki
N
Laboratory of Genomics and Proteomics, Faculty of Pharmacy and Pharmaceutical Science, Fukuyama University, 1 Gakuen-cho, Fukuyama 729-0292, Japan.
Ikuta
Megumi
M
Matsui
Takashi
T
D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins / Q000378:metabolism*; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D020033:Protein Isoforms; D012333:RNA, Messenger; C539738:RORA protein, human; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D004247:DNA; C078716:ROR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D057050:Receptor Tyrosine Kinase-like Orphan Receptors