Transcriptional regulation of the hypoxia inducible factor-2alpha (HIF-2alpha) gene during adipose differentiation in 3T3-L1 cells.
Adipose differentiation is regulated by coordination of several signaling pathways and transcription factors. We recently showed that Hypoxia inducible factor-2alpha (HIF-2alpha) plays several supporting roles in adipose differentiation and adipocytes functions including regulation of glucose uptake followed by lipid synthesis. HIF-2alpha expression is increased during adipogenesis, indicating that its up-regulation is necessary for execution of adipogenesis and maintenance of mature adipocytes functions. Therefore, in this study, to understand the mechanism by which HIF-2alpha expression is induced during adipogenesis, we investigated the promoter activity of HIF-2alpha gene during adipogenesis in 3T3-L1 cells. A comparison of HIF-2alpha promoter activity between preadipocytes and adipocytes revealed that the sequence -478/-445 is the putative core element that contributes to differentiation-dependent up-regulation of HIF-2alpha promoter activity. Electrophoretic mobility shift assays showed the presence of the specific nuclear factor bound to the sequence -478/-445 in both preadipocytes and adipocytes. Computer analysis revealed that this element contains several Sp1/Sp3 binding sites. Indeed, the presence of Sp1/Sp3 consensus oligonucleotides diminished the formation of the complexes composed of the sequence -478/-445 and the nuclear factor. Furthermore, specific retarded bands were supershifted with anti-Sp1 or -Sp3 antibodies. Binding of Sp1 and Sp3 to this element was also confirmed by chromatin immunoprecipitation analysis. The element encompassing -478/-445 favors Sp3 in preadipocytes and Sp1 in adipocytes. Finally, the activity of -478/-445 was increased by Sp1 but decreased by Sp3. Consequently, these results suggest that Sp1 and Sp3 are involved in transcriptional regulation of HIF-2alpha expression during adipogenesis in 3T3-L1 cells.
Wada
Taira
T
Department of Health Science, College of Pharmacy, Nihon University, Chiba, Japan.
Shimba
Shigeki
S
Tezuka
Masakatsu
M
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000273:Adipose Tissue / Q000254:growth & development* / Q000502:physiology; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D047468:Immunoprecipitation; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D000099162:Endothelial PAS Domain-Containing Protein 1; C497122:Sp3 protein, mouse