Functional role of VDR in the activation of p27Kip1 by the VDR/Sp1 complex.
Our previous study demonstrate that vitamin D3 induces the binding of vitamin D3 receptor (VDR) to Sp1 transcription factor and stimulates p27Kip1 expression via the Sp1 consensus sequences in the promoter. Both VDR and Sp1 are transcriptional activators, it is unclear which protein functions as the transcription component of the VDR/Sp1 complex. To address this issue, we constructed the AF-2 deletion mutant of VDR and tested the effect of vitamin D3 on p27Kip1 expression. In consistent with our previous results, we found that expression of wild-type VDR in SW620 colon cancer cells, which expressed very low level of endogenous VDR, increased vitamin D3-stimulated p27Kip1 promoter activity and protein expression. On the contrary, expression of AF-2 deletion mutant had little effect. DNA affinity precipitation assay (DAPA) showed that both wild-type and deletion mutant of VDR bound to the DNA probe corresponding to the Sp1 binding site in the p27Kip1 promoter in a vitamin D3-dependent manner indicating deletion of AF-2 domain does not affect the interaction between VDR and Sp1. Chromatin immunoprecipitation (CHIP) assay also confirmed that VDR and its AF-2 deletion mutant bound to p27Kip1 promoter in vivo. We found that deletion of AF-2 domain abolished the interaction of coactivators SRC-1 and DRIP205 with VDR. Taken together, our results suggest that VDR functions as the transactivation component of the VDR/Sp1 complex to trigger gene expression.
Cheng
Hsuen-Tsen
HT
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Chen
Jing-Yi
JY
Huang
Yu-Chun
YC
Chang
Hui-Chiu
HC
Hung
Wen-Chun
WC
D045744:Cell Line, Tumor; D002762:Cholecalciferol / Q000494:pharmacology*; D016384:Consensus Sequence; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D018167:Receptors, Calcitriol; D016329:Sp1 Transcription Factor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D002762:Cholecalciferol