1,25-dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells.

Upregulation of p27Kip1 protein in 1,25-dihydroxyvitamin D3-treated cancer cells is mediated via enhancement of gene transcription and reduction of protein degradation. 1,25-dihydroxyvitamin D3 inhibits the expression of p45Skp2, the F-box protein which is implicated in p27Kip1 degradation, to reduce turnover of p27Kip1 protein. In this study, we elucidate the underlying mechanism by which 1,25-dihydroxyvitamin D3 inhibits p45Skp2 in human LNCaP prostate cancer cells. Western blot and RT-PCR analysis suggest that 1,25-dihydroxyvitamin D3 suppresses p45Skp2 via transcriptional repression. Promoter activity assays indicate that 1,25-dihydroxyvitamin D3 directly inhibits p45Skp2 promoter activity. Deletion analysis shows that 1,25-dihydroxyvitamin D3 response element is localized at -447/-291 bp region from the translational start site of the p45Skp2 promoter. Mutation analysis suggests that two Sp1 sites localized at -386/-380 and -309/-294 bp region are required for transcriptional repression. Chromatin immunoprecipitation (CHIP) assay demonstrates that VDR indirectly binds to these Sp1 sites in vivo and this binding is increased after 1,25-dihydroxyvitamin D3 treatment. Re-CHIP assay suggests that VDR and Sp1 form a complex to bind to the Sp1 sites. DNA affinity precipitation assay (DAPA) shows that histone deacetylase 1 (HDAC1) is recruited to the Sp1 sites after 1,25-dihydroxyvitamin D3 stimulation. Re-CHIP assay verifies that binding of Sp1 and HDAC1 to p45Skp2 promoter is enhanced after 1,25-dihydroxyvitamin D3 treatment. HDAC inhibitor trichostatin A (TSA) reverses the inhibition of p45Skp2 promoter activity by 1,25-dihydroxyvitamin D3. Collectively, our results suggest that 1,25-dihydroxyvitamin D3 induces the formation of VDR/Sp1 complex and acts via a Sp1- and HDAC1-depedent pathway to inhibit p45Skp2 transcription.

Huang YC ，Hung WC 《JOURNAL OF CELLULAR PHYSIOLOGY》

Requirement of a specific Sp1 site for histone deacetylase-mediated repression of transforming growth factor beta Type II receptor expression in human pancreatic cancer cells.

Zhao S ，Venkatasubbarao K ，Li S ，Freeman JW ... -  《CANCER RESEARCH》

Vitamin D3 receptor/Sp1 complex is required for the induction of p27Kip1 expression by vitamin D3.

1alpha,25-dihydroxyvitamin D3 (vitamin D3) has been shown to upregulate p27Kip1 expression via Sp1 and NF-Y binding sites in the p27Kip1 promoter. However, whether vitamin D3 receptor (VDR) involves in this process is unclear. In this study, we demonstrated that expression of VDR in SW620 cells, which exhibited low level of endogenous VDR, increased vitamin D3-stimulated p27Kip1 promoter activity. On the contrary, suppression of Sp1 expression by small interference RNA reduced the stimulation of p27Kip1 promoter activity by vitamin D3 in LNCaP cells. DNA affinity precipitation assay and chromatin immunoprecipitation assay showed that VDR bound to the p27Kip1 promoter in vitro and in vivo. In addition, we also demonstrated that VDR interacted with Sp1 in vitro and in cells. Collectively, our results suggest that VDR is involved in the induction of p27Kip1 by vitamin D3 and may interact with Sp1 to modulate the expression of target genes that lack VDR response element (VDRE) in their promoters.

Huang YC ，Chen JY ，Hung WC 《ONCOGENE》

p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription.

Previous studies demonstrate that p16, a cyclin-dependent kinase inhibitor and a tumor suppressor, may inhibit matrix metalloproteinase-2 (MMP-2) expression in human cancer cells to suppress tumor invasion and metastasis. However, the detailed mechanism is still unclear. Our results show that p16 inhibits MMP-2 expression via transcriptional repression. Promoter deletion and mutation analysis indicates that p16 acts through the Sp1 transcription factor-binding site located between -72 and -64 bp region from the transcriptional start site of the human MMP-2 promoter to repress gene expression. DNA affinity precipitation assay (DAPA) and chromatin immuno-precipitation (CHIP) assay demonstrate that Sp1 proteins constitutively bind to this consensus sequence in vitro and in vivo. p16 attenuates Sp1 binding to the MMP-2 promoter to suppress gene transcription and overexpression of Sp1 may counteract p16-induced downregulation of MMP-2. CyclinA/CDK complex may directly phosphorylate Sp1 and enhance its DNA-binding activity. Thus, we investigated the effect of p16 on the interaction between cyclin A and Sp1. Our results indicate that p16 induces downregulation of cyclin A and CDK2, reduces the interaction between cyclin A and Sp1, and attenuates phosphorylation of Sp1. Ectoexpression of cyclin A counteracts p16-mediated inhibition of DNA binding of Sp1 and activates MMP-2 promoter activity and mRNA expression. Collectively, our results suggest that p16 suppresses MMP-2 by blocking Sp1-mediated gene transcription.

Wang CH ，Chang HC ，Hung WC 《JOURNAL OF CELLULAR PHYSIOLOGY》

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 HT ，Chen JY ，Huang YC ，Chang HC ，Hung WC ... -  《JOURNAL OF CELLULAR BIOCHEMISTRY》