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  • Title: 1,25-dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells.
    Author: Huang YC, Hung WC.
    Journal: J Cell Physiol; 2006 Nov; 209(2):363-9. PubMed ID: 16883603.
    Abstract:
    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.
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