Journal
JOURNAL OF CELLULAR PHYSIOLOGY
Volume 209, Issue 2, Pages 363-369Publisher
WILEY
DOI: 10.1002/jcp.20741
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Upregulation of p27(Kip1) protein in 1,25-dihydroxyvitamin D-3-treated cancer cells is mediated via enhancement of gene transcription and reduction of protein degradation. 1,25-dihydroxyvitamin D-3 inhibits the expression of p45(Skp2), the F-box protein which is implicated in p27(Kip1) degradation, to reduce turnover of p27(Kip1) protein. In this study, we elucidate the underlying mechanism by which 1,25-dihydroxyvitamin D-3 inhibits p45(Skp2) in human LNCaP prostate cancer cells. Western blot and RT-PCR analysis suggest that 1,25-dihydroxyvitamin D-3 suppresses p45(Skp2) via transcriptional repression. Promoter activity assays indicate that 1,25-dihydroxyvitamin D-3 directly inhibits p45(Skp2) Promoter activity. Deletion analysis shows that 1,25-dihydroxyvitamin D-3 response element is localized at -447/-291 bp region from the translational start site of the p45(Skp2) 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 D-3 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 D-3 stimulation. Re-CHIP assay verifies that binding of Sp1 and HDAC1 to p45(Skp2) promoter is enhanced after 1,25-dihydroxyvitamin D-3 treatment. HDAC inhibitor trichostatin A (TSA) reverses the inhibition of p45(Skp2) promoter activity by 1,25-dihydroxyvitamin D-3. Collectively, our results suggest that 1,25-dihydroxyvitamin D-3 induces the formation of VDR/Sp1 complex and acts via a Sp1- and HDAC1-depedent pathway to inhibit p45(Skp2) transcription.
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