title: 1,25-dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells.
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.
authors.affiliation: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, China.
authors.forename: Yu-Chun
authors.initials: YC
authors.lastname: Huang
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Hung
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002117:Calcitriol / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000473:pathology*; D011485:Protein Binding / Q000187:drug effects; D018167:Receptors, Calcitriol / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D020121:5' Untranslated Regions; D018167:Receptors, Calcitriol; D044786:S-Phase Kinase-Associated Proteins; D016329:Sp1 Transcription Factor; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases; D002117:Calcitriol