1,25-dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells
✍ Scribed by Yu-Chun Huang; Wen-Chun Hung
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 241 KB
- Volume
- 209
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Abstract
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. J. Cell. Physiol. 209: 363–369, 2006. © 2006 Wiley‐Liss, Inc.