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.

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See also:

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