Examination of VDR/RXR/DRIP205 Interaction, Intranuclear Localization, and DNA Binding in Ras-Transformed Keratinocytes and Its Implication for Designing Optimal Vitamin D Therapy in Cancer

摘要

Retinoid X receptor (RXR) occupies a central position within the nuclear receptor superfamily, serving as an obligatory partner to numerous other nuclear receptors, including vitamin D receptor (VDR). In the current study, we examined whether phosphorylation of RXR alpha at serine 260 affects VDR/RXR and VDR interacting protein (DRIP) 205 coactivator recruitment, interactions, and binding of the VDR/human RXR alpha(hRXR alpha)/DRIP205 complex to chromatin. Serine 260 is a critical amino acid on the hRXR alpha that is located in close spatial proximity to regions of coactivator and corepressor interactions. Using fluorescence resonance energy transfer and immunofluorescence studies, we showed that the physical interaction between hRXR alpha and DRIP205 coactivator was impaired in human keratinocytes with the ras oncogene (HPK1Aras) or transfected with the wild-type hRXR alpha. Furthermore, the nuclear colocalization of VDR/DRIP205, hRXR alpha/DRIP205, and VDR/hRXR alpha/DRIP205 complex binding to chromatin is impaired in the HPK1Aras cells when compared with the normal human keratinocytes (HPK1A cells). However, transfection with the nonphosphorylatable hRXRa (S260A) mutant or treatment with the mitogen-activated protein kinase (MAPK) inhibitor UO126 rescued their nuclear localization, interaction, and binding of the complex to chromatin in the HPK1Aras cells. In summary, we have demonstrated, using highly specific intracellular tagging methods in live and fixed cells, important alterations of the vitamin D signaling system in cancer cells in which the ras-raf-MAPK system is activated, suggesting that specific inhibition of this commonly activated pathway could be targeted therapeutically to enhance vitamin D efficacy.