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

Abstract

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 RXRα at serine 260 affects VDR/RXR and VDR interacting protein (DRIP) 205 coactivator recruitment, interactions, and binding of the VDR/human RXRα (hRXRα)/DRIP205 complex to chromatin. Serine 260 is a critical amino acid on the hRXRα 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 hRXRα and DRIP205 coactivator was impaired in human keratinocytes with the ras oncogene (HPK1Aras) or transfected with the wild-type hRXRα. Furthermore, the nuclear colocalization of VDR/DRIP205, hRXRα/DRIP205, and VDR/hRXRα/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 hRXRα (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.

Vitamin D and Breast Cancer: Mechanistic Update

“…Other factors that may impede vitamin D's antitumor effects include phosphorylation of VDR itself or its transcriptional partners, altered VDR subcellular localization, disruption of target genes rendering them non-functional or non-responsive to VDR regulation, etc. (74)(75)(76) Collectively, these observations demonstrate multiple mechanisms by which vitamin D signaling can be corrupted in cancers and are consistent with the demonstration that explants from breast cancers are less sensitive to 1,25D than explants from adjacent normal tissue or healthy breast epithelium. (77) Clarification of the physiologic, cellular, and molecular determinants of sensitivity to 1,25D-mediated anticancer actions remains a critical gap in translating preclinical findings into prevention/treatment strategies and population guidelines.…”

“…VDR is a member of the NR1I subfamily of nuclear hormone receptors, which also includes the pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3). These receptors and others (such as the retinoic acid receptor [RAR] isoforms and the peroxisome proliferator-activated receptor [PPAR] isoforms) dimerize with the α, β, or γ subtypes of retinoid X receptors (RXRs, (71,72,75) High VDR expression in differentiated epithelial cells. Increased hormonestimulated proliferation and branching in VDRKO glands in organ culture and in vivo compared with wild-type (WT) mice.…”

Vitamin D3 Modulates Impaired Crosstalk Between RANK and Glucocorticoid Receptor Signaling in Bone Marrow Cells After Chronic Prednisolone Administration

“…Mechanistically, ligand binding induces conformational changes in VDR that promote receptor heterodimerization with the retinoid X receptor (RXR). After ligand activation, it binds directly to the vitamin D response elements and recruits a variety of co-regulatory complexes that perform the additional functions to modify transcriptional activity ( 13 ). Importantly, VDR-mediated gene regulation requires the involvement of multiple modular enhancers at a range of locations many kilobases upstream, downstream, or within the transcription units ( 14 ).…”

New Roles for Vitamin D Superagonists: From COVID to Cancer

“…These include VDR Interacting Protein (DRIP205), also known as MED1 (mediator of RNA polymerase II transcription subunit 1), which directly interacts with the receptor. Additional interactions occurring with coactivators: SRC-1, AIB-1 and GRIP, have been described ( 38 ). Interestingly, many analogues displaying selective co-activator recruitment also demonstrate an enhanced tissue selectivity for activity.…”