Abstract
The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.
Highlights
HPK1Aras, a ras-transformed keratinocyte cell line, is resistant to the growth-inhibitory effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [6], as are several pancreatic [7] and breast carcinoma cell lines [8]
We demonstrate that coactivator recruitment to the VDR1⁄7RXR complex is disrupted in HPK1Aras cells and can be restored by treatment with a MAPK kinase (MAPKK) inhibitor or overexpression of a non-phosphorylable human retinoid X receptor (RXR)␣ mutant at serine 260
Inhibition of MAPKK Activity Results in a Reversal of the Partial Resistance of HPK1Aras Cells to the Transactivational Potential of Retinoids—We have shown that the transactivation of mouse osteopontin (mOP) by 1,25(OH)2D3 is partially inhibited in the MAPKKactivated HPK1Aras cells compared with that of HPK1A cells [18]
Summary
Cell Culture and Transfections—HPK1A and HPK1Aras cell lines were described previously [13, 14]. Cellular extracts were prepared by lysing HPK1A and HPK1Aras cells using a triple detergent lysis buffer (50 mM Tris/HCl, pH 8.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, Complete protease inhibitor tablet (Roche Applied Science)). Nuclear Extracts—Nuclear extracts were prepared by scraping the cells in phosphate-buffered saline, washing the cells with phosphate-buffered saline, and resuspending the cells in 500 l of lysis buffer (10 mM Tris (pH 7.9), 10 mM KCl, 0.1 mM EDTA, mM EGTA, 1 mM dithiothreitol, a mini Complete protease inhibitor tablet (Roche Applied Science), 1 mM sodium orthovanadate, 20 mM NaF) and incubation on ice for 15 min. In other experiments when HPK1A or HPK1Aras cells in 100-mm plates reached 60% confluency, medium was replaced with fresh DMEM containing 10% charcoal-stripped FBS and 1,25(OH)2D3 (10Ϫ7 M) or vehicle (ethanol) was added to cells for another 24 h. Cyclin-dependent kinase inhibitor (P21waf1/cip1) Cyclin C Vitamin D 24 hydroxylase (CYP24) Calmin (calponin-like, transmembrane) (CLMN)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
