Phosphorylation of the Human Retinoid X Receptor α at Serine 260 Impairs Coactivator(s) Recruitment and Induces Hormone Resistance to Multiple Ligands*

The retinoid X receptor α (RXRα) 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·RXRα heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRα 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 RXRα, inhibits signaling through several heterodimeric partners of the RXRα. 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 α ligands and LG1069 (an RXRα ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRα mutant at serine 260 (RXRα Ser-260 → Ala). Importantly, phosphorylation of RXRα at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR·RXRα 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 RXRα-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.

Ras activation has been detected in numerous cancers, including hepatocellular carcinoma, colorectal carcinomas, breast tumors, leukemias, and squamous tumors of the head and neck (1). Activation of the Ras-Raf-mitogen-activated protein kinase (MAPK-ERK) 2 signaling cascade leads to phospho-rylation of downstream targets, including some nuclear receptors, resulting in a mechanism for receptor control (2). Signaling through nuclear receptors is required in many aspects of cellular functions (3). Several nuclear receptors require heterodimerization with the retinoid X receptor (RXR) to fulfill their signaling functions (4). Upon dimerization, the receptors recognize and bind bipartite regions of promoters of target genes, known as response elements, involving a discrete DNA binding domain within the receptors (5).
HPK1Aras, a ras-transformed keratinocyte cell line, is resistant to the growth-inhibitory effects of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) (6), as are several pancreatic (7) and breast carcinoma cell lines (8). The phosphorylation of RXR␣ at serine 260 caused the resistance of the HPK1Aras cell line to the antiproliferative effects of 1,25(OH) 2 D 3 (9) as well as resistance to the antiproliferative effect of all-trans-retinoic acid (ATRA) in hepatocellular carcinoma cells (10) . This serine lies within a PSSP MAPK recognition sequence and the phosphorylation is MAPK kinase (MAPKK)-dependent (9). The latter study also raised the possibility that RXR phosphorylation on serine 260 induces conformational changes of the VDR⅐RXR complex. Ser-260 is located at a critical site in the omega loop between H1 and H3 helices of the ligand binding domain of RXR␣ (11) and in close spatial proximity to regions of potential coativatorcorepressor interactions with the RXR (12). In the present study we show that RXR␣ phosphorylation at Ser-260 affects several signaling pathways involving recruitment of partners other than the VDR. Furthermore, we demonstrate that coactivator recruitment to the VDR⅐RXR complex is disrupted in HPK1Aras cells and can be restored by treatment with a MAPKK inhibitor or overexpression of a non-phosphorylable human RXR␣ mutant at serine 260.

MATERIALS AND METHODS
Cell Culture and Transfections-HPK1A and HPK1Aras cell lines were described previously (13,14). The HPK1A cells are non-tumor forming, whereas HPK1Aras cells result in squamous cell carcinomas upon transplantation into nude mice (14). Cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS). Cells were plated at a density of 4 ϫ 10 3 cells per well in 24-well plates for growth curves, grown to 60% confluency in 6-well plates for chloramphenicol acetyl transferase (CAT) and ␤-galactosidase reporter assays, and grown to 60% confluency in 100-mm 2 plates for cell and nuclear extracts.
In some experiments cells in 100-mm 2 plates were transfected with the non-phosphorylable RXR␣ Ser-260 3 Ala (9) or vector alone (pCDNA 3.1). Human RXR␣ Ser-260 3 Ala (9) was subcloned into the EcoRI site of pCDNA 3.1 expression vector (Invitrogen). In some experiments wild-type and human RXR␣ Ser-260 3 Ala mutants were subcloned into the plasmid pCDNA3HA, which contains a plasmid encoding the HA epitope (9). Transfections were performed by incubating 5 g of plasmid DNA with 15 l of FuGene 6 transfection reagent (Roche Applied Science) in fresh serum-free Opti-MEM medium (Invitrogen) and adding 10% charcoal-stripped FBS after 6 h of incubation. After 24 h of transfection the medium was replaced with fresh DMEM containing 5% charcoalstripped FBS and treated with 1,25(OH) 2 D 3 at the concentrations indicated or with vehicle alone.
Nuclear Extracts-Nuclear extracts were prepared by scraping the cells in phosphate-buffered saline, washing the cells with phosphate-buffered saline, and then resuspending the cells in 500 l of lysis buffer (10 mM Tris (pH 7.9), 10 mM KCl, 0.1 mM EDTA,1 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. 25 l of 10% Nonidet P-40 was added, and the samples were vortexed for 15 s. The samples were spun, and the pellets were resuspended in a nuclear buffer (20 mM Tris (pH 7.9), 400 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, a mini Complete protease inhibitor tablet, 1 mM sodium orthovanadate, 20 mM NaF). The samples were placed in a microcentrifuge tube shaker for 1 h at 4°C, then spun, and the supernatant was aliquoted and stored at Ϫ80°C. In some experiments cells were transfected with the RXR␣ mutant RXR␣ Ser-260 3 Ala or the pCDNA vector alone driven by a cytomegalovirus promoter as described above. In other experiments when HPK1A or HPK1Aras cells in 100-mm 2 plates reached 60% confluency, medium was replaced with fresh DMEM containing 10% charcoal-stripped FBS and 1,25(OH) 2 D 3 (10 Ϫ7 M) or vehicle (ethanol) was added to cells for another 24 h. MAPKK inhibitors PD098059 (Sigma-Aldrich) or UO126 (Fisher Scientific, Mississauga, Ontario, Canada) or vehicle (Me 2 SO) were added 30 min prior to 1,25(OH) 2 D 3 addition and incubated for 24 h prior to extraction as described above.
Growth Assays-At 40% confluency, cells were placed under serum-free conditions to synchronize the cells. The medium was then replaced with DMEM 5% charcoal-stripped FBS in the presence or absence of increasing concentrations of 1,25(OH) 2 D 3 , LG1069 (Ligand Pharmaceuticals, a kind gift of E. Allegreto) or ATRA (Sigma-Aldrich) or TTNPB (Sigma). The cells were also pretreated for 1 h with vehicle or 25 M PD098059 (Sigma-Aldrich) and then treated with 25 M PD098059 or vehicle during the ligand treatment. Media was replaced after 48 h. After 96 h, cells were trypsinized and counted with a coulter counter (Coulter Electronics, Lufton, United Kingdom).
Reporter Assays-Cells were transfected using Fugene transfection reagent (Roche Applied Science) and 4.5 g of reporter plasmid, and 0.5 g of ␤-galactosidase plasmid as an internal control for transfection efficiency in 6-well plates. Cells were treated with increasing amounts of ligand with or without 25 M PD098059. The concentration of the MAPKK inhibitor used is consistent with its K d for the enzyme reported elsewhere (15). For all assays the cells were scraped after 24 h and washed with phosphate-buffered saline and lysed in lysis buffer (Roche Applied Science). The retinoic acid response element (RARE) reporter and the retinoid X response element (RXRE) consisted of three copies of the RAR␣ RARE or RXR␣ RXRE in front of a thymidine kinase reporter driving the expression of a CAT gene (Fisher Scientific, Nepean, Ontario, Canada), and assays were performed using a CAT enzyme-linked immunosorbent assay kit (Roche Applied Science). ␤-Galactosidase was measured in a ␤-galactosidase colorimetric assay.
Electrophoretic Mobility Gel Shift Assay-These assays were performed as previously described (9). Briefly, nuclear extracts (5 g) were incubated for 20 min on ice with 10 Ϫ7 M ligand and 1 g of poly(dIdC) in a binding buffer (25 mM Tris-HCl, pH 8, 5% glycerol, and 0.5 mM dithiothreitol). 5 fmol of 32 P-labeled response element oligonucleotide was added followed by 20 min of incubation at room temperature. In some cases, antibodies were added, and the mix was incubated for another 20 min at room temperature. The samples were resolved on a 5% non-denaturing polyacrylamide gel, dried, and exposed to Kodak XAR-5 film.
Streptavidin-Agarose Pulldown Assays Using Biotynylated DNA Probe-Biotinylated mOP was used as a probe to pull down the VDR⅐RXR⅐coactivator complex using a method previously published (16). We used this streptavidin-agarose bead pulldown assay for analyzing the recruitment of coactivators that bind to the biotinylated mOP.
Biotinylated mOP oligonucleotides were obtained from Invitrogen Canada (Burlington, Ontario, Canada). A non-relevant biotinylated probe was used as a negative control. The forward sequence (sense) of mOP was 5Ј-biotin/GTA CAA GGT TCA CGA GGT TCA CGT C-3Ј, and the reverse sequence (antisense) was 5Ј-biotin/GAC GTG AAC CTC GTG AAC CTT GTA C-3Ј. Double-stranded DNA was obtained by annealing the sense and antisense biotinylated sequences using heating at 85°C for 5-10 min and slowly cooling down.
The pulldown assay was performed by incubating nuclear extracts from cells treated with 1,25(OH) 2 D 3 (10 Ϫ7 M) or UO126 (10 Ϫ6 M) or both and prepared as described above under nuclear extracts preparation. Nuclear extracts (1000 g) were incubated with 2 g of poly(dIdC) and 1 g of double-stranded biotinylated mOP adjusted to a final volume of 500 l with HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCI, 5 mM MgCl 2 , 10% glycerol, 1 mM dithiothreitol, 1.5% Nodinet P-40, 1 mM sodium orthovanadate, 20 mM NaF with a complete protease inhibitor tablet (Roche Applied Science)) for 1 h at 4°C and then further incubated with 150 l of 4% streptavidin-agarose beads (Sigma-Aldrich) for another 3 h at 4°C. The optimal concentration of the biotinylated probe was assessed by first using increasing concentrations of the probe to reach maximal binding. All subsequent experiments were done using this probe concentration.
At the end of incubation the tube was centrifuged at 5000 ϫ g in a microcentrifuge for 30 s. The supernatant was removed, and the pellet washed four times with 1 ml of iced phosphate-buffered saline. The pellet was then resuspended in 500 l of Laemmli buffer and boiled for 5 min to elute protein and centrifuge at 5000 ϫ g in a microcentrifuge for 30 s. The sample was placed on a 6 -10% SDS-PAGE gel for further separation on a polyvinylidene difluoride nitrocellulose membrane (Bio-Rad) and immunoblotting using specific antibodies. Following incubation with horseradish peroxidase-conjugated secondary antibody, the proteins were viewed using Lumiglo chemiluminescence (KPL Laboratories, Washington D. C.). The membranes were exposed to Kodak XAR-5 film.
Chromatin Immunoprecipitation Assays-ChIP assays were performed using the Upstate Biotechnology Assay (Charlottesville, VA) according to the manufacturer's instructions. HPK1Aras cells were cultured in DMEM supplemented with 10% FBS to 40 -50% confluency. Cells were synchronized in serum-free DMEM overnight. Medium was then replaced with DMEM supplemented with 5% charcoal-stripped FBS and pretreated for 1 h with 10 Ϫ6 M UO126 (Promega, Madison, WI) or vehicle (Me 2 SO), before addition of 10 Ϫ7 M 1,25(OH) 2 D 3 for 4 h. The concentration of UO126 used is consistent with its K d for the enzyme reported elsewhere (15).
Genomic DNA was then cross-linked by adding 37% formaldehyde directly to the medium to a final concentration of 1%, and the incubation continued at 37°C for 15 min. Cells were washed with ice-cold phosphate-buffered saline containing a protease inhibitor (mini complete protease inhibitor tablet, Roche Applied Science), collected, and lysed in SDS lysis buffer containing a protease inhibitor. The bulk chromatin was then sonicated to obtain DNA fragments between 200 and 500 bp. Cellular debris was removed by centrifugation for 10 min at 13,000 rpm at 4°C, and the sonicated chromatin supernatant was diluted 10-fold in ChIP dilution buffer for the immunoprecipitation assay. An aliquot of the diluted supernatant was used to quantitate the amount of DNA input by PCR. To reduce the nonspecific background the diluted chromatin supernatant was pre-treated with protein A-agarose/salmon sperm DNA (50% v/v slurry) for 30 min at 4°C with agitation. The samples were centrifuged, and the recovered chromatin solutions were incubated with anti-VDR (C-20) or anti-DRIP205 rabbit polyclonal antibody (Santa Cruz Biotechnology) overnight at 4°C. Negative controls were prepared from pre-treated chromatin supernatant in which the VDR antibody is replaced with IgG. The antibody⅐chromatin complex was collected by adding 60 l of protein A-agarose/salmon sperm DNA (50% v/v slurry) and incubation continued for 1 h at 4°C with rotation. The protein A-agarose/antibody⅐chromatin complex was then washed sequentially 5 min in the following order: 1) low salt immune complex wash buffer; 2) high salt immune complex wash buffer; 3) LiCl immune complex was buffer, and 4) TE buffer. Finally, the chromatin complex was eluted with a freshly prepared elution buffer (1% SDS, 0.1 M NaHCO 3 ), and the cross-linking was reversed by adding NaCl to a final concentration of 0.2 M and incubating overnight at 65°C. The DNA was purified using the Qiagen QIA quick PCR purification kit prior to analysis.
PCR of Chromatin Templates-10 ng of immunoprecipitated DNA was used as a template for each PCR reaction using the following conditions as described previously (17): preincubation for 5 min at 94°C, denaturation by 40 cycles of 30 s at 95°C, annealing at primer specific temperature for 30 s (Table 1), and elongation for 30 s at 72°C. The PCR products were separated on a 2% agarose gel by electrophoresis, visualized with ethidium bromide, and quantified by laser densitometry.
Statistical Analysis-Statistical significance was determined by one-way analysis of variance or by Student's t test. A probability value of p Ͻ 0.05 was considered to be significant.

Expression of Heterodimeric Partners of RXR␣ and Coactivators in HPK1A and HPK1Aras
Keratinocytes-In this study, we performed Western blotting of whole cell extracts to identify the heterodimeric partners of RXR␣ and coactivators expressed in the keratinocyte cell lines (Fig. 1). In addition to VDR (panel A, lanes 1 and 2) and RXR␣ (panel A, lanes 3 and 4), RAR␣ (panel A, lanes 5 and 6), TR␤ (panel A, lanes 7 and 8), and PPAR␥ (panel A, lanes 9 and 10) were expressed in these cells. TR␣ and PPAR␣ protein expression were not found in the cell lines (data not shown). We next examined the nuclear expression of coactivators using specific antibodies and found that DRIP205 and RIP140 were expressed at high levels (panel B, lanes 1-4), whereas ACTR (SRC3/AIB1/TRAM1) was expressed at low levels (panel B, lanes 5 and 6) and SRC1 (panel B, lanes 7 and 8) and GRIP1 (SRC2/TIF2) (panel B, lanes 9 and 10) could not be detected. Several bands of higher and lower molecular weight were observed for some of the receptors or cofactors. In some cases (RIP140) a band of higher molecular weight  1-14). Addition of an antibody specific for PPAR␥ was used to identify the presence of PPAR␥ in the complex formed (lanes 2 and 8). A closed circle indicates the supershift in the presence of a PPAR␥ antibody, a closed diamond indicates a supershift in the presence of the RXR␣-N-terminal antibody, and a closed square a supershift in the presence of an RXR␣ LBD antibody. This figure is representative of three independent experiments performed under the same conditions. was observed in HPK1Aras but not in HPK1A cells. However, reprobing of these blots with only an anti-IgG antibody indicated that these bands were nonspecific. ␤-Tubulin expression of whole cell extracts (Abcam Inc., Cambridge, MA) was used as an indicator of protein loading.
Gel Mobility Shift Analysis of RXR-Heterodimeric Partner Binding-Our previous studies suggested that phosphorylation of RXR␣ on serine 260 induces a conformational change within the receptor (9), because the LBD antibody, which spans serine 260, was unable to supershift the VDR⅐RXR␣ complex, whereas an antibody directed at the NH 2 -terminal domain of RXR␣ could recognize this complex. In contrast an antibody directed against the VDR consistently recognized the VDR⅐RXR␣ complex in HPK1Aras cells. In the present study, we extended our observations to the interaction between the other heterodimeric partners of RXR␣ as well as the homodimeric RXR␣⅐RXR␣ complex formation using specific response element and ligands. Fig. 2 illustrates the ability of nuclear extracts from HPK1A and HPK1Aras to form complexes that recognize a 32 P-labeled response element specific for each receptor. Fig.  2A is shown as a positive control experiment using a 32 P-labeled mouse osteopontin (mOP) double-stranded oligonucleotide in the presence of 1,25(OH) 2 D 3. In Fig. 2A (lanes 1 and 7) a VDR⅐RXR␣ complex is observed in both HPK1A and HPK1Aras cells, which binds to the vitamin D-responsive element (VDRE), mOP, in the presence of 1,25(OH) 2 D 3 . The specificity of this complex is further demonstrated by addition of antibodies directed at the VDR and RXR␣ in HPK1A and HPK1Aras extracts. The binding of this complex to mOP results in a supershift following the addition of an antibody directed either against the C terminus portion of the VDR ( Fig.  2A, lanes 2 and 8) (17) or an antibody directed at the NH 2 terminus portion of RXR␣ ( Fig. 2A, lanes 3 and 9). In contrast an antibody directed at the LBD (C-terminal antibody) of the RXR␣ shifted the complex in HPK1A extracts ( Fig. 2A, lane 4) but not in HPK1Aras extracts ( Fig. 2A, lane 10). The supershift normally observed in HPK1A cells incubated with the RXR␣ LBD antibody (lanes 4 and 6) could be restored in the HPK1Aras cells by either the transient transfection of the nonphosphorylable mutant RXR␣ Ser-260 3 Ala (Fig. 2A, lane 14) or pre-treatment of HPK1Aras cells with the MAPKK inhibitor PD098059 (Fig. 2A, lane 12). The recognition of the VDR⅐RXR complex in HPK1Aras cell extracts by either a VDR antibody or an RXR antibody (directed against the N terminus of RXR away from Ser-260) indicate that heterodimerization of the VDR⅐RXR complex takes place. However, the absence of recognition of the VDR.RXR complex by an antibody against the C terminus of RXR␣ (LBD domain) also indicates that phosphorylation of RXR␣ at Ser-260 prevents the recognition of this complex by this antibody likely through conformational changes in this region. Furthermore, when phosphorylation of Ser-260 is prevented by either MAPKK inhibition or introduction of an alanine residue at position 260, the VDR⅐RXR complex is recognized by the C-terminal antibody against the LBD domain indicating that these conformational changes are reversible.
We next examined the formation of the homodimeric complex RXR␣⅐RXR␣ (Fig. 2B). Nuclear extracts from HPK1A and HPK1Aras cells formed a complex with a 32 P-labeled RXRE double-stranded oligonucleotide in the presence of the RXR␣ ligand LG1069 (Fig. 2B, lanes 1 and 6). This complex could be shifted by the addition of an antibody directed at the NH 2 terminus portion of the RXR␣ with both HPK1A and HPK1Aras extracts (Fig. 2B, lanes 2 and 7). In contrast, and similar to the observation with the VDR⅐RXR␣ complex formation, the antibody directed at the LBD of the RXR␣ shifted the RXR␣⅐RXR␣ complex in HPK1A extracts (Fig. 2B, lane 3) but not in HPK1Aras extracts (Fig. 2B, lane 8). The supershift could then be restored in HPK1Aras cells by either transient transfection with the mutant RXR␣ Ser-260 3 Ala (Fig. 2B, lane 12) or pre-treatment of HPK1Aras cells with the MAPKK inhibitor PD098059 (Fig. 2B, lane 10) as compared with vector alone (Fig.  2B, lane 8). As controls addition of PD098059 to HPK1A cells had no effect on either RXR␣⅐RXR␣ complex formation (Fig.  2B, lane 4) or its normal supershift (Fig. 2B, lane 5). These data therefore indicate that Ser-260 phosphorylation in HPK1Aras extracts do not affect homodimerization of the RXR⅐RXR complex but alters its recognition by a specific antibody raised against the LBD.
The next set of experiments was aimed at determining the consequences of RXR␣ phosphorylation at Ser-260 on the other heterodimeric partners of the RXR␣. Similar experiments were performed with specific response elements and antibodies directed at the RAR␣, TR␤, and PPAR␥. Addition of specific ligands to the RAR␣ (ATRA), TR␤ (T 3 ), and PPAR␥ (troglitazone) resulted in complex formation recognized by 32 P-labeled RARE (Fig. 2C, lanes 1 and 7), 32 P-labeled thyroid hormone response element (Fig. 2D, lanes 1 and 7), and 32 P-labeled PPARE (Fig. 2E, lanes 1 and 7). The specificity of the complexes was determined by addition of specific antibodies directed at RAR␣ (Fig. 2C, lanes 2 and 8), TR␤ (Fig. 2D, lanes 2 and 8), and PPAR␥ (Fig. 2D, lanes 2 and 8). Note that TR␤ recognizes the receptor A/B domain and abrogates the binding of the complex with TRE. In contrast, the RAR␣ and PPAR␥ antibodies induce a supershift of the heterodimeric complex. Similar to the observations in HPK1A and HPK1Aras extracts using an NH 2 -terminal antibody against the RXR␣, a supershift of the RAR␣⅐RXR␣ (Fig. 2C, lanes 3 and 9), TR␤⅐RXR␣ (Fig. 2D, lanes  3 and 9), and the PPAR␥⅐RXR␣ (Fig. 2E, lanes 3 and 9) complexes was observed. In contrast the antibody directed against the LBD of RXR␣ could shift the complexes in HPK1A extracts but was unable to do so in HPK1Aras extracts: RAR␣⅐RXR␣ (Fig. 2C, lanes 4 and 10) TR␤⅐RXR␣ (Fig. 2D, lanes 4 and 10), and PPAR␥⅐RXR␣ (Fig. 2E, lanes 4 and 10). Transient transfection of the non-phosphorylable RXR␣ Ser-260 3 Ala mutant or treatment with the MAPKK inhibitor PD090859 of HPK1Aras cells restored the RAR␣⅐RXR␣ (Fig. 2C, lanes 14 and 12), TR␤⅐RXR␣ (Fig. 2D, lanes 14 and 12), and PPAR␥⅐RXXR␣ (Fig.  2E, lanes 14 and 12) complexes. Similar to the effects on the VDR⅐RXR and RXR⅐RXR complexes, phosphorylation at Ser-260 does not affect heterodimerization of RXR with either RAR, TR, or PPAR but alters its recognition by a specific antibody raised against the LBD.
Inhibition of MAPKK Activity Results in a Reversal of the Partial Resistance of Ras-transformed Keratinocytes to the Growth Inhibitory Effects of Both Vitamin D and Retinoids-We previously demonstrated that the ras-transformed keratinocyte cell line, HPK1Aras, is partially resistant to the growth inhibitory effects of 1,25(OH) 2 D 3 (6) and LG1069 (9) compared with the control cell line HPK1A. We further demonstrated that the MAPKK inhibitor PD098059 reversed this partial resistance on cell growth with a maximal effect observed at 96 h (9). Here we extended these observations by examining the growth inhib-itory effect of ATRA (the natural ligand of RAR, Fig. 3C) and TTNPB (a highly specific synthetic ligand of RAR, Fig. 3D) as compared with LG1069 (Fig. 3B) and 1,25(OH) 2 D 3 (Fig. 3A) using HPK1A and HPK1Aras cells. A significant and dose-dependent growth inhibition was observed in HPK1A cells with both ATRA and TTNPB similar to the effect of 1,25(OH) 2 D 3 and LG1069. In contrast HPK1Aras cells were partially resistant to ATRA and TTNPB growth-inhibitory effects and required ϳ100-fold higher concentrations of the ligands to achieve the same effect. Addition of the MAPKK inhibitor PD098059 reversed the partial resistance to both ATRA and TTNPB in HPK1Aras cells restoring their growth-inhibitory effect similar to the one observed in HPK1A cells. For comparison the effect of 1,25(OH) 2 D 3 and LG1069 on HPK1Aras cells in the absence and presence of PD098059 are shown in panels A and B. These data therefore indicate that phosphorylation of RXR␣ in HPK1Aras cells by Ras Raf MAPK activation results in resistance to the growth inhibitory effects of vitamin D and retinoids and that this resistance can be reversed by inhibiting the MAPK pathway.

Transfection of the Non-phosphorylable hRXR␣ Mutant Reverses Partial Resistance to 1,25(OH) 2 D 3induced Growth Inhibition in Rastransformed
Keratinocytes-The HA-tagged wild-type (WT) hRXR␣, or HA-tagged Ser-260 mutant (Ala-260) or vector alone (pCDNA3.1) were transfected into HPK1Aras cells. HPK1Aras cells transfected with the WT hRXR␣ remained resistant to 1,25(OH) 2 D 3 similar to control cells (vector alone). In contrast HPK1Aras cells transfected with the mutant construct had increased sensitivity to 1,25(OH) 2 D 3 (Fig. 4A). Nuclear extracts of HPK1Aras cells transfected with the HA-tagged WT hRXR␣, HA-tagged hRXR␣ mutant or vector alone (pCDNA3) were analyzed by Western blotting with an anti-hRXR␣ antibody, anti-HA antibody, and an anti-(total) Rb antibody (loading control) (Fig. 4B). Blots probed with the antiRXR␣ antibody indicated that transient transfection of the hRXR␣ mutant approximately doubled total (both endogenous and exogenous) hRXR␣ expression as compared with cells transfected with vector alone. Blots probed with the HA-antibody show equal expression of the transfected exogenous wild-type and mutant hRXR␣. HPK1Aras extract transfected with pCDNA3 alone is shown as a negative control. Hence, expression of the nonphosphorylable mutant (but not WT hRXR␣) to levels approximately equal to endogenous hRXR␣ restores HPK1Aras sensitivity to growth inhibition by 1,25(OH) 2 D 3 .

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 mOP by 1,25(OH) 2 D 3 is partially inhibited in the MAPKKactivated HPK1Aras cells compared with that of HPK1A cells (18). Furthermore, this inhibition can be reversed by selectively inhibiting the MAPKK signaling pathway or expression of a non-phosphorylable Ser-260 3 Ala mutant. mOP3 activity increased ϳ120% above basal levels similar to the effect of 1,25(OH) 2 D 3 seen in HPK1A cells (9). By transfecting a reporter plasmid containing three repeats of the RARE ␤ cloned in front of a thymidine kinase-driven chloramphenicol acetyltransferase (CAT) gene, we now demonstrate that the resistance to the transactivation potential of increasing amounts of ATRA in HPK1Aras was reversed by the addition of 25 M PD098059, a MAPKK inhibitor (Fig. 5A). In addition, by transfecting a reporter plasmid containing three repeats of the RXRE cloned in front of a thymidine kinase-driven CAT gene, we determined that the resistance to the transactivation potential of increasing amounts of LG1069 existing within HPK1Aras cells was reversed by the addition of 25 M PD098059 (Fig. 5B). Similar results were observed following transfection of the nonphosphorylable Ser-260 3 Ala mutant of RXR␣ (Fig. 5, A and  B). These data indicate that phosphorylation of RXR␣ in HPK1Aras cells through activation of the Ras/Raf/MAPK pathway results in resistance to the transcriptional activation of retinoids and that this resistance can be reversed by inhibiting the MAPK pathway or specifically blocking phosphorylation on Ser-260.
Inhibition of MAPKK Activity Enhances the Effect of 1,25(OH) 2 D 3 on Cell Cycle Regulatory Proteins-p21 waf1/cip1 , a cyclin-dependent kinase inhibitor, is a key target of 1,25(OH) 2 D 3 antiproliferative effect (19) which directly up-regulates its expression. Here we demonstrate that in addition to reversing the resistance to 1,25(OH) 2 D 3 on cell growth, inhibition of MAPKK activity had significant effects on the expression of the cell cycle regulated proteins p21 waf1/cip1 . As shown in Fig. 6A pre-treatment of HPK1Aras cells with the MAPKK inhibitor UO126 enhanced the stimulatory effect of 1,25(OH) 2 D 3 on p21 waf1/cip1 expression (lanes 4 versus 2) in comparison to basal (lane 1). UO126 alone moderately increased p21 expression as compared with basal (lane 3).

Inhibition of MAPKK Activity Enhances the Interaction of the VDR⅐RXR Complex with 1,25(OH) 2 D 3 -responsive Promoter Regions as Well as Coactivator Recruitment to the Complex-
We screened several promoters for their interactions with the VDR⅐RXR complex and DRIP205 by ChIP assays using an antibody against either the VDR or DRIP205 following stimulation with 10 Ϫ7 M 1,25(OH) 2 D 3 . Representative agarose gels of the PCR products are shown in Fig. 6B. Comparable chromatin content of the samples was demonstrated by amplification of PCR products prior to immunoprecipitation (input lane).  2 versus 4). Taken together these data indicate that inactivation of MAPK significantly enhances the interaction of the VDR⅐RXR complex and its promoters as well as coactivator recruitment to the complex.
Disruption of Coactivators Interaction with the VDR⅐RXR Complex in HP1Aras Cells-Biotinylated mOP pulldown assays were used to assess VDR⅐RXR␣ coactivators interaction. With this methodology it is possible to characterize the coactivator recruitment to the VDR⅐RXR␣ complex after pulldown of the protein complex with a biotinylated mOP VDRE probe and subsequently identify specific coactivators in the complex by Western blot analysis.
The strength of the interaction was assessed by comparing the intensity of the specific bands prior to and following treatment of HPK1A and HPK1Aras cells with 1,25(OH) 2 D 3 (10 Ϫ8 M) (Fig. 7). Addition of 1,25(OH) 2 D 3 resulted in increased inter-

alone (panel A, lane 3 versus 4 and panel C, lane 3 versus 4).
In each of the experiments equal amounts of proteins were loaded onto the gel (a Western blot done with aliquots of HPK1A and HPK1Aras nuclear extracts prior to the pulldown assays is shown). There was no visible binding in pulldown assays when extracts were incubated with the non-relevant biotinylated probe (data not shown). Finally, we analyzed the specificity of serine 260 phosphorylation on the impairment of coactivator recruitment by expressing the non-phosphorylable RXR␣ Ser-260 3 Ala mutant or the empty vector in HPK1Aras cells and examining coactivator recruitment in the presence of 1,25(OH) 2 D 3 (Fig. 8). As predicted, transfection of the RXR␣ Ser-260 3 Ala mutant resulted in a sharp increase of coactivator recruitment (Fig. 8, lane 2 versus lane 1) in the presence of 1,25(OH) 2 D 3 similar to the effect observed with the MAPK inhibitor (Fig. 7, panels A and C, lane 4). Furthermore, addition of the MAPK inhibitor UO126 to hRXR␣ Ser-260 3 Ala transfected cells had no additive effect on coactivator recruitment (Fig. 8, lane 5 versus lane 2). These data therefore indicate that phosphorylation on Ser-260 of RXR␣ in HPK1Aras cells affect coactivator interaction with the VDR⅐RXR complex. Furthermore, when phosphorylation of Ser-260 is prevented by either MAPKK inhibition or introduction of a non-phosphorylable Ala residue at position 260, the VDR⅐RXR complex interaction with coactivators is restored.

DISCUSSION
In the present study we have further defined the role of RXR␣ phosphorylation at serine 260. Our previous studies had demonstrated that ras-transformed keratinocytes are partially resistant to the growth inhibitory effect of 1,25(OH) 2 D 3 (6). We then determined that the VDR⅐RXR complex was altered in CAT activity was assayed after 24 h and normalized for transfection efficiency by the corresponding ␤ gal production. B, HPK1Aras cells were transiently transfected with a RXRE-CAT reporter plasmid and a ␤-galactosidase plasmid and were treated with vehicle or increasing concentrations of LG1069 in the presence or absence of PD098059. CAT activity was assayed after 24 h and normalized for transfection efficiency by the corresponding ␤-galactosidase production. Each value represents the mean Ϯ S.D. of three determinations. An open circle represents a significant difference in cell number between hormone-treated cells and the vehicle-treated controls. An asterisk represents a significant difference between cells treated with PD098059 and hormone as compared with cells treated with hormone alone. This figure is representative of three independent experiments performed under the same conditions. exogenous mutant at levels comparable to the endogenous hRXR␣ is effective in re-establishing sensitivity to 1,25(OH) 2 D 3 -induced growth inhibition. Transfection of wildtype hRXR␣ induced similar changes in expression but had no effect on 1,25(OH) 2 D 3 resistance indicating that the observed effects were not a consequence of artifactual overexpression of the exogenous receptor. We also analyzed retinoid signaling in response to specific ligands and found that Ras-Raf-MAPK activation inhibited both the growth inhibitory response and transactivation to these ligands. Transactivation activity in response to these ligands could be restored by overexpression of a non-phosphorylable RXR␣ Ser-260 3 Ala mutant in ras-transformed keratinocytes similar to our previous observation that restored 1,25(OH) 2 D 3 -dependent transactivation (9). Our data therefore indicate that phosphorylation at Ser-260 of RXR␣ by the Ras-Raf-MAPK cascade not only affects the vitamin D signaling pathway but also the retinoid signaling pathways in ras-transformed keratinocytes.
We next examined in detail the mechanisms of this resistance. Our previous observations indicate that serine 260 phosphorylation likely resulted in conformational changes of the RXR␣ but does not alter VDR⅐RXR heterodimerization (9). This hypothesis was based on our observation that RXR␣ phosphorylation altered the recognition of the VDR⅐RXR␣ complex by a specific antibody raised against the LBD (C-terminal domain) of the RXR␣, which spans Ser-260 but not by a specific antibody raised against the N-terminal domain of RXR␣. This C-terminal antibody failed to recognize the complex in gel shift analysis when HPK1Aras extracts were used but recognized the VDR⅐RXR␣ complex in HPK1A extracts, which are not phosphorylated at serine 260 (9,18). In contrast, an antibody raised against regions of the NH 2terminal domain of RXR␣ remote from serine 260 was able to recognize the VDR⅐RXR␣ complex of HPK1Aras extracts as well as HPK1A extracts indicating that VDR⅐RXR heterodimerization occurred in HPK1Aras extracts, which are phoshorylated on Ser-260 (9). In the present study we confirmed the critical location of serine 260 phosphorylation in altering the conformation of RXR␣ by overexpressing the RXR␣ Ser-260 3 Ala mutant in HPK1Aras cells. Nuclear extracts from these cells were thus able to recognize this C-terminal antibody with an epitope spanning Ser-260 as indicated by a supershift of the VDR⅐RXR␣ complex (Fig. 2). Similarly, RXR␣⅐RXR␣, RAR␣⅐RXR␣, and PPAR␥⅐RXR␣ complexes in HPK1Aras nuclear extracts were recognized by this C-terminal antibody following overexpression of the RXR␣ Ser-260 3 Ala mutant. These data indicate that serine 260 phosphorylation does not alter dimerization of the RXR␣ with its heterodimeric partners but likely results in conformational changes of the RXR␣ complexes not only with VDR but also with RAR␣, RXR␣, as well as TR␤ and PPAR␥ (Fig. 9). As a general rule these receptors bind to their hormone-responsive elements in target genes as heterodimers rather than homodimers (24) It therefore raises the possibility that serine 260 is a key regulator of multiple signaling pathways mediated by the RXR␣. However, this would not exclude the possibility that, within the same cells, ligands interact preferentially with homodimers to modulate their biological response. Further studies are needed to fully evaluate the biological consequences of Ser-260 phosphorylation of RXR␣ on these other signaling pathways. In addition, more recent studies indicate that phosphorylation at serine 260 may affect cellular events unrelated to cancer biology. A recent study in HepG 2 liver cells indicates that the acute phase response in liver cells initiated by the proinflammatory cytokine interleukin-1␤ (IL-1␤) (25) directly phosphorylates RXR␣ and may be responsible for the suppression of RXR␣ signaling. Interestingly, the resistance to 1,25(OH) 2 D 3 in response to Ras-Raf-MAPK activation may be cell-dependent as reported in two osteoblastic cell lines MG-63 and MC3T3-E 1 (26). In these studies MAPKK activation inhibited vitamin D signaling in MC3T3-E 1 cells but not in MG-63 cells. Furthermore this "vitamin D resistance" was reversed by the MAPKK inhibitor UO126 in MC3T3-E1 cells. The critical importance of serine 260 of the human RXR␣ has also been reported with the mouse RXR␣ (27). In these studies F9 cells expressing phosphorylated wild-type RXR␣ on Ser-265 (Ser-265 of mouse RXR␣ is the equivalent of Ser-260 of human RXR␣) were resistant to ATRA signaling but not F9 cells expressing the RXR␣ Ser-265 3 Ala mutant highlighting the importance of this residue in both mouse and human RXR␣.
We next examined the consequences of MAPKK inhibition on the interaction between the VDR⅐RXR complex and the promoter regions of several 1,25(OH) 2 D 3 target genes and DRIP205 recruitment to the complex by ChIP assays. As predicted pre-treatment of ras-transformed keratinocytes with the MAPKK inhibitor UO126 significantly enhanced these interactions in response to 1,25(OH) 2 D 3 . In addition to CYP24, we examined several genes implicated in the regulation of the cell cycle, including p21 waf1/cip1 , cyclin C, and CLMN. In earlier studies it was demonstrated that p21 waf1/cip1 contains at least three VDREs, each of which show 1,25(OH) 2 D 3 -dependent recruitment of coactivator proteins (including DRIP205) to VDR-bound chromatin regions (23). A 2-fold increase in p21 waf1/cip1 binding between the VDR⅐RXR complex and VDREs and a 3-fold increase in DRIP205 recruitment was observed and suggested that vitamin D resistance is mediated at  (lanes 1, 3, and 4) 1-5). Coactivator binding to biotinylated mOP VDRE purified with streptavidin-agarose beads is described under "Materials and Methods." The lower panel represents the densitometric analysis of the DRIP205 bands (upper panel) from three separate experiments (mean Ϯ S.D.) and expressed as a percentage of basal activity in the absence of 1,25(OH) 2 D 3 and UO126 (HPK1Aras cells incubated with vehicle alone : lanes 1 and 3). WB represents Western blot analysis of DRIP205 using an aliquot of nuclear extracts prior to pulldown with biotinylated mOP VDRE to verify for DRIP205 loading. Equal protein loading was also verified with the nuclear protein TBP18 (Abcam) (data not shown). An asterisk indicates a significant difference from basal. An open circle indicates a significant difference between cells treated with a combination of 1,25(OH) 2 D 3 and UO126 versus cells treated with 1,25(OH) 2 D 3 alone. least in part by regulating this potent inhibitor of cell cycle progression. Similar results were obtained with cyclin C, a member of a cyclin protein superfamily activated by 1,25(OH) 2 D 3 , which controls cell cycle transition through activation of cyclin-dependent kinases (28). Our data therefore indicate that inhibition of the Ras-Raf-MAPK pathway reverses the resistance of 1,25(OH) 2 D 3 at least in part by enhancing the binding of the VDR⅐RXR complex and coactivator recruitment to the promoter region of cell cycle-regulated genes. It is important to note that the observed effects on the VDR⅐RXR binding to the promoter and coactivator recruitment are rapid (within 4 h) and precede the changes observed in transcriptional activation and cell growth. These data strongly support a causal relationship between RXR phosphorylation-induced alteration of coactivator recruitment and downstream biological events. Although re-ChIP assays were not used in the present study, previous studies using re-ChIP assays demonstrated the simultaneous association of VDR with RXR and coactivators (23, 28) on both p21 waf1/cip1 and cyclin C supporting the general model of coactivator recruitment on target genes (29).
Interestingly, UO126 binds with higher affinity to MAPKK as compared with PD098059 and has been shown to be effective in animal models in vivo in inhibiting MAPK activity without significant side effects (30). UO126 administration in vivo may therefore be useful in future cancer studies in combination with vitamin D compounds.
The importance of coactivator recruitment to steroid receptor signaling is now well established (31). Among these is steroid receptor coactivator 1 (SRC1), a major modulator of estrogen function (32) that belongs to the p160 family of coactivators and also includes SRC2 (GRIP1/TIF2) (33) and SRC3 (AIB1, ACTR, and TRAM1) (34). SRC1 and SRC2 have all been implicated in VDR-mediated transactivation (35,36), whereas SRC3 has been implicated in TR signaling (37). We examined the expression of SRC1, -2, and -3 in both immortalized and rastransformed keratinocytes and could not detect SRC1 or SRC2 (GRIP1), whereas SRC3 (activation of thyroid hormone receptors and retinoic acid receptors, ACTR) was detected at low levels in both cell lines (Fig. 1B). Previous studies also failed to detect SRC1 in human keratinocytes (38,39), whereas SRC2 expression was reported in both normal human keratinocytes and a human squamous carcinoma cell lines (38). It is possible that the expression of SRC-2 is cell line-specific. Low levels of expression of SRC-3 detected in our study (Fig. 1B) are in keeping with expression levels seen in human keratinocytes (38). We next examined the protein levels of DRIP205 using an antibody from Santa Cruz Biotechnology, which detects a major band at around 220 kDa. DRIP205 is an essential component of the DRIP complex critical for VDR activation (40), which binds to the LBD of VDR and initiates transcriptional activation by promoting interaction with vitamin D-responsive sequences (VDREs) on target genes. Using equal protein amount high and similar level of expression was observed in HPK1A and HPK1Aras cells. High levels of DRIP205 expression have also been reported in a human squamous cancer cell line and in proliferating keratinocytes (38). Finally, we analyzed expression of RIP140, a regulatory protein that acts as a co-repressor of the glucocorticoid receptor (41, 42) but also as a coactivator of vita-min D signaling (35). Our next objective was to analyze the recruitment of these coactivators to the VDR⅐RXR␣ complex. We focused our analyses to DRIP205 because of its importance in vitamin D signaling and on RIP140 because of its interesting dual activity and relatively high level of expression in our system as compared with coactivators of the SRC family. To quantitate coactivator recruitment we treated both immortalized and ras-transformed keratinocytes with 1,25(OH) 2 D 3 and subsequently pulled down the VDR⅐RXR⅐coactivator complex from nuclear extracts using a biotinylated mOP VDRE. Proteins bound to mOP VDRE were then extracted with agarose beads and analyzed by Western using specific antibodies against the coactivators. Using equal amounts of proteins (as verified by simultaneous Western analysis of nuclear extracts prior to extraction with biotinylated mOP VDRE) we could compare recruitment of coactivators to the VDR⅐RXR␣ complex in HPK1A cells versus HPK1Aras cells prior to and following treatment with 1,25(OH) 2 D 3 . Our study indicates that both DRIP205 and RIP140 are recruited to the complex; however, recruitment is significantly higher in HPK1A cells as compared with HPK1Aras cells (Fig. 7). Our next objective was then to determine the consequences of Ras-Raf-MAPK activation on coactivator recruitment. When ras-activated cells (HPK1Aras) were treated with the MAPK inhibitor UO126 in the presence of 1,25(OH) 2 D 3 recruitment of coactivators at least doubled in HPK1Aras cells reaching levels comparable to 1,25(OH) 2 D 3treated HPK1A cells. Furthermore, UO126 had no effect on coactivator recruitment in HPK1A cells. These data clearly indicate that MAPK activation inhibits coactivator recruitment likely through phosphorylation of MAPK consensus sites on the VDR⅐RXR⅐coactivator complex. To address the importance and specificity of serine 260 phosphorylation of RXR␣, we next expressed the non-phosphorylable RXR␣ Ser-260 3 Ala mutant into HPK1Aras cells treated or not with 1,25(OH) 2 D 3 . Our data indicate that expression of this mutant rescues coactivator recruitment to the complex (Fig. 8) similar to the effect observed with UO126. VDR is not a substrate for MAPK, but coactivators such as SRC1 and DRIP205 are possible targets (43,44). Therefore we cannot exclude the possibility that MAPK phosphorylation of coactivators may play a role in their recruitment to the VDR⅐RXR complex, although this contribution is likely minimal because overexpression of the non-phosphorylable RXR␣ Ser-260 3 Ala mutant restores their recruitment fully and similarly to the effect observed with UO126. Interestingly, DRIP205 phosphorylation by MAPK-ERK increases intrinsic DRIP205 transcriptional activity as well as TR-dependent transcription (44) as opposed to the negative effect of MAPK-ERK on RXR␣ mediated transactivation found here. This would suggest that in our model MAPK-ERK mediated inhibition of RXR␣ signaling predominates over a potential stimulatory effect of MAPK-ERK on DRIP205-mediated transactivation.
The one or more mechanisms by which phosphorylation of serine 260 of RXR␣ perturbs the recruitment of coactivator(s) to the VDR⅐RXR complex likely involve structural changes in the AF-2 domain, which appear to facilitate coactivator interaction (45,46). Serine 260 is located in the omega loop, which contacts the AF-2 domain (11), and its phosphorylation likely alters the conformation of this region and accessibility of coactivators and subsequent transactivation (Fig. 9). Specific residues in the omega loop (Pro-264 at the start of H3 and Asn-262 in the loop between H2 and H3) bond with specific residues in the AF-2 domain (Phe-450 and Glu-453) (11). Mutations of these AF-2 residues in hRXR␣ or the equivalent sequences in mRXR␣ result in suppression of the transactivation activities of both RXR⅐RXR homodimers and RXR/VDR heterodimer (47) presumably by abolishing/reducing interaction between RXR and coactivators. Interestingly, phosphorylation of the omega loop at the equivalent position on Ser-265 in the mouse RXR␣ also inhibits transcription of retinoic acid target genes (27). The omega loop is located between helices H1 and H3 of the LBD (11), and its sequence is identical between mouse and human RXR␣ (NMGLNPSSPNDPVTN). Consequently, its phosphorylation potentially affects recruitment of coactivators on the various RXR␣ complexes across species. Further studies will be needed to explore these possibilities.