Disruption of Vitamin D Receptor-Retinoid X Receptor Heterodimer Formation following ras Transformation of Human Keratinocytes*

Cynthia SolomonDagger §, Michael SebagDagger §, John H. White, Johng Rhimparallel , and Richard KremerDagger **

From the Departments of Dagger  Medicine and  Physiology, McGill University, Montreal, Quebec H3A 1A1, Canada and the parallel  Laboratory of Molecular Oncology, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A partial resistance to the growth inhibitory influence of 1,25-dihydroxyvitamin D3 is apparent when immortalized keratinocytes are transformed by the ras oncogene. The vitamin D receptor (VDR) was isolated, analyzed, and found to be identical in normal, immortalized, and ras-transformed keratinocytes. Subsequently, nuclear extracts from immortalized and ras-transformed keratinocytes were analyzed in gel mobility shift assays utilizing labeled vitamin D response elements or thyroid hormone response elements. A specific protein·DNA complex that was shown to contain VDR using an anti-VDR antibody was identified in both types of extracts; however, the addition of an anti-retinoid X receptor (RXR) antibody identified RXR in the complex of both normal and immortalized keratinocyte cell extracts, but not in ras-transformed keratinocytes. Furthermore, transfection of ras-transformed keratinocytes with wild-type human RXRalpha rescued VDR·RXR and thyroid hormone receptor·RXR complexes as demonstrated by a supershift in the presence of the anti-RXR antibody. Both cell lines were found to express RXRalpha message in equal amounts. Western blot analysis revealed that RXRalpha protein from ras-transformed keratinocytes was indistinguishable from that from immortalized keratinocytes and from control cells. These results suggest a causal relationship between resistance to the growth inhibitory influences of 1,25-dihydroxyvitamin D3 and disruption of the VDR·RXR complex in malignant keratinocytes.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3),1 the active metabolite of vitamin D, is a potent inhibitor of growth of keratinocyte cells and stimulates their differentiation (1, 2). It exerts these effects by binding to its receptor, found principally in the nuclei of its target cells, and then modulating the transcription of specific target genes involved in cell growth and differentiation, such as the proto-oncogenes c-myc, c-fos, and involucrin, a marker of keratinocyte differentiation (3-5). The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily, with which it shares structural homology in the DNA-binding domain, the hormone-binding domain, the dimerization domain, and the transactivation domain (6). Recent studies indicate that to effect gene transcription, VDR first requires protein-protein interaction with another, but distinct member of this receptor family, the retinoid X receptor (RXR) (7). Once dimerized, this VDR·RXR complex recognizes and binds to specific bipartite DNA sequences found on the promoter regions of vitamin D target genes known as vitamin D response elements (VDREs) (8). Bound to these elements, the VDR·RXR complex can have either a stimulatory or an inhibitory effect on gene transcription.

In previous studies, we have characterized the effects of 1,25-(OH)2D3 on human keratinocytes as they progress from the normal to the malignant phenotype (9). In our model of tumor progression, normal human keratinocytes were established as an immortal cell line by transfection with human papillomavirus type 16 and subsequently transformed with an activated ras oncogene. We determined that the malignant keratinocytes were resistant not only to the growth inhibitory effects of 1,25-(OH)2D3, but also to its transcriptional influences.

In this work, we have studied the mechanism responsible for vitamin D resistance in ras-transformed keratinocytes. We report the first incidence of vitamin D resistance caused by a disruption of the VDR·RXR complex.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Transfections-- The HPK1A cell line was established from normal human keratinocytes by stable transfection with human papillomavirus type 16. These cells have an indefinite life span in culture, but retain differentiation properties characteristic of normal keratinocytes and are nontumorigenic when injected into nude mice. These immortalized cells were then transformed into the malignant HPK1Aras cell line after transfection with a plasmid carrying an activated Ha-ras oncogene. In addition to forming colonies in soft agar, the malignant HPK1Aras cells produce invasive tumors when transplanted into nude mice (10). All cell lines were grown in Dulbecco's modified Eagle's medium (Gibco-BRL, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum and passaged once or twice weekly. Cells grown under these conditions were then seeded at a density of 1 × 104 cells/ml in either 6-well plates, for chloramphenicol acetyltransferase assays, or in 100-mm2 culture dishes, for transfections and nuclear extract preparation. Transfections were performed by incubating plasmid DNA (10 µg) with LipofectAMINE (10 µg; Gibco-BRL) for 20 h in serum-free Dulbecco's modified Eagle's medium and then replacing the medium with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Chloramphenicol Acetyltransferase Assays-- HPK1A and HPK1Aras cells were plated at a density of 1 × 104 cells/ml in 6-well plates and transfected with 5 µg of a mOP3 plasmid, containing three repeats of the mouse osteopontin vitamin D response element (CAAGGTTCACGAGGTTCAC) in front of a chloramphenicol acetyltransferase reporter gene and 2 µg of a beta -galactosidase plasmid (11). Cells were then treated with increasing concentrations of 1,25-(OH)2D3, (10-8-10-6 M) for 24 h. Following this incubation period, the cells were scraped, washed in phosphate-buffered saline, resuspended in phosphate-buffered saline containing 1.0 mM phenylmethylsulfonyl fluoride, and finally lysed by three freeze-thawing cycles. The cell lysate was centrifuged, and aliquots of cell extracts were used for chloramphenicol acetyltransferase assay. Assays were performed using a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay kit (5 Prime right-arrow 3 Prime, Inc., Boulder, CO). Statistical significance was determined by analysis of variance. A probability value of <0.01 was considered to be significant.

Nuclear Extracts-- African green monkey kidney cells (COS-7) and HPK1Aras cells at 50% confluence in 100-mm2 culture plates were transiently transfected with either 10 µg of plasmid DNAs expressing the human retinoid X receptor (hRXRalpha ) and the human vitamin D receptor (hVDR) (kind gifts of Drs. R. Evans and M. Haussler, respectively) or hRXRalpha alone using the LipofectAMINE technique, as described above. After 20 h, the medium was changed, and cells were further incubated for 48 h, washed with chilled phosphate-buffered saline, and collected. These cell pellets as well as cell pellets collected from untransfected HPK1A and HPK1Aras cells were processed in a Dounce tissue homogenizer (loose) in 2 volumes of buffer containing 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and one CompleteTM protease inhibitor mixture tablet (Boehringer Mannheim, Laval, Quebec, Canada). Nuclear pellets were obtained by centrifugation at 25,000 × g for 20 min at 4 °C; resuspended in 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol; and again Dounce-homogenized. Following a 20-min centrifugation at 25,000 × g, nuclear extracts (supernatant) were dialyzed for 5 h against 20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Protein content was determined with a protein assay kit (Bio-Rad, Mississauga, Ontario), and samples were then aliquoted and stored at -80 °C.

Gel Mobility Shift Analysis-- 2 µg of nuclear extracts were incubated for 20 min on ice with 200 nM 1,25-(OH)2D3 or 200 nM thyroxine and 1 µg of poly(dI·dC) in a binding buffer (250 mM Tris-HCl, pH 8.0, 50% glycerol, and 5 mM dithiothreitol). 5 fmol of the appropriate 32P-labeled DNA response element was then added and incubated for 20 min at room temperature. When required, the anti-RXR antibody (4X1D12, a monoclonal antibody recognizing a common region in the E-domain of all three types of RXR; a kind gift of Dr. P. Chambon) or the anti-VDR antibody (Affinity Bioreagents Inc., Neshanic, NJ) was added to the incubation. In some experiments, 1-5 pmol of baculovirus-expressed hVDR (Panvera, Madison, WI) were added to the above reactions. The samples were then electrophoresed on 5% nondenaturing polyacrylamide gels in 0.5× TBE (0.045 M Tris borate and 0.001 M EDTA, pH 8.0) at 6 V/cm. Following electrophoresis, the gels were dried and exposed to Kodak XAR-5 film without intensifying screens.

RNA Analysis-- Total cellular RNA was prepared using Trizol reagent (Gibco-BRL) and chloroform extractions. For Northern blot analysis, 5-10 µg of total RNA were electrophoresed on a formaldehyde-containing 1.1% agarose gel. RNA was transferred onto a nylon membrane (Nytran), air-dried, baked at 80 °C for 1 h, and then hybridized with a RXRalpha probe labeled with [32P]dCTP (ICN Biomedicals, Mississauga, Ontario, Canada) by the random primer method (Amersham Pharmacia Biotech, Ontario). After 24 h of incubation at 42 °C, filters were washed successively in 1× SSC and 1% SDS for 15 min at room temperature and in 0.1× SSC and 0.1% SDS twice for 30 min at 55 °C. Autoradiography of filters was carried out at -70 °C using Kodak XAR films and two intensifying screens. For reverse transcriptase-polymerase chain reaction (RT-PCR) first-strand cDNA synthesis, 1 µg of RNA in diethyl pyrocarbonate-treated water was denatured at 80 °C for 10 min, quick-cooled on ice, and reverse-transcribed from random hexamer primers (50 pmol; PdN6, Amersham Pharmacia Biotech) using 50 units of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL) in 1× PCR buffer (Perkin-Elmer, Ontario) containing 500 µmol/liter dNTPs (Perkin-Elmer) and 20 units of RNasin (Amersham Pharmacia Biotech). The reaction was carried out in a volume of 20 µl at 25 °C for 10 min and then at 41 °C for 30 min, followed by heating to 95 °C for 5 min. For amplification of first-strand cDNA derived from RXRalpha and RXRbeta mRNAs, each reverse transcription reaction was expanded to a volume of 100 µl to contain 1× PCR buffer, 200 mmol/liter dNTPs, and 50 pmol each of the upstream and downstream PCR primers for RXRalpha and RXRbeta . The reactions were heated to 72 °C and then supplemented with 2.5 units of Taq DNA polymerase (Gibco-BRL). The reactions were subjected to 30 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min), except for denaturation at 94 °C for 1 min, annealing for 1.5 min, and extension for 2 min during the first cycle and extension for 15 min during the last cycle. PCR products were resolved on a 1.5% agarose gel run in 1× TBE buffer and visualized with ethidium bromide.

Cloning and Sequencing-- PCR products were directly cloned in the vector pCRII (Invitrogen, San Diego, CA) according to the manufacturer's specifications. Plasmid clones of interest were identified by restriction endonuclease size analysis and/or PCR analysis, and the inserts were sequenced from primers complementary to the flanking T7 and SP6 RNA polymerase promoter sites using the T7 DNA sequencing kit (Amersham Pharmacia Biotech). The reaction mixtures, radiolabeled with 35S-dATP (ICN Biomedicals), were electrophoresed on 5% Long Ranger sequencing gel (AC Biochem, Malvern, PA) for 2 h at 60 watts. Gels were dried and exposed to Kodak XAR film at room temperature.

Western Blot Analysis-- 30 µg of nuclear proteins were separated on 10% SDS-polyacrylamide gels and transferred to Immobilon membranes (Millipore Corp., Bedford, MA) for 2 h at 200 mA in 25 mM Tris, pH 8.3, 0.19 M glycine, and 20% methanol. Blots were incubated for 1 h at room temperature in a blocking solution containing 5% nonfat dried milk in TBST (20 mM Tris, 140 mM NaCl, and 0.01% Tween 20), pH 7.5, followed by a 2-h incubation with an anti-RXRalpha polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted to 1:1000 in 5% milk in Tris-buffered saline (20 mM Tris and 140 mM NaCl). The blots were then washed three times for 10 min each in TBST and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G, Bio-Rad) diluted to 1:3000 in Tris-buffered saline with 5% milk. The blots were washed as described above and treated with LumiGlo (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Maryland) according to the manufacturer's specifications. Finally, the blots were exposed to Kodak XAR film at room temperature for 10 min.

Synthetic Oligonucleotides Used-- For gel shift experiments, the following synthetic oligonucleotides were used: mouse osteopontin (mOP) VDRE, 5'-GTACAAGGTTCACGAGGTTCACGTCTTA-3'; mutant mOP VDRE, 5'-GATCCGTACAAGGCCCACGAGGTTCACGTCTTA-3'; human osteocalcin VDRE, 5'-TTGGTGACTCACCGGGTGAACGGGGGCATT-3'; and thyroid hormone receptor response element (TRE) beta , 5'-GATCCTGGAGGTGACAGGAGGACAGCGATC-3'. For RT-PCR, the following synthetic oligonucleotides were used: VDR, 5'-ATGGCGGCCAGCACTTCCCTGCCTGAC-3' (upstream) and 5'-CTCCTCCTTCCGCTTCAGGATCATCTC-3' (downstream); RXRalpha , 5'-TGGCAAGGACCGGAACGAGAAT-3' (upstream) and 5'-TCCATAAGGAAGGTGTCAATGGG-3' (downstream); and RXRbeta , 5'-TGCGGGGACAGAAGCTCAGGCAAA-3' (upstream) and 5'-GTAGGTCTCCAGTGATGCATACAC-3' (downstream).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of 1,25-(OH)2D3 on Gene Transcription in HPK1A and HPK1Aras Cells-- To determine if the resistance of HPK1Aras cells to the growth inhibitory influence of 1,25-(OH)2D3 occurs at the transcriptional level, we transfected a reporter plasmid (mOP3) containing three repeats of the mouse osteopontin vitamin D response element cloned in front of a thymidine kinase-driven bacterial chloramphenicol acetyltransferase gene (Fig. 1). From the graph in Fig. 1, it was determined that the dose of 1,25-(OH)2D3 that gives the half-maximal transactivation effect in HPK1A cells was 8 × 10-9 M, whereas in HPK1Aras cells, that dose was ~10-fold higher at 6 × 10-8 M. A growth inhibition curve was constructed (9), and these data correlate well with the transactivation data; half-maximal inhibition was observed at 0.2 × 10-9 M in HPK1A cells and at 0.2 × 10-8 M in HPK1Aras cells. Therefore, 1,25-(OH)2D3 is ~10-fold less effective in the growth inhibition and transactivation of HPK1Aras cells as compared with HPK1A cells.


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Fig. 1.   Resistance to 1,25-(OH)2D3 action in ras-transformed keratinocytes. Cells were transfected with the reporter plasmid as described under "Materials and Methods." Cells were then treated with or without increasing concentrations of 1,25-(OH)2D3. Chloramphenicol acetyltransferase (CAT) activity was assayed and normalized for transfection efficiency by the corresponding beta -galactosidase activity. Results are expressed as the means ± S.E. *, significant differences from untreated cells; °, significant difference from corresponding HPK1Aras cells.

Cloning and Sequencing of the cDNA Encoding VDR from HPK1A and HPK1Aras Cells-- We previously reported that both the number of vitamin D receptors and their affinities for ligand were not significantly different in HPK1A and HPK1Aras cells (9). We concluded that the hormone-binding domain was functionally intact. Therefore, we first analyzed the DNA-binding domain of VDR by cloning the VDR cDNA from HPK1A and HPK1Aras cells using RT-PCR. After sequencing the cloned DNAs, we determined that sequences from both cell lines were identical to the known sequence of hVDR (data not shown).

Gel Mobility Shift Analysis of VDR·RXR Binding-- The ability of nuclear extracts derived from HPK1A and HPK1Aras cells to form specific VDR·RXR protein complexes interacting with known VDREs was next examined. We first determined that nuclear extracts from both HPK1A and HPK1Aras cells formed complexes with a 32P-labeled mOP VDRE probe (Fig. 2A). These complexes comigrated with those formed by control nuclear extracts (i.e. COS-7 cells cotransfected with plasmids encoding hVDR and hRXRalpha ). The addition of an anti-VDR antibody that targets the DNA-binding domain of VDR inhibited the formation of retarded complexes in all nuclear extracts. The extracts were also incubated with an anti-RXR antibody that recognizes the D- and E-domains of RXR and normally retards (supershifts) any complexes containing RXR. A supershift was evident with control COS-7 cell extracts as well as with extracts prepared from immortalized HPK1A cells. These results contrasted sharply with the absence of a supershift with HPK1Aras nuclear extracts in the presence of an anti-RXR antibody. Similar results were also observed using an osteocalcin VDRE as the labeled probe under the same conditions (data not shown).


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Fig. 2.   Gel shift analysis of VDR·RXR complex formation. A, a mOP VDRE was 32P-labeled and incubated with 2 µg of control extracts (COS-7 cells transfected with hVDR and hRXRalpha ) (lanes 3-5), HPK1A nuclear extracts (lanes 7-9), or HPK1Aras nuclear extracts (lanes 11-13), each in the presence or absence of either anti-VDR antibodies (alpha  VDR Ab) or anti-RXR antibodies (alpha  RXR Ab). The diamond indicates the presence of putative VDR·RXR complexes. The circle indicates the presence of supershifted complexes containing RXR. A mutant mOP VDRE was 32P-labeled and incubated with control COS-7 extracts (lane 2), HPK1A nuclear extracts (lane 6), and HPK1Aras nuclear extracts (lane 10). No VDR·RXR complexes were formed on this mutant probe. B, as an additional control, the labeled mOP VDRE was incubated with control COS-7 extracts (lanes 1-3), HPK1A nuclear extracts (lanes 4-6), and HPK1Aras nuclear extracts (lanes 7-9) in the presence of either 1 pg of unlabeled mutant mOP VDRE (Cold mut OP) or 1 pg of unlabeled mOP VDRE (Cold OP). The unlabeled mutant mOP VDRE did not affect VDR·RXR complex formation with the labeled mOP VDRE, whereas the unlabeled mOP VDRE competed out the binding of the VDR·RXR complex to the labeled mOP VDRE. C, the identification of VDR in the complexes formed from HPK1Aras nuclear extracts was confirmed by the addition of increasing concentrations of baculovirus-expressed hVDR to reactions. As noted, in the presence of the labeled mOP VDRE, nuclear extracts from HPK1A cells (lanes 6 and 7) and from HPK1Aras cells (lanes 1-4) both formed VDR complexes with dose-dependent increases when incubated with increasing concentrations of hVDR.

To verify the presence of VDR in the complexes formed by HPK1Aras nuclear extracts, purified hVDR expressed in baculovirus was added in increasing concentrations to HPK1Aras and HPK1A nuclear extracts (Fig. 2C). When hVDR was added to HPK1A or HPK1Aras nuclear extracts, a VDR-containing complex was formed on the VDRE with a dose-dependent increase. Consistent with the amount of starting material (2 µg of HPK1A and 1 µg of HPK1Aras nuclear extracts) used in this particular experiment, a lesser base-line complex formation was observed with HPK1Aras extracts (lane 1) as compared with HPK1A extracts (lane 6) in the absence of supplementation with hVDR. However, with the addition of 2 pmol of hVDR to the respective nuclear extracts, a similar 3-fold increase in the VDR-containing complex was observed with both the HPK1A extracts (lane 7) and the HPK1Aras extracts (lane 3). This dose-dependent increase in complex formation confirmed the presence of VDR in nuclear extracts prepared from HPK1Aras cells.

The specificity of complex formation was assessed through the competition of a 32P-labeled mOP probe with either an unlabeled mOP probe or an unlabeled mutant mOP probe (Fig. 2B). The protein·DNA complex was competed out by the unlabeled mOP probe; however, the mutant version of this probe left the complex unaffected in all cell extracts.

We next investigated the possibility that the level of RXR mRNA expression in HPK1Aras cells was low or absent. RT-PCR analysis confirmed the presence of RXRalpha message in both HPK1A and HPK1Aras cells and the absence of RXRbeta message in both of these cell lines (Fig. 3A). Furthermore, Northern blot analysis revealed an equal amount of RXRalpha mRNA in both of these cell lines (Fig. 3B).


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Fig. 3.   Analysis of RXRalpha and RXRbeta mRNAs. A, RNAs from HPK1A and HPK1Aras cells were prepared and used in RT-PCRs using primers designed to specifically amplify either RXRalpha (lanes 1-4) or RXRbeta (lanes 5-7). Positive controls were DNA plasmids containing either hRXRalpha (lane 2) or hRXRbeta (lane 5). Negative controls were carried out in the absence of either DNA or RNA (lane 8). PCR products were resolved on an ethidium bromide-stained 1.5% agarose gel with phi X174 HaeI DNA markers (lane 1). B, Northern blot analysis of RXRalpha was carried out using RNAs from HPK1A and HPK1Aras cells. Filters were hybridized with a 32P-labeled hRXRalpha probe. kb, kilobases.

We then examined the possibility that an inhibitory factor present only in HPK1Aras cells could prevent the formation of VDR·RXR complexes. To test this hypothesis, HPK1Aras nuclear extracts were supplemented with COS-7 cell extracts enriched with RXRalpha (Fig. 4A). The supershifted band in the presence of the anti-RXR antibody was restored (lane 6) with the addition of 1 µg of COS-7 extracts to the HPK1Aras extract. A dose-dependent increase in the intensity of this supershifted band was observed with the addition of increasing amounts of COS-7 extract to the HPK1Aras nuclear extract (data not shown).


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Fig. 4.   Rescue of VDR·RXR complexes in HPK1Aras nuclear extracts. A, HPK1A and HPK1Aras nuclear extracts incubated with a 32P-labeled mOP VDRE in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of an anti-RXRalpha antibody (alpha  RXR Ab). HPK1Aras nuclear extracts were supplemented with COS-7 cell extracts overexpressing hRXRalpha (COS-RXR; (lanes 5 and 6) before incubation with a 32P-labeled mOP VDRE oligonucleotide. Control reactions consisted of hRXRalpha -overexpressing COS-7 cell extracts incubated with or without the anti-RXRalpha antibody (lanes 7 and 8). B, extracts from HPK1Aras cells (lane 1) and from HPK1Aras cells transfected with a hRXRalpha plasmid (HPK1Aras-RXR; lanes 2 and 3) with or without the anti-RXRalpha antibody. Diamonds indicate the presence of VDR·RXR complexes; circles indicate the presence of supershifted complexes containing RXR.

In Fig. 2A, it was shown that the complex formed with the hRXRalpha -transfected COS-7 extracts (lane 3) was of similar intensity to the complex formed with the same amount of HPK1Aras nuclear extract (lane 11). In Fig. 4B, hRXRalpha was transiently transfected into HPK1Aras cells. For the transfection of both HPK1Aras and COS-7 cells, the same conditions were used as described under "Materials and Methods." However, the transfection efficiency of HPK1Aras cells was found to be 5-fold lower than that of COS-7 cells (determined through cotransfection with beta -galactosidase). The complexes formed in HPK1Aras cells were recognized by the anti-RXR antibody (lane 3), implying that such expression levels of wild-type hRXRalpha in HPK1Aras cells overcame the defect observed with RXR in these cells.

Finally, we examined the interaction of RXR with another dimerization partner, the thyroid hormone receptor (TR). To this effect, we analyzed the ability of TR to bind to its DNA recognition sequence or thyroid hormone response element (TRE) (Fig. 5). Results with a 32P-labeled TRE probe were similar to those observed with a VDRE. HPK1A and HPK1Aras cells formed complexes that comigrated in gel retardation assays. However, the complexes formed by HPK1Aras cells were not recognized by the anti-RXR antibody, such that no supershift was observed with this antibody. Overexpressing hRXRalpha in HPK1Aras cells restored the ability of the anti-RXR antibody to supershift complexes as previously observed with VDREs (Fig. 5).


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Fig. 5.   Gel shift analysis of RXR binding to a TRE. A TREbeta was labeled and incubated with nuclear extracts prepared from HPK1A cells (lanes 2 and 3), HPK1Aras cells (lanes 4 and 5), or HPK1Aras cells overexpressing hRXRalpha (HPK1Aras-RXR; lanes 6 and 7) in the presence (+) or absence (-) of the anti-RXRalpha antibody (alpha  RXR Ab) or a control preimmune IgG (lane 7). The diamond indicates the presence of TR·RXR complexes. The circle indicates the presence of supershifted complexes containing RXR.

Cloning and Sequencing of the cDNA Encoding hRXRalpha from HPK1A and HPK1Aras Cells-- The C-terminal domain of the RXRalpha cDNA derived from HPK1A and HPK1Aras cells was amplified and cloned into a sequencing vector. This area, from amino acids 706 to 1426, comprises the D- and E-domains of the receptor, domains that are responsible for ligand binding, but that also play a critical role in transactivation and dimerization (6, 12). No genetic alterations were found in the RXRalpha cDNA from HPK1Aras cells (data not shown).

Western Blot Analysis of RXRalpha -- Western blot analysis was performed using nuclear extracts prepared from HPK1A, HPK1Aras, and COS-7 cells transfected with hRXRalpha (Fig. 6). An antibody specific for hRXRalpha revealed a single protein complex in the control COS-7 extracts. A single protein complex was also observed in the HPK1A and HPK1Aras extracts (Fig. 6). No protein complex was observed in the presence of preimmune IgG.


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Fig. 6.   Western blot analysis of RXRalpha protein in HPK1Aras cells. COS-7 cell extracts overexpressing hRXRalpha (COS-RXR), HPK1A nuclear extracts, and HPK1Aras nuclear extracts were analyzed by Western blotting. The membranes were incubated either with a preimmune IgG for control purposes or with an anti-RXRalpha antibody (alpha  RXR Ab). The arrow indicates the presence of RXRalpha protein in each of the three types of extract as detected by the anti-RXRalpha antibody.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cultured keratinocytes have been shown to hydroxylate 25-(OH)D3 to its active metabolite, 1,25-(OH)2D3 (13), and its receptor (the vitamin D receptor) has been identified in skin and in keratinocyte cultures (14). It has therefore been proposed that 1,25-(OH)2D3 acts in an autocrine fashion to suppress growth and to stimulate the differentiation of keratinocytes, making it a crucial player in keratinocyte cell biology.

In this study, we first demonstrated that the vitamin D resistance observed in ras-transformed keratinocytes occurs at the level of gene transcription. Transfection experiments using a mOP VDRE attached to a chloramphenicol acetyltransferase reporter plasmid also supported the hypothesis that resistance in HPK1Aras cells is due to a disruption in the VDR signaling pathway. VDR isolated from HPK1Aras cells was present in amounts equal to those found in HPK1A cells and interacted normally with its ligand, 1,25-dihydroxyvitamin D3 (9). Consequently, we focused our attention on the DNA-binding domain of VDR.

To date, several mutations have been found in this region in the clinical condition described as type II vitamin D-resistant rickets (15, 16). The consequence of these mutations is a nonfunctional VDR, unable to modulate gene transcription. Our present study indicated that no such mutations are found in the VDR DNA-binding domain cloned from HPK1Aras cells. Furthermore, several clones were sequenced, and we ruled out the possibility of mutation in only one allele. Subsequently, we focused our attention on the interaction between VDR, RXR, and target DNA response elements.

Using gel mobility shift assays, we demonstrated that nuclear extracts prepared from HPK1A and HPK1Aras cells and control COS-7 cells enriched with VDR and RXR bind to a VDRE in a similar fashion. The addition of an anti-VDR antibody that targets the DNA-binding domain of the receptor confirmed the presence of VDR in the complex observed on gel retardation assays using nuclear extracts derived from both established and transformed keratinocytes. Further confirmation of the presence of VDR was obtained by the addition of exogenous baculovirus-produced hVDR to nuclear extracts. From these studies, we can therefore conclude that in HPK1Aras cells, VDR is present in the protein complex that retards a VDRE probe.

Recently, the dimerization partner of VDR was identified in gel retardation assays as RXR (7), and at least in vitro, RXR is required for VDR binding to a VDRE (16). Using an antibody that recognizes all three major types of RXR (alpha , beta , and gamma ), we determined that RXR was present in the VDR-containing complexes from control and HPK1A nuclear extracts. However, the same antibody did not bind to the protein complex obtained from ras-transformed keratinocytes. It is therefore possible that RXR is not present in VDRE-binding complexes formed with HPK1Aras nuclear extracts.

Much of the work supporting the notion that VDR has a weak affinity for a VDRE in the absence of RXR was executed using either bacterially expressed or yeast-expressed vitamin D receptors (16). However, this notion is still disputed, and some groups have proposed that VDR can indeed form homodimers and effectively bind to VDREs in the absence of RXR (17-19). Our present studies seem to indicate the lack of RXR in HPK1Aras complexes, but do not rule out the possibility that another, as of yet unidentified protein can participate in VDR binding to a VDRE. Further investigations are required to clarify this issue.

To further clarify RXR interaction in HPK1Aras cells, we next analyzed its interaction with another well characterized dimerization partner, TR (7). Our hypothesis that RXR interaction with this receptor was abnormal in ras-transformed keratinocytes was confirmed by the absence of a supershift in the presence of the anti-RXR antibody. As TR has been clearly shown to form transcriptionally active homodimers in vitro (20), it is not clear what the physiological implications are with respect to the absence of RXR. Further work must be done on HPK1Aras cells to identify any biological consequences contributed by the lack of RXR involvement in thyroid receptor heterodimerization.

Another potential mechanism of vitamin D resistance is the presence of interfering proteins preventing VDR·RXR interaction with VDREs. Such inhibitory accessory proteins have been previously identified for the thyroid hormone receptor (21, 22), and vitamin D-resistant New World primate cells were recently reported to contain a protein or complex of proteins capable of inhibiting VDR·RXR heterodimer binding to a VDRE (23). Consequently, we tested this possibility in our system by adding COS-7 extracts enriched with hRXRalpha to HPK1Aras nuclear extracts and observed heterodimer formation and binding to a VDRE. We observed that the same VDR-containing band was present, but also that the addition of this exogenous hRXRalpha rescued the supershift of this complex following the addition of the anti-RXR antibody. A similar rescue phenomenon was observed in gel shift experiments using nuclear extracts of HPK1Aras cells transfected with a plasmid overexpressing hRXRalpha . The presence of the expected VDR·RXR complex in these experiments ruled out the presence of an inhibitory protein in the HPK1Aras extracts. Similar results obtained using a TRE-labeled probe therefore indicate that the addition of exogenous hRXRalpha is sufficient to form both VDR·RXR and TR·RXR heterodimers in HPK1Aras cells.

RT-PCR analysis of RNA extracted from both HPK1A and HPK1Aras cells revealed that the predominant form of RXR expressed in these cell lines is RXRalpha . Although RXRbeta has been shown to increase and facilitate vitamin D receptor binding, it is RXRalpha that has the greatest effect on vitamin D-dependent transcription (24). The importance of RXRalpha in skin biology is underscored by previous reports that claim that it is the predominant form of RXR in skin and cultured normal human keratinocytes (25, 26). Northern blot analysis of RXRalpha message revealed that this receptor is expressed in equal amounts in both HPK1A and HPK1Aras cells, therefore eliminating low levels of RXRalpha expression as one potential mechanism of vitamin D resistance.

Another distinct possibility to explain the inability of RXRalpha obtained from HPK1Aras cells to heterodimerize with VDR could be explained by genetic alteration of the RXRalpha dimerization domain. We investigated this possibility by amplifying, cloning, and sequencing the D- and E-domains of RXRalpha . These regions contain functional domains responsible for ligand binding, transactivation, and dimerization (6, 12). No mutations in these regions were found.

Finally, we analyzed RXRalpha protein expression by Western blot analysis. Our data indicate that RXRalpha protein is expressed equally in both HPK1A and HPK1Aras cells. However, our present study did not exclude the possibility of post-translational modifications of the RXR protein in HPK1Aras cells. It should be noted that almost all nuclear receptors are known to be phosphoproteins, and an altered phosphorylation state of RXRalpha expressed in HPK1Aras cells as compared with RXRalpha from HPK1A cells could be responsible for the inability of RXRalpha to form complexes with the VDRE and TRE. In Fig. 4B, the lower intensity of the supershifted band of the hRXRalpha -enriched HPK1Aras nuclear extracts (lane 3) as compared with that of COS-7 extracts (Fig. 2A, lane 5) was likely mostly attributable to the 5-fold lower transfection efficiency relative to COS-7 cells. However, we cannot rule out that if post-translational modifications were occurring with endogenous RXR from HPK1Aras cells, hRXRalpha transfected in these cells was not also affected in this manner. This mechanism would also account for the less intense supershifted band recovered in these cells. Further studies are necessary to determine if this mechanism does indeed operate in ras-transformed keratinocytes.

Recently, it has been established that nuclear receptors transactivate their target genes by associating with several coactivator or corepressor proteins (27). Some of these coactivators, including SRC-1A (steroid receptor coactivator-1A), have been shown to possess an intrinsic histone acetyltransferase activity (27). SRC-1A binds to RXRalpha and enhances in vivo transcription by this nuclear receptor (29). It is thought that since RXR recruits SRC-1A to the receptor complex, this coactivator could increase the accessibility of response elements within the promoters of the target genes to the transcriptional machinery by virtue of its histone acetyltransferase activity. In several cancer cell lines, mutations causing over- or underexpression of such coactivators and corepressors have been implicated in tumor progression (28). Deregulated quantities of these proteins could greatly affect the transactivation of target genes normally regulated by the VDR·RXR nuclear receptor complex and could account for the partial "resistance" to 1,25-(OH)2D3 observed in the HPK1Aras cells. Further studies on these coactivators and corepressors are required to determine if their over- or underproduction contributes in any way to the resistance to 1,25-(OH)2D3 encountered in the HPK1Aras cells.

This study strongly suggests a causal relationship between resistance to vitamin D action and disruption of the VDR·RXR complex in ras-transformed keratinocytes. The crucial role of RXR in modulating VDR function is likely to expand our knowledge as to how malignant cells can evade normal growth-arresting signals.

    ACKNOWLEDGEMENTS

We thank V. Papavasiliou, P. Harakidas, I. Bolivar, H. Kubba, and K. Ammerman for excellent technical assistance and Drs. S. Rabbani and K. Meerovitch for helpful discussion.

    FOOTNOTES

* This work was supported by Grant MT10839 from the Medical Research Council of Canada. A preliminary report in abstract form was presented at the 8th Annual Meeting of the American Association for Cancer Research, Washington, D. C., April 22, 1996.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Calcium Research Lab., Royal Victoria Hospital, Rm. H4.67, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712.

1 The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; hVDR, human vitamin D receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; hRXRalpha ; human retinoid X receptor-alpha ; RT-PCR, reverse transcriptase-polymerase chain reaction; mOP, mouse osteopontin; TR, thyroid hormone receptor; TRE, thyroid hormone receptor response element.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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