From the Departments of Medicine and
¶ Physiology, McGill University, Montreal, Quebec H3A 1A1, Canada
and the
Laboratory of Molecular Oncology, NCI-Frederick Cancer
Research and Development Center, National Institutes of Health,
Frederick, Maryland 21702
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ABSTRACT |
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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 RXR 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 RXR
message in equal amounts. Western blot analysis revealed
that RXR
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.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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
-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
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 (hRXR) and the
human vitamin D receptor (hVDR) (kind gifts of Drs. R. Evans and M. Haussler, respectively) or hRXR
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 RXR 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 RXR
and
RXR
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 RXR
and
RXR
. 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-RXR 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) ,
5'-GATCCTGGAGGTGACAGGAGGACAGCGATC-3'. For
RT-PCR, the following synthetic oligonucleotides were used: VDR,
5'-ATGGCGGCCAGCACTTCCCTGCCTGAC-3' (upstream) and
5'-CTCCTCCTTCCGCTTCAGGATCATCTC-3' (downstream); RXR
,
5'-TGGCAAGGACCGGAACGAGAAT-3' (upstream) and 5'-TCCATAAGGAAGGTGTCAATGGG-3' (downstream); and RXR
,
5'-TGCGGGGACAGAAGCTCAGGCAAA-3' (upstream) and
5'-GTAGGTCTCCAGTGATGCATACAC-3' (downstream).
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RESULTS |
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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 × 109 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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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 hRXR). 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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Cloning and Sequencing of the cDNA Encoding hRXR from HPK1A
and HPK1Aras Cells--
The C-terminal domain of the RXR
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 RXR
cDNA from HPK1Aras cells (data not
shown).
Western Blot Analysis of RXR--
Western blot analysis was
performed using nuclear extracts prepared from HPK1A,
HPK1Aras, and COS-7 cells transfected with hRXR
(Fig.
6). An antibody specific for hRXR
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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DISCUSSION |
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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 (,
, and
), 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 hRXR 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 hRXR
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 hRXR
. 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
hRXR
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 RXR. Although RXR
has been shown
to increase and facilitate vitamin D receptor binding, it is RXR
that has the greatest effect on vitamin D-dependent
transcription (24). The importance of RXR
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 RXR
message revealed that this receptor is
expressed in equal amounts in both HPK1A and HPK1Aras cells,
therefore eliminating low levels of RXR
expression as one potential
mechanism of vitamin D resistance.
Another distinct possibility to explain the inability of RXR
obtained from HPK1Aras cells to heterodimerize with VDR
could be explained by genetic alteration of the RXR
dimerization
domain. We investigated this possibility by amplifying, cloning, and
sequencing the D- and E-domains of RXR
. These regions contain
functional domains responsible for ligand binding, transactivation, and
dimerization (6, 12). No mutations in these regions were found.
Finally, we analyzed RXR protein expression by Western blot
analysis. Our data indicate that RXR
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 RXR
expressed in
HPK1Aras cells as compared with RXR
from HPK1A cells
could be responsible for the inability of RXR
to form complexes with
the VDRE and TRE. In Fig. 4B, the lower intensity of the
supershifted band of the hRXR
-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, hRXR
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
RXR 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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;
hRXR; human retinoid X receptor-
; RT-PCR, reverse
transcriptase-polymerase chain reaction; mOP, mouse osteopontin; TR,
thyroid hormone receptor; TRE, thyroid hormone receptor response
element.
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REFERENCES |
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