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(Received for publication, August 30, 1995; and in revised form, November 14, 1995) From the
All-trans-retinoic acid (RA) markedly reduced the level
of intracellular fibronectin (FN) in a time- and
concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3
cells transformed by an activated Ha-ras oncogene. Pulse/chase
experiments indicated that RA affects FN biosynthesis rather than its
turnover rate. Steady state levels of FN transcripts did not change
after treatment of the cells with RA for various times or
concentrations, suggesting that RA acts at the translational level.
Similar effects were observed in other fibroblasts. In NIH-3T3
cells, RA had distinct effects on different receptors; it
down-modulated retinoic acid receptor (RAR) These studies have defined fibronectin
and RAR
All-trans- and 9-cis-retinoic acid are the
mediators of vitamin A action on growth and differentiation of normal,
premalignant, and malignant cells(1, 2) . Their
effects are mediated by two classes of nuclear receptors, the RARs ( FN is a large transformation-sensitive glycoprotein
composed of two non identical subunits of 220 kDa. It exists in the
extracellular matrix and in soluble form in the plasma. Cellular FN is
produced in large amounts by fibroblasts and is implicated in a wide
range of cellular processes including cell adhesion, migration,
morphology, differentiation, and
transformation(14, 15) . It is modulated by a variety
of effectors including cytokines like transforming growth factor The ras genes encodes a 21-kDa plasma membrane protein that binds guanine
nucleotides and is involved in signal transduction, cell growth, and
differentiation(18) . Many types of tumors express mutated
forms of the ras protein, resulting in constitutive activation
of this protein and altered gene expression(18) . In this
study, we identified fibronectin as target of RA action in NIH-3T3
cells but not in ras-transformed fibroblasts. We also
identified retinoid receptors involved in this process.
For the detection of RAR
Figure 1:
Regulation of intracellular FN protein
levels by RA in pSVneo and Ha-ras NIH-3T3 cells. A,
subconfluent cultures were treated for 24 h with 2 µM RA.
Cell monolayers were trypsinized to remove extracellular and cell
surface-associated proteins and lysed. Lysates were examined by
immunoblot analysis using specific polyclonal antibodies as under
``Experimental Procedures.'' B, subconfluent
fibroblasts were treated with 2 µM RA and examined for
intracellular FN. C, fibroblasts were treated for 48 h with
the indicated concentrations of RA. Intracellular FN was determined as
described above.
Figure 2:
Effect of RA on newly synthesized, labeled
FN in NIH-3T3 cells. A, subconfluent fibroblasts on 35-mm
dishes were treated with 2 µM RA for the indicated times.
Cells were pulsed with 80 µCi/ml
[
In pulse/chase experiments, the
rate of disappearance of labeled FN was not altered by RA treatment of
the cells (Fig. 2B). The time required to reduce the
amount of Northern blots of total cellular RNA from RA-treated and control
cells were hybridized to FN and GAPDH probes. The FN mRNA band
intensities, which represent relative steady-state level, were
normalized to GAPDH mRNA band intensities. The results (Fig. 3)
reveal that the accumulated levels of FN mRNA were not altered either
by different RA concentration or by times of RA treatment. RA failed to
alter FN mRNA in Ha-ras NIH-3T3 cells.
Figure 3:
Effect of RA on FN mRNA levels in pSVneo
and Ha-ras NIH-3T3 cells. Subconfluent cultures were treated
with 2 µM RA for 48 h (upper panels) or with the
indicated RA concentrations for 48 h (lower left panel) or
with 2 µM RA for the indicated times. Total RNA was
fractionated, transferred onto nitrocellulose, and hybridized to
RAR
Figure 4:
Effect of RA on the expression of RARs in
pSVneo and Ha-ras NIH-3T3 cells. A, effect of RA on
the expression of RAR
Figure 5:
FN
inhibition by RA in fibroblasts. NIH-3T3, C3H10T1/2, and primary mouse
skin fibroblast cells were treated with 3 µM RA for 24 h.
RAR
Figure 6:
Suppression of RA-induced transactivation
in NIH-3T3 cells by overexpression of the RAR dominant negative mutant,
RAR
RA failed to down-regulate the intracellular FN levels in the
LXRAR
Figure 7:
Role
of RARs in RA inhibition of RAR
Similar experiments were
performed, looking at the RA modulation of RAR
Figure 8:
Effect of RA on FN synthesis in NIH-3T3
cells overexpressing RAR
Figure 9:
Effect of RA on FN in Ha-ras NIH-3T3 overexpressing lXRAR
We have identified FN as a molecule whose biosynthesis is
down-regulated by RA in normal, but not in Ha-ras-transformed
NIH-3T3 cells. The inhibition of FN biosynthesis by RA is specific, as
neither collagen type IV nor laminin were affected by RA, and is RA
dose- and time-dependent. Two lines of evidence suggest that RA acts on
FN at a post-transcriptional level. First, the rate of newly
synthesized intracellular FN is reduced by RA treatment, an event not
due to an increased FN turnover rate. Second, RA did not alter the
levels of FN transcripts, consistent with the absence of RARE or RXRE
in the promoter region of the FN gene(37, 38) .
Effects of RA in other cell systems have been reported. FN mRNA and
protein levels were increased in primary hepatocytes from vitamin
A-deficient rats, while RA treatment caused a reduction of FN mRNA and
protein levels(39) . In C3H10T1/2 fibroblast cells, a complex
RA-dependent regulation of FN was observed as the cell surface levels
increased, while intracellular FN and FN mRNA decreased after
RA(19) . RA generally controls gene expression at the
transcriptional level. However, lipoprotein lipase enzyme expression in
3T3-L1 adipocytes was down-regulated, but mRNA levels were not affected
by RA treatment (40) . RA induction of differentiation of F9
cells is achieved by controlling the expression of various genes. Early
responsive genes are thought to be transcriptionally regulated, while
late responsive genes may be controlled both at the transcriptional and
post-transcriptional levels(41) . Laminin biosynthesis is
switched on, while synthesis and secretion of FN is switched off by RA
treatment of F9 cells (42, 43, 44) . Transformation of NIH-3T3 cells with an activated ras caused RAR RAR This work also shows that the
RA-dependent down-modulation of intracellular FN appears to be
RAR-mediated. The introduction of a mutated RAR In our RAR Overexpression of
the RAR
Volume 271,
Number 11,
Issue of March 15, 1996 pp. 6502-6508
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteins in NIH-3T3 Cells
BLOCK OF THIS RESPONSE BY ras TRANSFORMATION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
protein and
transcript levels, it up-regulated RAR
transcripts, and it had no
effect on RAR. Transformation of NIH-3T3 cells with an activated
Ha-ras oncogene down-modulated RAR expression and abolished
responsiveness to RA. We identified the retinoid signal transduction
pathways responsible for the effects of RA on FN and RAR
proteins
by the use of the retinoid X receptor-selective compound, SR11237, by
stable overexpression of a truncated form of the RAR gene,
RAR403, with strong RAR dominant negative activity, and by
overexpression of RAR. We conclude that: 1) RA-dependent FN
down-modulation is mediated by RARs, 2) retinoid X receptors mediate
the observed reduction of RAR by RA, and 3) the block of RA
responsiveness in Ha-ras cells cannot be overcome by
overexpression of RAR. as targets of RA in fibroblast cells and have shown that
oncogenic transformation renders the cells resistant to RA action.
)and RXRs, for which they function as respective ligands.
Distinct RAR- and RXR-dependent gene pathways exist, and individual
receptor subtypes may control distinct gene expression patterns by
interacting with RAREs, or RXREs, in the promoter region of different
responsive genes(3, 4) . RA has proven effective in
differentiation therapy of acute promyelocitic leukemia, a disease
characterized by a t(15;17) translocation with breakpoint in the
RAR gene(5) . In vitro, overexpression of
RAR has been shown to suppress transformation by v-myb in
monoblasts (6) and by polyoma virus in rat
fibroblasts(7, 8) . In addition RA treatment of
NIH-3T3 cells transformed by the introduction of an activated
Ha-ras oncogene inhibited focus formation(9) . Various
reports have shown opposing effects of RA and ras on the
regulation of the expression of different
genes(10, 11, 12, 13) , suggesting
that an interaction between the signal transduction pathways mediated
by RA and ras may take place. Therefore, studies of this
interaction may provide insight into the mechanism whereby RA inhibits
transformations.
and hormones like glucorticoids and RA. Loss of cell surface FN is a
hallmark of transformation, and it has been correlated with acquisition
of tumorigenic and metastatic potential. This effect has been observed
with many oncogenic stimuli, among which are the ras oncogenes(14, 16, 17) .
Cell Culture
NIH-3T3 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10%
calf serum, 400 units/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine. Cells were grown to subconfluence and treated
with retinoids. Activated Ha-ras-transfected cells were
generated and cultured as described(10) . All comparative
studies were done with matched pair clones, i.e. all the cells
were always transfected with vector plus/minus the activated ras construct. Furthermore identical results were obtained in four
NIH-3T3 clones compared to their ras-transformed counterparts.
C3H10T1/2 cells were obtained from ATCC (Rockville, MD) and cultured as
described(19) . Primary mouse skin fibroblasts were isolated
and cultured as described(20) .Retinoids
RA was obtained from Sigma. The
RXR-selective compound, SR11237(21) , was kindly provided by
Dr. A. Levin (Hoffman La Roche). RA and SR11237 were dissolved in
Me
SO at 10 mM and in ethanol at 1 mM,
respectively. The maximum concentration of the solvent used was 0.03%
for MeSO and 0.1% for ethanol. Subconfluent fibroblasts
were incubated in presence of the media containing the required
retinoids, and these media were replaced daily. For the last 16 h the
cells were serum-starved by adding serum-free media containing 0.1%
bovine serum albumin, with or without the effectors.Metabolic Labeling and Immunoprecipitation
RA- and
MeSO-treated cells were washed with PBS and serum-free DMEM
containing 0.1% bovine serum albumin, and 10% of the normal level of
methionine and cysteine was added. Cells were preincubated for 1 h
before adding 80 µCi/ml
[S]methionine/cysteine protein labeling mixture
(DuPont NEN). After 20 min of incubation, the cell monolayers were
washed with cold PBS and lysed with buffer A (40 mM Tris-HCl,
pH 8.5, 100 mM NaCl, 2 mM EDTA, 0.5% sodium
deoxycholate, 1.0% Nonidet P-40, 0.5% SDS, 2 mM phenylmethylsulfonyl fluoride, and 10 µM leupeptin).
Cell lysates were centrifuged at 15,000
g 5
min at 4 °C, and the insoluble pellets were discarded. Volumes
containing equal amounts of radioactivity (precipitation with 10%
trichloroacetic acid) were subjected to immunoprecipitation. Polyclonal
rabbit mouse FN antibodies (5 µl) were added. After 2 h of
incubation at room temperature, 25 µl of prewashed Pansorbin
(Calbiochem, La Jolla, CA) was added and the samples were incubated
overnight at 4 °C on a rotary shaker. The immunoprecipitates were
washed three times with buffer B (20 mM Tris-HCl, pH 8.5, 150
mM NaCl, 0.5% sodium deoxycholate), solubilized in SDS loading
buffer, and loaded onto 4-15% polyacrylamide gels(22) .Pulse/Chase
The reduced level of newly synthesized
FN in RA-treated cells may be due to either a reduced rate of
biosynthesis or an increased turnover rate. To explore these
possibilities we performed pulse/chase experiments. Fibroblasts were
pulsed for 20 min with [S]methionine and
[
S]cysteine and chased with unlabeled amino
acids. At the indicated time points, the cells were washed twice with
PBS and treated with 10 µg/ml trypsin for 5 min to remove
extracellular and cell surface FN. The reaction was stopped by adding 2
mg/ml trypsin inhibitor. The cells were washed with cold PBS containing
2 mM phenylmethylsulfonyl fluoride, lysed in buffer B, and
analyzed for labeled FN by immunoprecipitation.
Immunoblot Analysis
To determine intracellular Fn
levels, retinoid- and MeSO-treated cells were washed and
trypsinized as described in the pulse/chase section. Cells were lysed
in Laemmli buffer without reducing agent and bromphenol blue. Lysates
were boiled for 5 min and centrifuged to get rid of insoluble cell
debris. Protein concentration was determined by the bicinchoninic acid
method (23) (Pierce). -Mercapthanol and saturated solution
bromphenol blue were added to the samples at 1% and 3% final
concentrations, respectively. Equal amounts of protein were then loaded
onto 4-15% polyacrylamide gels. The proteins were transferred to
nitrocellulose (Schleicher & Schuell) on a Bio-Rad electroblot
apparatus. The membranes were stained with Ponceau stain. The blots
were incubated overnight at 4 °C in 5% milk in TTBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). Rabbit
polyclonal mouse FN antibodies (Life Technologies, Inc.) were used at
1:1250 dilution in TTBS, and blots were incubated at room temperature
for 1 h. They were rinsed four times with TTBS, and incubated 1 h with
horseradish peroxidase-labeled secondary antibodies (anti-rabbit IgG,
Amersham Corp.) at 1:5000 dilution in TTBS. The blots were washed five
times before determining immunoreactivity by a chemiluminescent method
using the ECL Western blotting system (Amersham). Rabbit polyclonal
mouse collagen type IV and laminin antibodies were from Becton
Dickinson (Bedford, MA). and
proteins, total SDS lysates were prepared from cells washed twice with
ice-cold PBS, as described above, except for the trypsin treatment.
Proteins were boiled and run immediately on 10% polyacrylamide gels.
They were transferred to nitrocellulose membranes which were then
blocked overnight in 5% milk in TBS (50 mM Tris-HCl, 150
mM NaCl). Polyclonal antibodies against the carboxyl termini
of RARs from Dr. Chambon's laboratory (24, 25) were diluted 1:1000 in 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1% bovine serum albumin,
and the membranes were incubated at room temperature for 2 h. The blots
were rinsed and washed five times in buffer C (50 mM Tris-HCl,
500 mM NaCl, 0.1% Tween 20) and incubated at room temperature
for 1 h in 5% nonfat dry milk in PBS containing a 1:3000 dilution of
the horseradish peroxidase-labeled IgG. The final wash sequence was
three washes with buffer C and two washes with buffer D (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% Tween
20)(26) . Blots were developed with ECL.
Stable Transfection
For stable transfection of
RAR cDNA, the fibroblasts were seeded at 6 10
cells/100-mm dish 24 h before transfection in regular DMEM
medium. Ten µg of the expression plasmid pSG5-RARf (27) and 1 µg of the dominant selective vector pSVneo (28) were cotransfected using the Lipofectamine (Life
Technologies, Inc.) methods according to the manufacturer's
instructions. Control cells were transfected with 10 µg of pSG5
empty vector and 1 µg of pSVneo vector. G418-resistant colonies
were selected in medium containing 1 mg/ml G418, and positive clones
(overexpressing RAR protein) were isolated and expanded.Northern Blot Analysis
Isolation of total RNA was
performed by using a Total RNA isolation kit (Tel-Test ``b''
Inc., Friendswood, TX). The full-length fragments of the mouse RARs
were excised from the expression plasmid
pSG5-RAR(29, 30) . The 1.4-kilobase human Fn fragment
was obtained from Life Technologies, Inc. The probes were labeled with
[P]dCTP using random primer labeling methods.
Total RNA (40 µg) was fractionated on a 1% agarose gel and blotted
overnight onto Schleicher & Schuell nitrocellulose. The membranes
were prehybridized for 2 h at 65 °C in a buffer of 5
SSC,
pH 7.0, 5 Denhardt's, 0.05 M sodium phosphate,
pH 6.8, 0.1% SDS, 5 mM EDTA, 20 µg/ml poly(A)
,
0.2 mg/ml denatured salmon sperm DNA, and 0.1 mg/ml denatured torula
yeast RNA. The probes (2 10
cpm/ml) were boiled and
added to the hybridization buffer (5 SSC, 1
Denhardt's solution, 0.02 M sodium phosphate, 5 mM EDTA, 0.2% SDS, 20 µg/ml poly(A), 0.2 mg/ml
denatured sperm DNA, and 0.1 mg/ml denatured yeast RNA. The membranes
were hybridized for 18 h at 65 °C, followed by two washes with 1
SSC, 0.1% SDS and two with 0.1 SSC, 0.15% SDS at 65
°C. Autoradiography on Kodak X-Omat AR film used double
intensifying screens. In order to reprobe the membranes with labeled
GAPDH, the blots were first stripped in 1% glycerol at 85 °C for 5
min. Densitometric evaluation of Northern blots was performed with NIH
Image 1.52 software (created by Wayne Rasband, National Institutes of
Health) running on a Macintosh Centris 650 using an XC-77/77CE CCD
video camera module.Infection of NIH-3T3 with Retroviral Vectors
The
retroviral vector LRAR403SN, in which a truncated RAR gene is
inserted into the retroviral vector LXSN, was a gift from Dr. S. J.
Collins(31, 32) . Cells were seeded at 50% confluence
into 100-mm dishes. The next day, they were infected with the LXSN and
LXRARSN retroviral vector in presence of 4 µg/ml
Polybrene(31) . After overnight incubation, the medium was
replaced and cells were grown for 36-48 h before G418 (1 mg/ml)
was added. G418-resistant cells were isolated.Transient Transfection with Constructs Containing
-RARE-tk-LUC-RARE-tk-LUC was a gift from Dr. S.
Minucci (National Institutes of Health)(33) . Fibroblasts (1.5
10/dish) were seeded in 35-mm dishes. After 24 h,
cells were cotransfected with 1 µg of the -RARE-tk-LUC and 1
µg of the cytomegalovirus--galactosidase reporter (34) using Lipofectamine reagent. Six hours post-transfection,
the cells were incubated in DMEM containing 10% serum for 16 h. The
cells were treated with 2 µM RA or solvent
(MeSO) for 24 h. Cells were lysed with reporter lysis
buffer (Promega, Madison, WI). Luciferase activity was determined in 1
µg of protein from each sample using the luciferase assay system
from Promega in a Monolight 2010 luminometer (Analytical Luminescence
Laboratory, San Diego, CA). -Galactosidase activity was quantified
by the FluoReporter LacZ/Galactosidase kit (Molecular Probes, Inc.,
Eugene, OR).
Inhibition of FN Biosynthesis by RA in NIH-3T3 but Not
in Ha-ras NIH-3T3 Cells
FN levels were markedly reduced by RA in
pSVneo, but not in Ha-ras-transfected cells (Fig. 1A). The effect was specific for FN, since
neither collagen type IV nor laminin B1 and B2 chains (the only ones to
be detected in NIH-3T3 cells) were affected. RA inhibition of FN was
time-dependent (Fig. 1B) and concentration-dependent (Fig. 1C). A 3-fold reduction of intracellular FN was
already observed after 12 h of treatment (Fig. 1B). RA
caused a 90% down-regulation at 2 10M.
Mechanism of RA Inhibition of Newly Synthesized
FN
RA-treated cells were metabolically labeled with a mixture of
[S]methionine/cysteine for 20 min, lysed, and
immunoprecipitated with specific anti-FN antibodies. The choice of the
labeling time was crucial because FN takes about 30 min to reach the
cell surface from the cytosolic site of synthesis (data not shown and (35) ). RA inhibited the newly synthesized FN in a
time-dependent fashion. An 80% reduction was observed after 4 h of RA
treatment (Fig. 2A).
S]methionine/cysteine protein labeling mixture,
and cell lysates were analyzed by immunoprecipitation as described
under ``Experimental Procedures.'' B, effect of RA
on the turnover rate of intracellular FN. Cells were treated with 2
mM RA for 3 h, pulsed with 100 µCi/ml
[
S]methionine/cysteine protein labeling mixture,
and chased for the indicated times with unlabeled amino acids. The
levels of intracellular labeled FN were determined by
immunoprecipitation as described under ``Experimental
Procedures.'' The experiment shown is representative of four
experiments.
S-FN by 50% was 28 ± 2 and 31 ± 3
min in RA- and Me
SO-treated cells, respectively. Therefore,
a reduction of FN biosynthetic rate likely accounts for the effects of
RA.
P-labeled probes for FN and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). The sizes of the detected mRNA were 9.1
and 1.6 kilobases for FN and GAPDH.
RA Modulation of RARs in pSVneo NIH-3T3 but Not in Ha-ras
NIH-3T3 Cells
In pSVneo NIH-3T3 cells, RAR transcripts were
constitutively expressed and slightly (30%) down-regulated by RA (data
not shown). RAR mRNAs were expressed to a lower extent than
RAR
mRNAs and were not substantially altered by RA. RAR
transcripts were induced in pSVneo cells to a similar extent (data not
shown) as in primary mouse skin fibroblasts(36) . In Ha-ras NIH-3T3 cells, the levels of RARs mRNA were generally lower than
in control cells and were not responsive to RA treatment.
protein levels were strongly down-regulated by RA in NIH-3T3 cells, but
not in Ha-ras cells (Fig. 4A). RAR
proteins were not altered by RA in either type of cells and could be
detected only after overexposing the blots (data not shown). We were
unable to detect RAR
proteins. The effect of RA on the levels of
RAR protein was time- and dose-dependent (Fig. 4B). A concentration of 0.1 nM RA was
sufficient to bring about a 80% inhibition of RAR protein, and a
40% inhibition could be already observed after 2 h of RA treatment.
protein levels. Subconfluent fibroblasts
were treated for 24 h with 3 µM RA. Total cellular protein
extracts were examined by immunoblot analysis using RAR mouse
polyclonal antibodies as described under ``Experimental
Procedures.'' A 2-h exposure detected RAR protein levels
(data not shown) as compared to 1 min for RAR
. B, time-
and concentration-dependent effect of RA on the expression of RAR
proteins. Subconfluent fibroblasts were treated for 24 h with RA (left panel) or for different times with 3 µM RA (right panel). Total cell proteins were analyzed for RAR
content by immunoblotting techniques as described
above.
RA Inhibition of FN and RAR
We investigated the effects of RA on FN and RAR Proteins in Fibroblast
Cells
proteins in C3H10T1/2 mouse embryo fibroblasts and in primary mouse
skin fibroblasts. RA markedly reduced the level of FN and RAR in
these two cells (Fig. 5).
proteins were determined by immunoblotting analysis as
described in Fig. 4A. Intracellular FN was determined
by immunoprecipitation analysis as in Fig. 2A.
Role of RAR Signal Transduction Pathways on RA Inhibition
of FN and RAR
SR11237, an RXR-selective compound,
specifically activates reporter genes fused to the RXR-responsive
element of the CRBPII promoter, to which only RXR-RXR homodimers bind,
and is unable to induce genes driven by an RAR-responsive element, like
the RARE of RAR Proteins and CRBPI(21) . The RAR-mediated signaling
pathways can be blocked by overexpressing a mutated form of the
RAR gene, RAR403, which shows strong dominant negative
activity(31, 32) . The truncated form of the RAR
gene, RAR403, inserted into the retroviral vector LXRARSN and
the corresponding control retrovirus LXSN were used. We infected
NIH-3T3 cells with these amphotrophic retroviruses containing the
neomycin resistance gene. Neomycin-resistant cells were isolated and
expanded. Northern blot analysis was performed to detect the expression
of the typical 4.7-kilobase retroviral transcripts containing the
RAR403 mRNA (Fig. 6). The dominant negative activity of
such mutated receptor was confirmed in RAR403 NIH-3T3 cells by
transient reporter assays. LXSN control and LXRAR403SN NIH-3T3
cells were transiently transfected with a vector carrying the
luciferase reporter gene fused to two copies of the RARE of RAR2
gene(33) , RA-dependent transactivation of luciferase was then
evaluated. Cells overexpressing RARa403 transcripts showed an almost
negligible 1.2-fold induction of luciferase activity in comparison to a
5-fold induction in control LXSN cells (Fig. 6B).
Similar data were obtained by others in tkNIH-3T3
cells infected with the very same retroviral vectors(31) .
403. A, RAR403 expression in
LXRAR403SN-infected cells. Total RNA from LXSN- and
LXRAR403SN-infected cells treated with 3 µM RA for 24
h were examined by Northern blot analysis using labeled RAR
probes. B, effect of RA on the transactivation of the
-RARE-tk-LUC construct in LXSN- and LXRAR403SN-infected
cells. After transfection, cells were treated with 3 µM RA
for 24 h and assayed for luciferase activities as described under
``Experimental Procedures.'' Results are representative of
four separate experiments. Relative luciferase activity is the rate of
activity in RA- versus MeSO-treated cells (after
correcting for transfection efficiency) in LXSN- and
LXRAR403SN-infected cells. 1, uninfected cells; 2, LXSN-infected cells; 3, LXRAR403SN-infected
cells.
403SN cells (Fig. 7B, left panel).
Furthermore, the RXR-selective compound, SR11237, also failed to reduce
FN protein (Fig. 7B, right panel). These
results indicate that an RAR-mediated signaling pathway likely accounts
for the RA-dependent FN down-regulation.
and FN proteins. A, left panel, effect of RA on RAR protein levels in LXSN
and LXRAR403SN-infected cells. Subconfluent cells were treated
with 3 µM RA for 24 h. Total cell extracts were analyzed
by immunoblotting for RAR protein. Right panel, effect of
SR11237 on the levels of RAR in NIH-3T3 cells. Cells were treated
with 2 µM RA, 1 µM SR11237, or the solvents
(control) for 24 h before determining the levels of RAR. B, right panel, effect of RA on intracellular FN in
LXSN and LXRAR403SN-infected cells. Subconfluent cultures were
treated with 2 µM RA for 48 h and intracellular FN was
determined as in Fig. 2A. Left panel, effect
of SR11237 on intracellular FN in NIH-3T3 cells. Fibroblasts were
exposed to 2 µM RA, 1 µM SR11237, or the
solvents (control) for 48 h, and intracellular FN was determined as
above.
(Fig. 7A). In LXRARSN NIH-3T3, treatment with RA
caused a marked reduction of the levels of RAR, ruling out the
involvement of RAR signal transduction pathways. This finding was
strengthened by the inhibition of RAR in cells treated with
SR11237 to a similar extent as that achieved by RA treatment (Fig. 7A, right panel). This suggests the
involvement of RXR in this effect.Effect of RA in NIH-3T3 Cells Overexpressing
RAR
To further investigate the role of RARs, we generated
cell lines overexpressing the RAR gene. Cotransfections of NIH-3T3
cells with the plasmid pSVneo, harboring the neomycin resistance gene,
and the pSG5RARaf vector, containing the full-length RAR gene,
were performed. G418-resistant clones were isolated, expanded, and
tested for RAR protein overexpression. Two representative clones
are shown in Fig. 8. Both express very high levels of RAR
protein, however, the levels of RAR were down-modulated by RA only
in clone 3A. Along the same trend, when FN sensitivity to RA was
determined, RA failed to reduce the level of FN in the resistant clone.
. Cells were co-transfected with
pSG5RARf and pSVneo plasmids. Neomycin-resistant clones, 13A and
3A, overexpressing RAR proteins were treated with 3 µM RA for 48 h. RAR protein levels and intracellular FN were
determined by immunoblot and immunoprecipitation analysis,
respectively, as described under ``Experimental
Procedures.''
Effect of RA on Ha-ras NIH-3T3 Cells Overexpressing
RAR
A similar series of experiments was
performed in Ha-ras NIH-3T3 cells. Since the levels of RARs
were generally lower than in control cells, we generated Ha-ras NIH-3T3 cell lines, which overexpressed RAR and RAR403. The RA effects
on FN biosynthesis were evaluated. Overexpression of RAR protein
was not sufficient to overcome the block on RA responsiveness (Fig. 9A). Similarly, disruption of the RAR signaling
pathways by the introduction of the dominant negative RAR construct,
RAR403, into Ha-ras NIH-3T3 cells, was ineffective in
altering the responsiveness of FN protein to RA.
403SN or RAR. A,
cells were transfected with pSG5RARf and pSVneo plasmids.
Neomycin-resistant colonies were isolated and treated with 2 µM RA for 48 h. RAR protein (upper panel) and
intracellular FN (lower panel) levels were determined as
described in Fig. 8. B, upper panel,
RAR403 expression in LXRAR403SN-infected cells. Total RNA
from LXSN- and LXRAR403SN-infected cells was analyzed by Northern
blot with radiolabeled RAR probes. Lower panel, effect of
RA on intracellular FN in LXSN- and LXRAR403SN-infected cells.
Subconfluent fibroblasts were treated with 3 µM RA for 48
h, and intracellular FN was determined as in Fig. 2A.
and to be expressed to lower levels than in
normal cells, while RA induction of RAR
was absent. In addition to
specific mutations in the RAR gene(5) , alteration of the
expression of RARs by transformation or in tumor derived cells has been
reported. In lung cancer cells, RAR and and RXRs were well
expressed; however, RA induction of RAR
was not
observed(45) . Estrogen receptor-negative human breast cancer
cells were insensitive to RA inhibition of cell growth, probably due to
a low level of RAR expression(46) . Primary keratinocytes
infected by v-ras showed lower levels of RAR
and proteins, which is accompanied by a reduction in RA-
induction of reporter genes fused to a RARE. (
)The molecular
mechanism of this down-regulation is not known yet, but it could have
important implications in explaining the variability in RA
responsiveness of different tumor cells. protein is
down-modulated by RA in a dose- and time-dependent fashion in NIH-3T3
cells. The modest inhibition (30%) of RAR mRNA is in agreement
with various reports in different cell lines and tissues, which,
however, had exclusively focused on the effect of RA at the level of
transcripts (36, 47, 48) . More recently,
RA-dependent down-modulation of RAR proteins was reported in
estrogen receptor-positive human breast cancer cells(46) .
Transcriptional regulation of retinoid receptors by RA has long been
known, and one of the first RARE was found in the promoter region of
the RAR gene(49) . gene, which has
strong dominant negative activity and blocks the RAR-mediated signaling
pathways, abolished the RA inhibition of FN. Furthermore the
RXR-selective compound, SR11237, was unable to mimic the action of RA
on the intracellular FN. We conclude that RAR down-modulation by
RA is likely mediated by RXRs, since RA reduces RAR levels, even
when we blocked RAR-mediated signaling pathways by the overexpression
of the RAR dominant negative gene, RAR403. In addition the
RXR-selective retinoid, SR11237, is as powerful as RA in
down-regulating RAR protein. overexpression
studies we isolated a clone, 13A, which appeared to be insensitive to
RA in that the two gene products, FN and RAR, were not responsive
to RA. The mechanism of the observed RA resistance is not clear. We
showed that action of RA on FN is mediated by RAR and is dependent on
RXR/RAR heterodimer, while RAR inhibition by RA is likely mediated
by RXR/RXR homodimer. The fact that RA resistance is observed for both
gene products argues against a defect localized entirely on the RAR
signaling pathway. If this were the case, RAR protein expression
should have remained sensitive to RA effects. Introduction of an
activated ras alters the responsiveness to RA and the level of
expression of RARs. Manipulation of RAR protein levels was utilized in
an attempt to correlate these two observations. gene in Ha-ras NIH-3T3 cells was not sufficient
to overcome the block in FN and RAR (data not shown)
responsiveness. Failure to regain RA sensitivity after constitutively
expressing RAR in lung cancer cells has been reported (45) . RA-dependent induction of a luciferase reporter gene
fused to the RARE of the RAR promoter was equally or more
efficient in Ha-ras NIH-3T3 than in normal cells (data not
shown). Caution should be used in evaluating the physiological
relevance of reporter gene assays, because the RARE is taken out of the
context of its natural promoter. Swisshelm et al.(50) showed that when a 1.5-kilobase region of the
RAR2 promoter was used in reporter gene constructs, instead of the
RARE, suppression of RA-induced activation was detected in human MCF-7
breast cancer cells. The observation that -RARE-tk-LUC can be
activated in Ha-ras NIH-3T3 cells suggests that necessary
factors for activating RARE are functional. The level of retinoid
receptor expression often does not correlate with RA-responsiveness.
RAR was expressed in most leukemia cells whether or not they were
responsive to RA(51, 52, 53) . In melanomas,
the level of RAR and RAR were similar in RA-sensitive and
-resistant cells (54) . These findings and the observed failure
of RAR
overexpression to confer RA responsiveness to Ha-ras cells suggests that other factors are required to mediate RA
action. These factors may be missing or not functional after ras transformation.
))
We thank Dr. Steven J. Collins for the retroviral
construct LRAR403SN and for reviewing the manuscript prior to
submission. We are also indebted to Drs. Keiko Ozato and Saverio
Minucci for their gift of constructs containing the
-RARE-tk-luciferase reporter gene. We thank Dr. Pierre
Chambon for the pSG5-RAR construct and the mouse-polyclonal RAR
antibodies.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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