(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) 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,
RAR
403, 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
.
These studies have defined fibronectin
and RAR as targets of RA in fibroblast cells and have shown that
oncogenic transformation renders the cells resistant to RA action.
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 ()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.
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
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) .
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 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.
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
[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.
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 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.
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 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.
RAR
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.
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 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.
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 proteins were determined by immunoblotting analysis as
described in Fig. 4A. Intracellular FN was determined
by immunoprecipitation analysis as in Fig. 2A.
Figure 6:
Suppression of RA-induced transactivation
in NIH-3T3 cells by overexpression of the RAR dominant negative mutant,
RAR403. A, RAR
403 expression in
LXRAR
403SN-infected cells. Total RNA from LXSN- and
LXRAR
403SN-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 LXRAR
403SN-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 Me
SO-treated cells (after
correcting for transfection efficiency) in LXSN- and
LXRAR
403SN-infected cells. 1, uninfected cells; 2, LXSN-infected cells; 3, LXRAR
403SN-infected
cells.
RA failed to down-regulate the intracellular FN levels in the
LXRAR403SN 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.
Figure 7:
Role
of RARs in RA inhibition of RAR and FN proteins. A, left panel, effect of RA on RAR
protein levels in LXSN
and LXRAR
403SN-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 LXRAR
403SN-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.
Similar experiments were
performed, looking at the RA modulation of RAR (Fig. 7A). In LXRAR
SN 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.
Figure 8:
Effect of RA on FN synthesis in NIH-3T3
cells overexpressing RAR. Cells were co-transfected with
pSG5RAR
f 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.''
Figure 9:
Effect of RA on FN in Ha-ras NIH-3T3 overexpressing lXRAR403SN or RAR
. A,
cells were transfected with pSG5RAR
f 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,
RAR
403 expression in LXRAR
403SN-infected cells. Total RNA
from LXSN- and LXRAR
403SN-infected cells was analyzed by Northern
blot with radiolabeled RAR
probes. Lower panel, effect of
RA on intracellular FN in LXSN- and LXRAR
403SN-infected cells.
Subconfluent fibroblasts were treated with 3 µM RA for 48
h, and intracellular FN was determined as in Fig. 2A.
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 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.
RAR 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) .
This work also shows that the
RA-dependent down-modulation of intracellular FN appears to be
RAR-mediated. The introduction of a mutated RAR 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, RAR
403. In addition the
RXR-selective retinoid, SR11237, is as powerful as RA in
down-regulating RAR
protein.
In our RAR 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.
Overexpression of
the RAR 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
RAR
2 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.