| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
The Burnham Institute Cancer Center, La Jolla, California 92037
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, ß, and 
). Recent studies have demonstrated that RARß plays
a critical role in mediating anticancer effects of retinoids. However,
how RARß exerts its potent anticancer effects remains largely
unknown. In this study, we investigated anti-Activator Protein-1 (AP-1)
activity of RARß. In a transient transfection assay, all three RAR
subtypes, RAR, RARß, and RAR, could effectively inhibit phorbol
ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1
activity and the activity of oncogenes c-Jun and
c-Fos on AP-1 containing reporter genes in the presence
of retinoic acid (RA). However, RARß showed a strong RA-independent
inhibition of AP-1 activity, whereas inhibition of AP-1 activity by
RAR and RAR was RA dependent. By using several hybrid receptors
that contain either the COOH-terminal portion or the
NH2-terminal portion of RARß, we demonstrated that the
NH2-terminal portion of RARß, the A/B domain, was mainly
responsible for the RA-independent inhibition of AP-1 activity. This
activity was not attributable to constitutive AF-1 activity of RARß,
because it did not activate several RA response element-containing
reporter genes. In addition, inhibition of histone deacetylase activity
by trichostatin A did not overcome the inhibitory effect of RARß. In
cancer cells, stable transfection of RARß exhibited strong inhibition
of AP-1 activity, even in the absence of RA. Moreover, expression of
endogenous AP-1-responsive gene collagenase I was strongly repressed in
cancer cells stably transfected with RARß. In studying the
antitransforming activity of RARß, we observed that the growth of
breast cancer MDA-MB231 cells in soft agar was significantly repressed
in a RA-independent manner when cells were stably transfected with
RARß but not RAR. Together, our results demonstrate that RARß
may exert its potent anticancer effect in part through its unique
anti-AP-1 activity. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, ß, and ) and are members of the steroid/thyroid hormone
receptor superfamily that function as ligand-activated transcription
factors (4, 5, 6)
. RARs and RXRs primarily function as
RXR/RAR heterodimers that bind to a variety of RAREs and regulate their
transactivation activities (4, 5, 6)
. Regulation of gene
expression by retinoid receptors requires interaction with additional
cofactors that appears to provide a direct link to the core
transcriptional machinery and to modulate chromatin structure
(7)
. In addition to their positive regulation of gene transcription, retinoid receptors also function as negative transcriptional factors (8) . One of the well-known transcriptional repressive effects of retinoid receptors is their inhibition of AP-1 activity (8) . Retinoid receptors, in response to their ligands, can inhibit the effect of tumor promoter TPA by repressing the transcriptional activity of AP-1 (9 , 10) . AP-1 is composed of proto-oncogenes c-Jun and c-Fos, the activity of which is often associated with cell proliferation and tumor progression (11) . AP-1 activity is regulated by growth factors, cytokines, oncogenes, and tumor promoters that activate protein kinase C. It induces transcriptional activation by binding to TRE (11) . The mechanism by which ligand-activated retinoid receptors repress AP-1 activity remains largely unknown, although a direct protein-protein interaction between retinoid receptors and AP-1 (9 , 10) and a competition for a common coactivator (12) have been proposed. Nevertheless, the interaction between membrane and retinoid receptor signaling pathways may represent an important mechanism by which retinoids exert their potent antineoplastic effect (8) . RA could prevent transformation of JB6 mouse epidermal cells promoted by TPA (13) and counteract the effect of TPA on expression of collagenase and stromelysin, presumably through inhibition of AP-1 activity (14) . In addition, synthetic retinoids that selectively inhibit AP-1 activity and cannot induce transactivation of RA-responsive genes were able to inhibit the growth of lung and breast cancer cells (15 , 16) .
Recent studies have demonstrated that RARß plays a critical role in mediating the anticancer effect of retinoids in many different types of cancer cells (17 , 18) , including breast cancer (19, 20, 21) , lung cancer (22, 23, 24, 25, 26, 27) , ovarian cancer (28) , cervical cancer (29) , prostate cancer (30) , neuroblastoma (31) , renal cell carcinoma (32) , pancreatic cancer (33) , liver cancer (34) , and head and neck cancer (35) . Expression of RARß in RARß-negative cancer cells restored RA-induced growth inhibition, whereas inhibition of RARß expression in RARß-positive cancer cells abolished RA effects (19 , 20) . In addition, transgenic mice expressing RARß antisense sequences showed increased incidence of lung tumor (25) , whereas suppression of RARß expression was responsible for diminished anticancer activities of retinoids in animal (36) , and up-regulation of RARß is associated with a positive clinical response to retinoids in patients with premalignant oral lesions (37) . Furthermore, loss of RARß was suggested to be an early event in carcinogenesis (26 , 38, 39, 40) and may be involved in liver cancer development (41) .
How RARß exerts its anticancer effects remains largely unknown.
We have demonstrated previously that expression of RARß in certain
breast cancer and lung cancer cells could induce apoptosis (20
, 22)
, suggesting that apoptosis induction could contribute to the
anticancer activities of RARß. However, expression of RARß does not
always induce apoptosis of cancer cells (20
, 22)
,
indicating that mechanism other than apoptosis induction mediates
anticancer effects of RARß. In this study, we evaluated anti-AP-1
activity of RARß. Unlike RAR and RAR which repressed AP-1
activity in a RA-dependent manner, RARß could repress TPA-induced
AP-1 activity and activity of oncogenes c-Jun and
c-Fos in a RA-independent manner. By using various hybrid
receptors and RARß deletion mutants, we demonstrated that the
NH2-terminal portion (A/B domain) of RARß is
mainly responsible for the RA-independent anti-AP-1 activity of RARß.
On several RAREs, RARß did not show any constitutive AF-1 activity
associated with the A/B domain. In addition, inhibition of histone
deacetylase activity by TSA did not relieve the inhibitory effect of
RARß. These observations suggest that competition for common
coactivator or the recruitment of receptor corepressor by RARß is
unlikely the mechanism for its effect. Furthermore, we found that
cancer cells constitutively expressing RARß, but not RAR,
exhibited reduced AP-1 activity, even in the absence of RA treatment.
Moreover, expression of endogenous AP-1 responsive gene collagenase I
and the growth of cancer cells in soft agar was significantly inhibited
in a RA-independent manner in cancer cells stably expressing RARß.
Together, our results demonstrate that RARß has a unique anti-AP-1
activity and that this unique RA-independent inhibition of AP-1
activity may represent one of the mechanisms by which RARß exerts its
potent anticancer activities.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasmid Constructions.
The CAT reporter constructs, -73Col-CAT, TRE-tk-CAT, and TREpal-tk-CAT,
have been described previously (9
, 42)
. Constructions of
expression vectors for RAR, RARß, RAR, c-Jun, and
c-Fos and of RAR hybrid receptors were described previously
(9
, 42, 43, 44)
. RARß/
AB was constructed by cloning
BamHI-flanked PCR product, using forward primer 5'-CGG GAT
CCC GAA TGT ACA AAC CCT GCT TCG TC-3' and reverse
T3 primer, into pcDNA3 vector. RARß/E was
generated by deleting the EcoRI fragment from the receptor.
Transient and Stable Transfection Assay.
For HeLa cells, they were plated at 1 x
105 cells/well in a 24-well plate 16–24 h before
transfection as described previously (43)
. For cancer
cells, 5 x 105 cells were seeded
in a six-well plate. A modified calcium phosphate precipitation
procedure was used for transient transfection and is described
elsewhere (43)
. Briefly, 250 ng of reporter plasmid, 100
ng of ß-galactosidase expression vector (pCH 110; Pharmacia), and
various amounts of expression vector were mixed with carrier DNA
(pBluescript) to 1000 ng of total DNA/well. CAT activity was normalized
for transfection efficiency to the corresponding ß-gal activity. For
stable transfection, the pRc/CMV-RARß recombinant plasmid was stably
transfected into SK-MES-1, H292, and MDA-MB231 cells using the calcium
phosphate precipitation method and screened using G418 (Life
Technologies, Inc., Grand Island, NY) as described (20
, 22)
. The integration and expression of transfected cDNA were
determined by Southern blotting and Northern blotting, respectively.
The stable clones obtained have been described (20
, 22)
.
Multi-RT-PCR Assay.
For multi-RT-PCR analysis, total RNAs were isolated and purified by
Qiagen RNeasy Mini kit. Expression of AP-1 responsive gene collagenase
I in cells was determined by the multi-RT-PCR, modified according to
the method described (45)
. Briefly, 1.5 µg total RNA
were used for reverse transcription in 20 µl of reaction mixture
containing of 500 ng of oligo (dT)12–18, 250
µM deoxynucleotide triphosphates, first strand buffer,
and 200 units of superscript II (Life Technologies, Inc.). PCR was
carried out with collagenase I primers and GAPDH primers in one
reaction system under optimized running conditions. PCR products were
analyzed on 2.5% agarose gel and visualized by ethidium bromide
staining. The primers used were as follows: collagenase I,
5'-ATTGGAGCAGCAAGAGGCTGGG-3' (sense);
5'-TTCCAGGTATTTCTGGACTAAGTCC-3'; GAPDH,
5'-CCATCACCATCTTCCAGGAG-3' (sense), 5'-CCTGCTTCACCACCTTCTTG-3'.
Soft-Agar Assay.
About 20,000 cells in culture medium containing 10% FCS, 0.3% agar
(Difco, Detroit, Michigan), and 10-7
M all-trans RA in a six-well plate were plated
onto an already hardened 0.6% agar underlayer in medium supplemented
with 10% FCS. The plates were incubated for 21 days with 6%
CO2. A colony was defined as >40 cells, and
colonies with >40 cells were counted with a microscope.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and RAR was studied.
The -73Col-CAT was transiently transfected into HeLa cells together
with each of RARs. Cells were then treated with or without 100 ng/ml
TPA in the presence or absence of 10-6
M RA. As shown in Fig. 1A
, treatment of HeLa cells with TPA strongly induced the
reporter transcription with about a 5-fold induction. Induction of the
collagenase promoter activity by TPA was mainly attributable to its
induction of the endogenous AP-1 activity (9)
. In the
absence of cotransfected RAR expression vector, RA treatment slightly
inhibited the TPA-induced reporter activity, with 27% inhibition, most
likely attributable to activation of endogenous retinoid receptor. When
we analyzed the effect of cotransfected RARs in the absence of RA, we
observed a strong inhibition of the TPA-induced reporter activity when
RARß expression vector was cotransfected. The RA-independent
inhibition of TPA activity by RARß was concentration dependent. About
60% inhibition was observed when 50 ng of RARß was cotransfected. RA
treatment further enhanced the effect of RARß. In contrast,
inhibition of the TPA-induced promoter activity by RAR or RAR
required RA treatment. In the absence of RA, we did not observe any
inhibition of the reporter activity when various concentrations of
RAR or RAR were transfected. These data demonstrated that RARß
could inhibit TPA activity in a RA-independent manner, whereas
inhibition of TPA activity by RAR and RAR requires their ligand
binding. To determine whether the AP-1 binding site in the collagenase
promoter was responsible for the observed effects, we used the
TRE-tk-CAT reporter, in which the consensus AP-1 binding site (TRE) was
linked with the tk promoter (42)
. About 6-fold induction
of reporter transcription was observed when HeLa cells were treated
with 100 ng/ml TPA (Fig. 1B
). The TPA-induced TRE-tk-CAT
reporter activity was significantly repressed when RARß expression
vector was cotransfected. In the absence of RA treatment,
cotransfection of 50 ng of RARß expression vector led to 
50%
inhibition of TPA-induced TRE-tk-CAT activity. In contrast, repression
of the reporter transcription by cotransfected RAR or RAR
occurred only when cells were treated with 10-6
M RA. Together, these results demonstrate that
RARß could inhibit TPA activity in a RA-independent manner and that
the inhibition is mediated by the AP-1 binding site.
|
) or a 5-fold (Fig. 1D
)
induction, respectively. Treatment of cells with RA only slightly
inhibited the reporter transcription in the absence of RAR
cotransfection. When RARß expression vector was cotransfected, the
reporter activity was strongly inhibited in a concentration-dependent
manner. In contrast, when RAR or RAR was cotransfected, the
reporter activity was inhibited only when cells were treated with RA.
Thus, expression of RARß could repress transcriptional activity of
oncogenes c-Jun and c-Fos in a ligand-independent
manner.
RARs can heterodimerize with RXR and may function as a RAR/RXR
heterodimer in cells (43)
. We therefore investigated
whether expression of RXR had any effect on RA-independent inhibition
of AP-1 activity. As shown in Fig. 1E
, cotransfection of
RXR expression vector did not show any effect on TPA-induced
-73Col-CAT reporter activity. However, when RXR was cotransfected
with RARß, RA-independent inhibitory effect of RARß was enhanced.
The enhancing effect of RXR on RARß activity was observed when
three different concentrations of RARß were used. The results
therefore demonstrate that RARß/RXR heterodimer could act as an
effective inhibitor of AP-1 activity in the absence of RA.
To study whether the observed inhibition of AP-1 activity by RARß in
the absence of RA treatment was attributable to a trace amount of
endogenous retinoids, we evaluated the activation function of RARß in
response to various concentrations of RA by using the TREpal-tk-CAT
reporter, which is known to be activated by RAR (43)
. For
comparison, activation by RAR was analyzed. As shown in Fig. 1F
, both RAR and RARß showed a very similar response to
various concentrations of RA in activating the TREpal-tk-CAT reporter.
The maximum activation of the reporter by both receptors was observed
when cells were treated with 10-6
M RA. These results demonstrate that activation
of gene transcription by RARß is RA dependent and suggest that
repression of AP-1 activity by RARß in the absence of exogenous RA is
unlikely because of the presence of trace amounts of RA in the cells.
The A/B Domain of RARß Is Responsible for RA-independent
Repression of AP-1 Activity.
To determine which domain of RARß is responsible for its
RA-independent anti-AP-1 activity, we analyzed the activity of several
RARß deletion mutants (Fig. 2A
). Cotransfection of the -73Col-CAT reporter with a RARß
mutant deleted with the E domain (RARß/E) significantly repressed
the c-Jun-induced reporter activity in the absence of RA
treatment (Fig. 2B
). The observation is similar to that
observed with the parental RARß receptor (Fig. 1A
),
although the degree of inhibition by RARß/E was reduced. Addition
of RA treatment showed slight enhancement of the inhibitory effect by
RARß/E, which is likely attributable to the effect by endogenous
RAR activity, because a similar degree of inhibition by RA was observed
in the absence of cotransfected receptor. In contrast, cotransfection
of a mutant lacking the A/B domain RARß/AB did not show any
inhibitory effect on c-Jun-induced reporter activity in the
absence of RA (Fig. 2B
), indicating that the A/B domain is
crucial for RA-independent inhibition of AP-1 activity. However, the
mutant strongly inhibited the reporter activity when cells were treated
with RA, demonstrating that the A/B domain is not required for
RA-dependent inhibition of AP-1 activity. When a mutant deleted with
the ABC domain (RARß/ABC) was analyzed, we found that both
RA-dependent and -independent activities were abolished, indicating
that the ligand-binding domain (E/F domain) alone is not sufficient to
confer RA-dependent inhibition of AP-1 activity and that the C domain
is also required. Together, these data demonstrate that the
RA-independent anti-AP-1 activity of RARß is likely mediated through
the A/B domain of the receptor, whereas the RA-dependent activity
requires both the DNA-binding domain and ligand-binding domain.
|
or RAR (Fig. 3A
). Inhibition of AP-1 activity by these hybrid receptors was
evaluated in HeLa cells using the -73Col-CAT reporter. As shown in
Fig. 3B
, cotransfection of hybrid receptors in which the
COOH-terminal portion of RARß was replaced with the corresponding
portion from either RAR (RARß/) or RAR (RARß/)
significantly inhibited TPA-induced reporter activity in a
RA-independent manner, similar to that observed with the wild-type
RARß (Fig. 1A
). In contrast, cotransfection of the hybrid
receptor in which the NH2-terminal domain of
RARß was replaced with RAR (RAR/ß) did not show any
inhibitory effect on TPA-induced AP-1 activity in the absence of RA
treatment. These data further demonstrate that the unique
RA-independent anti-AP-1 activity of RARß is mediated by the
NH2-terminal portion of RARß.
|
). Our results did not reveal any constitutive activation by RARß on
three RAREs examined. In contrast, RAR that did not show any
RA-independent inhibition of AP-1 acitivity (Fig. 1
) displayed a
significant RA-independent transactivation on these RAREs. The fact
that RARß lacks RA-independent transactivation function demonstrates
that RA-independent inhibition of AP-1 activity by RARß is unlikely
attributable to its sequestration of a coactivator.
|
, treatment of HeLa cells with TSA alone slightly enhanced the
-73Col-CAT reporter activity, probably because of inhibition of
corepressor activity. TSA did not show a clear effect on TPA-induced
reporter activity. When RARß expression vector was cotransfected, the
RA-independent inhibition of TPA-induced reporter activity was not
affected by TSA treatment. These data suggest that histone
deacetylase-associated activity is unlikely responsible for
transcriptional repression of AP-1 activity by unliganded RARß.
|
6-fold induction (Fig. 6
). The TPA-induced reporter activity was slightly reduced when cells
were treated with RA. When RARß was cotransfected, we observed a
RA-independent repression of the TPA-induced reporter activity in a
concentration-dependent manner. Cotransfection of 100 ng of RARß led
to 45% inhibition of the TPA-induced activity. In contrast, RAR
and RAR did not exhibit any RA-independent repression of AP-1
activity in the cells. They repressed the TPA-induced activity only
when cells were treated with RA.
|
, TPA strongly induced transcription of the reporter gene in
the parental SK-MES-1 cells, with 5-fold induction. However, the
ability of TPA to induce the reporter gene expression was completely
suppressed in the stable clones. We did not observe any induction of
the reporter transcription in SK-MES-1/RARß#6 and SK-MES-1/RARß#7
clones, either in the absence or presence of RA treatment. The reduced
ability of TPA to induce reporter transcription was specific to RARß
transfection because expression of the empty vector (Vector) showed a
similar response to TPA as compared with the parental cells. These data
suggest that expression of RARß could inhibit the ability of TPA to
induce transcription of AP-1-responsive genes in a RA-independent
manner.
|
(48)
. We therefore compared the effect of RARß and
RAR on the ability of c-Jun/c-Fos to induce
-73Col-CAT reporter transcription in the absence or presence of RA. As
shown in Fig. 7B
, c-Jun/c-Fos strongly
induced the reporter transcription in the wild-type MDA-MB231 cells,
with about a 5-fold induction. A similar degree of induction by
c-Jun/c-Fos was also observed in MDA-MB231 cells
stably transfected with the empty vector (Vector). In both MDA-MB231
and MB231/vector cells, c-Jun/c-Fos-induced
reporter activity was slightly inhibited when cells were treated with
RA, probably because of the presence of endogenous RARs
(20)
. When MDA-MB231 cells transfected with RARß or
RAR were analyzed, we observed that
c-Jun/c-Fos could still induce the reporter gene
activity in the RAR stable clones (RAR#2 and RAR#21). The
degree of induction was similar to that observed in the wild-type cells
or Vector cells. c-Jun/c-Fos-induced reporter
activity, however, was strongly repressed when cells were treated with
10-6 M RA, demonstrating
that the transfected RAR could repress
c-Jun/c-Fos-induced AP-1 activity in a
RA-dependent manner. In contrast, the ability of
c-Jun/c-Fos to induce the reporter gene
transcription was significantly reduced in RARß stable clones
(RARß#2 and RARß#3), with only a 2-fold induction. Treatment of the
RARß stable clones with RA further reduced the ability of
c-Jun/c-Fos to induce reporter transcription.
Thus, stably transfected RARß, but not RAR, could prevent
activation of AP-1-responsive genes by
c-Jun/c-Fos in the absence of RA.
Inhibition of Collagenase I Expression by Stable Expression of
RARß.
We next examined the effect of RARß expression in SK-MES-1 cells on
the ability of TPA to induce expression of endogenous collagenase I
gene expression by RT-PCR. Expression of collagenase 1 is known to be
induced by TPA through its activation of AP-1 that binds to a TRE
present in the promoter (14)
. As shown in Fig. 8A
, TPA strongly induced expression of collagenase I by
10-fold in SK-MES-1 cells, as reported previously (14)
.
Treatment of cells with RA slightly repressed induction of collagenase
I by TPA. For comparison, we did not observe any effect of TPA or RA on
expression of the GAPDH gene. Similar results were observed
in SK-MES-1 cells stably transfected with the empty vector
(Vector). However, the ability of TPA to induce collagenase I
expression was largely reduced in the RARß stable clones (RARß#6
and RARß#7), with only 3-fold induction. Treatment of the cells
with RA completely repressed the ability of TPA to induce collagenase I
expression. To further confirm the RA-independent anti-AP-1 effect of
RARß, we compared the inducibility of TPA on collagenase I expression
in H292 lung cancer cells and H292 cells stably expressing transfected
RARß. Our result (Fig. 8B
) showed that the ability of TPA
to induce collagenase I expression was significantly reduced by
overexpressing of RARß also in this cell line. These data further
confirm that RARß exerts a potent RA-independent anti-AP-1 activity.
|
). The growth of the wild-type MDA-MB231 cells in the soft agar was not
affected by RA treatment, as reported previously (20)
.
However, the growth of MDA-MB231 cells expressing stably transfected
RARß (MB231/RARß#3) was largely reduced, even in the absence of RA,
with 50% inhibition (Fig. 9
). Treatment of MB231/RARß#3 cells
with RA further inhibited the growth of the cells. In contrast,
inhibition of the growth of MDA-MB231 cells expressing transfected
RAR (MB231/RAR#2) was RA dependent. Our previous observation
(20
, 21)
that RARß was induced by RA in the
MB231/RAR#2 cells suggests the RA-dependent inhibition of
MB231/RAR#2 cell growth is in part mediated by the induced RARß.
The observed effect was specific because stable transfection of the
empty vector (Vector) did not show any effect on growth of the cells.
These results suggest that the potent and unique anti-AP-1 activity of
RARß may contribute to the anticancer activity of RARß.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, ß, and ). They
exert their anticancer activities by modulating proliferation,
differentiation, and apoptosis of cancer cells (1
, 2)
. A
growing literature has demonstrated that RARß is primarily
responsible for mediating the anticancer effect of retinoids
(17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
. Here, we provide evidence that RARß has an
unique RA-independent anti-AP-1 activity that is different from other
RAR subtypes, suggesting that RARß may exert its potent anticancer
effects in part through RA-independent inhibition of AP-1 activity.
The anticancer activity of retinoids is thought to be in part
attributable to their direct antiproliferative effects, which have been
observed in several transformed cell lines, including those for
mammary, melanoma, lymphoid, and fibroblastic cancers (3)
.
Studies in human breast cancer cells and bronchial epithelial cells
also suggest that retinoids may inhibit proliferation through
inhibition of transcription factor AP-1 activity (15
, 50
, 51)
, which is composed of c-Jun homodimers or
c-Jun/c-Fos heterodimers. Several lines of
evidence provided here demonstrate a unique RA-independent anti-AP-1
activity of RARß. In transient transfection assays, cotransfection of
RARß or RARß and RXR inhibited either TPA-induced AP-1
transcriptional activity or c-Jun/c-Fos
transcriptional activity in a RA-independent manner, whereas inhibition
of AP-1 transcriptional activity by RAR or RAR was RA dependent
(Fig. 1
). The RA-independent repression of AP-1 transcriptional
activity by RARß was observed in several cell lines, including HeLa
(Fig. 1
), SK-MES-1 lung cancer (Figs. 6
and 7A
), H292 lung
cancer (Fig. 8B
), and MDA-MB231 breast cancer (Fig. 7B
) cells, suggesting that it may function in various cell
types. The effect of RARß could be also observed in RARß-negative
cancer cells stably transfected with RARß. Stable expression of
RARß in RARß-negative SK-MES-1 and MDA-MB231 cells reduced the
ability of AP-1 to induce expression of AP-1-responsive genes,
including the transfected AP-1-responsive reporter genes (Fig. 7
) and
endogenous AP-1-responsive gene (Fig. 8
).
Previous studies investigating domain requirement of AP-1/RAR
interaction have shown that the ligand binding domain and DNA binding
domain are required for AP-1/RAR interaction (9
, 52)
.
Consistent with these previous results, our data (Figs. 2
and 3
)
demonstrate that both the NH2-terminal portion
(A/B and C domains) and COOH-terminal portion (E/F domain) of RARß
are involved in the inhibition of AP-1 transcriptional activity. In
addition, by using various RARß mutant receptors, we demonstrate that
the A/B domain of the RARß is responsible for the RA-independent
repression of AP-1 transcriptional activity (Fig. 2
). Cotransfection of
the NH2-terminal half of the RARß, but not the
COOH-terminal half of RARß, showed a RA-independent repression of
AP-1 activity (Fig. 2
). In addition, deletion of the A/B domain
completely abolished the RA-independent inhibition of AP-1
transcriptional activity. Furthermore, hybrid receptors containing the
NH2-terminal half of RARß, but not RAR or
RAR, showed the RA-independent anti-AP-1 effects (Fig. 3
). It is
likely that the A/B domain of RARß may directly interact with AP-1.
Alternatively, the A/B domain of RARß may interact with certain
cofactors that mediate AP-1/RARß interaction.
Recently, it was demonstrated that competition of liganded retinoid
receptors and AP-1 for common transcriptional coactivator CBP
may in part contribute to the mutual inhibition of their
transcriptional activity (12)
. However, RARß does not
possess RA-independent transactivation function on several RAREs in
HeLa cells (Fig. 4
), indicating that sequestration of a coactivator is
unlikely the mechanism for RA-independent inhibition of AP-1 activity.
Unliganded retinoid receptor is known to interact with receptor
corepressor (7)
. The fact that unliganded RARß could
repress AP-1 activity suggests that RARß may inhibit AP-1
transcriptional activity through the recruitment of a receptor
corepressor. Receptor corepressors are known to form a complex with
histone deacetylases to mediate transcriptional repression
(7)
. However, TSA, a specific inhibitor of histone
deacetylase (46)
, failed to relieve the transcriptional
repressive effect of unliganded RARß (Fig. 5
). This observation
demonstrates that histone deacetylase-associated activity is unlikely
the mechanism for RA-independent inhibition of AP-1 transcriptional
activity by RARß.
We have demonstrated previously that expression of RARß could
induce apoptosis of certain cancer cells in response to RA treatment
(20)
. Our present finding suggests that inhibition of AP-1
activity may also contribute to the growth-inhibitory effect of RARß.
Thus, RARß may exert its tumor-suppressive effect through different
mechanisms, such as the anti-AP-1 effect and apoptosis induction, which
are likely operated in a cell type-dependent manner. Our observation
that RARß could inhibit AP-1 activity in the absence of RA may
provide an explanation for the potent tumor-suppressive effect of
RARß. Thus, unlike other RAR subtypes, RARß could act as an
efficient tumor suppressor even in the absence of RA. This is clearly
seen in our growth inhibition assay (Fig. 9
), showing that expression
of RARß could repress the growth of MDA-MB231 cells in soft agar in a
RA-independent manner whereas the growth-inhibitory effect of RAR is
RA dependent. Previous studies have demonstrated that RARß exerts
tumor-suppressive effects in cancer cells (20
, 22,
and
49
) and that a low expression level of RARß may be an
important contributing factor in cancer development
(17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
. Our present finding suggests that the unique
anti-AP-1 activity of RARß may contribute to its tumor-suppressive
effect. The mitogenic stimulus, often generated by the autocrine
secretion of growth factors, is transmitted to cell nucleus to activate
the nuclear transcriptional factors c-Jun and
c-Fos, which often trigger cell proliferation. By inhibiting
AP-1 transcriptional activity, RARß may inhibit cell proliferation
often associated with cancer development. Loss of this negative growth
control mechanism likely plays a role in cancer development.
Interestingly, overexpression of AP-1 could abrogate the
growth-inhibitory effect of RA, resulting in retinoid resistance
(51
, 53)
. Thus, the cross-talk between AP-1 and retinoid
receptor is reciprocal, and a balance between RARß and AP-1 activity
may contribute to the maintenance of the proper growth of cells.
|
FOOTNOTES |
|---|
1 This work was supported in part by NIH Grants
CA60988 and CA51933 (to X-k. Z.), the Grant 6RT-0168 from the
Tobacco-Related Disease Research Program of California, Grant 3PB-0018
from the California Breast Cancer Research Program, and Grant
DAMD17-4440 from the United States Army Medical Research Program. 
2 To whom requests for reprints should be
addressed, at The Burnham Institute Cancer Center, 10901 North Torrey
Pines Road, La Jolla, CA 92037. Phone: (858) 646-3141; Fax:
(858) 646-3195; E-mail: xzhang{at}burnham-inst.org
3 The abbreviations used are: RAR, retinoic acid
receptor; RXR, retinoid X receptor; RARE, retinoic acid response
element; TPA, 12-O-tetradecanoylphorbol-13-acetate; AP,
activator protein; TRE, TPA response element; CAT, chloramphenicol
acetyltransferase; ß-gal, ß-galactosidase; RT-PCR, reverse
transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tk,
thymidine kinase; TSA, trichostatin A.
Received 9/10/99. Accepted 4/18/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and is abnormally down-regulated in cervical carcinoma cells. Cancer Res., 57: 1460-1467, 1997.l (RAR l) levels control RARß2 expression in SK-N-BE2 (c) neuroblastoma cells and regulate a differentiation-apoptosis switch. Mol. Cell. Biol., 18: 6482-6492, 1998. acquire sensitivity to growth inhibition by retinoids. J. Biol. Chem., 269: 21440-21447, 1994.This article has been cited by other articles:
|
|
 |
|
  Q. Shen, I. P. Uray, Y. Li, Y. Zhang, J. Hill, X.-C. Xu, M. R. Young, E. J. Gunther, S. G. Hilsenbeck, N. H. Colburn, et al. Targeting the Activator Protein 1 Transcription Factor for the Prevention of Estrogen Receptor-Negative Mammary Tumors Cancer Prevention Research, June 1, 2008; 1(1): 45 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. De-Castro Arce, E. Gockel-Krzikalla, and F. Rosl Retinoic Acid Receptor beta Silences Human Papillomavirus-18 Oncogene Expression by Induction of de Novo Methylation and Heterochromatinization of the Viral Control Region J. Biol. Chem., September 28, 2007; 282(39): 28520 - 28529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Lefebvre, C. Brand, S. Flajollet, and P. Lefebvre Down-Regulation of the Tumor Suppressor Gene Retinoic Acid Receptor {beta}2 through the Phosphoinositide 3-Kinase/Akt Signaling Pathway Mol. Endocrinol., September 1, 2006; 20(9): 2109 - 2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. P. Mongan and L. J. Gudas Valproic acid, in combination with all-trans retinoic acid and 5-aza-2'-deoxycytidine, restores expression of silenced RAR{beta}2 in breast cancer cells Mol. Cancer Ther., March 1, 2005; 4(3): 477 - 486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Yen, R. Fenning, R. Chandraratna, P. Walker, and S. Varvayanis A Retinoic Acid Receptor {beta}/{gamma}-Selective Prodrug (tazarotene) Plus a Retinoid X Receptor Ligand Induces Extracellular Signal-Regulated Kinase Activation, Retinoblastoma Hypophosphorylation, G0 Arrest, and Cell Differentiation Mol. Pharmacol., December 1, 2004; 66(6): 1727 - 1737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D.-C. Arce, U. Soto, J. van Riggelen, E. Schwarz, H. z. Hausen, and F. Rosl Ectopic Expression of Nonliganded Retinoic Acid Receptor {beta} Abrogates AP-1 Activity by Selective Degradation of c-Jun in Cervical Carcinoma Cells J. Biol. Chem., October 29, 2004; 279(44): 45408 - 45416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Suzui, M. Shimizu, M. Masuda, J. T. E. Lim, N. Yoshimi, and I. B. Weinstein Acyclic retinoid activates retinoic acid receptor {beta} and induces transcriptional activation of p21CIP1 in HepG2 human hepatoma cells Mol. Cancer Ther., March 1, 2004; 3(3): 309 - 316. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Shimizu, M. Suzui, A. Deguchi, J. T. E. Lim, and I. B. Weinstein Effects of Acyclic Retinoid on Growth, Cell Cycle Control, Epidermal Growth Factor Receptor Signaling, and Gene Expression in Human Squamous Cell Carcinoma Cells Clin. Cancer Res., February 1, 2004; 10(3): 1130 - 1140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Kolluri, N. Bruey-Sedano, X. Cao, B. Lin, F. Lin, Y.-H. Han, M. I. Dawson, and X.-k. Zhang Mitogenic Effect of Orphan Receptor TR3 and Its Regulation by MEKK1 in Lung Cancer Cells Mol. Cell. Biol., December 1, 2003; 23(23): 8651 - 8667. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-D. Wang Retinoids and Alcohol-Related Carcinogenesis J. Nutr., January 1, 2003; 133(1): 287S - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Lin, S. K. Kolluri, G.-q. Chen, and X.-k. Zhang Regulation of Retinoic Acid-induced Inhibition of AP-1 Activity by Orphan Receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor J. Biol. Chem., June 7, 2002; 277(24): 21414 - 21422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Sirchia, M. Ren, R. Pili, E. Sironi, G. Somenzi, R. Ghidoni, S. Toma, G. Nicolo, and N. Sacchi Endogenous Reactivation of the RAR{beta}2 Tumor Suppressor Gene Epigenetically Silenced in Breast Cancer Cancer Res., May 1, 2002; 62(9): 2455 - 2461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Smith, C. D. Capo-chichi, J. He, J. L. Smedberg, D.-H. Yang, A. H. Prowse, A. K. Godwin, T. C. Hamilton, and X.-X. Xu Disabled-2 Mediates c-Fos Suppression and the Cell Growth Regulatory Activity of Retinoic Acid in Embryonic Carcinoma Cells J. Biol. Chem., December 7, 2001; 276(50): 47303 - 47310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Lippman, J. J. Lee, D. D. Karp, E. E. Vokes, S. E. Benner, G. E. Goodman, F. R. Khuri, R. Marks, R. J. Winn, W. Fry, et al. Randomized Phase III Intergroup Trial of Isotretinoin to Prevent Second Primary Tumors in Stage I Non-Small-Cell Lung Cancer J Natl Cancer Inst, April 18, 2001; 93(8): 605 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Smith, J. L. Smedberg, M. E. Rula, T. C. Hamilton, and X.-X. Xu Disassociation of MAPK Activation and c-Fos Expression in F9 Embryonic Carcinoma Cells following Retinoic Acid-induced Endoderm Differentiation J. Biol. Chem., August 17, 2001; 276(34): 32094 - 32100. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or with
10-6 M of all-trans RA (
).
After 12 h, the cells were harvested, and CAT activity was
determined. The activities of cotransfected ß-gal were used as
reference values. B, inhibition of TPA-induced
TRE-tk-CAT activity by RAR. The TRE-tk-CAT reporter (250 ng) was
cotransfected with or without the indicated RAR expression vector (100
ng) into HeLa cells. After transfection, cells were incubated in DMEM
medium containing 0.5% charcoal-treated FCS for 24 h and treated
with either TPA (100 ng/ml) alone (
