(Received for publication, November 7, 1996, and in revised form, February 17, 1997)
From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, BP 163, 67404 Illkirch cedex, France
Human stromelysin-3 and interstitial collagenase are matrix metalloproteinases whose expression by stromal cells in several types of carcinomas has been associated with cancer progression. We compared here the regulation of the expression of both proteinases by retinoids in human fibroblasts. Physiological concentrations of retinoic acid were found to simultaneously induce stromelysin-3 and repress interstitial collagenase. In both cases, the involvement of a transcriptional mechanism was supported by run-on assays. Furthermore, in transient transfection experiments, the activity of the stromelysin-3 promoter was induced by retinoic acid through endogenous receptors acting on a DR1 retinoic acid-responsive element. The ligand-dependent activation of the receptors was also investigated by using selective synthetic retinoids, and we demonstrated that retinoic acid-retinoid X receptor heterodimers were the most potent functional units controlling both stromelysin-3 induction and interstitial collagenase repression. However, specific retinoids dissociating the transactivation and the AP-1-mediated transrepression functions of the receptors were found to repress interstitial collagenase without inducing stromelysin-3. These findings indicate that such retinoids may represent efficient inhibitors of matrix metalloproteinase expression in the treatment of human carcinomas.
Stromelysin-3, based on sequence homologies and its domain organization, belongs to the matrix metalloproteinase (MMP)1 family consisting of extracellular proteinases that are implicated in a variety of tissue remodeling processes. Stromelysin-3 expression has been associated with cutaneous wound healing (1), mammary gland involution (2), cycling endometrium (3), embryonic development (4), and metamorphosis (5), where its expression was predominantly found in cells of mesodermal origin. In human carcinomas, stromelysin-3 was the first MMP identified as being expressed by stromal cells (6, 7). Although human stromelysin-3 appears to be unable to degrade any major component of the extracellular matrix (8, 9) and exhibits unusual activation properties (10, 11), its role in cancer progression is supported by high expression levels, which are predictive of a poor clinical outcome (12, 13). Furthermore, we have demonstrated that stromelysin-3 facilitates the tumor take of cancer cells in nude mice (14). Following the identification of stromelysin-3, a number of other MMPs have also been found to be expressed by stromal cells of human carcinomas (15), indicating that the stromal cell production of MMPs represents a significant contribution to the overall proteolytic activities in malignant tumors (Refs. 15 and 16 and references therein). Despite the observation that most stromal MMPs are expressed by fibroblastic cells, no regulatory sequence that could account for this cell-specific expression pattern has yet been identified in the promoter of the corresponding genes.
While stromelysin-3 expression, like other MMPs, can be induced in human fibroblasts by agents such as phorbol ester (TPA) or growth factors (6, 17), very little is known about the mechanisms regulating its expression. We have recently isolated the stromelysin-3 gene (18) and shown that its proximal promoter differs from those of other MMPs by the absence of a consensus AP-1 (c-Jun/c-Fos) binding site and the presence of a retinoic acid-responsive element (RARE) of the DR1 type. This RARE can be transactivated by retinoid receptors (RARs/RXRs) in a ligand-dependent manner in COS-1 cells. In contrast, AP-1 binding sites were found to play a crucial role in controlling both the activation of other MMP gene promoters in response to growth factors and cytokines (19, 20) and their inhibition by retinoic acid (RA) (20-22). Gene transcription studies have shown that while RARs and RXRs can induce transcriptional activation through specific DNA binding sites, they can also interact indirectly with AP-1 through transcriptional mediators to repress gene transcription (23-25). In agreement with these findings, inhibition of base-line and TPA-induced RNA expression by RA has been reported for interstitial collagenase (20) and stromelysin-1 (21).
Retinoid effects are achieved through two classes of
ligand-dependent transactivators, the retinoic acid
receptors (RAR, -
, and -
and their isoforms) and retinoid X
receptors (RXR
, -
, and -
and their isoforms), which are
members of the nuclear receptor superfamily. RARs bind and are
activated by t-RA and 9C-RA, whereas RXRs bind and are activated by
9C-RA (26-28). Retinoids are known to regulate cell proliferation and
differentiation, and they are regarded as agents that may be used to
prevent or suppress human cancer (29-31). In addition, experimental
and clinical studies suggest that retinoids may also be therapeutically
useful in preventing connective tissue degradation caused by MMP
overproduction in arthritis (32, 33). However, despite extensive
knowledge of RA action at the molecular level, only a few RA target
genes have been identified.
In the present study, we investigate further the regulation of stromelysin-3 gene expression by RA in cells of mesodermal origin. We have found that while nanomolar concentrations of RA can induce the expression of both stromelysin-3 RNA and protein in human fibroblasts, they prevent the expression of interstitial collagenase. The involvement of a transcriptional control in RA action is supported by run-on analyses, showing that the elongation of stromelysin-3 nuclear RNA was up-regulated and that of interstitial collagenase was down-regulated by RA. Furthermore, using stromelysin-3 promoter-based plasmid constructs, we showed that the stromelysin-3 promoter can be activated in cells of mesodermal origin exposed to RA, without the addition of exogenous RARs/RXRs. The observation that both induction of the stromelysin-3 gene and repression of the interstitial collagenase gene were optimally achieved by combining selective RAR agonists with a pan-RXR retinoid indicates that combination of receptors of the RAR and RXR types are required for optimal transcriptional regulation of these genes.
t-RA was purchased from Sigma, and 9C-RA was
provided by P. F. Sorter, J. F. Grippo, and A. A. Levin (Hoffmann-La
Roche, Nutley, NJ). CD666 (34) was donated by B. Shroot (Centre
International de Recherches Dermatologiques Galderma, Valbonne,
France). Am80 (35) was provided by K. Shudo (University of Tokyo).
BMS649 (36) (originally known as SR11237) was provided by the
Bristol-Myers-Squibb Pharmaceutical Research Institute (Buffalo, NY).
The last retinoid BMS753 (37, 38), which is a pure RAR agonist, was
also provided by the Bristol-Myers-Squibb Pharmaceutical Research
Institute, where it is available to academic investigators upon
request.
Human fibroblasts (HFL1, CCL 153) and rhabdomyosarcoma tumor cell line (RD, CCL 136) were obtained from the American Tissue Culture Collection (Rockville, MD) and maintained in monolayer culture in Dulbecco's modified Eagle's medium with or without 5% calf serum. Retinoids t-RA, 9C-RA, BMS753, BMS649, Am80, and CD666 were dissolved in ethanol and added at desired concentrations for the time periods indicated in the figure legends.
RNA Extraction and Northern Blot AnalysisCell cultures
were washed with phosphate-buffered saline, and RNA extraction was
carried out by the guanidinium thiocyanate phenol/chloroform procedure
(39). 10-30 µg of total RNA was denatured at 65 °C for 5 min and
electrophoresed on 1% agarose gel prior to being transferred onto a
nylon membrane (Hybond-N; Amersham Corp.) as described previously (40).
Hybridization to cDNA probes [-32P]dCTP-labeled by
random priming was performed overnight at 42 °C in 40% formamide, 2 mM EDTA, 0.9 M NaCl, 50 mM
Na2HPO4/NaH2PO4, pH
6.5, 1% sodium dodecyl sulfate, 0.4 g/liter polyvinylpyrrolidone, 0.4 g/liter Ficoll, 50 g/liter dextran sulfate, and 50 mg/liter denatured
salmon sperm DNA. The nylon membranes were washed twice at room
temperature in 2 × standard sodium citrate (SSC), 0.1% SDS for
20 min, followed by a last wash under stringent conditions with
0.1 × SSC, 0.1% SDS at 56 °C for 1 h. The following
human cDNA fragments were used as probes: a 1.7-kb EcoRI
fragment for stromelysin-3 (ZIV, Ref. 18), a 1.3-kb
EcoRI-XbaI fragment for interstitial collagenase,
a 1.8-kb EcoRI fragment for stromelysin-1 (41), a 0.7-kb
PstI fragment for 36B4 (42), a 0.6-kb PstI fragment for RAR
, a 0.41-kb XhoI-EcoRI
fragment for RAR
(43), a 1.3-kb AvaI-BamHI
fragment for RAR
(44), and a 1.6-kb XhoI-XbaI fragment for RXR
(45). In the case of the RXR
probe, a 0.8-kb BamHI cDNA fragment and a 0.6-kb fragment (nucleotides
1057-1677) amplified by polymerase chain reaction were generated from
pTL1-hRXR
plasmid (46). Similarly, for RXR
, a 1.1-kb
ApaI-PstI cDNA fragment and a 0.4-kb fragment
(nucleotides 356-769) amplified by polymerase chain reaction were
generated from the pSG5-hRXR
plasmid (26). All human RAR and RXR
cDNA-containing plasmids were kindly provided by P. Kastner
(Institut de Génétique et de Biologie Moléculaire et
Cellulaire). Blots were autoradiographed for 1-4 days, and signal
quantification was performed using a bioimaging analyzer (BAS 2000;
Fuji Ltd).
Conditioned media from HFL1 fibroblasts were collected and centrifuged to eliminate cell debris, followed by a 100-fold concentration by 80% ammonium sulfate precipitation and dialysis against 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.005% Brij-35. Protein samples were then separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred onto nitrocellulose membranes, and revealed with monoclonal antibody 5ST-4C10 against the catalytic domain of stromelysin-3 by using enhanced chemiluminescence (ECL; Amersham) and a peroxidase-coupled anti-mouse IgG (Jackson) (11).
Nuclear Run-on Transcription AssaysControl cells and cells
treated with 9C-RA (1 µM) for 1-3 days were washed twice
with ice-cold phosphate-buffered saline, harvested, and centrifuged at
1300 × g at 4 °C for 5 min. The pellet was
resuspended in 4 ml of lysis buffer (10 mM Tris-HCl, pH
7.4, 10 mM NaCl, 3 mM MgCl2, 0.5%
(v/v) Nonidet P-40), incubated for 5 min on ice, and centrifuged at
1300 × g at 4 °C for 5 min. This procedure was
repeated twice. The final pellet containing the nuclei was resuspended
in storage buffer consisting of 50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, 40% (v/v)
glycerol, and aliquots of 2 × 107 nuclei were stored
at 80 °C before use. In vivo initiated RNA transcripts
from these 2 × 107 nuclei aliquots were elongated
in vitro for 30 min at 30 °C in the presence of 200 µCi
of [
-32P]UTP in a final volume of 200 µl containing
1 mg/ml heparin, 0.6% (v/v) sarkosyl, 0.4 mM
concentrations of ATP, CTP, and GTP, 2.5 mM dithiothreitol,
0.15 mM phenylmethylsulfonyl fluoride, 350 mM
(NH4)2SO4. The reaction was stopped
by the addition of DNase I-RNase free (800 units) in the presence of
1.8 mM CaCl2 for 10 min at 30 °C, followed
by protein digestion with proteinase K (100 µg/ml) in 50 mM Tris-HCl, pH 7.4, 20 mM EDTA, 1% SDS and incubation (45-90 min) at 42 °C until clear samples were obtained. RNA extraction was then performed with phenol/chloroform (1:1, v/v),
and the organic phase was further extracted with 10 mM
Tris-HCl, pH 7.4, 5 mM EDTA, 1% SDS. Pooled aqueous phases
were finally extracted with chloroform, and RNA precipitation was
carried out at 4 °C for 15 min after the addition of 1 volume of
20% trichloroacetic acid in the presence of 20 µg of tRNA as a
carrier. RNA pellets were washed 3 times in 5% trichloroacetic acid
and once with 80% ethanol. Dried pellets were then dissolved in
hybridization buffer (as described above) to a final specific activity
of 5 × 106 cpm/ml and hybridized to cDNAs
corresponding to human stromelysin-3 (ZIV in Ref. 18), human
interstitial collagenase (41), 36B4 (42), and the pBluescript II SK+
plasmid. These DNAs were denatured in the presence of 0.3 N
NaOH and immobilized onto Hybond nylon membranes (Amersham) by using a
slot blot apparatus. Prehybridization at 42 °C for 18 h and
hybridization to in vitro 32P-labeled elongated
RNAs at 42 °C for 3 days were carried out in the same hybridization
buffer. Filters were subjected to various washing conditions as
follows: twice in 2 × SSC, 1% SDS for 15 min at 22 °C; twice
in 0.1 × SSC, 0.1% SDS for 15 min at 52 °C; once in 2 × SSC, in the presence of RNase A (10 µg/ml) for 15 min at 37 °C;
twice in 2 × SSC, 1% SDS for 15 min at 22 °C; and finally,
once in 0.1 × SSC, 0.1% SDS for 15 min at 52 °C. Signal quantification was carried out as described for Northern blot analysis.
The DR1-tk-CAT, 0.29ST3-CAT,
0.45ST3-CAT, 1.47ST3-CAT, and 3.4ST3-CAT constructs have been
previously described (18). The RARE (DR5)-CAT construct (47) was
kindly provided by J-Y. Chen (Institut de Génétique et de
Biologie Moléculaire et Cellulaire). The 3.4ST3-CAT-
DR1
construct was generated by inserting the 3-kb SphI-XbaI 5
-fragment from the 3.4ST3-CAT
construct into the 0.29ST3-CAT construct digested with the same
restriction enzymes, thereby deleting a 0.16-kb promoter sequence
containing the DR1 RARE that is present at position
385 in the
stromelysin-3 gene promoter (18).
Human RD rhabdomyosarcoma
cells were transiently transfected by the calcium phosphate procedure
as described previously (18), except that the total amount of DNA
transfected in each 10-cm diameter culture dish was made up to 20 µg
with pBluescribe plasmid DNA. For a 4-day treatment with RA, cells were
first exposed to 1 µM 9C-RA for 2 days before
transfection, whereas for a 2-day 9C-RA treatment, cells were directly
transfected at 4 h after plating. In both cases, cells were
incubated in the presence of 1 µM 9C-RA for 2 days after
transfection. The -galactosidase expression vector pCH110
(Pharmacia) was used as a internal control to normalize for
transfection efficiency. Cell extracts containing 4 units of
-galactosidase activity were used for chloramphenicol acetyltransferase (CAT) assays, and the reaction products were separated by thin layer chromatography and visualized by
autoradiography. Signal quantification was performed as described for
Northern blot analysis.
Having previously identified a RARE that conferred
ST3 promoter inducibility in COS-1 cells in the presence of RA and its receptors (18), we decided to evaluate whether stromelysin-3 gene
expression was also regulated by RA in human fibroblasts. Time course
and dose response experiments were performed, and expression of the
stromelysin-3 gene was compared with that of interstitial collagenase
by Northern blot analysis in HFL1 fibroblasts exposed to 9C-RA in the
presence of 5% calf serum (Fig. 1).
As shown in Fig. 1A, in the presence of 1 µM
9C-RA, stromelysin-3 RNA levels progressively increased from day 1 to
day 4, with a 20-fold increase measured after 4 days of incubation. In contrast, the levels of interstitial collagenase RNA remained constant
when fibroblasts were exposed to 9C-RA for 1 day and rapidly decreased
to almost undetectable levels after 2 days of treatment. Dose response
experiments were conducted after incubation during 3.5 days with 9C-RA
concentrations ranging from 0.1 nM to 1 µM
(Fig. 1B). The effect of 9C-RA was
dose-dependent for both genes. Nevertheless, the repression
of interstitial collagenase expression was much more sensitive to 9C-RA
treatment than the induction of the ST3 gene. Indeed, the half-maximal
values for stromelysin-3 induction (EC50) and interstitial
collagenase repression (IC50) differed by a factor of about
100 (Fig. 1B; EC50 ~ 10 nM and
IC50 ~ 0.1 nM). Similar results were obtained
by using t-RA instead of 9C-RA or when the experiments were carried out
in serum-free conditions (data not shown). However, in the latter case,
the interstitial collagenase base line was much lower (Fig.
2), hampering analysis of its repression by RA
isomers.
Our results showing a significant induction of ST3 RNA levels in HFL1 fibroblasts in the presence of RA are in apparent contradiction with the observation recently made by Anderson et al. (48). Using the same fibroblasts as models, they found that TPA-mediated induction of stromelysin-3 RNA was inhibited by RA. As shown on Fig. 2, we could reproduce this inhibition. However, this effect was only observed for a 10 µM 9C-RA concentration, which by far exceeds the RA concentrations usually found in physiological conditions. Furthermore, we would like to point out that when using this 10 µM 9C-RA concentration, we could not obtain any repression of interstitial collagenase RNA expression, whereas this repression was observed at lower concentrations (Fig. 2), as previously noted by others (23).
Induction of Stromelysin-3 Protein Synthesis and Secretion by Retinoic Acid in FibroblastsTo find out whether stromelysin-3
protein synthesis and/or secretion were also increased by RA treatment,
conditioned media from HFL1 fibroblasts were analyzed by Western blot
(Fig. 3). In serum-free conditions, only low levels of
the mature stromelysin-3 form were detected at about 47 kDa. However,
when fibroblasts were exposed for 3 days to 1 µM of
either 9C-RA or t-RA, high levels of this form were detected together
with additional protein species. The highest molecular weight form
corresponds to the stromelysin-3 proform, which is known to be
converted by furin or furin-like enzymes into the mature form (10, 11),
which in turn can be processed further into another low molecular
weight species (Fig. 3 and Ref. 14).
Transcriptional Control of Stromelysin-3 and Interstitial Collagenase Genes by Retinoic Acid in Fibroblasts
To determine
whether a transcriptional mechanism was involved in controlling the
levels of stromelysin-3 and interstitial collagenase RNAs by RA, we
analyzed the nuclear RNAs of both MMPs by using run-on assays performed
on nuclei isolated from HFL1 fibroblasts after they had been treated
for 1-3 days with 1 µM 9C-RA. Radiolabeled RNAs
resulting from nascent nuclear RNA transcripts elongated in
vitro were hybridized to cDNAs cloned into the pBluescript II
SK+ plasmid and corresponding to interstitial collagenase, stromelysin-3, 36B4, or the plasmid alone as a control for nonspecific hybridization. The results presented on Fig. 4 show that
both MMP genes are constitutively transcribed in HFL1 fibroblasts. After 3 days in the presence of 9C-RA, interstitial collagenase transcription was no longer detectable. On the other hand, 9C-RA was
found to increase the rate of stromelysin-3 gene transcription by
2-fold, thereby reaching levels similar to those observed for the 36B4
gene, whose expression is not affected by 9C-RA (Fig. 4 and Ref. 37).
Shorter exposure times of HFL1 fibroblasts to 9C-RA (1 or 2 days) led
to either no increase or a very little increase in stromelysin-3 gene
transcription (data not shown).
Activation of the Human Stromelysin-3 Gene Promoter by Retinoic Acid via Endogenous Retinoid Receptors in Rhabdomyosarcoma Cells
HFL1 fibroblasts, like other nonimmortalized human diploid
fibroblasts, are difficult to use for promoter studies in transient transfection experiments. Therefore, we looked for an established cell
line expressing the stromelysin-3 gene that would be easier to use for
transfection studies. Since the stromelysin-3 gene is only weakly
expressed in human fibrosarcoma cell lines such as HT-1080 and cannot
be induced by TPA in these cells (17), we screened several human cell
lines of mesodermal origin based on their ability to respond to TPA and
RA. We thus identified a rhabdomyosarcoma tumor cell line (RD) that
exhibits a stromelysin-3 expression pattern very similar to that of
HFL1 fibroblasts. In particular, basal levels of stromelysin-3 RNA
expression as well as its induction by 9C-RA, which is maximal after 4 days of incubation, were found to be similar in both cell types (Fig.
5). However, we observed that these RD rhabdomyosarcoma
cells do not express the interstitial collagenase gene (Fig. 5), even
upon exposure to TPA (data not shown).
To further evaluate whether a transcriptional regulation was involved
in the induction of stromelysin-3 gene expression by RA, we analyzed
stromelysin-3 promoter activity in RD rhabdomyosarcoma cells exposed to
9C-RA for 4 days. RD rhabdomyosarcoma cells that had been preincubated
with 9C-RA for 2 days were transiently transfected by a CAT reporter
gene driven by various lengths of stromelysin-3 promoter and further
exposed to 9C-RA for an additional period of 2 days, before measurement
of CAT activities (Fig. 6 and data not shown). Upon the
addition of 9C-RA, the activities of all three stromelysin-3 promoter
constructs containing the DR1-RARE (0.45-, 1.47-, and 3.40-ST3-CAT)
were induced 2.8 ± 0.5-, 3.2 ± 0.6-, and 3.3 ± 0.5-fold (n = 3), respectively. Conversely, the absence
of the DR1-RARE in the 0.29ST3-CAT and the 3.40ST3-DR1 constructs
reduced 9C-RA inducibility to 1.2 ± 0.1- and 1.6 ± 0.1-fold
(n = 3), respectively. A similar remaining activation by RA was previously observed for the 0.29ST3-CAT construct when transfected into COS-1 cells (18). This may be attributed to the
presence of several widely spaced half-RARE motifs (PuG(G/T)TCA) present in this promoter region that have been shown to activate transcription in the presence of RA (49). The activation by 9C-RA was
also tested on the RAR
2 promoter, which contains a RARE of the DR5
type (47), and on the isolated DR1 element inserted upstream of the
herpes simplex virus thymidine kinase promoter. The activity of these
two constructs was induced 3.1 ± 0.2- and 4.9 ± 0.7-fold
(n = 3) by 9C-RA, respectively, thus to levels comparable with those observed for ST3 constructs. However, the transactivation of the DR1-tk-CAT construct was weaker (1.4 ± 0.3-fold, n = 2) when RD rhabdomyosarcoma cells were
exposed to 9C-RA for only 2 instead of 4 days, thereby suggesting that
some of the regulatory factors implicated in this activation are not constitutively expressed in these cells. Also, it should be noted that
these experiments were performed without the cotransfection of any
retinoid receptor, indicating that the observed effects were mediated
through endogenous receptors.
Retinoic Acid Receptor Expressions in Fibroblasts
To
determine the respective contribution of RARs and RXRs in mediating
stromelysin-3 induction and interstitial collagenase repression by RA,
their expression in HFL1 fibroblasts was first analyzed by Northern
blot. We found that untreated fibroblasts cultured in serum-free
conditions expressed similar levels of RAR, RAR
, and RXR
RNAs
(Fig. 7), with steady state levels relatively constant
over the time of culture (not shown). No expression was detected,
however, in untreated cells for RAR
, RXR
, and RXR
RNAs, even
when up to 30 µg of total RNA were loaded for analysis (Fig. 7 and
data not shown). These results are consistent with recent studies that
have shown that RAR
, RAR
, and RXR
are the predominant
receptors expressed in human skin (50) as well as in various human cell
lines (51-53). The expression of RXR
was only slightly increased
(less than 2-fold) in cells treated with either 9C-RA or t-RA, whereas
RAR
and RAR
levels remained unaffected. In contrast, RAR
RNA
levels increased from undetectable to high levels in cells exposed to
either of the RA isomers (Fig. 7). Similar results were obtained by
using fibroblasts cultured in 5% calf serum, although RAR
was
induced to a lower extent than in serum-free conditions (data not
shown).
Synergistic Activation of Stromelysin-3 Gene and Repression of Interstitial Collagenase Gene by Specific Synthetic Retinoids
We
then investigated whether the ligand-dependent activation
of both RARs and RXRs was required for inducing or repressing the
expression of stromelysin-3 and interstitial collagenase genes, respectively. We used the synthetic ligands Am80 (35) and CD666 (34),
which at appropriate concentrations selectively activate RAR and
RAR
, respectively (37), BMS753, a pure RAR
agonist (38), and
BMS649, a pan-RXR agonist (36). The expression levels of stromelysin-3
and interstitial collagenase RNAs in HFL1 fibroblasts cultured in 5%
calf serum were evaluated after 3.5 days of culture in the presence of
these synthetic retinoids and compared with those observed in the
presence of 9C-RA and t-RA.
Using these retinoids individually at low and/or selective
concentrations, either no induction or a very weak induction of stromelysin-3 was detected (Fig. 8, A and
B), while interstitial collagenase expression was reduced by
at least 50% (Fig. 8, C and D). At higher
concentrations (>10 nM), when Am80 and CD666 lose their
specificity and act as pan-RAR agonists (37), higher levels of
stromelysin-3 RNA were observed, while interstitial collagenase
expression was repressed further. Interestingly, very little
stromelysin-3 gene induction was noted with the pure RAR agonist
BMS753 and the pan-RXR agonist BMS649, even when these retinoids were
used at a 1 µM concentration. In marked contrast, the
combination of either Am80 (100 nM and 1 µM)
or CD666 (100 nM) with the pan-RXR ligand BMS649 (1 µM) resulted in a synergistic induction of the
stromelysin-3 gene, reaching expression levels close to those observed
with the natural ligands. A synergistic effect was also observed when
the BMS753 and BMS649 ligands were combined, although the expression
levels of stromelysin-3 RNA did not exceed 50% of those observed in
the presence of the natural ligands. However, any of these combinations
was found to fully repress interstitial collagenase gene expression. We
note that stromelysin-1 gene expression was similarly repressed in HFL1 fibroblasts,2 suggesting that the retinoids
used here may efficiently repress the expression of any AP-1-regulated
MMP.
Taken together, our observations indicate that while the selective
activation of RAR, RAR
, or RXRs substantially represses interstitial collagenase gene expression, the combination of RARs and
RXRs is required for optimal stromelysin-3 gene induction and for full
repression of interstitial collagenase.
We have previously shown that the stromelysin-3 gene promoter differed from most other MMP promoters by the absence of a functional AP-1 binding site and the presence of a RARE in its proximal region. In the present study, we have further investigated the regulation of stromelysin-3 gene expression by RA and compared this regulation with that of interstitial collagenase, another MMP. Stromelysin-3 and interstitial collagenase are both predominantly expressed by stromal cells of human carcinomas (15), and their high expression levels were found to be associated with a poor clinical outcome in some carcinomas (12, 13, 54). Considering that retinoids by themselves or when associated with other drugs such as tamoxifen, are regarded as potential new anticancer agents (29-31), it is important to elucidate the mechanisms by which the expression of MMPs implicated in cancer progression is regulated by RA. We demonstrate here that both natural RA isomers, 9C-RA and t-RA, strongly induce stromelysin-3 RNA and protein expression and simultaneously repress interstitial collagenase expression in human fibroblasts. In addition, we show that both genes are controlled by RA through a transcriptional mechanism, and we provide evidence indicating that RAR·RXR heterodimers are the functional units required for an optimal control of these genes by RA.
AP-1 and retinoid receptors are regarded as effectors of opposite
pathways of cell proliferation and differentiation, and they mutually
antagonize each other at the level of transactivation and DNA binding
(22-25). Indeed, MMP genes containing an AP-1 binding site in a
conserved position in their promoter or other genes like those for
tumor growth factor-1 (55) and interleukin-6 (56) are TPA-inducible,
while their expression is inhibited by RA. Knowing that, reciprocally,
AP-1 can inhibit transactivation by RARs and RXRs, the observation that
the stromelysin-3 gene is induced by both TPA and RA in a given cell
type is quite unexpected and represents an unusual example of a gene
up-regulated by both agents.
We have found that physiological concentrations of RA efficiently
induce both the expression of the stromelysin-3 gene and the repression
of the interstitial collagenase gene in HFL1 human fibroblasts, the
latter being observed at RA concentrations lower than those necessary
for stromelysin-3 induction. Interestingly, the IC50 for
interstitial collagenase and the EC50 for stromelysin-3 that we found were very similar to the values recently reported by Chen
et al. (25) in promoter studies. These authors have shown
that the repression of AP-1-induced transcription from the interstitial
collagenase promoter was about 100 times more sensitive than the
transactivation of a RARE-tk-CAT construct in the presence of RA. Since
these observations suggested that the regulation of both genes by RA
may be achieved through a transcriptional mechanism, we further
evaluated this possibility by measuring the transcriptional rate of
both genes in HFL1 fibroblasts in run-on assays. In the presence of
9C-RA, we observed a complete inhibition of interstitial collagenase
transcription, which is likely to result from an RAR/AP-1 interaction,
since this has been previously documented (24, 57). On the other hand,
a 2-fold increase in the stromelysin-3 gene transcriptional rate was
found when HFL1 fibroblasts were exposed to 9C-RA for 3 days. No clear
transcriptional activation could be detected for shorter exposure
times. Although it is difficult to determine whether this 2-fold
increase can fully account for the 20-fold increase in stromelysin-3
RNA levels observed after 4 days of RA treatment, we note that run-on
studies with other RA-inducible genes containing a RARE in their
promoter exhibited similar profiles. Thus, the RAR and the laminin
B1 RNAs were found to be induced at high levels by RA in F9 cells,
while no increase or only a moderate increase in transcriptional rates
could be detected for these genes by nuclear run-on assays (58, 59). In
all instances, the contribution of a transcriptional mechanism in
stromelysin-3 gene induction is further supported by our findings
showing that 9C-RA induces stromelysin-3 promoter activity in RD
rhabdomyosarcoma cells. By analyzing various lengths of this promoter
in transient transfection experiments, we observed a 3-fold induction
of stromelysin-3 promoter activity in the presence of 9C-RA, which was
strongly reduced in the constructs lacking the DR1-RARE. Interestingly, this transactivation was observed without the addition of retinoid receptors, indicating that the DR1-RARE was activated by functional endogenous retinoid receptors in these cells.
Previous studies have shown that while all RARs could potentially
mediate the induction of RA targets genes, the involvement of a given
receptor was dependent on many parameters including promoter context
and cell type (37, 47). When the expression of RARs and RXRs was
evaluated in HFL1 fibroblasts, we found that RAR, RAR
, and RXR
RNAs were constitutively expressed at high levels. In contrast, we
could not detect any RNA for RXR
and RXR
, while that for RAR
was strongly induced from barely detectable levels in untreated
fibroblasts to high levels in the presence of 9C-RA or t-RA. Similar
observations have been made in human dermal (60) and lung (61)
fibroblasts.
Since these observations suggested that specific retinoid receptors
could be involved in the regulation of stromelysin-3 and interstitial
collagenase expression by RA in HFL1 fibroblasts, we tested the
expression of both genes in the presence of selective retinoids. We
found that these retinoids, when used individually at concentrations at
which they selectively activate a given RAR (Am80, CD666, BMS753) or
all three RXRs (BMS649), led to very weak, if any, stromelysin-3 gene
induction, whereas they repress interstitial collagenase expression by
at least 50%. In marked contrast, a clear induction of the
stromelysin-3 gene was observed when any of the selective RAR ligands
was used in combination with the BMS649 RXR-specific ligand. We note,
however, that the combination BMS753-BMS649 (RAR·RXRs) was less
efficient than the other combinations. Since stronger inductions were
observed by combining the BMS649 RXR agonist with either Am80 or CD666 at concentrations at which they both promiscuously activate all three
RARs, it is reasonable to believe that either RAR
and/or RAR
could interact with RXRs for an optimal stromelysin-3 induction. In
keeping with the fact that RXR
seems to be the major RXR expressed in fibroblasts, we can conclude from our results that the two heterodimers RAR
·RXR
and/or RAR
·RXR
are likely to
represent the functional units required to induce the expression of the stromelysin-3 gene at physiological RA concentrations. This possibility is also consistent with in vitro studies that have shown
that heterodimers bind to RARE much more efficiently than the
respective homodimers (27, 28). In this respect, it is noteworthy that the activation of a single RAR or RXR was sufficient to substantially repress interstitial collagenase expression in HFL1 fibroblasts but
that the activation of both partners of heterodimers was necessary for
a full repression.
In summary, while transcription studies have demonstrated that RA regulates the expression of target genes by either activating RAREs or repressing AP-1 activity, we have looked at the regulation of two genes that belong to the MMP family and shown that they are differentially regulated by RA in human fibroblasts. Indeed, we have shown that physiological concentrations of RA induce stromelysin-3 expression but repress interstitial collagenase expression. Compared with the repression of interstitial collagenase, stromelysin-3 gene induction relies on more restricted conditions, based on a lower sensitivity to both natural and synthetic retinoids, and on a more restricted receptor requirement involving RAR·RXR heterodimers. In contrast, a substantial transcriptional repression of interstitial collagenase is achieved by retinoids activating only one type of receptors, although the involvement of RAR·RXR heterodimers is required for a full repression. Taking into account the ongoing efforts in designing potent MMP inhibitors to inhibit cancer progression, the finding that dissociating retinoids such as RXR-selective ligands can prevent the expression of some MMPs by an AP-1 transrepression mechanism without inducing stromelysin-3 gene expression may be of interest for therapeutic applications.
We thank P. Chambon for support, R. Taneja, H. Gronemeyer, and J.Y. Chen for stimulating discussions, R. Kannan for critical review of the manuscript, and I. Stoll for protein analyses. We are also grateful to P. Reczek (Bristol-Myers-Squibb Pharmaceutical Research Institute, Buffalo, NY) for the gifts of BMS649 and BMS753, to B. Shroot (Centre International de Recherches Dermatologiques Galderma, Valbonne, France) for CD666, and to K. Shudo (University of Tokyo) for Am80.