Stromelysin-3 Induction and Interstitial Collagenase Repression by Retinoic Acid
THERAPEUTICAL IMPLICATION OF RECEPTOR-SELECTIVE RETINOIDS DISSOCIATING TRANSACTIVATION AND AP-1-MEDIATED TRANSREPRESSION*

(Received for publication, November 7, 1996, and in revised form, February 17, 1997)

Eric Guérin , Marie-Gabrielle Ludwig , Paul Basset and Patrick Anglard Dagger

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

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 (RARalpha , -beta , and -gamma and their isoforms) and retinoid X receptors (RXRalpha , -beta , and -gamma 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.


MATERIALS AND METHODS

Ligands

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 RARalpha agonist, was also provided by the Bristol-Myers-Squibb Pharmaceutical Research Institute, where it is available to academic investigators upon request.

Cell Culture

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 Analysis

Cell 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 [alpha -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 RARalpha , a 0.41-kb XhoI-EcoRI fragment for RARbeta (43), a 1.3-kb AvaI-BamHI fragment for RARgamma (44), and a 1.6-kb XhoI-XbaI fragment for RXRalpha (45). In the case of the RXRbeta 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-hRXRbeta plasmid (46). Similarly, for RXRgamma , 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-hRXRgamma 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).

Protein Analysis

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 Assays

Control 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 [alpha -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.

CAT Reporter Constructs

The DR1-tk-CAT, 0.29ST3-CAT, 0.45ST3-CAT, 1.47ST3-CAT, and 3.4ST3-CAT constructs have been previously described (18). The beta 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-Delta 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).

Cell Transfection and CAT Assay

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 beta -galactosidase expression vector pCH110 (Pharmacia) was used as a internal control to normalize for transfection efficiency. Cell extracts containing 4 units of beta -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.


RESULTS

Stimulation of Stromelysin-3 and Inhibition of Interstitial Collagenase RNA Expressions by Retinoic Acid in Fibroblasts

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).


Fig. 1. Time course and dose response of stromelysin-3 and interstitial collagenase RNA expression after 9C-RA treatment of HFL1 fibroblasts. HFL1 fibroblasts were cultured in 5% calf serum with 1 µM 9C-RA for 1-4 days (A) or with 9C-RA concentrations ranging from 0.1 nM to 1 µM for 3.5 days (B). Levels of ST3 and interstitial collagenase (Int. Col.) transcripts were analyzed by Northern blot performed with 10 µg of total RNA, as described under "Materials and Methods." Hybridization signals were estimated by using a bioimaging analyzer (BAS 2000; Fuji Ltd.). In the quantitative representations, stromelysin-3 and interstitial collagenase RNAs are expressed relative to their highest expression levels (100%) after normalization with respect to 36B4 RNA levels.
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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.


Fig. 2. 9C-RA effect on TPA-stimulated stromelysin-3 and interstitial collagenase RNA expressions in HFL1 fibroblasts. HFL1 fibroblasts cultured in serum-free conditions were left untreated or pretreated for 24 h with 9C-RA concentrations as indicated and subsequently incubated in the presence of TPA (10 ng/ml) or vehicle alone for additional 24 h. Total RNA (10 µg) was analyzed by Northern blot for ST3, interstitial collagenase (Int. Col.) and 36B4 expression, as described under "Materials and Methods."
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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 Fibroblasts

To 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).


Fig. 3. Induction of stromelysin-3 synthesis and secretion by RA in HFL1 fibroblasts. Culture media conditioned by HFL1 fibroblasts exposed to either 1 µM 9C-RA or 1 µM t-RA in serum-free conditions for 3 days were collected prior to being concentrated 100-fold by ammonium sulfate precipitation. Western blot analysis was performed by using monoclonal antibody 5ST-4C10 raised against the stromelysin-3 catalytic domain and enhanced chemiluminescence, as described under "Materials and Methods." Molecular mass markers (kDa) are indicated on the left.
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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).


Fig. 4. Run-on transcription assays with nuclei prepared from HFL1 fibroblasts exposed to 9C-RA. Nuclei were isolated from cells incubated in the absence or presence of 1 µM 9C-RA for 3 days. Transcripts were labeled by in vitro elongation reaction in the presence of [alpha -32P]UTP, as described under "Materials and Methods." Equal amounts of newly synthesized RNA (5 × 106 cpm) were hybridized to cDNAs immobilized on nylon filters. These cDNAs were those for interstitial collagenase (Int. Col.), ST3, and 36B4 cloned into the pBluescript II SK+ vector and the pBluescript plasmid itself. The data are representative of one of three independent experiments.
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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).


Fig. 5. Comparative expression of stromelysin-3 and interstitial collagenase RNAs in response to 9C-RA in HFL1 fibroblasts and RD rhabdomyosarcoma cells. Total RNA (10 µg) from HFL1 and RD rhabdomyosarcoma cells grown in 5% calf serum and treated with 1 µM 9C-RA for the indicated times was analyzed by Northern blot for ST3, interstitial collagenase (Int. Col.), and 36B4 expression, as described under "Materials and Methods."
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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-Delta 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 RARbeta 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.


Fig. 6. Ligand-dependent transactivation of the stromelysin-3 gene promoter by endogenous retinoic acid receptors in RD rhabdomyosarcoma cells. RD rhabdomyosarcoma cells exposed for 2 days to 1 µM 9C-RA or ethanol vehicle were transfected with 10 µg of the indicated CAT reporter plasmids. Control and treated cells were then harvested after an additional incubation of 2 days in the presence of 1 µM 9C-RA. For the DR1-tk-CAT construct, the effect of 9C-RA was also determined after 2 days of treatment following transfection but without preincubation in the presence of 9C-RA. CAT activities were measured as described under "Materials and Methods" and are representative of one of three independent experiments.
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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 RARalpha , RARgamma , and RXRalpha 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 RARbeta , RXRbeta , and RXRgamma 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 RARalpha , RARgamma , and RXRalpha are the predominant receptors expressed in human skin (50) as well as in various human cell lines (51-53). The expression of RXRalpha was only slightly increased (less than 2-fold) in cells treated with either 9C-RA or t-RA, whereas RARalpha and RARgamma levels remained unaffected. In contrast, RARbeta 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 RARbeta was induced to a lower extent than in serum-free conditions (data not shown).


Fig. 7. Expression of RARs and RXRs in HFL1 fibroblasts. HFL1 fibroblasts were cultured in serum-free conditions in the presence or absence of 1 µM 9C-RA or t-RA for 3 days. Total RNA (20 µg) was analyzed by Northern blot for RARalpha , RARbeta , RARgamma , RXRalpha , and 36B4 expression, as described under "Materials and Methods." Autoradiographs were exposed for 16 h (RXRalpha ), 1.5 days (RARalpha and RARgamma ), 3 days (RARbeta ), and 4 h (36B4).
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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 RARalpha and RARgamma , respectively (37), BMS753, a pure RARalpha 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 RARalpha 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.


Fig. 8. Comparative expression of stromelysin-3 and interstitial collagenase genes in HFL1 fibroblasts exposed to selective retinoids. The effects of natural and synthetic retinoids on ST3 gene induction (A and B) and interstitial collagenase (Int. Col.) gene repression (C and D) were analyzed by Northern blot as described under "Materials and Methods." HFL1 fibroblasts cultured in 5% calf serum were treated for 3.5 days with RARalpha - (Am80 and BMS753; panels A and C) or RARgamma - (CD666; panels B and D) specific synthetic retinoids and/or the pan-RXR agonist BMS649, used at the indicated concentrations. The two natural isomers 9C-RA and t-RA were used at 1 µM. Relative levels of RNA transcripts, as evaluated by using a bioimaging analyzer (BAS 2000; Fuji Ltd.) for each blot after normalization with 36B4 RNA levels, are presented in the histograms. The induction of stromelysin-3 RNA is expressed relative to that observed in the presence of 9C-RA, while the repression of interstitial collagenase RNA is expressed relative to its level in untreated cells.
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Taken together, our observations indicate that while the selective activation of RARalpha , RARgamma , 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.


DISCUSSION

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-beta 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 RARbeta 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 RARalpha , RARgamma , and RXRalpha RNAs were constitutively expressed at high levels. In contrast, we could not detect any RNA for RXRbeta and RXRgamma , while that for RARbeta 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 (RARalpha ·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 RARbeta and/or RARgamma could interact with RXRs for an optimal stromelysin-3 induction. In keeping with the fact that RXRalpha seems to be the major RXR expressed in fibroblasts, we can conclude from our results that the two heterodimers RARbeta ·RXRalpha and/or RARgamma ·RXRalpha 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.


FOOTNOTES

*   This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Centre Hospitalier Universitaire Régional, the Bristol-Myers Squibb Pharmaceutical Research Institute, the BIOMED 2 (contract BMH4CT96-0017) and BIOTECH 2 (contract ERBBIO4CT96-0464) programs, the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer and the Comité du Haut-Rhin, the Fondation pour la Recherche Médicale Française, the Fondation de France, and a grant from the Fondation Jeantet (to P. Chambon).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP163, 67404 Illkirch cedex, France. Tel.: 33 388 653421 or 33 388 653425; Fax: 33 388 653201; E-mail: anglard{at}titus.u-strasbg.fr.
1   The abbreviations used are: MMP, matrix metalloproteinase; CAT, chloramphenicol acetyltransferase; DR1, directly repeated consensus hexameric half-sites (5'-PuG(G/T)TCA-3') separated by one base pair; DR5, directly repeated consensus hexameric half-sites (5'-PuG(G/T)TCA-3') separated by five base pairs; RA, retinoic acid; 9C-RA, 9-cis-retinoic acid; t-RA, all-trans-retinoic acid; RARE, retinoic acid-responsive element; RAR, retinoic acid receptor; RXR, retinoid X receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ST3, stromelysin-3; SSC, standard sodium citrate; kb, kilobase pair(s).
2   P. Anglard, unpublished results.

ACKNOWLEDGEMENTS

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.


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