©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Promoter Characterization of the Human Stromelysin-3 Gene (*)

(Received for publication, February 1, 1995; and in revised form, May 15, 1995)

Patrick Anglard (1)(§) Thomas Melot (2) Eric Guérin (1) Gilles Thomas (2) Paul Basset (1)

From the  (1)Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP163, 67404 Illkirch Cedex, C.U. de Strasbourg, France and the (2)Laboratoire de Génétique des Tumeurs, Unité U434 INSERM, Institut Curie, 26 rue d'Ulm, 75231 Paris Cedex 05, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the present study, we have isolated the human stromelysin-3 (ST3) gene which encodes a matrix metalloproteinase (MMP) expressed in fibroblastic cells of tissues associated with intense remodeling. The gene was found to span 11.5 kilobases (kb) including 8 exons and 7 introns. The genomic organization of ST3 gene exons is well conserved compared to other members of the MMP family, except for the 3 last exons corresponding to the hemopexin-like domain and to a long 3`-untranslated region. The transcription initiation site was located 31 nucleotides downstream of a TATA box. Analysis of 1.4 kb of 5`-flanking DNA sequence in the ST3 gene promoter revealed the presence of putative regulatory elements, but no consensus sequence for AP1-binding site in contrast to other MMP promoters. However, a specific cis-acting retinoic acid responsive element of the DR1 type was identified in the proximal region(-385) of the ST3 gene promoter. Transient transfection experiments demonstrated that a minimal promoter activity could be modulated by various sequences within the 3.4 kb of 5`-flanking region, and that the ST3 promoter was transactivated by retinoic acid receptors in the presence of retinoic acid. These findings indicate that the human ST3 gene promoter is characterized by structural and functional features which differ from those previously described in other MMP promoters, and further supports the possibility that ST3 gene expression is controlled by specific factors during tissue remodeling.


INTRODUCTION

The stromelysin-3 (ST3) (^1)gene was initially identified by differential screening of a breast cancer cDNA library, as a gene specifically expressed in fibroblastic cells surrounding invasive cancer cells of breast carcinomas(1) . More recently, its expression was identified in most other types of human invasive carcinomas, and also in some precursor lesions known to have a high probability to evolve toward invasion(2, 3) . Furthermore, ST3 expression was associated with tissue remodeling in physiological conditions such as embryonic development(4) , amphibian metamorphosis (5) , wound healing(6) , mammary gland involution(7) , and cycling endometrium(8) . Although ST3 belongs to the matrix metalloproteinase (MMP) family(9, 10) , its amino acid sequence suggests that it may be distinct in its properties from previously described MMPs(1, 11) . Indeed, ST3 has been found to exhibit specific enzymatic properties as well as a specific activation pattern suggesting that despite its name, ST3 should be considered as the first member of a new MMP subgroup(12, 13) .

Most MMP promoters including those of the interstitial collagenase(14, 15) , stromelysin-1 and -2(16) , matrilysin(17) , and gelatinase B (18) genes are characterized by the presence of an AP1-binding site, also called TRE (for TPA responsive element), which has been identified in a very conserved location within the first 80 bp upstream of the transcription start. Since this AP1 motif has been shown to confer responsiveness to various stimuli including oncogenes(19) , growth factors(20, 21) , and cytokines(22, 23, 24) , it has therefore been proposed to play a major role in the induction (or repression) of transcription from various MMP gene promoters(9, 10, 25) .

Although previous studies have shown that ST3 expression in tissues other than the placenta and the spinal cord during embryonic development was restricted to fibroblastic cells(1, 2, 3, 4, 5, 6, 7, 8) and could be induced in these cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) or by basic fibroblast growth factor(1) , very little is known on the mechanism by which this expression is regulated. Thus, in order to study the mechanism by which high levels of ST3 expression could be achieved in tissues, we first isolated the human ST3 gene together with its 5`-flanking sequence. We describe here the gene structure and organization as well as the gene promoter sequence in comparison to other MMP genes, and analyze the promoter region to identify putative regulatory elements that may play a role in the regulation of ST3 gene transcription. We show in the present study that the ST3 gene promoter markedly differs from previously described MMP promoters by the absence of a consensus AP1-binding site and the presence of a functional retinoic acid responsive element (RARE) which can be transactivated by retinoic acid receptors in the presence of retinoic acid (RA).


MATERIALS AND METHODS

Genomic Cloning and DNA Sequencing of the ST3 Gene

The human chromosome 22 specific library LL22NC01 (26) constructed in the Lawrence Livermore Laboratory (Livermore, CA) was screened with an [alpha-P]dCTP-labeled human ST3 cDNA probe (ZIV, nucleotides +360 to +2118 numbered from the transcription start). Two cosmids 111E10 and 77A2 were identified and mapped by restriction enzyme digestion and Southern blot hybridization using [-P]dATP-labeled oligonucleotides designed according to the known ST3 cDNA sequence. Relevant restriction fragments generated from these cosmids were subcloned into the pBluescript II SK+ plasmid vector. Minipreparations of plasmid DNAs which have been further purified by NaCl and polyethylene glycol 6000 precipitation were sequenced with Taq polymerase using universal and/or reverse primers, or internal primers. Dye-labeled ddNTP were used for detection on an Applied Biosystems 373A automated sequencer. Nucleic acid sequence homology searches were performed using the FASTA programs of the GCG sequence analysis package and the combined GenBank/EMBL data bases.

Southern Blot Analysis

DNA from normal human kidney and from chromosome 22 cosmids was extracted as described previously(27) . 10 µg of human total genomic DNA and 0.5 µg of cosmid DNA were digested with appropriate restriction enzymes, under the conditions recommended by the supplier (New England Biolabs). Digested DNA was separated by agarose gel electrophoresis and transferred to Hybond N nylon membranes (Amersham Corp.). Hybridization to [alpha-P]dCTP probes was then performed as described previously (27) and filters were washed under stringent conditions (0.1 standard sodium citrate and 0.1% sodium dodecyl sulfate) for 1 h at 60 °C, and exposed for autoradiography.

Primer Extension Analysis and S1 Nuclease Mapping

The transcriptional start site was mapped by primer extension and S1 mapping using a 20-mer oligodeoxynucleotide OU85 (5`-TGGAGCAGCAGCAGCAGCAT-3`) complementary to nucleotides +77 to +96 in the ST3 gene sequence (underlined in Fig. 4). In primer extension experiments, this [-P]dATP-labeled oligonucleotide was annealed at 30 °C with 10 µg of total human placenta RNA or 10 µg of yeast tRNA in 30 µl of hybridization buffer consisting of 40 mM PIPES, pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% formamide. After ethanol precipitation, the hybridized primer was extended at 42 °C for 1.5 h, using 40 units of avian myeloblastosis virus reverse transcriptase in 50 mM Tris-HCl, pH 8.3, 8 mM MgCl(2), 30 mM KCl, 2 mM of each dNTP, and 10 mM dithiothreitol. The extended product was incubated with 1 µg of RNase A for 30 min at 37 °C. In S1 nuclease protection assays, oligonucleotide OU85 was hybridized to single-stranded DNA derived from a 644-nucleotide BamHI genomic fragment spanning the first gene exon (Fig. 1) and subcloned into the pBluescriptII SK+ plasmid. Extension was carried out using the Klenow enzyme in the presence of [alpha-P]dCTP. The synthesized double-stranded DNA fragment was then digested with TaqI restriction enzyme at position -153 (Fig. 4) and the resulting 249-nucleotide single-stranded radioactive probe was isolated from a 6% denaturating polyacrylamide gel. For hybridization, this probe (100,000 cpm) was hybridized with 10 µg of total human placenta RNA at 50 °C for 16 h, and S1 nuclease reaction carried out at 25 °C for 1 h(28) . Yeast tRNA (10 µg) was used in parallel experiments as a control. The primer-extended products and the protected fragments from S1 nuclease assays were run on a sequencing gel along with sequencing reactions of the 644-bp BamHI genomic fragment, using the same oligonucleotide OU85 as a primer. Sequencing reactions were performed with Sequenase and 7-deaza-dGTP (U. S. Biochemical Corp.).


Figure 4: Nucleotide sequence of the 5`-flanking region of the stromelysin-3 gene. The numbering of nucleotides starts at the transcription initiation site (+1) which is indicated by a bent arrow. The TATA box and putative regulatory elements are boxed. The RARE consists of the direct repeat of 2 core motifs which are boxed. The orientation of each core motif PuGGTCA and that of additional identical motifs observed at position -1195, -730, and -215, is indicated by an arrow. Relevant restriction enzyme sites, the translation initiation site (ATG), the sequence of oligonucleotide OU85 used for primer extension and S1 mapping experiments, and complementary regions (nucleotides -1409 to -1189) and (nucleotides -731 to -511) are underlined.




Figure 1: Structure of the human stromelysin-3 gene. A, the relative positions of two overlapping genomic cosmids 77A2 and 111E10 are shown in the top two lines in relationship with the schematic structure of the ST3 gene in the bottom line. Vertical bars represent HindIII and XbaI restriction endonuclease sites as they are indicated in the bottom line. Exons are numbered from the 5`-end of the gene and depicted by black boxes. Closed and open boxes represent the coding and the non-coding regions, respectively. Introns as well as flanking regions are depicted by interconnecting solid lines. B, the restriction map of the cosmid 111E10 is represented in the upper line. This map is interrupted 3` to a HindIII restriction site located in the first intron and marked with an asterisk (*) in panels A and B. The lower line extends this map between the two indicated XbaI restriction sites.



Construction of Promoter-CAT Reporter Genes

Fragments from the ST3 gene 5`-flanking sequence were inserted into either pBLCAT6 (promoterless) or pBLCAT5 (with herpes simplex virus tk-promoter) vectors(29) . The 0.29ST3-CAT and 0.45ST3-CAT constructs were generated by polymerase chain reaction (PCR) amplification from a 5.5-kb XhoI/NotI genomic DNA template spanning the 5`-end of the ST3 gene (Fig. 1), using 5` primers corresponding to 5`-GACTCCTCTCAGACTCTAGACAAGTC-3` (nucleotides -304 to -278) and to 5`-ACACCACTGCACTCCTGCCTGGG-3` (nucleotides -560 to -538), respectively. In both cases, the same 3` PCR primer corresponding to 5`-AGCCATCCGCCTCGAGGCTGCTGGGCTT-3` (complementary to nucleotides +28 to +1) was used. The two amplified fragments were sequenced, and then digested with XbaI within the 5`-PCR primer and within the promoter sequence at nucleotide -450, for the 0.29- and 0.45-kb fragments respectively, and with XhoI within the 3` PCR primer, before subcloning into the pBLCAT6 vector digested with the same restriction enzymes. The 1.47ST3-CAT and 3.40ST3-CAT constructs were both generated by inserting genomic fragments 5` to the 0.45ST3-CAT construct. For the 1.47ST3-CAT construct, a 1-kb BamHI/XbaI fragment (nucleotides -1468 to -451) was first subcloned into pBluescript II, and then digested with HindIII (within the vector polylinker) and with XbaI, before subcloning into the 0.45ST3-CAT construct digested with the same restriction enzymes. For the 3.40ST3-CAT construct, a 2.95-kb SphI/XbaI fragment ending at nucleotide -451 was similarly subcloned into the 0.45ST3-CAT construct. The 1.02 ST3-tk/CAT, 1.93 ST3-tk/CAT, and 2.95 ST3-tk/CAT constructs were generated by subcloning the 1.02-kb HindIII/XbaI fragment described above, a 2-kb SphI/BamHI fragment ending at nucleotide -1469, and the 2.95-kb SphI/XbaI fragment ending at nucleotide -451 into the pBLCAT5 vector, respectively.

Polymerase Chain Reaction

The PCR reaction mixture contained 250 mM of each dNTP, 25 pmol of each amplimer, 10 ng of template DNA, 1 µl of Taq polymerase (Perkin Elmer Cetus), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl(2), in a total volume of 100 µl overlaid with 50 µl of mineral oil. The PCR reaction was performed (Perkin Elmer Thermal Cycler) with a first cycle of 94 °C for 3 min, 50 °C for 1 min and 72 °C for 1 min, followed by 4 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min. Amplification was further carried on with 20 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, with a last cycle having an additional 10-min extension time at 72 °C.

Cell Transfection and CAT Assays

Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% delipidated fetal calf serum (stripped of hormones). Calcium phosphate procedure was used to transfect the cells 4 h after plating. The amount of ST3-CAT reporter constructs and of receptor expression vectors used are indicated in the figure legends. beta-Galactosidase expression vector (pCH110, Pharmacia, 0.5 µg) was used as an internal control to normalize for transfection efficiency. The total amount of DNA transfected per 10-cm diameter culture dishes was made up to 15 µg with pBluescribe plasmid DNA. The culture medium was changed after 15 h and 9-cis-RA added to the concentrations indicated in the figure legends. After an additional period of 24 h, the cells were harvested and disrupted by 3 freeze-thaw cycles. Cell extracts containing 10-20 units of beta-galactosidase activity were used for CAT assays. The reaction products were separated by thin chromatography (TLC plastic sheets precoated with silica gel, Merck) and visualized by autoradiography. Densitometry analysis was carried out by using a Bio-Image Analyser (Fujix Bas 2000). RARalpha and RXRalpha expression vectors were provided by Dr. C. Stricker (Laboratoire de Génétique Moléculaire des Eucaryotes, Strasbourg), while c-fos and c-jun expression vectors were gifts from Dr B. Wasylyk (Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)). Other constructs including dnRARalpha, the human interstitial collagenase promoter (-517/+63-CAT) and the rat stromelysin-1 (84-CAT) constructs, and the DR1G-tk-CAT reporter plasmid were gifts from Dr. H. Gronemeyer (IGBMC).


RESULTS

Characterization of ST3 Genomic Clones

Two overlapping clones (77A2 and 111E10) have been isolated by screening a human chromosome 22 cosmid library (26) with a ST3 cDNA probe (ZIV) extending from nucleotides +360 to +2118. These cosmid clones were analyzed by restriction mapping and Southern blot hybridization using a variety of 5`-[-P]dATP-end-labeled oligonucleotides designed according to the known ST3 cDNA sequence. Cosmid 77A2 spanned 8 kb of ST3 genomic DNA, encompassing all exons except the first one (Fig. 1A). The cosmid 111E10 was shown to span about 26 kb of genomic DNA including the entire structural gene, 9 kb of 5`-flanking region, and 5 kb of 3`-flanking sequences. The restriction map of the ST3 genomic DNA as deduced from the analysis of the 2 cosmids is shown in Fig. 1B.

In order to further confirm the general organization of the ST3 gene and its flanking regions, we have compared the Southern blot hybridization pattern of digested cosmid 111E10 DNA to that of total genomic DNA from normal human tissue (Fig. 2). Total human genomic DNA and cosmid genomic DNAs were digested with HindIII (H), BglII (B), and XbaI (X) prior to being hybridized with a 0.5-kb genomic 5`-probe and a 1.76-kb cDNA probe. The 5`-genomic probe (BN0.5) was a BamHI-NotI restriction fragment overlapping the 5`-end of the first exon (Fig. 1A), while the 3`-probe was the ST3 ZIV cDNA fragment previously used for the library screening. The sizes of the restriction fragments revealed by both probes were identical for both DNAs, except for the HindIII fragment revealed by the cDNA probe showing a size of 11 kb in total human genomic DNA and of 10.5 kb in cosmid DNA (Fig. 2). While both of these HindIII fragments include the 3`-end of the gene, the cosmid HindIII DNA fragment was generated from a 3`-HindIII restriction site present in the cosmid polylinker, thereby explaining the 0.5-kb difference in size.


Figure 2: Southern blot hybridization of human stromelysin-3 gene fragments. 10 µg of total human genomic DNA and 0.5 µg of cosmid DNA (111E10) have been digested with the restriction enzymes HindIII (H), BglII (B), and XbaI (X) as indicated. The 5`-probe (BN0.5) used for hybridization was a 0.5-kb BamHI-NotI restriction fragment (nucleotides -450 to +51) containing 450 bp of the gene promoter and the first 50 bp of the first exon. The 3`-probe (ZIV) was a 1.76-kb cDNA fragment (nucleotides +360 to +2118) containing most of the ST3 cDNA from the third to the last exon, including part of the 3`-untranslated sequence. The sizes of restriction fragments detected with each probe are indicated in kb. They are identical to those defined by the restriction map in Fig. 1, except for the 10.5-kb HindIII fragment from cosmid 111E10 for which the 3` HindIII restriction site is located within the vector polylinker. Note that the 5.5-kb BglII DNA fragment hybridized with the ZIV probe corresponds to a fragment at the 3`-end of the ST3 gene which includes the last exon, and which is 3` to the 2.8-kb BglII fragment (see also Fig. 1B).



Exon/Intron Junctions

Relevant segments of the 2 cosmids were subcloned into the pBluescript II/SK+ vector in order to determine the exon/intron boundaries as well as their precise size by direct sequencing, using synthetic oligonucleotide primers which were synthesized on the basis of the cDNA sequence. The whole gene was sequenced except the first 6.1-kb intron whose size was determined by restriction mapping and which was only sequenced at both extremities. This analysis demonstrated that the entire gene spans 11.5 kb and consists of 8 exons and 7 introns (Fig. 1A and Table 1). The exons and introns range in size from 130 to 905 bp and from 92 bp to 6.1 kb, respectively. Based on this analysis, the size of the transcribed RNA is 2260 bp including a 5`-untranslated region of 22 bp and a large 3`-untranslated region of 773 bp.



Mapping of the Transcription Start Site and of Putative Regulatory Elements in the 5`-Flanking Gene Sequence

The transcription start site was determined by primer extension and S1 nuclease mapping with RNA recovered from human placenta, a tissue known to express high ST3 RNA levels(1) . The 20-mer oligonucleotide OU85 corresponding to genomic DNA between nucleotides +77 and +96 and complementary to the ST3 mRNA was used as a primer (see ``Materials and Methods''). Primer extension resulted in one band, indicating that one single site is used for the start of transcription (Fig. 3). This site (+1) is located 22 bp upstream from the translation initiation codon (ATG) and was further confirmed by the S1 nuclease experiment resulting in the protection of a fragment of the same size as that demonstrated by primer extension (Fig. 3). The sequence C(-1)A(+1) at the transcription start site (Fig. 4) has been found in the majority of eucaryotic promoters analyzed(30) .


Figure 3: Determination of the transcription initiation site of human stromelysin-3 gene. The transcription start site has been mapped by using both primer extension and S1 nuclease protection assays. For primer extension reaction, a 20-mer oligonucleotide (OU85) complementary to the human ST3 mRNA (nucleotides +77 to +96) was [-P]dATP-end-labeled, hybridized with 10 µg of total RNA from human placenta or 10 µg of yeast tRNA and reverse transcribed. For S1 mapping, a 249-nucleotide single strand DNA complementary to nucleotides -153 to +96 was generated as described under ``Materials and Methods,'' and hybridized with 10 µg of total RNA isolated from human placenta or 10 µg of yeast tRNA, before S1 nuclease reaction. The S1 nuclease protected fragment as well as the primer extended product were run on a sequencing gel along with the sequencing reactions of the BamHI ST3 gene fragment overlapping the first exon, using the oligonucleotide OU85 as a primer. Lane1, primer extension with tRNA, and lane 2, with human placenta total RNA. Lanes 3-6, the nucleotides of the sequencing reactions as they are indicated at the top of the Fig. 7, S1 nuclease reaction with human placenta total RNA, and lane 8, with tRNA. Arrows indicate the position of the transcription start site.




Figure 7: Comparison of exon structure of the human stromelysin-3 gene with those of other matrix metalloproteinases. Exons in the human gelatinase A(31) , human gelatinase B(18) , human interstitial collagenase(53) , rat stromelysin-1(54) , and human matrilysin (17) genes are indicated by boxes with their size in base pairs. Exon regions corresponding to homologous protein domains are aligned and consist, respectively, from left to right of those for peptide signal or pre-domain (solid boxes), pro-domain (light diagonallystriped boxes), catalytic domain (dark diagonallystriped boxes), and hemopexin-like domain (gray boxes). The exons corresponding to additional domains such as the fibronectin-like domain (light horizontallystriped boxes) of gelatinase A and B genes, to the collagen V domain of gelatinase B gene (light vertically striped box), and to carboxyl-terminal amino acids in the matrilysin gene (dark vertically striped box) are also indicated. Open boxes represent 5`- and 3`-untranslated sequences.



Previous analysis of MMP gene promoter sequences have shown that most of them share a TATA box as well as transcriptional regulatory elements such as PEA3 elements, transforming growth factor-beta inhibitory elements, and AP1-binding sites(17) . In the case of the ST3 gene promoter, a TATA box and one PEA3 consensus sequence (C/AGGAA/T) were identified clustered between nucleotides -39 and -27, but no consensus sequences for transforming growth factor-beta inhibitory elements or AP1-binding site were found within the first 1.4 kb of 5`-flanking region (Fig. 4). However, an AP1-like motif (TGTGTCA) differing by 1 bp from the consensus sequence was found at position -461 (Fig. 4). Also as was the case for the human gelatinase B and A gene promoters(24, 31) , several copies of GT (5`-GGGGTGGGG-3`) and GC (5`-GGGCGG-3`) boxes were found between nucleotides -425 and -20. Additional sequences similar to that of the nuclear factor-1 binding motif (5`-TGGN(7)CCA-3`)(32), at position -270, and a silencer binding site (3`-TTTTAATA-5`) (33) at position -815 were found in the ST3 gene promoter but have not been reported in other MMP gene promoters. Further upstream we identified a 221-bp region (nucleotides -1409 to -1189) showing 85% sequence complementarity with another proximal region (nucleotides -731 to -511). Finally, and most importantly, a RARE was observed at position -385, consisting of a direct repeat of the motif 5`-PuGGTCA (Pu stands for purine residue) (34) with one G intervening nucleotide. Three other copies of this motif were also found at positions -215, -730, and -1195 (Fig. 4).

Characterization of Putative Transcriptional Promoter Elements

We next analyzed ST3 gene promoter activity by a transient transfection method in order to define the DNA elements that direct gene expression. The reporter plasmid pBLCAT6 (promoter less) was used to examine the expression of the bacterial choline acetyltransferase (CAT) under the control of various lengths of ST3 gene 5`-flanking sequences. These chimeric constructs were transfected into fibroblastic COS-1 cells by the DNA-calcium phosphate co-precipitation method and CAT activities were determined in cellular extracts prepared 48 h after transfection. The lowest basal CAT activity was observed for the smallest construct 0.29ST3-CAT, indicating the presence of a minimal promoter in the first 290 bp of this 5`-flanking region (Fig. 5A). There was a 3-fold stimulation of CAT activity recovered from COS-1 cells transfected with the largest construct 3.40ST3-CAT, over the basic promoter activity observed with the 0.29ST3-CAT construct. In this 3.4-kb DNA sequence, we found two regions whose deletion resulted in a decrease in CAT activity. The first region of 1.93 kb (nucleotides -3400 to -1469) encompassed the most distal region of the 3.4-kb DNA fragment and the second region of 0.16 kb (nucleotides -451 to -290) contains the RARE. In contrast, another 1.02-kb DNA fragment (-1469 to -451) does not seem to correspond to a positive regulatory region since almost no change in CAT activity was observed between the 1.47ST3-CAT and the 0.45ST3-CAT constructs (Fig. 5A).


Figure 5: Human stromelysin-3 gene promoter activity and identification of regulatory regions. A, schematic representation of DNA constructions containing various lengths of ST3 promoter linked to the CAT gene (pBLCAT6) and B, linked to the tk/CAT promoter (pBLCAT-5). Each DNA fragment subcloned into CAT reporter plasmids is defined by its position in the ST3 gene promoter relative to the transcription start (+1). 5 µg of each construct was transfected into COS-1 cells by the calcium phosphate co-precipitation method. Forty-eight hours after transfection, cells were harvested and CAT activity was determined as described under ``Materials and Methods.'' Averaged values of the resulting CAT activities from four experiments are presented in the corresponding histograms.



To further characterize these cis-acting regulatory DNA sequences, we examined whether they could modulate the activity of the heterologous thymidine kinase (tk)-promoter present in the pBLCAT5 vector. The highest level of CAT expression relative to the tk-promoter activity was found for the 1.93ST3-tk/CAT construct, confirming the presence of an enhancer in the distal region located upstream to nucleotide position -1469 (Fig. 5B). Interestingly, the 1.02-kb fragment nucleotides (-1469 to -450) which did not by itself modify the tk-promoter activity could abolish the enhancer activity present in the 1.93-kb region when ligated at its 3`-end to generate the 2.95ST3-tk/CAT construct (Fig. 5B), indicating the presence of negative regulatory sequences in the 1.02-kb region between -1469 and -450. Very similar relative CAT activities have been observed by transfecting the ST3 gene promoter constructs into epithelial HeLa cells (data not shown).

Since the AP1-like motif (TGTGTCA) found at position -461 (Fig. 4) has been shown to have a binding affinity for AP1 proteins although much lower than that observed for the consensus sequence(35) , we attempted to find out whether the various lengths of ST3 gene promoter inserted into pBLCAT-6 and pBLCAT-5 vectors could differentially respond to TPA when transfected into COS-1 or HeLa cells. We also performed experiments where c-fos and c-jun expressing vectors were cotransfected with the ST3-CAT constructs and used the 2 AP1-containing promoters of the interstitial collagenase (nucleotides -517 to +63) (14) and of the stromelysin-1 (nucleotides -84 to +8) (36) genes as positive controls. Significant stimulation of the interstitial collagenase and stromelysin-1 promoters were observed in the transfected cells exposed to TPA or cotransfected with c-fos and c-jun expression vectors. However, no response to TPA and no transactivation by c-fos and c-jun were observed for the ST3 gene promoter. Importantly, the absence of 3.40ST3-CAT response to TPA was also observed using HFL1 human fibroblasts known to express the endogenous ST3 gene(1) , while the interstitial collagenase promoter (nucleotides -517 to +63) was responding to TPA in these fibroblasts (data not shown).

Functional Characterization of a Retinoic Acid Responsive Element

DNA constructs containing various lengths of ST3 gene promoter inserted into the pBLCAT6 vector were tested for RA inducibility following transfection into COS-1 cells together with an expression vector expressing retinoid X receptor-alpha (RXRalpha)(37, 38) . Activity of the 3.40ST3-CAT construct was induced (3.5 ± 0.6-fold; mean of four independent experiments) by the addition of 10M 9-cis-RA (Fig. 6). The two constructs, 1.47ST3-CAT and 0.45ST3-CAT, were also inducible (2.3 ± 0.3- and 2.0 ± 0.3-fold, respectively). However, the deletion of an additional 160 bp containing the RARE almost eliminated RA inducibility as shown by the CAT expression observed for the O.29ST3-CAT construct (1.3 ± 0.3-fold). No RA inducibility was found for the parental promoterless CAT expression vector pBLCAT6, while in contrast, a synthetic RARE consisting of 2 repeated motifs, 5`-PuGGTCA, separated by 1 G inserted upstream of the tk-promoter (DR1G-tk-CAT) (39) was induced 5 ± 0.4-fold by 10M 9-cis-RA in the presence of RXRalpha (Fig. 6). The 0.45ST3-CAT construct was similarly induced by 9-cis-RA in the presence of RARalpha (2.3 ± 0.2-fold) as it was with RXRalpha. In contrast, no transactivation was observed with the receptor dnRARalpha which has lost its ligand binding domain(40) . Finally, the effect of various 9-cis-RA concentrations ranging from 10 to 5 10M was tested on the level of CAT activity obtained from the 0.45ST3-CAT construct cotransfected with RXRalpha expression vector. The stimulation of 0.45ST3-CAT activity induced by 9-cis-RA in COS-1 cells was concentration dependent, having a median effective dose of 10M (data not shown).


Figure 6: Stimulation of stromelysin-3 gene promoter activity by retinoic acid. COS-1 cells were transfected with 3 µg of DR1-tk-CAT (positive control), or 5 µg of various ST3-CAT constructs (see Fig. 5for definition) in the presence of 0.5 µg of RXRalpha or RARalpha or dnRARalpha (which has lost its ligand binding domain) expression plasmids. After transfection, cells were treated with 9-cis-RA for 24 h and CAT activity was determined. Representative CAT assays as well as histograms corresponding to averaged values from at least three independent experiments are shown (open boxes, no RA; filled boxes, 10M 9-cis-RA). Statistical evaluation was performed using Student's t test for stimulation folds in the presence of 9-cis-RA over absence of RA; *, p < 0.1;**, p < 0.001).




DISCUSSION

While high levels of ST3 gene expression have been associated with a number of physiological and pathological conditions(1, 2, 3, 4, 5, 6, 7, 8) , the molecular mechanism by which this expression is regulated remains unknown. In order to initiate the study of elements involved in this regulation, we have isolated the whole ST3 gene with its 5`-flanking region. We describe here the structural organization of the gene and the first characterization of the ST3 gene promoter in comparison to other members of the MMP gene family. The transcription start site has been determined and promoter sequences that drive the basic transcription of the ST3 gene have been located between positions -290 and +13. In addition, we have shown that this basic promoter activity can be modulated by various 5`-flanking regions and more specifically by a RARE conferring promoter inducibility in the presence of RA together with its receptors.

The comparison of the structural organization of the ST3 gene to that of other members of the MMP family demonstrates both differences and similarities. While all MMP genes, except the matrilysin gene which is the smallest and most fundamental member of the family(17) , are composed of 10 or more exons, the ST3 gene has only 8 exons with a size of 11.5 kb including an unusually large first intron of 6.1 kb. In addition, the ST3 gene has a large last exon of 905 bp which includes a 3`-untranslated region of 773 bp. This region is not conserved among MMP genes but is very similar in size to that of the gelatinase A gene (31) . Despite these differences, the ST3 gene belongs to the MMP family based on structural similarities of gene parts which encode homologous protein domains (Fig. 7). The 3 amino-terminal protein domains (pre-, pro- and catalytic domains) are contained within the first 5 exons, and are conserved in all members of the MMP family for which the genomic structure has been determined. In the ST3 gene, as in the other MMP genes, exon 1 contains the pre-domain and a portion of the pro-domain, while exon 2 encodes the remainder of the pro-domain and the amino-terminal portion of the catalytic domain which is spread over exons 2 to 5. In contrast, the genomic structure of the carboxyl-terminal hemopexin-like domain present in the ST3 gene markedly differs from that of its counterparts. In other MMP genes except the matrilysin gene, this domain is encoded by 5 exons of relatively conserved sizes with the exception of the 3`-untranslated region. In the case of the ST3 gene, although the hemopexin-like domain has a similar size to that of other MMPs, it is encoded by only 4 exons including 2 exons of larger sizes and 2 other exons which are shared with the catalytic domain and the 3`-untranslated region, respectively. Since the MMP family members are supposed to arise by the shuffling of relatively conserved exons into duplicated genomic sequences(41, 42) , the specific structure of the ST3 hemopexin domain suggests that it might have a different origin.

Sequence analysis of the 5`-flanking region of the ST3 gene revealed that it contains various putative regulatory elements providing insights into the pattern of regulation of ST3 gene expression. Of these elements, the GT boxes and the PEA3 motif have been shown to play a role in the regulation of some MMP genes. Several copies of GT box have been recently identified by Sato et al.(24) in the gelatinase B gene promoter, and they seem to be essential for promoter transactivation by the v-sarc oncogene(24) . The PEA3 motif is a binding site for products of the Ets gene family and has been demonstrated to mediate activation by oncoproteins such as Ha-Ras in interstitial collagenase and stromelysin-1 gene promoters(19, 43) . However, no transactivation by c-Ets-1 was found for the PEA3 containing 0.29ST3-CAT construct. (^2)

Besides the identification of these candidate regulatory elements, we have also shown that the minimal ST3 promoter activity contained between nucleotides -290 and +13 could be modulated by various 5`-flanking regions, including two positive and one negative regulatory regions. The first positive region confined between nucleotide -450 and -290 contains a RARE, while the second is more distal (nucleotides -3400 to -1470). This latter region can be silenced by its contiguous 1.02-kb 3`-flanking DNA fragment whose silencing activity may result from the presence of a ``Silencing Binding Site 2,'' originally identified as a negative regulatory element in the promoter of the major histocompatibility complex class 1 gene PD1, and at a similar position with respect to the transcription start(33) . Alternatively, the existence in this 1.02-kb fragment of two almost complementary regions of 221 bp each, by having the potential to generate a cruciform structure, may also play a role in the silencing activity of this 1.02-kb DNA fragment(44) . Sequence homology searches revealed that one of these complementary regions (nucleotides -731 to -510) has 85% homology to a sequence found in the human gelatinase B gene promoter (nucleotides -1650 to -1430)(24) , and is also homologous to a variety of ``Alu-type'' DNA sequences.

Although ST3 gene expression could be induced in HFL1 human fibroblasts by TPA(1) , no consensus sequence for the AP1-binding site (TGA(G/C)TCA) has been found in the first 1.4 kb of the ST3 promoter, and no response to TPA has been observed for the first 3.4 kb of the ST3 promoter analyzed in COS-1, HeLa, or HFL1 cells. In addition, the absence of any ST3 promoter transactivation by c-fos and c-jun suggests that the AP1-like motif present at position -461 is not functional and that no additional AP1-binding site is present within the 3.4 kb of 5`-flanking sequence examined in the present study. Although an AP1-binding site may be present outside of this DNA region, other possibilities must be considered to explain ST3 induction by TPA in human fibroblasts. Thus, several recent reports have indicated that MMP gene induction by TPA such as that observed for interstitial collagenase(45, 46, 47) , gelatinase B(48) , or stromelysin-1 (43, 49) cannot be entirely explained by an AP1 dependent mechanism but requires additional cis-acting elements. In addition, recent observations have indicated that besides transcriptional regulation, interstitial collagenase, stromelysin-1, and gelatinase A gene expressions in human fibroblasts are also regulated by a post-transcriptional mechanism involving enhanced mRNA stability (46, 50, 51) . Whether ST3 gene expression could also be regulated by similar mechanisms remains an important issue that will have to be addressed in the future.

Besides its role in MMP gene induction by TPA or growth factors, the AP1-binding site has also been described as a target for inhibition by RA in the regulation of interstitial collagenase and stromelysin-1 gene expression(23, 36) . In vitro experiments have suggested that an interaction between RAR and AP1 proteins results in mutual loss of their DNA binding activity(52) . In contrast, we have observed that ST3 gene promoter activity was increased by RA through the presence of a RARE in the 5`-flanking region of the ST3 gene, which has not been reported for any other MMP promoter. This RARE of the DR1 type(34, 39) has been previously suggested by binding and transactivation studies to correspond to a specific binding site for RXRs(37, 38) . In agreement to these studies, we have shown that RXRalpha can transactivate DR1/RARE-containing ST3-CAT constructs into COS-1 cells exposed to 9-cis-RA, and that this transactivation was dependent of RA concentrations. The possibility that the RARE present in the ST3 gene promoter is also operating in vivo is supported by Northern blot analyses showing that ST3 RNA levels are increased by RA in HFL1 human fibroblasts.^2

In conclusion, the present study has shown that the ST3 gene promoter is characterized by the presence of a RARE and the absence of a functional AP1-binding site in the 3.4 kb of 5`-flanking DNA sequence. Although further studies are required to better define the molecular mechanisms controlling the specific expression of the ST3 gene during tissue remodeling processes, our findings support previous observations suggesting that these mechanisms differ from those regulating the expression of other MMP genes(2, 8) .


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 Hôspitalier Universitaire Régional, the Groupement de Recherches et d'Etudes sur les Génomes (Grants 94/50 and 93/09), the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer, the Fondation pour la Recherche Médicale, 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. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence should be addressed: IGBMC, BP163 67404 Illkirch cedex, C.U. de Strasbourg, France. Tel.: 33-88-65-34-21 or 88-65-34-25; Fax: 33-88-65-32-01; anglard{at}titus.u-strasbg.fr.

(^1)
The abbreviations used are: ST3, stromelysin-3; MMP, matrix metalloproteinase(s); PIPES, 1,4-piperazinediethanesulfonic acid; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid responsive element; bp, base pair(s); kb, kilobase(s); tk, thymidine kinase; PCR, polymerase chain reaction; CAT, choline acetyltransferase; DR1, RARE consisting of the direct repeat of the core motif PuGGTCA separated by 1 bp; TPA, 12-O-tetradecanoylphorbol-13-acetate.

(^2)
P. Anglard, unpublished results.


ACKNOWLEDGEMENTS

We are indebted to P. Chambon for his support and interest in this study. We are most grateful to J. M. Garnier, H. Gronemeyer, O. Lefèbvre, O. Podhajcer, M. Schmitt, C. Stricker, and B. Wasylyk, for helpful discussions, S. Vicaire and A. Staub for DNA sequencing, and to P. De Jong for the chromosome 22 library.


REFERENCES

  1. Basset, P., Bellocq, J. P., Wolf, C., Stoll, I., Hutin, P., Limacher, J. M., Podhajcer, O. L., Chenard, M. P., Rio, M. C., and Chambon, P. (1990) Nature 348,699-704 [CrossRef][Medline] [Order article via Infotrieve]
  2. Wolf, C., Rouyer, N., Lutz, Y., Adida, C., Loriot, M., Bellocq, J. P., Chambon, P., and Basset, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,1843-1847 [Abstract]
  3. Rouyer, N., Wolf, C., Rio, M. C., Chambon, P., Bellocq, J. P., and Basset, P. (1994) Invasion Metastasis 14,269-275 [Medline] [Order article via Infotrieve]
  4. Lefebvre, O., Regnier, C., Chenard, M. P., Wendling, C., Chambon, P., Basset, P., and Rio, M. (1995) Development 121,947-955 [Abstract/Free Full Text]
  5. Patterton, D., Hayes, W. H., and Shi, Y. B. (1995) Dev. Biol. 167,252-262 [CrossRef][Medline] [Order article via Infotrieve]
  6. Wolf, C., Chenard, M. P., Durand de Grossouvre, P., Bellocq, J. P., Chambon, P., and Basset, P. (1992) J. Invest. Dermatol. 99,870-872 [Abstract]
  7. Lefebvre, O., Wolf, C., Limacher, J. M., Hutin, P., Wendling, C., Le Meur, M., Basset, P., and Rio, M. C. (1992) J. Cell Biol. 119,997-1002 [Abstract]
  8. Rodgers, W. H., Matrisian, L. M., Giudice, L. C., Dsupin, B., Cannon, P., Svitek, C., Gorstein, F., and Osteen, K. G. (1994) J. Clin. Invest. 94,946-953 [Medline] [Order article via Infotrieve]
  9. Matrisian, L. M. (1990) Trends Genet. 6,121-125 [CrossRef][Medline] [Order article via Infotrieve]
  10. Birkedal-Hansen, H., Moore, W. G., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., De Carlo, A., and Engler, J. A. (1993) Crit. Rev. Oral. Biol. Med. 4,197-250 [Abstract]
  11. Murphy, G. J., Murphy, G., and Reynolds, J. J. (1991) FEBS Lett. 289,4-7 [CrossRef][Medline] [Order article via Infotrieve]
  12. Murphy, G., Segain, J. P., O'Shea, M., Cockett, M., Ioannou, C., Lefebvre, O., Chambon, P., and Basset, P. (1993) J. Biol. Chem. 268,15435-15441 [Abstract/Free Full Text]
  13. Pei, D., Majmudar, G., and Weiss, S. J. (1994) J. Biol. Chem. 269,25849-25855 [Abstract/Free Full Text]
  14. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C., Herrlich, P., and Karin, M. (1987) Cell 49,729-739 [Medline] [Order article via Infotrieve]
  15. Jonat, C., Rahmsdorf, H. J., Park, K. K., Cato, A. C., Gebel, S., Ponta, H., and Herrlich, P. (1990) Cell 62,1189-1204 [Medline] [Order article via Infotrieve]
  16. Windsor, L. J., Grenett, H., Birkedal-Hansen, B., Bodden, M. K., Engler, J. A., and Birkedal-Hansen, H. (1993) J. Biol. Chem. 268,17341-17347 [Abstract/Free Full Text]
  17. Gaire, M., Magbanua, Z., McDonnell, S., McNeil, L., Lovett, D. H., and Matrisian, L. M. (1994) J. Biol. Chem. 269,2032-2040 [Abstract/Free Full Text]
  18. Huhtala, P., Tuuttila, A., Chow, L. T., Lohi, J., Keski-Oja, J., and Tryggvason, K. (1991) J. Biol. Chem. 266,16485-16490 [Abstract/Free Full Text]
  19. Wasylyk, B., Hahn, S. L., and Giovane, A. (1993) Eur. J. Biochem. 211,7-18 [Abstract]
  20. Kerr, L. D., Olashaw, N. E., and Matrisian, L. M. (1988) J. Biol. Chem. 263,16999-17005 [Abstract/Free Full Text]
  21. McDonnell, S. E., Kerr, L. D., and Matrisian, L. M. (1990) Mol. Cell. Biol. 10,4284-4293 [Medline] [Order article via Infotrieve]
  22. Frisch, S. M., and Ruley, H. E. (1987) J. Biol. Chem. 262,16300-16304 [Abstract/Free Full Text]
  23. Lafyatis, R., Kim, S. J., Angel, P., Roberts, A. B., Sporn, M. B., Karin, M., and Wilder, R. L. (1990) Mol. Endocrinol. 4,973-980 [Abstract]
  24. Sato, H., Kita, M., and Seiki, M. (1993) J. Biol. Chem. 268,23460-23468 [Abstract/Free Full Text]
  25. Hu, E., Mueller, E., Oliviero, S., Papaioannou, V. E., Johnson, R., and Spiegelman, B. M. (1994) EMBO J. 13,3094-3103 [Abstract]
  26. Zucman, J., Delattre, O., Desmaze, C., Plougastel, B., Joubert, I., Melot, T., Peter, M., De Jong, P., Rouleau, G., Aurias, A., and Thomas, G. (1992) Genes Chrom. Cancer 5,271-277 [Medline] [Order article via Infotrieve]
  27. Anglard, P., Trahan, E., Liu, S., Latif, F., Merino, M. J., Lerman, M. I., Zbar, B., and Linehan, W. M. (1992) Cancer Res. 52,348-356 [Abstract]
  28. Davis, A. M., Dibner, M. D., and Battely, J. F. (1986) Basic Methods in Molecular Biology, pp. 276-284, Elsevier Science Publishing Co., Inc., New York
  29. Boshart, M., Kluppel, M., Schmidt, A., Schutz, G., and Luckow, B. (1992) Gene (Amst.) 110,129-130 [CrossRef][Medline] [Order article via Infotrieve]
  30. Bucher, P. (1990) J. Mol. Biol. 212,563-578 [Medline] [Order article via Infotrieve]
  31. Huhtala, P., Chow, L. T., and Tryggvason, K. (1990) J. Biol. Chem. 265,11077-11082 [Abstract/Free Full Text]
  32. Jones, K. A., Kadonaga, J. T., Rosenfeld, P. J., Kelly, T. J., and Tjian, R. (1987) Cell 48,79-89 [Medline] [Order article via Infotrieve]
  33. Weissman, J. D., and Singer, D. S. (1991) Mol. Cell. Biol. 11,4217-4227 [Medline] [Order article via Infotrieve]
  34. Leid, M., Kastner, P., and Chambon, P. (1992) Trends Biochem. Sci. 17,427-433 [CrossRef][Medline] [Order article via Infotrieve]
  35. Risse, G., Jooss, K., Neuberg, M., Bruller, H. J., and Muller, R. (1989) EMBO J. 8,3825-3832 [Abstract]
  36. Nicholson, R. C., Mader, S., Nagpal, S., Leid, M., Rochette-Egly, C., and Chambon, P. (1990) EMBO J. 9,4443-4454 [Abstract]
  37. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355,446-449 [CrossRef][Medline] [Order article via Infotrieve]
  38. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J. Y., Staub, A., Garnier, J. M., Mader, S., and Chambon, P. (1992) Cell 68,377-395 [Medline] [Order article via Infotrieve]
  39. Nagpal, S., Saunders, M., Kastner, P., Durand, B., Nakshatri, H., and Chambon, P. (1992) Cell 70,1007-1019 [Medline] [Order article via Infotrieve]
  40. Durand, B., Saunders, M., Leroy, P., Leid, M., and Chambon, P. (1992) Cell 71,73-85 [Medline] [Order article via Infotrieve]
  41. Collier, I. E., Bruns, G. A., Goldberg, G. I., and Gerhard, D. S. (1991) Genomics 9,429-434 [Medline] [Order article via Infotrieve]
  42. Matrisian, L. M. (1992) Bioessays 14,455-463 [Medline] [Order article via Infotrieve]
  43. Buttice, G., and Kurkinen, M. (1993) J. Biol. Chem. 268,7196-7204 [Abstract/Free Full Text]
  44. Klungland, H., Andersen, O., Kisen, G., Alestrom, P., and Tora, L. (1993) Mol. Cell. Endocrinol. 95,147-154 [CrossRef][Medline] [Order article via Infotrieve]
  45. Auble, D. T., and Brinckerhoff, C. E. (1991) Biochemistry 30,4629-4635 [Medline] [Order article via Infotrieve]
  46. Shapiro, S. D., Doyle, G. A., Ley, T. J., Parks, W. C., and Welgus, H. G. (1993) Biochemistry 32,4286-4292 [Medline] [Order article via Infotrieve]
  47. Chamberlain, S. H., Hemmer, R. M., and Brinckerhoff, C. E. (1993) J. Cell. Biochem. 52,337-351 [Medline] [Order article via Infotrieve]
  48. Sato, H., and Seiki, M. (1993) Oncogene 8,395-405 [Medline] [Order article via Infotrieve]
  49. Buttice, G., Quinones, S., and Kurkinen, M. (1991) Nucleic Acids Res. 19,3723-3731 [Abstract]
  50. Overall, C. M., Wrana, J. L., and Sodek, J. (1991) J. Biol. Chem. 266,14064-14071 [Abstract/Free Full Text]
  51. Delany, A. M., and Brinckerhoff, C. E. (1992) J. Cell. Biochem. 50,400-410 [Medline] [Order article via Infotrieve]
  52. Yang-Yen, H. F., Zhang, X. K., Graupner, G., Tzukerman, M., Sakamoto, B., Karin, M., and Pfahl, M. (1991) New. Biol. 3,1206-1219 [Medline] [Order article via Infotrieve]
  53. Collier, I. E., Smith, J., Kronberger, A., Bauer, E. A., Wilhelm, S. M., Eisen, A. Z., and Goldberg, G. I. (1988) J. Biol. Chem. 263,10711-10713 [Abstract/Free Full Text]
  54. Breathnach, R., Matrisian, L. M., Gesnel, M. C., Staub, A., and Leroy, P. (1987) Nucleic Acids Res. 15,1139-1151 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.