(Received for publication, February 1, 1995; and in revised form, May 15, 1995)
From the
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.
The stromelysin-3 (ST3) ()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).
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.
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).
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- 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-
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
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).
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).
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 RXR or RAR
or dnRAR
(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, 10
M 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).
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. ()
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 RXR 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.
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) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].