v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements. A mechanism regulating gene expression independent of that by inflammatory cytokines.

The 92-kDa type IV collagenase (matrix metalloproteinase-9; MMP-9) is frequently expressed in cells showing an invasive nature during physiological and pathological processes, and the expression is strictly controlled by a variety of trans-membrane signals. Binding sites for NF-kB, Sp-1, and AP-1 are reportedly required for induction of MMP-9 gene expression by tumor necrosis factor-alpha or 12-O-tetradecanoylphorbol-13-acetate. Comparison of the sequence of the newly cloned mouse MMP-9 promoter region with our previous human isolate revealed that, in addition to the above mentioned elements, four units of GGGG(T/A)GGGG sequence (GT box) were conserved between the two species. In this study, we have demonstrated that one of the GT boxes located downstream of the AP-1 site is essential along with the AP-1 site for the activation of the promoter by v-Src but not by tumor necrosis factor-alpha or 12-O-tetradecanoylphorbol-13-acetate. Gel mobility-shift assays revealed that binding proteins for retinoblastoma control element, including Sp-1 family protein, can bind specifically to GT boxes. Thus, the v-Src signals to the AP-1 site and to the GT box homologous to retinoblastoma control element acted synergistically in transcriptional activation. These results suggest that certain v-Src-mediated signals are propagated along pathways that are independent of inflammatory cytokines.

The 92-kDa type IV collagenase (matrix metalloproteinase-9; MMP-9) is frequently expressed in cells showing an invasive nature during physiological and pathological processes, and the expression is strictly controlled by a variety of trans-membrane signals. Binding sites for NF-KB, Sp-1, and AP-1 are reportedly required for induction of MMP-9 gene expression by tumor necrosis factor-a or 12-0-tetradecanoylphorbol-13-acetate. Comparison of the sequence of the newly cloned mouse MMP-9 promoter region with our previous human isolate revealed that, in addition to the above mentioned elements, four units of GGGG(T/ A)GGGG sequence (GT box) were conserved between the two species. In this study, we have demonstrated that one of the GT boxes located downstream of the AP-1 site is essential along with the AP-1 site for the activation of the promoter by v-Src but not by tumor necrosis factor-a or 12-O-tetradecanoylphorbol-13acetate. Gel mobility-shift assays revealed that binding proteins for retinoblastoma control element, including Sp-1 family protein, can bind specifically to GT boxes. Thus, the v-Src signals to the AP-1 site and to the GT box homologous to retinoblastoma control element acted synergistically in transcriptional activation. These results suggest that certain v-Src-mediated signals are propagated along pathways that are independent of inflammatory cytokines.
Matrix metalloproteinases (MMP)' are a family of zinccontaining endopeptidases that are collectively capable of degrading most or all of the constituent macromolecules of the extracellular matrix. This enzyme family includes the two *This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan and by a special coordination fund for promoting science and technology from the Ministry of Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We previously examined the mechanism of MMP-9 gene activation by TNF-a as a model system for signal transduction involved in the gene regulation of MMPs during inflammation (26). Unlike interstitial collagenase and stromelysin-1, our data showed that transcription of MMP-9 promoter was stimulated by TNF-a through binding to sites for not only AP-1 but also NF-KB and Sp-1.
MMP-9 is often expressed by malignant tumor cells, but its expression is not necessarily coordinated with that of interstitial collagenase and stromelysin-l (27). The expression of MMP-9 is thought to potentiate the invasive character of the producer cells through its capacity to degrade type IV collagen, a major component of basement membrane. In fact expression of MMP-9 mRNA correlates well with tumorigenicity and metastatic ability of tumor cells (28). In this context, it is important to understand the regulatory mechanism of the expression of this gene in malignant tumor cells. Thus, we examined the effect on MMP-9 gene expression of oncogene products which are potent inducers of malignant tumors.
We show here that expression of v-Src induces the synthesis of MMP-9, which is mediated by alterations in activity of binding factors for the AP-1 site and the sequence motif GGGGTGGGG (GT box). This GT box is homologous to the so-called retinoblastoma (Rb) control element (RCE) (29,30), and Rb can produce an anti-oncogene or tumor suppressor gene product (31)(32)(33)(34)(35)(36)(37)(38) which is involved in regulating transcription of certain genes. However, the mechanism through which Rb functions to suppress tumorigenisis is unknown, although Rb also regulates transcription of the c-fos, c-myc, and transforming growth factor 01 (TGF-01) promoters in either a positive or negative manner depending on cell type (30). This action is mediated through a common motif termed RCE (29,30).
Although the AP-1 site is essential for response to TNF-a and TPA, transactivation of human MMP-9 promoter by these mediators can only be attained with the cooperation of the binding site for either NF-KB or Sp-1 but not the GT box. Based on a comparative sequence analysis of the human and murine promoter regions of the MMP-9 gene, the above mentioned motifs are all well conserved in both genes. These findings suggest the existence of distinct mechanisms by which the transcription of the MMP-9 gene is activated during both oncoprotein-mediated malignant transformation and inflammation.

MATERIALS AND METHODS
Determination of the Nucleotide Sequence of the Mouse MMP-9 Gene-A genomic DNA clone for the mouse MMP-9 gene was isolated from a Charon28 phage genomic library of BALB/C mouse obtained from the Japanese Cancer Research Resources Bank by using human MMP-9 cDNA as a probe. The DNA sequence was determined by the dideoxy chain termination method (39).
Cell Culture and DNA Transfections-The human tumor cell lines, fibrosarcoma HT1080 and hepatoma HepGZ, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected as described previously (26). In general, cells were incubated with calcium phosphate-DNA coprecipitates containing the various expression and reporter plasmids, as indicated in the figure legends. Expression vector pSG5 lacking an insert was used in control plates, as appropriate, such that all plates were transfected with equivalent amounts of expression plasmid. Cells were harvested 24 h after transfection and were assayed for chloramphenicol acetyltransferase (CAT) activity as described previously (26).
Plusrnids-The reporter plasmid constructs were generated as described previously (26). Expression plasmids were all driven by the SV40 early promoter in the pSG5 vector.
Gel-shift Assays-Nuclear extracts were prepared from HT1080 and HepG2 cells using the rapid method (40). The double-stranded synthetic oligonucleotides were end-labeled with [Y-~*P]ATP and polynuceotide kinase. Binding reactions and analyses on 4% polyacrylamide gel electrophoresis in 0.25 X TBE buffer (1 X TBE = 89 mM Tris borate, 89 mM boric acid, 2 mM EDTA) were performed as described previously (26).

Sequence
Comparison of the 5'-End of the Mouse and Human MMP-9 Genes-To approach to identification of important regulatory sequences in the human MMP-9 gene, we compared human and mouse MMP-9 gene sequences (Fig. 1). Binding sites for AP-1, Sp-1, and NF-xB, which had previously been identified as essential elements for the induction of human MMP-9 gene by TPA and TNF-a (26), were conserved in the 5'-flanking regions of the human and mouse genomes. In addition to the elements described above, a CA repeat region lying upstream of the TRE, several copies of GGGGAGGGG and related sequences were conserved in the mouse genome.
Induction of MMP-9 by Oncoproteins-We initially examined whether MMP-9 gene promoter was activated by the following oncoproteins: c-Jun, JunB, c-Fos, v-Src, Ha-Ras, normal ErbB-2, ErbB-2(N), and the activated form of ErbB-2, ErbB-e(A). This was done by transfecting the expression plasmid of each oncoprotein with the reporter plasmid containing the promoter linked to the CAT gene ( Fig. 2.4). The transfections were repeated at least four times with more than two different preparations of plasmid DNA to ensure repro-

C C T C C T T C C C T T T C C C A C~Q T C T Q C A Q T T T Q C A Q~C T~C C C T Q A
QTTCTCTQQTTTCCTQTQQQTCTQQQQQTCCTQCCTQACTTQQCMTQGG -2 1 9 QQACCTQTQQQCAQQQCATAAQQQAQQQQQTAQTQT~CACACACACACA -1 1 9 ducibility of the results. As described previously, c-Jun stimulated the MMP-9 promoter and c-Fos enhanced c-Jun activity (26). JunB, which cannot activate the interstitial collagenase promoter (41), did however stimulate the MMP-9 promoter. Moreover transcription from the MMP-9 promoter was activated by v-Src but not by Ha-Ras or both forms of ErbB-2.

T Q C~Q O C A Q C Q T T A Q C C A Q M Q C T~Q Q T C C T C A C C~A Q T C C C T Q Q
To confirm the stimulatory effect of v-Src on MMP-9 gene expression, the effect of v-Src expression on the synthesis of the enzyme was examined by a gelatin-substrate gel analysis (Fig. 2B). Culture fluids from HT1080 cells transfected with v-Src expression plasmid and control plasmid were harvested and analyzed for the synthesis of MMP-9. Transfection of v-Src expression plasmid enhanced the synthesis of MMP-9 by 2-3-fold (lane 21, which is a significant induction considering the inherent efficiency of transfection by the calcium phosphate co-precipitation method. Mapping of the v-Src-responsive Element in the MMP-9 Promoter-To determine the basis of enhanced MMP-9 transcript caused by v-Src, a series of plasmids containing MMP-9 promoter mutants linked to the CAT gene were transfected with v-Src-expression or control plasmids, and the effect of v-Src was compared to that of c-Jun and TPA (Fig. 3). Stimulation of transcription from the promoter of MMP-9 gene by TPA is mediated through binding sites for not only AP-1 but also for either Sp-1 or NF-KB (26). Transcription from the promoter was markedly stimulated by c-Jun and v-Src until the deletion extended to nucleotide position -90 (-90-CAT). In contrast, transcription from the promoter was drastically lost by deleting the AP-1 site (-73-CAT); however, only v-Src still significantly stimulated the promoter lacking the AP-1 site. To confirm the role of the AP-1 site, a double point mutation was introduced into the sequence (TGAGTCA t o T E G T C A ) in the -634-CAT construct. The mutant promoter did not significantly respond to either c-Jun or TPA but still had a residual response to v-Src. Mutation or deletion of the possible binding sites for Sp-1 and NF-KB did not significantly alter the response to either c-Jun or v-Src. These results suggest that the AP-1 site is cis-element essential for the MMP-9 promoter response to stimuli. In addition to the AP-1 site, another sequence within nucleotide position -73 is essential for v-Src inducibility.
Apart from the elements described above, three copies of GA box and a CACA box located upstream of AP-1 site are conserved in the human and mouse promoter regions. Deletion of the -531 to -91 region from the -634-CAT reporter plasmid containing three copies of GA box and a CACA box did not have any significant effect on basal level or stimulated expression (HinfI-90-CAT).
v-Src-responsive Element Includes the GT Box-Based on comparative sequence analysis of the human and murine genes, the GGGGTGGGG sequence located between the AP-1 site and TATA box in the human gene corresponds to a homologous GC-rich motif (GGGGAGGGGCGGGG) in the mouse gene. We introduced mutations in this element of the -90-CAT construct to determine whether this element contributes to the regulation of MMP-9 gene expression (Fig.  4A). Since the binding site for AP-1 is included in this promoter region, the effects of AP-1 family members c-Jun and JunB were compared with that of v-Src. Mutating the GGGGTGGGG sequence to GMGTGGGG (-9Ogtmut.l-CAT) or GGGGTMGG (--SOgtmut.Z-CAT) completely abolished transcriptional response to v-Src, whereas only a slightly decreased basal level and response to c-Jun and JunB was observed. When the GGGGTGGGG sequence was changed to GGGGCGGGG (a possible binding sequence for Sp-1 protein, -90gtmut.3-CAT), the promoter was activated by c-Jun and JunB, but less effectively by v-Src than for the wild type promoter. Mutation of the GGGGTGGGG sequence to GGGGAGGGG (GA box) (-9Ogtmut.4-CAT), which is the same as in the mouse promoter, did not have any significant effect on the response to v-Src.
Next we examined the role of the GT box in the promoter lacking the AP-1 site (-73-CAT) (Fig. 4B). Mutation of the wild type sequence to GAJGTGGGG (-73gtmut.l-CAT) or GGGGTMGG (-73gtmut.2-CAT) abolished the response to v-Src, suggesting that the GT box on its own confers v-Src inducibility, which is synergistically enhanced by AP-l-mediated transactivation.
GT Box Is Not Essential for TPA Inducibility-To compare the mechanism of induction by TPA and v-Src, the effect of TPA on induction of the transcription from the MMP-9 promoter by v-Src was examined (Fig. 5). The promoter containing the G T box and binding sites for AP-1, NF-KB, and Sp-1 was activated by v-Src and TPA, but simultaneous induction by v-Src and TPA did not show either synergistic or additive effects. Mutation of the GT box, which abolished the response to v-Src, did not have a significant effect on TPA inducibility (-634gtmut.2-CAT), and v-Src did not affect TPA inducibility of the promoter. TPA not only failed to activate transcription from the reporter plasmid lacking binding sites for NF-KB and Sp-1 (-90-CAT) but also repressed the trancriptional activation induced by v-Src.
Augmentation of AP-1 -mediated Transactivatwn of MMP-9 Promoter by v-Src-Transforming oncogene products Ha-Ras and v-Src reportedly activate c-Jun by phosphorylating its transcription-activation domain (42,43). This led us to examine the contribution of this pathway to the transactivation of the MMP-9 promoter by v-Src. As shown above, overexpression of c-Jun and JunB stimulated the promoter regardless of the GT box, and transactivation by c-Jun and JunB was further augmented by v-Src (Fig. 6). Thus, the G T box was not essential for the augmentation of c-Jun-and JunB-mediated transactivation by v-Src. However, a stimulatory effect of v-Src on the promoter lacking a G T box was not observed in the absence of exogeneous c-Jun and JunB expression as mentioned above. Augmentation of c-Jun-and JunB-mediated activation by v-Src was not due to increased expression of these proteins by v-Src, because the SV40 early promoter used to control these genes was not significantly affected by v-Src. Moreover, v-Src could stimulate the activity of c-Jun and JunB expressed at saturated levels (data not TPA (50 ng/ml) was included in the culture medium after transfection of control plasmid (+TPA). APmu, SPrnu, and kBmu are the mutations in the TRE, Sp-1, and KB consensus sequences, respectively, as described previously (26). = +Src shown). As described above Ha-Ras on its own could not induce the transcription from MMP-9 promoter, but it did cooperate with c-Jun and JunB to activate the promoter. These results suggest that augmentation of the activity of c-Jun and JunB may not be the main pathway for the activation of the MMP-9 promoter by v-Src. TPA which is also known to activate c-Jun failed to augment transactivation by c-Jun and JunB (data not shown). Reportedly v-Src also induces expression of c-fos which forms heterodimers with members of the Jun family and enhances AP-1-mediated transactivation (44). Expression of c-fos enhanced c-Jun and JunB activity, but this was less extensive than that observed with v-Src.

5"Flanking Sequence
Characterization of Nuclear Factors Which Bind to the GGGGTGGGG Sequence-As demonstrated above, v-Src-responsive elements in the human MMP-9 promoter were delimited to the AP-1 site and the GT box located between the AP-1 site and TATA box. To characterize the nuclear factors which bind to the GT box, nuclear extracts were prepared from HT1080 cells which constitutively synthesize the enzyme and non-producer HepG2 cells. DNA binding activity was analyzed by gel retardation assays using a 32P-labeled double-stranded oligonucleotide DNA encompassing the GGGGTGGGG sequence (nucleotide positions -64 to -39) as a probe (Fig. 7). Two major retarded bands, A and B, were observed with the extract from HT1080 cells (lane 1  B (lane 7). The oligonucleotide gtmut.4 (GGGGAGGGG) showed the same competition pattern as the wild type g t oligonucleotide, and a faster migrating band C was observed at the lower competitor concentration (lanes 8 and 9). A nuclear extract from HepG2 cells also generated the two major retarded bands A and B, but the faster migrating band B was much weaker than that seen with HT1080 cells (lane 10). Formation of complexes A and B was interfered with by unlabeled gt oligonucleotide, and in this case a faster migrating band C was detected (lanes 11 and 12) 13 and 14, respectively).

GT Box Is Homologous to RCE-A GT box or closely related
sequence has been identified in the promoter regions of the c-myc, c-fos, TGF-B, and insulin-like growth factor 11 genes and has been mapped as a RCE (29,30). We therefore compared c-fos RCE binding factors with those of the GT box. As shown in Fig. 8, a c-fos RCE oligonucleotide formed similar complexes with nuclear factors as the g t oligonucleotide probe (lane I ) , which were specifically abolished by excess gt oligonucleotide (lane 4 ) . Oligonucleotides gtmut.1 and gtmut.2, pattern to the GT box distinct from that of HT1080 cells, which led us to examine the effect of v-Src in HepGZ cells. Transcription from the MMP-9 promoter (-634-CAT), which contains binding sites for NF-rtB, Sp-1, and AP-1, was stimulated by Jun and TPA in both HT1080 and HepGZ cells.

Activation of 92-kDa Type IV Collagenase Promoter by v-Src
However, the promoter was transactivated by v-Src only in HT1080 cells and not in HepGZ cells (Fig. 9). In contrast, the collagenase promoter was activated by these effectors including v-Src in both cells.

DISCUSSION
v-Src Can Induce Expression of MMP-9-Oncoproteinmediated malignant transformation affects gene expression, the products of which can directly modulate the malignant phenotype of tumor cells, e.g. potentiating their invasive ability. Since the expression of MMP-9 is thought to potentiate the invasive character of the producer cells, it was of interest to examine the effect of oncogene products on the expression of this gene. In this communication we have demonstrated that the v-Src gene product stimulates the expression of the MMP-9 gene and determined which cis-acting elements respond to v-Src induction. These elements are distinct from those responsible for induction by TNF-a, an inflammatory cytokine, and TPA (26).
Transactivation of MMP-9 Promoter by v-Src Is Mediated via an AP-1 Site and a GT Box-A number of studies have shown that v-Src can affect expression of various genes such as c-myc, c-fos, c-jun, junB, EGR-1, TGF-8, collagenase, and stromelysin-1 (25,(45)(46)(47)(48)(49). The v-Src protein was reported to activate the collagenase, stromelysin-1 and TGF-8 genes through an AP-1 element in their promoters (25,45,49). Transfection with the v-src oncogene enhances not only gene expression of c-jun but also the transcriptional activity of the c-Jun protein (43). v-Src was also reported to transactivate promoters of the c-fos and junB genes, although cis-acting elements responding to v-Src in these promoters were different not only from AP-1 but also from each other (44,471. Transforming oncoproteins such as v-Src, Ha-Ras, and Raf-1 stimulate AP-1 activity by phosphorylating the activation domain of c-Jun in a signaling cascade where Ha-Ras acts downstream to v-Src and upstream to Raf-1 (43). Thus, TPAinducible MMP genes such as interstitial collagenase and stromelysin-1 are induced by these oncoproteins (25,43,45). In contrast, the MMP-9 promoter was transactivated by v-Src but not by Ha-Ras, although both v-Src and Ha-Ras cooperate with c-Jun and JunB during transactivation of MMP-9 promoter through an AP-1 site. This suggests that v-Src may modulate signaling pathways independently of the ras gene product. We have also shown that the GT box, which is homologous to RCE (see below), participates synergistically with the AP-1 site in the response of the MMP-9 promoter to v-Src. Thus, the effect of v-Src may involve transcriptional and/or post-transcriptional augmentation of AP-1 activity which alone may not be strong enough to stimulate the promoter and thus require synergistic cooperation with GT box-mediated activation. The particular mechanism by which the v-Src signal stimulates G T box-mediated activation remains to be determined. Interestingly, the location of the GT box is important, because the G(A/C) box located upstream of the AP-1 site does not contribute to activation by v-Src.
v-Src reportedly alleviates repression of c-Jun by a cellspecific inhibitor (50); however, this is not the case during transactivation of the MMP-9 promoter by v-Src, because v-Src can activate the promoter in NIH3T3 cells which lack an inhibitor (data not shown). """""""""""""""""""""""-  U-Src and TPA Stimulate the MMP-9 Promoter by Different mRNA, rather than junB mRNA, repressed the response to Mechanisms-The AP-1 site is also essential to confer induc-both v-Src and TPA (data not shown). This suggests that cibility by TPA and TNF-a, but transactivation of MMP-9 Jun is involved in the activation by v-Src and TPA. Induction promoter by TPA and TNF-a can only be attained with the of c-jun by the protein kinase C agonist TPA is thought to cooperation of the binding site for either NF-KB or Sp-1, but result from a positive autoregulatory mechanism (21) and to not the GT box. Expression of anti-sense RNA for c-jun involve dephosphorylation of preexisting c J u n a t inhibitory phosphorylation sites next to its DNA binding domain (51). The fact that v-Src cooperates with c-Jun but not with TPA suggests that v-Src can not stimulate c-Jun which is induced and activated by TPA, i e . , c-Jun activated by signals from protein kinase C requires different cofactors for transactivation of the MMP-9 promoter than from v-Src. In contrast, transcription of interstitial collagenase and stromelysin-1 promoters is activated by both TPA and oncogenes via binding sites for AP-1 and Ets (25,45).

Activation of 92-kDa Type IV Collagenase Promoter by u-Src
GT Box Is Homologous to RCE-The sequence motif 'GGGGTGGGG' or closely associated sequence has been identified in the promoter regions of c-myc, c-fos, TGF-P, and insulin-like growth factor I1 (30, 52). Interestingly, these sequences have been mapped as RCE in these promoters (Fig.  lo), and GC-rich sequences in junB and EGR-1 gene promoters were also identified as RCE. Oligonucleotides encompassing the GT box competed specifically for the formation of c-fos RCE complex, indicating that the GT box of the MMP-9 promoter is homologous to RCE. Kim et al. (52) have shown that Sp-1 can bind to, and stimulate transcription from, these promoters via RCE and that Rb can positively regulate RCE-mediated transcriptional activity by Sp-1. Recently, Kingsley and Winoto (53) have cloned new members of the Sp-1 family which bind to both GC/T boxes. Expression of the c-myc, c-fos, TGF-8, junB, and EGR-1 genes, all of whose promoters contain RCE, has been reported to be induced by v-Src; however, a contribution of RCE has not yet been demonstrated (44, [46][47][48][49]. An attempt to detect a specific effect of expression of the Rb gene or anti-sense RNA for the Rb gene on the MMP-9 promoter was unsuccessful. However, the possibility cannot be ruled out that anti-oncogene product Rb can regulate the expression of the MMP-9 gene either negatively or positively through RCE with consequent modulation of the metastatic potential of tumor cells. Daily et al. (54) purified a nuclear factor, H4TF1, which binds to the GGGGAGGG sequence motif but not to the GC box, binding site for Sp-1, and stimulates transcription from the histone H4 promoter. A GA box or closely related motif has also been identified in the promoter region of c-myc, type IV collagen, laminin B1, laminin B2, and fibronectin (55,56).
One of GT box-binding factors we detected here by gel mobility shift assays, which specifically bound to GT/A but not to the GC box, was probably H4TF1. A few copies of the GA/ T box are conserved upstream of the AP-1 site in human and mouse promoters, but their role is not clear at present. The binding protein or protein complex which specifically binds to the GT/A box is more abundant in MMP-9-producing HT1080 cells than in non-producing HepG2 cells, in which v-Src failed to transactivate MMP-9 promoter. In contrast, the Sp-1 family protein exists at a comparable level in both cell types. This suggests that a GT/A box-specific binding factor may participate in regulation of MMP-9 gene expression not only at a basal level but also in response to v-Src. Substitution of GT for the GC box, which is located upstream of AP-1 site and is essential for induction by TPA, abolished TPA inducibility (data not shown). These results suggest distinct roles for GT/A and GC boxes in transcriptional regulation of the MMP-9 promoter. Consistent with this, Bmggeman et al. (56) demonstrated that nuclear extracts isolated from tissues that variably express type Iv collagen showed distinctly different gel shift patterns with respect to GA box binding characteristics. It is important to note that promoters of MMP-9 and the major structural components of epidermal growth factor, e.g. type IV collagen, laminin B1, laminin B2, and fibronectin, share GT/A box, or homologous elements. Alterations in matrix protein metabolism are critical to the process of tumor metastasis, suggesting a specific role of the GT box homologous to RCE in the regulation of genes associated with tumor invasion.