Regulation of 92 Kda Type IV Collagenase Expression by the Jun Aminoterminal Kinase- and the Extracellular Signal-Regulated Kinase- Dependent Signaling Cascades

Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase- dependent signaling cascades

R Gum1, H Wang1, E Lengyel2, J Juarez1 and D Boyd1

1Department of Head & Neck Surgery/Tumor Biology, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030, USA; 2Department of Obstetrics and Gynecology, Technical University, Munich, Germany

The 92 kDa type IV collagenase (MMP-9), which activity of a promoter driven by either the MMP-9 degrades type IV collagen, has been implicated in tissue promoter or three tandem AP-1 repeats. Conversely, remodeling. The purpose of the current study was to treatment of UM-SCC-1 cells with PMA, which determine the role of Jun amino-terminal kinase (JNK)- activates c-Raf-1, increased 92 kDa gelatinolysis. These and extracellular signal-regulated kinase- (ERK)-depen- data suggest that MMP-9 expression in UM-SCC-1 dent signaling cascades in the regulation of MMP-9 cells, is regulated by JNK- and ERK-dependent signaling expression. Towards this end, we ®rst determined the pathways. transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid Keywords: MMP-9; type IV collagenase; JNK; ERK; secretor of this collagenase. Transfection of these cells AP-1 with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning 7144 to 773 for Introduction optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides The 92 kDa type IV collagenase (gelatinase B also 791 to 768 and containing a consensus AP-1 motif at known as MMP-9) plays a major role in cell 779. Mutation of this AP-1 motif practically abolished migration in both physiological and pathological the activity of the MMP-9 promoter-driven CAT processes (Hurwitz et al., 1993; Bernhard et al., reporter. Mobility shift assays indicated c-Fos and Jun- 1994; Matrisian, 1992) such as tumor cell invasion by D bound to this motif and transfection of the cells with a facilitating the destruction of the type IV collagen- mutated c-Jun, which quenches the function of endogen- containing basement membrane which separates the ous Jun and Fos proteins, decreased MMP-9 promoter epithelial and stromal compartments (Tryggvason et activity by 80%. UM-SCC-1 cells contained a constitu- al., 1987). The 92 kDa type IV collagenase is secreted tively activated JNK and the expression of a kinase- as a proenzyme (Wilhelm et al., 1989) and subse- de®cient JNK1 reduced the activity of a CAT reporter quently activated by multiple enzymes including driven either by the MMP-9 promoter or by three cathepsin G, trypsin, stromelysin 1 (Okada et al., tandem AP-1 repeats upstream of a thymidine kinase 1992; Shapiro et al., 1995) and 72 kDa gelatinase A minimal promoter. Conditioned medium collected from (MMP-2) (Fridman et al., 1995) by the removal of 73 UM-SCC-1 cells transfected with the dominant negative amino acids from the amino terminus of the protease. JNK1 expression vector diminished 92 kDa gelatinolysis. The active enzyme, which is capable of digesting Similarly, interfering with MEKK, which lies upstream native type I, III, IV and V collagens at non- of JNK1, using a dominant negative expression vector denaturing temperatures (Wilhelm et al., 1989; Tracey reduced MMP-9 promoter activity over the same and Cerami, 1994), consists of ®ve domains; the concentration range which repressed the AP-1-thymidine amino-terminal and zinc-binding domains shared by kinase CAT reporter construct. UM-SCC-1 cells also all members of the metalloproteinase family, a contained a constitutively activated ERK1. MMP-9 collagen-binding ®bronectin-like domain, a carboxy- expression, as determined by CAT assays and by terminal hemopexin-like domain and a unique 54 zymography, was reduced by the co-expression of a amino acid long proline-rich domain homologous to kinase-de®cient ERK1. Interfering with MEK1, which is the a2 chain of type V collagen (Wilhelm et al., an upstream activator of ERK1, either with PD 098059, 1989). which prevents the activation of MEK1, or with a MMP-9 is encoded by a 7.7 kb gene which spans 13 dominant negative expression construct, reduced 92 kDa exons (Collier et al., 1991; Huhtala et al., 1991). gelatinolysis and MMP-9 promoter activity respectively. Transcription of the gene, which yields a 2.3 kb c-Raf-1 is an upstream activator of MEK1 and a kinase- mRNA (Wilhelm et al., 1989), is regulated by 670 de®cient c-Raf-1 expression construct decreased the base pairs of upstream sequence (Sato and Seiki, 1993) which includes motifs corresponding to AP-1 (7533, 779), NF-kB(7600), PEA3 (PEA3 is an Ets family member) (7540) and Sp1 binding sites (7558). Indeed, one or more of these binding sites have been implicated in mediating the eects of a diverse set of agents which Correspondence: D Boyd Received 2 August 1996; revised 18 November 1996; accepted 19 includes TNF-a (Sato and Seiki, 1993), EGF, TGF-a, November 1996 phorbol ester (Lyons et al., 1993) v-src (Sato et al., Regulation of MMP-9 gene expression by MAPKs RGumet al 1482 1993) and Ha-Ras (Gum et al., 1996). For example, we Results recently showed that the stimulation of MMP-9 expression by Ha-Ras required juxtaposed PEA3 and Activation of the MMP-9 promoter in UM-SCC-1 cells AP-1 motifs located 540 and 533 base pairs upstream requires a proximal AP-1 motif of the transcriptional start site respectively (Gum et al., 1996). Sato and co-workers (Sato et al., 1993) We previously (Lengyel et al., 1995) demonstrated demonstrated that the stimulation of the MMP-9 that the squamous cell carcinoma cell line UM-SCC-1 promoter by v-src was mediated through an AP-1 was an avid secretor of MMP-9. To determine the motif and a GT box located 79 and 54 nucleotides transcriptional requirements for activation of the upstream of the transcriptional start site respectively. promoter the following experiments were carried out. On the other hand, the increased expression of this First, UM-SCC-1 cells were transiently transfected collagenase brought about by phorbol ester and by with a CAT reporter driven by 670 base pairs of 5' TNF-a involved an NF-kB and an Sp1 binding site at ¯anking sequence or 5' deleted fragments of the 7600 and 7558 respectively in addition to the AP-1 promoter and subsequently assayed for CAT activity motif at 779 (Sato and Seiki, 1993). (Figure 1a). The CAT reporter fused to 670 While the studies cited above have focused on nucleotides of regulatory sequence was strongly determining the transcriptional requirements for activated in UM-SCC-1 cells. While progressive MMP-9 expression, there is relatively little informa- deletions of sequences from the 5' end resulted in a tion on the signaling pathways which connect the cell gradual decrease in promoter activity, a dramatic (12- surface to the nuclear transcriptional machinery fold) reduction in CAT activity was apparent with 73 regulating the expression of this collagenase. The base pairs (construct 773) when compared with 144 activity and/or synthesis of several transcription base pairs (construct 7144) of 5' ¯anking sequence. factors in the AP-1, Ets and Rel/NF-kB (Whitmarsh These results indicated that while the maximal et al., 1995; Cavigelli et al., 1995; Hibi et al., 1993; activation of the promoter required sequences spread Minden et al., 1994b; Meyer et al., 1996) families have over the length of the MMP-9 promoter, a nucleotide recently been shown to be regulated by Jun amino sequence residing between 7144 and 773 was critical terminal kinases (JNKs also referred to as SAPKs) for promoter stimulation. To identify the protein- (Derijard et al., 1994). The JNKs represent a subset of binding sequences in this region of the promoter, the mitogen-activated protein kinase (MAPK) family. DNase I footprinting was performed. UM-SCC-1 The JNKs are activated by the dual activity kinase nuclear extract was incubated with a radiolabeled JNK kinase (JNKK1 also known as MKK4) (Lin et fragment of the MMP-9 promoter spanning nucleo- al., 1995a) which, in turn, is stimulated by a serine- tides 7144 to +1 and subsequently with DNase I threonine kinase MEKK (Minden et al., 1996). The (Figure 1b). At least two regions of this sequence were latter transduces the signals from both Ras-dependent footprinted. The ®rst of these spanned nucleotides (Minden et al., 1994a) and Ras-independent (Winston 791 to 768 (thus including the region shown to be et al., 1995) (e.g. TNF-a) growth factor/cytokine cell required for optimal MMP-9 promoter activity ± see surface receptors. Figure 1a) and contained a consensus AP-1 motif Alternatively, the synthesis and/or activity of (TGAGTCA) at position 779. The second protected transcription factors in the AP-1 (Gille et al., 1992; region (764 to 744) contained a GT-box at 754. Pulverer et al., 1991; Bogoyevitch et al., 1995; Agarwal Site-directed mutagenesis of these motifs dramatically et al., 1995), Ets (Agarwal et al., 1995; Rao and Reddy, reduced promoter activation in UM-SCC-1 cells 1993) and NF-kB (Li and Sedivy, 1993) families can be (Figure 1c). Both of these transcription factor-binding regulated by an extracellular signal-regulated kinase sites have been previously implicated in the stimula- (ERK)-dependent signaling cascade. The ERKs are a tion of MMP-9 expression by Ha-Ras (Gum et al., subgroup of the MAPK family which are distantly 1996) and by v-src (Sato et al., 1993). In fact, to the related to the JNKs (Hibi et al., 1993; Derijard et al., knowledge of the authors, all studies to date have 1994). However, the regulation of ERK activity is demonstrated a critical requirement of this AP-1 motif distinct from that of the JNKs. Thus, the activity of in the transcriptional regulation of MMP-9 expression ERK1 and ERK2 is dependent on their phosphoryla- this being regardless of cell type or stimulus (Sato et tion by the dual activity protein kinase MEK1 (also al., 1993; Gum et al., 1996; Sato and Seiki, 1993). referred to as MAPKK) (Minden et al., 1994a; Crews Mutation of NF-kB and Sp1 motifs at 7600 and et al., 1992). MEK1, in turn, is stimulated by the 7558 respectively, both of which are important for serine-threonine kinase c-Raf-1 (Huang et al., 1993). In the stimulation of MMP-9 promoter activity by contrast, the activity of MEK1 is relatively unaected phorbol ester (Sato and Seiki, 1993), also impaired by MEKK (responsible for JNKK1 activation) activation of the MMP-9 promoter in UM-SCC-1 cells (Minden et al., 1994a). c-Raf-1 is a down-stream although to a lesser extent. Interestingly, while we eector of Ras (Kolch et al., 1991; Kyriakis et al., recently reported that juxtaposed PEA3/AP-1 motifs 1992; Jelinek et al., 1994) and protein kinase C (Sozeri located at 7540 and 7533, respectively, were critical et al., 1992). for the stimulation of MMP-9 gene expression by Ha- Considering that MMP-9 expression has been Ras in OVCAR-3 ovarian cancer cells (Gum et al., previously shown to require DNA motifs recognized 1996), mutation of either of these binding sites had by transactivators which themselves are substrates for only a minor eect on the constitutive activation of the JNKs and ERKs, we undertook a study to this promoter in UM-SCC-1 cells (Figure 1c). determine the role of JNK- and ERK-dependent Considering the importance of the proximal AP-1- signaling modules in the regulation of expression of binding motif in the regulation of MMP-9 promoter this collagenase. activity in UM-SCC-1 and other cell types (Sato et al., Regulation of MMP-9 gene expression by MAPKs RGumet al 1483 1993; Gum et al., 1996; Sato and Seiki, 1993), mutated (mt competitor). Incubation of the reaction electrophoretic mobility shift assays (EMSA) were mixture with an antibody speci®c for Jun-D super- carried out to identify UM-SCC-1 derived nuclear shifted the oligonucleotide-containing complex. Simi- factors bound to this site. Nuclear extract from UM- larly, addition of a c-Fos-speci®c antibody to the SCC-1 cells was incubated with an end-labeled reaction mixture supershifted the oligonucleotide- oligonucleotide spanning nucleotides 791 to 760 bound complex indicating the presence of this thus containing the AP-1 motif located at 779. The transcription factor. In contrast, antibodies speci®c reaction mixture was then subjected to electrophoresis for Fra-1, Fra-2, Jun-B, c-Jun and ATF-2 did not in a polyacrylamide gel (Figure 2a). A binding complex discernibly alter complex formation., These data of the oligonucleotide and nuclear extracted proteins indicated that the proximal AP-1 motif at 779 in the was observed (arrow). This oligonucleotide-containing MMP-9 promoter binds Jun-D and c-Fos transcription complex was speci®c for the AP-1 motif since the factors and it is possible that transactivation of this retarded band could be eliminated with a 100-fold collagenase in UM-SCC-1 cells is achieved by a excess of an unlabeled wild type oligonucleotide (wt heterodimer of these transcription factors similar to competitor) but not with an equivalent amount of an that reported for human papilloma virus type 18 oligonucleotide in which the AP-1 motif had been transcription (Thierry et al., 1992).

a c

b UMSCC-1 Nuclear Extract (µg protein) G

G+A 0 0 13 26 39 52 65 78 90 110 0 Figure 1 The constitutive activity of the MMP-9 promoter in UM-SCC-1 cells requires an AP-1 motif and a GT box at 779 and 754, respectively. (a) UM-SCC-1 cells were transiently transfected at 70% con¯uency with 8 mg of a CAT reporter driven by the full length (670 base pairs) MMP-9 promoter or an equimolar amount of a reporter fused to the 5' deleted fragments of the MMP-9 promoter. All transfections were done in the presence of 4 mgofab-galactosidase-expressing vector. As a

G -44

A control, cells were also transfected with no DNA (mock). Cells C 

C were shocked with DMSO, cultured for 48 h, harvested and C

C  assayed for b-galactosidase activity. Cell extracts, corrected for

A GT-box C C  dierences in transfection eciency, were incubated at 378C with C 14 C [ C]chloramphenicol and acetyl coenzyme A for 8 h. The mixture T C was extracted with ethyl acetate and subjected to thin layer C T chromatography. (b) A fragment of the MMP-9 promoter T G spanning 7144 to +1 was end-labeled with Klenow and A C incubated with UM-SCC-1 nuclear extract. DNase I was A -64 A G subsequently added and the material electrophoresed in a DNA -68 G T sequencing gel. The G and G+A ladders generated by chemical

C degradation are shown. The footprinted sequences on the non-

T 

G coding strand are indicated. (c) UM-SCC-1 cells were transiently A 

C transfected, as described above, with a CAT reporter driven by

T AP-1 motif C  670 base pairs of wild type (wt), point-mutated (mt) or deleted A G (del) fragments of the MMP-9 promoter. Construct names G G indicate the binding site which was mutated or deleted and its G T position relative to the transcription start site. All mutants and C A deletions are in context of 670 base pairs of 5' ¯anking sequence. G G The conversion of [14C]chloramphenicol to acetylated derivatives G T G was determined with a 603 Betascope. The data are representative -91 of six, three and two separate experiments for a, b and c respectively Regulation of MMP-9 gene expression by MAPKs RGumet al 1484 a ++++++++++– nuclear extract –+–––––––––wt competitor ––+–––––––– mt competitor –––+––––––– c-Jun Ab ––––+––––––Jun B Ab –––––+–––––Jun-D Ab ––––––+––––ATF2 Ab –––––––+–––c-Fos Ab ––––––––+––Fra-1 Ab –––––––––+–Fra-2 Ab

b c

Figure 2 MMP-9 promoter activity in UM-SCC-1 cells is repressed by the co-expression of a transactivation domain-lacking c-Jun. (a) UM-SCC-1 nuclear extract (10 mg) was incubated with 5 fmoles of a 32P end-labeled oligonucleotide (spanning 791 to 760) in the presence, or the absence, of a 100-fold excess of the wild type sequence (wt competitor) or the oligonucleotide (mt competitor) which had been mutated (TttGTCA) in the AP-1 motif. The indicated antibody (Ab) was subsequently added and binding complexes resolved in a 5% polyacrylamide gel. The gel was dried and autoradiographed. (b) UM-SCC-1 cells were transiently transfected, as described in the legend to Figure 1a using a CAT reporter driven by 670 base pairs of 5' ¯anking sequence of the MMP-9 gene (MMP-9 CAT), a b-galactosidase-expressing vector and the indicated amount (where 26 represents a twofold molar excess of the TAM-67 with respect to MMP-9 CAT) of an expression vector encoding a transactivation domain-lacking c-Jun protein (TAM-67) or the empty vector (CMV vector). (c) UM-SCC-1 cells were transiently transfected, as described in the legend to b, but using a CAT reporter driven by the 5' deleted fragments of the MMP-9 promoter and, where indicated (+), a twofold molar equivalent (relative to the MMP-9 CAT) of TAM-67 or the empty vector (CMV vector). The amount of [14C]chloramphenicol converted to acetylated derivatives was determined with a 603 Betascope. Mock, no DNA was added. The data are typical of at least three experiments

through a quenching mechanism by inhibiting the Expression of a transactivation domain-lacking c-Jun function of endogenous Jun and/or Fos proteins represses MMP-9 promoter activity (Brown et al., 1994). Expression of TAM-67 caused a To further investigate the role of the AP-1 binding dose-dependent repression of the MMP-9 promoter in transcription factors in the regulation of MMP-9 UM-SCC-1 cells. A threefold molar excess of the expression, UM-SCC-1 cells were co-transfected with TAM-67, with respect to the reporter construct, a CAT reporter fused to 670 base pairs of MMP-9 inhibited MMP-9 promoter activity by over 80%. regulatory sequence (Figure 2b) and an expression The ability of the mutated Jun expression construct to construct (TAM-67) encoding a c-Jun which lacks decrease CAT activity was retained using 241 base amino acids 3 ± 122 (Brown et al., 1993). The mutated pairs of MMP-9 regulatory sequence in which the protein inhibits AP-1-dependent gene expression upstream AP-1 motif (at 7533) is absent (Figure 2c). Regulation of MMP-9 gene expression by MAPKs RGumet al 1485 These data taken together with the observation that makes it unlikely that the AP-1 sequence at 7533 is mutation of the distal AP-1 motif had only a minor being utilized in UM-SCC-1 cells. eect on MMP-9 promoter activation (Figure 1c) In view of the requirement for AP-1-binding

a b

GST-c-Jun ¨ NIH3T3 M-SCC-1 U

c d 2× 3×       CMV Vector JNK1mt CMV Vector JNK1mt

92 kDa ¨

72 kDa ¨

Figure 3 A dominant negative JNK1 expression vector represses MMP-9 promoter activity and gelatinolytic activity in UM-SCC- 1. (a) Equal amounts (100 mg) of extracted protein were incubated with an anti-JNK antibody. Protein A agarose beads were 32 subsequently added to the mixture, the beads rinsed and then incubated with [g P]ATP, a GST-c-Jun (1 ± 79) protein, and 20 mM unlabeled ATP. The mixture was heated to 1008C, centrifuged and the supernatant electrophoresed in a 12% polyacrylamide gel. The gel was dried and subjected to autoradiography. (b) UM-SCC-1 cells were transiently transfected, as described in the legend to Figure 1a using a CAT reporter driven by 670 base pairs of upstream regulatory sequence of the MMP-9 gene (MMP-9 CAT), a b- galactosidase-expressing vector and the indicated amount (where 36 represents a threefold molar excess of the eector plasmid relative to the reporter construct) of an expression vector encoding a dominant negative JNK1 protein (JNK1mt) or the empty vector (CMV vector). The cells were harvested and cell extracts assayed for CAT activity after correction for varying transfection eciencies. The conversion of [14C]chloramphenicol to acetylated derivatives was determined with a 603 Betascope. (c) UM-SCC-1 cells were transiently transfected, as described in the legend to b using 2.5 mg of a CAT reporter driven by three AP-1 tandem repeats upstream of a thymidine kinase minimal promoter (36AP-1 pBLCAT) or by the minimal promoter (pBLCAT) alone, a b- galactosidase-expressing vector and the indicated amount (where 36 corresponds to the mg amount of 36 used in b) of the dominant negative JNK1 protein (JNK1mt) or the empty vector (CMV vector). Incubations with [14C]chloramphenicol were carried out for 1 h. (d) UM-SCC-1 cells were transiently transfected, as described in the legend to Figure 1a with the exception that no CAT reporter construct was added. The amounts of JNK1mt expression vector and the empty vector (CMV vector) used correspond to those stated in (b). The cells were changed to serum-free media 16 h after being shocked and the conditioned medium collected 48 h later. Conditioned medium, normalized for dierences in transfection eciency, was assayed for gelatinolysis using a 7.5% polyacrylamide gel containing 0.1% gelatin. Proteolysis was detected as a white zone in a dark ®eld. The data shown are typical of three or more dierent experiments Regulation of MMP-9 gene expression by MAPKs RGumet al 1486 transcription factors in the regulation of MMP-9 Carter et al., 1993; Derijard et al., 1995). Consequently, promoter activity, we undertook studies to identify we determined the eect of a dominant negative upstream signaling molecules regulating MMP-9 MEKK expression vector (Minden et al., 1994a) expression in UM-SCC-1 cells. The role of the JNK (refer to Figure 4) on MMP-9 promoter activity. and ERK subsets of MAPKs in the regulation of UM-SCC-1 cells were co-transfected with the MMP-9 MMP-9 expression was determined since these kinases promoter-driven CAT reporter and varying amounts of regulate the synthesis and/or activity of transcription the MEKK mutant (MEKKmt) or an equivalent factors which bind to the AP-1 motif (Adler et al., amount of the empty expression vector (Sra Vector). 1994; Cavigelli et al., 1995; Hibi et al., 1993; Minden et Expression of an equimolar amount of the MEKKmt al., 1994b; Gille et al., 1992; Bogoyevitch et al., 1995). repressed MMP-9 promoter activity by approximately 50% when compared with the empty vector (Sra Vector) (Figure 5a). A twofold molar excess of the Repression of MMP-9 expression by an expression MEKKmt was slightly more active in this regard. vector encoding a mutated JNK1 Similarly, both a one- and twofold molar excess of the JNK proteins immunoprecipitated with an anti-JNK MEKKmt expression vector inhibited the activation of antibody reactive with human and murine JNK1 and the 3X AP-1 pBLCAT reporter construct indicating JNK2 were highly ecient at phosphorylating a GST- regulation of AP-1-dependent gene expression (Figure c-Jun (amino acids 1 ± 79) fusion protein indicating that 5b). Put together, these data indicate a role for a JNK- UM-SCC-1 cells contain an activated JNK(s) (Figure dependent signaling module in the regulation of MMP- 3a). In contrast, NIH3T3 cells, previously shown to 9 expression. contain little activated JNK (Minden et al., 1996), had substantially less kinase activity. UM-SCC-1 cells were Repression of MMP-9 expression by a mutant ERK1 then co-transfected with the MMP-9 promoter-driven expression construct CAT reporter and an expression vector (JNK1mt) (see Figure 4) encoding a dominant negative JNK1 protein Considering that ERK1 is constitutively activated in (Derijard et al., 1994). Expression of a threefold molar UM-SCC-1 cells (Lengyel et al., 1996), we also excess of the mutated JNK1 repressed MMP-9 investigated the role of this MAPK in the regulation promoter activity by over 50% (Figure 3b) compared of MMP-9 expression. ERK1 increases the synthesis of with the empty expression vector (CMV vector). To c-Fos via the phosphorylation of p62Tcf/Elk (Gille et al., con®rm that the JNK1mt expression vector was indeed 1992). To determine if MMP-9 promoter activity was impairing AP-1-dependent gene expression, UM-SCC-1 regulated by this MAPK, we made use of a construct

cells were co-transfected with a CAT reporter driven by (mt ERK1 pCEP4) encoding a kinase-inactive ERK1

either a thymidine kinase minimal promoter (pBLCAT) (Frost et al., 1994). Expression of the mtERK1 pCEP4 or three AP-1 tandem repeats upstream of the minimal inhibited the activity of the MMP-9 promoter-driven promoter (3X AP-1 pBLCAT). The thymidine kinase CAT reporter with a twofold molar excess reducing minimal promoter was only weakly activated in UM- [14C]chloramphenicol acetylation by 70% (Figure 6a). SCC-1 cells (Figure 3c). However, a strong activity was Parallel experiments using a CAT reporter driven by a evident with the thymidine kinase minimal promoter minimal promoter consisting of three tandem AP-1 ¯anked by three AP-1 tandem repeats (Figure 3c). repeats upstream of a thymidine kinase promoter were Transfection of the UM-SCC-1 cells with amounts of conducted to con®rm that the mutated ERK1 the dominant negative JNK1mt expression vector, expression construct was indeed repressing AP-1 which repressed the MMP-9 promoter, similarly activity. Transfection of the kinase-defective ERK1 diminished CAT activity from the 3X AP-1 pBLCAT expression construct into UM-SCC-1 cells reduced the construct. activity of the AP-1-thymidine kinase minimal promo- To determine if the mutated JNK1 expression vector ter (Figure 6b) over the same concentration range that modulated endogenous MMP-9 expression, we transi- attenuated MMP-9 promoter activity. UM-SCC-1 cells ently transfected UM-SCC-1 cells with amounts of the were then transiently transfected with the mutant

JNK1mt construct which repressed the MMP-9 CAT ERK1 or the empty expression vector (pCEP4). The reporter construct. After 24 h, the cells were changed to transfected cells were changed to serum-free medium serum-free medium and incubated for an additional 2 24 h later and incubated for an additional 48 h. The days. The conditioned medium was subsequently conditioned medium was subsequently subjected to collected and subjected to zymography using gelatin as zymography using gelatin as substrate (Figure 6c). A substrate (Figure 3d). A gelatinase activity, indistin- reduced gelatinolysis at the 92 kDa position was guishable in size (92 kDa) from MMP-9, was detected apparent with the conditioned medium from the in the conditioned medium from UM-SCC-1 cells ERK1 mutant-transfected cells compared with cells

transfected with the empty expression vector (CMV made to express the empty vector (pCEP4). vector). However, the intensity of the band was reduced in UM-SCC-1 cells transfected with amounts of the Reduction in MMP-9 enzyme activity/promoter activity mutated JNK1 expression construct which reduced by the MEK1-speci®c inhibitor (PD 098059) and by a MMP-9 promoter activity in the CAT assays. dominant negative expression vector Since ERK1 is a downstream eector of the dual Expression of a dominant negative MEKK represses activity protein kinase MEK1 (Crews et al., 1992), we MMP-9 promoter activity tested the possibility that MMP-9 expression was The JNKs are, via JNKK1, activated by the serine- regulated through this ERK1 activator. Two experi- threonine kinase MEKK (Lin et al., 1995a; Lange- ments were carried out. First, UM-SCC-1 cells were Regulation of MMP-9 gene expression by MAPKs RGumet al 1487 treated for 4 days with dierent concentrations of a Second, we asked whether transfection of UM-SCC- speci®c inhibitor of MEK1 (PD 098059 ± refer to 1 cells with a dominant negative MEK1 expression Figure 4) (Dudley et al., 1995; Alessi et al., 1995) or vector (Mansour et al., 1994) altered MMP-9 promoter an equivalent amount of the DMSO carrier, the activity. UM-SCC-1 cells were co-transfected with the conditioned medium collected and assayed for gelati- MMP-9 promoter-driven CAT reporter and dierent nolysis (Figure 7a). Concentrations of the agent, which amounts of an expression vector encoding either the have been shown to block MEK1 (Alessi et al., 1995; wild type (MEK1 wt) or a mutated (MEK1 K97M) Lin et al., 1995b), were highly eective at reducing MEK1 sequence. A threefold molar excess of the 92 kDa gelatinolysis (compared with the paired dominant negative expression vector reduced MMP-9 amount of carrier alone). In this particular experiment promoter activity by approximately 50% relative to an we also detected a 72 kDa gelatinase, which was equivalent amount of the MEK1 wt expression indistinguishable in size from MMP-2 (gelatinase A) construct (Figure 7b). (Huhtala et al., 1990). However, this enzyme activity was not diminished by the PD 098059 (compared with Expression of a kinase-de®cient c-Raf-1 represses the the equivalent amount of DMSO) indicating speci®city MMP-9 promoter of this drug for 92 kDa gelatinolysis. The concentrations of PD 098059 used in the current study did not The activity of MEK1 is controlled by the serine- retard the growth of the cells. threonine kinase c-Raf-1 and we determined the eect of a kinase-de®cient c-Raf-1 expression construct (Raf C4) (Bruder et al., 1992) on the regulation of MMP-9 promoter activity in UM-SCC-1 cells. Towards this end, UM-SCC-1 cells were co-transfected with varying amounts of the Raf C4 expression vector or the empty vector (Vector) and a CAT reporter driven by either the MMP-9 promoter (Figure 8a) or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter (Figure 8b). Raf C4 expression in UM-SCC- 1 cells repressed, in a dose-dependent manner, MMP-9 promoter activity (Figure 8a). The reduced MMP-9 promoter activity coincided with the diminished activity of the AP-1-regulated CAT reporter (Figure 8b). We then asked whether stimulation of c-Raf-1 by phorbol ester treatment was inductive for MMP-9 Figure 4 Diagrammatic representation of the targets of the expression. The phorbol ester PMA (phorbol 12- dominant negative expression constructs and PD 098059. The myristate 13-acetate) binds to protein kinase C which JNK and ERK-dependent signaling cascades leading to altered in turn activates c-Raf-1 (Sozeri et al., 1992). synthesis and/or activity of AP-1-binding proteins are shown. An arrow with an X indicates the molecule targeted by the dominant Treatment of UM-SCC-1 cells with PMA caused a negative expression constructs or by PD 098059 dose-dependent increase in MMP-9 activity (Figure

a b

Figure 5 Repression of MMP-9 promoter activity by a dominant negative MEKK expression vector in UM-SCC-1 cells. UM- SCC-1 cells were transiently transfected and assayed for CAT activity, as described in the legend to Figure 3b using the indicated amount (where 26 represents a twofold molar excess of the eector plasmid over the reporter construct) of an expression vector encoding a dominant negative MEKK1 protein (MEKKmt) or the empty vector (SRa vector). Transfections and CAT assays were as described in the legend to Figure 3c. The data are typical of triplicate experiments 1488 elnme a sae o eaioyi.Tepstoso h 2ad7 D eaiae r niae.( indicated. carrier are or gelatinases 098059 in kDa dierence PD 72 any of and for presence 92 normalized or days, the medium absence 2 Conditioned of After the counted. positions alone. in cells The (DMSO) same the gelatinolysis. carrier and the for the later with assayed of days replenished was amount 2 and equivalent number collected medium an cell was or serum-free medium DMSO with Conditioned in washed alone. 098059 were PD ( cells of the respectively. concentrations indicated vector the expression without MEK1 negative dominant 7 Figure pCEP ERK1 (mt vector expression ( ERK1 3c. negative ( dominant Figure activity. a to CAT with legend for 3d, assayed the Figure were to in eciency, pCEP4) legend ERK1 described transfection (mt the in as protein in dierences ERK1 out described for negative as dominant corrected carried a extracts, transfected, were encoding Cell vector transfections (pCEP4). expression vector Transient an of empty construct) the reporter or the to relative plasmid a reporter, CAT ( cells. 1 6 Figure n he eaaeeprmnsfor experiments separate three and A ciiy h muto [ of amount The activity. CAT neumlraon wt epc oterpre osrc)o nepeso etrecdn oiatngtv R1protein ERK1 negative dominant a or sequence encoding MEK1 vector wt) expression (MEK1 an type of wild 1 construct) or K97M) (where reporter (MEK1 amount the pCEP mutated indicated to a ERK1 the respect encoding (mt with (with vector 3b amount expression Figure an equimolar to of an reporter) legend CAT the MMP-9 in the described as transfected, transiently a eetda ht oei ak®l.Tedt r ersnaieo rpiaeexperiments triplicate of representative Proteolysis are gelatinolysis. data for The assayed ®eld. eciency, dark transfection a in dierences in on zone based white amounts a and as later, detected h was 48 collected was medium same 7 9 ab 2 kDa 2 kDa m mutcrepnigwt 2 with corresponding amount g a MSC1clswr rninl rnfce,a ecie ntelgn oFgr buiga M- promoter-driven MMP-9 an using 3b Figure to legend the in described as transfected, transiently were cells UM-SCC-1 ) oiatngtv R1epeso etrrpessMP9pooe ciiyadgltnltcatvt nUM-SCC- in activity gelatinolytic and activity promoter MMP-9 represses vector expression ERK1 negative dominant A niiino eaioyi n M- rmtratvt yaceia niio fMK P 909 n ya by and 098059) (PD MEK1 of inhibitor chemical a by activity promoter MMP-9 and gelatinolysis of Inhibition ¨ ¨

.1 .6 0.13 0.065 0.013 –––  b 4

risepyvco (pCEP vector empty its or )

glcoiaeepesn etradteidctdaon wee2 (where amount indicated the and vector -galactosidase-expressing euaino M- eeepeso yMAPKs by expression gene MMP-9 of Regulation

 a b

1510 –– –– 

]hoapeio ctltdwsdtrie iha63Btsoe h aaaetpclo two of typical are data The Betascope. 603 a with determined was acetylated C]chloramphenicol  a  and 6

in b 

a respectively  (pCEP vector empty the or )

.Cl xrcs orce o ieecsi rnfcinecec,wr sae for assayed were eciency, transfection in dierences for corrected extracts, Cell ).  RGum PD 098059( DMSO (%v/v) a % Chloramphenicol converted al et MSC1clswr ltdi eu-otiigmdu ih or with, medium serum-containing in plated were cells UM-SCC-1 ) 10 15 20 30 35 40 45 25 0 5 µ M –– –+– ––––– ––+ ––––– –––1X–3X––––1X–3X––– +++ –++++ ) 4 n a and ) b glcoiaeepesn etr Conditioned vector. -galactosidase-expressing c 6 72 kDa 92 kDa c MSC1clswihwr transiently were which cells UM-SCC-1 ) satoodmlrecs fteeector the of excess molar twofold a is ¨ ¨ 6 steaon qioa with equimolar amount the is pCEP4 b MSC1clswere cells UM-SCC-1 ) mt ERK1 pCEP4 pCEP mt ERK1pCEP MEK1 K97M MEK1 wt MMP-9 CAT 4 4 (the ) 4 b ) Regulation of MMP-9 gene expression by MAPKs RGumet al 1489 ab

72 kDa 92 kDa ¨ ¨

200 

20  PMA (nM) 2  DMSO

Figure 8 Repression of MMP-9 promoter activity by the expression of a kinase-inactive c-Raf-1. (a) UM-SCC-1 cells were transiently transfected, as described in the legend to Figure 3 using a CAT reporter driven by either the wild type MMP-9 promoter (MMP-9 CAT) (a) or 3 tandem AP-1 repeats upstream of a thymidine kinase minimal promoter (36AP-1 pBLCAT) (b) and the indicated amount (where 26 is a twofold molar excess of the eector plasmid relative to the reporter construct) of an expression vector encoding a dominant negative c-Raf-1 protein (Raf C4) or the empty vector (Vector). Cell extracts, normalized for dierences in transfection eciencies, were assayed for CAT activity. (c) UM-SCC-1 cells at 75% con¯uency were changed to serum free medium supplemented with, or without, the indicated concentration of PMA and cultured for 48 h. After this time, the conditioned medium was harvested and centrifuged to remove cellular debris. Cell numbers were determined. Aliquots of the conditioned medium, corrected for dierences in cell number, were assayed for MMP-9 activity by zymography as described in the legend to Figure 3d. The experiment was carried out three times

8c). Together these data indicate a role for an ERK- the AP-1-binding transcription factor(s) targeted by dependent signaling pathway in the regulation of either of these signaling cascades. Certainly, a likely MMP-9 expression in UM-SCC-1 cells. candidate is c-Fos since the gel shift assays revealed this transcription factor bound to the AP-1 motif at 779. Indeed, the synthesis of c-Fos is regulated both Discussion by the JNK-(Whitmarsh et al., 1995; Cavigelli et al., 1995) and the ERK- (Gille et al., 1992) dependent The 92 kDa type IV collagenase plays a critical role in signaling modules via the phosphorylation of p62Tcf/Elk. tumor cell invasion (DeClerck et al., 1992; Anderson et In fact, Whitmarsh and co-workers (Whitmarsh et al., al., 1996; Cha et al., 1996) by virtue of its ability to 1995) have concluded that the serum-response element hydrolyze type IV collagen present in basement (in the c-Fos promoter) represents a point of membranes. While studies by other investigators have convergence of the JNK and ERK-dependent signal- identi®ed a diverse set of exogenous agents including ing modules. It should be emphasized, however, that EGF, TNF-a, the v-src oncogene as well as phorbol our studies do not rule out the possibility that the ester which induce the synthesis of this enzyme there regulation of MMP-9 expression by the JNK- and have been no studies investigating the signaling events ERK-dependent signaling cascades also requires connecting the cell surface to the transcriptional transcription factors binding to non-AP-1 motifs machinery regulating the expression of this gene. To necessary for optimal promoter activation in UM- the knowledge of the authors, this is the ®rst SCC-1 cells (e.g. the GT box at 754 or the NF-kB demonstration of a role for JNK- and ERK- motif at 7600). Nevertheless, the observations that dependent signaling cascades in the regulation of MMP-9 promoter activity is reduced substantially MMP-9 expression. either by the co-expression of a construct (TAM-67), Our data are consistent with the notion that the which interferes with the ability of endogenous Fos regulation of MMP-9 expression in UM-SCC-1 cells and Jun proteins to transactivate AP-1 controlled by the JNK- and ERK-dependent signaling modules genes, or by mutation of the AP-1 motif at 779 is, at least in part, through the modulation in the strongly suggest a role for AP-1-binding proteins in synthesis and/or activity of AP-1-binding proteins. the regulation of expression of this collagenase in However, we can only speculate as to the identity of UM-SCC-1 cells. Regulation of MMP-9 gene expression by MAPKs RGumet al 1490 We were initially surprised by the requirement of a ERK and JNK members) (Liu et al., 1995; Alessi et al., JNK in the regulation of MMP-9 expression since the 1993; Chu et al., 1996) to counter this induction. In ®rst member of this MAPK subgroup (JNK1) was contrast, interfering with the ERK activator MEK1 by originally identi®ed as being rapidly induced in employing a dominant negative MEK1 expression response to ultraviolet light or osmotic stress vector had little eect on the induction of MMP-9 (Derijard et al., 1994). However, in the pioneering promoter activity by Ha-Ras suggesting the involve- studies (Derijard et al., 1994), while the authors ment of MAPK(s) other than ERK1/ERK2. demonstrated ultraviolet light to be the most eective The idea that the expression of inducible genes can stimulus for JNK activation, a threefold induction of be regulated by multiple signaling modules which this kinase was observed with EGF. Indeed, it is now terminate in activated AP-1-binding transcription becoming apparent that this signaling kinase may be factors is not unprecedented. Zohn and others (1995) implicated in the regulation of expression of at least reported that angiotensin II, like EGF, was capable of some inducible genes. For example, Xie and Hersch- activating both the ERK and JNK subsets of MAPK man (1995) recently reported that the induction of in GN4 rat liver epithelial cells albeit with dierent prostaglandin synthase 2 gene expression by v-src was potencies. Further, in a report by Khosravi-Far et al. via activation of JNK1 and through the c-Jun (1995) it was demonstrated that the activity of a transcription factor. Further, Khosravi-Far and co- weakly transforming c-Raf-1 was enhanced 35-fold by workers demonstrated that the oncogenic transforma- the co-expression of a constitutively activated Rac1 the tion of NIH3T3 cells by Ras could be antagonized by latter being an upstream activator of the JNK pathway the co-expression of Rac1 (Khosravi-Far et al., 1995). (Minden et al., 1996; Olson et al., 1995). Finally, Rac1 is, via MEKK, an upstream activator of JNK Bogoyevitch and others (1995) reported that endothe- (Minden et al., 1996). Our ®ndings could provide an lin-1, a known activator of the ERKs, also transiently explanation as to how a diverse set of stimuli including stimulated JNKs two ± ®vefold in ventricular myocytes. the v-src oncogene (Sato et al., 1993), IL-1 (Hurwitz et We can only speculate that the ability of the cell to al., 1993; Lyons et al., 1993; Unemori et al., 1991) and employ more than one signaling module to control TNF-a (Sato and Seiki, 1993) all induce MMP-9 MMP-9 expression may provide it with a means of expression. Recent studies have shown that IL-1 responding to a diverse set of growth factors/cytokines treatment increases JNK activity (Bagrodia et al., and/or environmental conditions. 1995) and TNF-a also causes a rapid stimulation of In conclusion, the study herein, suggests that MMP- this kinase while only weakly inducing the ERK subset 9 expression in UM-SCC-1 cells is regulated by JNK- of MAPK at least in HL 60 cells (Westwick et al., and ERK-dependent signaling modules. These ®ndings 1995). Likewise, v-src is a potent inducer of JNK1 (Xie raise the exciting possibility that interfering with either, and Herschman, 1995). Since our studies demonstrated or both, of these pathways could reduce MMP-9 a clear involvement of a JNK(s) in the regulation of synthesis in invasive cancer (Pyke et al., 1992; MMP-9 expression it may very well be that the ability Nakajima et al., 1993; Rao et al., 1993) and in of these cytokines and the v-src oncogene to induce the in¯ammatory disorders such as rheumatoid arthritis expression of this collagenase is mediated through this (Unemori et al., 1991). MAPK subset. On the other hand, phorbol ester, which is also a potent stimulus for MMP-9 expression is, via c-Raf-1 (Sozeri et al., 1992), an ERK1, but not a JNK (Cavigelli et al., 1995; Bogoyevitch et al., 1995) Materials and methods activator. Thus, it is more likely that its inductive eects on synthesis of this collagenase is propagated Materials along an ERK-dependent signaling cascade. Indeed, The following antibodies used in mobility shift assays were this contention is given further support by the studies purchased from Santa Cruz Biotechnology: # SC 6056 of Sato and co-workers showing dierent transcription which is speci®c for Fra-1; # SC 526 which reacts factor binding site requirements for MMP-9 induction speci®cally with c-Fos; # SC 6046 which speci®cally by v-src and by phorbol ester (Sato and Seiki, 1993; reacts with Fra-2; # SC 8226 directed against c-Jun; # Sato et al., 1993). In those studies, the authors SC 466 which speci®cally recognizes Jun-B; and # SC 1876 which reacts with ATF-2. The speci®c anti-Jun- demonstrated that while the induction of MMP-9 D antibody was kindly provided by Dr Moshe Yaniv, expression by v-src was mediated through a 90 base (Pasteur Institute, Paris, France). pair sequence containing the proximal AP-1 (779) motif and a GT box (754), increased promoter activity brought about by PMA treatment required Cell culture the AP-1 (779), NF-kB(7600) and Sp1 (7558) UM-SCC-1 cells derived from a squamous cell carcinoma motifs but not the GT box at 754 (Sato and Seiki, of the oral cavity were obtained from Dr Thomas Carey at 1993; Sato et al., 1993). the University of Michigan. All cells were maintained in The current study may also shed light onto the McCoy's 5A medium containing 10% FBS with the mechanism by which Ha-Ras activates MMP-9 exception of conditioned medium collection which was performed using McCoy's 5A medium supplemented with expression in the OVCAR-3 ovarian cancer cells as 4 mg/ml transferrin, 5 mg/ml insulin and 10 ng/ml EGF previously reported by this laboratory (Gum et al., (serum-free medium). 1996). In that study, we demonstrated that the induction of MMP-9 expression in OVCAR-3 cells by Ha-Ras utilized a MAPK-dependent signaling pathway DNA constructs as evidenced by the ability of a phosphatase (CL100), The TAM-67 vector, encoding a c-Jun protein lacking the which inactivates multiple MAPK members (including transactivation domain (amino acids 3 ± 122 absent) of the Regulation of MMP-9 gene expression by MAPKs RGumet al 1491 molecule, has been described elsewhere (Brown et al., 1993; DNA was labeled with Klenow then electrophoresed in a Domann et al., 1994). The Raf C4 expression vector 5% polyacrylamide gel. The DNA fragment was isolated, encodes a mutated c-Raf-1 lacking the kinase domain of and puri®ed by sodium acetate/ethanol precipitation. End- the serine-threonine kinase (Bruder et al., 1992). The labeled DNA (15 ng) was mixed with poly (dI-dC), nuclear ERK1 construct encodes the ERK1 cDNA in which the extract and transcription buer (20 mM HEPES, conserved lysine at codon 71 was changed to arginine thus 100 mM KCl, 2 mM EDTA, 5 mM MgCl2, 1.5 mM DTT) in impairing the catalytic eciency (Frost et al., 1994). The a total volume of 20 ml. Binding was allowed to proceed for dominant negative JNK1 used herein includes the coding 15 min at room temperature after which DNase I (®nal sequence in which threonine 183 and tyrosine 185 are concentration 10 mg/ml) was added. A stop solution (100 ml) substituted with alanine or phenylalanine respectively containing 10 mM EDTA, 0.1% SDS and 50 mg/ml of (Derijard et al., 1994). The mutated MEKK expression proteinase K was added 30 s later and the mixture construct encodes a MEKK cDNA harboring a lysine to incubated at 378C for 30 min. The mixture was extraced methionine substitution at residue 432 and lacking the Nco with phenol/chloroform, precipitated, washed with 70% fragment from the aminoterminus (Minden et al., 1994a). ethanol and dried. The material was dissolved in The MEK1 K97M expression construct encodes the MEK1 denaturing solution containing formamide and electrophor- sequence in which a lysine at codon 97 is replaced with a esed in a DNA sequencing gel. methionine residue (Mansour et al., 1994). The 36AP1 pBLCAT construct consists of three AP-1 tandem repeats upstream of a thymidine kinase minimal promoter-CAT Electrophoretic mobility shift assays (EMSA) reporter (pBLCAT) (Angel et al., 1988). The wild type, mutated and 5' deleted MMP-9 promoter fragments have Nuclear extract was prepared as described previously been described elsewhere (Sato and Seiki, 1993; Sato et al., (Schreiber et al., 1989). Cultured cells were collected by 1993; Gum et al., 1996). centrifugation, washed and resuspended in a buer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM- Chloramphenicol acetyl transferase (CAT) assays PMSF. After 15 min on ice, the cells were vortexed in the presence of 0.6% Nonidet NP-40. The nuclear pellet was UM-SCC-1 cells were co-transfected at 70% con¯uency then collected by centrifugation and extracted in a buer with CAT reporter constructs and 4 mg of an expression containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, vector bearing the b-galactosidase gene. DNA dissolved in 1mMEDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF 1ml of HBS buer (25mMHEPES, 1 mM MgCl , 2 for 15 min at 48C. The extract was centrifuged and the 0.1 mM CaCl ,0.1MNaCl, 5 mM KCl and 0.7 mM 2 supernatant frozen at 7808C until needed. Na HPO , pH 7.44) was mixed with 50 mg hexadimethrine 2 4 Nuclear extract (10 mg) was incubated at 218C for 10 min, bromide (Polybrene, Aldrich Chemicals, WI) and added to with shaking, in a buer (25 mM HEPES buer pH 7.9, the cells in 5 ml of 10% FBS. After 6 h the cells were 0.5 mM EDTA, 0.5 mM DTT, 0.05 M NaCl and 2.5% glycer- shocked for 4 min with 34% DMSO and cultured for an ol) containing 2 mg of poly dI/dC and 5 fmoles (36104 additional 48 h. The cells were then harvested and lysed by c.p.m.) of a Klenow end-labeled [a32P]ATP oligonucleotide repeated freeze-thaw cycles in a buer containing (spanning nucleotides 791 to 760 relative to the transcrip- 0.25 M Tris-Cl pH 7.8. Transfection eciencies were tional start site) in the absence, or presence, of a 100-fold determined by assaying for b-galactosidase activity. CAT excess of the wild type or mutated competitor sequence. After activity was subsequently measured by incubating cell this time 1 mg of the indicated antibody was added and the lysate (normalized for transfection eciency) at 378Cfor incubation continued for 10 min. The reaction mixture was 8 h (unless otherwise stated) with 4 mM electrophoresed in a 5% polyacrylamide gel, containing 5% [14C]chloramphenicol and 1 mg/ml acetyl coenzyme A. glycerol using 0.56TBE (89 mM Tris, 89 mM boric acid and After 4 h, the acetyl coenzyme A was replenished. The 1mMEDTA) running buer. The gel was rinsed with water, acetylated products were extracted with ethyl acetate and dried and exposed to X-ray ®lm overnight. were subjected to thin layer chromatography using chloroform/methanol (95:5) as the mobile phase. The amount of acetylated [14C]chloramphenicol was determined using a 603 Betascope. JNK activity assays JNK activity was determined as described elsewhere (Coso et al., 1995). Cell extracts prepared in a buer containing DNase I footprinting (1% Triton X-100, 25 mM HEPES pH 7.6, 300 mM NaCl,

Nuclear extracts were prepared according to methods 1.5 mM MgCl2,0.2mMEGTA, 0.5 mM DTT, 0.5% sodium previously published (Dignam et al., 1983). Brie¯y, cells deoxycholate, 0.1% SDS, 20 mM b-glycerophosphate, (^109) were collected, washed with PBS and the packed 20 mg/ml aprotinin, and 20 mg/ml leupeptin, 1 mM PMSF, cell volume determined. The cells were resuspended in 1mMsodium vanadate) were incubated at 48C for 2 h with buer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 1.5 mg of an anti-JNK antibody (Santa Cruz Biotechnology 10 mM KCl, 2 mM DTT) and lysed with a Dounce # sc-474) which is crossreactive with mouse and human homogenizer. Nuclei were collected by centrifugation and JNK1 and JNK2. Protein A agarose beads were then resuspended in a 1/2 volume of a buer (C) containing added to the mixture and incubated for an additional 1.5 h 20 mM HEPES (pH 7.9), 20% glycerol, 0.6 M NaCl, at 48C. The beads were rinsed several times as described

1.5 mM MgCl2,0.2mMEDTA, 0.5 mM PMSF and elsewhere (Coso et al., 1995) and then incubated in 30 mlof

2mMDTT. Nuclei were fractured using a Dounce pestle, kinase buer (12.5 mM MOPS (pH 7.5), 7.5 mM MgCl2, stirred for 30 min at 48C and then centrifuged. The 12.5 mM b-glycerophosphate, 0.5 mM EGTA, 0.5 mM NaF, supernatant (nuclear extract) was retained and dialyzed 0.5 mM sodium vanadate) with 1 mCi [g32P]ATP, 2 mgGST- against 100 volumes of a buer containing 20 mM HEPES c-Jun protein (amino acid residues 1 ± 79 of c-Jun) and pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 20 mM cold TP at 308C. The reaction was terminated

5mMMgCl2 and 2 mM DTT. The extract was centrifuged 20 min later with 26Laemmli's buer, the reaction for 20 min and aliquots frozen at 7808C until required. mixtureheatedto1008C for 5 min and the beads removed An MMP-9 promoter CAT reporter plasmid containing by centrifugation. The supernatant (65 ml) was electro- nucleotides +1 to 7144 (Sato and Seiki, 1993) of the phoresed in a 12% polyacrylamide gel. The gel was dried promoter was digested with Asp718 and PstI. The digested and autoradiographed for 6 h. Regulation of MMP-9 gene expression by MAPKs RGumet al 1492 Zymography Melanie Cobb (Southwestern Medical Center, Dallas, Texas) for the mutated ERK1 expression vector. We These were performed as described previously (Gum et al., would like to express our thanks to Dr Ulf Rapp, 1996). Culture supernates were denatured in the absence of Wurzburg Germany for the Raf-1 C4 expression vector. reducing agent and electrophoresed in a 7.5% polyacryla- We are indebted to Dr Michael Birrer (Biomarkers and mide gel containing 0.1% (w/v) gelatin. The gel was Prevention Research Branch, NIH, Bethesda, Maryland) incubated at room temperature for 2 h in the presence of for supplying the TAM-67 expression construct. The work 2.5% Triton X-100 and subsequently at 378C overnight in would not have been possible without the wild type and 5' a buer containing 10 mM CaCl ,0.15MNaCl, and 2 deleted MMP-9 promoter fragment-CAT reporter con- 50 mM Tris (pH 7.5). The gel was then stained for protein structs kindly provided by Dr Motoharu Seiki (Cancer with 0.25% Coomassie and photographed on a light box. Research Institute, Kanazawa University, Ishikawa, Ja- Proteolysis was detected as a white zone in a dark ®eld. pan). The mutated JNK and MEKK expression vectors were kindly provided by Dr Michael Karin, (University of California at San Diego, California) and by Dr Gary Acknowledgements Johnson respectively (National Jewish Center for Immu- Supported by National Institute of Health (R01DE10845 nology and Respiratory Medicine, Denver, Colorado). We and R01CA58311) and Council for Tobacco Research would like to thank Dr Alan Saltiel at Parke Davis (Ann (3438) grants. We are grateful to Dr Natalie Ahn Arbor, Michigan) for providing the PD 098059. Finally, we (University of Colorado, Denver, Colorado) for kindly would like to thank Dr Xian-Jun Fang for the GST-c-Jun providing the MEK1 wt and MEK1 K97M expression fusion protein and Drs Audrey Minden and Gordon Mills vectors. We are also indebted to Drs Je Frost and for advice and helpful discussions.

References

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