Regulation of 92 Kda Type IV Collagenase Expression by the Jun Aminoterminal Kinase- and the Extracellular Signal-Regulated Kinase- Dependent Signaling Cascades
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Oncogene (1997) 14, 1481 ± 1493 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 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).