Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades.
The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)- and extracellular signal-regulated kinase- (ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we first determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning -144 to -73 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides -91 to -68 and containing a consensus AP-1 motif at -79. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinase-deficient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-deficient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinase-deficient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK- and ERK-dependent signaling pathways.
Gum
R
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Department of Head & Neck Surgery/Tumor Biology, M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Wang
H
H
Lengyel
E
E
Juarez
J
J
Boyd
D
D
D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D017364:Collagenases / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020780:Matrix Metalloproteinase 9; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one