Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene.
Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon-intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5'-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5'-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.
Lohi
J
J
Department of Virology, Haartman Institute, University of Helsinki, Finland. jlohi@helsinki.fi
Lehti
K
K
Valtanen
H
H
Parks
W C
WC
Keski-Oja
J
J
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes / Q000235:genetics; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D008156:Luciferases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate