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 m atrix components directly and indirectly by activation of other matrix meta lloproteinases (MMPs). In the present study, we have isolated and character ized 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 tw o exon-intron splicing sites are distinct from the preserved positions amon g other known MMP genes. Primer extension and RNAse and S1 nuclease protect ion analyses indicated that there are four major and several minor transcri ption start sites. The 5'-flanking sequence of the gene contains putative r egulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas t here is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter construct s 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-di rected 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 structura l and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and patholo gical processes. (C) 2000 Elsevier Science B.V. All rights reserved.