Abstract

Mammalian extracellular matrix metalloproteinases, MMPs, are a family of enzymes capable of degrading components of the connective tissue. The in vivo regulation of the cell lineage-specific expression of MMPs, however, is not well known. This study used transgenic mice to identify cell-specific elements in the upstream regulatory regions of MMP-2 and MMP-9. Transgenic mice were generated by pronuclear microinjections into fertilised oocytes using lacZ as a reporter gene. The reporter gene constructs containing varying lengths of the MMP-9 5'-upstream region revealed an area that allowed for expression in osteoclasts and migrating keratinocytes, the cells that also express MMP-9 in vivo. The sequence driving the cell specific expression included the nucleotides from -2722 to -7745.

When the same upstream regulatory fragment of MMP-9 was used to drive the expression of the human tissue specific inhibitor of MMPs, TIMP-1, instead of lacZ, the transgenic mice developed normally and the animals were fertile with normal post-embryonic growth. However, cutaneous wound healing was remarkably retarded, but not totally prevented, and the migration of keratinocytes over the wound was slow. The mice expressed the human TIMP-1 in keratinocytes during wound healing and in situ zymography revealed a total blockage of the gelatinolytic activity of MMP-2 and MMP-9, the main gelatinases active in the healing wound tissues.

By using a sequence of 6500 base pairs from the 5'-upstream regulatory region of the MMP-2 gene it was possible to drive the expression of lacZ in mesenchymal cells of the developing transgenic mouse embryo. The expression pattern was similar to that found in previous in situ hybridization studies, following the different stages of tissue morphogenesis and being present in the areas of basement membrane degradation and epithelial cell invasion. Computer analyses of the sequence revealed three regulatory upstream regions conserved between human, mouse, and rat, and possibly responsible for the cell-and tissue specificity. New transgene constructs containing fragments of the conserved regions will provide a more detailed profile of the in vivo MMP-2 regulation in the future.

This study defined a fragment in the upstream regulatory region of MMP-9 that is essential for expression in osteoclasts and migrating keratinocytes. Furthermore, the keratinocyte derived MMPs, including MMP-9, were found to play important role in epithelial cell migration in the area of the healing wound.