REGULATION OF MATRIX METALLOPROTEINASE ACTIVATION

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The invasion of tumor cells depends on a cascade of proteolytic enzymes capable of degrading extracellular matrix components. The 72 kDa (MMP-2) and 92 kDa (MMP-9) enzymes are two members of the matrix metalloproteinase family which have been associated with tumor invasion and metastasis. The long term objective of this proposal is to unveil the biochemical and biological mechanisms controlling the activation and inhibition of MMP-2 and MMP-9 and their relevance to the degradation of ECM in human diseases. The activity of the 72 and 92 kDa enzymes is regulated by the generation of active species and their interactions with the tissue inhibitors of metalloproteinases (TIMPs). We and other have shown that the C-terminal end of these proteinases is the TIMP binding domain in the proenzyme form and is necessary for the plasma membrane- dependent activation of MMP-2. Preliminary studies suggest that the activation process generates active forms lacking the C-terminal end. However, the biochemical and biological properties of these species is poorly understood. We hypothesize that the C-terminal end of the 72 and 92 kDa enzymes is a key regulator of activation and enzymatic activity and its cleavage generates active enzymes with a reduced sensitivity to TIMP inhibition. To define the functional properties of the active species and the significance of the C-terminal domain in the regulation of these enzymes we propose (1) to study the molecular properties of the active species of MMP-2 and MMP-9 formed after cleavage of the C-terminal domain using analytical and biochemical methods; (2) to express a secreted MMP-2 C- terminal end in a vaccinia expression system and in tumor cells to study the role of this domain on activation, TIMP-2 inhibition and in vitro cell invasion and, (3) to construct and express chimeric MMP-2 and MMP-9 enzymes with a heterologous C-terminal end in a vaccinia expression system and in tumor cells to determine the importance of the C-terminal domain in membrane activation and interactions with TIMPs. The proposed studies will provide fundamental information on the regulation of MMP activity and inhibition and may contribute to the development of specific inhibitors to control the activity of these enzymes in connective tissue diseases and in malignant processes.