Locatization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus - Metalloproteinase inhibition is associated with proapoptotic activity

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four se creted inhibitors of matrix metalloproteinases (MIMPs). Recently, additiona l functions have been attributed to the TIMPs, including cell growth and in hibition of angiogenesis. In particular, we demonstrated that TIMP-3 overex pression using gene transfer induces apoptosis in a variety of cell types a nd can inhibit vascular neointima formation in vivo. However, little is kno w about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using bo th purified recombinant proteins and novel adenoviral vectors we demonstrat e that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins pr omoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furth ermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhib itory activity of TIMP-3 and also failed to promote apoptosis. This study d emonstrates, using multiple apoptosis assays, that the predeath function of TIMP-3 is located within the N-terminal three loops and the presence of fu nctional metalloproteinase-inhibitory activity is associated With the induc tion of apoptosis.