The importance of apoptosis in normal development and pathogenesis has been well recognized, and explosive progress towards dissecting its commitment step has been made during the past decade. Mitochondria, Apaf-l, caspase, and bcl-2 family members play central roles in the commitment step. However, it is still unclear how upstream cell survival pathways regulate apoptosis. It is also unknown whether the bcl-2 family members have any effect on the upstream survival pathways. Preliminary studies of this application demonstrate that the anti-apoptotic gene product bcl-2 greatly induces expression of the tissue inhibitor of metalloproteinase-1 (TIMP-1) in human breast epithelial cells. Surprisingly, we found that TIMP-1, like bcl-2, is a potent inhibitor of apoptosis induced by a variety of stimuli. Functional studies indicate that TIMP-1 inhibits a classical apoptotic pathway mediated by caspases, and that focal adhesion kinase (FAK)/PI 3-kinase and mitogen activated protein kinase (MAPK) are critical for TIMP-1-mediated cell survival. Preliminary studies show specific association of TIMP-1 with the cell surface. Consistently, a 150-kDa surface protein was identified in MCF10A cells that specifically binds TIMP- 1. Taken together, we hypothesize that TIMP-1 binding on the cell surface induces a cell survival pathway that regulates the common apoptosis commitment step. To test our working hypothesis of a positive feedback loop between bcl-2 and TIMP-1 in apoptosis regulation, we propose to (1) investigate TIMP-1 induction of the FAK/PI 3-kinase/Akt and MAPK survival pathways, (2) determine the mechanism by which TIMP-1 inhibits caspase activation, (3) establish the structural basis for the anti-apoptotic activity of TIMP-1, and (4) study the cell surface binding of TIMP-1 and characterize its binding partner in MCF10A cells. The results of these studies will address a new paradigm in the regulation of apoptosis by an extracellular molecule TIMP-I, and also greatly enhance our understanding of TIMP-1's pleiotropic activity in many physiological and pathological processes. This information may also be useful in designing more rational therapeutic interventions aimed at modulating the anti-apoptotic activity of TIMP-1.
