TIMP-1 and Apoptosis

描述（申请人提供）：细胞凋亡在正常发育和发病机制中的重要性已得到充分认识，在过去的十年中，在解剖其承诺步骤方面取得了爆炸性进展。线粒体、apaf - 1、caspase和bcl-2家族成员在承诺步骤中发挥核心作用。然而，尚不清楚上游细胞存活途径如何调节细胞凋亡。目前尚不清楚bcl-2家族成员是否对上游生存途径有任何影响。该应用的初步研究表明，抗凋亡基因产物bcl-2可显著诱导人乳腺上皮细胞中组织金属蛋白酶-1 （TIMP-1）的表达。令人惊讶的是，我们发现TIMP-1和bcl-2一样，是多种刺激诱导的细胞凋亡的有效抑制剂。功能研究表明，TIMP-1抑制了由caspase介导的经典凋亡途径，并且局灶黏附激酶(FAK)/PI 3-激酶和丝裂原活化蛋白激酶（MAPK）对TIMP-1介导的细胞存活至关重要。初步研究表明TIMP-1与细胞表面有特异性关联。一致地，在MCF10A细胞中发现了一个150 kda的表面蛋白特异性结合TIMP- 1。综上所述，我们假设TIMP-1在细胞表面的结合诱导了一个细胞存活途径，该途径调节了常见的凋亡承诺步骤。为了验证bcl-2和TIMP-1在细胞凋亡调节中存在正反馈回路的工作假设，我们提出(1)研究TIMP-1诱导FAK/PI 3-激酶/Akt和MAPK存活通路，(2)确定TIMP-1抑制caspase激活的机制，(3)建立TIMP-1抗凋亡活性的结构基础，(4)研究TIMP-1在MCF10A细胞中的细胞表面结合及其结合伙伴的特征。这些研究结果将为细胞外分子TIMP-1调控细胞凋亡提供一个新的范式，并极大地增强了我们对TIMP-1在许多生理和病理过程中的多效性的理解。这一信息也可能有助于设计更合理的治疗干预措施，旨在调节TIMP-1的抗凋亡活性。

DESCRIPTION (provided by applicant): 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.