Metalloproteinase Regulation of Neuronal Death

描述(申请人提供)：在多种细胞类型中，细胞表面的金属蛋白酶活性可以强烈地影响细胞对外源性死亡信号的敏感性，而不是生存信号，然而金属蛋白酶和受体信号在神经系统中的趋同在很大程度上仍未被探索。金属蛋白酶组织抑制因子-3(TIMP-3)是一种独特的天然金属蛋白酶抑制因子，是所有已知的金属蛋白酶脱落酶的有效抑制剂，已被证明在多种非神经细胞类型的受体介导的细胞死亡调节中发挥作用。TIMP-3通过抑制针对死亡受体及其配体的脱落酶的能力，在许多细胞类型中发挥促凋亡作用。我们对金属蛋白酶在卒中后中枢神经系统损伤中的作用的兴趣促使我们研究了TIMP-3和金属蛋白酶脱落酶-3在大鼠局灶性脑缺血模型中的表达。我们发现，尽管TIMP-3和MMP-3在成人脑中的表达水平很低，甚至无法检测到，但在缺血后，它们的表达显著上调，特别是在经历延迟性凋亡死亡的皮质神经元中。采用组织培养的方法，我们发现TIMP-3和MMP-3在培养的胚胎皮质神经元中有结构性表达，并调节神经元对化疗药物阿霉素(Dox)诱导的受体介导的细胞凋亡的敏感性。TIMP-3对金属蛋白酶的抑制是Dox诱导细胞凋亡所必需的，而外源性活性基质金属蛋白酶-3的加入则显著减弱细胞表面的凋亡和钝化死亡受体-配体之间的相互作用。这些观察结果强烈暗示TIMP-3和基质金属蛋白酶-3在神经系统受体介导的死亡调节中的生理学作用。虽然金属蛋白酶活性与脑缺血后的血管损伤有关，但金属蛋白酶及其抑制物影响神经元对缺血应激的易感性的能力尚未被研究。在拟议的研究中，利用体外和体内的脑缺血损伤模型，结合药理学和基因缺失的方法，旨在建立金属蛋白酶活性在脑缺血后受体介导的神经元死亡调节中的作用，并探索潜在的机制。

DESCRIPTION (provided by applicant): Metalloproteinase activity at the cell surface can strongly influence cell sensitivity to extrinsic death vs. survival signals in a variety of cell types, yet the convergence of metalloproteinases and receptor signaling in the nervous system remains largely unexplored. TIMP-3 (tissue inhibitor of metalloproteinase-3) is a unique natural metalloproteinase inhibitor, in that it is a potent inhibitor of all known metalloproteinase sheddases, and has been shown to play a role in the regulation of receptor-mediated cell death in various non-neuronal cell types. TIMP-3 plays a pro-apoptotic role in many cell types through its ability to inhibit sheddases that target death receptors and their ligands. Our interest in the role of metalloproteinases in CNS damage following stroke led us to investigate the expression of TIMP-3 and the metalloproteinase sheddase, MMP-3, in a rodent model of focal cerebral ischemia. We found that while TIMP-3 and MMP-3 are expressed at low to non-detectable levels in the adult brain, their expression becomes markedly upregulated following ischemia, particularly in cortical neurons undergoing delayed apoptotic death. Using a tissue culture approach, we found that TIMP-3 and MMP-3 are constitutively expressed by embryonic cortical neurons in culture and modulate neuronal sensitivity to receptor-mediated apoptosis induced by the chemotherapeutic drug, doxorubicin (Dox). Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced apoptosis, whereas addition of exogenous active MMP-3 markedly attenuated apoptosis and blunted death receptor-ligand interactions at the cell surface. These observations strongly implicate a physiologic role for TIMP-3 and MMP-3 in the regulation of receptor-mediated death in the nervous system. While metalloproteinase activity has previously been implicated in vascular damage following cerebral ischemia, the ability of metalloproteinases and their inhibitors to influence neuronal vulnerability to ischemic stress has not been studied. In the proposed studies, experiments are designed to establish a role for metalloproteinase activity in the regulation of receptor-mediated neuronal death following cerebral ischemia, and to explore underlying mechanisms, utilizing both in vitro and in vivo models of ischemic injury, coupled with pharmacological and gene deletion approaches.