Metalloproteinase Regulation of Neuronal Death

描述（由申请人提供）：在细胞表面的金属蛋白酶活性可以强烈影响细胞对各种细胞类型的外源性死亡和生存信号的敏感性，但金属蛋白酶和神经系统受体信号的趋同在很大程度上仍未被探索。TIMP-3 （tissue inhibitor of metalloproteinase-3）是一种独特的天然金属蛋白酶抑制剂，它是所有已知金属蛋白酶脱落酶的有效抑制剂，并已被证明在多种非神经元细胞类型的受体介导的细胞死亡调节中发挥作用。TIMP-3通过抑制靶向死亡受体及其配体的脱落酶，在许多细胞类型中发挥促凋亡作用。我们对金属蛋白酶在中风后中枢神经系统损伤中的作用感兴趣，因此我们研究了局灶性脑缺血啮齿动物模型中TIMP-3和金属蛋白酶脱落酶MMP-3的表达。我们发现，虽然TIMP-3和MMP-3在成人大脑中的表达水平很低，甚至无法检测到，但缺血后它们的表达明显上调，特别是在经历延迟性凋亡死亡的皮质神经元中。通过组织培养方法，我们发现胚胎皮质神经元在培养中组成性表达TIMP-3和MMP-3，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作用是dox诱导细胞凋亡的必要条件，而外源性活性MMP-3的加入可显著减弱细胞凋亡并减弱细胞表面死亡受体与配体的相互作用。这些观察结果强烈暗示TIMP-3和MMP-3在调节神经系统受体介导的死亡中的生理作用。虽然金属蛋白酶活性先前与脑缺血后的血管损伤有关，但金属蛋白酶及其抑制剂影响神经元对缺血应激易感性的能力尚未得到研究。在本研究中，实验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控中的作用，并利用体外和体内缺血损伤模型，结合药理学和基因缺失方法探索其潜在机制。

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.