Metalloproteinase Regulation of Neuronal Death

描述（由申请人提供）：细胞表面的金属蛋白酶活性可强烈影响多种细胞类型中细胞对外源性死亡与存活信号的敏感性，但神经系统中金属蛋白酶和受体信号传导的融合仍在很大程度上未被探索。TIMP-3（金属蛋白酶组织抑制剂-3）是一种独特的天然金属蛋白酶抑制剂，因为它是所有已知金属蛋白酶脱落酶的有效抑制剂，并且已显示在各种非神经元细胞类型中调节受体介导的细胞死亡中起作用。TIMP-3通过其抑制靶向死亡受体及其配体的脱落酶的能力在许多细胞类型中起促凋亡作用。我们对金属蛋白酶在卒中后CNS损伤中的作用的兴趣使我们研究了TIMP-3和金属蛋白酶脱落酶MMP-3在啮齿动物局灶性脑缺血模型中的表达。我们发现，虽然TIMP-3和MMP-3在成人脑中以低至不可检测的水平表达，但它们的表达在缺血后显著上调，特别是在经历延迟性凋亡死亡的皮质神经元中。使用组织培养的方法，我们发现，TIMP-3和MMP-3组成型表达的胚胎皮层神经元的文化和调节神经元的敏感性受体介导的化疗药物，阿霉素（Dox）诱导的细胞凋亡。金属蛋白酶抑制剂TIMP-3被认为是必要的Dox诱导的细胞凋亡，而另外的外源性活性MMP-3显着衰减细胞凋亡和钝化死亡受体-配体的相互作用在细胞表面。这些观察结果强烈暗示了TIMP-3和MMP-3在神经系统中调节受体介导的死亡中的生理作用。虽然金属蛋白酶活性先前已被牵连在脑缺血后的血管损伤，金属蛋白酶及其抑制剂的能力，影响神经元的脆弱性，缺血应激尚未研究。在拟议的研究中，实验的目的是建立一个作用的金属蛋白酶活性的受体介导的神经元死亡的调节脑缺血后，并探讨潜在的机制，利用在体外和体内模型的缺血性损伤，加上药理学和基因删除的方法。

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.