Muir D
Department of Pediatrics and Neuroscience, University of Florida College of Medicine, Gainesville 32610.
Exp Cell Res. 1994 Feb;210(2):243-52. doi: 10.1006/excr.1994.1036.
In order to assess the requirement for matrix metalloproteinases in neuronal regeneration, in vitro neurite outgrowth by chick dorsal root ganglionic neurons (DRGn) was examined within a reconstituted extracellular matrix. For these studies, cultured neurons were treated with a synthetic peptide inhibitor of metalloproteinases (spIMP), LMHKPRCGVPDVGG. spIMP inhibited all neuronal metalloproteinase activities in zymography and substrate-release assays and was used to examine the role of metalloproteinases in neurite outgrowth by DRGn. Cultures of dissociated DRGn rapidly extended neurites on planar extracellular matrix substrates and this rate of outgrowth was not affected by adding NGF or spIMP. In contrast, neurite extension within a three-dimensional gel of extracellular matrix increased nearly threefold after adding NGF. The NGF-induced neurite penetration was negated in the presence of spIMP but not by control peptide. Similar results were obtained using explanted dorsal root ganglia. These findings suggested that NGF-induced neurite outgrowth within an extracellular matrix involves metalloproteinase activity. Zymographic analysis of media conditioned by NGF-treated DRGn revealed a pair of gelatinolytic bands with apparent molecular masses 72 and 66 kDa, which comigrated as a single 66-kDa band after activation with an organomercurial agent. The gelatinase activities were calcium- and zinc-dependent and were absent from zymograms developed in the presence of spIMP, indicating that NGF-treated DRGn release and activate a 72-kDa metalloproteinase. Samples from DRGn cultures treated with low levels of NGF contained similar amounts of latent and activated metalloproteinase, while high levels of NGF induced an apparent increase in total metalloproteinase secretion and a substantially greater proportion of activated enzyme. Western blot analysis showed this metalloproteinase was immunologically similar to 72-kDa type IV collagenase and immunoassays revealed that this matrix metalloproteinase was increased threefold by high NGF. Furthermore, after high NGF treatment, DRGn media contained sixfold more metalloproteinase activity in assays of matrix degradation. In summary, these results indicate that NGF enhanced metalloproteinase-dependent neurite outgrowth of DRGn within a reconstituted extracellular matrix. Also, NGF increased the expression and activation of 72-kDa type IV collagenase, suggesting a role for this matrix-degrading metalloproteinase in neuronal regeneration.
为了评估神经元再生过程中基质金属蛋白酶的需求，在重组细胞外基质中检测了鸡背根神经节神经元（DRGn）的体外神经突生长情况。在这些研究中，用金属蛋白酶的合成肽抑制剂（spIMP），即LMHKPRCGVPDVGG处理培养的神经元。spIMP在酶谱分析和底物释放试验中抑制了所有神经元金属蛋白酶的活性，并用于研究金属蛋白酶在DRGn神经突生长中的作用。解离的DRGn培养物在平面细胞外基质底物上能迅速延伸神经突，添加NGF或spIMP均不影响这种生长速度。相比之下，在添加NGF后，细胞外基质三维凝胶内的神经突延伸增加了近三倍。在存在spIMP的情况下，NGF诱导的神经突穿透受到抑制，但对照肽则无此作用。使用外植背根神经节也获得了类似结果。这些发现表明，细胞外基质内NGF诱导的神经突生长涉及金属蛋白酶活性。对经NGF处理的DRGn条件培养基进行酶谱分析，发现有一对明胶酶解带，表观分子量分别为72 kDa和66 kDa，在用有机汞试剂激活后，它们合并为一条单一的66 kDa带。明胶酶活性依赖于钙和锌，在存在spIMP的情况下酶谱中未出现，这表明经NGF处理的DRGn释放并激活了一种72 kDa的金属蛋白酶。用低水平NGF处理的DRGn培养物样本中，潜伏型和激活型金属蛋白酶的含量相似，而高水平的NGF则导致总金属蛋白酶分泌明显增加，激活型酶的比例大幅提高。蛋白质印迹分析表明，这种金属蛋白酶在免疫上与72 kDa的IV型胶原酶相似，免疫测定显示，高浓度NGF可使这种基质金属蛋白酶增加三倍。此外，在高浓度NGF处理后，DRGn培养基在基质降解试验中的金属蛋白酶活性增加了六倍。总之，这些结果表明，NGF增强了重组细胞外基质中DRGn依赖金属蛋白酶的神经突生长。此外，NGF增加了72 kDa IV型胶原酶的表达和激活，表明这种基质降解金属蛋白酶在神经元再生中发挥作用。
