Kao Biological Science Laboratories, 2606 Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan.
J Biol Chem. 2010 Dec 17;285(51):39819-27. doi: 10.1074/jbc.M110.161547. Epub 2010 Sep 28.
Although human skin fibroblast (HSF) elastase has been characterized as a membrane-bound metalloproteinase, little is known about its structure, amino acid sequence, and encoding gene. As there are similarities in the molecular weights and inhibitory profiles of HSF elastase and neprilysin (neutral endopeptidase 24.11 (NEP)), in this study we tested the hypothesis that they are identical using immunoprecipitation and transfection methods. An immunoprecipitation study demonstrated that HSF elastase activity co-immunoprecipitated with anti-NEP in lysates of cultured HSF. Transfection of an NEP cDNA expression vector into COS-1 cells elicited the expression of HSF elastase and NEP activities in the transfected cells. These findings strongly suggest that HSF elastase is identical to NEP, which functions mainly in neuron-associated cells to degrade neuropeptides. Analysis of the expression pattern of NEP revealed that its expression was remarkably up-regulated at the gene, protein, and enzymatic activity levels during the replicative senescence of cultured HSF. Further, the activity of NEP was markedly enhanced in a pattern similar to elastase activity during the intrinsic aging of mouse skin, in UVA-exposed HSF as well as in HSF treated with conditioned medium from UVB-exposed human keratinocytes. Analysis of the cytokine profile for the stimulation of NEP and HSF elastase activities in HSF demonstrated that among the 11 cytokines tested, IL-1α, IL-1β, IL-6, IL-8, and GM-CSF had the potential to significantly stimulate both activities similarly, again supporting the identity of HSF elastase and NEP.
尽管人皮肤成纤维细胞 (HSF) 弹性蛋白酶已被鉴定为一种膜结合金属蛋白酶，但对其结构、氨基酸序列和编码基因知之甚少。由于 HSF 弹性蛋白酶和 Neprilysin（中性内肽酶 24.11（NEP））的分子量和抑制谱存在相似性，因此在本研究中，我们使用免疫沉淀和转染方法检验了它们是相同的假设。免疫沉淀研究表明，HSF 弹性蛋白酶活性与培养的 HSF 裂解物中的抗 NEP 共沉淀。将 NEP cDNA 表达载体转染到 COS-1 细胞中，可在转染细胞中诱导 HSF 弹性蛋白酶和 NEP 活性的表达。这些发现强烈表明 HSF 弹性蛋白酶与主要在神经元相关细胞中降解神经肽的 NEP 相同。对 NEP 表达模式的分析表明，在培养的 HSF 的复制性衰老过程中，其基因、蛋白质和酶活性水平的表达显著上调。此外，在小鼠皮肤的内在老化过程中、在 UVA 暴露的 HSF 中以及在接受 UVB 暴露的人角质形成细胞条件培养基处理的 HSF 中，NEP 的活性以与弹性蛋白酶活性相似的模式显著增强。对刺激 HSF 中 NEP 和 HSF 弹性蛋白酶活性的细胞因子谱的分析表明，在测试的 11 种细胞因子中，IL-1α、IL-1β、IL-6、IL-8 和 GM-CSF 具有相似的潜力，可显著刺激两种活性，再次支持 HSF 弹性蛋白酶和 NEP 的同一性。
