Peracchia F, Tamburro A, Prontera C, Mariani B, Rotilio D
Istituto di Ricerche Farmacologiche Mario Negri, G. Paone, Department of Vascular Medicine and Pharmacology, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy.
Arterioscler Thromb Vasc Biol. 1997 Nov;17(11):3185-90. doi: 10.1161/01.atv.17.11.3185.
Matrix metalloproteinases (MMPs) are a multigene family of enzymes secreted by a variety of cells, including human umbilical vein endothelial cells (HUVECs). Because metalloproteinases are potentially destructive agents, their production is tightly controlled at several levels. Rather little is known about the presence and regulation of MMPs in endothelial cells. In this study, we investigated the expression and regulation of MMP-2 and membrane type-matrix metalloproteinase (MT-MMP1), a membrane metalloproteinase strictly related to MMP-2 activation. Zymographic analysis of conditioned medium (CM) of HUVECs showed the presence of gelatinolytic activity mainly at 72 and 64 and 62 kD. The 64- and 62-kD bands, respectively, represent the intermediate and the completely active forms of MMP-2. When HUVECs were treated with forskolin (FK) (100 and 25 mumol/l), there was a decrease in the appearance of the 64 to 62 kDa doublet, suggesting an inhibition of the fully activated form of MMP-2. FK raises intracellular cAMP in HUVECs. The same data were obtained using dibutyryl-cAMP. Northern analysis revealed that the expression of MMP-2 increased slightly after treatment with FK, in contrast with gelatin zymography results. Taking into consideration the mechanism of activation of MMP-2, we tested the hypothesis that this compound could modulate MT-MMP1. As expected, FK was able to decrease MT-MMP1 expression. These data correlate with experiments using membranes of FK-treated HUVECs and incubated with control CM. Zymography revealed that when CM was incubated with membranes prepared from FK-treated HUVECs, there was a decrease in the appearance of the 64-kDa band, suggesting that the expression of MT-MMP1 was negatively modified. These results correlate with the MT-MMP1 protein level, negatively modified after FK treatment.
基质金属蛋白酶（MMPs）是一个多基因家族的酶，由多种细胞分泌，包括人脐静脉内皮细胞（HUVECs）。由于金属蛋白酶具有潜在的破坏作用，其产生在多个水平上受到严格控制。关于内皮细胞中MMPs的存在和调节知之甚少。在本研究中，我们研究了MMP-2和膜型基质金属蛋白酶（MT-MMP1）的表达和调节，MT-MMP1是一种与MMP-2激活密切相关的膜金属蛋白酶。对HUVECs条件培养基（CM）的酶谱分析显示，主要在72、64和62 kD处存在明胶酶解活性。64 kD和62 kD的条带分别代表MMP-2的中间形式和完全活性形式。当用福司可林（FK）（100和25 μmol/l）处理HUVECs时，64至62 kDa双峰的出现减少，表明MMP-2的完全活化形式受到抑制。FK可提高HUVECs中的细胞内cAMP水平。使用二丁酰-cAMP也获得了相同的数据。Northern分析显示，与明胶酶谱结果相反，用FK处理后MMP-2的表达略有增加。考虑到MMP-2的激活机制，我们测试了该化合物可能调节MT-MMP1的假设。正如预期的那样，FK能够降低MT-MMP1的表达。这些数据与使用FK处理的HUVECs膜并与对照CM孵育的实验相关。酶谱分析显示，当CM与由FK处理的HUVECs制备的膜孵育时，64 kDa条带的出现减少，表明MT-MMP1的表达被负向修饰。这些结果与FK处理后MT-MMP1蛋白水平的负向修饰相关。
