Hao J L, Nagano T, Nakamura M, Kumagai N, Mishima H, Nishida T
Department of Ophthalmology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
Exp Eye Res. 1999 May;68(5):565-72. doi: 10.1006/exer.1998.0637.
We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.
我们研究了基质金属蛋白酶（MMP）的合成肽基异羟肟酸酯抑制剂加拉迪恩（GM6001）对在含有白细胞介素1α（IL-1α）和/或纤溶酶原的培养基中于I型胶原凝胶中三维培养的兔角膜基质成纤维细胞（角膜细胞）胶原降解的抑制作用。通过羟脯氨酸的释放来测量培养过程中胶原纤维的降解，并且还通过明胶酶谱法和蛋白质印迹法分析MMP的激活。使用合成底物测量纤溶酶活性。在没有纤溶酶原的情况下，在胶原凝胶中用IL-1α处理细胞极大地增强了前MMP-1、-3和-9的产生，但未检测到胶原的明显降解。在有纤溶酶原存在的情况下，IL-1α以剂量依赖性方式刺激角膜细胞的胶原降解。这是由于纤溶酶原激活物-纤溶酶系统依赖性激活前MMP-1、-3和-9所致。无论是否存在IL-1α，加拉迪恩在有纤溶酶原存在的情况下均以剂量依赖性方式抑制胶原降解。加拉迪恩抑制前MMP-3的激活，并且还阻止前MMP-9的激活以及MMP-1中间体向完全活性的MMP-1的转化。加拉迪恩不影响纤溶酶活性。目前的结果表明，加拉迪恩在有纤溶酶原存在的情况下抑制IL-1α刺激的胶原降解，这不仅是由于抑制活性MMP，还由于阻止前MMP向活性MMP的转化。
