Guo Chun, Jiang Juwei, Elliott J Martin, Piacentini Lucia
Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK.
J Cell Biochem. 2005 Feb 15;94(3):446-59. doi: 10.1002/jcb.20272.
Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.
基质金属蛋白酶-2（MMP-2）和膜型1-基质金属蛋白酶（MT1-MMP）的激活与细胞迁移相关，细胞迁移是伤口愈合和组织重塑中的关键细胞事件。我们之前已经证明，I型胶原可在心脏成纤维细胞中诱导弗林蛋白酶依赖性的MMP-2和MT1-MMP激活。为了解心脏成纤维细胞中潜在的非基质因子对MMP-2和MT1-MMP激活的调控机制，在本研究中，我们检测了包括伴刀豆球蛋白A（ConA）在内的多种试剂的作用，ConA是一种可产生蛋白水解表型的试剂。我们发现，用ConA处理细胞可激活前MMP-2，且这种激活与细胞内MT1-MMP和TIMP-2水平升高同时发生。从ConA处理的细胞制备的一部分细胞内蛋白质中存在活性MT1-MMP以及43 kDa和36 kDa的MT1-MMP加工形式，这表明MT1-MMP的不同形式可能发生了内化。在从ConA处理的细胞制备的条件培养基中出现36 kDa的MT1-MMP加工形式，表明进一步加工的MT1-MMP片段可能发生了细胞外释放。在ConA处理的细胞中抑制弗林蛋白酶可减弱前MT1-MMP的加工以及细胞内TIMP-2水平，并且它还能以时间依赖性方式降低细胞释放的活性MMP-2。这些结果表明弗林蛋白酶参与了ConA诱导的MT1-MMP和MMP-2激活。此外，与ConA处理的细胞相关的弗林蛋白酶抑制剂不敏感的前MMP-2和活性MMP-2种类的存在意味着，一种独立于弗林蛋白酶的机制可能解释了MMP-2种类与细胞的结合。本文的补充材料可在http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif上找到。
