Stanton H, Gavrilovic J, Atkinson S J, d'Ortho M P, Yamada K M, Zardi L, Murphy G
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
J Cell Sci. 1998 Sep;111 ( Pt 18):2789-98. doi: 10.1242/jcs.111.18.2789.
We have assessed the effect of fibronectin and laminin-1 on the expression of molecules involved in the activation pathway of MMP-2, a key proteinase in tissue remodelling. HT1080 fibrosarcoma cells cultured on fibronectin were shown to activate endogenous MMP-2, to a level comparable with that elicited by treatment with phorbol ester. In contrast, the MMP-2 expressed by HT1080 cells cultured on laminin-1 was mainly in the pro- (inactive form). Culture of the cells on peptide fragments of fibronectin derived from the central cell binding domain also promoted MMP-2 activation, indicating that signals via fibronectin binding to integrin receptors may be involved. HT1080 cells cultured on immobilised antibodies to the alpha5 and beta1 integrin subunits secreted levels of active MMP-2 similar to those observed for full length fibronectin, whereas cells cultured on an antibody to the alpha6 integrin subunit secreted mainly proMMP-2. The data demonstrate that the activation of MMP-2 by HT1080 cells is regulated by the nature of the extracellular matrix, and that signals via the alpha5beta1 integrin receptor may be involved in the fibronectin induced up-regulation of MMP-2 activation. We then assessed the effect of fibronectin on the components of the putative MT1-MMP/TIMP-2 'receptor' complex implicated in MMP-2 activation. Levels of TIMP-2 protein expressed by HT1080 cells did not vary detectably between cells cultured on fibronectin or laminin-1. However, the expression of MT1-MMP protein was up-regulated when the cells were cultured on fibronectin, which could be attributed to an increase in levels of a truncated 45 kDa form. Parallel studies using gelatin zymography demonstrated that the up-regulation of the production of the 45 kDa band was concomitant with MMP-2 activation. Inhibitor studies revealed that the truncation of MT1-MMP to a 45 kDa form is MMP mediated, although not inhibited by TIMP-1. In vitro, the 45 kDa form could be generated by cleavage of membrane-bound native MT1-MMP with several recombinant MMPs, including both active MT1-MMP and MMP-2. The implication that either MMP-2 or MT1-MMP can process MT1-MMP to 45 kDa, raises the possibility that truncation of MT1-MMP represents a self-regulatory end-point in the activation pathway of MMP-2.
我们评估了纤连蛋白和层粘连蛋白-1对参与基质金属蛋白酶-2（MMP-2，组织重塑中的一种关键蛋白酶）激活途径的分子表达的影响。在纤连蛋白上培养的HT1080纤维肉瘤细胞可激活内源性MMP-2，激活水平与佛波酯处理所引发的水平相当。相比之下，在层粘连蛋白-1上培养的HT1080细胞所表达的MMP-2主要处于前体（无活性形式）。在源自中央细胞结合域的纤连蛋白肽片段上培养细胞也能促进MMP-2的激活，这表明可能涉及通过纤连蛋白与整合素受体结合的信号。在固定有α5和β1整合素亚基抗体的表面培养的HT1080细胞分泌的活性MMP-2水平与全长纤连蛋白培养的细胞所观察到的水平相似，而在α6整合素亚基抗体上培养的细胞主要分泌前体MMP-2。数据表明，HT1080细胞对MMP-2的激活受细胞外基质性质的调节，并且通过α5β1整合素受体的信号可能参与纤连蛋白诱导的MMP-2激活上调。然后，我们评估了纤连蛋白对与MMP-2激活相关的假定MT1-MMP/TIMP-2“受体”复合物成分的影响。在纤连蛋白或层粘连蛋白-1上培养的HT1080细胞所表达的TIMP-2蛋白水平在检测上没有差异。然而，当细胞在纤连蛋白上培养时，MT1-MMP蛋白的表达上调，这可归因于截短的45 kDa形式水平的增加。使用明胶酶谱法的平行研究表明，45 kDa条带产生的上调与MMP-2的激活同时发生。抑制剂研究表明，MT1-MMP截短为45 kDa形式是由MMP介导的，尽管不受TIMP-1抑制。在体外，45 kDa形式可通过几种重组MMP（包括活性MT1-MMP和MMP-2）切割膜结合的天然MT1-MMP产生。MMP-2或MT1-MMP均可将MT1-MMP加工成45 kDa这一现象，增加了MT1-MMP截短代表MMP-2激活途径中一个自我调节终点的可能性。
