Analysis of a molecular mechanism by which JNK-binding proteins regulate turnover of focal adhesions and formAnalysis of a molecular mechanism by which JNK-binding proteins regulate turnover of focal adhesions and formation of cell polarity

膜型基质金属蛋白酶1(MT1-MMPs)与肿瘤的侵袭转移密切相关。我们先前报道，MT1-MMP胞外基质降解通过调节持续的整合素介导的信号来调节细胞迁移。在这项研究中，MT1-基质金属蛋白酶表达细胞被放置在纤维连接蛋白涂层的平板上，并监测细胞-基质黏附的形成和纤维连接蛋白的降解。纤维连接蛋白的降解和去除与细胞迁移轨迹一致。迁移细胞呈极化形态，通过细胞-基质黏附集中的前沿与纤维连接蛋白的边缘接触。通过与粘着斑激酶(FAK)的粘着斑靶向(FAT)结构域融合，靶向细胞-基质粘连的MT1-MMPs的表达促进了粘连蛋白在粘连后即刻在细胞周围的初始裂解。这些结果表明，位于细胞-基质粘附处的MT1-MMP能降解纤维连接蛋白，这些粘附物集中在迁移细胞的前沿。Lb在细胞-基质黏附中抑制MT1-MMPs，MT1-MMPs的主要阴性形式(MT1-Pex)通过与脂肪结构域的融合(MT1-Pex-FAT)靶向于细胞与基质的黏附。与亲本MT1-Pex相比，MT1-Pex-脂肪在细胞-基质粘连处堆积，并更有效地抑制纤维连接蛋白降解和FAK磷酸化。MT1-PEX-FAT对肿瘤细胞三维胶原凝胶侵袭的抑制作用也强于MT1-PEX。这些结果提示，MT1-MMP介导的细胞外基质在细胞-基质粘附处的裂解诱导了细胞极性的建立，从而促进了细胞-基质的黏附周转和随后的细胞迁移。该模型强调了MT1-MMPs在迁移细胞前沿的作用。

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion and metastasis. We previously reported that extracellular matrix degradation by MT1-MMP regulates cell migration via modulating sustained integrin-mediated signals. In this study, MT1-MMP-expressing cells were plated onto fibronectin-coated plates and monitored for cell-matrix adhesion formation and fibronectin degradation. The fibronectin was degraded and removed in line with the cell migration track. The migrating cells showed a polarized morphology and were in contact with the edge of fibronectin through the leading edge in which cell-matrix adhesions are concentrated. Expression of MT1-MMP targeted to cell-matrix adhesions by fusing with the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) promoted the initial fibronectin lysis at the cell periphery immediately after adhesion. These results suggest that fibronectin is degraded by MT1-MMP located at cell-matrix adhesions which are concentrated at the leading edge of the migrating cells. lb inhibit MT1-MMP at cell-matrix adhesion, the dominant negative form of MT1-MMP (MT1-Pex) was targeted to the cell-matrix adhesion by fusing with the FAT domain (MT1-Pex-FAT). MT1-Pex-FAT accumulated at cell-matrix adhesions and inhibited fibronectin degradation as well as FAK phosphorylation more effectively than parental MT1-Pex. MT1-Pex-FAT was also shown to suppress the invasion of tumor cells into 3-dimensional collagen gel more strongly than MT1-Pex. These results suggest that MT1-MMP-mediated extracellular matrix lysis at cell-matrix adhesions induces the establishment of cell polarity, which facilitates cell-matrix adhesion turnover and subsequent cell migration. This model highlights the role of MT1-MMP at the leading edge of migrating cells.