Dufour Antoine, Sampson Nicole S, Zucker Stanley, Cao Jian
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, USA.
J Cell Physiol. 2008 Dec;217(3):643-51. doi: 10.1002/jcp.21535.
The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non-proteolytic activities of MMPs are just beginning to be understood. Herein, we evaluated the role of proMMPs in cell migration. Employing a Transwell chamber migration assay, we demonstrated that transfection of COS-1 cells with various proMMP cDNAs resulted in enhancement of cell migration. Latent MMP-2 and MMP-9 enhanced cell migration to a greater extent than latent MMP-1, -3, -11 and -28. To examine if proteolytic activity is required for MMP-enhanced cell migration, three experimental approaches, including fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant MMP cDNAs, and addition of hydroxamic acid-derived MMP inhibitors, were employed. We demonstrated that the proteolytic activities of MMPs are not required for MMP-induced cell migration. To explore the mechanism underlying MMP-enhanced cell migration, structure-function relationship of MMP-9 on cell migration was evaluated. By using a domain swapping approach, we demonstrated that the hemopexin domain of proMMP-9 plays an important role in cell migration when examined by a transwell chamber assay and by a phagokinetic migration assay. TIMP-1, which interacts with the hemopexin domain of proMMP-9, inhibited cell migration, whereas TIMP-2 had no effect. Employing small molecular inhibitors, MAPK and PI3K pathways were found to be involved in MMP-9-mediated cell migration. In conclusion, we demonstrated that MMPs utilize a non-proteolytic mechanism to enhance epithelial cell migration. We propose that hemopexin homodimer formation is required for the full cell migratory function of proMMP-9.
基质金属蛋白酶（MMPs）的生物学功能不仅限于细胞外基质降解。MMPs的非蛋白水解活性才刚刚开始被了解。在此，我们评估了前MMPs在细胞迁移中的作用。采用Transwell小室迁移试验，我们证明用各种前MMP cDNA转染COS-1细胞会导致细胞迁移增强。潜伏性MMP-2和MMP-9比潜伏性MMP-1、-3、-11和-28更能增强细胞迁移。为了研究MMP增强细胞迁移是否需要蛋白水解活性，我们采用了三种实验方法，包括荧光底物降解试验、用催化失活的突变体MMP cDNA转染细胞以及添加异羟肟酸衍生的MMP抑制剂。我们证明MMP诱导的细胞迁移不需要MMPs的蛋白水解活性。为了探索MMP增强细胞迁移的潜在机制，我们评估了MMP-9在细胞迁移中的结构-功能关系。通过使用结构域交换方法，我们证明在前MMP-9通过Transwell小室试验和吞噬动力学迁移试验检测时，其血色素结合蛋白结构域在细胞迁移中起重要作用。与前MMP-9的血色素结合蛋白结构域相互作用的TIMP-1抑制细胞迁移，而TIMP-2没有作用。使用小分子抑制剂，发现MAPK和PI3K途径参与MMP-9介导的细胞迁移。总之，我们证明MMPs利用非蛋白水解机制增强上皮细胞迁移。我们提出前MMP-9的完整细胞迁移功能需要血色素结合蛋白同二聚体的形成。
