Tissue inhibitors of metalloproteinase 2 inhibits endothelial cell migration through increased expression of RECK

The antiangiogenic function of the tissue inhibitors of metalloproteinases (TIMPs) has been attributed to their matrix metalloproteinase inhibitory activity. Here we demonstrate that TIMP-1 but not Ala+TIMP-1 inhibits both basal and vascular endothelial growth factor (VEGF)-stimulated migration of human microvascular endothelial cells (hMVECs), suggesting that this effect is dependent on direct inhibition of matrix metalloproteinase (MMP) activity. In contrast, TIMP-2 and mutant Ala+TIMP-2, which is devoid of MMP inhibitory activity, block hMVEC migration in response to VEGF-A stimulation. TIMP-2 and Ala+TIMP-2 also suppress basal hMVEC migration via a time-dependent mechanism mediated by enhanced expression of RECK, a membrane-anchored MMP inhibitor, which, in turn, inhibits cell migration. TIMP-2 treatment of hMVECs increases the association of Crk with C3G, resulting in enhanced Rap1 activation. hMVECs stably expressing Rap1 have increased RECK expression and display reduced cell migration compared with those expressing inactive Rap1(38N). RECK-null murine embryo fibroblasts fail to demonstrate TIMP-2-mediated decrease in cell migration despite activation of Rap1. TIMP-2-induced RECK decreases cell-associated MMP activity. Anti-RECK antibody increases MMP activity and reverses the TIMP-2-mediated reduction in cell migration. The effects of TIMP-2 on BECK expression and cell migration were confirmed in A2058 melanoma cells. These results suggest that TIMP-2 can inhibit cell migration via several distinct mechanisms. First, TIMP-2 can inhibit cell migration after VEGF stimulation by direct inhibition of MMP activity induced in response to VEGF stimulation. Secondly, TIMP-2 can disrupt VEGF signaling required for initiation of hMVEC migration. Third, TIMP-2 can enhance expression of RECK via Rap1 signaling resulting in an indirect, time-dependent inhibition of endothelial cell migration.