Purpose: M¨¹ller cells (MC) are known to undergo functional and morphological changes and to produce matrix metalloproteinases (MMPs) during retinal angiogenic diseases. In previous studies we have demonstrated that MC expresses the ¦Á2-Macroglobulin (¦Á2M) receptor, LDL receptor-related protein 1 (LRP1), which mediates the ¦Á2M-induced MMP-2 activity and cell migration. In addition, it is well known that the trimolecular complex MT1-MMP/TIMP-2/proMMP-2 plays a key role in the migratory regulation of cells. Herein we investigated the subcellular distribution of MT1-MMP/TIMP-2/proMMP-2 complex and actin cytosqueleton remodeling in the MIO-M1 M¨¹ller cell line stimulated with a2M. In addition, we evaluated the cell migration induced by ¦Á2M on different matrix-protein-coated surfaces.
Methods: Transient transfection of the human MIO-M1 cells with the vector MT1-MMP-GFP was performed using Lipofectamine 2000. Transfected cells cultured in the presence of a2M were maintained in culture using Dulbecco¡¯s modified Eagle¡¯s medium (DMEM). The effect of a2M on subcellular distribution of the trimolecular complex proteins and cytosqueleton of actin was evaluated by immunofluorescence (IF) and confocal microscopy. The cell migration of MIO-M1 was determined by wound-healing assay on collagen or laminin-coated surface using time-lapse video microscopy.
Results: Under a2-M stimulation MT1-MMP was localized at the cell surface of MC. In agreement with a role for MMP-2 in cell motility, its pericellular expression was principally associated with actin motile structures such as filopodia and lamellipodia. Furthermore, MMP-2 and TIMP-2 partially colocalized at the periphery of the MC, a pattern that was prevented by RAP, a ligand binding antagonist of LRP1. Finally, this MMPs regulation was also accompanied by an increase of cell motility on collagen- or laminin-coated surfaces.
Conclusion: Altogether, these data demonstrate that a2M/LRP1 system is involved in the MIO-M1 cell migration, which could act as a linker between pericellular proteolysis and the actin cytoskeleton.