2-Macroglobulin Promotes MMP-2 Activation, Actin Cytosqueleton Remodeling and Cell Migration in the MIO-M1 Müller Cell Line Mediated by LRP1

Abstract

Purpose: : Müller 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 α₂-Macroglobulin (α₂M) receptor, LDL receptor-related protein 1 (LRP1), which mediates the α₂M-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 Müller cell line stimulated with α₂M. In addition, we evaluated the cell migration induced by α₂M 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 α₂M were maintained in culture using Dulbecco’s modified Eagle’s medium (DMEM). The effect of α₂M 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 α₂-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.

Conclusions: : All together, these data demonstrate that α₂M/LRP1 system is involved in the MIO-M1 cell migration, which could act as a linker between pericellular proteolysis and the actin cytoskeleton.

Keywords: Muller cells • neovascularization • retinal degenerations: cell biology