α2-MACROGLOBULIN PROMOTES MMP-2 ACTIVATION, ACTIN CYTOSQUELETON REMODELING AND CELL MIGRATION IN THE MIO-M1 MÜLLER CELL LINE MEDIATED BY LRP1

BARCELONA PF; CHIABRANDO GA; SANCHEZ MC

Fort lauderdale

Congreso; ARVO´s 2010 Annual Meeting (The Association for Research in Vision and Ophthalmology).; 2010

Purpose: M¨¹ller cells (MC) are known to undergo functional and morphological changes and to produce matrix metalloproteinases (MMPs) during retinal angiogenic diseases. is a large endocytic receptor expressed in most cell types. In previous studies we have demonstrated that MC express the alpha 2-Macroglobulin (¦Á2-M) receptor, LRP-1, and the interaction with its ligand, ¦Á2-M, is able to induce MMP-2 activity and cell migration. It is known that fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). Herein we investigated the subcellular distribution of this trimolecular complex at the migration front as well as the cytosqueleton organization in a human M¨¹ller cell line after a2-M stimulation. In addition we evaluated the cell migration 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 were cultured in the absence or presence of 60 nM a2-M were maintained in culture using Dulbecco¡¯s modified Eagle¡¯s medium (DMEM). The effect of a2-M 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 induction 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 LRP1 antagonist namely RAP. Finally, this MMPs regulation was also accompanied by an increase of cell motility on coated surfaces. Conclusion: Altogether, these data demonstrate the a2-M/LRP1 system implication in MIO-M1 cell migration, which could act as a linker between pericellular proteolysis and the actin cytoskeleton.