ACTIVATED ALPHA-2 MACROGLOBULIN REGULATES THE MT1-MMP AND LRP1 INTRACELLULAR TRAFFIC TOWARD THE PLASMA MEMBRANE BY DIFFERENT RECYCLING PATHWAYS AND INDUCES CELLULAR MOTILITY OF HUMAN MÜLLER GLIAL CELLS

JALDIN FINCATI JR; BARCELONA PF; SÁNCHEZ MC; CHIABRANDO GA

Puerto Natales

Workshop; Current advances in membrane trafficking: Implications for polarity and diseases. EMBO Workshop.; 2014

EMBO Workshop.

In retinal proliferative diseases, included Diabetic Retinopathy and Retinopathy of Prematurity, Müller glial cells (MGC) acquire an enhanced cellular motility. These diseases are associated with increased activities of matrix metalloprotease-2 (MMP-2) and membrane type 1-MMP (MT1-MMP) together with high levels of the protease inhibitor α2-Macroglobulin (α2M) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). The protease activated form of α2M, α2M*, induces different intracellular signaling pathways and MMP gene expression via LRP1. In the present work, we investigated the role of α2M*, LRP1 and MT1-MMP activity on MGC motility. The human cell line, MIO-M1 (MGC-derived cells), was used. To study cell motility and MMP activity, wound-scratch migration and zymography assays were performed. The cellular distribution of LRP1 and MT1-MMP was analyzed by fluorescence confocal microscopy. The intracellular traffic of MT1-MMP and LRP1 to plasma membrane (PM) was evaluated by biotinylation assays. Silencing techniques for LRP1 and MT1-MMP (siRNA and shRNA) were used. Different GFP and RFP wild-type or dominant-negative (DN) mutant Rabs plasmids were expressed by transient transfections. The α2M* induced cellular motility and proMMP-2 activation in MGC, which was blocked by LRP1 and MT1-MMP silencing. Time course experiments showed that α2M* increases LRP1 and MT1-MMP localization in early endosomes (EEA1+). Nevertheless, MT1-MMP was mainly localized in endocytic recycling compartments (Rab11+), whereas LRP1 was not detected in Rab11+ vesicles after α2M* stimulation. Both MT1-MMP and LRP1 increased the intracellular traffic toward PM in MIO-M1 cells stimulated with α2M*. Rab11-DN-transfected cells abrogated the MT1-MMP trafficking to PM as well as the cellular motility and proMMP-2 activation under α2M* stimulation, whereas LRP1 trafficking was unaffected by the inhibition of this recycling pathway. Hence, α2M* induces MGC motility by MT1-MMP endocytic recycling through Rab11-dependent pathway. α2M* induces MIO-M1 cell migration through LRP1 α2M* increases the intracellular association between LRP1 and MT1-MMP MT1-MMP and LRP1 are colocalizated in early endosomes (eea1-positive vesicles) in MIO-M1 cells stimulated with α2M*α2M* increases the colocalization of MT1-MMP with Rab11 without changing the distribution of the LRP1 in recycling compartments