Purpose: M¨¹ller cells (MC) are known to undergo functional and morphological changes and to produce matrix metalloproteinases (MMPs) during retinal proliferative diseases. Alpha 2- macroglobulin (¦Á2-M) receptor (LRP-1) is a large endocytic receptor expressed in most cell types. In previous studies we have demonstrated that MC express LRP-1 and its ligand, ¦Á2-M, is able to induce MMP-2 activity. However, the biochemical mechanism by which ¦Á2-M/LRP-1 system may control this activity is unclear, which could involve both endocytic and intracellular pathways activation. Herein we investigate the putative mechanisms of ¦Á2-M/LRP-1 system on the MMPs regulation and cell migration in a M¨¹ller cell line.
Methods: The human MIO-M1 was cultured in the absence or presence of 60 nM ¦Á2-M for different time periods. The effect of ¦Á2-M on MMPs activity was evaluated in MC supernatants by zymography. The expression level of MT1-MMP was analyzed by RT-PCR, Western blot (WB) and immunofluorescence (IF) analysis. MMP-2 and TIMP-2 expression were also examined by RT-PCR. Cell migration was evaluated on collagen or laminin-coated surface using time-lapse video microscopy. The ¦Á2-M induced signalling pathways were evaluated by WB. Cell binding and internalization of ¦Á2-M were evaluated by flow cytometry using anti LRP-1:RPE and Alexa fluor 488-conjugated ¦Á2-M.
Results: By zymography we observed that ¦Á2-M increased MMP-2 activity whereas MMP-9 activity was not modified. ¦Á2-M also increased MT1-MMP and TIMP-2 mRNA and MT1-MMP protein, which was associated with the MMP-2 activity. Under ¦Á2-M induction MT1-MMP was localized at the cell surface of MC. This MMPs regulation was also accompanied by an increase of cell motility on coated surfaces. Finally, using pharmacological inhibitors of signaling pathways and endocytosis we observed that MMPs regulation and MIO-M1 cell migration were dependent on the intracellular signal activation induced by the ¦Á2-M/LRP-1 system.
Conclusion: The present study demonstrates that the ¦Á2-M/LRP-1 system regulates the proteolytic activity of MMP-2 in MIO-M1 cells by inducing the MT1-MMP and TIMP-2 expression, suggesting that MAPK and PKB pathways are involved.
