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Pablo F. Barcelona, Javier R. Jaldin, Gustavo A. Chiabrando, Maria C. Sanchez; The Intracellular Traffic Of Mt1-mmp Is Regulated By α2-macroglobulin/lrp1 System In müLler Glial Cell.. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2017.
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© ARVO (1962-2015); The Authors (2016-present)
Müller cells (MC) are known to undergo functional and morphological changes related with the matrix metalloproteinases (MMPs) production during retinal proliferative disorders. Previously, we demonstrated that MC express the alpha2-Macroglobulin (α2M) receptor, LDL receptor-related protein 1 (LRP1), and that under α2M treatment, LRP1 regulates the MMP-2 activity and cell migration on different matrix-protein-coated surfaces, which suggest the participation of MT1-MMP. Herein, we evaluated whether LRP1 regulates the MT1-MMP function in MC stimulated by α2M. In addition, we investigated the subcellular distribution of MT1-MMP and LRP1 and evaluated the cell migration induced by α2M on different matrix-protein-coated surfaces.
Transient transfection of the MIO-M1 cells with MT1-MMP-GFP vector was performed using Lipofectamine 2000. These cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) were stimulated with α2M (60 nM) for different times. The cellular distribution of MT1-MMP and LRP1 was examined by confocal microscopy using GFP-conjugated MT1-MMP, specific antibodies against LRP1 and intracellular compartments of endocytosis. The molecular association of MT1-MMP/LRP1 was analyzed by immunoprecipitation (IP). The protein silencing of LRP1 was performed using siRNA LRP1 with siPORT Neo FX Transfection agent.
MIO-M1 cells, under α2M treatment, showed that LRP1 and MT1-MMP were mainly co-localized in endosomal compartments characterized as EEA1 early endosomes. By IP assay we showed a molecular association between MT1-MMP and LRP1, which was increased by α2M stimulation. Finally, the LRP1 silencing abrogated the MMP2 activation and cell migration capacity, promoted by α2M, in this cells.
These data demonstrated that MT1-MMP is associated with LRP1 at intracellular level in MIO-M1 cells stimulated with α2M, which suggest that this receptor is regulating the intracellular traffic of MT1-MMP. In addition, the MT1-MMP/LRP1 interaction induced by α2M, regulates the MMP-2 activation and cellular migration of MC.
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