Purpose: Glial Muller cells (MC) are known to undergo functional and morphological changes and to produce matrix metalloproteinases (MMPs) during retinal proliferative disorders. In previous in vivo and in vitro studies we have demonstrated that MC express the alpha 2- macroglobulin (α2-M) receptor (LRP-1) and α2-M induces MMP-2 activity in primary cultures of MC. Herein we investigated the biochemical mechanism of a2-M effect on MMPs activity regulation, in an spontaneously immortalized human Müller cell line that express LRP-1.
Methods: The human MIO-M1 (a kind gift from G. A. Limb, Institute of Ophthalmology and Moorfields, Eye Hospital, London, UK) was cultured in the absence or presence of 60 nM a2-M for different periods of time. The effect of a2-M on MMPs activity was evaluated in aliquots of MC supernatants by zymographic analysis. The expression level of MT1-MMP was analyzed by RT-PCR, Western blot and immunofluorescence analysis. MMP-2 and TIMP-2 expressions were also examined by RT-PCR. To evaluate intracellular signaling pathways induced by a2-M, MIO-M1 cells were pre-treated for 30 min with 20 µM PD98059, 50 µM SP600125, 25 µM SB203580 or 10 µM LY294002 and then treated with a2-M for 1h.
Results: a2-M increased MMP-2 activity whereas MMP-9 activity was not modified. By zymographic analysis was observed that the inhibition of JNK and Akt/PI3K, but not MAPK-ERK1/2, modified the a2-M-induced MMP-2 activity. In addition, a2-M also increased MT1-MMP and TIMP-2 mRNA and MT1-MMP protein, which was associated with the MMP-2 activity. Finally, we demonstrated by immunofluorescences that under a2-M induction MT1-MMP was predominantly localized at the cell surface and in the cellular processes of glial cells.
Conclusion: The present study demonstrates that the a2-M/LRP-1 system regulates the extracellular proteolytic activity of MMP-
