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AP Surguchov, RE Palazzo, JM Sivak, ME Fini, I Surgucheva; Transcriptional Activation of Matrix Metalloproteinase-9 in Retinoblastoma Y79 Cells Overexpressing Gamma- Synuclein . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3638.
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Purpose: Gamma synuclein (g-syn) is a small cytoplasmic protein implicated in neurodegenerative diseases and cancer, however, the mechanism of its involvement in diseases is not clear. One of the important structures both for tumor progression and for the development of neurodegenerative diseases is extracellular matrix. Remodeling of the extracellular matrix and its degradation are controlled by matrix metalloproteinases (MMPs). Here we explore a possible role of members of MMP family in the mechanism of g-syn implication in diseases. Methods: To generate stable clones overexpressing g-syn we used mammalian vector pCI-neo with inserted g-syn cDNA. Activity of MMPs was determined in gels containing gelatin (for MMP2 and MMP9) or casein (MMP1 and MMP3) as substrates, the quantity of these proteins was analyzed by Western blots. Effect of g-syn on transcriptional regulation of MMP9 was measured using CAT reporter constructs. Constructs containing the full length MMP9 promoter with both intact AP-1 binding sites (Pr12),as well as constructs with deleted (Pr14) or mutated AP-1 binding sites (Pr18) were used. Results: Western blots and zymography data demonstrate significant elevation of the amount of MMP2 and MMP9 in stable clones overexpressing g-syn. According to the results of CAT system,overstatement of g-syn increases the activity of MMP9 promoter in several independent stable clones. This increment of promoter activity is mediated by AP-1 binding site(s), since point mutations in these sites (Pr18), as well as the elimination of one of these sites (distal AP-1, Pr 14) reduces the increment of promoter activity. Conclusion: The increase in enzymatic activity of MMP9 observed in g-syn overexpressing clones is due not to the activation of the pre-existing enzyme, but most probably because of the activation of MMP9 gene on transcriptional level via the activation of AP-1.
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