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Rachel Exler, John G Flanagan, Darren Chan, Jeremy M Sivak; Mechanical Insult Abrogates the Anti-Apoptotic Role of PEA15. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2268.
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To determine the role of increased levels of astrocytic PEA15, and it’s associated changes in phosphorylation and function in the mechanism directing reactive gliosis. Phosphoprotein enriched in astrocytes (PEA15) was found following proteomic analysis on human optic nerve head astrocytes exposed to biomechanical insult. PEA15 is a molecular adaptor implicated in cell proliferation, apoptosis, and ECM remodelling, and depending on the phosphorylation state, it can bind and alter the function of several proapoptotic factors and ERK1/2.
Primary rat retinal astrocytes and a rat optic nerve astrocyte cell line (A7 cells) were stretched to induce pathologically relevant biomechanical insult. This was combined with PEA15 overexpression and knockdown techniques to better understand the role of PEA15 in apoptosis and the regulation of MMP expression. Levels of apoptosis were evaluated using flowing cytometry and western blot, while levels of MMPs were determined via zymography.
Levels of PEA15 protein were seen to increase at 1, 6, and 12 hours following mechanical insult while ERK and Caspase 8 activation were seen to peak at 6 hours. PEA15 knockdown revealed a two-fold increase in levels of apoptosis compared to control cells. Conversely, transfected PEA15 caused a two-fold decrease in rate of apoptosis. When mechanical insult was combined with PEA15 misexpression, the rate of apoptosis doubled compared to acquiescent cells, however there was no difference in the level of apoptosis between PEA15 overexpression, knockdown and mechanically insulted controls. Zymography analysis of culture media following mechanical insult revealed increased levels of MMPs 2 and 9 after 6 hours of insult. PEA15 knockdown also increased levels of MMP2 and MMP9, while PEA15 overexpression decreased MMP2 and MMP9 compared to control.
PEA15 is antiapoptotic and regulates secreted levels of MMPs 2 and 9 in quiescent astrocytes, however this activity is abrogated when cells undergo mechanical insult. Additional experiments are planned to investigate the mechanism for this effect.
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