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Jeremy M Sivak, Rachel Exler, John G Flanagan; Biomechanical Insult Initiates a Novel Molecular Switch of the Adapter Protein PEA15 to Promote Matrix Remodeling and Apoptosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4822.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that expression and phosphorylation of the small molecular adaptor, PEA15 (Phosphoprotein Enriched in Astrocytes), were strongly induced by pathologically relevant biomechanical insult in optic nerve head (ONH) astrocytes. Here we investigate the mechanism and function for this protein under conditions of biomechanical stretch.
12% equiaxial stretch was applied to primary retinal and ONH astrocytes using a FlexercellTM system as previously described. Phorphorylation of PEA15 was measured by immunoprecipitation coupled with detection of phospho-serine residues. PEA15 vectors with mutated phosphorylation sites were generated by standard molecular biology methods. PEA15 knockdown was achieved by transient transfection with siRNAs and controls. Early apoptosis was measured by annexin V labeling coupled with flow cytometry. MMP2 and MMP9 secretion and activity were measured by gelatin zymography.
Under resting conditions, PEA15 overexpression was strongly anti-apoptotic (p<0.005), and inhibited Erk-mediated secretion of MMPs 2 and 9 (p<0.05). Conversely, PEA15 knockdown increased apoptosis (p<0.005), and had no effect on MMPs. However, application of 12% biomechanical stretch rapidly increased PEA15 phosphorylation (p<0.01), and surprisingly resulted in a dramatic reversal of its effects. PEA15 overexpression was uncoupled from apoptosis, and instead promoted a 2-fold increase in the secretion of MMPs (p<0.05). Mutation of the PEA15 phosphorylation sites at serine 104 rescued its activities under biomechanical stretch.
We have demonstrated a novel mechanostransduction switch that dramatically alters the function of PEA15 compared to resting conditions. We propose that this mechanism may underlie broader pathologic changes in the optic nerve head by altering the function of established signaling pathways.
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