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M. Aga, M.J. Kelley, T.S. Acott; MAP Kinases Regulate Aqueous Humor Outflow and FAK Activation in Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1870.
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Trabecular meshwork (TM) cells sense increased intraocular pressure (IOP) as mechanical stretch and likely maintain IOP homeostasis by regulating cytoskeletal reorganization and initiating extracellular matrix turnover. Extracellular signal–regulated kinase1/2 (ERK1/2), a member of the mitogen activated protein (MAP) kinase family, and focal adhesion kinase (FAK) have been shown to regulate extracellular matrix turnover and matrix metalloproteinases in other systems. In the present study, we evaluated the involvement of ERK and FAK in regulating IOP and aqueous humor outflow facility in response to mechanical stretch.
Cultured porcine TM cells were stretched for 0–60 min or 24 hrs. U0126 (10 µM), an inhibitor of MEK (the kinase that activates ERK1/2), or control buffer was added to the cell culture media 30 min prior to initiating stretch. Cellular extracts were analyzed by western immunoblotting using phospho–specific antibodies. Aqueous humor outflow facility was determined in perfused human and porcine anterior segments.
Exposure of cells to U0126 augmented phosphorylation of FAK at Tyr–925 as early as 5 minutes, which was maintained until 60 min as compared to stretch. No change in basal phosphorylation of FAK at Tyr–861 was observed in response to either stretch or U0126 within 0–60 min. In contrast, prolonged stretch (24 hrs) increased the phosphorylation of FAK at Tyr–861 but not at Tyr–925. Treatment with U0126 for 24 hrs potentiated FAK phosphorylation at Tyr–925 and Tyr–861. A doubling of aqueous humor outflow was observed in response to an increase in perfusion pressure to 15 mm as compared to the baseline outflow rates (7 mm pressure). Sustained application of 15 mm pressure led to a further increase in outflow over time (24–48 hrs). However, no substantial increase in outflow rates was observed upon sustained perfusion of eyes at 15 mm in the presence of U0126. No changes in baseline outflow rates were observed in U0126 treated eyes.
Mechanical stretching of TM cells triggered differential phosphorylation of FAK at Tyr–925 and Tyr–861. Increased FAK phosphorylation was observed after addition of U0126 alone or in conjunction with mechanical stretch. Inhibition of ERK activation in perfused anterior segments resulted in reduced aqueous humor outflow, suggesting a critical role for this MAP kinase in the regulation of outflow facility. Overall, these studies suggest that ERK1/2 activity is associated with changes in FAK that coordinate the aqueous humor outflow facility following an increase in the intraocular pressure.
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