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J. A. Faralli, D. M. Peters; Effect of Substrate Stiffness and Pressure on ERK and FAK Phosphorylation in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1625.
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Increasing evidence suggest that mechanical cues which are inherent properties of the extracellular matrix affect cellular behavior by altering signal transduction events. In this study, we determined if the signaling response of human trabecular meshwork (HTM) cells exposed to either normotensive (15mmHg) or elevated (40mmHg) pressure is dependent on the stiffness and/or elasticity of the substratum.
HTM cells grown until confluency and then maintained for one week after confluency, were plated into 24 well plates at 2x105 cells/well onto various substrates (see below) for 24hrs. After replating, the cells were serum starved for 24hrs and then exposed to 15 or 40mmHg of pressure for 10, 20, or 30 min by fitting tubes filled with 25mM Hepes buffered low glucose DME into each well. Cells were lysed and the proteins were resolved on a 10% SDS-PAGE and transferred to a PDVF membrane for western blotting. Changes in signaling pathways were evaluated by probing membranes with antibodies against ERK1/2, phosphorylated ERK1/2, FAK or FAK pY397. Relative band densities were compared using Image J software. To test a stiff substrate, cells were plated directly into plates. Softer, flexible substrates were created by either polymerizing 400µl of 1, 2 or 4mg/ml rat tail collagen I gels in the wells or by using a piece of stretchable silicone membrane glued onto the bottom of the plate. The silicone membrane was coated overnight at 37°C with 80nM collagen I to promote cell binding.
Cells plated directly into wells showed a time dependent decrease in ERK phosphorylation with a maximum decrease of 66% (p< 0.025) after 30 min exposure to 40mmHg as compared to no pressure. There was no change in ERK phosphorylation when exposed to 15mmHg. There was no significant change in ERK phosphorylation in cells plated on collagen gels, regardless of gel concentration or pressure level. Cells plated on stretchable silicone membrane exhibited a 17% increase (p< 0.02) in ERK phosphorylation after 30 min exposure to 40mmHg pressure compared to no pressure, but no change at 15mmHg. In all conditions tested, there was no significant change in phosphorylation of FAK at Y397.
HTM cells respond to pressure differently depending on the stiffness of the substrate and the amount of pressure. Stiff substrates cause an initial decrease in ERK activity at elevated pressure while flexible substrates may increase ERK activity. This suggests that changes in the mechanical properties of the TM may influence how HTM cells respond to pressure changes and hence regulate outflow facility.
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