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J.A. Peterson, S. Dedhar, D.M. Peters; Integrin Linked Kinase (ILK) Modulates the Organization of the Actin Cytoskeleton in Human Trabecular Meshwork (HTM) Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3706.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the role of ILK and its downstream effectors in regulating the organization of the actin cytoskeleton. Methods: Quiescent, confluent HTM cells were serum starved for 24 h and plated on the III 7–10 repeats of FN (RGD cell binding domain; 47nM) in the absence or presence of the ILK inhibitor KP392 (100µM) for 3 h. In some experiments, the soluble Hep II domain of FN (472nM) or the phosphoinositol 3–kinase (PI3K) inhibitor LY294002 (25µM) was added. Immunofluorescence microscopy studies used phalloidin and vinculin antibodies to visualize stress fiber and focal adhesion formation respectively. The percentage of spread cells was determined by counting spread vs. non–spread cells from 8–12 fields of view from each experiment and averaged. Results: Quantitation from immunofluorescence images showed that 86.5% of control cells spread on the III 7–10 repeats of FN showed a polygonal morphology exhibiting cortical actin structures. The addition of the ILK inhibitor KP392 decreased the number of spread cells with cortical actin structures to 38% (p<0.027). Adding the soluble HepII domain in the presence of the ILK inhibitor reversed the effect of the inhibitor and increased cell spreading to 69%. The effect of adding soluble Hep II domain could be blocked by treating the HTM cells with the PI3K inhibitor LY29400. In this case, 28% (p<0.008) of cells spread when treated with the HepII domain and both the PI3K and ILK inhibitors. The PI3K inhibitor by itself decreased HTM cell spreading to 41.5% while cell spreading was reduced to 17.5% (p<0.001) in the presence of both the PI3K and ILK inhibitors. Conclusions: Both ILK and PI3K activity regulate the organization of the actin cytoskeleton in HTM cells. The Hep II domain of FN uses PI3K activity to circumvent ILK inhibition. This suggests that HTM cells may regulate IOP by altering ILK and PI3K activity to control cell contacts and contractility.
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