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J. M. Gonzalez, Jr., Y. Hu, B. T. Gabelt, P. L. Kaufman, D. M. Peters; Integrin-Binding Site in HepII Domain of Fibronectin Disrupts Actin Filaments in Human Trabecular Meshwork (TM) Cells and Increases Outflow Facility (OF) in Cultured Monkey Anterior Segments. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2054.
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© ARVO (1962-2015); The Authors (2016-present)
Determine the active site in HepII and its putative receptor that mediates the disruption of actin filaments in TM cells and regulates OF.
Cells were incubated for 24h with or without HepII. Changes in cell morphology were monitored using phase microscopy and a colorimetric adhesion assay. Changes in the actin cytoskeleton were determined by immunofluorescence microscopy using Alexa 488-phalloidin. The role of cell surface heparan sulfate proteoglycans (HSPGs) was determined using a HepII mutant (RK HepII) unable to bind HSPGs or chlorate (30mM) treated TM cells lacking cell surface HSPGs. In some experiments, cells were treated with integrin binding peptides (IDAPS and PRARI) from HepII or an anti-α4ß1 activating antibody, BU49. An antibody to the ectodomain of N-cadherin, GC-4, was used as a negative control. OF was determined by two-level constant pressure perfusion in anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with 100 µg/ml of HepII or 500 µg/ml of PRARI. The contralateral segment was exchanged with DMEM.
Cells treated with HepII (125 µg/ml), and the anti-α4ß1 antibody BU49 (1 µg/ml) appeared rounded and lacked actin filaments. The anti-N-cadherin antibody had no effect. Treatment with either the IDAPS or PRARI peptides (2 mg/ml) by themselves did not alter the organization of the actin cytoskeleton. When used in combination with a non-disrupting concentration of HepII (15 µg/ml), however, treatment with the PRARI peptide caused cells to round up suggesting an additive relationship. In cultured anterior segments, 100 µg/ml of HepII increased OF by 31±13% (n=9, p<0.05) and PRARI increased OF by 24±9% (n=8, p<0.05) after an overnight infusion. Mutation of the heparan sulfate binding site in RK HepII failed to block the ability of HepII to disrupt the actin cytoskeleton. Treatment with chlorate to prevent sulfation of HSPGs also failed to block the ability of the HepII domain to disrupt the actin cytoskeleton suggesting that HepII does not require HSPGs to induce the disassembly of the actin cytoskeleton in TM cells.
The active site in the HepII domain may be the putative α4ß1 integrin binding sequence PRARI. Both HepII and PRARI increased OF in monkey organ-cultured anterior segments while activation of α4ß1 integrins triggered the disassembly of the cytoskeleton in TM cells. This suggests that the PRARI site in fibronectin and α4ß1 integrins may play a role in regulating OF.
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