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Mark S. Filla, Marie K. Schwinn, Amanda K. Nosie, Ross W. Clark, Donna M. Peters; Dexamethasone-Associated Cross-Linked Actin Network Formation in Human Trabecular Meshwork Cells Involves β3 Integrin Signaling. Invest. Ophthalmol. Vis. Sci. 2011;52(6):2952-2959. doi: 10.1167/iovs.10-6618.
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© 2015 Association for Research in Vision and Ophthalmology.
To determine whether cross-linked actin networks (CLANs) formed in dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells are structurally similar to those formed after β3 integrin activation and involve αvβ3 integrin signaling.
Two HTM cell strains and an αvβ3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-pretreated HTM cells were plated on fibronectin with or without β3 integrin-activating mAb AP-5. Immunofluorescence microscopy was used to identify phalloidin-labeled CLANs and to ascertain the presence of α-actinin, PIP2, and syndecan-4 within them. β3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. αvβ3 Integrin expression levels and the β3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy.
CLANs associated with either DEX treatment or β3 integrin activation contained syndecan-4, PIP2, and α-actinin. In the absence of mAb AP-5, LY294002 did not affect DEX-associated CLAN formation, whereas NSC23766 decreased the percentage of CLAN-positive cells by 80%. In the presence of mAb AP-5, both inhibitors decreased DEX-associated CLAN formation. DEX pretreatment increased β3 integrin-induced CLAN formation nearly sixfold and the level of αvβ3 integrin expression and activation threefold compared with control cells. Activated β3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. αvβ3 Integrin overexpression in TM-1 cells increased CLAN formation twofold.
DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of αvβ3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced β3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism.
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