Dexamethasone-Associated Cross-Linked Actin Network Formation in Human Trabecular Meshwork Cells Involves β3 Integrin Signaling

PURPOSE. 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 beta 3 integrin activation and involve alpha v beta 3 integrin signaling. METHODS. Two HTM cell strains and an alpha v beta 3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-retreated HTM cells were plated on fibronectin with or without beta 3 integrin-activating mAb AP-5. Immunofluorescence microscopy was used to identify phalloidin-labeled CLANs and to ascertain the presence of alpha-actinin, PIP 2, and syndecan-4 within them. beta 3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. alpha v beta 3 Integrin expression levels and the beta 3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy. RESULTS. CLANs associated with either DEX treatment or beta 3 integrin activation contained syndecan-4, PIP 2, and alpha-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 beta 3 integrin-induced CLAN formation nearly sixfold and the level of alpha v beta 3 integrin expression and activation threefold compared with control cells. Activated beta 3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. alpha v beta 3 Integrin overexpression in TM-1 cells increased CLAN formation twofold. CONCLUSIONS. DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of alpha v beta 3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced beta 3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism. (Invest Ophthalmol Vis Sci. 2011;52:2952-2959) DOI:10.1167/iovs.10-6618