title: Dexamethasone-associated cross-linked actin network formation in human trabecular meshwork cells involves β3 integrin signaling.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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, PIP(2), 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.
  
  
  RESULTS
  CLANs associated with either DEX treatment or β3 integrin activation contained syndecan-4, PIP(2), 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin-Medical School, Madison, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Filla
authors.forename: Marie K
authors.initials: MK
authors.lastname: Schwinn
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Nosie
authors.forename: Ross W
authors.initials: RW
authors.lastname: Clark
authors.forename: Donna M
authors.initials: DM
authors.lastname: Peters
mesh_terms: D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010957:Plasmids; D015398:Signal Transduction; D053671:Syndecan-4 / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D005938:Glucocorticoids; D039302:Integrin alphaVbeta3; D019269:Phosphatidylinositol 4,5-Diphosphate; D000081082:Phosphoinositide-3 Kinase Inhibitors; D053671:Syndecan-4; D000185:Actinin; D003907:Dexamethasone; D020830:rac1 GTP-Binding Protein