Abstract
Purpose: :
To determine if cross-linked actin networks (CLANs) induced by dexamethasone (DEX) in human trabecular meshwork (HTM) cells are structurally similar to those formed following β3 integrin activation and involve αvβ3 integrin signaling.
Methods: :
Two HTM cell strains and the immortalized TM-1 cell line overexpressing αvβ3 integrin were used. DEX- or ethanol-pre-treated HTM cells were treated with cycloheximide to block de novo protein synthesis prior to being re-plated on fibronectin with or without β3 integrin-activating mAb AP-5. Fluorescence 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/Trio (NSC23766) inhibitor. αvβ3 integrin expression levels and the β3 integrin activation state were determined by FACS analysis and fluorescence microscopy.
Results: :
CLANs induced by either DEX or activating β3 integrin contained syndecan-4, PIP2, and α-actinin. PI3-kinase inhibition did not affect DEX-induced CLAN formation while Rac1/Trio inhibition decreased the percentage of CLAN-positive cells by 80%. In contrast, inhibition of PI3-kinase or Rac1/Trio decreased β3 integrin-induced CLAN formation in the presence of DEX. DEX pre-treatment increased β3 integrin-induced CLAN formation ~ 6-fold and the level of αvβ3 integrin expression and activation 3-fold relative to control cells. Focal adhesions containing activated β3 integrin also increased ~ 5-fold in DEX-treated cells. Finally, the overexpression of αvβ3 integrin in TM-1 cells increased CLAN formation 2-fold.
Conclusions: :
DEX-induced CLAN formation in HTM cells involved αvβ3 integrin activation and the Rac1/Trio signaling pathway previously shown to be associated with αvβ3-induced CLAN formation. Together these data suggest that steroid-induced CLAN formation may involve enhanced β3 integrin signaling in HTM cells possibly via an inside-out signaling mechanism. However, we can not exclude a role for β1 integrin signaling.
Keywords: trabecular meshwork • cytoskeleton • corticosteroids