Abstract
Purpose: :
Recently we identified Annexin II, a calcium-, phospholipid- and actin-binding molecule as one of the major cytoskeletal proteins in the trabecular meshwork ( TM ) tissue and cells. Here we explored the role of annexin II in actin-based cytoskeletal reorganization in the context of aqueous humor outflow through the TM.
Methods: :
TM cells were isolated from human eyes and cultured to confluence. We studied the regulation of annexin II activity using physiological and pharmacological agents known to modulate annexin II expression and activity in other cell types, including dexamethasone, withaferin A and insulin. Serum starved confluent cultures of TM cells were treated with different concentrations of dexamethasone, withaferin A and insulin for 2 to 24 hrs. Cell lysates derived from these treatments were assessed for changes in protein levels of annexin II, tyrosine phosphorylated proteins and phosphorylated cofilin by immunoblot analysis. Confluent cultures of cells seeded on glass coverslips were also treated as described above in preparation for immunocytochemistry-based analysis of changes in actin cytoskeletal organization and tyrosine phosphorylated proteins.
Results: :
Annexin II was found to be distributed equally to the cytosol and membrane fraction of TM cells. Both dexamethasone and withaferin A increased total annexin II protein, tyrosine phosphorylation of annexin II and cofilin phosphorylation in a dose dependent manner. Dexamethasone increases actin stress fibers and cross-linked actin networks (CLANs) in TM cells in a dose-dependent manner as evidenced by immunocytochemical analyses. Treatment with withaferin A and insulin, on the other hand, decreases actin stress fibers in association with increased tyrosine phosphorylation compared to controls.
Conclusions: :
The data derived from these studies demonstrate that dexamethasone, a known intraocular pressure (IOP) increasing agent, induces increases in annexin II protein levels in TM cells in association with increased CLANs formation. Withaferin A, a commonly used plant component of Ayurvedic medicine and a known annexin II binding agent, induces actin depolymerization in TM cells. Further, insulin, which is known to activate annexin II activity via tyrosine phosphorylation, increases actin depolymerization and tyrosine phosphorylation in TM cells. These studies collectively reveal annexin II as a potential molecular target for the novel treatment of glaucoma and IOP.
Keywords: outflow: trabecular meshwork • cytoskeleton • signal transduction: pharmacology/physiology