NFATc1 activity regulates the expression of myocilin induced by dexamethasone.

Mutations in the myocilin gene (MYOC) account for 10% of juvenile open-angle glaucoma cases and 3-4% of adult onset primary open-angle glaucoma cases. It is a secreted glycoprotein found in many ocular and non-ocular tissues and has been linked to elevated intraocular pressure. In human trabecular meshwork (HTM) cells, MYOC expression can be induced by the glucocorticoid dexamethasone (DEX). In this study we examined the role of the calcineurin/NFATc1 (Nuclear Factor of Activated T-cells) pathway in the DEX induction of MYOC in HTM cells. In post-confluent HTM cells treated with either 500 nM DEX or 0.1% ethanol (EtOH; vehicle control) for 0-6 days both protein and mRNA levels of MYOC were increased while DEX was present. The protein and mRNA levels remained elevated for an additional 12 days after the removal of DEX. Only 1 day of DEX treatment was sufficient to trigger a sustained increase in MYOC mRNA that lasted for 4 days after the removal of DEX. Similar to other studies, myocilin protein expression was not seen until the second day of DEX treatment while mRNA increased within one day of DEX indicating that this is a secondary glucocorticoid response. To determine if MYOC gene expression was regulated by calcineurin/NFATc1, HTM cells were pre-treated for 1 h with the calcineurin inhibitors cyclosporin A or INCA-6 prior to the addition of DEX or EtOH for 2 days. NFATc1 siRNA was used to determine if NFATc1 was required for MYOC mRNA expression. Cells were also treated with the ionophone ionomycin to determine if increased cytosolic calcium affected MYOC expression. These studies showed that the DEX induced increase in MYOC mRNA could be inhibited with either cyclosporin A or INCA-6 or by transfection with NFATc1 siRNA and that ionomycin was unable to increase MYOC mRNA. Immunofluorescence microscopy was also performed to determine if DEX caused the nuclear translocation of NFATc1. Immunostaining showed that NFATc1 relocated to the nucleus within 15 min of DEX treatment and remained there for up to 2 h. The data suggest that the DEX-induced increase in MYOC expression activates a calcineurin and NFATc1 pathway in a calcium independent mechanism.

Dexamethasone-associated cross-linked actin network formation in human trabecular meshwork cells involves ß3 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 ß3 integrin activation and involve αvß3 integrin signaling. METHODS: Two HTM cell strains and an αvß3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-pretreated HTM cells were plated on fibronectin with or without ß3 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. ß3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. αvß3 Integrin expression levels and the ß3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy. RESULTS: CLANs associated with either DEX treatment or ß3 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 ß3 integrin-induced CLAN formation nearly sixfold and the level of αvß3 integrin expression and activation threefold compared with control cells. Activated ß3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. αvß3 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 αvß3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced ß3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism.