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We also treated cells with the Ca2+ ionophore known as ionomycin as a positive control since calcineurin can be activated by an increase in intracellular calcium

We also treated cells with the Ca2+ ionophore known as ionomycin as a positive control since calcineurin can be activated by an increase in intracellular calcium. of DEX. Only 1 1 day of DEX treatment was sufficient to trigger a sustained increase in 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 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 mRNA expression. Cells were also treated with the ionophone ionomycin to determine if increased cytosolic calcium affected expression. These studies showed that the DEX induced increase in mRNA could be inhibited with either CsA or INCA-6 or by transfection with NFATc1 siRNA and that ionomycin was unable to increase mRNA. Immunofluorescence Taurine 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 Taurine increase in expression activates a calcineurin and NFATc1 pathway in a calcium independent mechanism. (myocilin)(WD Repeat Domain 36)(optineurin)and (neutrophin-4) (Fan and Wiggs, 2010; Takamoto and Araie, 2014). MYOC was one of the first proteins to be linked to glaucoma. It was originally identified because its expression in human trabecular meshwork (HTM) cells can be increased with the glucocorticoid dexamethasone (DEX) (Nguyen et al., 1998; Polansky et al., 1997). Thus, it is thought to play a role in both POAG and steroid-induced glaucoma which clinically mirrors POAG. Mutations in occur in 10% of juvenile open-angle glaucoma cases and in 3C4% of adult onset POAG cases (Fingert et al., 1999; Fingert et al., 2002; Kwon et al., 2009; Rabbit Polyclonal to GJC3 Stone et al., 1997). Increasing evidence suggests that mutations in the gene cause glaucoma through a gain of pathogenic function (Kim et al., 2001; Lam et al., 2000) which prevents MYOC from being secreted from the cell. As a result MYOC accumulates within the endoplasmic reticulum of the cell where it causes endoplasmic reticulum stress, impairing trabecular meshwork cell function and viability (Joe et al., 2003; Wang et al., 2007; Zode et al., 2011). MYOC is a secreted glycoprotein that is expressed in many structures of the eye, including the trabecular meshwork, iris, ciliary body, sclera, choroid, cornea, lamina cribosa, retina and optic nerve (Adam et al., 1997; Kubota et al., 1997; Ortego et al., 1997; Ricard et al., 2001; Tamm et al., 1999). The function of MYOC is not clear but it may play a role in cell-extracellular matrix interactions (Goldwich et al., 2009; Peters et al., 2005), cell migration (Kwon and Tomarev, 2011) and mitrochondrial function (Sakai et al., 2007). In skeletal muscle, MYOC is part of the dystrophin-associated protein complex by binding 1-syntrophin and may play a role as a regulator of muscle hypertrophy pathways (Joe et al., 2012). Taurine Recently, it was shown that MYOC can bind and activate ErbB2/ErbB3 in the sciatic nerve implicating a role for MYOC in myelination in the peripheral nervous system (Kwon et al., 2013). In addition to DEX, expression can also Taurine be induced in HTM cells with transforming growth factor-1 (TGF-1) (Tamm et al., 1999), optineurin (Park et al., 2007), and mechanical stretch (Tamm et al., 1999). The induction of by both DEX and TGF-1 is a delayed response, taking days rather than hours to see both mRNA and protein levels increase (Shepard et al., 2001; Tamm et al., 1999). This delayed response to stimuli is thought to be a secondary response as it requires new protein synthesis of an unidentified factor(s) for induction. Analysis of nucleotides upstream of the transcription start site support this idea because it failed to identify a functional glucocorticoid response element (Kirstein et al., 2000; Shepard et al., 2001). Recent studies examining how DEX regulates the expression of proteins in the TM show that MYOC is not the only protein up regulated as a result of a secondary glucocorticoid response. The 3 integrin subunit in HTM cells is also up regulated by DEX (Faralli et al., 2013) and this study showed that a calcineurin/NFAT (nuclear factor of activated T-cells) pathway may be involved. Calcineurin is a serine/threonine phosphatase that.