Cellular Processing of Myocilin

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 CsA 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.

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“…MYOC protein levels in TM cells appear to be short-lived and decreased about 50% after 3 h of cycloheximide treatment. (Qiu et al, 2014). …”

Section: Discussionmentioning

confidence: 99%

NFATc1 activity regulates the expression of myocilin induced by dexamethasone

Faralli

Clark

Filla

et al. 2015

Experimental Eye Research

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“…These have been extensively reviewed elsewhere so we shall only provide a brief overview of their findings (Liton, 2016; Liton et al, 2009a; Qiu et al, 2014; Sirohi and Swarup, 2016; Ying and Yue, 2016). All of these studies suggest that the pathways work in tandem to clear proteins from cells.…”

Section: Protein Degradation Pathways and Other Glaucoma-associatementioning

confidence: 99%

“…Myocilin is the archetypal glaucoma-associated gene and mutant myocilin is known to accumulate in the endoplasmic reticulum, ultimately inducing cell death (Stothert et al, 2016). In normal cells, myocilin was found to be cleared by both the UPS and AL pathways (Qiu et al, 2014). However, mutant myocilin was preferably cleared via autophagic degradation.…”

Section: Protein Degradation Pathways and Other Glaucoma-associatementioning

confidence: 99%

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma

Keller

Wirtz

2017

Experimental Eye Research

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Evidence is accumulating to suggest that mutations in the Ankyrin and SOCS Box-containing protein-10 (ASB10) gene are associated with glaucoma. Since its identification in a large Oregon family with primary open-angle glaucoma (POAG), ASB10 variants have been associated with disease in US, German and Pakistani cohorts. ASB10 is a member of the ASB family of proteins, which have a common structure including a unique N-terminus, a variable number of central ankyrin (ANK) repeat domains and a suppressor of cytokine signaling (SOCS) box at the C-terminus. Mutations in ASB10 are distributed throughout the entire length of the gene including the two alternatively spliced variants of exon 1. A homozygous mutation in a Pakistani individual with POAG, which lies in the center of the SOCS box, is associated with a particularly severe form of the disease. Like other SOCS box-containing proteins, ASB10 functions in ubiquitin-mediated degradation pathways. The ANK repeats bind to proteins destined for degradation. The SOCS box recruits ubiquitin ligase proteins to form a complex to transfer ubiquitin to a substrate bound to the ANK repeats. The ubiquitin-tagged protein then enters either the proteasomal degradation pathway or the autophagic-lysosomal pathway. The choice of pathway appears to be dependent on which lysine residues are used to build polyubiquitin chains. However, these reciprocal pathways work in tandem to degrade proteins because inhibition of one pathway increases degradation via the other pathway. In this publication, we will review the literature that supports identification of ASB10 as a glaucoma-associated gene and the current knowledge of the function of the ASB10 protein. In addition, we present new data that indicates ASB10 expression is up-regulated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1α. Finally, we will describe the emerging role of other SOCS box-containing proteins in protein degradation pathways in ocular cells.

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“…It is therefore possible that decrease in proteasome activity in TM cells from older donors (Caballero et al, 2004) and the increased mitochondrial ROS production in glaucomatous TM primary cultures (He et al, 2008) might result from the poor autophagic turnover of proteasomes and mitochondria, respectively. Also, as it will be discussed elsewhere in this Special Issue, clearance of mutant myocilin can be also compromised since the turnover of myocilin involves ubiquitin-proteasomes and lysosomal pathways (Qiu et al, 2014). …”

Section: Autophagy In Outflow Pathway Pathophysiology: Implicationmentioning

confidence: 99%

The autophagic lysosomal system in outflow pathway physiology and pathophysiology

Liton

2016

Experimental Eye Research

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Malfunction of the trabecular meshwork (TM)/schlemm’s canal (SC) conventional outflow pathway is associated with elevated intraocular pressure (IOP) and, therefore, increased risk of developing glaucoma, a potentially blinding disease affecting more than 70 million people worldwide. This TM/SC tissue is subjected to different types of stress, including mechanical, oxidative, and phagocytic stress. Long-term exposure to these stresses is believed to lead to a progressive accumulation of damaged cellular and tissue structures causing permanent alterations in the tissue physiology, and contribute to the pathologic increase in aqueous humor (AH) outflow resistance. Autophagy is emerging as an essential cellular survival mechanism against a variety of stressors. In addition to performing basal functions, autophagy acts as a cellular survival pathway and represents an essential mechanism by which organisms can adapt to acute stress conditions and repair stress-induced damage. A decline in autophagy has been observed in most tissues with aging and has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms. Dysfunction in the autophagy pathway is associated with several human diseases, from infectious diseases to cancer and neurodegeneration. In this review, we will summarize our current knowledge of the emerging roles of autophagy in outflow tissue physiology and pathophysiology, including novel evidence suggesting compromised autophagy in the glaucomatous outflow pathway.

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Cellular Processing of Myocilin

Cited by 28 publications

References 66 publications

NFATc1 activity regulates the expression of myocilin induced by dexamethasone

NFATc1 activity regulates the expression of myocilin induced by dexamethasone

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma

The autophagic lysosomal system in outflow pathway physiology and pathophysiology

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