Browsing by Subject "myocilin"
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Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Mutation in myocilin affect it's processing and secretion in the trabedular meshwork cell(2003-05-01) Jacobson, Nasreen; Robert Wordinger; Richard Easom; Neeraj AgarwalJacobson, Nasreen, Mutations in myocilin affect it secretion and processing in the cell. Doctor of Philosophy (Cell Biology and Ginetics), May 2003, 157 pp., 6 tables, 46 illustrations, 17 movies. Introduction. Myocilin is the protein product of the glaucoma gene MYOC whose function is unknown. Structural predictions of the protein indicate myocilin is secreted. This study uses several techniques to determine whether myocilin is synthesized and processed through the secretory pathway. Methods. Agents known to disrupt the secretory pathway at specific organelles will be used to examine the effect on myocilin secretion. Also, constructs for chimeric myocilin and fluorescent proteins (myoc.504DsRED and myoc.504EGFP) will be used in conjunction with EGFP directed to specific organelles to determine colocalization of myocilin in the cell. The disruption of wild type and disease-causing mutants (myocQ368X.DsRED, myocG364V.504DsRED and myocY437H.504DsRED) of myocilin will be compared. Then in vivo studies will be used to try to determine if myocilin is associated with increased intraocular pressure (IOP). Results. Myocilin appears as a doublet on SDS-PAGE western blots when probed with anti-myocilin antibody (AB129). Treatment of cells with tunicamycin prevents secretion of the upper band of the myocilin doublet, but not secretion of the lower band. Brefelden A prevents secretion of both bands of the myocilin doublet indicating that both bands are processed in the Golgi. Monensin treatment indicates there is no post-Golgi processing of myocilin prior to secretion. Colocalization of fluorescent myocilin with cellular organelles tagged with EGFP indicated that myocilin travels through the ER, Golgi and is secreted from the cell. Disease-causing mutations in myocilin are not secreted. The Q368X associates with wild type myocilin and appears to be degraded. The G364V and Y437H mutants can apparently be retained in the ER and also are closely associated with peroxisomes. Experiments designed to determine if myocilin can be correlated with increased IOP suggest an association of myocilin with increased IOP in an ex vivo human anterior segment perfusion system, but in vivo experiments gave inconclusive results. Conclusions. Myocilin is a secreted glycoprotein in the TM. Glaucomatous mutations in myocilin cause non-secretion. TM cells handle different myocilin mutations differently.