The trabecular meshwork (TM) is a major site for regulation of the aqueous humor outflow. Cells in the TM are essential for maintenance of the normal aqueous outflow. Aberrations of cell integrity may be a key step toward obstruction of the outflow, intraocular pressure elevation, and glaucomatous conditions. We have instituted a program in our laboratory to define the TM biology and have accumulated a significant body of information regarding the basic characteristics of TM cells and the dynamics of TM cell-matrix interactions. During the past grant period, we have concentrated on studies of myocilin, a gene linked directly to open angle glaucomas. We have shown that myocilin is localized at both intra- and extra-cellular sites. When overexpressed in TM cells, or when present extracellularly in the matrix, myocilin causes a loss of actin stress fibers and focal adhesions, and elicits a de-adhesion activity. The alteration of the actin organization has led us to hypothesize that the myocilin effects may be mediated via signaling of the small GTPase Rho family. Members of the Rho family, Rho, Rac, and Cdc42, are well documented to be critical regulators of actin dynamics. In this renewal application, we propose to examine the Rho hypothesis in specific aim 1. The Rho GTPase activities in control and myocilin-transfected human TM cells will be compared. The activator and inhibitor of Rho, and constitutively active or dominant negative Rho, Rac, and Cdc42 expression vectors, will be used to determine whether Rho modulation reverts or modifies the myocilin-triggered phenotype. The involvement of Rho GTPases in mediating the effects of extracellular myocilin will be deciphered by comparing the phenotype displayed by Rho, Rac, and Cdc42 transfected and nontransfected TM cells on recombinant eukaryotic myocilin- or fibronectin/ myocilin-coated plates. The events associated with truncated and mutated myocilin will be explored. Studies will also be extended to perfusion organ cultures.
In specific aim 2, we will pursue a theory that myocilin mutants may be misprocessed and/or misfolded to provoke unfolded protein response (UPR). This theory has been formulated based on findings that certain mutated forms of myocilin are not secreted and are retained inside the cells. We will study the intracellular trafficking, degradation and oligomerization of endogenous and overexpressed wild type and mutated myocilins in TM cells. The ubiquitination of myocilins will be examined and the chaperones associated with myocilin proteins will be identified. Results from cells transfected to overexpress wild type, truncated or mutated myocilin will be compared to assess whether the mutant processing is defective and UPR is induced by the mutants. The central hypothesis is that overexpressed and/or mutated myocilin may be deleterious to TM cells via different signaling pathways or mechanisms. The overall goal of our proposal is to illustrate the mechanisms to provide a deeper understanding regarding the pathogenesis of glaucomas.
| O'Brien, E Timothy; Wang, Yanhong; Ying, Hongyu et al. (2014) Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal. J Ocul Pharmacol Ther 30:291-9 |
| Qiu, Ye; Shen, Xiang; Shyam, Rajalekshmy et al. (2014) Cellular processing of myocilin. PLoS One 9:e92845 |
| Shen, Xiang; Ying, Hongyu; Yue, Beatrice Y J T (2012) Wnt activation by wild type and mutant myocilin in cultured human trabecular meshwork cells. PLoS One 7:e44902 |
| Ying, Hongyu; Yue, Beatrice Y J T (2012) Cellular and molecular biology of optineurin. Int Rev Cell Mol Biol 294:223-58 |
| Ying, Hongyu; Shen, Xiang; Yue, Beatrice Y J T (2012) Establishment of inducible wild type and mutant myocilin-GFP-expressing RGC5 cell lines. PLoS One 7:e47307 |
| Shen, Xiang; Ying, Hongyu; Qiu, Ye et al. (2011) Processing of optineurin in neuronal cells. J Biol Chem 286:3618-29 |
| Yue, Beatrice Y J T (2011) Myocilin and Optineurin: Differential Characteristics and Functional Consequences. Taiwan J Ophthalmol 1:6-11 |
| Ying, Hongyu; Shen, Xiang; Park, BumChan et al. (2010) Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene. PLoS One 5:e9168 |
| Park, BumChan; Ying, Hongyu; Shen, Xiang et al. (2010) Impairment of protein trafficking upon overexpression and mutation of optineurin. PLoS One 5:e11547 |
| Knepper, Paul A; Samples, John R; Yue, Beatrice Yjt (2010) Biomarkers of primary open-angle glaucoma. Expert Rev Ophthalmol 5:731-742 |
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