The aims of this proposal are to confirm that the thiol repair systems of glutathione (GSH)-dependent thioltransferase (TTase) and NADPH-dependent thioredoxin (TRx) protect against oxidative stress-induced cataract formation by their ability to repair oxidation damaged proteins and by their redox regulating functions of controlling cell proliferation and differentiation. Emphasis is placed on proving our hypothesis that oxidative stress-induced protein-thiol mixed disulfide (thiolation) formation with GSH or cysteine is the initial protein damage that leads to protein disulfide aggregation and lens opacification. We hypothesize that TTase and TRx can restore the functions of these thiolated lens proteins via dethiolation, and control growth factor-stimulated cell proliferation and differentiation via regulating redox signaling. With the acquired genetic mouse models of TTase knockout (KO), TTase transgene (TG), and Grx2 KO (TTase mitochondrial isozyme), we can achieve our research goals.
The specific aims are 1). Validate TTase's role in maintaining lens clarity by comparing the effect of TTase KO and TG on cataract formation. Examine TTase regulation of PDGF-induced actin elongation. 2). Study the mechanisms of Grx2 deletion-induced cataract and the altered cell denucleation and differentiation. Identify Grx2 target proteins and Grx2's role in mitochondria. 3) Study TRx's mitogenic function via interaction with TRPC channel protein. Morphological and biochemical studies will be done on TG and wild type mice for aging (2-22 m) and UVB-induced cataracts. Lens epithelial cells cultured from the 3 genetic mouse models will be used to study TTase and Grx2 functions. TTase-dependent actin elongation will be clarified by measuring G/F-actin ratio in PDGF-stimulated cells with and w/o TTase. Altered lens fiber formation in Grx2 KO mice will be studied by lens explants, activity assays of denucleation enzymes (DNase II? and ubiquitin) and bFGF-specific phosphatases (PTEN and Shp2). Target proteins for Grx2 will be identified by mass spectrometry, and Grx2 mitochondrial function by O2 consumption. Calcium imaging and BrdU assay will be used to study TRx-TRPC interaction using TRPC overexpressed, knockdown and control cells. Results from these studies will provide mechanisms of how thiol repair systems maintain lens clarity and regulate cell proliferation and differentiation. Successful completion of the aims will offer therapeutic strategies for oxidation- induced ocular diseases.

Public Health Relevance

Human age-related cataract is the major cause for blindness in the world, yet the reason for cataract formation is not well understood. This proposed research is based on the hypothesis that cataract is induced by oxidative damage, thus the research will focus on the oxidative damage repair systems in the lens so that a better strategy can be developed for preserving lens clarity, and for protecting against other oxidation-induced ocular diseases.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (BVS)
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Araj, Houmam H
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University of Nebraska Lincoln
Veterinary Sciences
Schools of Earth Sciences/Natur
United States
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Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli et al. (2015) Expression and distribution of thiol-regulating enzyme glutaredoxin 2 (GRX2) in porcine ocular tissues. Exp Eye Res 130:58-65
Wu, Hongli; Yu, Yibo; David, Larry et al. (2014) Glutaredoxin 2 (Grx2) gene deletion induces early onset of age-dependent cataracts in mice. J Biol Chem 289:36125-39
Wei, Min; Xing, Kui-Yi; Fan, Yin-Chuan et al. (2014) Loss of thiol repair systems in human cataractous lenses. Invest Ophthalmol Vis Sci 56:598-605
Yu, Yibo; Xing, Kuiyi; Badamas, Rilwan et al. (2013) Overexpression of thioredoxin-binding protein 2 increases oxidation sensitivity and apoptosis in human lens epithelial cells. Free Radic Biol Med 57:92-104
Zhang, Jie; Yan, Hong; Lofgren, Stefan et al. (2012) Ultraviolet radiation-induced cataract in mice: the effect of age and the potential biochemical mechanism. Invest Ophthalmol Vis Sci 53:7276-85
Wang, Yin; Xing, Kui-Yi; Lou, Marjorie F (2011) Regulation of cytosolic phospholipase A2 (cPLA2alpha) and its association with cell proliferation in human lens epithelial cells. Invest Ophthalmol Vis Sci 52:8231-40
Wu, HongLi; Lin, LiRen; Giblin, Frank et al. (2011) Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells. Free Radic Biol Med 51:2108-17
Pan, Qing; Qiu, Wen-Ya; Huo, Ya-Nan et al. (2011) Low levels of hydrogen peroxide stimulate corneal epithelial cell adhesion, migration, and wound healing. Invest Ophthalmol Vis Sci 52:1723-34
Xing, Kui-Yi; Lou, Marjorie F (2010) Effect of age on the thioltransferase (glutaredoxin) and thioredoxin systems in the human lens. Invest Ophthalmol Vis Sci 51:6598-604
Wu, Hongli; Xing, Kuiyi; Lou, Marjorie F (2010) Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria. Biochim Biophys Acta 1797:1705-15

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