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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY010595-18A1
Application #
8576078
Study Section
Special Emphasis Panel (BVS)
Program Officer
Araj, Houmam H
Project Start
1994-02-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
18
Fiscal Year
2013
Total Cost
$409,259
Indirect Cost
$127,981
Name
University of Nebraska Lincoln
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
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
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
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
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
Huo, Yanan; Qiu, Wen-Ya; Pan, Qing et al. (2009) Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing. Exp Eye Res 89:876-86
Wang, Yin; Lou, Marjorie F (2009) The regulation of NADPH oxidase and its association with cell proliferation in human lens epithelial cells. Invest Ophthalmol Vis Sci 50:2291-300
Lofgren, Stefan; Fernando, M Rohan; Xing, Kui-Yi et al. (2008) Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse. Invest Ophthalmol Vis Sci 49:4497-505
Liyanage, Namal P M; Fernando, M Rohan; Lou, Marjorie F (2007) Regulation of the bioavailability of thioredoxin in the lens by a specific thioredoxin-binding protein (TBP-2). Exp Eye Res 85:270-9

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