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N. Fatma, E. Kubo, D.A. Beier, D.P. Singh; Impaired Homeostasis and Phenotypic Abnormalities of Reactive Oxygen Species in AOP2–/– Mice Lens Epithelial Cells: Increased Expression and Activation of TGF ß1 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1024.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Reactive oxygen species (ROS) are implicated in many cellular metabolic and signaling processes. Antioxidant protein 2 (AOP2), a member of peroxiredoxins family, protects cells by removing H2O2. In the present study, we demonstrated that AOP2 offsets deleterious effects of ROS on LECs or lenses and regulates gene expression by limiting its levels to maintain cellular integrity. Methods: mLECs were generated from lenses of AOP2–/– and AOP2+/+ mice. ROS levels were monitored with fluorescent dye, H2DCFH–DA. Western and real time quantitative PCR were carried out to assess the expression of TGFß1 and its inducible genes, LEDGF, hsp27 and αB–crystalline. Gelmobility assay was conducted to assess DNA binding affinity of LEDGF to stress response and heat shock elements. The transfection–system was utilized to monitor the TGF ß1–induced modulation of LEDGF, Hsp27 and αB–crystalline promoters activity. Bioactive TGFß1 was determined using Emax Immuno Assay System (Promega). GFP–AOP2 and/or AOP2 protein linked to protein transduction domain were used to supply AOP2 to AOP2–/– LECs. Results: Mice lenses and/or LECs lacking the AOP2 had abnormal phenotypes and biochemical abnormalities and altered gene expression patterns. Biochemical assays showed an enhanced generation of ROS and higher prevalence of bioactive TGFß1 in AOP2–/– cells. Molecular analysis indicated a reduced expression of LEDGF and its inducible–genes: α B–crystalline, hsp27, and increased expressions of TGF1 and its inducible genes (ß1gh3), α–SM–actin. Intrinsic as well as extrinsic supply of AOP2 to AOP2–/–cells could restore the survival function(s) of LEDGF. Moreover, treatment of AOP2–/– LECs with SOD mimetic (MnTBAP) or AOP2 reversed the several aspects of null phenotypes and could maintain the cellular integrity. Conclusions: Taken together, our findings suggest that AOP2 is not only antioxidant, but also the regulator of ROS–mediated signaling. We propose a rheostat role for AOP2 that controls gene expression by controlling ROS level for maintaining cellular homeostasis.
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