Hypoxia-regulated activity of PKCepsilon in the lens.

PURPOSE: To show that hypoxia is necessary to prevent opacification of the lens. Protein kinase C (PKC)-epsilon serves a role that is distinct from PKC-gamma when both PKC isoforms are expressed in the lens. PKCepsilon serves a very important role in hypoxic conditions, helping to prevent opacification of the lens. METHODS: Digital image analysis, confocal microscopy, dye transfer assay, coimmunoprecipitation, Western blot analysis, and enzyme activity assays were used, respectively, to study opacification of the lens, intercellular communications, cellular localization of connexin-43 (Cx43), and the interactions between PKCepsilon, PKCgamma, and Cx43 in the lens epithelial cells. RESULTS: Hypoxic conditions (1%-5% of oxygen) were very important in maintaining clarity of the lenses of wild-type (WT) mice. Normoxic conditions induced opacification of the WT lens. Lenses from the PKCepsilon-knockout mice underwent rapid opacification, even in hypoxic conditions. Hypoxia did not induce apoptosis in the lens epithelial cells, judging by the absence of active caspase-3, and it did not change intercellular communication and did not affect the number and localization of junctional Cx43 plaques in the lens epithelial cell culture. Hypoxia activated PKCepsilon, whereas phorbol ester (TPA), oxidation (H(2)O(2)), and insulin-like growth factor-1 (IGF-1) activated PKCgamma and decreased the activity of PKCepsilon. Hypoxia did not induce the phosphorylation of the Cx43. CONCLUSIONS: Hypoxia-induced activation of PKCepsilon is very important in surviving hypoxia and maintaining the clarity of the lens. However, PKCgamma is utilized in the control of Cx43 gap junctions.

Expression of superoxide dismutase in whole lens prevents cataract formation.

PURPOSE: Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress. METHODS: Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 degrees C in a CO2 cell culture chamber with 95% air and 5% CO2. SOD1 expression was determined by western blotting and SOD enzyme activity. Lenses with or without overexpression of SOD1 were treated with H2O2 and cataract formation was examined. SOD1 regulation of protein kinase Cgamma (PKCgamma) was determined by PKCgamma enzyme activity assay. Intact lens gap junctions were determined by dye transfer assay. RESULTS: In the lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which allow detection from endogenous SOD1. Incubation of intact lenses with plasmid DNA produced EYFP:SOD1 fusion proteins as determined by western blot using anti-GFP or anti-SOD1 antibodies. This caused significant increases in SOD enzyme activity. Data indicated that SOD1 plasmid DNA can be expressed as a functional enzyme in intact lenses in culture. Lenses overexpressing SOD1 remained clear after H2O2 treatment at 100 muM for 24 h, similar to control. Overexpression of SOD1 diminished the effect of H2O2 on PKCgamma activation and subsequent inhibition of gap junctions, indicating that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H2O2 effect on cataract formation. CONCLUSIONS: Overexpression of SOD1 in whole lens prevents H2O2-induced oxidative damage (cataract formation) to the lens and subsequent control of gap junctions by protein kinase Cgamma.