Expression of superoxide dismutase in whole lens prevents cataract formation.
OBJECTIVE
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.
Lin
Dingbo
D
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Barnett
Micheal
M
Grauer
Laura
L
Robben
Jerry
J
Jewell
Annie
A
Takemoto
Larry
L
Takemoto
Dolores J
DJ
D000818:Animals; D015153:Blotting, Western; D002386:Cataract / Q000139:chemically induced / Q000201:enzymology / Q000517:prevention & control*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000633:toxicity; D007908:Lens, Crystalline / Q000201:enzymology*; D008297:Male; D009924:Organ Culture Techniques; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014162:Transfection
C000606290:SOD1 protein, human; D006861:Hydrogen Peroxide; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C087857:protein kinase C gamma; D011493:Protein Kinase C