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V. Akoyev, L. Grauer, D. Takemoto; Hypoxia Regulates the Activity of Pkc in the Lens. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1897.
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Lens is a naturally hypoxic and avascular tissue. The purpose of this study is to demonstrate that hypoxic conditions are important for PKCε activation through the hypoxia-induced phosphorylation of PKCε on Ser-729. These data support the hypothesis that activated and phosphorylated Ser729-PKCε interacts with cytochrome C oxidase component IV and protects mitochondria against apoptosis.
Hypoxic conditions were created with the help of Proox C21 hypoxic chamber (BioSpherix, NY). Immediately after surgery mouse lenses were incubated in the DMEM (10% serum) for 12 hours under different (3%, 5%, 7% and 10%) oxygen concentration to parallel those changes observed during vitreous detachment/vitrectomy. Normoxic conditions were considered as 21% of oxygen, 5% CO2, at 370C and 100% of relative humidity. After hypoxia or normoxia treatments, 2 types of experiments were conducted 1) lenses were photographed and the level of the opacity was estimated. 2) Lenses were subjected to solubilization and homogenization. Whole lens homogenate was used for western blot analysis with anti-phospho-Ser-729-PKCε antibody.
We found that: 1) hypoxia or normoxia conditions did not change the level of PKC-ε in the lens; 2) The highest level of Ser-729-PKCε was found in the lenses right after surgery. 3) The lowest level of Ser729-PKCε was found at 21% O2. 4) The level of phosphorylation is gradually decreased concurrently with changes in oxygen concentration from 3% to 10% and 21%. 5) Normoxic conditions induce the highest rate of opacification of lenses. 6) The lowest rate of opacification was found under hypoxic (3-5% O2) conditions.
Hypoxia induces and regulates the phosphorylation/activation of PKCε on Ser-729. This may indicate that PKCε activity is reduced during vitreous detachment/vitrectomy. Hypoxia is important for transparency of the lens.
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