Hypoxia-regulated activity of PKCepsilon in the lens.
OBJECTIVE
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.
Akoyev
Vladimir
V
Department of Biochemistry, Kansas State University, Manhattan, Kansas.
Das
Satyabrata
S
Jena
Snehalata
S
Grauer
Laura
L
Takemoto
Dolores J
DJ
D000818:Animals; D015153:Blotting, Western; D002386:Cataract / Q000517:prevention & control; D018929:Cell Culture Techniques; D018031:Connexin 43 / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D011817:Rabbits; D012815:Signal Processing, Computer-Assisted; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018031:Connexin 43; D007334:Insulin-Like Growth Factor I; D006861:Hydrogen Peroxide; C087857:protein kinase C gamma; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate