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V. Akoyev, L. Grauer, L. L. Willard, D. J. Takemoto; Lens PKC and PKC Have Opposing Interactions With Cx43: Role of Hypoxia. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4215.
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Regulation of intercellular communications (ICC) via gap junction coupling is extremely important for maintaining cell growth and surviving under hypoxia and oxidative stress. Such combination of growth and stress conditions is typical for lens epithelial cells (LEC). Previously, we have shown that insulin-like growth factor-1 (IGF1) and stress factors (SF) such as phorbol-12-myristate-13-acetate (TPA) or oxidative stress inhibited ICC in LEC via direct activation of PKCγ followed by phosphorylation on Serine-368 of the major gap junction protein Cx43 which results in the disassembly of gap junction plaques. Aside from PKCγ, there is also the ε isoform of PKC in LEC. It has been shown that PKCε plays a key role in cardiac protection against hypoxia. In the heart, growth factors (GF) and SF activate PKCε and induce interaction with and phosphorylation of Cx43. This mechanism has been shown to be involved in the reduction of the size of the infarct area. The functional role of PKCε in the LEC is still unclear. The purpose of this study is to investigate interactions between PKCε and Cx43 in the LEC and show how SF and GF regulate them.
LEC were treated at 37 0C for 30 minutes with SF: H2O2 (100 µM), TPA (300 nM), hypoxia, and GF such as FGF2, EGF and IGF1 (25 ng/mL). Then, cells were subjected to immunoprecipitation (IP), western blots (WB), confocal image analysis (CIA), and enzyme activity assays.
1). WB and IP analyses showed that stress factors (TPA and H2O2) and FGF or IGF1 causes opposite effects: a decreased association of Cx43 with PKCε and increased association with PKCγ. 2) CIA showed a colocalization of Cx43 and PKCε and the absence of colocalization with PKCγ under control conditions. 3) SF caused the disassembly of Cx43 plaques, the loss of colocalization with PKCε and the induction of membrane colocalization with PKCγ. 4) SF also have opposite effects on enzyme activity: PKCγ was activated and PKCε was inhibited by SF. In this study, GF had no effect on PKCε enzyme activity. 5) Lenses from PKCε knockout mice are smaller and have an elongated shape.
1). PKCγ and PKCε have opposing effects on lens Cx43. PKCγ is activated by SF or GF and this activation increases its interaction with Cx43. SF causes inhibition of PKCε activity and a decreased association with Cx43. 3) Analysis of PKCε knockouts suggest that PKCε is required for lens growth and it is regulated differently than PKCγ.
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