Lin Dingbo, Barnett Micheal, Lobell Samuel, Madgwick Daniel, Shanks Denton, Willard Lloyd, Zampighi Guido A, Takemoto Dolores J
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
J Exp Biol. 2006 Nov;209(Pt 21):4371-8. doi: 10.1242/jeb.02524.
Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cgamma (PKCgamma) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCgamma control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCgamma knockout and control mice as our models. The results demonstrate that PKCgamma knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCgamma knockout mice, suggesting that the deletion of PKCgamma causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H(2)O(2) significantly induced lens opacification (cataract) in the PKCgamma knockout mice when compared to controls. Biochemical and structural results also demonstrated that H(2)O(2) activation of endogenous PKCgamma resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCgamma altered the arrangement of gap junctions on the cortical fiber cell surface, and completely abolished the inhibitory effect of H(2)O(2) on lens gap junctions. Data suggest that activation of PKCgamma is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCgamma knockout mice may cause those lenses to have increased susceptibility to oxidative damage.
白内障，即晶状体混浊，是全球失明的主要原因。白内障会随着年龄增长和环境损伤（如氧化应激）而增多。晶状体的内环境稳定依赖于功能性缝隙连接。晶状体缝隙连接蛋白Cx46或Cx50的基因敲除或错义突变会导致小鼠发生白内障。我们之前已经证明蛋白激酶Cγ（PKCγ）调节晶状体上皮和皮质中的缝隙连接。在当前研究中，我们以PKCγ基因敲除小鼠和对照小鼠为模型，进一步确定PKCγ对缝隙连接的控制是否能在体外保护晶状体免受氧化应激诱导的白内障形成。结果表明，与对照相比，PKCγ基因敲除小鼠在出生后2天时晶状体正常。然而，在6周龄的PKCγ基因敲除小鼠的晶状体中观察到细胞损伤，但无明显白内障，这表明PKCγ的缺失使晶状体更容易受到损伤。此外，与对照相比，体外培养或用H₂O₂对PKCγ基因敲除小鼠的晶状体进行氧化应激处理会显著诱导晶状体混浊（白内障）。生化和结构结果还表明，H₂O₂激活内源性PKCγ会导致对照小鼠晶状体中Cx50磷酸化，随后抑制缝隙连接，但在基因敲除小鼠中则不会。PKCγ的缺失改变了皮质纤维细胞表面缝隙连接的排列，并完全消除了H₂O₂对晶状体缝隙连接的抑制作用。数据表明，PKCγ的激活是调节晶状体纤维细胞中由缝隙连接通道介导的通讯通路关闭的重要机制。PKCγ基因敲除小鼠中缺乏这种调节机制可能导致这些晶状体对氧化损伤的易感性增加。
