Tamada Yoshiyuki, Nakajima Emi, Nakajima Takeshi, Shearer Thomas R, Azuma Mitsuyoshi
Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, OR 97006, USA.
Brain Res. 2005 Jul 19;1050(1-2):148-55. doi: 10.1016/j.brainres.2005.05.048.
Our previous studies in retina on the mechanism for hypoxia-induced cell death suggested activation of a class of calcium-activated proteases known as calpains. This conclusion was based on data showing proteolysis of a calpain substrate alpha-spectrin, autolysis of activated calpain, and reduction of cell damage by calpain inhibitor SJA6017. Less is known about changes in downstream pathways after calpain activation. Thus, the purpose of the present investigation was to measure proteolysis of neuronal cytoskeletal proteins and apoptotic cell signaling factors during hypoxia-induced retinal cell death. Rat retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. Hypoxia was induced with 95% N2/5% CO2 without glucose. Immunoblotting was used to detect activation of calpain and proteolysis of substrates. Amounts of mRNA for calpain 1 and 2 were determined by quantitative PCR. Twelve times more calpain 2 mRNA than calpain 1 was present in retinas. Activation of calpain 2 and production of a calpain-specific alpha-spectrin breakdown product at 150 kDa were confirmed in hypoxic retinas. Further, pro-caspase-3 at 32 kDa was proteolyzed to a fragment at 30 kDa, tau protein was lost, and p35 was proteolyzed to p25 suggesting prolonged activation of cdk5. SJA6017 partially inhibited the production of these fragments. During hypoxia in rat retinas, calpains may be major proteases causing breakdown of neuronal proteins involved in apoptotic cell death. Calpain inhibitor SJA6017 may have potential for testing as a therapeutic agent against retinal pathologies such those caused by glaucoma, although future studies such as testing in in vivo animal models are required.
我们之前在视网膜上关于缺氧诱导细胞死亡机制的研究表明，一类名为钙蛋白酶的钙激活蛋白酶被激活。这一结论基于以下数据：钙蛋白酶底物α-血影蛋白的蛋白水解、活化钙蛋白酶的自溶以及钙蛋白酶抑制剂SJA6017对细胞损伤的减轻。关于钙蛋白酶激活后下游通路的变化，人们了解较少。因此，本研究的目的是测量缺氧诱导视网膜细胞死亡过程中神经元细胞骨架蛋白和凋亡细胞信号因子的蛋白水解情况。将大鼠视网膜置于含有葡萄糖和95% O₂/5% CO₂的RPMI培养基中孵育，以为视网膜细胞存活提供充足的氧气。用不含葡萄糖的95% N₂/5% CO₂诱导缺氧。采用免疫印迹法检测钙蛋白酶的激活和底物的蛋白水解情况。通过定量PCR测定钙蛋白酶1和2的mRNA含量。视网膜中钙蛋白酶2的mRNA含量是钙蛋白酶1的12倍。在缺氧的视网膜中证实了钙蛋白酶2的激活以及产生了150 kDa的钙蛋白酶特异性α-血影蛋白降解产物。此外，32 kDa的前半胱天冬酶-3被蛋白水解为30 kDa的片段，tau蛋白丢失，p35被蛋白水解为p25，提示细胞周期蛋白依赖性激酶5的持续激活。SJA6017部分抑制了这些片段的产生。在大鼠视网膜缺氧期间，钙蛋白酶可能是导致参与凋亡细胞死亡的神经元蛋白分解的主要蛋白酶。钙蛋白酶抑制剂SJA6017可能有潜力作为治疗青光眼等视网膜病变的治疗药物进行测试，不过还需要进行如体内动物模型测试等进一步研究。
