Department of Pathophysiology, Key Laboratory of Neurological Disease of National Education Ministry, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Neurochem. 2013 Jul;126(2):234-42. doi: 10.1111/jnc.12285. Epub 2013 May 30.
Glycogen synthase kinase-3 beta (GSK-3β) dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. Some studies have shown that GSK-3β activity is up-regulated under oxidative stress. This study evaluated how oxidative stress regulates GSK-3β activity in human embryonic kidney 293 (HEK293)/Tau cells treated with hydrogen peroxide (H₂O₂). Here, we show that H₂O₂ induced an obvious increase of GSK-3β activity. Surprisingly, H₂O₂ dramatically increased phosphorylation of GSK-3β at Ser9, an inactive form of GSK-3β,while there were no changes of phosphorylation of GSK-3β at Tyr216. Moreover, H₂O₂ led to a transient Ca²⁺ elevation, and simultaneously increased the truncation of GSK-3β into two fragments of 40 kDa and 30 kDa, whereas inhibition of calpain decreased the truncation and recovered the activity of GSK-3β. Furthermore, tau was hyperphosphorylated at Ser396, Ser404, and Thr231, three most common GSK-3β targeted sites after 100 μM H₂O₂ administration in HEK293/Tau cells, whereas inhibition of calpain blocked the tau phosphorylation. In addition, we found that there were no obvious changes of Cyclin-dependent kinase 5 (CDK5) expression (responsible for tau phosphorylation) and of p35 cleavage, the regulatory subunit of CDK5 in H₂O₂-treated HEK293/Tau cells. In conclusion, Ca²⁺-dependent calpain activation leads to GSK-3β truncation, which counteracts the inhibitory effect of Ser9 phosphorylation, up-regulates GSK-3β activity, and phosphorylates tau in H₂O₂-treated HEK293/Tau cells.
糖原合成酶激酶-3β（GSK-3β）功能障碍可能在精神疾病、代谢性疾病、神经退行性疾病的发病机制中发挥重要作用，而这些疾病同时存在氧化应激。一些研究表明，GSK-3β活性在氧化应激下上调。本研究评估了过氧化氢（H₂O₂）处理的人胚肾 293（HEK293）/Tau 细胞中氧化应激如何调节 GSK-3β活性。在这里，我们表明 H₂O₂诱导 GSK-3β活性明显增加。令人惊讶的是，H₂O₂显著增加了 GSK-3β在 Ser9 上的磷酸化，这是 GSK-3β 的无活性形式，而 GSK-3β 在 Tyr216 上的磷酸化没有变化。此外，H₂O₂导致瞬时[Ca²⁺]（i）升高，同时增加 GSK-3β截短为 40 kDa 和 30 kDa 两个片段，而钙蛋白酶抑制剂减少截短并恢复 GSK-3β活性。此外，tau 在 Ser396、Ser404 和 Thr231 上过度磷酸化，这是 GSK-3β 靶向的三个最常见位点，在 100 μM H₂O₂处理后 HEK293/Tau 细胞中，而钙蛋白酶抑制剂阻断了 tau 磷酸化。此外，我们发现 H₂O₂处理的 HEK293/Tau 细胞中细胞周期蛋白依赖性激酶 5（CDK5）表达（负责 tau 磷酸化）和 CDK5 调节亚基 p35 切割没有明显变化。总之，Ca²⁺依赖性钙蛋白酶激活导致 GSK-3β截短，抵消了 Ser9 磷酸化的抑制作用，上调 GSK-3β活性，并在 H₂O₂处理的 HEK293/Tau 细胞中磷酸化 tau。
