Pentose phosphate pathway activation via HSP27 phosphorylation by ATM kinase: A putative endogenous antioxidant defense mechanism during cerebral ischemia-reperfusion
Pentose phosphate pathway activation via HSP27 phosphorylation by ATM kinase: A putative endogenous antioxidant defense mechanism during cerebral ischemia-reperfusion
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DOI：
10.1016/j.brainres.2018.03.001
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发表时间：
2018-05
期刊：
影响因子：
2.9
通讯作者：
Yusuke Yamamoto;K. Hosoda;T. Imahori;J. Tanaka;K. Matsuo;T. Nakai;Y. Irino;M. Shinohara;Naoko Sa
中科院分区：
文献类型：
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作者：
Yusuke Yamamoto;K. Hosoda;T. Imahori;J. Tanaka;K. Matsuo;T. Nakai;Y. Irino;M. Shinohara;Naoko Sa
缺血性卒中的分子机制仍然知之甚少。我们先前报道，在脑缺血时，磷酸戊糖途径(PPP)中的葡萄糖6-磷酸脱氢酶(G6PD)活性是通过热休克蛋白27(HSP27)丝氨酸85(S85)的磷酸化被共济失调性毛细血管扩张突变(ATM)激酶激活的。这一机制可能是内源性抗氧化系统。为了确定该系统在再灌注期间是否也起作用，我们采用大脑中动脉阻塞(MCAO)的方法，对再灌注对大鼠大脑皮质代谢的影响进行了比较代谢分析。用气相色谱/质谱仪分析的代谢谱显示，代谢状态的改变依赖于再灌流时间。浓缩分析显示，在缺血-再灌流过程中，PPP显著上调。再灌流后6-磷酸果糖和5-磷酸核酮糖显着升高也提示PPP增强。与PPP相比，缺血再灌注24小时后，HSP27转录本的表达增加了69倍。免疫印迹显示HSP27蛋白逐渐增加，HSP27磷酸化显著增加(S85)，且呈时间依赖性(再灌注24小时后增加4.5倍)。G6PD活性在MCAO后1h显著升高(20%)，再灌流1h后降低，以后逐渐升高，再灌流后明显升高。NADPH/NAD+比值呈类似的上升趋势。侧脑室注射血管紧张素转换酶抑制剂(KU-55933)可显著降低热休克蛋白27的磷酸化和G6PD活性，显著增加蛋白羰基含量，并使脑缺血再灌注后24 h的梗塞面积增加(100%)。因此，通过ATMK对HSP27的磷酸化激活G6PD可能是缺血再灌注时激活的内源性抗氧化防御神经保护机制的一部分。这些发现对卒中的治疗具有重要意义。
Molecular mechanism underlying ischemic stroke remains poorly understood. We previously reported glucose 6-phosphate dehydrogenase (G6PD) activity in pentose phosphate pathway (PPP) is activated via heat shock protein 27 (HSP27) phosphorylation at serine 85 (S85) by ataxia telangiectasia mutated (ATM) kinase during cerebral ischemia. This mechanism seems to be endogenous antioxidative system. To determine whether this system also works during reperfusion, we performed comparative metabolic analysis of reperfusion effect on metabolism in rat cortex using middle cerebral artery occlusion (MCAO). Metabolic profiling using gas-chromatography/mass-spectrometry analysis showed changes in metabolic state that depended on reperfusion time. Enrichment analysis showed PPP was significantly upregulated during ischemia-reperfusion. Significant increases in fructose 6-phosphate and ribulose 5-phosphate after reperfusion also suggested enhancement of PPP. In relation to PPP, ischemia-reperfusion induced an increase of up to 69-fold in HSP27 transcripts after 24-h reperfusion. Immunoblotting showed gradual increase in HSP27 protein and marked increase in HSP27 phosphorylation (S85) that were time-dependent (4.5-fold after 24-h reperfusion). G6PD activity was significantly elevated after 1-h MCAO (20%), reduced after 1-h reperfusion, increased gradually thereafter and significantly elevated after 24-h reperfusion. The NADPH/NAD+ratio displayed similar increasing pattern. Intracerebroventricular injection of ATM kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD activity, significantly increased protein carbonyl, and resulted in increase in infarct size (100%) 24-h after reperfusion following 90-min MCAO. Consequently, G6PD activation via HSP27 phosphorylation by ATM kinase may be part of endogenous antioxidant defense neuroprotection mechanism that is activated during ischemia-reperfusion. These findings have important implications for treatment of stroke.