氧化应激通过激活Cdk5抑制FOXO1的神经元损伤及其机制
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摘要
目的探究氧化应激通过激活细胞周期蛋白依赖性激酶5(cyclin-dependent kinase 5, Cdk5)抑制叉头框转录因子O亚家族蛋白1(forkhead box O1, FOXO1)的神经元损伤的具体机制。方法在HEK细胞中,共同转染过表达P39/Cdk5和FOXO1-WT或者FOXO1-S249A突变体,利用特异性磷酸化抗体pS249-FOXO1能够特异识别FOXO1的Ser249位点磷酸化特点,通过Western blot检测Cdk5/p39对FOXO1的磷酸化。通过体外培养HEK细胞中过表达Cdk5、p39和FOXO1,免疫共沉淀探究Cdk5和FOXO1以及Cdk5和p39的相互作用。在原代神经元过表达不同的Cdk5激活因子(p25、p35、p39)以及Cdk5激酶复合物,使用双荧光素酶实验探究其不同组合对FOXO1转录活性的影响。用Cdk5抑制剂rocovitine干预,观察其对FOXO1出入的影响;同时通过核质分离的方法对比了Cdk5杂合小鼠和野生型大脑皮层组织的FOXO1核质定位。分别用H_2O_2、glutamate处理神经元,通过Western blot观察氧化应激对Cdk5的活性调节,同时用双荧光素酶实验探究其对FOXO1转录因子的影响;最后通过caspase-3染色观察H_2O_2、glutamate的不同时间梯度处理对神经元凋亡的影响。结果 Cdk5和p39能够直接与FOXO1相互作用,并磷酸化FOXO1的Ser249位点。激活Cdk5能够显著抑制FOXO1的转录活性,而用Cdk5抑制剂(roscovitine)能够显著提高FOXO1的转录活性并诱导FOXO1入核。与野生型对比,在Cdk5杂合子中,FOXO1蛋白在细胞核中定位显著提高。H_2O_2和glutamate处理诱导p35切割形成p25,同时抑制FOXO1的转录活性,而敲低Cdk5能够缓解H_2O_2和glutamate对FOXO1的转录活性的抑制。通过H_2O_2和glutamate处理的原代神经元,caspase3染色显著增加。结论 Cdk5/p39可以磷酸化FOXO1-ser249位点,并抑制FOXO1的转录活性;而氧化应激能够通过激活Cdk5,抑制FOXO1的转录活性,并诱导神经元凋亡。
Objective To investigate the role of oxidative stress in inhibiting forkhead box O1(FOXO1) through activating cyclin-dependent kinase 5(Cdk5) in neuronal damage. Methods Cdk5 was coexpressed with p39 in HEK cells together with wild-type FOXO1 or a nonphosphorylatable FOXO1-S249 A mutant; FOXO1 phosphorylation was immunoblotted through pS249-FOXO1 antibody which specific recognized FOXO1 Ser249 phosphorylation. Cdk5 or p39 was coexpressed in HEK cells together with FOXO1 and their interaction was explored through Co-IP. In primary cortical culture neurons, various Cdk5 activators(p25, p35, and p39) were overexpressed together with Cdk5. FOXO1 transcriptional activity was tested by luciferase assay. We detected the effect of Cdk5 inhibitor roscovitine on FOXO1 transcriptional activity in primary neurons. We compared FOXO1 subcellular localization of wild type with Cdk5 heterozygous mice cortical brain tissues through cytoplasmic and nuclear extracts. In addition, we administered oxidative stress(H_2O_2 and glutamate treatment) in primary neurons and examined the effect on the cdk5 activity by Western blot,FOXO1 transcriptional activity by luciferase assay,and neuronal damage through stained caspase3.Results(1) Identification of Cdk5/p39 was capable of phosphorylating the transcription factor FOXO1 at Ser249,and associated with it.(2) Activated Cdk5 dramatically inhibited FOXO1 transcriptional activity.In contrast,roscovitine,Cdk5 inhibitor,significantly increased FOXO1 transcriptional activity and led to FOXO1 translocation into the nucleus.(3) FOXO1 protein was found predominately in the nucleus in the Cdk5 heterozygous mice.(4) H_O_2 or glutamate treatment promoted p35 to be cleaved to p25,and inhibited FOXO1 transcriptional activity.Importantly,this effect of oxidative stress was abolished when Cdk5 was silenced by shRNA.(5) Oxidative stress dramatically increased caspase3 immunostaining in primary neurons.Conclusion Cdk5/p39 inhibits FOXO1 activity through phosphorylation and association.Moreover,oxidative stress inhibits FOXO1 activity through activating Cdk5,and leads to neuronal death.
Objective To investigate the role of oxidative stress in inhibiting forkhead box O1(FOXO1) through activating cyclin-dependent kinase 5(Cdk5) in neuronal damage. Methods Cdk5 was coexpressed with p39 in HEK cells together with wild-type FOXO1 or a nonphosphorylatable FOXO1-S249 A mutant; FOXO1 phosphorylation was immunoblotted through pS249-FOXO1 antibody which specific recognized FOXO1 Ser249 phosphorylation. Cdk5 or p39 was coexpressed in HEK cells together with FOXO1 and their interaction was explored through Co-IP. In primary cortical culture neurons, various Cdk5 activators(p25, p35, and p39) were overexpressed together with Cdk5. FOXO1 transcriptional activity was tested by luciferase assay. We detected the effect of Cdk5 inhibitor roscovitine on FOXO1 transcriptional activity in primary neurons. We compared FOXO1 subcellular localization of wild type with Cdk5 heterozygous mice cortical brain tissues through cytoplasmic and nuclear extracts. In addition, we administered oxidative stress(H_2O_2 and glutamate treatment) in primary neurons and examined the effect on the cdk5 activity by Western blot,FOXO1 transcriptional activity by luciferase assay,and neuronal damage through stained caspase3.Results(1) Identification of Cdk5/p39 was capable of phosphorylating the transcription factor FOXO1 at Ser249,and associated with it.(2) Activated Cdk5 dramatically inhibited FOXO1 transcriptional activity.In contrast,roscovitine,Cdk5 inhibitor,significantly increased FOXO1 transcriptional activity and led to FOXO1 translocation into the nucleus.(3) FOXO1 protein was found predominately in the nucleus in the Cdk5 heterozygous mice.(4) H_O_2 or glutamate treatment promoted p35 to be cleaved to p25,and inhibited FOXO1 transcriptional activity.Importantly,this effect of oxidative stress was abolished when Cdk5 was silenced by shRNA.(5) Oxidative stress dramatically increased caspase3 immunostaining in primary neurons.Conclusion Cdk5/p39 inhibits FOXO1 activity through phosphorylation and association.Moreover,oxidative stress inhibits FOXO1 activity through activating Cdk5,and leads to neuronal death.
引文
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[22] YUAN Z,BECKER EB,MERLO P,et al.Activation of FOXO1 by Cdk1 in cycling cells and postmitotic neurons[J].Science,2008,319(5870):1665-1668.
[23] GROSS DN,VAN DEN HEUVEL AP,BIRNBAUM MJ.The role of FoxO in the regulation of metabolism[J].Oncogene,2008,27(16):2320-2336.
[24] GILLEY J,COFFER PJ,HAM J.FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[J].J Cell Biol,2003,162(4):613-622.
[25] SHAO D,ZHAI P,DEL RE DP,et al.A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response[J].Nat Commun,2014,5:3315.
[2] LANG JD,MCARDLE PJ,O'REILLY PJ,et al.Oxidant-antioxidant balance in acute lung injury[J].Chest,2002,122(6 Suppl):314S-320S.
[3] OH TY,LEE JS,AHN BO,et al.Oxidative stress is more important than acid in the pathogenesis of reflux oesophagitis in rats[J].Gut,2001,49(3):364-371.
[4] ZHU X,RAINA AK,LEE HG,et al.Oxidative stress signalling in Alzheimer’s disease[J].Brain Res,2004,1000(1-2):32-39.
[5] PACE GW,LEAF CD.The role of oxidative stress in HIV disease[J].Free Radic Biol Med,1995,19(4):523-528.
[6] KAMENETZ F,TOMITA T,HSIEH H,et al.APP processing and synaptic function[J].Neuron,2003,37(6):925-937.
[7] GOMEZ-LAZARO M,GALINDO MF,MELERO-FERNANDEZ DE MERA RM,et al.Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate[J].Mol Pharmacol,2007,71(3):736-743.
[8] LEE MS,KWON YT,LI M,et al.Neurotoxicity induces cleavage of p35 to p25 by calpain[J].Nature,2000,405(6784):360-364.
[9] PATZKE H,TSAI LH.Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29[J].J Biol Chem,2002,277(10):8054-8060.
[10] SU SC,TSAI L-H.Cyclin-dependent kinases in brain development and disease[J].Ann Rev Cell Dev Biol,2011,27(1):465-491.
[11] WANG J,LIU S,FU Y,et al.Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors[J].Nat Neurosci,2003,6(10):1039-1047.
[12] DHAVAN R,TSAI LH.A decade of CDK5[J].Nat Rev Mol Cell Biol,2001,2(10):749-759.
[13] MAIESE K.FoxO proteins in the nervous system[J].Anal Cell Pathol (Amst),2015,2015:569392.
[14] WANG Z,YU T,HUANG P.Post-translational modifications of FOXO family proteins (Review)[J].Mol Med Rep,2016,14(6):4931-4941.
[15] ZHOU J,LI H,LI X,et al.The roles of Cdk5-mediated subcellular localization of FOXO1 in neuronal death[J].J Neurosci,2015,35(6):2624-2635.
[16] PATRICK GN,ZUKERBERG L,NIKOLIC M,et al.Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration[J].Nature,1999,402(6762):615-622.
[17] TARRICONE C,DHAVAN R,PENG J,et al.Structure and regulation of the CDK5-p25(nck5a) complex[J].Mol Cell,2001,8(3):657-669.
[18] KUSAKAWA G,SAITO T,ONUKI R,et al.Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25[J].J Biol Chem,2000,275(22):17166-17172.
[19] NATH R,DAVIS M,PROBERT AW,et al.Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells[J].Biochem Biophys Res Commun,2000,274(1):16-21.
[20] ARDEN KC.FoxO:linking new signaling pathways[J].Mol Cell,2004,14(4):416-418.
[21] HUANG H,REGAN KM,LOU Z,et al.CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage[J].Science,2006,314(5797):294-297.
[22] YUAN Z,BECKER EB,MERLO P,et al.Activation of FOXO1 by Cdk1 in cycling cells and postmitotic neurons[J].Science,2008,319(5870):1665-1668.
[23] GROSS DN,VAN DEN HEUVEL AP,BIRNBAUM MJ.The role of FoxO in the regulation of metabolism[J].Oncogene,2008,27(16):2320-2336.
[24] GILLEY J,COFFER PJ,HAM J.FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[J].J Cell Biol,2003,162(4):613-622.
[25] SHAO D,ZHAI P,DEL RE DP,et al.A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response[J].Nat Commun,2014,5:3315.