PKC-ERK通路在热损伤诱导单核细胞凋亡中的作用

应用流式细胞检测术、Western印迹、激酶活性测定等技术,检测PKC与ERK在热损伤诱导单核细胞株Raw264.7细胞凋亡中的作用。结果显示热损伤导致PKC短暂激活,PKC激活剂佛波脂(PMA)与热损伤联合作用导致PKC持续活化;并且PKC的持续激活抑制热损伤诱导的Raw264.7细胞凋亡,而PKC的抑制可促进细胞凋亡;ERK活性检测显示热损伤抑制ERK磷酸化,而PMA激活ERK磷酸化活化,并且这种激活作用通过PKC;进一步细胞凋亡检测显示ERK抑制剂PD098059可解除PMA对热损伤诱导Raw264.7细胞凋亡的抑制作用,从而提示PKC通过ERK负调控热损伤诱导的Raw264.7细胞凋亡。     

IRE1/TRAF2诱导细胞凋亡的分子机制

在细胞内质网应激中,IRE1/TRAF2/ASK1复合物可激活JNK信号通路,诱导细胞凋亡。IRE1-Lys828可被E3连接酶CHIP泛素化而激活。而TRAF2本身也具有RING结构域的E3泛素连接酶活性,可结合于IRE1的泛素化位点Lys828,促进IRE1磷酸化活化。IRE1/TRAF2复合物可募集ASK1,进而磷酸化激活JNK信号通路。另外,IRE1/TRAF2也可激活MAPK/p38、NF-κB以及caspase-12等信号途径,促进细胞凋亡。ASK1也是内质网应激IRE1/TRAF2诱导激活细胞凋亡所必需的。因此,TRAF2在调控细胞内质网应激,激活IRE1途径,诱导细胞凋亡中具有重要的作用,可作为一个靶点进行药物开发。     

细胞衰老过程中JNK、p38和Akt的活性变化

研究发现衰老成纤维细胞抗凋亡. 为探明其机制,检测与凋亡相关的信号传导通路JNK、p38和Akt在细胞衰老过程中是否发生改变.在本研究中,UV被用作JNK和p38传导通路的诱导剂,胎牛血清用作Akt通路的诱导剂.结果表明: p38和Akt在年轻和衰老细胞中均能被相应的诱导剂活化;相反, UV照射却不能有效激活衰老细胞中JNK的活性.结果提示:衰老细胞对凋亡诱导不敏感可能与JNK不能被有效活化有关.     

酸性鞘磷脂水解酶和MAPK信号通路在UVA诱导细胞凋亡中的作用

为了探讨酸性鞘磷脂水解酶 (ASM)和MAPK信号通路在UVA诱导的细胞凋亡中的作用 ,用DNA梯形条带 (DNAladder)和荧光显微镜鉴定细胞凋亡 ,Western印迹分析MAPK信号通路的激活情况 .结果显示 :①经UVA照射 ,正常的淋巴母细胞JY出现严重的细胞凋亡 ,而ASM遗传性缺陷的淋巴母细胞MS1 4 1 8出现轻微凋亡 ;给予ASM特异性抑制剂NB6 ,UVA诱导的JY细胞凋亡明显减轻 ,表明UVA诱导的细胞凋亡依赖于ASM .②UVA照射后 ,磷酸化ERK含量在MS1 4 1 8细胞中明显升高 ,在JY细胞中受到抑制 ;UVA照射前给予NB6 ,JY细胞中磷酸化ERK含量上升 ,表明ASM能抑制ERK的激活 .③UVA照射后 ,磷酸化JNK含量在MS1 4 1 8细胞中几乎没有变化 ,而在JY细胞中含量升高 ;UVA照射前给予NB6 ,JY细胞中磷酸化JNK含量没有明显升高 ,表明ASM激活JNK通路 .④NB6对UVA激活的p38MAPK信号通路没有影响 ,表明p38的激活与ASM关系不大 .研究表明 ,UVA诱导的细胞凋亡是通过激活ASM、激活JNK信号通路并抑制ERK信号通路来完成的     

PKCδ与JNK共同调控喜树碱诱导的白血病细胞凋亡

采用流式细胞术、蛋白质免疫印迹法检测了喜树碱诱导白血病细胞凋亡过程中蛋白激酶Cδ(protein kinase Cδ,PKCδ)与c-Jun氨基末端激酶(c-Jun N-termital kinase,JNK)的作用。结果发现,50 nmol/L喜树碱诱导处理U937细胞24、36或48 h后,细胞发生明显凋亡,并且PKCδ和JNK均被激活。用化学抑制剂rottlerin抑制PKCδ的活化可以降低喜树碱诱导细胞凋亡过程中JNK的磷酸化,进而抑制细胞凋亡;而用化学抑制剂SP600125抑制JNK的磷酸化也会降低PKCδ的剪切活化,进而一定程度地阻断细胞凋亡;同时,JNK抑制剂SP600125也可以阻断过表达PKCδ活性片段诱导的细胞凋亡。这些结果提示,PKCδ和JNK介导的信号通路可以相互调控,共同促进细胞凋亡。该研究对理解细胞凋亡的精细调控机制以及肿瘤的治疗都有一定的借鉴意义。     

赭曲霉毒素A对体外培养人肾小管上皮细胞凋亡的作用

探讨赭曲霉毒素A(ochratoxin A,OA)对体外培养的人肾小管上皮细胞(HKC)凋亡的影响.体外培养HKC,给予不同浓度OA处理24 h后,分别采用流式细胞定量检测术(FCM)检测细胞的凋亡率,应用免疫细胞化学染色(SP法)和免疫蛋白印迹(Western 印迹)方法,观察凋亡相关蛋白caspase-3的表达以及JNK的表达水平和磷酸化水平(p-JNK).FCM检测结果表明,经OA处理24 h,HKC的凋亡率明显升高.SP法和Western印迹结果表明,OA处理组HKC caspase-3的表达以及JNK的磷酸化水平(p-JNK)均明显高于空白对照组和溶剂对照组,但各组间细胞JNK的表达水平无明显变化.OA处理可促进体外培养的人肾小管上皮细胞发生凋亡,JNK激活以及caspase-3可能参与OA诱导HKC凋亡的过程.     

Grx1 抑制高糖诱导的H9c2细胞中Caspase-8/3和JNK /c-Jun凋亡通路的激活

谷氧还蛋白1（glutaredoxin 1,Grx1）作为一种重要的抗氧化剂,通过响应多种重要蛋白质的活动和功能调节细胞的关键过程．了解Grx1功能,对寻求糖尿病和心肌病等,以凋亡失调和氧化还原稳态改变为发病机制的疾病的新颖治疗策略至关重要．为研究Grx1对高糖诱导的大鼠心肌细胞凋亡的抑制作用及相关信号机制,本研究以高糖诱导大鼠心肌细胞H9c2建立高糖损伤模型,采用免疫印迹实验检测caspase-3、8、9蛋白活性片段的表达和凋亡信号蛋白JNK/c-Jun的磷酸化水平．结果显示,与正常对照组相比,高糖组caspase家族中,剪切的caspase-3、caspase-8、caspase-9相对含量均显著增多,JNK和c-Jun蛋白的磷酸化水平均显著上调. 但给予外源性Grx1保护后,剪切的caspase-3和caspase-8相对含量均显著降低,JNK和c-Jun蛋白的磷酸化水平均显著下调．上述结果表明,高糖通过介导caspase-8/3和caspase-9/3凋亡通路,并激活凋亡相关信号通路JNK/c-Jun诱导H9c2心肌细胞凋亡．给予外源性Grx1保护后,可通过抑制caspase-8/3凋亡通路和JNK /c-Jun信号通路的激活,拮抗高糖诱导的心肌细胞凋亡．     

Nmp4促进TNF-α诱导的成骨细胞凋亡

核基质蛋白4(nuclear matrix protein4,Nmp4)是一种具有核质穿梭功能的结构性转录因子.主要通过负调控调节成骨细胞分化和增殖,抑制骨密度及骨量增加,而Nmp4是否调节成骨细胞凋亡,还未有相关报道.本课题通过分离Nmp4基因敲除(Nmp4-KO)和野生型(WT)小鼠原代成骨细胞,以肿瘤坏死因子(TNF-α)为凋亡诱导手段,研究了Nmp4对成骨细胞凋亡的影响及其作用机制.体外细胞实验发现,Nmp4-KO显著抑制TNF-α诱导的成骨细胞内caspase-3激活.Nmp4-KO促进细胞外信号调节激酶(Erk)和丝氨酸/苏氨酸蛋白激酶(Akt)信号途径的激活,抑制c-Jun氨基末端激酶(JNK)磷酸化,从而对抗成骨细胞凋亡.TNF-α诱导处理可增强成骨细胞核因子NFκB磷酸化及其核转位,但Nmp4基因缺失无进一步促进作用.未经诱导处理的Nmp4-KO细胞内NFκB磷酸化水平显著高于WT对照.此外,TNF-α诱导处理促使线粒体途径信号分子Bad磷酸化及Bcl-xl表达水平适当升高,但在两种细胞表型间无显著差异.这些结果证实,Nmp4基因敲除可促进相关抗凋亡信号分子的激活和表达,抑制促凋亡信号的激活,进而抑制成骨细胞凋亡的发生.     

热激蛋白70研究进展

热激蛋白70（HSP70）是广泛存在且高度保守的蛋白,作为伴侣分子能够促进蛋白折叠;HSP70可以通过阻止细胞色素c从线粒体释放,与凋亡诱导因子结合使其不能入核,或者抑制JNK激酶活性调节细胞凋亡;HSP70可以调节细胞周期进程,促进细胞生长,阻止细胞衰老;免疫功能研究表明HSP70是有效的免疫佐剂,可激发抗原特异性的CTL反应,同时细胞外HSP70和膜结合HSP70可激发非特异性免疫反应。     

TNF-α通过激活蛋白磷酸酶4调节HepG2细胞中JNK磷酸化

目的 探讨蛋白磷酸酶4（protein phosphatase 4,PP4）在肿瘤坏死因子（tumor necrosis factorα,TNF-α）诱导JNK磷酸化中的作用.方法 TNF-α处理人肝癌细胞HepG2,免疫印迹检测JNK磷酸化水平和PP4表达水平,免疫沉淀结合磷酸酶活性测定法分析PP4活性变化.构建PP4磷酸酶活性缺失突变体 PP4-RL,转染HepG2细胞,经过G418筛选获得稳定表达FLAG-PP4-RL的克隆,进一步观察PP4对TNF-α诱导的JNK磷酸化的影响.结果 TNF-α处理后迅速引起HepG2细胞JNK的磷酸化,在20 min时达到峰值.PP4的表达在TNF-α处理后60 min内没有显著变化,但活性迅速增强,在20 min时达到峰值.在稳定表达FLAG-PP4-RL的HepG2细胞中,TNF-α诱导的JNK磷酸化被PP4-RL阻断.结论 TNF-α通过激活蛋白磷酸酶4调节HepG2细胞中JNK磷酸化.     

Phosphorylation of Bcl-associated death protein (Bad) by erythropoietin-activated c-Jun N-terminal protein kinase 1 contributes to survival of erythropoietin-dependent cells

The glycoprotein erythropoietin (Epo) is a hematopoietic cytokine necessary for the survival of erythrocytes from immature erythroid cells. The mitogen-activated c-Jun N-terminal kinase 1 (JNK1) plays an important role in the proliferation and survival of erythroid cells in response to Epo. However, the precise mechanism of JNK1 activation promoting erythroid cell survival is incompletely understood. Here, we reported that JNK1 is required for Epo-mediated cell survival through phosphorylation and inactivation of the pro-apoptotic, Bcl-2 homology domain 3 (BH3)-only Bcl-associated death protein (Bad). Upon Epo withdrawal, HCD57 cells, a murine Epo-dependent cell line, displayed increased apoptotic cell death that was associated with decreased JNK1 activity. Epo withdrawal-induced apoptosis was promoted by inhibition of JNK1 activity but suppressed by expression of a constitutively active JNK1. Furthermore, Epo-activated JNK1 phosphorylated Bad at threonine 201, thereby inhibiting the association of Bad with the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-X(L)). Replacement of threonine 201 by alanine in Bad promoted Epo withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK1 contributes to the survival of erythroid cells.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

Role of JNK activation in apoptosis： A double-edged sword

JNK is a key regulator of many cellular events, including programmed cell death (apoptosis). In the absence of NF-κB activation, prolonged JNK activation contributes to TNF-α induced apoptosis. JNK is also essential for UV induced apoptosis. However, recent studies reveal that JNK can suppress apoptosis in IL-3-dependent hematopoietic cells via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Thus, JNK has pro- or antiapoptotic functions, depending on cell type, nature of the death stimulus, duration of its activation and the activity of other signaling pathways.     

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1)

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.     

Regulation of c-Myc through phosphorylation at Ser-62 and Ser-71 by c-Jun N-terminal kinase.

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.     

Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis

Phosphorylation of BCL-2 family member BAD at different residues triggers different physiological effects, either inhibiting or promoting apoptosis. The recently identified phosphorylation site at Ser-128 enhances the apoptotic activity of BAD. We here show that BAD becomes phosphorylated at Ser-128 in the mitotic phase of the cell cycle in NIH3T3 cells. We also show that BAD-S128 is phosphorylated in taxol-treated mouse fibroblasts and MDA-MB-231 human breast cancer cells. However, expression of a phosphorylation-defective dominant negative BAD mutant did not block taxol-induced apoptosis. These data support the view that the phosphorylation of BAD Serine 128 exerts cell-specific effects on apoptosis. Whereas the BAD Serine 128 phosphorylation induces apoptosis in neuronal cells, it does not appear to promote apoptosis in proliferating non-neural cells during mitosis or upon exposure to the antineoplastic agent taxol.     

JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD

JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending on the cell type and stimulus used. The precise mechanism of JNK action, under conditions when it promotes cell survival, is not entirely clear. Here, we report that JNK is required for IL-3-mediated cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family protein BAD. IL-3 withdrawal-induced apoptosis is promoted by inhibition of JNK but suppressed by expression of a constitutively active JNK. JNK phosphorylates BAD at threonine 201, thereby inhibiting BAD association with the antiapoptotic molecule BCL-X(L). IL-3 induces BAD phosphorylation at threonine 201, and replacement of threonine 201 by alanine generates a BAD mutant, which promotes IL-3 withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK contributes to cell survival.