PRKAA1 Promotes Proliferation and Inhibits Apoptosis of Gastric Cancer Cells Through Activating JNK1 and Akt Pathways

PRKAA1 (protein kinase AMP-activated catalytic subunit 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC- 823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.     

Transforming growth factor-β during carcinogenesis: the shift from epithelial to mesenchymal signaling

Transforming growth factor-β (TGF-β) activates not only TGF-β type I receptor (TβRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While the TβRI/pSmad3C pathway inhibits growth of normal epithelial cells, JNK/pSmad3L-mediated signaling is involved in invasion by activated mesenchymal cells. During sporadic human colorectal carcinogenesis, TGF-β signaling confers a selective advantage on tumor cells by shifting from the TβRI/pSmad3C pathway characteristic of mature epithelial cells to the JNK/pSmad3L pathway, which is more characteristic of the state of flux shown by the activated mesenchymal cells. JNK acts as a regulator of TGF-β signaling by increasing the basal level of pSmad3L available for action in the nuclei of the invasive adenocarcinoma, in the meantime shutting down TGF-β-dependent nuclear activity of pSmad3C. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. From the viewpoint of TGF-β signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal colorectal epithelial cells at the expense of effects promoting aggressive behavior of the adenocarcinoma. Specific inhibitors of the JNK/pSmad3L pathway might prove useful in this respect. In the case of molecularly targeted therapy for human cancer, pSmad3L and pSmad3C could be assessed as biomarkers to evaluate the likely benefit from specific inhibition of the JNK/pSmad3L pathway.     

JNK信号通路对NaAsO2诱导NIH3T3细胞中hnRNP K蛋白聚集体形成的影响

 目的：观察核不均一核糖核蛋白(hnRNP)K的表达与定位以及亚砷酸钠（NaAsO2）刺激NIH3T3细胞的反应情况及氧化应激变化,探讨JNK信号通路在hnRNP K蛋白聚集体形成中的作用。方法：构建重组载体pcDNA3-hnRNP K-HA,转染NIH3T3细胞,荧光显微镜观察内源性hnRNP K在细胞中的表达与定位,以及在NaAsO2不同刺激时间该蛋白聚集体的形成情况,并利用活性氧（ROS）检测试剂盒测定胞内ROS水平;给予JNK、MEK、PI3K/Akt、NF-κB和核转运等5种信号通路的抑制剂预处理后,观察聚集体的变化情况。结果：重组质粒构建正确,荧光显微镜观察显示hnRNP K主要定位于胞核中,而重组质粒转染NIH3T3细胞后,可见胞内有蛋白聚集体形成并随NaAsO2刺激时间的延长而递增,且过表达hnRNP K可抑制NaAsO2刺激细胞生成ROS;JNK信号通路抑制剂SP600125明显抑制聚集体的形成。结论：hnRNP K主要定位在NIH3T3细胞的胞核中,胞浆少量分布;NaAsO2可诱导NIH3T3细胞形成hnRNP K蛋白聚集体,此聚集体可抑制胞内ROS生成,且该聚集体的形成有赖于JNK介导的信号通路。     

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PP...     

尿酸诱导树突状细胞成熟的信号转导机制研究

目的: 观察树突状细胞(DCs)成熟过程中MAPKs和NF-κB信号分子表达情况,探讨尿酸刺激DCs成熟的分子机制。方法: 用尿酸体外刺激大鼠未成熟 DCs,在不同时点(0~45 min),免疫印迹方法检测p- p38、p-ERK1/2、p-JNK和NF-κB p65表达情况;以MAPKs和NF-κB信号分子抑制剂分别联合尿酸刺激DCs 48 h后,用流式细胞术检测表面分子CD83、CD86、IA/IE的表达;ELISA法测定IL-12 p70的水平。结果: (1)尿酸刺激后15 min,DCs p- p38、p-ERK1/2、p-JNK和NF-κB p65表达量明显增加,30 min时达到最大值;使用相应信号分子抑制剂后,相关蛋白表达均不能测出。(2)经p38、JNK、NF-κB p65等信号通路抑制剂预处理后,与单独尿酸刺激相比,DCs CD83、CD86、IA/IE等表面分子表达及IL-12 p70分泌水平均出现下降(P＜0.05或P<0.01),ERK1/2抑制剂预处理者,则出现表达上升(P＜0.05)。结论: 尿酸可以调节DCs p38、ERK1/2、JNK、 NF-κB等信号分子的活化,从而促进DCs表面分子表达及IL-12 p70分泌。这可能为尿酸能诱导DCs成熟及提高免疫功能的分子机制之一。     

补青颗粒联合石决明水提液对白内障大鼠眼组织细胞中TFAR19和JNK3蛋白表达的影响

目的探讨补青颗粒联合石决明水提液对白内障大鼠眼组织细胞中TF-1细胞凋亡相关基因19(TFAR19)和c-Jun氨基末端激酶(JNK3)蛋白表达的影响。方法选择无特定病原体(SPF)级SD大鼠48只,雌雄各半,根据随机数字表法分为4组:正常组、白内障模型组、阳性对照组和实验组,每组各12只。正常组采用生理盐水滴眼,其余3组均通过亚硒酸钠诱导白内障模型,模型建立后白内障模型组采用生理盐水滴眼,阳性对照组采用莎普爱思滴眼和实验组采用0.25%石决明水提液滴眼+2 g/mL补青颗粒水溶液灌胃,灌胃给药每日2次,滴眼每日1次,每次1滴,持续干预30 d。分别于干预3、10、20、30 d后,蛋白质印迹法检测各组大鼠晶状体组织中TFAR19、JNK3以及pJNK3的表达,检测各组大鼠血清氧化应激指标丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、总超氧化物歧化酶(T-SOD)含量。结果干预3、10、20、30 d后,阳性治疗组和实验组晶状体组织中TFAR19相对表达量和p-JNK3/JNK3比值均随治疗时间延长而显著降低(P<0.05)。干预20~30 d后,4组大鼠晶状体组织中TFAR19相对表达量为:正常组<实验组<阳性对照组<白内障模型组,且组间两两比较,差异有统计学意义(P<0.05)。干预30 d后,4组大鼠视网膜组织中p-JNK3/JNK3水平比较:正常组<实验组<阳性对照组<白内障模型组,且组间两两比较差异有统计学意义(P<0.05)。各组大鼠血清中MDA水平比较:正常组<实验组<阳性对照组<白内障模型组,GSH-Px和T-SOD则为:白内障模型组<阳性对照组<实验组<正常组,且组间两两比较差异均有统计学意义(P<0.05)。结论补青颗粒联合石决明水提液可通过抑制大鼠晶状体TFAR19蛋白表达和视网膜JNK3磷酸化,并进一步调节氧化应激反应水平,从而对白内障大鼠发挥治疗作用,且在本研究时间范围内其作用效果随时间增加而增强。     

理中汤合四神丸对脾肾阳虚型溃疡性结肠炎大鼠JNK/Beclin 1/Bcl-2信号通路相关蛋白及基因表达的影响

目的观察以温补脾肾法为指导的理中汤合四神丸对脾肾阳虚型溃疡性结肠炎(UC)大鼠的影响,探讨其可能的作用机制。方法制备脾肾阳型UC大鼠模型,将成模大鼠随机分为模型组、柳氮磺吡啶组(SASP组)、肠胃宁组和理中汤合四神丸低、中、高剂量组,每组16只,同时设空白组16只。各给药组予相应药物灌胃,空白组和模型组予等体积蒸馏水灌胃,连续21 d。观察大鼠结肠黏膜组织损伤情况,对结肠黏膜损伤指数(CMDI)进行评分;HE染色观察大鼠结肠组织病理变化;免疫组化和RT-qPCR分别检测大鼠结肠组织JNK1、Beclin 1、Bcl-2、LC3B蛋白和m RNA表达;Western blot检测大鼠结肠组织JNK1、p-JNK1、Bcl-2、p-Bcl-2、Beclin 1和LC3B蛋白表达,并计算LC3BⅡ/LC3BⅠ比值。结果与空白组比较,模型组大鼠CMDI评分显著升高(P<0.01),结肠组织腺体排列紊乱,黏膜层消失,有大量炎性细胞浸润,黏膜基层与基底层分界不清;结肠组织JNK1、Beclin 1、Bcl-2、LC3B mRNA和蛋白表达显著升高(P<0.01),p-JNK1、p-Bc...     

The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells

The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.     

SP600125对大鼠脑缺血再灌注神经元的保护作用

目的探讨SP600125-JNK特异性抑制剂对大鼠脑缺血再灌注神经元损伤的保护性作用及其作用机制。方法雄性SD大鼠54只,体重230～250g,随机分成假手术组（SH组）,缺血再灌注组（IR组）和JNK抑制剂SP600125组（SP组）,每组根据再灌注时间分为30min、24h和72h3个亚组,每亚组6只动物。采用4-VO法建立SD大鼠脑缺血模型,三组于缺血前30min侧脑室注射DMSO,DMSO及JNK抑制剂SP600125（溶媒采用DMSO）,容积均为10μl;脑缺血再灌注后30min、24h、72h免疫组织化学方法测定各时间点海马CA1区Bcl-2和Bax蛋白表达阳性细胞数量,TUNEL法检测CA1区凋亡细胞。结果缺血再灌注使海马CA1区Bcl-2和Bax阳性锥体细胞数目表达增加,再灌注24h阳性锥体细胞数目表达至高峰（P〈0.01）,再灌注24～72h可见阳性锥体细胞数目表达减少（P〈0.05）。其中SP组Bcl-2阳性锥体细胞数目显著多于IR组（P〈0.05）,而Bax阳性锥体细胞数目显著小于IR组（P〈0.05）。脑缺血再灌注后海马CA1区神经元存活数目SP组高于IR组（P〈0.01）,凋亡细胞数目低于IR组（P〈0.01）。结论 SP600125对大鼠脑缺血再灌注神经元损伤具有保护作用。