氧化型低密度脂蛋白通过Akt/mTOR/p70S6K通路诱导血管内皮细胞自噬

目的 探讨氧化型低密度脂蛋白(oxidized low-density lipoprotein, oxLDL)对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)自噬和Akt/mTOR/p70S6K信号通路的影响.方法 将培养的HUVECs分为oxLDL组和对照组,分别用100 μg/ml oxLDL和等体积磷酸盐缓冲液处理.在培养6 h和12 h后收集细胞.应用透射电子显微镜观察自噬小体,实时荧光定量聚合酶链反应检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3)和p62 mRNA表达,应用蛋白质印迹法检测LC3、p62、p-Akt/Akt、p-mTOR/mTOR和p-p70S6K/p70S6K蛋白表达.结果 与对照组相比,oxLDL处理组细胞内的自噬小体数量明显增多(P<0.05),LC3 mRNA及蛋白表达水平均显著增高(P均<0.05),而p62 mRNA和蛋白表达水平均显著降低(P均<0.05).此外,oxLDL处理组Akt、mTOR、p70S6K磷酸化蛋白表达水平均较对照组显著降低(P均<0.01),但Akt、mTOR和p70S6K总蛋白表达水平与对照组无显著差异.结论 oxLDL可通过抑制Akt/mTOR/p70S6K信号通路诱导HUVECs自噬.     

肾癌组织中mTOR、P70S6K蛋白的表达变化及意义

目的：观察肾癌组织中哺乳动物雷帕霉素靶蛋白（ mTOR）及其底物激酶核糖体蛋白S6激酶（ P70S6K）的蛋白表达水平,并探讨其意义。方法采用免疫组化（ S-P法）及Western blotting法检测肾癌组织、正常肾脏组织中mTOR及P70S6K蛋白表达,并观察两者表达与肾癌临床病理参数的关系。结果 mTOR蛋白在肾癌、正常肾组织中的表达水平分别为1．223±0．125、0．667±0．106,两者比较,P＜0．01;P70S6K在肾癌、正常肾组织的表达水平分别为1．108±0．258、0．489±0．21,两者比较,P＜0．01。 mTOR、P70S6K蛋白阳性表达与肾癌的组织分化程度、淋巴结转移和临床分期有关（P均＜0．05）。肾癌组织中mTOR与P70S6K蛋白表达呈正相关（r＝0．478,P＝0．002）。结论肾癌组织中mTOR、P70S6K蛋白表达升高,mTOR/P70S6K信号通路可能在肾癌的发生、发展中起重要作用。     

mTOR/P70S6K信号通路在肝细胞肝癌中的表达及临床意义

目的:研究mTOR/P70S6K信号通路在肝细胞肝癌(HCC)中的表达,探讨其在HCC发生发展中的作用及意义.方法:用逆转录聚合酶链反应(RT-PCR)技术检测120例HCC患者癌组织、癌旁肝组织以及10例正常肝组织中mTOR及P70S6K mRNA表达情况;并分析mTOR及P70S6K mRNA的表达与相关临床参数的关系.结果:mTOR及P70S6K mRNA在HCC组织中的表达水平显著高于在癌旁肝组织和正常肝组织中的表达水平(mTOR mRNA:0.594±0.218 vs 0.437±0.156.0.594±0.218 vs 0.383±0.081,均P＜0.05;P70S6K mRNA:0.610±0.147 vs 0.486±0.162.0.610±0.147 vs 0.440±0.141,均P＜0.05).mTOR mRNA和P70S6KmRNA在HCC组织中的表达呈正相关(r=0.548,P=0.012),且两者在癌旁肝组织及正常肝组织中的表达亦正相关性(r=0.607,0.737,P=0.005,0.015).mTOR及P70S6K mRNA在HCC组织中的表达水平与病理分期、门静脉癌栓等明显相关,而与肿瘤直径、血清AFP水平、性别等无明显关系.结论:mTOR/P70S6K信号通路在HCC中特异性激活.mTOR/P70S6K信号通路可能在肝细胞肝癌的发生、发展中起重要作用.     

良恶性子宫内膜病变组织中p-mTOR及p-P70S6K的表达及意义

目的 探讨磷酸化雷帕霉素靶蛋白（p-roTOR）及磷酸化40S核糖体蛋白6s激酶（P-P70S6K）在良恶性子宫内膜病变组织中的表达情况。方法选取49例子宫内膜腺癌组织（A组）、11例子宫内膜不典型增生组织（B组）、11例子宫内膜单纯性增生组织（C组）、10例子宫内膜增生期组织（D组）。用免疫组化sP法染色及显微图像分析系统检测四组中的P．mTOR、P—P70S6K。结果A组P．mTOR、P．P70S6K的表达强度均明显高于B、C、D组（P均〈0．05）。子宫内膜腺癌病理分期不同,其p-mTOR、P-P70S6K表达亦不同（P〈0．05）。结论p-mTOR、P—P70s6K可能参与了子宫内膜腺癌发生发展过程。     

肾透明细胞癌组织中AKT／mTOR的表达变化及意义

目的观察磷酸化蛋白激酶B（p-AKT）和磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）因子在肾透明细胞癌组织中的表达变化,为mTOR及AKT抑制剂应用于肾透明细胞癌的治疗提供依据。方法用免疫组化染色测定肾透明细胞癌和正常肾组织中p-AKT和p-mTOR的表达情况。结果p-AKT在肾透明细胞癌和正常肾组织中的阳性表达率分别为61．3％、26．1％（P〈0．01）,p-mTOR的阳性表达率分别为56．0％、28．3％（P〈0．05）。P—AKT表达与肿瘤临床分期明显相关,临床分期越高,其阳性表达率越高（P〈0．05）。p-mTOR蛋白表达与肿瘤临床分期也具有相关性（P〈0．05）。肾透明细胞癌组织中p-AKT与p-mTOR的表达呈正相关（r=0．953,P=0．000）。结论AKT／mTOR信号通路在肾透明细胞癌组织中被过度激活。理论上,应用mTOR及AKT抑制剂可治疗肾透明细胞癌。     

抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制

目的 探讨抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬中的影响。方法 分离培养纯种新西兰兔腹腔原代巨噬细胞并分为4组,加入磷脂酰肌醇3激酶（PI3K）抑制剂LY294002（10 μmol/L）组、哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂雷帕霉素（10 nmol/L）组、蛋白激酶B（Akt）抑制剂曲西立滨组（20 μmol/L）以及空白对照组。共培养4 h、12 h后分别收集细胞,运用透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法检测微管相关蛋白轻链3Ⅱ（LC3Ⅱ）分子的表达,Western blot检测 Akt、mTOR、磷酸化Akt（p-Akt）、磷酸化mTOR（p-mTOR）及自噬相关蛋白Beclin-1和自噬蛋白Atg5-Atg12连接体的表达,单丹酰尸胺（MDC）染色法观察自噬溶酶体的变化。结果 与空白对照组相比,透射电镜下LY294002组自噬体、自噬空泡、髓磷脂图像等自噬标记物明显减少,雷帕霉素组、曲西立滨组明显增多;激光共聚焦显微镜下LY294002组LC3Ⅱ表达显著减少,雷帕霉素组、曲西立滨组表达显著增多;Western blot结果显示LY294002组Beclin-1及Atg5-Atg12蛋白表达水平显著下降,p-mTOR、p-Akt蛋白表达显著减少;雷帕霉素组、曲西立滨组Beclin-1及Atg5-Atg12蛋白表达水平明显上调,共培养4 h后p-Akt表达增多,雷帕霉素组p-mTOR表达增多,曲西立滨组减少;共培养12 h后雷帕霉素组、曲西立滨组p-mTOR表达显著减少,雷帕霉素组p-Akt表达显著增多,曲西立滨组显著减少;MDC染色显示LY294002组自噬溶酶体明显减少,雷帕霉素组、曲西立滨组明显增多。结论 抑制PI3K/Akt/mTOR信号通路能促进兔原代巨噬细胞自体吞噬,抑制PI3K能减少兔原代巨噬细胞自体吞噬,可能是不同类型的PI3K分子通过其他通路起作用。     

Akt和磷酸化Akt1在胰腺癌组织中的表达及意义

目的 了解Akt和磷酸化Akt1(p-Akt1)蛋白在胰腺癌组织中的表达情况,探讨其临床意义.方法 应用免疫组织化学法检测74例胰腺癌组织、10例正常胰腺组织中Akt和p-Akt1蛋白的表达,并分析其与临床病理学特征及预后的关系.结果 Akt和p-Akt1蛋白在胰腺癌组织中的阳性表达率分别为87.8%和83.8%,而正常胰腺组织均阴性表达,相差非常显著(P<0.05).癌组织中Akt和p-Akt1蛋白的表达呈正相关性(r=0.274,P=0.018).Akt和p-Akt1蛋白表达与年龄、性别、肿瘤生长部位、肿瘤大小、病理学分级、淋巴结转移、临床分期及神经浸润均无显著相关性(P>0.05),但p-Akt1蛋白高表达与病理学T分期及TNM分期相关(P=0.002).Akt和p-Akt1蛋白高表达者的中位生存期分别为(16.0±5.7)个月和(23.0±5.5)个月,明显高于低表达者的(9.3±0.2)个月和(11.1±1.8)个月(P分别为0.007和0.004).结论 胰腺癌组织中p-Akt1蛋白表达的程度与良性预后有关,检测胰腺癌中p-Akt1蛋白的表达具有一定的临床意义.     

中药肝复康对肝星状细胞mTOR/P70S6K信号通路的干预作用

目的观察中药肝复康含药血清对乙醛刺激的肝星状细胞mTOR/P70S6K信号通路的影响,以探讨肝复康抗纤维化的分子生物学机制。方法体外培养大鼠HSC-T6细胞,用肝复康对乙醛刺激的HSCs进行干预,采用RT-PCR法测定HSCs中Ⅰ、Ⅲ型胶原、mTOR、P70S6K的表达。结果空白对照组肝星状细胞各指标表达量呈相对较低水平,乙醛刺激组细胞Ⅰ型胶原、mTOR、P70S6K和Ⅲ型胶原表达量明显升高(P<0.05),而肝复康干预对于细胞Ⅰ型胶原、mTOR和P70S6K的升高有明显的抑制作用(P<0.01)。结论 mTOR/P70S6K信号通路在HSCs活化过程中起到重要作用,肝复康发挥抗纤维化的作用机制与阻断mTOR/P70S6K信号通路有关。     

运动干预对大鼠骨骼肌mTOR与P70^S6K磷酸化的影响

运动干预增加大鼠骨骼肌核糖体蛋白S6激酶(P70S6K)及哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平,改善P70S6KmRNA表达,降低血液胰岛素浓度(P均<0.05).提示运动干预可能通过增加mTOR与P70S6K活性,提高骨骼肌对胰岛素的敏感性.     

叉头转录因子O1在冬虫夏草预防糖尿病模型大鼠对比剂肾病中的机制

目的探讨叉头转录因子(Fox)O1在冬虫夏草预防糖尿病模型大鼠对比剂肾病中的机制。方法 SD大鼠80只,随机选取20只为正常对照组(N组),其余60只腹腔注射链脲佐菌素(60 mg/kg)建立糖尿病模型,造模成功后再随机分为对比剂肾病组(M组)、冬虫夏草组(D组)、普罗布考组(P组)。10 w后,N组和M组给予生理盐水灌胃,D组和P组分别给予冬虫夏草(3.0 g/kg)和普罗布考(500 mg/kg)灌胃。7 d后,麻醉状态下,除N组外,其他三组给予碘克沙醇320(2.0 g/kg)。取血标本测血清肌酐(Scr)、尿素氮(BUN),取尿标本测尿中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、肾损伤因子(KIM)-1、白细胞介素(IL)-18;肾脏标本用于肾脏病理研究、氧化应激指标检测、蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)/P70核糖体蛋白S6激酶(P70S6K)通路及FoxO1的研究。结果与M组比较,D组Scr、BUN、NAGL、KIM-1、IL-18水平均明显降低(P<0.05),P组BUN、Scr、NAGL、IL-18含量明显降低(P<0.05),而KIM-1无明显变化(P>0.05)。与M组比较,D组丙二醛(MDA)含量明显降低,而一氧化氮(NO)、一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)和总抗氧化能力(T-AOC)含量明显升高;P组MDA含量亦明显降低,NO、NOS和SOD含量明显升高(P<0.05),T-AOC无明显变化。与M组比较,D组和P组磷酸化(p-Akt)、p-mTOR、p-P70S6K mRNA及蛋白表达水平均明显升高,p-FoxO1 mRNA水平和蛋白表达明显降低,p-FoxO1/FoxO1比值显著降低。结论冬虫夏草可通过上调信号通路Akt/mTOR/P70S6K蛋白表达水平,进而促使FoxO1高表达,减少其磷酸化水平,减轻氧化应激和肾细胞凋亡,对糖尿病对比剂肾病大鼠的肾脏具有较好的保护作用。     

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN^+/− mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.     

Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.     

Follicle-stimulating hormone increases tuberin phosphorylation and mammalian target of rapamycin signaling through an extracellular signal-regulated kinase-dependent pathway in rat granulosa cells

FSH-mediated regulation of mammalian target of rapamycin (mTOR) signaling in proliferating granulosa cells and the effect of dihydrotestosterone (DHT) on this pathway were examined. Inhibiting mTOR activation using rapamycin significantly reduced the FSH-mediated increase in cyclin D2 mRNA expression, suggesting that mTOR plays a role in the FSH-mediated increase in granulosa cell proliferation. FSH treatment of granulosa cells showed a 2-fold increase in phosphorylation of p70S6 kinase (p70S6K), the downstream target of mTOR. The increase in p70S6K phosphorylation by FSH treatment was abolished by prior exposure to DHT, suggesting that DHT inhibits FSH-mediated activation of mTOR signaling in cultured granulosa cells. The effect of FSH and DHT treatment on tuberin (TSC2), the upstream regulator of mTOR, was then examined. FSH treatment increased TSC2 phosphorylation, and pretreatment with DHT for 24 h reduced this stimulation. These results indicate that reduced p70S6K phosphorylation observed in DHT-treated cells might be the result of reduced TSC2 phosphorylation. Because Akt is the upstream activator of TSC2 phosphorylation, the effect of Akt inhibition was examined to test whether FSH-mediated TSC2 phosphorylation proceeds through an Akt-dependent pathway. Our results show that inhibiting Akt phosphorylation did not block FSH-stimulated TSC2 phosphorylation, whereas ERK inhibition reduced FSH-mediated stimulation. These results demonstrate the involvement of ERK rather than Akt in FSH-mediated TSC2 phosphorylation in granulosa cells. Based on these observations, we conclude that in granulosa cells, FSH uses a protein kinase A-/ERK-dependent pathway to stimulate TSC2 phosphorylation and mTOR signaling, and DHT treatment significantly reduces this response.     

Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance

The mammalian target of rapamycin (mTOR) pathway integrates insulin and nutrient signaling in numerous cell types. Recent studies also suggest that this pathway negatively modulates insulin signaling to phosphatidylinositol 3-kinase/Akt in adipose and muscle cells. However, it is still unclear whether activation of the mTOR pathway is increased in obesity and if it could be involved in the promotion of insulin resistance. In this paper we show that basal (fasting state) activation of mTOR and its downstream target S6K1 is markedly elevated in liver and skeletal muscle of obese rats fed a high fat diet compared with chow-fed, lean controls. Time-course studies also revealed that mTOR and S6K1 activation by insulin was accelerated in tissues of obese rats, in association with increased inhibitory phosphorylation of insulin receptor substrate-1 (IRS-1) on Ser636/Ser639 and impaired Akt activation. The relationship between mTOR/S6K1 overactivation and impaired insulin signaling to Akt was also examined in hepatic cells in vitro. Insulin caused a time-dependent activation of mTOR and S6K1 in HepG2 cells. This was associated with increased IRS-1 phosphorylation on Ser636/Ser639. Inhibition of mTOR/S6K1 by rapamycin blunted insulin-induced Ser636/Ser639 phosphorylation of IRS-1, leading to a rapid (approximately 5 min) and persistent increase in IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation. These results show that activation of the mTOR pathway is increased in liver and muscle of high fat-fed obese rats. In vitro studies with rapamycin suggest that mTOR/S6K1 overactivation contributes to elevated serine phosphorylation of IRS-1, leading to impaired insulin signaling to Akt in liver and muscle of this dietary model of obesity.     

mTOR／S6K1信号通路在高脂饮食诱导小鼠胰岛素抵抗发生中的作用

目的探讨mTOR及其下游效应因子S6K1在胰岛素抵抗（IR）发生中的作用。方法C57BL／6雄性小鼠20只随机平均分为正常饮食组（NC组）和高脂饮食组（HF组）。后者喂养14周后建立IR模型。检测两组血清胰岛素水平和葡萄糖耐量,显微镜下观察胰岛形态学变化;检测骨骼肌中mTOR和S6K1mRNA的表达,以及mTOR、S6K1和其磷酸化水平蛋白的表达。结果与NC组相比,HF组小鼠表现出明显的IR症状,其体重和空腹胰岛素分别升高了21．99％（P〈0．05）和181．82％（P〈0．01）;胰岛8细胞团面积也显著增加,糖耐量明显受损,骨骼肌细胞中mTOR mRNA和蛋白分别升高了25．61％（P〈0．05）和37．41％（P〈0．01）,S6K1 mRNA和蛋白分别升高了54．98％（P〈0．01）和37．36％（P〈0．01）,S6K1磷酸化水平蛋白表达在高脂饮食后升高了680．15％（P〈0．01）。结论mTOR／S6K1信号通路与高脂饮食诱导IR的发生密切相关。     

Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.     

Immunohistochemical analysis of the mTOR pathway in intrahepatic cholangiocarcinoma

The aim of the study was to evaluate the expression of activated mammalian rapamycin (mTOR) and its downstream effectors, phosphorylated p70 ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1), in intrahepatic cholangiocarcinomas (ICC), in order to strengthen the rationale for targeted therapy using mTOR inhibitors in patients with ICC. p-mTOR (Ser 2448), p-4EBP1 (Thr 70) and p-p70S6K (Thr 389) were detected in 77 primary ICC tumors by immunohistochemistry. High levels of p-mTOR, p-4EBP1 and p-p70S6K expression were defined in 48.1% (37/77), 50.6% (39/77) and 51.9% (40/77) of all tumors, respectively. No significant correlation was observed between mTOR pathway proteins overexpression with clinicopathological characteristics and patient's prognosis, except that high p-p70S6K expression correlated with the poorly differentiated subtype, and high expression of p-4EBP1 predicted poor prognosis in ICC patients and retained an independent prognostic factor in multivariate analysis. In conclusion, our results showed high prevalence of activation of mTOR pathway in ICC tumors, suggesting that a high proportion of ICC patients might benefit from mTOR pathway targeted therapies. In addition, p-4EBP1 phosphorylation at Thr 70 could be a useful prognostic biomarker for ICC patients.     

PPIs抑制P13K/Akt／mTOR信号通路逆转胃癌细胞的化疗多药耐药

背景：前期实验显示质子泵抑制剂（PPIs）可抑制空泡型质子泵（V-H＋-ATPases）和多药耐药蛋白P—gP、MRP1表达,增强胃癌细胞的化疗敏感性。目的：探讨PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药与P13K／Akt／mTOR信号通路的关系。方法：应用不同浓度埃索美拉唑或泮托拉唑预处理人胃腺癌细胞敏感株SGC7901和多药耐药株SGC7901／MDR,或以V—H＋-ATPasessiRNA干扰SGC7901／MDR细胞内的V-H＋-ATPases表达,或以雷帕霉素阻断mTOR表达,以蛋白质印迹法检测经不同方式处理的细胞内V—H＋ATPases、P—SP、MRPl蛋白表达以及P13K／Akt/mT0R／HIF—1α信号通路及其信号旁路TSCl／2-Rheb中的相关蛋白表达;以免疫荧光法检测经埃索美拉唑预处理的SGC7901／MDR细胞内的V-H＋ATPases、P—gP蛋白表达和定位。结果：PPIs可呈浓度依赖性地抑制SGC7901／MDR细胞内的V—H＋ATPases、P13K、Akt、roTOR、HIF-1仅、TSCI、TSC2、Rheb、P—gP、MRP1表达以及Akt底物和TSC2磷酸化,改变V-H＋ATPases、P—gP的胞内定位,对SGC7901细胞则无上述影响。以V—H＋-ATPasessiRNA抑制SGC7901／MDR细胞内的V—H＋-ATPases表达,作用与PPIs预处理相似。以雷帕霉素阻断mTOR可呈浓度依赖性地抑制SGC7901／MDR细胞内的HIF-1α、P—gP表达。结论：PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药的机制与抑制P13K／Akt／mTOR信号通路有关。     

Growth factor-stimulated phosphorylation of Akt and p70(S6K) is differentially inhibited by LY294002 and Wortmannin

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.     

Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1

OBJECTIVE: Insulin-responsive adipogenic signaling molecules include insulin receptor substrates (IRS)-1 and -2, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB; also known as Akt). Mammalian target of rapamycin (mTOR) is a PKB substrate, and regulates p70 S6 kinase (p70 S6K). Since p70 S6K is an insulin-responsive kinase downstream of PI3K and PKB, its potential role in adipogenic insulin signaling was investigated. DESIGN: We measured the effect of rapamycin, a specific inhibitor of mTOR, on insulin-induced 3T3-L1 adipogenesis and on insulin-stimulated p70 S6K activation. RESULTS: Rapamycin partially reduced differentiation, measured by Oil Red O staining, triacylglycerol accumulation (by up to 46%), and peroxisome proliferator-activated receptor gamma protein expression (by 50%). In contrast, rapamycin completely inhibited insulin-stimulated p70 S6K activation, assessed by phosphorylation of p70 S6K and its substrate, S6. Expression of a constitutively activated form of p70 S6K did not promote 3T3-L1 adipogenesis. The considerable residual differentiation in the presence of rapamycin, despite the complete blockade of p70 S6K activation, prompted us to measure the phosphorylation of another rapamycin-sensitive protein, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Insulin-stimulated 4E-BP1 phosphorylation in 3T3-L1 preadipocytes was only partially affected by rapamycin, consistent with the differentiation data. Phosphorylation of eIF4E itself, an expected consequence of 4E-BP1 phosphorylation, was also only partially inhibited. CONCLUSION: Our data suggest that adipogenic mTOR signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70 S6K.