分枝菌酸诱导巨噬细胞泡沫细胞化对细胞自噬的影响

研究分枝菌酸(mycolic acid,MA)诱导的巨噬细胞泡沫细胞化对巨噬细胞自噬的影响,探讨MA促巨噬细胞泡沫细胞化的机制。构建LC3真核表达质粒(p EGFP-LC3B),转染RAW264.7细胞后获得其稳定转染细胞株(RAW264.7/p EGFP-LC3B);用MA诱导RAW264.7/p EGFP-LC3B获得RAW264.7/p EGFP-LC3B泡沫细胞。实验分为三组:RAW264.7细胞组、RAW264.7/p EGFP-LC3B细胞组及MA诱导的RAW264.7/p EGFP-LC3B泡沫细胞组。通过RT-PCR法检测各组细胞中自噬相关基因Becn1、LC3B的转录水平,Western blot法检测各组细胞自噬标志蛋白LC3B II/I的表达水平。RTPCR检测结果显示,RAW264.7/p EGFP-LC3B泡沫细胞组的Becn1基因及LC3B基因的转录水平明显较其他两组低(P0.05)。Western blot检测结果显示,RAW264.7/p EGFP-LC3B泡沫细胞组LC3B II/I比值明显较其他两组低(P0.05)。由此可见,当巨噬细胞被MA诱导成为泡沫巨噬细胞后,其自噬功能显著降低。该研究证明,MA可能通过抑制巨噬细胞自噬,引起脂代谢失衡,进而发生巨噬细胞泡沫细胞化。     

细叶远志皂苷在Aβ23-35诱导SH-SY5Y 细胞氧化损伤中的作用及机制研究

为研究细叶远志皂苷(tenuifolin,TEN)在Aβ25-35诱导SH-SY5Y细胞氧化损伤中的作用,并探讨其作用机制。建立Aβ25-35诱导的细胞损伤模型,细叶远志皂苷以及自噬抑制剂3-MA进行干预,显微镜观察细胞形态变化,试剂盒检测细胞氧化应激水平,RT-qPCR和Westernblot检测细叶远志皂苷以及自噬抑制剂干预前后Beclin-1、LC3、mTOR、AMPK和ULK1mRNA及蛋白水平变化。结果发现,TEN改善Aβ25-35诱导的SH-SY5Y细胞形态损伤和细胞活力下降;降低ROS和MDA浓度,并提高SOD、GSH-Px及过氧化氢酶的活性;增加AMPK和ULK1的表达,减少mTOR的表达及增加Beclin-1和LC3-Ⅱ/Ⅰ的表达水平。而加入3-MA会拮抗TEN的作用。总之,TEN可能通过调控AMPK/mTOR/ULK1通路,增加Beclin-1及LC3-Ⅱ/Ⅰ蛋白水平激活自噬,进而改善Aβ25-35诱导的细胞形态损伤和细胞活力下降,提高细胞抗氧化应激能力,发挥神经保护作用。     

基于PI3K/AKT/mTOR通路的三百棒促进脂多糖诱导的RAW 264.7细胞自噬并抑制炎症CSCD

三百棒来源于芸香科植物飞龙掌血Toddalia asiatica(L.)Lam的根,是一种天然土家族中草药,具有抗炎、抗风湿、抗肿瘤、抗微生物等药理活性。其毒副作用小,疗效显著的特点使之成为当前研究热点。许多天然产物已被证明可通过靶向PI3K/AKT/mTOR介导的自噬来抑制炎症及自身免疫性疾病,本项研究通过调节PI3K/AKT/mTOR信号通路来研究三百棒醇提物(Toddalia asiatica alcohol extract,TAAE)对自噬的影响,用脂多糖(LPS)诱导单核巨噬细胞(RAW 264.7)建立炎症模型,通过细胞毒性检测试剂盒检测TAAE对细胞活力的影响,并筛选出药物的浓度及干预时间,透射电镜和单丹磺酰尸胺染色检测巨噬细胞的生物学功能,酶联免疫吸附法检测上清液中相关炎症因子水平,Western blot检测自噬和通路相关蛋白的表达水平;并采用自噬早期抑制剂(3-MA)和通路PI3K激动剂(740Y-P)进一步验证自噬对炎症和信号通路的影响。实验结果表明TAAE可能通过抑制PI3K/AKT/mTOR信号通路,促进自噬泡的形成、自噬体溶酶体融合和降解,降低LPS处理的RAW 264.7细胞中炎性细胞因子的表达和分泌。总体而言,本研究结果为三百棒的抗炎机制的研究提供了新的线索,并为临床更好的应用三百棒治疗炎症性疾病提供理论依据。     

克柔假丝酵母菌通过Dectin-1/Syk信号通路活化巨噬细胞自噬

目的探究Dectin-1/Syk信号通路在克柔假丝酵母菌激活RAW264.7细胞自噬中的作用。方法以特异性抗体封闭RAW264.7细胞表面TLR-2、TLR-4及Dectin-1受体,免疫蛋白印记检测克柔假丝酵母菌刺激后LC3II的表达量;通过白皮杉醇及Raf-1抑制剂分别阻断RAW264.7细胞Syk及Raf-1磷酸化,观察对克柔假丝酵母菌激活细胞自噬的影响;采用SiMi Transfection Reagents转染Atg5siRNA,检测不同时间段RAW264.7细胞对克柔假丝酵母菌的杀菌率。结果封闭细胞膜Dectin-1、阻断Syk磷酸化显著抑制克柔假丝酵母菌诱导RAW264.7细胞LC3II的表达,而封闭细胞膜TLR-2或TLR-4,以及阻断Raf-1磷酸化对于克柔假丝酵母菌刺激下LC3II的表达无显著影响。敲低Atg5后RAW264.7细胞在感染6h后对克柔假丝酵母菌的杀菌率显著降低。结论 Dectin-1/Syk信号通路介导了克柔假丝酵母菌激活RAW264.7细胞自噬,并且自噬功能参与了该细胞对克柔假丝酵母菌的杀灭作用。     

HIF-1α/BNIP3信号通路对BCG诱导巨噬细胞自噬的影响

【背景】缺氧诱导因子1α(hypoxia-inducible factor 1-alpha,HIF-1α)是响应细胞低氧反应的关键因子,在红细胞生成、血管形成、能量代谢及调节宿主免疫代谢中发挥着重要作用。【目的】探讨HIF-1α/Bcl-2-腺病毒E1B相互作用蛋白3(Bcl-2-adenovirus E1B 19-kDa interacting protein 3,BNIP3)信号通路对牛分枝杆菌卡介苗(Bacillus Calmette-Guérin,BCG)诱导小鼠巨噬细胞RAW 264.7自噬的影响。【方法】构建HIF-1α的小干扰RNA (siHIF-1α),转染RAW 264.7细胞后,结合BCG感染,采用流式细胞仪检测细胞自噬率,用Western blotting或免疫荧光技术检测HIF-1α、BNIP3、LC3、Beclin 1、Rheb和mTOR的表达水平。【结果】BCG感染显著上调巨噬细胞中LC3和HIF-1α的表达,用siHIF-1α结合BCG感染后显著下调巨噬细胞中HIF-1α、BNIP3、LC3、Beclin 1和细胞自噬率水平,并促进Rheb和p-mTOR的表达。【结论】在BCG感染RAW 264.7细胞过程中,干扰HIF-1α表达抑制了HIF-1α/BNIP3信号通路,进而激活了mTOR途径,抑制BCG感染诱导的细胞自噬。     

红毛五加多糖单一组分AHP-Ⅲ对巨噬细胞的免疫调节作用及其机制

探讨红毛五加多糖(Acanthopanax giraldii Hams polysaccharide)单一组分AHP-Ⅲ(Acanthopanax giraldii Hams polysaccharideⅢ)对小鼠巨噬细胞RAW 264.7的激活作用及机制。不同浓度AHP-Ⅲ作用RAW 264.7细胞,中性红试验检测细胞吞噬能力;ELISA和Griess法检测其IL-6、TNF-α和NO的释放量;RT-qPCR检测iNOS、TNF-α和IL-6 mRNA相对表达水平;Western blot检测NF-κB信号通路相关蛋白磷酸化水平。在实验浓度范围内,AHP-Ⅲ可显著增强RAW 264.7细胞的吞噬能力(P<0.05);促进RAW 264.7分泌NO、TNF-α和IL-6(P<0.05或P<0.001);并显著增加RAW 264.7细胞中IL-6、TNF-α和iNOS mRNA的表达量,呈剂量依赖性;Western blot结果表明,AHP-Ⅲ作用RAW 264.7细胞后,NF-κB中的p65、IKKβ、IκBα磷酸化水平明显升高。结果显示红毛五加多糖AHP-Ⅲ对小鼠巨噬细胞RAW 264.7具有显著激活作用。     

阻断Kv1.3通道可增强RAW264.7巨噬细胞的吞噬功能

本研究旨在阐明电压门控性钾通道1.3(Kv1.3)在巨噬细胞吞噬功能中的作用.利用RAW264.7巨噬细胞吞噬鸡红细胞的半定量检测系统及吞噬异硫氰酸荧光素标记的大肠杆菌(E.coli)k-12的流式细胞术定量检测系统测定巨噬细胞的吞噬功能.研究发现,用海葵神经毒素(Sh K)(100 pmol/L)选择性阻断Kv1.3通道能显著增强处于静息状态的和被脂多糖(LPS)激活的RAW264.7巨噬细胞吞噬鸡红细胞的能力;Sh K也可增强静息RAW264.7细胞吞噬大肠杆菌的能力,但由于LPS刺激吞噬的效应近乎饱和,Sh K并不能进一步增加被LPS激活的RAW264.7细胞吞噬大肠杆菌的数量.Sh K促进LPS激活的RAW264.7细胞释放一氧化氮(NO),但并不增加静息RAW264.7细胞的NO释放.Sh K(100 pmol/L)自身并不影响静息RAW264.7细胞释放细胞因子,但能抑制LPS激活的RAW264.7细胞释放白细胞介素-1?.Sh K(100 pmol/L)对RAW264.7细胞的活力无明显影响.RAW264.7细胞表达Kv1.3通道蛋白;LPS使RAW264.7细胞的Kv1.3蛋白表达下调,菲律宾菌素Ⅲ(小凹蛋白依赖性内吞途径抑制剂)使Kv1.3蛋白表达上调,细胞松弛素D对Kv1.3蛋白表达无明显影响.研究表明,RAW264.7细胞表达Kv1.3蛋白;阻断Kv1.3通道可增强RAW264.7细胞的吞噬能力和NO生成.结果提示,Kv1.3通道可能是RAW264.7细胞吞噬活动的负调节因子,有可能成为治疗巨噬细胞吞噬功能异常相关疾病的一个靶点.     

不同形态的烟曲霉对巨噬细胞自噬水平的影响

目的探究烟曲霉静息孢子、膨胀孢子以及菌丝对巨噬细胞自噬水平的影响。方法培养烟曲霉并收获静息孢子,在沙保弱液体培养基中振荡不同时间获得膨胀孢子及菌丝。以3种形态的烟曲霉分组处理RAW264.7细胞,免疫印迹法检测LC3BⅡ蛋白的表达量,逆转录PCR检测自噬相关蛋白Atg5、Atg7、Atg12 mRNA的转录水平。观察不同形态的烟曲霉刺激GFP-LC3B-RAW264.7细胞后GFP-LC3B的表达与定位。结果膨胀的烟曲霉孢子及菌丝刺激巨噬细胞后LC3BⅡ表达水平升高;Atg5与Atg12 mRNA的转录均明显增高,Atg7转录水平无显著变化;膨胀孢子诱导LC3B呈斑点样聚集并与之共定位,烟曲霉菌丝刺激后自噬体增多。结论烟曲霉膨胀孢子与菌丝能显著提高巨噬细胞自噬水平,静息的分生孢子不能引起巨噬细胞自噬功能的应答。     

嗜肺军团菌重组免疫原蛋白(IP)对RAW264.7细胞自噬的影响

【目的】观察嗜肺军团菌重组免疫原蛋白(immunogenic protein,IP)对RAW264.7细胞自噬流及自噬相关因子表达水平的影响并探讨其作用机制。【方法】采用His标签蛋白纯化试剂盒纯化嗜肺军团菌重组免疫原蛋白(IP),并用CCK-8法检测IP对RAW264.7细胞半数抑制浓度(half maximal inhibitory concentration,IC₅₀)。采用低浓度(0.05×IC₅₀)、中浓度(0.1×IC₅₀)、高浓度(0.2×IC₅₀) IP与RAW264.7细胞进行体外共培养1、3、6、12 h,并设细胞对照组,应用自噬双标质粒pmCherry-C1-EGFP-LC3B检测巨噬细胞自噬流的变化,筛选最佳浓度进行后续实验;最佳浓度IP与RAW264.7细胞进行体外共培养6、12、24 h,并设细胞对照组,RT-qPCR检测各组自噬相关蛋白Beclin1、微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3,LC3)、SQSTM 1 (sequestosome 1,p62)及组蛋白去乙酰化酶6 (histone deacetylase 6,HDAC6)的mRNA表达水平;Western blotting法检测各组自噬相关因子的蛋白表达水平;免疫荧光检测各组自噬相关因子的表达。【结果】根据CCK-8结果计算IC₅₀为0.26μg/μL。自噬双标质粒pmCherry-C1-EGFP-LC3B检测自噬流结果显示,与对照组相比,中浓度(0.026μg/μL) IP与RAW264.7细胞进行体外共培养后自噬流抑制显著。RT-qPCR及Western blotting检测结果显示,与对照组相比,IP与RAW264.7细胞共培养6 h,P62表达水平升高,LC3B、HDAC6、Beclin1表达水平均降低,P<0.05;与6 h组相比,12 h组LC3B表达水平降低,P62、HDAC6及Beclin1表达水平均升高,P<0.05;与12 h组相比,24 h组Beclin1表达水平升高,P<0.05。IP一定程度上以时间依赖的方式降低了LC3-Ⅱ/LC3-Ⅰ比值并升高了P62蛋白的表达水平,P<0.05。免疫荧光结果基本与RT-qPCR及Western blotting检测结果一致。【结论】嗜肺军团菌IP抑制巨噬细胞自噬,可能与通过影响自噬小体-溶酶体融合途径,干扰自噬小体及自噬溶酶体的形成、成熟等过程有关。     

ESAT6通过mTOR抑制自噬并促进BCG增殖

目的研究mTOR在结核杆菌毒力因子ESAT6诱导的自噬抑制以及促进BCG增殖中的作用。方法 PCMV-HA-ESAT6质粒转染Raw264.7细胞,用蛋白免疫印迹检测LC3、P62、P-mTOR和P-70S6K表达水平;用mTOR阻断剂Torin1联合ESAT6转染以及分别作用于Raw264.7细胞后,免疫印迹检测P62和P-mTOR表达水平,LysoTracker Red染色观察溶酶体变化,BCG增殖实验计数各组菌落数。结果 ESAT6转染细胞后,细胞P62、P-mTOR和P-70S6K表达水平显著增高,LC3I完成向LC3II的转化;联合Torin1的ESAT6转染组和Torin1处理组的P-mTOR和P62无显著变化,溶酶体无变化,BCG菌落数减少。结论 ESAT6诱导的自噬抑制和BCG的增殖依赖于mTOR的活化。     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity

Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1_S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.     

The association of AMPK with ULK1 regulates autophagy

Autophagy is a highly orchestrated intracellular bulk degradation process that is activated by various environmental stresses. The serine/threonine kinase ULK1, like its yeast homologue Atg1, is a key initiator of autophagy that is negatively regulated by the mTOR kinase. However, the molecular mechanism that controls the inhibitory effect of mTOR on ULK1-mediated autophagy is not fully understood. Here we identified AMPK, a central energy sensor, as a new ULK1-binding partner. We found that AMPK binds to the PS domain of ULK1 and this interaction is required for ULK1-mediated autophagy. Interestingly, activation of AMPK by AICAR induces 14-3-3 binding to the AMPK-ULK1-mTORC1 complex, which coincides with raptor Ser792 phosphorylation and mTOR inactivation. Consistently, AICAR induces autophagy in TSC2-deficient cells expressing wild-type raptor but not the mutant raptor that lacks the AMPK phosphorylation sites (Ser722 and Ser792). Taken together, these results suggest that AMPK association with ULK1 plays an important role in autophagy induction, at least in part, by phosphorylation of raptor to lift the inhibitory effect of mTOR on the ULK1 autophagic complex.     

Cigarette smoke exposure induces ROS-mediated autophagy by regulating sestrin,AMPK, and mTOR level in mice

Many pathological conditions linked to cigarette smoking are caused by the production of reactive oxygen species (ROS). The present study was conducted to analyze the effect of ROS on the lungs of Swiss mice exposed to cigarette smoking, focusing on autophagy-mediated mechanisms, and investigate the involvement of SESN2, AMPK, and mTOR signaling. Mice were exposed to cigarette smoke (CS) for 7, 15, 30, 45, and 60 days; the control group was not exposed to CS. Only mice exposed to CS for 45 days were selected for subsequent N-acetylcysteine (NAC) supplementation and smoke cessation analyses. Exposure to CS increased the production of ROS and induced molecular changes in the autophagy pathway, including an increase in phosphorylated AMPK and ULK1, reduction in phosphorylated mTOR, and increases in SESN2, ATG12, and LC3B levels. NAC supplementation reduced ROS levels and reversed all molecular changes observed upon CS treatment, suggesting the involvement of oxidative stress in inducing autophagy upon CS exposure. When exposure to CS was stopped, there were decreases in the levels of oxidative stress, AMPK and ULK1 phosphorylation, and autophagy-initiating molecules and increase in mTOR phosphorylation. In conclusion, these results suggest the involvement of ROS, SESN2, AMPK, and mTOR in the CS-induced autophagic process in the lung.     

AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.     

Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy

Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.     

microRNA-17-5p Modulates Bacille Calmette-Guerin Growth in RAW264.7 Cells by Targeting ULK1

To explore the potential roles of miRNAs in controlling the survival of mycobacteria in macrophages, miR-17-5p in the regulation of Bacillus Calmette-Guérin(BCG)growth in the macrophage RAW264.7 cells was interrogated. Our results reveal that an infection of BCG shows a time-dependent up-regulation of miR-17-5p in RAW264.7 cells in early phase; importantly, excessive expression of miR-17-5p in these cells exhibits an increased propagation of intracellular BCG. Mechanistically, the Unc-51 like autophagy activating kinase 1 (ULK1), an initial molecular of autophagy are identified as novel target of miR-17-5p, the miR-17-5p is capable of targeting down-regulating the expression of ULK1 protein. In addition, an overexpression of miR-17-5p in RAW264.7 cells is correlated with repression of ULK1 and the autophagosome related proteins LC3I/II. These results imply that miR-17-5p may be able to arrest the maturation of mycobacterial phagosomes in part by targeting ULK1, subsequently reduces the ability of host cells to kill intracellular BCG.     

The serine/threonine kinase ULK1 is a target of multiple phosphorylation events

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.