mTOR复合物在糖尿病肾病小鼠肾组织中的不同分布和表达

目的探讨哺乳动物雷帕霉素靶分子复合物(mTORC)在糖尿病肾病(DN)小鼠肾组织中的分布、表达。方法 14只C57BL/6小鼠随机分成对照组和DN组,每组各7只。DN组小鼠予以链脲菌素腹腔注射建立小鼠DN模型,采用生化技术检测小鼠血、尿肌酐以及白蛋白水平,组织学染色检测肾脏病理变化,免疫荧光以及免疫印迹技术检测肾组织中mTOR、mTOR第2448位丝氨酸磷酸化修饰后mTOR(p-mTOR)、mTORC1(Raptor)、mTORC2(Rictor)以及mTOR信号通路下游的效应蛋白磷酸化S6K1(p-S6K1)的分布和表达。结果 DN组小鼠血糖、尿白蛋白/肌酐比值明显增加(P0.01),肾小球明显增大(P0.05)。mTOR、Raptor以及Rictor在正常以及DN小鼠肾皮质和髓质中均有表达,主要表达在肾小球系膜区、毛细血管袢、皮质近曲小管以及外髓和内髓集合管上皮细胞中。其中正常小鼠内髓肾组织中未见p-S6K1表达,正常以及DN小鼠肾小球中未见p-mTOR表达。免疫印迹检测表明,DN小鼠肾组织中mTOR、p-mTOR、Raptor、Rictor以及p-S6K1均明显上升(P0.05)。结论 mTORC广泛分布于小鼠肾组织且参与DN的发生发展,但mTOR第2448位丝氨酸磷酸化并不直接参与高血糖介导的肾小球损伤。     

mTORC1 信号促进前成骨细胞MC3T3-E1 的分化成熟

目的:研究mTORC1 信号对前成骨细胞MC3T3-E1 向成骨细胞分化成熟的调控作用。方法:通过向MC3T3-E1 转染pcDNA3.1-Raptor,对mTORC1 信号相关蛋白Raptor 进行过表达。向MC3T3-E1 转染Raptor siRNA,对mTORC1 信号蛋白Raptor 进行基因沉默。通过Real-time PCR 方法测定Raptor 的基因表达,通过蛋白免疫印迹法测定Raptor 蛋白水平,并通过茜素红染色检测成骨矿化情况,以测定成骨分化程度。通过Real-time PCR 检测成骨分化指标的基因表达。结果:与对照组相比,Raptor 过表达组的Raptor mRNA 和蛋白水平明显增加;茜素红染色结果显示Raptor 过表达组染色更深,说明成骨矿化程度更高;荧光定量PCR 结果显示,Raptor 过表达组的成骨分化标记基因以及成骨转录因子的表达量均高于对照组。与对照组相比,Raptor siRNA 组的Raptor mRNA 和蛋白水平明显降低;茜素红染色结果显示Raptor siRNA 组染色更浅,说明成骨矿化程度更低;荧光定量PCR 结果显示,Raptor siRNA 组的成骨分化标记基因以及成骨转录因子的表达量均低于对照组。结论:mTORC1 信号促进前成骨细胞MC3T3-E1 向成骨细胞分化成熟。     

Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells

Activation of stress signaling pathways normally leads to inhibition of the mammalian target of rapamycin complex 1 (mTORC1); however, human cytomegalovirus (HCMV) infection maintains mTORC1 activity in the presence of numerous types of stress. We previously demonstrated that HCMV infection maintains mTORC1 activity during amino acid deprivation through a Ras-related GTP-binding (Rag) protein-independent mechanism. This depends on the colocalization of mTOR and its activator, Rheb (Ras homology enriched in brain)-GTP, to a perinuclear position that corresponds to the viral cytoplasmic assembly compartment (AC). The data presented here show that the HCMV-induced, amino acid depletion-resistant perinuclear localization and activation of mTORC1 occurs as early as 8 h post-infection, prior to AC formation. We show that the molecular motor dynein is required for perinuclear localization of mTORC1 in both uninfected and HCMV-infected cells. Association between dynein and mTOR is shown by coimmunoprecipitation, and inhibition of dynein function using RNAi or the small molecule inhibitor ciliobrevin A inhibits mTORC1 activity in both uninfected and HCMV-infected cells. The data suggest that mTORC1 activation requires dynein-dependent transport to a position in the cell where it can be activated. Thus, the HCMV commandeers a cellular dynein-dependent mTORC1 activation mechanism to maintain stress-resistant mTORC1 activity during infection and to form the AC.     

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction

The mammalian target of rapamycin (mTOR) is a serine‐threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B‐cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short‐lived plasma cells, long‐lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock‐out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre‐ and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long‐lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.     

Rtp801 Suppression of Epithelial mTORC1 Augments Endotoxin-Induced Lung Inflammation

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke–induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801^−/− mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801^−/− mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.In the past decade, increasing scientific attention has been devoted to the role of the mechanistic (formerly mammalian) target of rapamycin (mTOR) in the maintenance of cellular homeostasis.¹ mTOR signaling occurs via two well-defined, evolutionarily conserved complexes named mTORC1 and mTORC2. mTORC1, the best characterized of the mTOR complexes, primarily functions in the transduction of cell growth signals. mTORC1 signaling is activated during states of nutrient (eg, amino acids, lipids, and glucose) availability and growth factor stimulation. Activated mTORC1, in turn, activates downstream targets such as ribosomal S6 kinase, promoting protein synthesis and cell growth.² Accordingly, nutrient deprivation and environmental stress attenuate mTORC1 signaling, mediated by several upstream pathways that converge on the mTORC1 inhibitory complex TSC-1 (hamartin)/TSC-2 (tuberin).Acute respiratory distress syndrome (ARDS) is a common and morbid critical illness characterized by the diffuse and rapid onset of neutrophilic pulmonary inflammation in response to either a direct (eg, pneumonia) or an indirect (eg, nonpulmonary sepsis) pulmonary insult.³ Several known modifiers of mTOR signaling have been implicated in the pathophysiological characteristics of ARDS, such as oxidative stress and localized hypoxia (both of which inhibit mTORC1^4,5), as well as local inflammatory cytokine expression (associated with mTORC1 activation⁶). Interestingly, published reports conflict regarding the impact of mTORC1 signaling in ARDS.^7–10 Because these conflicting studies were on the basis of the systemic use of the pharmacological mTORC1 inhibitor rapamycin, their discrepant results could reflect varied effects of mTORC1 across the complex multicellular microenvironment of the injured alveolus. Furthermore, few studies have addressed the endogenous regulation of mTORC1 signaling during the onset and progression of lung injury.In mice, cigarette smoke inhalation increases pulmonary expression of Rtp801 (alias Redd1 or Ddit4), an endogenous activator of TSC-2, and, consequently, inhibitor of mTORC1.⁴ Within the lungs, Rtp801 is primarily localized to type II alveolar epithelial cells.⁴ Rtp801-mediated inhibition of epithelial mTORC1 activity during cigarette smoke inhalation augments alveolar injury, contributing to emphysema formation. Concordantly, RTP801 expression is increased in lung samples collected from human subjects with emphysema.⁴ These findings indicate that Rtp801/mTORC1 signaling functions to sense environmental stressors, integrating epithelial injury responses within the lungs.Given that bacterial-originated lipopolysaccharide (LPS) might be recognized as an environmental threat, we hypothesized that Rtp801 expression would increase in a mouse LPS model of ARDS, with the ensuing suppression of epithelial mTORC1 consequently augmenting lung inflammation. We investigated this hypothesis by using a model of direct (intratracheal LPS) lung injury in wild-type (WT) mice and Rtp801-knockout (Rtp801^−/−) mice. These investigations were complemented by the development of transgenic mice that overexpressed Rtp801, to determine whether mTORC1 inhibition would lower the threshold for LPS-induced lung injury. Neutrophilic inflammation was correlated to cell-specific measures of mTORC1 activity, demonstrating the complex cellular heterogeneity of mTORC1 activity within the injured lung.     

Crystal structure of the Gtr1p-Gtr2p complex reveals new insights into the amino acid-induced TORC1 activation

The target of rapamycin (TOR) complex 1 (TORC1) is a central cell growth regulator in response to a wide array of signals. The Rag GTPases play an essential role in relaying amino acid signals to TORC1 activation through direct interaction with raptor and recruitment of the TORC1 complex to lysosomes. Here we present the crystal structure of the Gtr1p–Gtr2p complex, the Rag homologs from Saccharomyces cerevisiae, at 2.8 Å resolution. The heterodimeric GTPases reveal a pseudo-twofold symmetric organization. Structure-guided functional analyses of RagA–RagC, the human homologs of Gtr1p–Gtr2p, show that both G domains (N-terminal GTPase domains) and dimerization are important for raptor binding. In particular, the switch regions of the G domain in RagA are indispensible for interaction with raptor, and hence TORC1 activation. The dimerized C-terminal domains of RagA–RagC display a remarkable structural similarity to MP1/p14, which is in a complex with lysosome membrane protein p18, and directly interact with p18, therefore recruiting mTORC1 to the lysosome for activation by Rheb. Our results reveal a structural model for the mechanism of the Rag GTPases in TORC1 activation and amino acid signaling.     

Lentivirus-based RNA Silencing of Nemo-like Kinase (NLK) Inhibits the CAL 27 Human Adenosquamos Carcinoma Cells Proliferation and Blocks G0/G1 Phase to S Phase

Background: The Nemo-like kinase (NLK) is a serine/threonine-protein kinase that involved in a number of signaling pathways regulating cell fate. Variation of NLK has been shown to be associated with the risk of cancer. However, the function of NLK in oral adenosquamous carcinoma cells line CAL-27 is unknown.Methods: In this study, we evaluated the function of NLK in CAL-27 cells by using lentivirus-mediated RNA silence. The targeted gene expression, cell proliferation and cell cycle are investigated by RT-PCR, western-blot, MTT method, colony forming assay and flow cytometry analysis respectively.Results: After NLK silencing, the number of colonies was significantly reduced (54±5 colonies/well compared with 262±18 colonies/well in non-infected or 226±4 colonies/well in negative control group (sequence not related to NLK sequence with mismatched bases). Using crystal violet staining, we also found that the cell number per colony was dramatically reduced. The RNA silencing of NLK blocks the G0/G1 phase to S phase progression during the cell cycle.Conclusions: These results suggest that NLK silencing by lentivirus-mediated RNA interference would be a potential therapeutic method to control oral squamous carcinoma growth.     

Pharmacological inhibition of mTORC1 activity protects against inflammation-induced apoptosis of nucleus pulposus cells

Lumbar disc herniation is a common disease characterized by the degeneration of intervertebral discs (IVDs), accompanied by imbalance of metabolic and inflammatory homeostasis. Current studies establish that IVD degeneration is induced by increased apoptosis of nucleus pulposus (NP) cells. However, the underlying mechanisms of NP cell survival/apoptosis are not well elucidated. Here, we reveal a novel mechanism by which mTORC1 signaling controls NP cell survival through regulating metabolic homeostasis. We demonstrated that hyperactivated mTORC1 activity induced by inflammatory cytokines engenders the apoptosis of NP cells, whereas pharmacological inhibition of mTORC1 activity promotes NP cell survival. Using an integrative approach spanning metabolomics and biochemical approaches, we showed that mTORC1 activation enhanced glucose metabolism and lactic acid production, and therefore caused NP cell apoptosis. Our study identified mTORC1 in NP cells as a novel target for IVD degeneration, and provided potential strategies for clinical intervention of lumbar disc herniation.     

mTORC1信号通路对能量代谢调控作用及其机制

哺乳动物雷帕霉素靶蛋白（mTORC1）通路是细胞内最主要的能量感受器,能感受营养和激素、与能量需求相关的多个细胞功能的调节相关的上游调节信号。调节下游信号从而影响不同的细胞代谢,从蛋白质和脂质合成到线粒体活性等不同方面调节细胞代谢。作为对细胞代谢过程的调节,mTORC1的活性在外周激活有利于脂肪细胞活化,脂肪生成,葡萄糖摄取和β细胞数量的增加。本文综述了现有的知识对mTORC1的作用在能量平衡和能量代谢的调节,特别是旨在提供有关mTORC1在功能细胞中心能够整合不同激素的作用的背景下的研究进展进行了综述。     

GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2

Mammalian cells possess two amino acid-sensing kinases: general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). Their combined effects orchestrate cellular adaptation to amino acid levels, but how their activities are coordinated remains poorly understood. Here, we demonstrate an important link between GCN2 and mTORC1 signaling. Upon deprivation of various amino acids, activated GCN2 up-regulates ATF4 to induce expression of the stress response protein Sestrin2, which is required to sustain repression of mTORC1 by blocking its lysosomal localization. Moreover, Sestrin2 induction is necessary for cell survival during glutamine deprivation, indicating that Sestrin2 is a critical effector of GCN2 signaling that regulates amino acid homeostasis through mTORC1 suppression.     

miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway

Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway.     

Constitutive activation with overexpression of the mTORC2-phospholipase D1 pathway in uterine leiomyosarcoma and STUMP: morphoproteomic analysis with therapeutic implications

The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for therapy and drug development. Biologically, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR complex 1 (mTORC1) which is predominantly cytoplasmic in subcellular localization and mTOR complex 2 (mTORC2) which is both cytoplasmic and nuclear. mTORC1 is sensitive to the selective inhibitor rapamycin. By contrast, mTORC2 is relatively resistant to rapamycin. Moreover, its putative downstream effector, Akt phosphorylated on serine 473 represents a signal transduction pathway for tumor survival. Phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as an activator of mTOR signaling, including the direct phosphorylative activation of p70S6K atthreonine 389. The latter promotes cell cycle progression. In this study, we investigated the activation status and subcellular localization of mTOR and the relative expression of PLD1, as well as their downstream effectors in a spectrum of uterine smooth muscle tumors using normal myometria as controls. The results show significant activation with overexpression of phosphorylated mTORC2 complex in uterine leiomyosarcoma (ULMS) and smooth muscle tumors of uncertain malignant potential (STUMP) as evidenced by nuclear localization of p-mTOR (Ser 2448) in ULMS>STUMP>uterine leiomyoma and normal myometria (p<0.05) and with overexpression of PLD1(p<0.05). Cor-relatively, there are overexpressions of nuclear p-Akt (Ser 473) and nuclear p-p70S6K (Thr 389) in ULMS and STUMP (p<0.05). The activation with overexpression of components of the mTORC2-PLD1 pathway in ULMS and to a lesser degree in STUMP provides insight into their tumorigenic mechanisms. Thus the development of therapies designed to target mTORC2 and PLD1 activity may be beneficial in treating ULMS.     

Metformin Induces G1 Cell Cycle Arrest and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma Cells

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP‐activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1–50 mM of metformin in a dose‐ and time‐dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666‐1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time‐dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)‐binding protein 1 (4E‐BP1) (T37/46) and S6 (S235/236) in C666‐1 cells. In a summary, metformin prevents proliferation of C666‐1 cells by down‐regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK‐mediated inhibition of mTORC1 signaling may be involved in this process. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

mTOR links oncogenic signaling to tumor cell metabolism

As a key regulator of cell growth and proliferation, the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) has been the subject of intense investigation for its role in tumor development and progression. This research has revealed a signaling network of oncogenes and tumor suppressors lying upstream of mTORC1, and oncogenic perturbations to this network result in the aberrant activation of this kinase complex in the majority of human cancers. However, the molecular events downstream of mTORC1 contributing to tumor cell growth and proliferation are just coming to light. In addition to its better-known functions in promoting protein synthesis and suppressing autophagy, mTORC1 has emerged as a key regulator of cellular metabolism. Recent studies have found that mTORC1 activation is sufficient to stimulate an increase in glucose uptake, glycolysis, and de novo lipid biosynthesis, which are considered metabolic hallmarks of cancer, as well as the pentose phosphate pathway. Here, we focus on the molecular mechanisms of metabolic regulation by mTORC1 and the potential consequences for anabolic tumor growth and therapeutic strategies.     

Characterization of p70 S6 kinase 1 in early development of mouse embryos

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one‐cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M‐phase promoting factor (MPF) and delayed the first mitotic cleavage. Down‐regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one‐cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho‐S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos. Developmental Dynamics 238:3025–3034, 2009. © 2009 Wiley‐Liss, Inc.