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Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency. [ABSTRACT FROM AUTHOR]

Canine soft tissue sarcoma (STS) is relatively common in dogs and is the generic term for tumours that originate from mesenchymal cells. While histopathological grade and immunolabelling with Ki-67 have been used for estimating prognosis, additional indicators are needed for predicting prognosis. Aberrant cell signalling pathways may contribute to disease activity and, therefore, prognostic markers. However, their role in canine STS remains poorly understood. The aim of this study was to investigate expression of phosphorylated Akt (phospho-Akt) and phosphorylated S6 (phospho-S6) as potential prognostic indicators. Immunohistochemical labelling was conducted on clinical samples of canine STS (n = 67). We found that phospho-Akt expression was positively correlated with histopathological grade (P = 0.001) and Ki-67 index (P <0.01). There was no apparent relationship between the type of STS and the expression of phospho-Akt. The number of cases that expressed phospho-S6, which is the downstream molecule of the Akt signalling pathway, was higher in immunopositive phospho-Akt cases than in immunonegative phospho-Akt cases (P <0.0001). Furthermore, phospho-Akt expression was significantly higher in recurrent and metastatic cases. We also confirmed that phosphorylation of Akt occurred in conjunction with S6 phosphorylation in three canine STS cell lines. These results suggest that immunolabelling for phospho-Akt, phospho-S6 and Ki-67 could potentially be used as a prognostic indicator and therapeutic target in canine STS. [ABSTRACT FROM AUTHOR]

Environmental contaminant exposures occur due to the widespread use of synthetic chemicals. Tributyltin (TBT), dibutyltin (DBT), and pentachlorophenol (PCP) are each used in a variety of applications, including antifouling paints and stabilizers in certain plastics. Each of these compounds has been found in human blood, as well as other tissues, and they have been shown to stimulate pro-inflammatory cytokine production in human immune cells, Inflammatory cytokines mediate response to injury or infection. However, if their levels are increased in the absence of an appropriate stimulus, chronic inflammation can occur. Chronic inflammation is associated with a number of pathologies including cancer. Stimulation of pro-inflammatory cytokine production by these toxicants is dependent on activation of ERK 1/2 and/or p38 MAPK pathways. MAPK pathways have the capacity to regulate translation by increasing phosphorylation of key translation regulatory proteins. There have been no previous studies examining the effects of TBT, DBT, or PCP on translation. The current study shows that ribosomal protein S6 (S6), eukaryotic initiation factor 4B (eIF4B), and eIF4E are phosphorylated (activated) and/or their total levels are elevated in response to each of these compounds at concentrations found in human blood. Activation/increased levels of translational proteins occurred at concentrations of the compounds that have been shown to elevate pro-inflammatory cytokine production, but where there is no increase in mRNA for those proteins was seen. Compound-stimulated increases in translation appear to be part of the mechanism by which they elevate protein production in immune cells. [ABSTRACT FROM AUTHOR]

Background: Although abnormal cell proliferation and apoptosis are associated with the pathogenesis of oral lichen planus (OLP), the exactly mechanism of which is not yet known. It has been reported that glutamine (Gln) can promote cell proliferation and inhibit apoptosis of various tumor cells. This study aims to evaluate the effect of Gln metabolism on the balance of proliferation and apoptosis in epithelial cells of OLP. Methods: Thirty human OLP specimens and 11 normal controls were stained by immunohistochemistry to detect the levels of proliferation and Gln metabolism related proteins. Then, the critical role of Gln in cell proliferation and apoptosis was determined by Gln deprivation or treatment with glutaminase inhibitor (CB‐839) to intervene Gln metabolism in human gingival epithelial cells. Cell proliferation was detected using CCK8, p‐mTOR and p‐S6 proteins were detected using Western Blot, cell apoptosis and cell cycle were detected using flow cytometry, and cell stress was detected using immunofluorescence. Results: Compared with normal controls, OLP specimens showed higher levels of Ki‐67 and Gln metabolism‐related proteins, including Gln transporter (ASCT2), glutaminase (GLS), and pathway proteins (p‐mTOR and p‐S6). In vitro, Gln promoted cell proliferation and simultaneously upregulated the activity of mTOR/S6 pathway. Moreover, rapamycin, an mTOR pathway inhibitor, could effectively block the Gln‐induced cell proliferation. MHY1485, an mTOR pathway agonist, could effectively reverse the decline of cell proliferation under Gln deprivation. In addition, inhibiting Gln metabolism caused the accumulation of intracellular radical oxygen species (ROS) and induced cell apoptosis. However, N‐acetylcysteine reversed this state and then decreased cell apoptosis by eliminating intracellular ROS. Conclusion: Gln metabolism is essential to maintain the balance of proliferation and apoptosis in oral epithelial cells, and inhibition of Gln metabolism may have a beneficial effect on OLP treatment. [ABSTRACT FROM AUTHOR]

Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL. [ABSTRACT FROM AUTHOR]

The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1. [ABSTRACT FROM AUTHOR]

Background and Hypothesis Schizophrenia is characterized by a complex interplay between genetic and environmental risk factors converging on prominent signaling pathways that orchestrate brain development. The Akt/GSK3β/mTORC1 pathway has long been recognized as a point of convergence and etiological mechanism, but despite evidence suggesting its hypofunction, it is still not clear if this is already established during the first episode of psychosis (FEP). Study Design Here, we performed a systematic phosphorylation analysis of Akt, GSK3β, and S6, a mTORC1 downstream target, in fresh peripheral blood mononuclear cells from drug-naive FEP patients and control subjects. Study Results Our results suggest 2 distinct signaling endophenotypes in FEP patients. GSK3β hypofunction exhibits a promiscuous association with psychopathology, and it is normalized after treatment, whereas mTORC1 hypofunction represents a stable state. Conclusions Our study provides novel insight on the peripheral hypofunction of the Akt/GSK3β/mTORC1 pathway and highlights mTORC1 activity as a prominent integrator of altered peripheral immune and metabolic states in FEP patients. [ABSTRACT FROM AUTHOR]

Short- or mid-term fasting, full or partial, triggers metabolic response known to have in turn health effects in an organism. At central level, the metabolic stimulus triggered by fasting is known to be perceived firstly by hypothalamic neurons. In the field of neuroscience, ribosomal protein S6 (S6) phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signalling activation or as a marker for neuronal activity. The aim of this study is addressed to evaluate whether the phosphorylation of S6 occurs in the central neurons of zebrafish exposed to four (short-term) and seven (mid-term) days of complete fasting. Group-housed adult zebrafish were exposed to four and seven days of complete food withdrawal. At the end of the experimental period, Western blotting analyses were carried out to measure the expression levels of the phosphorylated S6 (pS6) by comparing the two experimental conditions versus the control group. The same antibody was then used to identify the distribution pattern of pS6 immunoreactive neurons in the whole brain and in the taste buds. We did not observe increased pS6 levels expression in the brain of animals exposed to short-term fasting compared to the control, whereas the expression increased in brain homogenates of animals exposed to mid-term fasting. pS6 immunoreactivity was reported in some hypothalamic neurons, as well as in the dorsal area of telencephalon and preoptic area, a neurosecretory region homolog to the mammalian paraventricular nucleus. Remarkably, we observed pS6 immunostaining in the sensory cells of taste buds lining the oral epithelium. Taken together, our data show that in zebrafish, differently from other fish species, seven days of fasting triggers neuronal activity. Furthermore, the immunostaining on sensory cells of taste buds suggests that metabolic changes may modulate also peripheral sensory cells. This event may have valuable implications when using zebrafish to design metabolic studies involving fasting as well as practical consequences on the animal welfare, in particularly stressful conditions, such as transportation. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive degeneration of motor neurons. Mislocalization of TAR DNA-binding protein 43 (TDP-43) is an early event in the formation of cytoplasmic TDP-43-positive inclusions in motor neurons and a hallmark of ALS. However, the underlying mechanism and the pathogenic impact of this mislocalization are relatively unexplored. We previously reported that abnormal AMPK activation mediates TDP-43 mislocalization in motor neurons of humans and mice with ALS. In the present study, we hypothesized that other nuclear proteins are mislocalized in the cytoplasm of motor neurons due to the AMPK-mediated phosphorylation of importin-α1 and subsequently contribute to neuronal degeneration in ALS. To test this hypothesis, we analyzed motor neurons of sporadic ALS patients and found that when AMPK is activated, importin-α1 is abnormally located in the nucleus. Multiple integrative molecular and cellular approaches (including proteomics, immunoprecipitation/western blot analysis, immunohistological evaluations and gradient analysis of preribosomal complexes) were employed to demonstrate that numerous RNA binding proteins are mislocalized in a rodent motor neuron cell line (NSC34) and human motor neurons derived from iPSCs during AMPK activation. We used comparative proteomic analysis of importin-α1 complexes that were immunoprecipitated with a phosphorylation-deficient mutant of importin-α1 (importin-α1-S105A) and a phosphomimetic mutant of importin-α1 (importin-α1-S105D) to identify 194 proteins that have stronger affinity for the unphosphorylated form than the phosphorylated form of importin-α1. Furthermore, GO and STRING analyses suggested that RNA processing and protein translation is the major machinery affected by abnormalities in the AMPK-importin-α1 axis. Consistently, the expression of importin-α1-S105D alters the assembly of preribosomal complexes and increases cell apoptosis. Collectively, we propose that by impairing importin-α1-mediated nuclear import, abnormal AMPK activation in motor neurons alters the cellular distribution of many RNA-binding proteins, which pathogenically affect multiple cellular machineries in motor neurons and contribute to ALS pathogenesis.

The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1.

Jingren Shi,1 Wenli Zhang,2 Lu He,2 Fanhong Kong,2 Meichen Pan,1 Jingjing Guo,1 Xinmin Xu,1 Jie Guo,1 Huizhu Wang,1 Yajie Wang11Department of Clinical Laboratory, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, People’s Republic of China; 2Department of Clinical Laboratory, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, People’s Republic of ChinaAim: To investigate the anticancer effects of Jinlong capsule (JLC) against human glioblastoma cells and the possible underlying mechanism.Methods: Cell Counting Kit-8 and colony formation assay were adopted for the analysis of cell viability. Cell invasion and migration were evaluated by transwell and wound healing assays. Then, the expression level of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), S6 and phosphorylated S6 (p-S6) were determined by western blotting.Results: The results showed that JLC significantly inhibited human glioblastoma cell proliferation, invasion and migration in a dose-dependent manner. The expressions of p-mTOR and p-S6 were dramatically suppressed by JLC. Furtherly, inhibition of mTOR reduced the cell migration and invasion, while the mTOR agonist (MHY1485) could partially reverse the anti-migration and anti-invasion activity of JLC.Conclusion: The above results suggested that JLC would be a potential candidate for the treatment of glioblastoma.Keywords: Jinlong capsule, invasion, migration, glioblastoma, mTOR, S6

Early-life stress (ELS) predisposes individuals to psychiatric disorders, including anxiety and depression, and cognitive impairments later in life. However, the underlying molecular mechanisms are not completely understood. Developmental deficits in hippocampal synaptic plasticity are among the primary detrimental alterations in brain function induced by ELS. Impaired synaptic plasticity is usually accompanied by decreased synaptic proteins, such as postsynaptic density 95 (PSD95) and synaptophysin, which are important for synaptic function. The mTOR signaling pathway plays a vital role in regulating protein translation, and mTOR activation is functionally associated with synaptic protein synthesis. In the present study, we observed whether ELS impacts synaptic protein synthesis and mTOR signaling, which is involved in synaptic plasticity. Herein, we established a maternal separation (MS) and chronic restraint stress (CRS) model and evaluated anxiety-like behavior and cognitive function (e.g., learning and memory) in adulthood through behavioral examination and analyzed hippocampal expression levels of PSD95 and synaptophysin. To explore whether the mTOR signaling pathway was associated with ELS, we also examined the activity of mTOR and s6. The behavior tests indicated that maternally separated mice showed increased anxiety-like behavior and cognitive impairments. PSD95 and synaptophysin mRNA and protein expression levels were decreased in the hippocampus, and phosphorylated mTOR and phosphorylated s6 were significantly decreased in maternally separated mice vs. those not exposed to MS. Our data demonstrate that MS impairs synaptic plasticity and inhibits mTOR signaling, specifically via s6. Therefore, we speculate that ELS decreased synaptic plasticity via the inhibition of the mTOR pathway in the hippocampus, which may underlie vulnerability to stress and mental disorders in adulthood.

Early-life stress (ELS) predisposes individuals to psychiatric disorders, including anxiety and depression, and cognitive impairments later in life. However, the underlying molecular mechanisms are not completely understood. Developmental deficits in hippocampal synaptic plasticity are among the primary detrimental alterations in brain function induced by ELS. Impaired synaptic plasticity is usually accompanied by decreased synaptic proteins, such as postsynaptic density 95 (PSD95) and synaptophysin, which are important for synaptic function. The mTOR signaling pathway plays a vital role in regulating protein translation, and mTOR activation is functionally associated with synaptic protein synthesis. In the present study, we observed whether ELS impacts synaptic protein synthesis and mTOR signaling, which is involved in synaptic plasticity. Herein, we established a maternal separation (MS) and chronic restraint stress (CRS) model and evaluated anxiety-like behavior and cognitive function (e.g., learning and memory) in adulthood through behavioral examination and analyzed hippocampal expression levels of PSD95 and synaptophysin. To explore whether the mTOR signaling pathway was associated with ELS, we also examined the activity of mTOR and s6. The behavior tests indicated that maternally separated mice showed increased anxiety-like behavior and cognitive impairments. PSD95 and synaptophysin mRNA and protein expression levels were decreased in the hippocampus, and phosphorylated mTOR and phosphorylated s6 were significantly decreased in maternally separated mice vs. those not exposed to MS. Our data demonstrate that MS impairs synaptic plasticity and inhibits mTOR signaling, specifically via s6. Therefore, we speculate that ELS decreased synaptic plasticity via the inhibition of the mTOR pathway in the hippocampus, which may underlie vulnerability to stress and mental disorders in adulthood. [ABSTRACT FROM AUTHOR]

The altered wiring of signaling pathways downstream of antigen receptors of T and B cells contributes to the dysregulation of the adaptive immune system, potentially causing immunodeficiency and autoimmunity. In humans, the investigation of such complex systems benefits from nature’s experiments in patients with genetically defined primary immunodeficiencies. Disturbed B-cell receptor (BCR) signaling in a subgroup of common variable immunodeficiency (CVID) patients with immune dysregulation and expanded T-bethighCD21low B cells in peripheral blood has been previously reported. Here, we investigate PI3K signaling and its targets as crucial regulators of survival, proliferation and metabolism by intracellular flow cytometry, imaging flow cytometry and RNAseq. We observed increased basal but disturbed BCR-induced PI3K signaling, especially in T-bethighCD21low B cells from CVID patients, translating into impaired activation of crucial downstream molecules and affecting proliferation, survival and the metabolic profile. In contrast to CVID, increased basal activity of PI3K in patients with a gain-of-function mutation in PIK3CD and activated PI3K delta syndrome (APDS) did not result in impaired BCR-induced AKT-mTOR-S6 phosphorylation, highlighting that signaling defects in B cells in CVID and APDS patients are fundamentally different and that assessing responses to BCR stimulation is an appropriate confirmative diagnostic test for APDS. The active PI3K signaling in vivo may render autoreactive T-bethighCD21low B cells in CVID at the same time to be more sensitive to mTOR or PI3K inhibition.

Ribosomal protein S6 kinase beta-1 (S6K1) is a serine/threonine kinase downstream of the mechanistic target of rapamycin (mTOR) pathway, and plays crucial roles in immune regulation. Although remarkable progress has been achieved with a mouse model, how S6K1 regulates adaptive immunity is largely unknown in early vertebrates. In this study, we identified an S6K1 from Nile tilapia Oreochromis niloticus (OnS6K1), and further investigated its potential regulatory role on the adaptive immunity of this fish species. Both sequence and structure of OnS6K1 were highly conserved with its homologs from other vertebrates and invertebrates. OnS6K1 was widely expressed in immune tissues, and with a relative higher expression level in the liver, spleen and head kidney. At the adaptive immune stage of Nile tilapia that infected with Aeromonas hydrophila , mRNA expression of OnS6K1 and its downstream effector S6 was significantly up-regulated in spleen lymphocytes. Meanwhile, their phosphorylation level was also enhanced during this process, suggesting that S6K1/S6 axis participated in the primary response of anti-bacterial adaptive immunity in Nile tilapia. Furthermore, after spleen lymphocytes were activated by the T cell-specific mitogen PHA or lymphocytes agonist PMA in vitro, mRNA and phosphorylation levels of S6K1 were elevated, and phosphorylation of S6 was also enhanced. Once S6K1 activity was blocked by a specific inhibitor, both mRNA and phosphorylation levels of S6 were severely impaired. More importantly, blockade of S6K1/S6 axis reduced the expression of T cell activation marker IFN-γ and CD122 in PHA-activated spleen lymphocytes, indicating the essential role of S6K1/S6 axis in regulating T cell activation of Nile tilapia. Together, our study suggests that S6K1 and its effector S6 regulate lymphocyte activation of Nile tilapia, and in turn promote lymphocyte-mediated adaptive immunity. This study enriched the mechanism of adaptive immune response in teleost and provided useful clues to understand the evolution of adaptive immune system. • S6K1 from Nile Tilapia O. niloticus is evolutionary conserved. • OnS6K1 takes part in primary response of the adaptive immunity. • OnS6K1 is essential for OnS6 transcription and phosphorylation. • OnS6K1-controlled S6 activity is indispensable for lymphocyte activation. [ABSTRACT FROM AUTHOR]

The receptor deleted in colorectal cancer (DCC) and its ligand netrin-1 are essential for axon guidance during development and are expressed by neurons in the mature brain. Netrin-1 recruits GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and is critical for long-term potentiation (LTP) at CA3-CA1 hippocampal Schaffer collateral synapses, while conditional DCC deletion from glutamatergic neurons impairs hippocampal-dependent spatial memory and severely disrupts LTP induction. DCC co-fractionates with the detergent-resistant component of postsynaptic density, yet is enriched in axonal growth cones that differentiate into presynaptic terminals during development. Specific presynaptic and postsynaptic contributions of DCC to the function of mature neural circuits have yet to be identified. Employing hippocampal subregion-specific conditional deletion of DCC, we show that DCC loss from CA1 hippocampal pyramidal neurons resulted in deficits in spatial memory, increased resting membrane potential, abnormal dendritic spine morphology, weaker spontaneous excitatory postsynaptic activity, and reduced levels of postsynaptic adaptor and signaling proteins; however, the capacity to induce LTP remained intact. In contrast, deletion of DCC from CA3 neurons did not induce detectable changes in the intrinsic electrophysiological properties of CA1 pyramidal neurons, but impaired performance on the novel object place recognition task as well as compromised excitatory synaptic transmission and LTP at Schaffer collateral synapses. Together, these findings reveal specific pre- and post-synaptic contributions of DCC to hippocampal synaptic plasticity underlying spatial memory. [ABSTRACT FROM AUTHOR]

Bitter melon seed oil (BMSO) was found to have an advantageous effect on anti-obesity. Up to date, the mechanisms underlying this process have been extensively investigated. However, there are very few reports focusing on the roles of central nervous system (CNS) involved. In this study, Golgi-Cox staining and western blotting assays were used to examine the hypothalamic spine density and the expression levels of NMDA-2B receptor and P-S6 protein, respectively. A significant reduction concerning hypothalamic spine density was observed in HFD mice, a phenomenon that could be partially rescued by the BMSO administration. Furthermore, it was found that BMSO could also reverse the changes upon the phosphorylation levels of S6, a typical protein involved in mTOR signaling pathway, indicating that mTOR signaling potentially participated in this metabolism regulation. Besides, NMDA-2B levels were up-regulated in HFD mice, which could not be considerably influenced by BMSO. In summary, this study first proposed aberrant hypothalamic plasticity as CNS's roles in BMSO's fat-reducing effects, favoring the better recognition and treatment of the intractable hypothalamic obesity.

RAS is frequently mutated in various tumors and known to be difficult to target. NRASQ61K/R are the second most frequent mutations found in human skin melanoma after BRAFV600E. Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRASQ61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRASQ61K mouse melanoma that can be used in vitro and in vivo in an immune‐competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRASQ61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses. [ABSTRACT FROM AUTHOR]

Oestrogen receptor (ER)-positive, metachronous, contralateral breast cancer (MCBC) sometimes develops during or soon after completion of hormone therapy (HT), but it is uncertain whether it is HT-resistant. We examined the association between ER-positive second cancer and activation of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways, which are associated with HT resistance. We examined the treatment-free interval (time after completion of HT for initial cancer) in 41 patients with ER-positive MCBC with a history of adjuvant HT for initial cancer (HT group), and initial-to-second period duration (time after operation of initial cancer to onset of second cancer) in 17 patients with ER-positive MCBC in whom adjuvant HT was not applied to the initial tumour (control group or no HT group). Phosphorylated S6 (pS6) and phosphorylated MAPK (pMAPK) were used as indicators of PI3K/Akt/mTOR and MAPK pathway activity, respectively. Tumours were classified as showing negative, positive or strongly positive staining, and the correlation between staining and treatment-free interval or initial-to-second period duration was evaluated using the Spearman's rank correlation coefficient (ρ). Treatment-free interval and pS6 staining showed a negative correlation (ρ=−0.5355; P=0.0003) in the HT group. There was no correlation between initial-to-second period duration and pS6 staining in the no HT group (ρ=−0.0814; P=0.756). There was no correlation between pMAPK signalling and the treatment-free interval in the HT group (ρ=−0.1560; P=0.330) or the initial-to-second period duration in the no HT group (ρ=−0.0116; P=0.965). Development of a second ER-positive cancer during or soon after completion of HT for the initial cancer may be associated with activation of the PI3K/Akt/mTOR pathway. Care should be taken during follow-up and when selecting adjuvant therapy for second cancer. [ABSTRACT FROM AUTHOR]

The red-eared slider turtle (Trachemys scripta elegans), has developed remarkable adaptive mechanisms for coping with decreased oxygen availability during winter when lakes and ponds become covered with ice. Strategies for enduring anoxia tolerance include an increase in fermentable fuel reserves to support anaerobic glycolysis, the buffering of end products to minimize acidosis, altered expression in crucial survival genes, and strong metabolic rate suppression to minimize ATP-expensive metabolic processes such as protein synthesis. The mammalian target of rapamycin (mTOR) is at the center of the insulin-signaling pathway that regulates protein translation. The present study analyzed the responses of the mTOR signaling pathway to 5 (5H) or 20 h (20H) of anoxic submergence in liver and skeletal muscle of T. scripta elegans with a particular focus on regulatory changes in the phosphorylation states of targets. The data showed that phosphorylation of multiple mTOR targets was suppressed in skeletal muscle, but activated in the liver. Phosphorylated mTORSer2448 showed no change in skeletal muscle but had increased by approximately 4.5-fold in the liver after 20H of anoxia. The phosphorylation states of upstream positive regulators of mTOR (p-PDK-1Ser241, p-AKTSer473, and protein levels of GβL), the relative levels of dephosphorylated active PTEN, as well as phosphorylation state of negative regulators (TSC2Thr1462, p-PRAS40Thr246) were generally found to be differentially regulated in skeletal muscle and in liver. Downstream targets of mTOR (p-p70 S6KThr389, p-S6Ser235, PABP, p-4E-BP1Thr37/46, and p-eIF4ESer209) were generally unchanged in skeletal muscle but upregulated in most targets in liver. These findings indicate that protein synthesis is enhanced in the liver and suggests an increase in the synthesis of crucial proteins required for anoxic survival. [ABSTRACT FROM AUTHOR]

Peutz-Jeghers syndrome (PJS) is a rare hereditary disease caused by mutations in serine threonine kinase 11 (STK11) and characterized by an increased risk of developing cancer. Inactivation of STK11 has been associated with the mammalian target of rapamycin (mTOR) pathway. Hyperactivation and phosphorylation of the key downstream target genes ribosomal protein S6 kinase 1 (S6K1) and S6 promote protein synthesis and cell proliferation. To better understand the effects of STK11 dysfunction in the pathogenesis of PJS, genomic DNA samples from 21 patients with PJS from 11 unrelated families were investigated for STK11 mutations in the present study. The results revealed 6 point mutations and 2 large deletions in 8 (72.7%, 8/11) of the unrelated families. Notably, 3 novel mutations were identified, which included 2 missense mutations [c.88G>A (p.Asp30Asn) and c.869T>C (p.Leu290Pro)]. Subsequent immunohistochemical analysis revealed staining for phosphorylated-S6 protein in colonic hamartoma and breast benign tumor tissues from patients with PJS carrying the two respective missense mutations. Additionally, the novel missense STK11 mutants induced phosphorylation of S6K1 and S6, determined using western blot analysis, and promoted the proliferation of HeLa and SW1116 cells, determined using Cell Counting Kit-8 and colony formation assays. Collectively, these findings extend the STK11 mutation spectrum and confirm the pathogenicity of two novel missense mutations. This study represents a valuable insight into the molecular mechanisms implicated in the pathogenesis of PJS. [ABSTRACT FROM AUTHOR]

The spatial boundaries of tissue response to wounding are unknown. Here, we show that in mammals, the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury, forming a zone of activation surrounding the region of the initial insult. This p-rpS6-zone forms within minutes after wounding and is present until healing is complete. The zone is a robust marker of healing as it encapsulates features of the healing process, including proliferation, growth, cellular senescence, and angiogenesis. A mouse model that is unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in impaired healing, identifying p-rpS6 as a modulator but not a driver of healing. Finally, the p-rpS6-zone accurately reports on the status of dermal vasculature and the effectiveness of healing, visually dividing an otherwise homogeneous tissue into regions with distinct properties. [Display omitted] • Wounding induces phosphorylation of rpS6, leading to formation of the p-rpS6-zone • The zone is induced by DAMPs and layers cell death responses • The zone spatially and temporally delineates the process of healing • Healing processes are limited to the zone, including cellular senescence and angiogenesis Ring and Dworak et al. describe the discovery of an immediate and persistent phosphorylation of rpS6 in response to wounding, forming the p-rpS6-zone. The zone spatially and temporally delineates the processes of healing, appearing within minutes of wounding, encompassing all healing processes (including cellular senescence and angiogenesis) and disappearing upon complete healing. [ABSTRACT FROM AUTHOR]

Background The purpose of this study was to investigate the clinical relevance of phosphorylated ribosomal protein S6 (p-S6), a surrogate marker of mammalian target of rapamycin (mTOR) activation, and p21 in a series of 125 patients with oral squamous cell carcinomas (OSCCs). Methods Immunohistochemical analysis was performed to ascertain the phosphorylation status of p-S6 at Ser235/236 and Ser240/244, p21, and p53 protein expression. Results Expression of phosphorylated S6 protein on either serine 235/236 or serine 240/244 was detected in 83% and 88% tumors, respectively, and both of them were inversely and significantly correlated with the tumor size and local infiltration. Positive p21 expression was found in 91.5% of the cases, and was inversely correlated with tumor size. In OSCC, p21 expression correlates with p-S6 levels, a surrogate marker of mTOR activation, independently of p53 status. Conclusion Expression of both p21 and p-S6 was found to inversely associate with tumor size but not survival outcomes in patients with OSCC. [ABSTRACT FROM AUTHOR]

Summary TOR (Target Of Rapamycin) signalling coordinates cell growth and division in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.

Objectives The aim of this study was to investigate the expression of upstream and downstream molecules of the oncogenic mTOR signaling pathway in intra-oral minor salivary gland tumors ( SGTs). Materials and Methods Tissue samples consisted of 39 malignant and 13 benign minor SGTs, and 8 controls of normal minor salivary glands ( NMSG). An immunohistochemical analysis for phosphorylated Akt, 4 EBP1 and S6 (total and phosphorylated), and eIF4E was performed. Results Expression of pAkt and 4 EBP1 was observed in all SGTs and in most NMSG. p4 EBP1 was detected in almost all SGT cases, NMSG being negative. S6 immunoreactivity was observed in 37.5% of NMSG, 92.3% of benign and 100% of malignant SGTs, while pS6 expression was observed in 77% of benign and 95% of malignant SGTs, but not in NMSG. Finally, eIF4E was expressed in 12.5% of NMSG, 69.2% of benign, and 76.9% of malignant tumors. All molecules studied had statistically significantly lower expression in NMSG compared with SGTs. Moreover, malignant neoplasms received higher scores compared with benign tumors for all molecules with the exception of eIF4E. Conclusion The mTOR signaling pathway is activated in SGTs, especially in malignancies. Therefore, the possible therapeutic role of targeting the mTOR pathway by rapamycin analogs in SGTs needs further investigation. [ABSTRACT FROM AUTHOR]

Ras homolog enriched in brain (Rheb1) is a small GTPase and is known to be a direct activator of mTORC1. Dysregulation of Rheb1 has been shown to impair the cellular-energetic state and cell homeostasis. However, the role of Rheb1 in monocytes/macrophages differentiation and maturation is not clear. Here, we investigate the role of Rheb1 in mouse myelopoiesis using a Rheb1 conditional deletion murine model. We found that the absolute number of macrophages decreased in the bone marrow (BM) of Rheb1-deficient mice. Loss of Rheb1 inhibited the monocyte-to-macrophage differentiation process. Additionally, Rheb1 deletion reduced phagocytosis ability of macrophages by inhibiting the mTORC1 signaling pathway. Furthermore, 3BDO (an activator of mTORC1) rescued the phagocytosis ability of Rheb1-deficient macrophages. Thus, Rheb1 is critical for macrophage production and phagocytosis and executes these activities possibly via mTORC1-dependent pathway. [ABSTRACT FROM AUTHOR]

Here we report that focal adhesion kinase (FAK) is required for optimal signalling to the Akt-p70S6K-S6 pathway in squamous cell carcinoma (SCC) cells. Specifically, in SCCs that are genetically deficient for FAK, there is reduced phosphorylation of Akt, p70S6K and S6, and signalling to Akt-p70S6K-S6 is more sensitive to inhibition by multiple agents that suppress the pathway. By contrast, mTOR is unaffected. Indeed, pharmacological agents that inhibit the Akt-p70S6K-S6 pathway, and PDK1 that lies upstream of Akt, also impair the autophagic targeting of activated c-Src (p-Src) in FAK deficient cells. This is associated with loss of a complex between p-Src and the autophagy protein LC3, a biochemical surrogate of impaired Src-selective autophagy. In keeping with a vital role for p70S6K, inhibition by a selective inhibitor and specific siRNA also impaired Src-selective autophagy. Finally, components of the PDK1-Akt-p70S6K signalling pathway were co-located with p-Src at autophagosomes, and Src and p70S6K co-exist in the same biochemical complex. We therefore deduce that the FAK-regulated signalling module PDK1-Akt-p70S6K that controls Src's intracellular trafficking operates at Src-containing autophagosomes. [ABSTRACT FROM AUTHOR]

Protein kinase D1 (PKD1) is increasingly implicated in multiple biological and molecular events that regulate the proliferation or invasiveness in several cancers. However, little is known about the expression and functions of PKD1 in non-small cell lung cancer (NSCLC). In the present study, 34 pairs of human NSCLC and matched normal bronchiolar epitheliums were enrolled and evaluated for PKD1 expression by quantitative real-time PCR. We showed that PKD1 was downregulated in 26 of 34 cancer tissues in comparison with matched normal epitheliums. Moreover, patients with venous invasion or lymph node metastasis showed significant lower expression of PKD1. Exposure of NSCLC A549 and H520 cells to the PKD family inhibitor kb NB 142-70(Kb), at concentrations that inhibited PKD1 activation, strikingly potentiated S6K1 phosphorylation at Thr 389 and S6 phosphorylation at Ser 235/236 in response to phorbol ester (PMA). Knockdown of PKD1 with siRNAs strikingly enhanced S6K1 phosphorylation whereas constitutively active PKD1 resulted in the S6K1 activity inhibition. Furthermore, the PI3K inhibitors LY294002, BKM120 and MEK inhibitors U0126, PD0325901 blocked the enhanced S6K1 activity induced by Kb. Collectively, our results identify decreased expression of the PKD1 as a marker for NSCLC and the loss of PKD1 expression increases the malignant potential of NSCLC cells. This may be due to the function of PKD1 as a negative regulator of mTORC1-S6K1. Our results suggest that re-expression or activation of PKD1 might serve as a potential therapeutic target for NSCLC treatment. [ABSTRACT FROM AUTHOR]

Although the mechanisms that contribute to the development of traumatic brain injury (TBI)-related deficits are not fully understood, it has been proposed that altered energy utilization may be a contributing factor. The tuberous sclerosis complex, a heterodimer composed of hamartin/Tsc-1 and tuberin/Tsc-2, is a critical regulatory node that integrates nutritional and growth signals to govern energy using processes by regulating the activity of mechanistic Target of Rapamycin complex 1 (mTORC1). mTORC1 activation results in enhanced protein synthesis, an energy consuming process. We show that mice that have a heterozygous deletion of Tsc2 exhibit elevated basal mTORC1 activity in the cortex and the hippocampus while still exhibiting normal motor and neurocognitive functions. In addition, a mild closed head injury (mCHI) that did not activate mTORC1 in wild-type mice resulted in a further increase in mTORC1 activity in Tsc2+/KO mice above the level of activity observed in uninjured Tsc2+/KO mice. This enhanced level of increased mTORC1 activity was associated with worsened cognitive function as assessed using the Morris water maze and context discrimination tasks. These results suggest that there is a threshold of increased mTORC1 activity after a TBI that is detrimental to neurobehavioral performance, and interventions to inhibit excessive mTORC1 activation may be beneficial to neurocognitive outcome. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50 μM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin complex 1 ( mTORC1) is an intracellular kinase complex that regulates energy homeostasis and transcription. Modulation of mTORC1 has proven beneficial in experimental spinal cord injury, making this molecular target a candidate for therapeutic intervention in spinal cord injury. However, both inactivation and activation of mTORC1 have been reported beneficial for recovery. To obtain a more complete picture of mTORC1 activity, we aimed to characterize the spatiotemporal activation pattern of mTORC1 and identify activation in particular cell types after contusion spinal cord injury in rats. To be able to provide a spatial characterization of mTORC1 activation, we monitored activation of downstream target S6. We found robust mTORC1 activation both at the site of injury and in spinal segments rostral and caudal to the injury. There was constitutive mTORC1 activation in neurons that was biphasically reduced caudally after injury. We found biphasic mTORC1 activation in glial cells, primarily activated microglia/macrophages. Furthermore, we found mTORC1 activation in proliferating cells, suggesting this may be a function affected by mTORC1 modulation. Our results reveal potential windows of opportunity for therapeutic interference with mTORC1 signaling and immune cells as targets for inhibition of mTORC1 in spinal cord injury. [ABSTRACT FROM AUTHOR]

Objectives EGFR inhibitors are ineffective against most EGFR wild-type non-small cell lung cancer, for which novel treatment strategies are needed. AKT signalling is essential for mediating EGFR survival signals in NSCLC. We evaluated the combination of gefitinib and two different AKT inhibitors, the allosteric inhibitor AKTi-1/2 and the ATP-competitive pan-AKT inhibitor AZD5363, in EGFR-mutant (HCC-827 and PC-9) and -wild-type (NCI-H522, NCI-H1651), non-small cell lung cancer cell lines. Materials and methods Drug interaction was studied in two EGFR mutant and two EGFR wild-type non-small cell lung cancer cell lines by calculating combination index (CI) using median effect analysis. The effects on p-EGFR, p-ERK, p-AKT, p-S6 and apoptosis were studied by Western blot analysis. Results The combination of gefitinib and AKTi-1/2 or AZD5363 showed synergistic growth inhibition in all cell lines. CI values for the combination of gefitinib and AKTi-1/2 were 0.35 (p=0.0048), 0.56 (p=0.036), 0.75 (p=0.13) and 0.64 (p=0.0003) in NCI-H522, NCI-H1651, HCC-827 and PC-9 cell lines, respectively; CI values of 0.45 (p=0.0087) and 0.22 (p<0.0001) were observed in NCI-H522 and PC-9 cells, respectively, when gefitinib was combined with AZD5363. Additive inhibition of signalling output through AKT and key downstream proteins (S6) and increased apoptosis were demonstrated. Conclusion Dual inhibition of EGFR and AKT may be a useful up-front strategy for patients with EGFR-mutant and -wild-type non-small cell lung cancer. [ABSTRACT FROM AUTHOR]

The altered wiring of signaling pathways downstream of antigen receptors of T and B cells contributes to the dysregulation of the adaptive immune system, potentially causing immunodeficiency and autoimmunity. In humans, the investigation of such complex systems benefits from nature's experiments in patients with genetically defined primary immunodeficiencies. Disturbed B-cell receptor (BCR) signaling in a subgroup of common variable immunodeficiency (CVID) patients with immune dysregulation and expanded T-bethighCD21low B cells in peripheral blood has been previously reported. Here, we investigate PI3K signaling and its targets as crucial regulators of survival, proliferation and metabolism by intracellular flow cytometry, imaging flow cytometry and RNAseq. We observed increased basal but disturbed BCR-induced PI3K signaling, especially in T-bethighCD21low B cells from CVID patients, translating into impaired activation of crucial downstream molecules and affecting proliferation, survival and the metabolic profile. In contrast to CVID, increased basal activity of PI3K in patients with a gain-of-function mutation in PIK3CD and activated PI3K delta syndrome (APDS) did not result in impaired BCR-induced AKT-mTOR-S6 phosphorylation, highlighting that signaling defects in B cells in CVID and APDS patients are fundamentally different and that assessing responses to BCR stimulation is an appropriate confirmative diagnostic test for APDS. The active PI3K signaling in vivo may render autoreactive T-bethighCD21low B cells in CVID at the same time to be more sensitive to mTOR or PI3K inhibition. [ABSTRACT FROM AUTHOR]

Highlights: [&#x2022;] Ferulic acid protects brain tissues against cerebral ischemic injury. [&#x2022;] Ferulic acid prevents brain injury-induced decrease of phospho-mTOR. [&#x2022;] Ferulic acid prevents brain injury-induced decrease of phospho-p70S6 kinase. [&#x2022;] Ferulic acid prevents brain injury-induced decrease of phospho-S6. [ABSTRACT FROM AUTHOR]

Targeted therapy in advanced clear cell renal cell carcinomas (RCC) is now an established modality. The latter is in stark contrast to non-clear cell subtypes. We explored the translational support for the use of antagonists of the mammalian target of rapamycin (mTOR) and the vascular endothelial growth factor pathways in chromophobe RCC. The immunoexpression of PTEN, phos-AKT, phosphorylated S6 (phos-S6), 4EBP1, p27, c-MYC, and HIF-1a was evaluated in 33 patients with chromophobe RCC who were treated by partial/radical nephrectomy without adjuvant therapy. PTEN was lower in tumor than in normal kidney (P < .001), and loss of PTEN expression was found in 67% of the tumors. In tumor tissues, phos-S6 and 4EBP1 were higher than in normal kidney (P ≤ .005). Conversely, scores of p27 were lower in tumor than in normal kidney (P < .001). Finally, scores of phos-AKT, c-MYC, and HIF-1a were not significantly different in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 9% and 0%, respectively. Multifocal tumors had higher levels of PTEN, phos-AKT, and HIF-1a (P ≤ .01). None of the clinicopathologic variables (age, ethnicity, gender, pT stage, tumor size, multifocality, and positive surgical margins) was associated with outcome. Similarly, none of the tested biomarkers predicted overall mortality, either in unadjusted or adjusted Cox regression models. In summary, our study provides new evidence of dysregulation of the mTOR pathway in chromophobe RCC. Immunohistochemistry for mTOR pathway and hypoxiainduced pathway members lacked prognostic significance in our cohort. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The IGF system exerts systemic and local actions during development. We previously demonstrated that fetal cerebral cortical IGF1 is reduced at 0.5 gestation in our IUGR baboon nonhuman primate model. We hypothesized that by term protein expression of several key IGF system stimulatory peptide pathway components and downstream nutrient signaling effectors of IGF, mammalian target of rapamycin (mTOR) and S6, would decrease, indicating reduced cellular nutrient uptake and protein synthesis. Design: We fed 7 control baboons ad libitum while 6 baboons ate a globally reduced diet (70% of feed eaten by controls) from 0.16 gestation through pregnancy that produces IUGR. Fetuses were removed at Cesarean section at 0.9 gestation. Frontal cortex sections were stained for IGFI, IGFII, IGFRI, IGFR2, IGFBP2, 3, 5 and 6, and mTOR and ribosomal protein S6 and double stained with NeuN a neuron-specific nuclear antigen. Results: All proteins stained neuronal cytoplasm except IGFRI which showed only glial cell cytoplasmic and blood vessel staining. IUGR fetuses showed decreased frontal cortical immunoreactive IGFI, IGFII, IGFRI, IGFBP2, 5 and 6, and mTOR and S6 (p<0.05). IGFBP3 increased (p<0.05) and IGFR2 was unchanged (p>0.05). There were no differences between male and female fetal brains. Conclusions: When fetal nutrient availability is decreased, IUGR down regulates the IGF system and its mTOR signaling pathway in the fetal frontal cortex coincident with slowed growth. These findings emphasize the importance of the local tissue IGF system in fetal primate brain development. [Copyright &y& Elsevier]

Abstract: Intracerebroventricular (ICV) injection of ouabain, a specific Na/K-ATPase inhibitor, induces behavioral changes in rats in a putative animal model of mania. The binding of ouabain to Na/K-ATPase affects signaling molecules in vitro, including ERK1/2 and Akt, which promote protein translation. We have also reported that ERK1/2 and Akt in the brain are involved in the ouabain-induced hyperactivity of rats. In this study, rats were given an ICV injection of ouabain, and then their frontal cortices were examined to determine the effects of ouabain on the mTOR/p70S6K/S6 signaling pathway and protein translation, which are important in modifications of neural circuits and behavior. Rats showed ouabain-induced hyperactivity up to 8h following injection, and increased phosphorylation levels of mTOR, p70S6K, S6, eIF4B, and 4E-BP at 1, 2, 4, and 8h following ouabain injection. Immunohistochemical analyses revealed that increased p-S6 immunoreactivity in the cytoplasm of neurons by ouabain was evident in the prefrontal, cingulate, and orbital cortex. These findings suggested increased translation initiation in response to ouabain. The rate of protein synthesis was measured as the amount of [3H]-leucine incorporation in the cell-free extracts of frontal cortical tissues, and showed a significant increase at 8h after ouabain injection. These results suggest that ICV injection of ouabain induced activation of the protein translation initiation pathway regulated by ERK1/2 and Akt, and prolonged hyperactivity in rats. In conclusion, protein translation pathway could play an important role in ouabain-induced hyperactivity in a rodent model of mania. [Copyright &y& Elsevier]

Abstract: Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5mg/mL. We name this threshold concentration C FR, the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C FR conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions. [Copyright &y& Elsevier]

Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictornull cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCɑ protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation. [ABSTRACT FROM AUTHOR]

Dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways has been consistently identified in clear cell renal cell carcinomas. However, experience with non-clear cell renal cell carcinoma subtypes is scant. In this study, we evaluated the immunohistochemical expression of upstream (PTEN and phosphorylated AKT) and downstream (phosphorylated S6 and 4EBP1) effectors of the mammalian target of rapamycin pathway, as well as related cell-cycle proteins (p27 and c-MYC), and a member of the hypoxia-induced pathway (HIF-1a) in 54 patients with papillary renal cell carcinoma treated by nephrectomy. PTEN was lower in tumor than in normal kidney, and loss of PTEN expression was found in 48% of the patients. In tumor tissues, phosphorylated S6, 4EBP1, and HIF-1a were higher than in normal kidney. Conversely, scores of p27 were lower in tumor than in normal kidney. Finally, scores of c-MYC and phosphorylated AKT were similar in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 24% and 11%, respectively. Tumor progression was observed in 17% of the patients. None of the tested biomarkers predicted cancer-specific mortality or tumor progression. As expected, patients with high T-stage tumors had higher hazard ratios for cancer-specific mortality (hazard ratio, 6.9) and tumor progression (hazard ratio, 6.7). Patients with higher Fuhrman grades also had higher risks for cancer-specific mortality (hazard ratio, 11.4) and tumor progression (hazard ratio, 4.5). In summary, our study provides evidence of dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways in papillary renal cell carcinoma. Immunohistochemistry for members of the mammalian target of rapamycin pathway and for HIF-1a lacked prognostic significance in our cohort. [ABSTRACT FROM AUTHOR]

The protein kinase p70 S6K1 is regulated in response to cytokines, nutrients and growth factors, and plays an important role in the development of a variety of human diseases. Mammalian target of rapamycin (mTOR) is known to phosphorylate and thereby activate p70 S6K1. p70 S6K1 phosphorylates different cytoplasmic and nuclear substrates involved in the regulation of protein synthesis, cell cycle, cell growth and survival. Recently, we have shown that mTOR-mediated phosphorylation of p70 S6K1 at T389 also regulates its nucleocytoplasmic localization. Since this phosphorylation is associated with its kinase activity the question whether p70 S6K1 phosphorylation or kinase activity is essential for its proper localization remained elusive. Recently, the chemical compound PF-4708671 has been demonstrated to block p70 S6K1 kinase activity while inducing its phosphorylation at T389. This potential of PF-4708671 to separate p70 S6K1 activity from its T389 phosphorylation allowed us to demonstrate that the proper nucleocytoplasmic localization of this kinase depends on its mTOR-mediated phosphorylation but not on its kinase activity. These findings provide important insights into the regulation of p70 S6K1 and allow a more detailed understanding of subcellular enzyme localization processes. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Uterine leiomyosarcoma generally has an unfavorable response to standard chemotherapy. The loss of PTEN which results in constitutive AKT-mTOR activation causes an increase in leiomyosarcoma formation in mice. The active ingredient derived from the herb Curcuma longa, curcumin, shows antitumor properties in a variety of cancer cell lines by altering a number of oncogenic pathways. To explore the possibility of curcumin as an alternative to standard chemotherapy, we decided to investigate curcumin''s antitumor effect on uterine leiomyosarcoma cells. Methods: Human leiomyosarcoma cell lines, SKN and SK-UT-1, were cultured for in vitro experiments. Rapamycin or curcumin was added in different doses and their effect on cell growth was detected by MTS assay. The influence of rapamycin or curcumin on AKT, mTOR, p70S6 and S6 phosphorylation and protein expression was detected by Western Blotting. The ability of rapamycin or curcumin to induce apoptosis was determined by Western blotting using cleaved-PARP specific antibody, Caspase-3 activity assay and TUNEL assay. Results: Both rapamycin and curcumin significantly reduced SKN cell proliferation. Curcumin inhibited mTOR, p70S6 and S6 phosphorylation similar with rapamycin. Cleaved PARP, caspase-3 activity and DNA fragmentation increased proportional with curcumin concentration. At a high concentration, curcumin significantly induced apoptosis in SKN cells, but not rapamycin. Conclusions: Curcumin inhibited uterine leiomyosarcoma cells'' growth by targeting the AKT-mTOR pathway for inhibition. However, rapamycin, a specific mTOR inhibitor, did not induce apoptosis in SKN cells unlike curcumin that also has a pro-apoptotic potential in SKN cells. [Copyright &y& Elsevier]

Background: The mammalian target of rapamycin (mTOR) pathway plays an important role in the regulation of cell growth and increasing evidence suggests its dysregulation in tumors. It also implements many other critical cellular functions, including protein degradation and angiogenesis. To date, a correlation between the mTOR pathway in human glioma and patients'' prognosis has not been reported. Methods: To address this question, we carried out an immunohistochemical study of the mTOR upstream and downstream targets phosphorylated Akt (pAkt), phosphorylated S6 ribosomal protein (pS6), and p27, as well as phosphatase and tensin homologue (PTEN) using biopsies from 96 patients with primary glioma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. Results: Immunostaining revealed that the mTOR pathway was significantly associated with the Karnofsky performance scale (KPS) score and World Health Organization (WHO) grade of patients with glioma. Especially, the positive expression rates of pAkt, cytoplasmic p27, and pS6 were significantly higher in patients with higher grade (P = 0.002, 0.001 and 0.002) and lower KPS score (P = 0.007, 0.005, and 0.008), which were opposite to the nuclear p27 and PENT expression. Statistical analysis showed that patients with glioma expressing pAkt, PTEN, cytoplasmic p27, nuclear p27, and pS6 have different overall survival rates relative to those not expressing these proteins. Cox multi-factor analysis showed that KPS (P = 0.02), WHO grade (P = 0.005), pAkt (P = 0.009), PTEN (P = 0.006), cytoplasm p27 (P = 0.008), nuclear p27 (P = 0.01), and pS6 (P = 0.003) were independent prognosis factors for human glioma. Conclusion: These results provide convincing evidence for the first time that the mTOR pathway correlated closely with overall survival of patients with glioma and might be a novel prognostic marker. [Copyright &y& Elsevier]

Abstract: Circadian (24-h) rhythms influence virtually every aspect of mammalian physiology. The main rhythm generation center is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and work over the past several years has revealed that rhythmic gene transcription and post-translational processes are central to clock timing. In addition, rhythmic translation control has also been implicated in clock timing; however the precise cell signaling pathways that drive this process are not well known. Here we report that a key translation activation cascade, the mammalian target of rapamycin (mTOR) pathway, is under control of the circadian clock in the SCN. Using phosphorylated S6 ribosomal protein (pS6) as a marker of mTOR activity, we show that the mTOR cascade exhibits maximal activity during the subjective day, and minimal activity during the late subjective night. Importantly, expression of S6 was not altered as a function of circadian time. Rhythmic S6 phosphorylation was detected throughout the dorsoventral axis of the SCN, thus suggesting that rhythmic mTOR activity was not restricted to a subset of SCN neurons. Rather, rhythmic pS6 expression appeared to parallel the expression pattern of the clock gene period1 (per1). Using a transgenic per1 reporter gene mouse strain, we found a statistically significant cellular level correlation between pS6 and per1 gene expression over the circadian cycle. Further, photic stimulation triggered a coordinate upregulation of per1 and mTOR activation in a subset of SCN cells. Interestingly, this cellular level correlation between mTOR activity and per1 expression appears to be specific, since a similar expression profile for pS6 and per2 or c-FOS was not detected. Finally, we show that mTOR activity is downstream of the ERK/MAPK signal transduction pathway. Together these data reveal that mTOR pathway activity is under the control of the SCN clock, and suggests that mTOR signaling may contribute to distinct aspects of the molecular clock timing process. [Copyright &y& Elsevier]

Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells. [ABSTRACT FROM AUTHOR]

The ribosomal protein S6 is essential for the formation of the subunits of higher eukaryotic ribosomes, and S6 heterozygosity leads to early embryonal lethality in mice. S6 is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. So far, the S6Kinases, S6K1 and S6K2 are the only proven S6 S240/244 phosphorylating enzymes in mammalian cells. The activity of these S6Kinases is strictly regulated via the mammalian target of rapamycin (mTOR) enzyme complex with raptor, named mTORC1. In time course experiments with the mTORC1 inhibitor rapamycin we here demonstrate rapamycin-resistant phosphorylation of the ribosomal protein S6 at S240/244. Serum-restimulation experiments further demonstrated that this rapamycin-resistant S6 240/244 phosphorylation is induced via serum factors in a cell cycle-dependent manner. Our data allow new insights into the regulation of S6 phosphorylation and provide evidence for the existence of rapamycin-resistant S6 phosphorylating kinase activities. [ABSTRACT FROM AUTHOR]

EGb 761 is an extract of Gingko biloba that exhibits neuroprotective effects against cerebral ischemia. The mammalian target of rapamycin (mTOR) is a critical downstream effector of Akt and a central regulator of ribosomal biogenesis and protein synthesis. We investigated whether EGb 761 regulates Akt downstream targets, including mTOR, p70S6 kinase, and S6 phosphorylation. Adult male rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24 hours after MCAO and the cerebral cortex regions were examined. We previously showed that EGb 761 significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Ischemic brain injury induces a decrease in Akt up-stream target, PDK1 phosphorylation. The levels of phospho-mTOR, phospho-p70S6 kinase, and phospho-S6 are subsequently decreased in regions affected by ischemic injury. However, EGb 761 prevented injury-induced decreases in these protein levels. We confirmed that EGb 761 inhibits injury-induced decreases in the number of positive cells for phospho-p70S6 kinase and phospho-S6. The results of this study provide evidence that EGb 761 protects neuronal cells against ischemic brain injury by preventing injury-induced decreases in p70S6 kinase and S6 phosphorylation. [ABSTRACT FROM AUTHOR]

The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96-residue P54-55, and the structure of its 101-residue parent protein S6wt in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6wt, the overall solution structures of both P54-55 and S6wt show a β-sheet of four antiparallel β-strands with two α-helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6wt reveals a disordered loop in the region between β-strands 2 and 3 (Leu43-Phe60) instead of a well-ordered stretch and associated hydrophobic mini-core observed in the crystal structure. Moreover, the data for P54-55 show that the joined wild-type N- and C-terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6wt structure is relatively insensitive to mutational perturbations, and why P54-55 is more stable than S6wt: the permutant incision at Lys54-Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild-type N- and C-termini is stabilizing. [ABSTRACT FROM AUTHOR]

Abstract: Using B3PW91/6-31+G(3d) method based on the density functional theory (DFT) we have investigated the potential energy hypersurface (PES) of hexasulphur (S6). Eight stable isomeric structures are located and connected by eight transition states on the PES. Compared with previous theoretical results, the results of our method are in better agreement with the experimental ones. Especially, we have also studied two possible dissociation channels of S6 based on the rule of bond breaking in unimolecular reaction [R.G. Pearson, Symmetry Rules for Chemical Reaction, Orbital Topology and Elementary Processes, A Wiley–Interscience Publication, 1976]. One is from the chair-shaped structure to 2S3 through a transition state and the other is that the prism-shaped isomer dissociate to 3S2 directly. [Copyright &y& Elsevier]

Abstract: Germline mutations in LKB1 cause Peutz–Jeghers syndrome (PJS), an autosomal dominant disorder with a predisposition to gastrointestinal polyposis and cancer. Hyperactivation of mTOR-signaling has been associated with PJS. We previously reported that rapamycin treatment of Lkb1 +/− mice after the onset of polyposis reduced the polyp burden. Here we evaluated the preventive efficacy of rapamycin on Peutz–Jeghers polyposis. We found that rapamycin treatment of Lkb1 +/− mice initiated before the onset of polyposis in Lkb1 +/− mice led to a dramatic reduction in both polyp burden and polyp size and this reduction was associated with decreased phosphorylation levels of S6 and 4EBP1. Together, these findings support the use of rapamycin as an option for chemoprevention and treatment of PJS. [Copyright &y& Elsevier]