Activation Of P70 S6 Kinase Research Articles

Chronic mild stress leads to anxiety-like behavior and decreased p70 S6K1 activity in the hippocampus of male mice

Major affective disorders are highly prevalent, however, current treatments are limited in their effectiveness due to a lack of understanding of underlying molecular mechanisms. Recent studies have shown that reduced activity of p70 S6 kinase 1 (S6K1), a downstream target of the mechanistic target of rapamycin complex 1 (mTORC1), is linked to anxiety-like behavior in both humans and rodents. The purpose of this study was to investigate the relationship between S6K1 and anxiety-like behavior following chronic mild stress (CMS) and drug-induced inhibition of S6K1. Following CMS, anxiety-like behavior was evaluated using an open field (OF) and elevated plus maze (EPM) in adult male C57/Bl6 mice. After behavior analysis, samples of the hippocampus were harvested for quantification of S6K1, S6 ribosomal protein, glycogen synthase kinase-3 β (GSK3β), and beta tubulin via western blot. Our results demonstrate that CMS mice exhibit anxiety-like behavior in the OF and EPM and reduced activity of S6K1 in the hippocampus (HPC). We measured phosphorylation levels of GSK3β and found that GSK3β phosphorylation was also reduced following CMS compared to control mice. Furthermore, pharmacological inhibition of S6K1 with PF-4708671 in male mice was sufficient to produce anxiety-like behavior in the OF and EPM. These results further support the significant role of S6K1 in the pathogenesis of anxiety and affective disorders.

Low rumen degradable starch promotes the growth performance of goats by increasing protein synthesis in skeletal muscle via the AMPK-mTOR pathway.

Since starch digestion in the small intestine provides more energy than digestion in the rumen of ruminants, reducing dietary rumen degradable starch (RDS) content is beneficial for improving energy utilization of starch in ruminants. The present study tested whether the reduction of rumen degradable starch by restricting dietary corn processing for growing goats could improve growth performance, and further investigated the possible underlying mechanism. In this study, twenty-four 12-wk-old goats were selected and randomly allocated to receive either a high RDS diet (HRDS, crushed corn-based concentrate, the mean of particle sizes of corn grain=1.64mm, n=12) or a low RDS diet (LRDS, non-processed corn-based concentrate, the mean of particle sizes of corn grain >8mm, n=12). Growth performance, carcass traits, plasma biochemical indices, gene expression of glucose and amino acid transporters, and protein expression of the AMPK-mTOR pathway were measured. Compared to the HRDS, LRDS tended to increase the average daily gain (ADG, P=0.054) and decreased the feed-to-gain ratio (F/G, P<0.05). Furthermore, LRDS increased the net lean tissue rate (P<0.01), protein content (P<0.05) and total free amino acids (P<0.05) in the biceps femoris (BF) muscle of goats. LRDS increased the glucose concentration (P<0.01), but reduced total amino acid concentration (P<0.05) and tended to reduce blood urea nitrogen (BUN) concentration (P=0.062) in plasma of goats. The mRNA expression of insulin receptors (INSR), glucose transporter 4 (GLUT4), L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in BF muscle, and sodium-glucose cotransporters 1 (SGLT1) and glucose transporter 2 (GLUT2) in the small intestine were significantly increased (P<0.05) in LRDS goats. LRDS also led to marked activation of p70-S6 kinase (S6K) (P<0.05), but lower activation of AMP-activated protein kinase (AMPK) (P<0.05) and eukaryotic initiation factor 2α (P<0.01). Our findings suggested that reducing the content of dietary RDS enhanced postruminal starch digestion and increased plasma glucose, thereby improving amino acid utilization and promoting protein synthesis in the skeletal muscle of goats via the AMPK-mTOR pathway. These changes may contribute to improvement in growth performance and carcass traits in LRDS goats.

Loss of glyoxalase 2 alters the glucose metabolism in zebrafish

Glyoxalase 2 is the second enzyme of the glyoxalase system, catalyzing the detoxification of methylglyoxal to d-lactate via SD-Lactoylglutathione. Recent in vitro studies have suggested Glo2 as a regulator of glycolysis, but if Glo2 regulates glucose homeostasis and related organ specific functions in vivo has not yet been evaluated. Therefore, a CRISPR-Cas9 knockout of glo2 in zebrafish was created and analyzed. Consistent with its function in methylglyoxal detoxification, SD-Lactoylglutathione, but not methylglyoxal accumulated in glo2−/− larvae, without altering the glutathione metabolism or affecting longevity. Adult glo2−/− livers displayed a reduced hexose concentration and a reduced postprandial P70–S6 kinase activation, but upstream postprandial AKT phosphorylation remained unchanged. In contrast, glo2−/− skeletal muscle remained metabolically intact, possibly compensating for the dysfunctional liver through increased glucose uptake and glycolytic activity. glo2−/− zebrafish maintained euglycemia and showed no damage of the retinal vasculature, kidney, liver and skeletal muscle. In conclusion, the data identified Glo2 as a regulator of cellular energy metabolism in liver and skeletal muscle, but the redox state and reactive metabolite accumulation were not affected by the loss of Glo2.

S6 kinase 1 at the central node of cell size and ageing.

Genetic evidence in living organisms from yeast to plants and animals, including humans, unquestionably identifies the Target Of Rapamycin kinase (TOR or mTOR for mammalian/mechanistic) signal transduction pathway as a master regulator of growth through the control of cell size and cell number. Among the mTOR targets, the activation of p70 S6 kinase 1 (S6K1) is exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant flies and mice reveals that S6K1 predominantly regulates cell size versus cell proliferation. Here we review the putative mechanisms of S6K1 action on cell size by considering the main functional categories of S6K1 targets: substrates involved in nucleic acid and protein synthesis, fat mass accumulation, retrograde control of insulin action, senescence program and cytoskeleton organization. We discuss how S6K1 may be involved in the observed interconnection between cell size, regenerative and ageing responses.

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties.

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt pathway: An experimental study using rat model.

Hypobaric hypoxic stress leads to oxidative stress, inflammation, and disturbance in protein turnover rate. Aggregately, this imbalance in redox homeostasis is responsible for skeletal muscle protein loss and a decline in physical performance. Hence, an urgent medical need is required to ameliorate skeletal muscle protein loss. The present study investigated the efficacy of ursolic acid (UA), a pentacyclic triterpene acid to ameliorate hypobaric hypoxia (HH)-induced muscle protein loss. UA is a naturally occurring pentacyclic triterpene acid present in several edible herbs and fruits such as apples. It contains skeletal muscle hypertrophy activity; still its potential against HH-induced muscle protein loss is unexplored. To address this issue, an in vivo study was planned to examine the beneficial effect of UA supplementation on HH-induced skeletal muscle loss. Male Sprague Dawley rats were exposed to HH with and without UA supplementation (20 mg/kg; oral) for 3 continuous days. The results described the beneficial role of UA as supplementation of UA with HH exposure attenuated reactive oxygen species production and oxidative protein damage, which indicate the potent antioxidant activity. Furthermore, UA supplementation enhanced Akt, pAkt, and p70S6kinase activity (Akt pathway) and lowered the pro-inflammatory cytokines in HH exposed rats. UA has potent antioxidant and anti-inflammatory activity, and it enhanced the protein content via upregulation of Akt pathway-related proteins against HH exposure. These three biological activities of UA make it a novel candidate for amelioration of HH-induced skeletal muscle damage and protein loss.

Stromal-MDM2 Promotes Lung Cancer Cell Invasion through Tumor-Host Feedback Signaling.

Tumor-host interactions play a major role in malignancies' initiation and progression. We have reported in the past that tumor cells attenuate genotoxic stress-induced p53 activation in neighboring stromal cells. Herein, we aim to further elucidate cancer cells' impact on signaling within lung cancer stroma. Primary cancer-associated fibroblasts were grown from resected human lung tumors. Lung cancer lines as well as fresh cultures of resected human lung cancers were used to produce conditioned medium (CM) or cocultured with stromal cells. Invasiveness of cancer cells was evaluated by transwell assays, and in vivo tumor growth was tested in Athymic nude mice. We found CM of a large variety of cancer cell lines as well as ex vivo-cultured lung cancers to rapidly induce protein levels of stromal-MDM2. CM of nontransformed cells had no such effect. Mdm2 induction occurred through enhanced translation, was mTORC1-dependent, and correlated with activation of AKT and p70 S6 Kinase. AKT or MDM2 knockdown in fibroblasts reduced the invasion of neighboring cancer cells, independently of stromal-p53. MDM2 overexpression in fibroblasts enhanced cancer cells' invasion and growth of inoculated tumors in mice. Our results indicate that stromal-MDM2 participates in a p53-independent cancer-host feedback mechanism. Soluble cancer-originated signals induce enhanced translation of stromal-MDM2 through AKT/mTORC1 signaling, which in turn enhances the neighboring cancer cells' invasion ability. The role of these tumor-host interactions needs to be further explored. IMPLICATIONS: We uncovered a novel tumor-stroma signaling loop, which is a potentially new therapeutic target in lung cancer and possibly in additional types of cancer.

Activation of ribosomal p70 S6 kinase by selective serotonin (5-HT)&lt;sub&gt;2B&lt;/sub&gt; receptor agonist BW723C86 is mediated by epidermal growth factor/transforming growth factor-α receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Activation of ribosomal p70 S6 kinase by selective serotonin (5-HT)&lt;sub&gt;2B&lt;/sub&gt; receptor agonist BW723C86 is mediated by epidermal growth factor/transforming growth factor-α receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Influence of level of inclusion of Azolla leaf meal on growth performance, meat quality and skeletal muscle p70S6 kinase α abundance in broiler chickens

The interest in biodiesel production from oil-bearing seeds rather than soybean necessitates the scientific validation of other good quality protein sources that could substitute soybean meal in animal diets, particularly, broiler chickens where soybean meal constitutes a large portion of their diet. Therefore, the present study was conducted to investigate the effect of sun-dried Azolla leaf meal (ALM) as an unconventional dietary protein source in broiler chicken diet on growth performance, meat quality, skeletal muscle cell growth and protein synthesis through regulation of ribosomal protein S6 kinase (p70S6 kinase α). A total of 120 male Ross 308 broiler chicks were randomly allocated to three dietary treatments. Each treatment had four cages (i.e. replicates) with 10 birds/cage. The control group was fed with a corn–soy-based diet, the AZ5 group was supplemented with 5% ALM and the AZ10 group was supplemented with 10% ALM for 37 days. A 5-day trial was also conducted to measure the apparent nutrient digestibility. Growth performance parameters were measured weekly. At the end of the experiment, 12 birds from each group (3/cage) were euthanized and used for samplings. Inclusion of ALM tended to improve BW gain (P = 0.06) and increased feed intake (P < 0.01). Additionally, ALM decreased the percentage of breast meat cooking loss linearly (P < 0.01). In addition, ALM at a dose of 5% increased the production of propionate in the cecum (P = 0.01). Activation of breast muscle p70S6 kinase was higher when ALM was included in a dose-dependent manner (P < 0.01). The inclusion of ALM increased breast meat redness (P < 0.01); however, the lightness was within the normal range in all groups. Findings from our study suggest that ALM could be included in a broiler chicken diet up to 5% without any major negative effect on meat quality or performance, and it regulates muscle protein synthesis through activation of mammalian target of rapamycin/6S kinase signaling.

MEK inhibition drives anti-viral defence in RV but not RSV challenged human airway epithelial cells through AKT/p70S6K/4E-BP1 signalling

BackgroundThe airway epithelium is a major target tissue in respiratory infections, and its antiviral response is mainly orchestrated by the interferon regulatory factor-3 (IRF3), which subsequently induces type I (β) and III (λ) interferon (IFN) signalling. Dual specificity mitogen-activated protein kinase kinase (MEK) pathway contributes to epithelial defence, but its role in the regulation of IFN response in human primary airway epithelial cells (AECs) is not fully understood. Here, we studied the impact of a small-molecule inhibitor (MEKi) on the IFN response following challenge with two major respiratory viruses rhinovirus (RV2) and respiratory syncytial virus (RSVA2) and a TLR3 agonist, poly(I:C).MethodsThe impact of MEKi on viral load and IFN response was evaluated in primary AECs with or without a neutralising antibody against IFN-β. Quantification of viral load was determined by live virus assay and absolute quantification using qRT-PCR. Secretion of cytokines was determined by AlphaLISA/ELISA and expression of interferon-stimulated genes (ISGs) was examined by qRT-PCR and immunoblotting. A poly(I:C) model was also used to further understand the molecular mechanism by which MEK controls IFN response. AlphaLISA, siRNA-interference, immunoblotting, and confocal microscopy was used to investigate the effect of MEKi on IRF3 activation and signalling. The impact of MEKi on ERK and AKT signalling was evaluated by immunoblotting and AlphaLISA.ResultsHere, we report that pharmacological inhibition of MEK pathway augments IRF3-driven type I and III IFN response in primary human AECs. MEKi induced activation of PI3K-AKT pathway, which was associated with phosphorylation/inactivation of the translational repressor 4E-BP1 and activation of the protein synthesis regulator p70 S6 kinase, two critical translational effectors. Elevated IFN-β response due to MEKi was also attributed to decreased STAT3 activation, which consequently dampened expression of the transcriptional repressor of IFNB1 gene, PRDI-BF1. Augmented IFN response translated into inhibition of rhinovirus 2 replication in primary AECs but not respiratory syncytial virus A2.ConclusionsOur findings unveil MEK as a key molecular mechanism by which rhinovirus dampens the epithelial cell’s antiviral response. Our study provides a better understanding of the role of signalling pathways in shaping the antiviral response and suggests the use of MEK inhibitors in anti-viral therapy against RV.

Signalling Effects Induced by Acid Ceramidase in Human Epithelial Or Leukemic Cell Lines.

Recent studies indicated that an inhalation treatment of cystic fibrosis mice with acid ceramidase prevents and eliminates infections with Pseudomonas aeruginosa and Stapyhlococcus aureus. Inhalation of acid ceramidase facilitated the elimination of P. aeruginosa in acutely- or chronically-infected mice with cystic fibrosis. Thus, inhalation of acid ceramidase might be a preventive and/or curative treatment for patients with cystic fibrosis suffering from pneumonia. We treated cultured epithelial cells or leukemic T-lymphocytes (Jurkat cells) with purified acid ceramidase and determined intracellular signalling events, proliferation and cell survival. Specifically, we measured the activity of AKT, p38-kinase and p70S6-kinase using activation-specific phospho-antibodies in western blot studies. Trypan Blue staining served to analyze proliferation and cell survival. Our studies indicate that treatment of Chang epithelial cells or Jurkat T lymphocytes with purified acid ceramidase results in a dose dependent activation of AKT, p38-kinase and p70S6-kinase, while tyrosine phosphorylation of intracellular proteins remains largely unchanged. Acid ceramidase treatment did not change expression of tight junction proteins such as ZO-1, ZO-2 and occludin. Cellular viability and proliferation were not affected by acid ceramidase treatment. Our data suggest that treatment of epithelial cells and lymphocytes with acid ceramidase results in activation of distinct pathways, in particular AKT- and p38K-dependent pathways, while no global activation or cell death was observed.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

Gingerenone A Sensitizes the Insulin Receptor and Increases Glucose Uptake by Inhibiting the Activity of p70 S6 Kinase.

The bioactive constituents in ginger extract are responsible for anti-hyperglycemic effects and the underlying mechanisms are incompletely understood. Gingerenone A (Gin A) has been identified as an inhibitor of p70 S6 (S6K1), a kinase that plays a critical role in the pathogenesis of insulin resistance. This study aims to evaluate if Gin A can sensitize the insulin receptor by inhibiting S6K1 activity. Western blot analysis reveals that Gin A induces phosphatidylinositide-3 kinase (PI3K) feedback activation in murine 3T3-L1 adipocytes and rat L6 myotubes, as evidenced by increased AKTS473 and S6K1T389 but decreases S6S235/236 and insulin receptor substrate 1 (IRS-1)S1101 phosphorylation. Western blot and immunoprecipitation analysis reveal that Gin A increases insulin receptor tyrosine phosphorylation in L6 myotubes and IRS-1 binding to the PI3K in 3T3-L1 adipocytes. Confocal microscopy reveals that Gin A enhances insulin-induced translocation of glucose transporter 4 (GLUT4) into the cell membrane in L6 cells. 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) Fluorescent assay reveals that Gin A enhances insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 myotubes. Gin A overcomes insulin resistance and increases glucose uptake by inhibiting S6K1 activity. Gin A or other plant-derived S6K1 inhibitors could be developed as novel antidiabetic agents.

Association of polybrominated diphenyl ethers in two fat compartments with increased risk of insulin resistance in obese individuals

BackgroundPolybrominated diphenyl ethers (PBDEs), a widely utilized class of flame retardants in various commercial products, represent a prominent source of environmental contaminants. PBDEs tend to accumulate in adipose tissue, potentially altering the function of this endocrine organ and increasing risk of insulin resistance. The aim of this study was to compare levels of PBDEs in adipose tissues from two metabolically distinct obese groups; the insulin sensitive (IS) and the insulin resistant (IR). MethodsLevels of 28 PBDE congeners were assessed in subcutaneous and omental adipose tissues from 34 obese Qatari individuals (11 IS and 23 IR) using gas chromatography (Trace GC Ultra) coupled to a TSQ Quantum triple Quadrupole mass spectrometer. Correlations of identified PBDEs and mediators of metabolic disease were established and effects of PBDEs treatment on insulin signaling in primary omental preadipocytes were determined. ResultsOut of 22 detectable PBDEs in subcutaneous and omental adipose tissues, PBDEs 28, 47, 99 and 153 were predominant in omental adipose tissues from obese Qatari subjects. PBDEs 99, 28, and 47 were significantly higher in IR individuals compared to their IS counterparts. Significant positive correlations were identified between PBDEs 28 and 99 in the omental tissues and with fasting insulin levels. When considering PBDEs congeners, penta congeners were also higher in IR compared to IS individuals, while no significant differences were detected in mono, tri, tertra, hexa, hepta and octa congeners between the two studied groups. Treatment of human omental preadipocytes from insulin sensitive individuals with PBDE28 caused inhibition of phosphorylation of GSK3 α/β (Ser21/Ser9), mTOR (Ser2448), p70 S6 kinase (Thr389) and S6 ribosomal protein (Ser235/Ser236) and activation of PTEN (Ser380) phosphorylation, suggesting inhibition of insulin signaling. ConclusionThis pilot data suggests that accumulation of specific PBDEs in human adipose tissues is associated with insulin resistance in obese individuals. Further investigation of the functional role of PBDEs in the pathology of insulin resistance should help developing therapeutic strategies targeting obese individuals at higher risk.

Extinction of auditory threat memory triggers activation of p70 S6 kinase 1 in the basolateral nucleus of the amygdala

Extinction of auditory threat memory triggers activation of p70 S6 kinase 1 in the basolateral nucleus of the amygdala

Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

Environmental enrichment improves hippocampal function in aged rats by enhancing learning and memory, LTP, and mGluR5-Homer1c activity

Previous studies from our laboratory have shown that environmental enrichment (EE) in young rats results in improved learning ability and enhanced metabotropic glutamate receptor–dependent long-term potentiation (mGluR-dependent LTP) resulting from sustained activation of p70S6 kinase. Here, we investigated whether 1-month EE is sufficient to improve hippocampus-dependent learning and memory and enhance hippocampal LTP in 23–24 month-old Fischer 344 male rats. Aged rats were housed in environmentally enriched, socially enriched, or standard housing conditions. We find that aged rats exposed to 1-month of EE demonstrate enhanced learning and memory relative to standard housed controls when tested in the Morris water maze and novel object recognition behavioral tasks. Furthermore, we find that environmentally enriched rats perform significantly better than socially enriched or standard housed rats in the radial-arm water maze and display enhanced mGluR5-dependent hippocampal LTP. Enhanced hippocampal function results from activity-dependent increases in the levels of mGluR5, Homer1c, and phospho-p70S6 kinase. These findings demonstrate that a short exposure of EE to aged rats can have significant effects on hippocampal function.

Differential impact of treadmill training on stroke-induced neurological disorders

ABSTRACTObjective: Physical exercise contributes to improving stability against nerve injury caused by ischaemic stroke. Here we aimed to preliminarily investigate the effects of continuous endurance training (CET) and high-intensity interval training (HIT) on stroke-associated anxiety, locomotion, neurological assessments and P70S6 Kinase (P70S6K) activation as well. To do this, rats were trained according to HIT and CET protocols for 2 months prior to being subject to middle cerebral artery occlusion surgery.Methods: Twenty-four hours later behavioural examination was performed by elevated plus maze (EPM) testing, open field and neurological scoring followed by cortical and hippocampal P70S6Ks immunoblotting.Results: According to the obtained data pre-ischaemic HIT and CET similarly improved neurological performance, anxiety levels and locomotion in EPM and open field tests following ischaemic stroke while there was a remarkable rise in hippocampal and cortical P70S6K activation in the HIT group compared to the CET counterparts.Conclusion: Behavioral and molecular data suggest that interval training is more beneficial rather than CET, but the distinct mechanisms of CET and HIT on memory are still topics to be discovered.

RET Signaling in Prostate Cancer.

Purpose: Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its coreceptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Because both GDNF and GFRα1 are secreted by nerves, we examined the role of RET signaling in prostate cancer.Experimental Design: Expression of RET, GDNF, and/or GFRα1 was assessed. The impact of RET signaling on proliferation, invasion and soft agar colony formation, perineural invasion, and growth in vivo was determined. Cellular signaling downstream of RET was examined by Western blotting.Results: RET is expressed in all prostate cancer cell lines. GFRα1 is only expressed in 22Rv1 cells, which is the only line that responds to exogenous GDNF. In contrast, all cell lines respond to GDNF plus GFRα1. Conditioned medium from dorsal root ganglia contains secreted GFRα1 and promotes transformation-related phenotypes, which can be blocked by anti-GFRα1 antibody. Perineural invasion in the dorsal root ganglion assay is inhibited by anti-GFRα antibody and RET knockdown. In vivo, knockdown of RET inhibits tumor growth. RET signaling activates ERK or AKT signaling depending on context, but phosphorylation of p70S6 kinase is markedly increased in all cases. Knockdown of p70S6 kinase markedly decreases RET induced transformed phenotypes. Finally, RET is expressed in 18% of adenocarcinomas and all three small-cell carcinomas examined.Conclusions: RET promotes transformation associated phenotypes, including perineural invasion in prostate cancer via activation of p70S6 kinase. GFRα1, which is secreted by nerves, is a limiting factor for RET signaling, creating a perineural niche where RET signaling can occur. Clin Cancer Res; 23(16); 4885-96. ©2017 AACR.