Activation Of S6 Kinase Research Articles

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.

Fermented ginseng extract, BST204, disturbs adipogenesis of mesenchymal stem cells through inhibition of S6 kinase 1 signaling

BackgroundThe biological and pharmacological effects of BST204, a fermented ginseng extract, have been reported in various disease conditions. However, its molecular action in metabolic disease remains poorly understood. In this study, we identified the antiadipogenic activity of BST204 resulting from its inhibition of the S6 kinase 1 (S6K1) signaling pathway. MethodsThe inhibitory effects of BST204 on S6K1 signaling were investigated by immunoblot, nuclear fractionation, immunoprecipitation analyses. The antiadipogenic effect of BST204 was evaluated by measuring mRNA levels of adipogenic genes and by chromatin immunoprecipitation and quantitative real-time polymerase chain reaction analysis. ResultsTreatment with BST204 inhibited activation and nuclear translocation of S6K1, further decreasing the interaction between S6K1 and histone H2B in 10T1/2 mesenchymal stem cells. Subsequently, phosphorylation of H2B at serine 36 (H2BS36p) by S6K1 was reduced by BST204, inducing an increase in the mRNA expression of Wnt6, Wnt10a, and Wnt10b, which disturbed adipogenic differentiation and promoted myogenic and early osteogenic gene expression. Consistently, BST204 treatment during adipogenic commitment suppressed the expression of adipogenic marker genes and lipid drop formation. ConclusionOur results indicate that BST204 blocks adipogenesis of mesenchymal stem cells through the inhibition of S6K1-mediated histone phosphorylation. This study suggests the potential therapeutic strategy using BST204 to combat obesity and musculoskeletal diseases.

DPP-4 Inhibitor Sitagliptin Improves Cardiac Function and Glucose Homeostasis and Ameliorates β-Cell Dysfunction Together with Reducing S6K1 Activation and IRS-1 and IRS-2 Degradation in Obesity Female Mice.

Background Chronic overnutrition leads to cardiac dysfunction and insulin (INS) resistance. Dipeptidyl peptidase-4 (DPP-4) improves glucose metabolism and insulin sensitivity in both human and animal models. In this study, we explored whether DPP-4 inhibitor sitagliptin (SIT) is involved in the protection of cardiac function and β-cell function using an obesity female mouse model. Methods Six-week-old C57BL6/J mice were fed a high fat and fructose Western diet with DPP-4 inhibitor SIT for 12 weeks. Cardiac function was examined by echocardiography. Body weight, plasma glucose, and insulin concentrations were measured. The contents of total S6 kinase 1 (S6K1), phosphorylation of S6K1 activation, and INS docking proteins INS receptor substrates 1 and 2 (IRS-1, IRS-2) were assayed, and histology of heart tissue was performed. Results Chronic Western diet consumption elevated plasma glucose and insulin and caused obesity, diastolic dysfunction, and β-cell dysfunction. DPP-4 inhibition with SIT resulted in reduction in body weight, fasting glucose, and plasma insulin, and improved cardiac diastolic dysfunction. SIT also decreased mTOR/S6K1 activation and prevented the degradation of IRS-1 and IRS-2. Conclusions This study revealed pleiotropic protective effects of DPP-4 inhibitor SIT on cardiac function, glycemia, and β-cell function together with reducing S6K1 activation and IRS-1 and IRS-2 degradation in the obesity female mouse model.

Abstract 1828: Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers

Abstract Background: Tumor genomic profiling has identified patients with cancers harboring activating ERBB2 (HER2) mutations that are sensitive to HER2 targeted therapies. In the SUMMIT phase II ‘basket' trial, a subset of patients with ERBB2 mutant cancers have exhibited significant clinical benefit from treatment with the pan-HER irreversible tyrosine kinase inhibitor (TKI) neratinib. However, durable responses to neratinib are few, suggesting mechanisms of de novo and acquired drug resistance. Thus, we sought to identify druggable mechanisms of resistance to neratinib. Methods: We utilized 5637 bladder cancer (with HER2S310F) and OVCAR8 ovarian cancer (with HER2G776V) cells. Drug resistant cells were developed by exposing cells to increasing concentrations of neratinib over 6 months (5637, 600 nM; OVCAR8, 2 µM). Neratinib resistant H1781 lung cancer cells (with HER2G776>VC) and MCF7 breast cancer cells (with L755S or V777L) knock-in mutations are currently being developed. For immunoblot and drug sensitivity assays, neratinib resistant cells were maintained drug-free for 96 hours and then retreated with neratinib and other inhibitors. Candidate pathways/genes driving neratinib resistance were identified by performing RNA sequencing and whole exome sequencing in drug-resistant and -sensitive cells. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis of both cells treated with neratinib showed effective suppression of HER2, EGFR and HER3 phosphorylation. However, they exhibited a striking increase in S6 kinase (S6K) activity and S6 phosphorylation compared to drug-sensitive parental cells, which was maintained in the presence of supra-pharmacological levels of neratinib (1 µM). S6 phosphorylation and viability of drug resistant cells was completely ablated by the combination of neratinib and the mTORC1 inhibitor everolimus, but not with the PI3Kα inhibitor alpelisib, the pan-PI3K inhibitor buparlisib, or the AKT inhibitor MK-2206, suggesting PI3K- and AKT-independent activation of mTORC1. Gene set enrichment analysis (GSEA) of RNA seq data from the drug-resistant cells revealed significant enrichment of K-Ras pathway components in addition to mTORC1 pathway. Consistent with these results, whole exome sequencing revealed activating alterations of the Ras pathway including a truncating mutation in RASA2 and a P200L mutation in PIK3CA Ras binding domain; thereby suggesting potential Ras mediated mTOR activation driving neratinib resistance. Studies are underway to confirm the contribution of Ras pathway in mTOR mediated neratinib resistance. Conclusions: These data suggest that hyperactivation of mTORC1 promotes acquired resistance to neratinib across histologically distinct ERBB2-mutant cancers. Citation Format: Dhivya R. Sudhan, Ariella B. Hanker, Angel Guerrero-Zotano, Luigi Formisano, Yan Guo, Qi Liu, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Richard Bryce, Alan Auerbach, Carlos L. Arteaga. Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1828.

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

Short- and long-term effects of leucine and branched-chain amino acid supplementation of a protein- and energy-reduced diet on muscle protein metabolism in neonatal pigs

The objective of this study was to determine if enteral leucine or branched-chain amino acid (BCAA) supplementation increases muscle protein synthesis in neonates who consume less than their protein and energy requirements, and whether this increase is mediated via the upregulation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway or the decrease in muscle protein degradation signaling. Neonatal pigs were fed milk replacement diets containing reduced energy and protein (R), R supplemented with BCAA (RBCAA), R supplemented with leucine (RL), or complete protein and energy (CON) at 4-h intervals for 9 (n = 24) or 21 days (n = 22). On days 9 and 21, post-prandial plasma amino acids and insulin were measured at intervals for 4h; muscle protein synthesis rate and activation of mTOR-related proteins were determined at 120min post-feeding in muscle. For all parameters measured, the effects of diet were not different between day 9 or day 21. Compared to CON and R, plasma leucine and BCAA were higher (P ≤ 0.01) in RL- and RBCAA-fed pigs, respectively. Body weight gain, protein synthesis, and activation of S6 kinase (S6K1), 4E-binding protein (4EBP1), and eukaryotic initiation factor 4 complex (eIF4E·eIF4G) were decreased in RBCAA, RL, and R relative to CON (P < 0.01). RBCAA and RL upregulated (P ≤ 0.01) S6K1, 4EBP1, and eIF4E·eIF4G compared to R. In conclusion, when protein and energy are restricted, both leucine and BCAA supplementation increase mTOR activation, but do not enhance skeletal muscle protein synthesis and muscle growth in neonatal pigs.

MTORC1 signaling regulates vascular endothelial function via reactive oxygen species and NFκB signaling

The mechanistic target of rapamycin complex 1 (mTORC1) is an important intracellular energy sensor that regulates protein synthesis through its downstream signaling components the S6‐kinase and the ribosomal S6 protein. Recently, our laboratory has demonstrated a critical role of the mTORC1 signaling pathway in the cardiovascular regulation with implications for obesity and hypertension. In this study, we tested the hypothesis that the mTORC1 signaling pathway in the vasculature is involved in the regulation of vascular endothelial function and that dysregulation of this pathway contributes to the cardiovascular disorders associated with obesity. We began by assessing the effects of activating mTORC1 signaling in cultured mouse lung endothelial cells (MLECs) using leucine (10 mM; 16hrs) or a constitutively active S6‐kinase adenoviral construct (Ad‐S6KCA; 48hrs). Activated mTORC1 signaling was confirmed by the increased phosphorylated levels of the ribosomal S6 protein (2–4 fold; p&lt;0.05). Increasing mTORC1 signaling elevated mRNA expression of oxidative stress markers (NOX1 and NOX2; 1.5–5 fold; p&lt;0.05), decreased mRNA expression of superoxide dismutase 2 (0.5 fold; p&lt;0.05) and increased reactive oxygen species (ROS) generation (via dihydroethidium staining; 1.5 fold; p&lt;0.05) in MLECs demonstrating a pro‐oxidant gene environment evoked by activation of mTORC1 signaling. Blockade of the IKKβ subunit of the NFκB transcriptional complex (BMS‐345541; 300nM) prevented mTORC1 signaling induced ROS generation in MLECs demonstrating a critical role of NFκB signaling in the cellular response to mTORC1 activation. Next, we used leucine and Ad‐S6KCA to test the consequence of enhancing mTORC1 signaling on endothelial function in aortic rings isolated from wildtype mice. Both leucine and an adenovirus expressing a constitutively active form of S6K (Ad‐S6KCA) impaired endothelial dependent acetylcholine‐induced relaxation (~10–15%; p&lt;0.05) without changing endothelial‐independent relaxation responses evoked by sodium nitroprusside indicating endothelial but not smooth muscle dysfunction in response to increased mTORC1 signaling. Blockade of mTORC1 signaling using a dominant negative S6K adenoviral construct (Ad‐S6KDN) as well as inhibition of ROS signaling by Tempol (non‐specific free radical scavenger) ameliorated mTORC1‐induced endothelial dysfunction (p&lt;0.05). To determine the involvement of mTORC1 signaling in the endothelial dysfunction associated with obesity, we utilized diet‐induced obese mice that display vascular endothelial dysfunction as compared to lean controls. Obese mice exhibited increased mTORC1 signaling in the aorta and mesenteric artery indicated by the elevated phosphorylated levels of the ribosomal S6 protein (1.5–3 fold; p&lt;0.05) and this was associated with increases in NOX2 expression (1.5 fold; p&lt;0.05) further indicating ROS signaling in the vascular pathology associated with obesity. We conclude that mTORC1 signaling is a novel regulator of vascular endothelial function through its effects on the NFκB complex and ROS signaling. Our data also indicate that dysregulation of the mTORC1 signaling pathway may be involved in the endothelial dysfunction associated with obesity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Arginase-II activates mTORC1 through myosin-1b in vascular cell senescence and apoptosis

Type-II L-arginine:ureahydrolase, arginase-II (Arg-II), is shown to activate mechanistic target of rapamycin complex 1 (mTORC1) pathway and contributes to cell senescence and apoptosis. In an attempt to elucidate the underlying mechanism, we identified myosin-1b (Myo1b) as a mediator. Overexpression of Arg-II induces re-distribution of lysosome and mTOR but not of tuberous sclerosis complex (TSC) from perinuclear area to cell periphery, dissociation of TSC from lysosome and activation of mTORC1-ribosomal protein S6 kinase 1 (S6K1) pathway. Silencing Myo1b prevents all these alterations induced by Arg-II. By overexpressing Myo1b or its mutant with point mutation in its pleckstrin homology (PH) domain we further demonstrate that this effect of Myo1b is dependent on its PH domain that is required for Myo1b-lysosome association. Notably, Arg-II promotes association of Myo1b with lysosomes. In addition, we show that in senescent vascular smooth muscle cells with elevated endogenous Arg-II, silencing Myo1b prevents Arg-II-mediated lysosomal positioning, dissociation of TSC from lysosome, mTORC1 activation and cell apoptosis. Taken together, our study demonstrates that Myo1b mediates the effect of Arg-II in activating mTORC1-S6K1 through promoting peripheral lysosomal positioning, that results in spatial separation and thus dissociation of TSC from lysosome, leading to hyperactive mTORC1-S6K1 signaling linking to cellular senescence/apoptosis.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

Automatic Quantitative Segmentation of Myotubes Reveals Single-cell Dynamics of S6 Kinase Activation.

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.

Inhibition of Gli1‑mediated prostate cancer cell proliferation by inhibiting the mTOR/S6K1 signaling pathway

Ectopic activation of the canonical Hedgehog signaling pathway is involved in the development and progression of prostate cancer, which is one of the leading causes of cancer-associated mortality in males worldwide. However, the role of the non-canonical Hedgehog signaling pathway in prostate cancer remains generally unexplored. In the present study, it was identified that Gli (glioma-associated oncogene)1 and Gli2 were highly expressed at the protein level in the androgen-independent prostate cancer cell lines PC3 and DU145, but not in the androgen-dependent cancer cell line LNCaP. Silencing of Gli1 using small interfering RNA markedly decreased PC3 cell viability and liquid colony formation in vitro. The Gli1/2-specific inhibitor GANT61 markedly decreased cell viability by inducing cell apoptosis in PC3 and DU145 cells. GANT61 also alleviated liquid colony formation efficiency in PC3 and DU145 cells, suggesting that the activity of Gli1 is required for prostate cancer cell survival. To explore further the upstream signaling pathway involved in the regulation of Gli1 expression, it was identified that tumor necrosis factor α-triggered mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase 1 (S6K1) activation was required for Gli1 expression. Pharmacological and genetic inhibition of S6K1 activation markedly decreased Gli1 and its downstream target gene mRNA expression. In addition, the phosphoinositide 3-kinase/mTOR inhibitor BEZ235 markedly decreased in vitro PC3 cell proliferation. The results of the present study indicate that the non-canonical Hedgehog pathway (mTOR/S6K1/Gli1) contributes to the development and progression of prostate cancer and that Gli1 is a potential therapeutic target in the treatment of prostate cancer.

Male hormones activate EphA2 to facilitate Kaposi's sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi's sarcoma.

There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells.

Abstract P209: Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling Generates Reactive Oxygen Species in Mouse Endothelial Cells via NFkB Signaling

Obesity is associated with vascular endothelial dysfunction and reactive oxygen species (ROS) signaling. We have previously demonstrated a novel role for the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in the regulation of cardiovascular reactivity. In this study, we assessed the ability of mTORC1 signaling to generate ROS in mouse endothelial cells (MEC) and investigated the role of NF κ B signaling as a potential transcriptional regulator. We used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling in MEC. Infection of MEC (48hrs) with the Ad-CAS6K resulted in increased mRNA levels of NADPH oxidase 1 (3.4±0.7 fold) and 2 (3.6±0.6 fold) and decreased superoxide dismutase 2 expression (0.5±0.2 fold) compared to an adenoviral GFP (Ad-GFP) control (p&lt;0.05). Next, we used dihydroethidium staining as a measure of ROS signaling in MEC. Infection of MEC with the Ad-CAS6K enhanced ROS signaling compared to the Ad-GFP (1.5±0.1 fold; p&lt;0.05). Conversely, infection with a dominant negative S6K construct (Ad-DNS6K) decreased ROS generation (0.6±0.1; p&lt;0.05). Notably, infection of aortic rings of wildtype mice with the Ad-CAS6K resulted in reduced acetylcholine induced vasorelaxation compared to Ad-GFP (Max. relaxation: 67±5 vs. 81±3%; p&lt;0.05) with no change in relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%). The reduced endothelial-mediated vasorelaxation was rescued with Tempol (Max. relaxation: 74±2%; p&lt;0.05) indicating ROS signaling as a mechanism of mTORC1 signaling induced endothelial dysfunction. Using immunoprecipitation assays, we uncovered a physical interaction between the mTOR subunit and the IKKβ subunit of the NF κ B complex. We also found that blockade of the IKKβ subunit with BMS-345541 (300nM) prevented the increased ROS generation in MEC in response to the Ad-CAS6K (1.4±0.1 vs 0.8±0.1 fold; p&lt;0.05) implicating NF κ B in underlying mTORC1 signaling induced ROS generation. Our data demonstrate that enhanced mTORC1 signaling in endothelial cells impair endothelial function via ROS production and NF κ B signaling complex and may play a critical role in the endothelial dysfunction associated with various conditions including obesity.

Hepatic S6K1 Partially Regulates Lifespan of Mice with Mitochondrial Complex I Deficiency.

The inactivation of ribosomal protein S6 kinase 1 (S6K1) recapitulates aspects of caloric restriction and mTORC1 inhibition to achieve prolonged longevity in invertebrate and mouse models. In addition to delaying normative aging, inhibition of mTORC1 extends the shortened lifespan of yeast, fly, and mouse models with severe mitochondrial disease. Here we tested whether disruption of S6K1 can recapitulate the beneficial effects of mTORC1 inhibition in the Ndufs4 knockout (NKO) mouse model of Leigh Syndrome caused by Complex I deficiency. These NKO mice develop profound neurodegeneration resulting in brain lesions and death around 50–60 days of age. Our results show that liver-specific, as well as whole body, S6K1 deletion modestly prolongs survival and delays onset of neurological symptoms in NKO mice. In contrast, we observed no survival benefit in NKO mice specifically disrupted for S6K1 in neurons or adipocytes. Body weight was reduced in WT mice upon disruption of S6K1 in adipocytes or whole body, but not altered when S6K1 was disrupted only in neurons or liver. Taken together, these data indicate that decreased S6K1 activity in liver is sufficient to delay the neurological and survival defects caused by deficiency of Complex I and suggest that mTOR signaling can modulate mitochondrial disease and metabolism via cell non-autonomous mechanisms.

Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in KrasG12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Downregulated Translation Initiation Signaling Predisposes Low-Birth-Weight Neonatal Pigs to Slower Rates of Muscle Protein Synthesis.

Low-birth-weight (LBWT) neonates experience restricted muscle growth in their perinatal life. Our aim was to investigate the mechanisms that contribute to slower skeletal muscle growth of LBWT neonatal pigs. Twenty-four 1-day old male LBWT (816 ± 55 g) and normal-birth-weight (NBWT; 1,642 ± 55 g) littermates (n = 12) were euthanized to collect blood and longissimus dorsi (LD) muscle subsamples. Plasma glucose, insulin, and insulin-like growth factor-I (IGF-I) were lower in LBWT compared with NBWT pigs. Muscle IGF-I mRNA expression were lower in LBWT than NBWT pigs. However, IGF-I receptor mRNA and protein abundance was greater in LD of LBWT pigs. Abundance of myostatin and its receptors, and abundance and phosphorylation of smad3 were lower in LBWT LD by comparison with NBWT LD. Abundance of eukaryotic initiation factor (eIF) 4E binding protein 1 and mitogen-activated protein kinase-interacting kinases was lower in muscle of LBWT pigs compared with NBWT siblings, while eIF4E abundance and phosphorylation did not differ between the two groups. Furthermore, phosphorylation of ribosomal protein S6 kinase 1 (S6K1) was less in LBWT muscle, possibly due to lower eIF3e abundance. In addition, abundance and phosphorylation of eIF4G was reduced in LBWT pigs by comparison with NBWT littermates, suggesting translation initiation complex formation is compromised in muscle of LBWT pigs. In conclusion, diminished S6K1 activation and translation initiation signaling are likely the major contributors to impaired muscle growth in LBWT neonatal pigs. The upregulated IGF-I R expression and downregulated myostatin signaling seem to be compensatory responses for the reduction in protein synthesis signaling.

Abstract 5259: Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related mortality in the United States. Most patients present with an advanced disease and the majority die within five years, many surviving less than six months. Cytotoxic chemotherapy including Gemcitabine (Gem), FOLFIRINOX, nab-paclitaxel offer modest improvement in survival, albeit at the cost of increased side effects and unwanted toxicities. Therefore, developing novel chemotherapeutic agents for PDAC treatment is critical to improve survival. Ellagic acid/ellatitannins are abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin A (UA). Oral administration of UA has shown to be highly bioavailable and non-toxic. UA inhibits multiple kinases that are known to be involved in PDAC progression and metastasis. Therefore, we hypothesized that UA would elicit potent anti-cancer therapeutic potential in PDAC. The effect of UA on kinase activity was assessed . Inhibition of AKT (downstream of PI3K/PDK1), p70 S6 Kinase (PS6K) and STAT3 activation was quantified in PDAC cells treated with UA in dose-dependent manner. The mechanism of action was validated for UA’s activity on PI3K/PDK1, PS6K and STAT3 activation using immunoblot analysis. MiaPaCa2 cells were treated with specific inhibitors for either AKT (MK2206) or STAT3 (AZD1480) and analyzed for tumorigenicity. UA treated PDAC cells were analyzed for cell proliferation, cell invasion and colony formation. Cell cycle analysis and cell apoptosis were measured by flow cytometry. To test the efficacy of UA in vivo, cells were implanted subcutaneously in athymic nude mice. The animals received UA daily and tumor volume was measured for 5 weeks. Next, we assessed tumor growth and overall survival (OS) in PKT (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) mice, an aggressive genetically engineered PDAC mouse model, in response to UA and/or Gem treatment. Tissues from the xenografts and PKT mice treated with vehicle or UA were analyzed for cell proliferation (Ki67) and apoptosis (cleaved Caspase 3) by immunohistochemistry. High expression levels of activated STAT3 or AKT correlate with decreased survival in PDAC. UA treated MiaPaCa2 cells showed significant dose-dependent increase in apoptosis and decrease in anchorage-independent growth. UA inhibited AKT, PS6K and STAT3 signaling. As a single agent, UA effectively reduced in vivo PDAC tumor growth. Immunohistochemistry of UA treated tissues from tumor xenografts and PKT mice showed inhibition of Ki67 positive tumor cells and increased cleaved caspase 3 staining. PKT mice treated with UA showed a decrease in tumor size and an increased OS when compared to vehicle or Gem treated mice alone. These findings show that UA is a novel inhibitor/modulator/regulator for multiple signaling pathways in PDAC. These results suggest UA has potential for pre-clinical development in pancreatic cancer. Citation Format: Supriya Srinivasan, Venkatakrishna Jala, Kumar Honnenahally, Jason Castellanos, Praveen Kumar Vermula, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli. Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5259. doi:10.1158/1538-7445.AM2017-5259

Activation of a novel p70 S6 kinase 1-dependent intracellular cascade in the basolateral nucleus of the amygdala is required for the acquisition of extinction memory.

Repeated presentations of a previously conditioned stimulus lead to a new form of learning known as extinction, which temporarily alters the response to the original stimulus. Previous studies have shown that the consolidation of extinction memory requires de novo protein synthesis. However, the role of specific nodes of translational control in extinction is unknown. Using auditory threat conditioning in mice, we investigated the role of mechanistic target of rapamycin complex 1 (mTORC1) and its effector p70 S6 kinase 1 (S6K1) in the extinction of auditory threat conditioning. We found that rapamycin attenuated the consolidation of extinction memory. In contrast, genetic deletion and pharmacological inhibition of S6K1, a downstream effector of mTORC1, blocked within session extinction, indicating a role for S6K1 independent of protein synthesis. Indeed, the activation of S6K1 during extinction required ERK activation in the basolateral nucleus of the amygdala (BLA) and was necessary for increased phosphorylation of the GluA1 (Thr840) subunit of the AMPA receptor following extinction training. Mice exposed to brief uncontrollable stress showed impaired within session extinction as well as a downregulation of ERK and S6K1 signaling in the amygdala. Finally, using fiber photometry we were able to record calcium signals in vivo, and we found that inhibition of S6K1 reduces extinction-induced changes in neuronal activity of the BLA. These results implicate a novel ERK-S6K1-GluA1 signaling cascade critically involved in extinction.