S6K1 Activation Research Articles

The necessary role of mTORC1 in central nervous system axon regeneration.

Permanent loss of vital functions after central nervous system (CNS) injury, e.g., blindness in traumatic optic nerve (ON) injury or paralysis in spinal cord injury, occurs in part because axons in the adult mammalian CNS do not regenerate after injury. Growth failure is due to the diminished intrinsic regenerative capacity of mature neurons and the inhibitory environment of the adult CNS. Neutralizing extracellular inhibitory molecules genetically or pharmacologically yields only limited regeneration and functional recovery, highlighting the critical importance of neuron-intrinsic factors. To explore the intrinsic regenerative signaling molecules, a relatively simple but robust in vivo model that replicates CNS traumatic injury and permits straightforward interpretation is required. Mouse retinal ganglion cell (RGC) and ON provide a valuable in vivo neural injury system to study intrinsic growth mechanisms in adult CNS neurons (Figure 1). Retina and ON are CNS structures that are essential for visual functions. RGC is the only neuronal type to relay visual information from retina to brain. The ON is formed by the projection axons sent exclusively from RGCs; it has the simplicity of a unidirectional axon pathway, which insures that any nerve fibers observed distal to a complete crush injury have regenerated and do not represent spared axons that underwent collateral sprouting or efferent axons from the brain to the retina. Its easy access allows adeno-associated viruses to be injected directly into the vitreous chamber of the eye and express transgenes specifically and efficiently in adult RGCs. This spatially and temporally controlled genetic manipulation allows us to overcome developmental issues associated with germ line manipulation and to test interventions that can potentially be translated to therapies. Exploiting the anatomical and technical advantages of the RGC/ON crush model to understand the intrinsic mechanisms of regenerative failure led us to the finding that deletion of phosphatase and tensin homolog (PTEN) promoted significant ON regeneration (Park et al., 2008). PTEN, a lipid phosphatase, is a major negative regulator of the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin complex 1 (mTORC1) pathway. Similar axon regeneration phenotypes after PTEN deletion have been reported for mouse cortical motor neurons (Liu et al., 2010), drosophila sensory neurons (Song et al., 2012) and C. elegans motor neurons (Byrne et al., 2014), presumably through activating PI3K-mTORC1-controlled cell growth (Figure 2). Direct activation of mTORC1 also promotes axon regeneration in dopaminergic neurons (Kim et al., 2011) and peripheral nerves (Abe et al., 2010), further support for the critical role of mTORC1 in axon regeneration.

Effects of resveratrol on growth and skeletal muscle physiology of juvenile southern flounder

Resveratrol is a naturally occurring antioxidant that has been widely studied in mammals due to its potential to extend lifespan. However, antioxidants may also limit protein damage and therefore reduce rates of protein degradation, providing a potential avenue for enhancing growth in an aquaculture setting. The present study tested the hypotheses that in Southern flounder, Paralichthys lethostigma, resveratrol would decrease protein carbonylation and 4-HNE (indicators of protein and lipid oxidative damage, respectively), levels of ubiquitinylation and LC3 (indicators of non-lysosomal and lysosomal protein degradation, respectively), while having no effect on S6K activation (indicator of protein synthesis). These effects were predicted to increase growth rate. Mitochondrial volume density was also examined since resveratrol may lead to the proliferation of mitochondria, which are the principal source of reactive oxygen species (ROS) that cause oxidative damage. Juvenile fish (n=142) were fed a control diet or a diet supplemented with 600μg resveratrol per g of food for 16weeks. Fish treated with resveratrol had a 9% greater length and 33% greater body mass than control fish after 16weeks. Additionally, there was lower protein carbonylation and lipid 4-HNE within the muscle tissues of treated fish, indicating decreased oxidative damage, and reduced protein ubiquitinylation in the resveratrol fed flounder, indicating less protein degradation. However, there was not a significant difference in LC3, S6K activation, or mitochondrial volume density. These results suggest that resveratrol has positive effects on growth due to its antioxidant properties that reduce non-lysosomal protein degradation.

Melittin exerts antitumorigenic effects in human MM1.S multiple myeloma cells through the suppression of AKT/mTOR/S6K1/4E-BP1 signaling cascades

Although melittin, a water-soluble 26-amino acid peptide derived from bee venom of Apis mellifera, is known to exert anti-proliferative effects on various tumor cell lines, very little is known about its potential molecular mechanism(s) of action. In the present study, we investigated the effects of melittin on the AKT/mTOR/S6K1/4E-BP1 activation, associated gene products, cellular proliferation, and apoptosis in several tumor cells. We found that melittin inhibited both constitutive phosphorylation of AKT and mTOR and exerted a significant time-dependent anti-proliferative effect on MM1.S cells as compared to other types of tumor cells. Indeed, melittin clearly suppressed the constitutive activation of AKT/mTOR/S6K1/4E-BP1 signaling cascades, which correlated with the induction of apoptosis. Melittin can cause broad-spectrum inhibition of AKT/mTOR/S6K1/4E-BP1 axes in multiple myeloma cells when compared with various pharmacological AKT/mTOR inhibitors. Aberrant AKT activation by pcDNA3-myr-HA-AKT1 plasmid could not prevent the observed suppressive effect of melittin on constitutive mTOR, S6K1, and 4E-BP1 activation and overexpression of Bcl-2 also attenuated melittin-mediated apoptosis in the cells. Our results clearly indicate that melittin can interfere with multiple signaling cascades involved in carcinogenesis and thereby used as a potential drug candidate for both the prevention and treatment of cancer.

A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis.

ABSTRACTAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions.We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression.The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.

Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. Invitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase.

Abstract 2431: Downregulation of PHLPP promotes glycolysis in colon cancer cells

Abstract PHLPP belongs to a novel family of Ser/Thr protein phosphatases. Our previous studies have shown that PHLPP can antagonize multiple signaling pathways by negatively regulating signaling molecules such as Akt, S6K, and PKC. Functionally, PHLPP serves as a tumor suppressor to inhibit cell proliferation, promote apoptosis and increase therapeutic sensitivity in colon cancer cells. Here, we further examined the role of PHLPP in regulating metabolic pathways in colon cancer. Control and PHLPP knockdown cells colon cancer cells, as well as wild-type and Phlpp knockout MEF cells, were subjected to bioenergetic phenotype analysis using Seahorse XF96 analyzer. The mitochondrial respiration measurements revealed that decreased PHLPP expression resulted in an increase in oxygen consumption rate (OCR) suggesting enhanced cellular respiration. In addition, PHLPP depletion significantly stimulated glycolysis as indicated by increased extracellular acidification rate (ECAR). Consistently, the rates of glucose consumption and lactate production were significantly increased in PHLPP knockdown cells. Furthermore, the numbers of apoptotic cells were increased in PHLPP knockdown cells compared to control cells when cultured in glucose-free medium, suggesting that loss of PHLPP expression render cells more sensitive to glucose deprivation as they became relied on glycolysis. To begin determining the underlying molecular mechanism, we found that the activity of both Akt and S6K was largely induced in PHLPP-knockdown cancer cells and PHLPP-knockout MEF cells. Since both kinases are known to play important roles in re-programming metabolic networks to favor tumor growth, it is likely that PHLPP inhibits glycolysis by negatively regulating Akt- and S6K-mediated metabolic effects. Taken together, our studies identified PHLPP as a novel regulator of glucose metabolism in colon cancer cells. Downregulation of PHLPP may promote tumorigenesis by providing metabolic advantages in colon cancer Citation Format: Yang-an Wen, Xiaopeng Xiong, Tianyan Gao. Downregulation of PHLPP promotes glycolysis in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2014-2431

Abstract LB-181: The impact of Ras/MAPK/S6K signaling pathway on prediction of clinical outcome in metastatic Her-2 positive breast cancer patients treated with trastuzumab

Abstract Background: The overexpression of Her-2 (c-erbB2/Neu) in breast cancer is associated with poor prognosis, tumor recurrence and shortened survival. The administration of the trastuzumab significantly improves patients prognosis. However, in spite of these successful results, trastuzumab is effective only in 30-50% of cases. PI3K/Akt and Ras/MAPK signaling pathways are activated through Her-2 receptor and other growth factors’ receptors and both play important role in tumor behavior. Methods: The study included 76 women with verified Her-2+ metastatic breast cancer (MBC) who were treated with trastuzumab based palliative chemotherapy. Immunohistochemistry was performed on formalin fixed, paraffin embedded tissue sections with antibodies against S6K, p-S6K-Ser235/236, MAPK, p-MAPK-Thr202/Tyr204, GSK3β, p-GSK3β-Ser9, mTOR, p-mTOR-Ser2448. The cytoplasmatic and nuclear fractions of the staining were assessed separately. Results: Patients whose tumors showed cytoplasmic (c) and nuclear (n) expression of p- GSK3β-Ser9 exhibited worse PFS compared to tumors with negative p-GSK3β-Ser9 (PFS 5,1 vs 9,1 months; P=0,006). Similar results were also found in p-S6K kinase activity, with the difference that it was possible to observe the dependence on the p-S6K kinase compartmentalization. Patients whose tumors showed p-S6K-Ser235/236 expression accompanied with only cytoplasmatic (c) or nuclear and cytoplasmatic (n+c) staining exhibited worse PFS compared to tumors with negative p-S6K-Ser235/236 expression (negat) (c vs negat: 6,3 vs 16,1 months, P=0,006; n+c vs negat: 7,8 vs 16,1 months, P=0,025). Of the remaining kinases, we showed no effect of their expression on treatment outcome. Conclusions: This study confirms that prediction of the response or resistance to trastuzumab treatment depends on the S6K and GSK3β kinase activity. Patients, whose tumors had high level of p-S6K or p-GSK3β, had poorer benefit from trastuzumab based therapy. These patients are candidates for targeted blockade of PI3K/Akt and/or RAS/MAPK signaling pathways. Supported by the Internal Grant Agency of Ministry of Health, Czech Republic, No.NT/14599 and Biomedreg CZ.1.05/2.1.00/01.0030. Citation Format: Marek Svoboda, Marta Khoylou, Peter Grell, Jiri Navratil, Pavel Fabian, Jaroslav Juracek, Marketa Palacova, Rostislav Vyzula, Marian Hajduch. The impact of Ras/MAPK/S6K signaling pathway on prediction of clinical outcome in metastatic Her-2 positive breast cancer patients treated with trastuzumab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-181. doi:10.1158/1538-7445.AM2014-LB-181

Abstract 315: Regulation of the transcription factor TFEB and the autophagic/lysosomal network by GSK3 in pancreatic cancer cells

Abstract We recently demonstrated that prolonged inhibition of GSK3 activity induces JNK-dependent cell death in human pancreatic cancer cells. Surprisingly, we observed that, concomitant to apoptosis, GSK3 inhibition also induces autophagy. Preventing this autophagic response sensitizes pancreatic cancer cells to the GSK3 inhibition-induced apoptosis suggesting a protective role for autophagy against cell death caused by prolonged inhibition of GSK3. Nevertheless, the mechanisms underlying the control of autophagy by GSK3 remain unknown. The aim of this study was to determine the molecular mechanism involved in the regulation of autophagy by GSK3 in pancreatic cancer cells. Methods: Experiments were performed in pancreatic cancer cells (PANC1, MIA PaCa2 and BxPC3), and HEK293T cells. GSK3 activity was inhibited using shRNA directed against GSK3beta and/or GSK3alpha or by using the GSK3 specific inhibitors CHIR99021 and SB216763. The JNK inhibitor SP600125 and the mTOR specific inhibitor Torin1 were also used. Results: 1) Inhibition of the JNK-cJun pathway prevented the apoptotic response but not the autophagic response upon GSK3 inhibition excluding a contribution of the JNK-cJUN pathway in the autophagy induced by GSK3 inhibition. We therefore analyzed the activity of the mTOR pathway since this pathway is well known to modulate autophagy. 2) Inhibition of GSK3 activity reduced the activity of S6K1, a direct substrate of the mTOR complex suggesting an inhibition of the mTOR pathway upon GSK3 inhibition. The mTOR pathway was recently shown to regulate autophagy through its impact on the transcription factor TFEB, a master regulator of an autophagic/lysosomal gene network. 3) Interestingly, treatment of cells with GSK3 inhibitors or a mTOR inhibitor both led to the nuclear translocation of TFEB, 4) and correlated with the expression of a lower molecular weight TFEB protein. 5) Pre-treatment of cells with the serine/threonine phosphatase inhibitor calyculin A prevented the molecular weight shift of TFEB induced upon GSK3 inhibition suggesting that GSK3 impacts on TFEB phosphorylation. 6) Furthermore, either GSK3 or mTOR inhibition correlated with increased activity of a TFEB-luciferase reporter gene, 7) and with an increased staining of lysosomes (lysotracker). 8) Interestingly, shRNA-mediated reduction of TFEB expression sensitized pancreatic cancer cells to the apoptosis induced upon GSK3 inhibition. Conclusion: Our results demonstrate that GSK3 inhibition promotes the dephosphorylation of TFEB that correlates with increased transcriptional activity of this master regulator of autophagy and lysosomal biogenesis. Furthermore, our results support a protective role against cell death assumed by TFEB upon GSK3 inhibition. Citation Format: Benoît Marchand, Alexandre Raymond-Fleury, Marie-Josée Boucher. Regulation of the transcription factor TFEB and the autophagic/lysosomal network by GSK3 in pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 315. doi:10.1158/1538-7445.AM2014-315

Transducin-like enhancer of split 3 (TLE3) in adipose tissue is increased in situations characterized by decreased PPARγ gene expression.

Transgenic overexpression of adipose tissue (AT) transducin-like enhancer of split 3 (TLE3) mimicked peroxisome proliferator-activated receptor gamma (PPARγ) agonists, improving insulin resistance in mice. This study aimed to investigate TLE3 gene expression (qRT-PCR) and protein (Western blot) in subjects with a wide spectrum of obesity and insulin sensitivity and in an independent cohort of obese subjects following surgery-induced weight loss. TLE3 was analyzed in human adipocytes and after treatment with rosiglitazone. Given the findings in humans, TLE3 was also investigated in mice after a high-fat diet (HFD) and in PPARγ knockout mice. Subcutaneous (SC) AT TLE3 was increased in subjects with type 2 diabetes (T2D). In fact, SC TLE3 was associated with increased fasting glucose (r = 0.25, p = 0.015) and S6K1 activity (r = 0.671, p = 0.003), and with decreased Glut4 (r = -0.426, p = 0.006) and IRS-1 expression (-31 %, p = 0.007) and activation (P-IRS-1/IRS-1, -17 %, p = 0.024). TLE3 was preferentially expressed in mature adipocytes and increased during in vitro differentiation in parallel to PPARγ. Weight loss led to improved insulin sensitivity, increased AT PPARγ and decreased TLE3 (-24 %, p = 0.0002), while rosiglitazone administration downregulated TLE3 gene expression in fully differentiated adipocytes (-45 %, p < 0.0001). The concept that TLE3 may act as a homeostatic linchpin in AT was also supported by its increased expression in HFD-fed mice (39 %, p = 0.013) and PPARγ knockout (74 %, p = 0.001). In summary, increased AT TLE3 in subjects with T2D and in AT from HFD-fed and PPARγ knockout mice suggest that TLE3 may play an adaptive regulatory role that improves AT function under decreased PPARγ expression. TLE3 is expressed in mature adipocytes concomitantly with PPARγ. Subcutaneous adipose TLE3 is increased in T2D patients. Adipose TLE3 is upregulated in genetically ablated PPARγ and HFD-fed mice. TLE3 may be a homeostatic linchpin in insulin resistance and defective PPARγ.

Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: a tale of two complexes.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

Acute rapamycin treatment improved glucose tolerance through inhibition of hepatic gluconeogenesis in rainbow trout (Oncorhynchus mykiss).

Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were first treated with a single intraperitoneal injection of rapamycin or vehicle and then submitted to a second intraperitoneal administration of glucose 4 h later. Our results revealed that intraperitoneal administration of glucose induced hyperglycemia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish at 8 and 17 h, whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle-treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt on Thr-308 or Ser-473. Despite the lack of significant variation in phosphoenolpyruvate carboxykinase mRNA abundance, mRNA abundance for glucokinase (GK), glucose 6-phosphatase (G6Pase) I and II, and fructose 1,6-bisphosphatase (FBPase) was reduced by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppression of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1-dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout.

Requirement of Mammalian target of rapamycin complex 1 downstream effectors in cued fear memory reconsolidation and its persistence.

Memory retrieval, often termed reconsolidation, can render previously consolidated memories susceptible to manipulation that can lead to alterations in memory strength. Although it is known that reconsolidation requires mammalian target of rapamycin complex 1 (mTORC1)-dependent translation, the specific contributions of its downstream effectors in reconsolidation are unclear. Using auditory fear conditioning in mice, we investigated the role of eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interactions and p70 S6 kinase polypeptide 1 (S6K1) in reconsolidation. We found that neither 4EGI-1 (2-[(4-(3,4-dichlorophenyl)-thiazol-2-ylhydrazono)-3-(2-nitrophenyl)]propionic acid), an inhibitor of eFI4E-eIF4G interactions, nor PF-4708671 [2-((4-(5-ethylpyrimidin-4-yl)piperazin-1-yl)methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole], an inhibitor of S6K1, alone blocked the reconsolidation of auditory fear memory. In contrast, using these drugs in concert to simultaneously block eIF4E-eIF4G interactions and S6K1 immediately after memory reactivation significantly attenuated fear memory reconsolidation. Moreover, the combination of 4EGI-1 and PF-4708671 further destabilized fear memory 10 d after memory reactivation, which was consistent with experiments using rapamycin, an mTORC1 inhibitor. Furthermore, inhibition of S6K1 immediately after retrieval resulted in memory destabilization 10 d after reactivation, whereas inhibition of eIF4E-eIF4G interactions did not. These results indicate that the reconsolidation of fear memory requires concomitant association of eIF4E to eIF4G as well as S6K1 activity and that the persistence of memory at longer intervals after memory reactivation also requires mTORC1-dependent processes that involve S6K1. These findings suggest a potential mechanism for how mTORC1-dependent translation is fine tuned to alter memory persistence.

Fluorescence resonance energy transfer based quantitative analysis of feedforward and feedback loops in epidermal growth factor receptor signaling and the sensitivity to molecular targeting drugs.

The Ras-ERK and PI3K-mTOR pathways are hyperactivated in various malignant tumors. Feedforward (FF) and feedback (FB) regulations between the Ras-ERK and the PI3K-mTOR pathways have been suggested to attenuate sensitivity to drugs targeting these pathways and confer tumor resistance to therapies. However, because analyses of such regulations require measurements and perturbations with high temporal resolution, the quantitative roles played by FF and FB regulations in the intrinsic resistance to molecular targeting drugs still remain unclear. To address this issue, we quantified FF and FB regulations of the epidermal growth factor receptor (EGFR) signaling pathway by Förster/fluorescence resonance energy transfer (FRET) imaging. EGF-induced activation of EGFR, Ras, extracellular-signal-regulated kinase and S6K with or without inhibitors was measured by FRET imaging, and analyzed by semi-automatic image processing. Based on the imaging data set and kinetic parameters determined by our previous studies, we identified the roles played by a coherent FF regulation and two negative FB regulations, one of which was not recognized previously. The systems analyses revealed how these FF and FB regulations shape the temporal dynamics of extracellular-signal-regulated kinase activity upon EGF stimulation. Furthermore, the simulation model predicts the response of molecular targeting drugs applied solely or in combination with each other to BRaf- or KRas-mutated cancer cell lines, indicating the validity of a quantitative model integrating FF and FB regulations.

Sulforaphane suppresses LPS-induced or TPA-induced downregulation of PDCD4 in RAW 264.7 cells.

Sulforaphane is a natural chemopreventive isothiocyanate and abundantly found in various cruciferous vegetables. Although chemopreventive activity of sulforaphane is well documented, the detailed biochemical mechanism(s), underlying how it regulates the protein translation process to antagonize pro-inflammatory responses are largely unclear. In the present study, we show that lipopolysaccharide (LPS) or 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment reduces cellular levels of PDCD4, and this event is mediated by affecting both transcription and proteolysis in RAW 264.7 cells. We show that LPS-mediated or TPA-mediated PDCD4 downregulation is catalyzed by the activation of intracellular Akt1 or S6K1 kinases and that sulforaphane suppresses LPS-induced or TPA-induced Akt1 or S6K1 activation, thereby resulting in the attenuation of PDCD4 downregulation in RAW 264.7 cells. We propose that sulforaphane suppression of PDCD4 downregulation serves as a novel molecular mechanism to control proliferation in response to pro-inflammatory signals.

Mis-regulation of Mammalian Target of Rapamycin (mTOR) Complexes Induced by Albuminuria in Proximal Tubules

High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease.

The cytoplasmic tail of FPC antagonizes the full-length protein in the regulation of mTOR pathway.

FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.

Long term exposure to L-arginine accelerates endothelial cell senescence through arginase-II and S6K1 signaling.

L-arginine supplementation is proposed to improve health status or as adjunct therapy for diseases including cardiovascular diseases. However, controversial results and even detrimental effects of L-arginine supplementation are reported. We investigate potential mechanisms of L-arginine-induced detrimental effects on vascular endothelial cells. Human endothelial cells were exposed to a physiological (0.1 mmol/L) or pharmacological (0.5 mmol/L) concentration of L-arginine for 30 minutes (acute) or 7 days (chronic). The effects of L-arginine supplementation on endothelial senescence phenotype, i.e., levels of senescence-associated beta-galactosidase, expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, eNOS-uncoupling, arginase-II expression/activity, and mTORC1-S6K1 activity were analyzed. While acute L-arginine treatment enhances endothelial NO production accompanied with superoxide production and activation of S6K1 but no up-regulation of arginase-II, chronic L-arginine supplementation causes endothelial senescence, up-regulation of the adhesion molecule expression, and eNOS-uncoupling (decreased NO and enhanced superoxide production), which are associated with S6K1 activation and up-regulation of arginase-II. Silencing either S6K1 or arginase-II inhibits up-regulation/activation of each other, prevents endothelial dysfunction, adhesion molecule expression, and senescence under the chronic L-arginine supplementation condition. These results demonstrate that S6K1 and arginase-II form a positive circuit mediating the detrimental effects of chronic L-arginine supplementation on endothelial cells.

Mo1783 Class III PI3K (HVPS34) Is Involved in AMPK-Regulated Neurotensin Secretion Independent of mTORC1 Signaling

Class III PI3K, also known as hVps34, along with its associated regulatory subunit, hVps15 kinase, are critical components of nutrient sensing, and act as upstream factors required for activation of S6K1, a downstream effector of mTOR complex 1 (mTORC1). The hVps34/ hVps15 complex regulates a variety of cellular functions such as intracellular vesicle trafficking. In addition, AMPK regulates the hVps34-containing complex in response to changes in energy state. Neurotensin (NT), a gut hormone produced and stored in N cells of the distal small bowel, has multiple physiologic effects, including facilitation of fatty acid translocation from the intestinal lumen, coordination of gut motility and stimulation of pancreaticobiliary secretion. Since we have previously found that AMPK and mTORC1 positively and negatively regulate NT release, respectively, the purpose of the present study was to investigate whether hVps34 and hVps15 are required in AMPK or mTORC1-mediated secretion. METHODS. i) The human endocrine cell line, BON, was used for all experiments. BON cells synthesize and secrete NT peptide and process the NT peptide in a manner analogous to that of N cells in the small bowel, thus serving as a useful model for enteroendocrine cell secretion. AMPK activation was induced by Aicar or 2-DG, and NT secretion measured using an NT EIA kit. ii) To determine the involvement of hVps34 and hVps15 in AMPK-mediated effects on NT secretion, endogenous expression of hVps34 and hVps15 was inhibited by siRNA-mediated knockdown. Stable BON cell lines overexpressing either the control vector or hVps34/hVps15 (hVps34 and hVps15 are expressed in one vector but regulated by individual promoters) were established. iii) The physical association of hVps34 with AMPK was evaluated by co-immunoprecipitation (co-IP). RESULTS. NT secretion (induced by Aicar or 2-DG) was attenuated in BON cells transfected with siRNAs targeting hVps34 and hVps15 compared to non-targeting control (NTC) siRNA. Overexpression of hVps34/hVps15 increased both basal and Aicarand 2-DG-stimulated NT secretion. Moreover, we detected the physical association of hVps34 with AMPK by co-IP. hVps34 was detected in both AMPKα1and α2-precipitated complexes with increased hVps34 bound in the AMPKα2 complex. CONCLUSIONS. These results identify a novel regulatory mechanism for the hVps34/hVps15 complex in NT peptide secretion, providing a direct connection between AMPK and hVps34 which is independent of mTORC1/S6K1 signaling.

Mo1787 Valproic Acid (VPA), a Histone Deacetylase Inhibitor That Reduces Intraabdominal Adhesions Modulates Peritoneal Plasma Extravasation and Genes That Regulate Fibrin Deposition and Stability

Class III PI3K, also known as hVps34, along with its associated regulatory subunit, hVps15 kinase, are critical components of nutrient sensing, and act as upstream factors required for activation of S6K1, a downstream effector of mTOR complex 1 (mTORC1). The hVps34/ hVps15 complex regulates a variety of cellular functions such as intracellular vesicle trafficking. In addition, AMPK regulates the hVps34-containing complex in response to changes in energy state. Neurotensin (NT), a gut hormone produced and stored in N cells of the distal small bowel, has multiple physiologic effects, including facilitation of fatty acid translocation from the intestinal lumen, coordination of gut motility and stimulation of pancreaticobiliary secretion. Since we have previously found that AMPK and mTORC1 positively and negatively regulate NT release, respectively, the purpose of the present study was to investigate whether hVps34 and hVps15 are required in AMPK or mTORC1-mediated secretion. METHODS. i) The human endocrine cell line, BON, was used for all experiments. BON cells synthesize and secrete NT peptide and process the NT peptide in a manner analogous to that of N cells in the small bowel, thus serving as a useful model for enteroendocrine cell secretion. AMPK activation was induced by Aicar or 2-DG, and NT secretion measured using an NT EIA kit. ii) To determine the involvement of hVps34 and hVps15 in AMPK-mediated effects on NT secretion, endogenous expression of hVps34 and hVps15 was inhibited by siRNA-mediated knockdown. Stable BON cell lines overexpressing either the control vector or hVps34/hVps15 (hVps34 and hVps15 are expressed in one vector but regulated by individual promoters) were established. iii) The physical association of hVps34 with AMPK was evaluated by co-immunoprecipitation (co-IP). RESULTS. NT secretion (induced by Aicar or 2-DG) was attenuated in BON cells transfected with siRNAs targeting hVps34 and hVps15 compared to non-targeting control (NTC) siRNA. Overexpression of hVps34/hVps15 increased both basal and Aicarand 2-DG-stimulated NT secretion. Moreover, we detected the physical association of hVps34 with AMPK by co-IP. hVps34 was detected in both AMPKα1and α2-precipitated complexes with increased hVps34 bound in the AMPKα2 complex. CONCLUSIONS. These results identify a novel regulatory mechanism for the hVps34/hVps15 complex in NT peptide secretion, providing a direct connection between AMPK and hVps34 which is independent of mTORC1/S6K1 signaling.

Mo1786 HIF-1α Plays a Gut-Injurious Role in Interferon-γ Induced Intestinal Epithelial Barrier Dysfunction

Class III PI3K, also known as hVps34, along with its associated regulatory subunit, hVps15 kinase, are critical components of nutrient sensing, and act as upstream factors required for activation of S6K1, a downstream effector of mTOR complex 1 (mTORC1). The hVps34/ hVps15 complex regulates a variety of cellular functions such as intracellular vesicle trafficking. In addition, AMPK regulates the hVps34-containing complex in response to changes in energy state. Neurotensin (NT), a gut hormone produced and stored in N cells of the distal small bowel, has multiple physiologic effects, including facilitation of fatty acid translocation from the intestinal lumen, coordination of gut motility and stimulation of pancreaticobiliary secretion. Since we have previously found that AMPK and mTORC1 positively and negatively regulate NT release, respectively, the purpose of the present study was to investigate whether hVps34 and hVps15 are required in AMPK or mTORC1-mediated secretion. METHODS. i) The human endocrine cell line, BON, was used for all experiments. BON cells synthesize and secrete NT peptide and process the NT peptide in a manner analogous to that of N cells in the small bowel, thus serving as a useful model for enteroendocrine cell secretion. AMPK activation was induced by Aicar or 2-DG, and NT secretion measured using an NT EIA kit. ii) To determine the involvement of hVps34 and hVps15 in AMPK-mediated effects on NT secretion, endogenous expression of hVps34 and hVps15 was inhibited by siRNA-mediated knockdown. Stable BON cell lines overexpressing either the control vector or hVps34/hVps15 (hVps34 and hVps15 are expressed in one vector but regulated by individual promoters) were established. iii) The physical association of hVps34 with AMPK was evaluated by co-immunoprecipitation (co-IP). RESULTS. NT secretion (induced by Aicar or 2-DG) was attenuated in BON cells transfected with siRNAs targeting hVps34 and hVps15 compared to non-targeting control (NTC) siRNA. Overexpression of hVps34/hVps15 increased both basal and Aicarand 2-DG-stimulated NT secretion. Moreover, we detected the physical association of hVps34 with AMPK by co-IP. hVps34 was detected in both AMPKα1and α2-precipitated complexes with increased hVps34 bound in the AMPKα2 complex. CONCLUSIONS. These results identify a novel regulatory mechanism for the hVps34/hVps15 complex in NT peptide secretion, providing a direct connection between AMPK and hVps34 which is independent of mTORC1/S6K1 signaling.