mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.     

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway

The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein synthesis, a blockade in ribosome component biosynthesis, and G1 cell cycle arrest. G1 arrest may occur by inhibiting the protein synthesis of critical factors required for cell cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT activity are relatively resistant. Our previous data suggest that low AKT activity induces resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-Myc proteins. In support of this notion, the current study demonstrates that the human cyclin D1 mRNA 5' untranslated region contains an internal ribosome entry site (IRES) and that both this IRES and the c-myc IRES are negatively regulated by AKT activity. Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as specific inhibitors of these pathways reduce IRES-mediated translation and protein levels under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation initiation may permit cell cycle progression upon mTOR inactivation in cells in which AKT kinase activity is relatively low.     

Porcine Circovirus Type 2 Induces Autophagy via the AMPK/ERK/TSC2/mTOR Signaling Pathway in PK-15 Cells

Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.     

Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.     

Activation of mammalian target of rapamycin (mTOR) by insulin is associated with stimulation of 4EBP1 binding to dimeric mTOR complex 1

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.     

A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin

Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.     

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA-protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.     

The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.     

Mammalian Target of Rapamycin (mTOR) and S6 Kinase Down-regulate Phospholipase D2 Basal Expression and Function

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nm rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration.     

S6K1 phosphorylates and regulates fragile X mental retardation protein (FMRP) with the neuronal protein synthesis-dependent mammalian target of rapamycin (mTOR) signaling cascade

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.