Hypothesis: cell signalling influences age‐related risk of colorectal cancer

We propose that ageing is linked to colonic carcinogenesis through crosstalk between Wnt activity and signalling pathways related to ageing and senescence: progerin, klotho and mTOR. Mutations in the Wnt signalling pathway are responsible for the majority of colorectal cancers (CRCs); however, hyperactivation of Wnt signalling by butyrate, a breakdown product of dietary fibre, induces CRC cell apoptosis. This effect of butyrate may in part explain the protective action of fibre against CRC. Hutchinson–Gilford progeria syndrome is a premature ageing disorder caused by accumulation of the progerin protein; however, healthy individuals also produce progerin in the course of their normal ageing. Progerin activates expression of the Wnt inhibitors HES1 and TLE1. Thus, we hypothesize that with age, the increasing expression of progerin suppresses butyrate‐mediated Wnt hyperactivation and apoptosis, leading to increased CRC risk. Wild‐type klotho contributes to a significantly increased lifespan; however, Klotho gene variants differ significantly between newborns and elderly. Klotho inhibits basal Wnt signalling activity; thus, the protein may function as a tumour suppressor for CRC. However, similar to progerin, klotho variants associated with lifespan differences may repress butyrate‐mediated Wnt hyperactivation, and thus increase the risk of CRC. Finally, mTOR signalling has also been linked to human ageing, and crosstalk between Wnt and mTOR signalling may influence colonic tumourigenesis. Understanding how progerin, klotho and mTOR link ageing with colonic neoplastic development may lead to novel preventive and therapeutic strategies against CRC associated with age.     

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.     

Reducing Igf-1r Levels Leads To Paradoxical and Sexually Dimorphic Effects in HD Mice

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called “proteinopathies” include Alzheimer’s disease (AD) and Huntington’s disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

Combinatorial Approach Using Caenorhabditis elegans and Mammalian Systems for Aging Research

Aging is associated with functional and structural declines in organisms over time. Organisms as diverse as the nematode Caenorhabditis elegans and mammals share signaling pathways that regulate aging and lifespan. In this review, we discuss recent combinatorial approach to aging research employing C. elegans and mammalian systems that have contributed to our understanding of evolutionarily conserved aging-regulating pathways. The topics covered here include insulin/IGF-1, mechanistic target of rapamycin (mTOR), and sirtuin signaling pathways; dietary restriction; autophagy; mitochondria; and the nervous system. A combinatorial approach employing high-throughput, rapid C. elegans systems, and human model mammalian systems is likely to continue providing mechanistic insights into aging biology and will help develop therapeutics against age-associated disorders.     

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors)

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.     

Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function

Lysozymes are antibacterial enzymes widely distributed among organisms. Within the animal kingdom, mainly three major lysozyme types occur. Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type lysozyme is typical for invertebrate organisms, and hence its name. Since their discovery in 1975, numerous research articles report on the identification of i-type lysozymes in a variety of invertebrate phyla. This review describes the current knowledge on i-type lysozymes, outlining their distribution, molecular mechanism and in vivo function taking the representative from Venerupis philippinarum (formerly Tapes japonica) (Vp-ilys) as a model. In addition, invertebrate g-type and ch-type (chalaropsis) lysozymes, which have been described in molluscs and nematodes, respectively, are also briefly discussed.     

Optimization of a Method for Chromatin Immunoprecipitation Assays in the Marine Invertebrate Chordate Ciona

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.     

MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster

Background

MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. We have analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development.

Methodology and Principal Findings

Using loss-of-function mutants and over-expression conditions we find that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). We also show that MAP4K3 display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor.

Conclusions and Significance

We suggest that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability.     

Microarray analyses of the effects of NF-κB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: Synergistic effects of rapamycin on LPS-induced MMP9-overexpression

Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-κB and the MAPK cascade, but recent evidence suggests that LPS stimulation also activates the PI3K pathway. To unravel the specific roles of both pathways in LPS signalling and gene expression profiling, we investigated the effects of different inhibitors of NF-κB (BAY 11-7082), PI3K (wortmannin and LY294002) but also of mTOR (rapamycin), a kinase acting downstream of PI3K/Akt, in LPS-stimulated RAW264.7 macrophages, analyzing their effects on the LPS-induced gene expression profile using a low density DNA microarray designed to monitor the expression of pro-inflammatory genes. After statistical and hierarchical cluster analyses, we determined five clusters of genes differentially affected by the four inhibitors used. In the fifth cluster corresponding to genes upregulated by LPS and mainly affected by BAY 11-7082, the gene encoding MMP9 displayed a particular expression profile, since rapamycin drastically enhanced the LPS-induced upregulation at both the mRNA and protein levels. Rapamycin also enhanced the LPS-induced NF-κB transactivation as determined by a reporter assay, phosphorylation of the p38 and Erk1/2 MAPKs, and counteracted PPAR activity. These results suggest that mTOR could negatively regulate the effects of LPS on the NF-κB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARγ), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38^MAPK), that were able to counteract the rapamycin mediated overexpression of mmp9 in response to LPS. Our results suggest a new pathway involving mTOR for regulating specifically mmp9 in LPS-stimulated RAW264.7 cells.     

The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.     

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.     

Invertebrate models of age-related muscle degeneration

Functional and structural deterioration of muscles is an inevitable consequence of ageing in a wide variety of animal species. What underlies these changes is a complex network of interactions between the muscle-intrinsic and muscle-extrinsic factors, making it very difficult to distinguish between the cause and the consequence. Many of the genes, structures, and processes implicated in mammalian skeletal muscle ageing are preserved in invertebrate species Drosophila melanogaster and Caenorhabditis elegans. The absence in these organisms of mechanisms that promote muscle regeneration, and substantially different hormonal environment, warrant caution when extrapolating experimental data from studies conducted in invertebrates to mammalian species. The simplicity and accessibility of these models, however, offer ample opportunities for studying age-related myopathologies as well as investigating drugs and therapies to alleviate them.     

Helicobacter pylori promotes eukaryotic protein translation by activating phosphatidylinositol 3 kinase/mTOR

The innate immune response elicited by Helicobacter pylori in the human gastric mucosa involves a range of cellular signalling pathways, including those implicated in metabolism regulation. In this study, we analysed H. pylori-induced PI3K/Akt/mTOR signalling, which regulates glycolysis and protein synthesis and associates thereby with cellular energy- and nutrients-consuming processes such as growth and proliferation. The immunohistochemical analysis demonstrated that Akt kinase phosphorylation is abundant in gastric biopsies obtained from gastritis, gastric adenoma and adenocarcinoma patients. Infection with H. pylori led to the phosphorylation of Akt effectors mTOR and S6 in a type 4 secretion system (T4SS)-independent manner in AGS cells. We observed that the activation of these molecules was dependent on PI3K and the Src family tyrosine kinases. Furthermore, H. pylori induced the phosphorylation of 4E-BP1 and eIF4E and suppressed the phosphorylation of eEF2, which are important regulators of protein synthesis. Inhibition of PI3K and Akt kinase prevented the phosphorylation of 4E-BP1, suggesting that PI3K signalling is involved in the regulation of translation initiation during H. pylori infection. Metabolic labelling showed that infected cells had higher rates of [³⁵S]methionine/cysteine incorporation, and this effect could be prevented using LY294002, an PI3K inhibitor. Thus, H. pylori activates PI3K/Akt signalling, mTOR, eIFs and protein translation, which might impact H. pylori-related gastric pathophysiology.     

Death and dessert: nutrient signalling pathways and ageing

Reduction in nutrient intake without malnutrition can delay ageing and extend healthy life in diverse organisms from yeast to primates. This effect can be recapitulated by genetic or pharmacological dampening of the signal through nutrient signalling pathways, making them a promising target for intervention into human ageing and age-related diseases. Here we review the current knowledge of the interactions between nutrient signalling pathways and ageing, focusing on the findings emerged in the past few years.     

A phototaxis signalling complex in Dictyostelium discoideum

Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains – the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.