Negative feedback regulation of the ERK1/2 MAPK pathway

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.

Role of fibroblast growth factor receptor in regulation of membrane traffic

Several studies show that membrane transport mechanisms are regulated by signalling molecules. Recently, genome-wide screen analyses in C.elegans have enabled scientists to identify novel regulators in membrane trafficking and also signalling molecules which are found to couple with this machinery. Fibroblast growth factor (FGF) via binding to fibroblast growth factor receptor (FGFR) mediate signals which are essential in the development of an organism, patterning, cell migration and tissue homeostasis. Impaired FGFR-mediated signalling has been associated with various developmental, neoplastic, metabolic and neurological diseases and cancer. In this study, the potential role of FGFR-mediated signalling pathway as a regulator of membrane trafficking was investigated. The GFP-tagged yolk protein YP170-GFP trafficking was analysed in worms where 1) FGFR signalling cascade components were depleted by RNAi and 2) in mutant animals. From these results, it was found that the disruption of the genes egl-15 (FGFR), egl-17(FGF), let-756(FGF), sem-5, let-60, lin-45, mek-2, mpk-1 and plc-3 lead to abnormal localization of YP170-GFP, suggesting that signalling downstream of FGFR via activation of MAPK and PLC-γ pathway is regulating membrane transport. The route of trafficking was further investigated, to pinpoint which membrane step is regulated by worm FGFR, by analysing a number of GFP-tagged intracellular membrane markers in the intestine of Wild Type (WT) and FGFR mutant worms. FGFR mutant worms showed a significant difference in the localisation of several endosomal membrane markers, suggesting its regulatory role in early and recycling steps of endocytosis. Finally, the trafficking of transferrin in a mammalian NIH/3T3 cell line was investigated to identify the conservation of these membrane trafficking regulatory mechanisms between organisms. Results showed no significant changes in transferrin trafficking upon FGFR stimulation or inhibition.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

Microscale mesoarrays created by dip-pen nanolithography for screening of protein-protein interactions

Using microarrays to probe protein-protein interactions is becoming increasingly attractive due to their compatibility with highly sensitive detection techniques, selectivity of interaction, robustness and capacity for examining multiple proteins simultaneously. The major drawback to using this approach is the relatively large volumes and high concentrations necessary. Reducing the protein array spot size should allow for smaller volumes and lower concentrations to be used as well as opening the way for combination with more sensitive detection technologies. Dip-Pen Nanolithography (DPN) is a recently developed technique for structure creation on the nano to microscale with the capacity to create biological architectures. Here we describe the creation of miniaturised microarrays, 'mesoarrays', using DPN with protein spots 400× smaller by area compared to conventional microarrays. The mesoarrays were then used to probe the ERK2-KSR binding event of the Ras/Raf/MEK/ERK signalling pathway at a physical scale below that previously reported. Whilst the overall assay efficiency was determined to be low, the mesoarrays could detect KSR binding to ERK2 repeatedly and with low non-specific binding. This study serves as a first step towards an approach that can be used for analysis of proteins at a concentration level comparable to that found in the cellular environment.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Interventions against obesity through increased lipolysis in adipose tissue

Obesity is a disease of excess adiposity affecting> 17% of men and >20% of women in Britain. Clinically, it is defined by a Body Mass Index (BMI, kg/m2) of 2:30. Obesity is a confounding factor that promotes insulin resistance, hyperinsulinaemia and type 2 diabetes. Type 2 diabetes accounts for >90% of all cases of diabetes, with a prevalence of 2-6% of adults in most western societies, a majority of which are overweight or obese. Weight loss in obese patients reduces the risk of developing diabetes by >50%. This thesis has investigated the first part of a two-stage therapeutic intervention against obesity in which adipose tissue lipolysis will be combined with increased energy expenditure: the approach is also designed to consider agents that will benefit glycaemic control in coexistent obesity and diabetes by improving insulin sensitivity. Rodent and human in vitro models of adipocyte biology and skeletal muscle have been developed, characterised and evaluated. They include isolated epididymal and parametrial adipocytes of lean and obese diabetic ob/ob mice, cultured 3T3-Ll preadipocytes, isolated human omental and subcutaneous adipocytes and rat L6 cultured muscle cells. Compounds investigated for anti-obesity and anti-diabetic properties include M2 (sibutramine metabolite), 3-guanidinopropionic acid and mazindol. In vivo studies were undertaken to investigate these compounds further in lean and ob/ob mice. In vivo studies indicated that M2 and 3-guanidinopropionic acid reduced body weight gain in ob/ob mice. The three compounds increased lipolysis in adipocytes isolated from lean and ob/ob mice and human adipose depots. The direct action of these compounds was mediated via a pathway involving the f3 adrenoceptors and components of the lipolytic signalling pathway, including protein kinase A and p38 MAP kinase. In addition, M2 and mazindol were capable of increasing glucose uptake into insulin sensitive tissues. M2 and mazindol can act directly on adipose tissue and skeletal muscle to increase glucose uptake via a pathway involving new protein synthesis and activation of the glucose transporters. The M2-stimulated pathway is activated by the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate by phosphatidylinositol 3-kinase. Thus, M2, mazindol and 3-GPA showed pharmacodynamic properties which suggested they might be potential therapeutic treatments for obesity and diabetes.

Investigation into the mechanisms responsible for muscle atrophy in cancer cachexia

Cachexia inducing tumours are known to produce a glycoprotein called proteolysis inducing factor (PIF), which induces skeletal muscle atrophy via increased protein degradation and decreased protein synthesis. The objective of this study was to investigate the signalling pathway by which PIF reduces protein synthesis in skeletal muscle and to determine the link, if any, to the ability to induce protein degradation. In murine myotubes PIF induced an increase in expression of the active form of the dsNRA dependent protein kinase (PKR), as well as the phosphorylated form of the translation initiator elF2a, possibly through the release of calcium, at the same concentration as that inhibiting protein synthesis. Inhibition of PKR reversed the inhibition of protein synthesis by PIF and also the induction of protein degradation through the ubiquitin-proteasome pathway by a reduction in the nuclear migration of NK-?B. The expression of phosphorylated forms of PKR and elF2a was also increased in the muscle of cancer patients experiencing weight loss, and in gastrocnemius muscle of mice bearing the cachexia inducing MAC16 tumour, as well as in the tumour itself. Treatment of mice bearing the MAC16 tumour with a PKR inhibitor attenuated muscle atrophy and inhibited tumour growth, through the inactivation of PKR and the consequent reduction of nuclear accumulation of NF-?B. A decreased translational efficiency of the elF-4F complex of initiation factors through dephosphorylation of 4E-BP1 and an increase eEF2 phosphorylation was seen in response to PIF in vitro. The same pattern of events also occurred in gastrocnemius muscle of mice bearing the MAC16 tumour demonstrating weight loss, where a depression of mTOR and p70S6K activation was also observed as weight loss increased.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

Background - The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results - Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions - These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

Ageing, adipose tissue, fatty acids and inflammation

A common feature of ageing is the alteration in tissue distribution and composition, with a shift in fat away from lower body and subcutaneous depots to visceral and ectopic sites. Redistribution of adipose tissue towards an ectopic site can have dramatic effects on metabolic function. In skeletal muscle, increased ectopic adiposity is linked to insulin resistance through lipid mediators such as ceramide or DAG, inhibiting the insulin receptor signalling pathway. Additionally, the risk of developing cardiovascular disease is increased with elevated visceral adipose distribution. In ageing, adipose tissue becomes dysfunctional, with the pathway of differentiation of preadipocytes to mature adipocytes becoming impaired; this results in dysfunctional adipocytes less able to store fat and subsequent fat redistribution to ectopic sites. Low grade systemic inflammation is commonly observed in ageing, and may drive the adipose tissue dysfunction, as proinflammatory cytokines are capable of inhibiting adipocyte differentiation. Beyond increased ectopic adiposity, the effect of impaired adipose tissue function is an elevation in systemic free fatty acids (FFA), a common feature of many metabolic disorders. Saturated fatty acids can be regarded as the most detrimental of FFA, being capable of inducing insulin resistance and inflammation through lipid mediators such as ceramide, which can increase risk of developing atherosclerosis. Elevated FFA, in particular saturated fatty acids, maybe a driving factor for both the increased insulin resistance, cardiovascular disease risk and inflammation in older adults.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Cellular signalling pathways involved in muscle atrophy in cancer cachexia

Cancer cachexia encompases severe weight loss, characterised by the debilitating atrophy of adipose and skeletal muscle mass. Skeletal muscle proteolysis in cancer cachexia is mediated by a sulphated glycoprotein with a relative molecular mass of 24kDa, termed Proteolysis-Inducing Factor (PIF). PIF induced a significant increase in protein degradation, peaking at 4.2nM PIF (p<0.001), ‘chymotrypsin-like’ activity of the proteasome (p<0.001) and increased expression of components of the ATP-ubiquitin dependent proteolytic pathway. This was attenuated in vitro by pre-incubation with the PKC inhibitor calphostin C (100µM) and NF-kB the inhibitors SN50 (18µM), curcumin (50µM) and resveratrol (30µM), 2 hours prior to the addition of PIF. In vivo studies found the IKK inhibitor resveratrol (1mg/kg) to be successful in attenuating protein degradation (p<0.001) and upregulation of ubiquitin-dependent proteolysis in MAC16 tumour bearing mice. C2C12 myoblasts transfected with mutant IkBα and PKCα inserts did not elicit a PIF-induced response, suggesting the importance of the transcription factor NF-kB and PKC  involvement in PIF signal transduction. 15(S)-HETE acts as an intracellular mediator of PIF and exerts an effect through the activation of PKC and subsequently IKK, which phosphorylates IkBα and allows NF-kB to migrate to the nucleus. This effect was negated with the PKC inhibitor calphostin C (300nM). A commercially produced PIF receptor antibody was raised in rabbits immunised with a peptide containing the partial N-terminal sequence of the PIF receptor. The PIF receptor antibody was successful in attenuating the PIF-induced increase in skeletal muscle catabolism and protein degradation in vitro at 10µg/ml (p<0.001) and 3.47mg/kg in vivo (p<0.001). The data suggest great potential in the development of this antibody as a therapy against cancer cachexia.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast

Background and Purpose The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. Experimental Approach We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. Key Results The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. Conclusions and Implications These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.