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.

Myopia: pathways to therapy

Editorial

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Molecular pathways leading to cancer cachexia

Loss of body weight in cancer patients strongly influences morbidity and mortality. Recent studies have suggested that both tumor and host factors play a major role in tissue catabolism in cachexia, leading to upregulation of degradative pathways in both skeletal muscle and adipose tissue. ©2005 Int. Union Physiol. Sci./Am. Physiol. Soc.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin-proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA 2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression. © 2003 Cancer Research UK.

Suppression pathways saturate with contrast for parallel surrounds but not for superimposed cross-oriented masks

Contrast masking from parallel grating surrounds (doughnuts) and superimposed orthogonal masks have different characteristics. However, it is not known whether the saturation of the underlying suppression that has been found for parallel doughnut masks depends on (i) relative mask and target orientation, (ii) stimulus eccentricity or (iii) surround suppression. We measured contrast-masking functions for target patches of grating in the fovea and in the periphery for cross-oriented superimposed and doughnut masks and parallel doughnut masks. When suppression was evident, the factor that determined whether it accelerated or saturated was whether the mask stimulus was crossed or parallel. There are at least two interpretations of the asymptotic behaviour of the parallel surround mask. (1) Suppression arises from pathways that saturate with (mask) contrast. (2) The target is processed by a mechanism that is subject to surround suppression at low target contrasts, but a less sensitive mechanism that is immune from surround suppression ‘breaks through’ at higher target contrasts. If the mask can be made less potent, then masking functions should shift downwards, and sideways for the two accounts, respectively. We manipulated the potency of the mask by varying the size of the hole in a parallel doughnut mask. The results provided strong evidence for the first account but not the second. On the view that response compression becomes more severe progressing up the visual pathway, our results suggest that superimposed cross-orientation suppression precedes orientation tuned surround suppression. These results also reveal a previously unrecognized similarity between surround suppression and crowding (Pelli, Palomares, & Majaj, 2004).

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells:role of AMPK and MAPK kinase pathways

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production.

Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

Advances in the generation and interpretation of proteomics data have spurred a transition from focusing on protein identification to functional analysis. Here we review recent proteomics results that have elucidated new aspects of the roles and regulation of signal transduction pathways in cancer using the epidermal growth factor receptor (EGFR), ERK and breakpoint cluster region (BCR)-ABL1 networks as examples. The emerging theme is to understand cancer signalling as networks of multiprotein machines which process information in a highly dynamic environment that is shaped by changing protein interactions and post-translational modifications (PTMs). Cancerous genetic mutations derange these protein networks in complex ways that are tractable by proteomics.

TRial of an Educational intervention on patients' knowledge of Atrial fibrillation and anticoagulant therapy, INR control, and outcome of Treatment with warfarin (TREAT)

Background Atrial fibrillation (AF) patients with a high risk of stroke are recommended anticoagulation with warfarin. However, the benefit of warfarin is dependent upon time spent within the target therapeutic range (TTR) of their international normalised ratio (INR) (2.0 to 3.0). AF patients possess limited knowledge of their disease and warfarin treatment and this can impact on INR control. Education can improve patients' understanding of warfarin therapy and factors which affect INR control. Methods/Design Randomised controlled trial of an intensive educational intervention will consist of group sessions (between 2-8 patients) containing standardised information about the risks and benefits associated with OAC therapy, lifestyle interactions and the importance of monitoring and control of their International Normalised Ratio (INR). Information will be presented within an 'expert-patient' focussed DVD, revised educational booklet and patient worksheets. 200 warfarin-naïve patients who are eligible for warfarin will be randomised to either the intervention or usual care groups. All patients must have ECG-documented AF and be eligible for warfarin (according to the NICE AF guidelines). Exclusion criteria include: aged < 18 years old, contraindication(s) to warfarin, history of warfarin USE, valvular heart disease, cognitive impairment, are unable to speak/read English and disease likely to cause death within 12 months. Primary endpoint is time spent in TTR. Secondary endpoints include measures of quality of life (AF-QoL-18), anxiety and depression (HADS), knowledge of AF and anticoagulation, beliefs about medication (BMQ) and illness representations (IPQ-R). Clinical outcomes, including bleeding, stroke and interruption to anticoagulation will be recorded. All outcome measures will be assessed at baseline and 1, 2, 6 and 12 months post-intervention. Discussion More data is needed on the clinical benefit of educational intervention with AF patients receiving warfarin. Trial registration ISRCTN93952605