An mTORC1-Dependent Mouse Model for Cardiac Sarcoidosis.

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c+ cells (TSC2fl/flCD11c-Cre; termed TSC2KO) and controls (TSC2fl/fl) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2KO animals were treated with either everolimus, an mTOR inhibitor, or Bay11-7082, a nuclear factor-kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c+ cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life-threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68+ macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis. Conclusions To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis

The desmosome and pemphigus

Desmosomes are patch-like intercellular adhering junctions (“maculae adherentes”), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required

CB1R-dependent activation of Fyn tyrosine kinase and protein kinase C Delta, PKCδ, in lipid rafts

Cannabinoid 1 receptors (CB1Rs) are heptahelical transmembrane receptors which may exert their effects through the activation of the extracellular signal-regulated kinases (ERKs). We have previously shown that stably overexpressed CB1R in neuroblastoma cells (SH-SY5Y-CB1R cell line) is coupled to ERK activation via a mechanism that involves cannabinoid-induced transactivation of the EGF receptor and PKC activation. In a new line of experiments, EGFR transactivation by cannabinoid agonists was further supported by assessments of Ras activity. Ras assays revealed elevated Ras activity after Methanandamide treatment, which was abolished by the EGFR inhibitor AG1478. In analyzing this mechanism, we investigated the subcellular trafficking of the CB1R in basal conditions and in response to agonist stimulation in SH-SY5Y-CB1R cells. We found that under basal conditions, CB1R was mainly distributed in subcellular fractions which contain plasma-membrane, mitochondria or ER membranes, whereas after treatment with the CB1 agonist Methanandamide we observed redistribution of the receptor into the lipid rafts fractions. Moreover, we found that the activated (phosphorylated) species of EGFR also appeared in the lipid rafts after Methanandamide and importantly this effect was completely abolished by AG1478. To address what molecular events couple CB1R activation to ERK activation, we investigated whether members of Src family tyrosine kinases mediate this coupling. We found that PP1 and PP2 inhibitors of the Src family of tyrosine kinases in particular of Fyn kinase, abolish the methanandamide-dependent ERK activation. Furthermore, Methanandamide treatment induced tyrosine phosphorylation, an event that was inhibited by PP1, as well as by inhibitors of novel PKCs. In addition, Methanandamide-induced phosphorylation- activation of PKCs was also partially inhibited by Fyn and PKC inhibitors. Next, using immunoprecipitations we found that the novel PKC isoform delta, PKCδ, was tyrosine-phosphorylated in response to Methanandamide treatment and that this tyrosine phosphorylation was abolished by PP1 and Ro31-8220 (an inhibitor of classic and novel PKCs) but not by Go6976 (an inhibitor of classic PKC isoforms). These results suggest that in CB1R signaling a) Fyn activation may lie upstream of PKCδ, and b) a novel PKC isoform other than PKCδ activates Fyn which in turn activates PKCδ. ©PHARMAKON-Press

Transactivation of epidermal growth factor receptor EGFR by CB1 cannabinoid receptor agonists

Cannabinoids have long been used as therapeutic agents. The recent identification of the endogenous cannabinoid system in the brain, namely the abundance of the CB1 receptor (CB1R) in the CNS and the discovery of the endogenous ligands, has suggested that the cannabinoid neuromodulatory system plays important roles in many physiological processes, such as in memory, as well as holds therapeutic promise in a wide range of diseases ranging from mood and anxiety disorders to movement disorders and spinal cord injury. The CB1 receptor exerts its effects through several signalling events, which include the activation of the extracellular signal-regulated kinase, or ERKs. The mechanism by which this G-protein coupled receptor triggers ERK activation is not, however, well understood. To investigate the molecular events coupling CB1 activation to PKC and ERK activation, we generated clonal SHSY-5Y neuroblastoma cell lines which stably overexpress EGFP-CB1R and assessed whether this coupling could be mediated via transactivation of growth factor receptors with intrinsic tyrosine kinase activity. We first established using both immunoprecipitation and immunocytochemistry and GFP fluorescent imaging that the EGFP-CB1R was properly glycosylated and targeted to plasma membranes. We now report that the CB1R agonist methanandamide specifically stimulated, in a time-dependent manner, tyrosine phosphorylation of several cellular proteins, including phosphorylation of the EGF receptor and Src kinase on tyrosine residues 1148 and 416, respectively, and of ERK2. These effects were abolished by the specific CB1 antagonist AM251 and by the EGFR antagonist AG1478. These data provide first evidence that in a neuronal cellular background, the CB1R intracellular signalling engages the specific transactivation of the tyrosine kinase EGF receptor to regulate ERK activation and downstream transcriptional events. ©ΦApmakon-Túttoς

Role of Lipid Rafts and the Underlying Filamentous-Actin Cytoskeleton in Cannabinoid Receptor 1 Signaling

The cannabinoid 1 receptor, CB1, has evolved as a major regulatory molecule for almost all known aspects of the development and function of the central nervous system (CNS), with biological actions ranging from proper CNS cellularity to complex behaviors such as fear, appetite, and addiction. It is therefore critical to understand the mechanisms and intracellular signal transduction pathways that CB1 utilizes for its acute and long-term actions, in particular activation of the major effector extracellular signal-regulated kinase (ERK) in discrete amplification waves. This highly complex regulation of CB1 signaling to ERK is facilitated by specialized membrane microdomains, the lipid rafts. Integral components of rafts are required for proper CB1 presentation at the plasma membrane as shown by confocal analysis, while the dynamic and hierarchic activation of its major proximal effectors protein kinase C?, Src, and Fyn requires raft integrity, additionally causing fibroblast growth factor receptor transactivation. Thus, lipid rafts constitute the plasma membrane platform on which CB1 signaling is initiated and organized. © 2016 Elsevier Inc. All rights reserved

A dangerous hobby? Erysipelothrix rhusiopathiae bacteremia most probably acquired from freshwater aquarium fish handling.

Erysipelothrix rhusiopathiae is a facultative anaerobic Gram-positive rod that occurs widely in nature and is best known in veterinary medicine for causing swine erysipelas. In humans, infections are rare and mainly considered as occupationally acquired zoonosis. A case of E. rhusiopathiae bacteremia most likely associated with home freshwater aquarium handling is reported. The route of transmission was probably a cut with the dorsal fin of a dead pet fish. A short review of clinical presentations, therapeutic considerations and pitfalls of E. rhusiopathiae infections in humans is presented

Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C epsilon-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a prerequisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation

Long-term changes in the ghrelin-CB1R axis associated with the maintenance of lower body weight after sleeve gastrectomy

OBJECTIVES: In the hypothalamus, the molecular actions of receptors for growth hormone secretagogue (ghrelin) receptor-GHSR, leptin receptor-b (LEPRb), Melanocortin-4 receptor (MC4R) and Cannabinoid-1 receptor (CB1R) regulate energy homeostasis and body weight. We hypothesized that the acute loss of stomach tissue upon sleeve gastrectomy (SG), performed to treat obesity, imposes modulations on the expression of these receptors in the brain to sustain weight loss. METHODS: Rats, induced to obesity with high-fat diet were randomized to SG- or sham-operation groups and killed at 30 or 90 days post surgery, when the expression of Ghrl, Mboat4 and Cnr1 in the stomach, and Ghsr, Leprb, Mc4r and Cnr1 in distinct brain areas was assessed by reverse transcription-PCR and western blotting. RESULTS: SG acutely reduced body weight and fat mass and suppressed the remnant stomach mRNA levels of preproghrelin and ghrelin O-acyltransferase, which correlated well with long-term decreases in CB1R mRNA. In the hypothalamus, increases in GHSR and decreases in CB1R and LEPRb by 30 days were followed by further downregulation of CB1R and an increase in MC4R by 90 days. CONCLUSIONS: Post SG, acyl-ghrelin initiates a temporal hierarchy of molecular events in the gut-brain axis that may both explain the sustained lower body weight and suggest intervention into the cannabinoid pathways for additional therapeutic benefits. © 2014 Macmillan Publishers Limited All rights reserved