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

PKC and Ras differentially regulate gene expression of the dopaminergic marker TH and noradrenergic marker DBH

We have successfully established for the first time in the literature cell lines from human fetal cells derived from the amniotic fluid (AF) and have documented that these cells may progress and stably express many of the same genes which define progenitor dopaminergic neurons. AF cells (AFCs) may thus provide an excellent model for studying the development of dopaminergic neurons. The dopaminergic and noradrenergic transcriptional program is highly regulated during development and in the adult, in response to activation of membrane receptor signalling cascades. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, is known to be regulated by receptors that act through protein kinase C (PKC) or Ras signaling. Therefore, we pharmacologically or genetically manipulated each signalling molecule by downregulating PKC with long term (24 h) exposure of cells to the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), by overexpressing Ras with transfection, or by downregulating activated Ras by overxepressing the GRDI domain of the neurofibromin protein, a potent Ras GAP. We found that treatment with TPA increased transcription of both TH and Nurr1 (a transcriptional 'hub' for the acquisition of a dopaminergic phenotype) by over 80%, whereas GRDI blocked almost all TH expression. Moreover, while Ras overexpression had no effect on these two genes, it induced the de novo expression of the noradrenergic phenotype marker DBH. Expression of VMAT2 increased with all molecular manipulations, while expression of several neuronal markers, namely Tau, b-tubulin, and syntaxin, was not affected by any condition. Interestingly, treatment of AFCs with cytochalasin, which disrupts microfilaments, caused a 50% decrease in Nurr1 transcript compared to control, a 4-fold increase in VMAT2 message, and a 40% decrease in Ptx3 message. Taken together, these studies suggest that PKC and Ras have important yet differential roles in regulating gene expression of the dopaminergic marker TH and noradrenergic marker DBH during neuronal progression. ©ΦAPMAKON-TÚTTOς

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

PKCε signalling activates ERK1/2, and regulates aggrecan, ADAMTS5, and miR377 gene expression in human nucleus pulposus cells

The protein kinase C (PKC) signaling, a major regulator of chondrocytic differentiation, has been also implicated in pathological extracellular matrix remodeling, and here we investigate the mechanism of PKCε-dependent regulation of the chondrocytic phenotype in human nucleus pulposus (NP) cells derived from herniated disks. NP cells from each donor were successfully propagated for 25+ culture passages, with remarkable tolerance to repeated freeze-and-thaw cycles throughout long-term culturing. More specifically, after an initial downregulation of COL2A1, a stable chondrocytic phenotype was attested by the levels of mRNA expression for aggrecan, biglycan, fibromodulin, and lumican, while higher expression of SOX-trio and Patched-1 witnessed further differentiation potential. NP cells in culture also exhibited a stable molecular profile of PKC isoforms: throughout patient samples and passages, mRNAs for PKC α, δ, ε, ζ, η, ι, and μ were steadily detected, whereas β, γ, and θ were not. Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan. More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan. Therefore, PKCε activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKCε/ERK/CREB/AP- 1-dependent transcriptional program that includes upregulation of both chondrogenic genes and microRNAs. Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks. © 2013 Tsirimonaki et al

Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-beta III, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta 3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons. (c) 2006 Wiley-Liss, Inc