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Function and Regulation of Cannabinoid Receptors in Primary Monocytes and Osteoclasts

English title Function and Regulation of Cannabinoid Receptors in Primary Monocytes and Osteoclasts
Applicant Gertsch Jürg
Number 120672
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pharmazeutische Wissenschaften ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Pharmacology, Pharmacy
Start/End 01.05.2008 - 30.04.2011
Approved amount 234'725.00
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All Disciplines (3)

Discipline
Pharmacology, Pharmacy
Cellular Biology, Cytology
Organic Chemistry

Keywords (9)

Cannabinoid Receptors; Monocytes; Osteoclasts; Endocannabinoid System; GPCR trafficking; Immune System; Bone formation; Inflammation; Cannabinoids

Lay Summary (English)

Lead
Lay summary
Cannabinoid receptors are G-protein coupled receptors that are expressed in the central nervous system (predominantly CB1 receptors) and in peripheral tissues like spleen and immune cells (CB1 and CB2 receptors). In vivo, CB receptors are activated by arachidonic acid-derived endocannabinoids, such as 2-arachidonoyl ethanolamine (anandamide or AEA) and 2-arachidonoylglycerol (2-AG). While cannabinoid research in the last 20 years has mainly focused on the function and regulation of centrally expressed CB1 receptors, the role of peripherally expressed CB receptors and the endocannabinoid system is still poorly understood. In the last 5 years, peripherally expressed CB receptors (in particular CB2) have been shown to be a potential therapeutic target for the treatment of diseases as diverse as inflammation (gut, liver, brain), chronic peripheral pain, atherosclerosis, and osteoporosis. In these studies, the potential therapeutic use of both CB1 and CB2 agonists and/or antagonists was mainly deduced from animal experiments and a mechanistic understanding at the level of receptor pharmacology is still widely lacking. In osteoclasts, CB receptors are assumed to play a prominent role for osteoclast formation and the regulation of bone resorption. However, data obtained in animal models with different CB1 and CB2 ligands are contradictory. A major problem is that different research groups frequently employ distinct CB receptor agonists, inverse agonists and/or antagonists, which may trigger distinctly different signals in different tissues upon receptor activation. Moreover, data on human osteoclasts are still widely lacking. Since osteoclasts can be generated from monocytes and CB receptor surface expression is high in primary CD14+ monocytes, as well as different CD14+ cancer cell lines, we focus our research on primary monocytes/macrophages and osteoclasts in order to study the role of the endocannabinoid system in immune and bone destructing cells. In particular, it is till unclear how CB receptor ligand interactions can modulate cellular function. The project also aims at studying the regulation of surface expression of CB receptors in monocytes and osteoclasts as receptor density critically controls GPCR signalling. A better understanding of how CB receptor surface expression is regulated in monocytes/macrophages and osteoclasts and how CB ligands influence the function of these cells with regard to therapeutic intervention is of key important for drug discovery and the development of new therapies as e.g. related to inflammatory bone desctruction.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
141174 Entstehung und Funktion von Peptid Endocannabinoiden 01.05.2012 Project funding (Div. I-III)
116324 The role of the endocannabinoid system in atherosclerosis 01.05.2007 Project funding (Div. I-III)
141174 Entstehung und Funktion von Peptid Endocannabinoiden 01.05.2012 Project funding (Div. I-III)

Abstract

Cannabinoid receptors are G-protein coupled receptors that are expressed in the central nervous system (CB1 receptors) and in peripheral tissues (CB1 and CB2 receptors). The CB2 receptor is often referred to as the peripheral cannabinoid receptor, but also CB1 is expressed in the periphery. In vivo, CB receptors are activated by arachidonic acid-derived endocannabinoids, such as 2-arachidonoyl ethanolamine (anandamide or AEA) and 2-arachidonoylglycerol (2-AG). While cannabinoid research in the last 20 years has mainly focused on the function and regulation of centrally expressed CB1 receptors, the role of peripherally expressed CB receptors and the endocannabinoid system is still poorly understood. In the last 4 years, peripherally expressed CB receptors (in particular CB2) have been shown to be potential therapeutic targets for diseases as diverse as inflammation, pain, artherosclerosis, and osteoporosis. In these studies, the potential therapeutic use of both CB1 and CB2 agonists and/or antagonists was mainly deduced from animal experiments and a mechanistic understanding at the level of receptor pharmacology is still widely lacking. In osteoclasts, CB receptors are assumed to play a prominent role for osteoclast formation and the regulation of bone resorption. However, data obtained in animal models with different CB1 and CB2 ligands are controversial. A major problem is that different research groups frequently employ distinct CB receptor agonists, inverse agonists and antagonists, which may trigger distinctly differential signals upon receptor activation. Moreover, data on human osteoclasts are still widely lacking. Since osteoclasts can be generated from monocytes and CB receptor surface expression is high in primary CD14+ monocytes, as well as different CD14+ cancer cell lines, we focus our research on primary monocytes/macrophages and osteoclasts in order to study the role of the endocannabinoid system in immune and bone destructing cells. We have recently found a new mechanism of CB receptor trafficking in primary monocytes and osteoclasts, showing that CB receptors are internalized and externalized in a tyrosine phosphatase-specific manner. Since CB receptor density appears to be a key regulatory mechanism for the transduction of ligand-mediated signals, regulation of CB receptor surface expression is likely to be of physiological importance. Objectives of Project Proposal• To characterize the differential functional effects of chemically diverse CB receptor ligands, including different endogenous and synthetic agonists, inverse agonists, and true antagonists. In an initial phase, a series of 30 distinct CB ligands shall be screened for their activation of CB-mediated G-protein effectors (cAMP, intracellular calcium, and activation of the kinases p38, JNK, and Erk in monocytes). This will allow a first comprehensive classification of the potential differences of distinct ligands belonging to the same group (agonists, inverse agonists, antagonists) within the same assays.By sub-grouping the ligands (e.g. partial agonists vs. full agonists) based on the readouts generated, we hope to be able to answer the question why CB ligands apparently belonging to the same group show differential CB-dependent pharmacological effects in vitro and in vivo. Preliminary data in our group show that CB2 ligands differentially modulate osteoclastogenesis from monocyte precursor cells and so inhibit or enhance bone resorption in vitro. E.g. the endocannabinoid 2-AG stimulates bone resorption while anandamide has no effect. The only difference between the two ligands is that 2-AG is a full CB2 agonist and anandamide is a partial agonist at CB2. In our laboratory, non-selective CB ligands stimulate osteoclastogenesis while some CB2-selective inverse agonists appear to inhibit this process. However, due to a lack of data, no conclusive statement about a specific type of CB ligand with a specific type of effect can be made so far. It will be interesting to see whether CB1 and CB2 receptors mediate similar or differential effects in this system. Based on the experimental data generated in the first part of the project, we will be able to get insight into the type of CB receptor ligand, which shows potentially useful therapeutic applications, such as e.g. inhibition of osteoclastogenesis and bone resorption or inhibition of inflammation (e.g. inhibition of TNF-alpha expression).Once the receptor pharmacology of the distinct CB ligands is characterized, the underlying signals and the mechanisms related to CB receptor interaction in osteoclasts shall be studied in more detail. It is e.g. possible that CB agonists indirectly modulate osteoclastogenesis by modulating the expression of cytokines and or growth factors known to influence osteoclast formation (e.g. RANKL, TNF-alpha, TGF-beta, IL-4, and IL-10). This will be analyzed by measuring changes in the intracellular production and release of those factors from differentiating monocytes and osteoclasts upon CB1 and CB2 receptor activation. Alternatively, CB receptor activation can directly modulate the fate of differentiating monocytes by interfering with the activation of transcription factors critically involved in osteoclast formation (e.g. NF-ATc1 variants and c-fos). The effects on transcription factors and gene expression will be analyzed by comparative gene expression profiling (receptor-activated vs. resting cells) and direct analysis of transcription factor recruitment.• Based on the new finding (preliminary data obtained in our laboratory) that PKC-active phorbol esters (PMA) lead to rapid desensitization and internalization of CB receptors in human primary monocyes and osteoclasts, we want to continue our investigations on the kinases and phosphatases involved in CB receptor trafficking (internalization and externalization). Data obtained in our laboratory show that tyrosine-phosphatase inhibitors can either induce or inhibit CB receptor internalization, depending on the cell type. We therefore want to screen siRNAs for tyrosine phosphatases expressed in these cells, in order to find the “molecular switch” for CB receptor trafficking. This work will be carried out at the RNAi Image based Screening Centre (RISC, ETHZ). As PMA is a non-natural stimulus we are interested to find endogenous modulators of CB trafficking in monocytes. Since diacylglycerols share at least the functional pathways leading to PKC activation we will first test whether naturally occurring diacylgylcerols are able to trigger CB receptor trafficking in vitro. Moreover, we want to screen for CB ligands that influence receptor internalization. CB receptor expression in monocytes and osteoclasts appears to be regulated by trafficking rather than transcription or translation, thus a better understanding of these processes should help to understand the overall role of CB receptors in the periphery. Moreover, CB receptor density also critically influences the action of ligands. • The project will enable us to participate in a planned and approved clinical ex vivo study (directed by Dr. Meliha Karsak in the group of Prof. Dr. Andreas Zimmer, University of Bonn, Germany). It is our aim to challenge the already established hypothesis that CB2 receptors modulate osteoclastogenesis, and that single nucleotide polymorphisms (SNPs) in Cnr2 leads to altered osteoclastogenesis and bone resorption. Our already established in vitro osteoclastogenesis and bone resorption model with primary monocytes will serve as a biological assay to explore the role of the peripherally expressed CB2 receptor (Cnr2) in osteoclastogenesis and bone resorption. If the CB2 receptor is important for bone formation its regulation should be critical. Thus, it will be interesting to analyse CB2 receptor trafficking in monocytes isolated from patients carrying Cnr2 SNPs.
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