ABSTRACT
The cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB1 and CB2 receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB2 receptors form oligomers and heterodimers with CB1 receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB2 receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression.
Subject(s)
Receptors, Cannabinoid/metabolism , Animals , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Mice , NIH 3T3 Cells , Protein TransportABSTRACT
The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(2), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of 2-AG (250-700 nM). We show that residual 2-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, 2-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove 2-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.
Subject(s)
Cannabinoid Receptor Modulators/metabolism , Cannabinoid Receptor Modulators/pharmacology , Endocannabinoids , Fetal Blood/chemistry , Macrophages/drug effects , Monocytes/drug effects , Osteoclasts/drug effects , Animals , Arachidonic Acids/metabolism , Arachidonic Acids/pharmacology , Cattle , Cell Movement/drug effects , Cells, Cultured , Flow Cytometry , Gas Chromatography-Mass Spectrometry , Glycerides/metabolism , Glycerides/pharmacology , Humans , Interleukin-6/metabolism , Lipids/analysis , Lipids/isolation & purification , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/metabolism , Monocytes/cytology , Monocytes/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , RANK Ligand/pharmacology , Receptors, Cannabinoid/metabolism , U937 CellsABSTRACT
The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.
