ABSTRACT
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.
