title: The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.
abstract: The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Marie
authors.initials: M
authors.lastname: Smith
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Wilson
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Sally
authors.initials: S
authors.lastname: O'Brien
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Cristina
authors.initials: C
authors.lastname: Tufarelli
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Susan I
authors.initials: SI
authors.lastname: Anderson
authors.affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
authors.forename: Saoirse Elizabeth
authors.initials: SE
authors.lastname: O'Sullivan
mesh_terms: D000943:Antigens, Differentiation / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D063388:Endocannabinoids / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005989:Glycerides / Q000494:pharmacology*; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism; D050916:TRPV Cation Channels / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; D001095:Arachidonic Acids; C588309:CNR2 protein, human; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D063388:Endocannabinoids; D005989:Glycerides; D053284:Polyunsaturated Alkamides; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C094503:glyceryl 2-arachidonate; D048049:Extracellular Signal-Regulated MAP Kinases; C078814:anandamide