title: Cannabinoid receptor 1 regulates ERK and GSK-3β-dependent glucocorticoid inhibition of osteoblast differentiation in murine MC3T3-E1 cells.
abstract: Supraphysiological glucocorticoid administration accelerates loss of survival and differentiation in osteoblastic cells, thereby increasing the risks of osteopenic or osteonecrotic disorders. Neuroendocrine component type 1 cannabinoid receptor (CB1) is found to regulate bone mass. This study characterized the biological role of CB1 in glucocorticoid-induced suppression of osteoblast differentiation. Murine MC3T3-E1 osteoblasts were incubated under osteogenic conditions in the presence or absence of 1 μM glucocorticoid, RNA interference, CB1 antagonist AM251, and agonist WIN55212-2. Cell survival was detected by formazan synthesis and TUNEL staining. Osteoblast differentiation was quantified by mineralized matrix accumulation and expression of the osteogenic factors Runx2 and osteocalcin. Expression of signaling molecules was assessed by immunoblotting. Glucocorticoid increased CB1 expression in association with decreased osteocalcin expression and mineralized nodule deposition. CB1 RNA interference and AM251 attenuated the deleterious actions of glucocorticoid treatment on survival and osteogenic activities, whereas activating CB1 by WIN55212-2 impaired osteoblast differentiation. CB1 signaling regulated JNK, ERK, GSK-3β, and Akt activation as well as Runx2 and IGF-I expression. Inhibition of GSK-3β by the kinase-inactive GSK-3β mutant or activation of ERK by the active MEK-1 mutant abrogated glucocorticoid-induced inhibition of osteoblast differentiation. Glucocorticoid-induced CB1 expression occurred via glucocorticoid receptor-dependent transcriptional and translational regulation. Gain of Runx2 function and loss of MKP-1 action attenuated glucocorticoid-induced enhancement of CB1 expression. Taken together, CB1 regulation of ERK and GSK-3β-dependent pathways participates in glucocorticoid inhibition of Runx2 signaling and osteoblast differentiation. Runx2 reciprocally regulates glucocorticoid-induced promotion of CB1 signaling. Our findings provide new insights into the role of the neuroendocrine component CB1 in glucocorticoid-induced osteoblast dysfunction.
authors.affiliation: Department of Orthopedic Surgery, Koahsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
authors.forename: Re-Wen
authors.initials: RW
authors.lastname: Wu
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Lin
authors.forename: Jih-Yang
authors.initials: JY
authors.lastname: Ko
authors.forename: Da-Wei
authors.initials: DW
authors.lastname: Yeh
authors.forename: Ming-Wen
authors.initials: MW
authors.lastname: Chen
authors.forename: Huei-Ching
authors.initials: HC
authors.lastname: Ke
authors.forename: Shin-Long
authors.initials: SL
authors.lastname: Wu
authors.forename: Feng-Sheng
authors.initials: FS
authors.lastname: Wang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D048588:Benzoxazines; D001857:Bone Matrix / Q000187:drug effects / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects; D063387:Cannabinoid Receptor Antagonists; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005938:Glucocorticoids / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D051379:Mice; D008954:Models, Biological; D009025:Morpholines; D050505:Mutant Proteins / Q000378:metabolism; D009281:Naphthalenes; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D034622:RNA Interference / Q000187:drug effects; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043882:Receptors, Cannabinoid / Q000378:metabolism*
chemical_list: D048588:Benzoxazines; D063387:Cannabinoid Receptor Antagonists; D050659:Core Binding Factor Alpha 1 Subunit; D005938:Glucocorticoids; D009025:Morpholines; D050505:Mutant Proteins; D009281:Naphthalenes; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043882:Receptors, Cannabinoid; C493716:Runx2 protein, mouse; C103505:AM 251; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3