title: Afamin stimulates osteoclastogenesis and bone resorption via Gi-coupled receptor and Ca2+/calmodulin-dependent protein kinase (CaMK) pathways.
abstract: BACKGROUND
  Afamin was recently identified as a novel osteoclast-derived coupling factor that can stimulate the in vitro and in vivo migration of preosteoblasts.
  
  
  OBJECTIVE
  In order to understand in more detail the biological roles of afamin in bone metabolism, we investigated its effects on osteoclastic differentiation and bone resorption.
  
  
  METHODS
  Osteoclasts were differentiated from mouse bone marrow cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were considered as osteoclasts, and the resorption area was determined by incubating the cells on dentine discs. The intracellular cAMP level was determined using a direct enzyme immunoassay. Signaling pathways were investigated using western blot and RT-PCR. Recombinant afamin was administered exogenously to bone cell cultures.
  
  
  RESULTS
  Afamin stimulated both osteoclastogenesis and in vitro bone resorption. Consistently, the expressions of osteoclast differentiation markers were significantly increased by afamin. Although afamin mainly affected the late-differentiation stages of osteoclastogenesis, the expression levels of receptor activator of nuclear factor-κB ligand (RANKL)-dependent signals were not changed. Afamin markedly decreased the levels of intracellular cAMP with reversal by pretreatment with pertussis toxin (PTX), a specific inhibitor of Gi-coupled receptor signaling. In addition, PTX almost completely blocked afamin-stimulated osteoclastogenesis. Furthermore, pretreatment with KN93 and STO609 - Ca2+/cal - mo dulin-dependent protein kinase (CaMK) and CaMK kinase inhibitors, respectively - significantly prevented decreases in the intracellular cAMP level by afamin while attenuating afamin-stimulated osteoclastogenesis.
  
  
  CONCLUSIONS
  Afamin enhances osteoclastogenesis by decreasing intracellular cAMP levels via Gi-coupled receptor and CaMK pathways.
authors.affiliation: Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2- Dong, Songpa-Gu, Seoul 138-736, Korea.
authors.forename: B J
authors.initials: BJ
authors.lastname: Kim
authors.forename: Y S
authors.initials: YS
authors.lastname: Lee
authors.forename: S Y
authors.initials: SY
authors.lastname: Lee
authors.forename: S Y
authors.initials: SY
authors.lastname: Park
authors.forename: H
authors.initials: H
authors.lastname: Dieplinger
authors.forename: K
authors.initials: K
authors.lastname: Yea
authors.forename: S H
authors.initials: SH
authors.lastname: Lee
authors.forename: J M
authors.initials: JM
authors.lastname: Koh
authors.forename: G S
authors.initials: GS
authors.lastname: Kim
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000418:Albumins / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D001862:Bone Resorption / Q000188:drug therapy* / Q000378:metabolism; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006023:Glycoproteins / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: C576771:Afm protein, mouse; D000418:Albumins; D006023:Glycoproteins; D007527:Isoenzymes; D016328:NF-kappa B; D053245:RANK Ligand; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C505689:Tnfsf11 protein, mouse; D000242:Cyclic AMP; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; C517598:Camk1 protein, mouse; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase