title: Osteoprotegerin induces podosome disassembly in osteoclasts through calcium, ERK, and p38 MAPK signaling pathways.
abstract: Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca(2+) concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca(2+) concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca(2+) concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix.
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Xuezhong
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zou
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Dai
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Lulian
authors.initials: L
authors.lastname: Yao
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Gu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China.
authors.forename: Jianchun
authors.initials: J
authors.lastname: Bian
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, PR China. Electronic address: liuzongping@yzu.edu.cn.
authors.forename: Zongping
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D021961:Cell Membrane Structures / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053244:Osteoprotegerin / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D002070:Butadienes; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009570:Nitriles; D053244:Osteoprotegerin; D011725:Pyridines; C113580:U 0126; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole; D002118:Calcium
keywords: Calcium; MAPKs; Osteoclast; Osteoprotegerin; Podosome