NADPH oxidase-derived reactive oxygen species are essential for differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts.
Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) homologues have been suggested to regulate osteoclast differentiation. However, no bone abnormalities have been documented in Nox1 deficient, Nox2 deficient, or Nox3 mutant mice. During receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts, mRNA levels of Nox enzymes (Nox1-4) and their adaptor proteins were monitored by real-time reverse transcriptase PCR. RAW264.7 cells constitutively expressed abundant Nox2 mRNA and small amounts of Nox1 and Nox3 transcripts. RANKL markedly attenuated Nox2 mRNA expression in association with reciprocal up-regulation of Nox1 and Nox3 transcripts. Introduction of small interference RNA targeting p67(phox) or p22(phox) into RAW264.7 cells effectively down-regulated ROS generation and significantly suppressed the RANKL-stimulated differentiation, which was assessed by appearance of tartrate resistant acid phosphatase (TRAP)-positive, multinucleated cells having an ability to form resorption pits on calcium phosphate thin film-coated disks, and by expression of osteoclast marker genes (TRAP, cathepsin K, Atp6i, ClC-7, and NFATc1). Our results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RANKL-induced osteoclast differentiation.
Sasaki
Hideyuki
H
Departments of Stress Science, Institute of Health Biosciences, the University of Tokushima Graduate School, Japan.
Yamamoto
Hironori
H
Tominaga
Kumiko
K
Masuda
Kiyoshi
K
Kawai
Tomoko
T
Teshima-Kondo
Shigetada
S
Rokutan
Kazuhito
K
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D053245:RANK Ligand; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C505689:Tnfsf11 protein, mouse; C079789:neutrophil cytosol factor 67K; D009247:NADH, NADPH Oxidoreductases; C488842:Cybb protein, mouse; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C484511:Nox3 protein, mouse; C515614:Cyba protein, mouse