Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6).
This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-kappaB signaling.


BACKGROUND
A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL.


METHODS
Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-kappaB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IkappaB kinase 2 (IKK(DN)) gene and mitogen-activated protein kinase kinase 7 (MKK7(DN)) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis.


CONCLUSIONS
Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-kappaB pathway by IKK(DN) overexpression, but not that of the JNK pathway by MKK7(DN) overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-kappaB-mediated downregulation is essential for efficient osteoclast differentiation.
Ogasawara
Toru
T
Department of Sensory and Motor System Medicine, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.
Katagiri
Mika
M
Yamamoto
Aiichiro
A
Hoshi
Kazuto
K
Takato
Tsuyoshi
T
Nakamura
Kozo
K
Tanaka
Sakae
S
Okayama
Hiroto
H
Kawaguchi
Hiroshi
H
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle; D002454:Cell Differentiation; D002478:Cells, Cultured; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000096:biosynthesis; D015536:Down-Regulation; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000502:physiology; D048688:MAP Kinase Kinase 7 / Q000235:genetics / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009000:Monocytes / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B
D002352:Carrier Proteins; D016213:Cyclins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; C495912:Cdk6 protein, mouse; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7