JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts.
BACKGROUND
RANKL is known to be important not only for differentiation and activation of osteoclasts but also for their survival. Experimentally, apoptosis of osteoclasts is rapidly induced by the deprivation of RANKL. RANKL activates Elk-related tyrosine kinase (ERK), p38, c-Jun N-terminal kinase (JNK), and NF-kappaB pathways through TRAF6 in osteoclasts and the precursor cells. It has been shown that ERK is critical for regulation of osteoclast survival. However, an involvement of other RANKL signaling pathways such as JNK signaling in survival of osteoclasts has not been fully understood yet.


METHODS
Osteoclasts derived from primary mouse bone marrow cells by soluble RANKL (sRANKL) were treated with a JNK inhibitor, SP600125, or infected with adenovirus carrying dominant-negative (DN)-c-jun, DN-c-fos, mitogen-activated protein kinase kinase 1 (MEKK1), I-kappaBalpha mutant, or NF-kappaB components, p50 and p65. Osteoclasts were cultured with or without sRANKL, and apoptotic phenotype was determined by TUNEL assay, DAPI staining, and expression of cleaved caspase 3 followed by TRACP staining.


RESULTS
Overexpression of TRAF6 activated JNK and NF-kappaB signaling pathways and clearly prevented osteoclasts from apoptosis caused by abrogation of sRANKL. An anti-apoptotic effect of RANKL/RANK/TRAF6 signaling on osteoclast was inhibited by JNK-specific inhibitor SP600125 and by overexpression of dominant-negative JNK1, c-jun, and c-fos. Also, overexpression of MEKK1 inhibited apoptosis of osteoclasts even in the absence of sRANKL along with activation of JNK/c-jun signaling. On the other hand, blockade of NF-kappaB signaling by I-kappaBalpha mutant or overexpression of NF-kappaB components showed a marginal effect on apoptosis of osteoclasts.


CONCLUSIONS
An important role of RANKL-induced activation of MEKK1/JNK/c-jun signaling in the regulation of apoptosis in osteoclasts was shown. Our study suggests that c-fos plays a role as a partner of activator protein-1 factor, c-jun, during the regulation of apoptosis in osteoclasts.
Ikeda
Fumiyo
F
Department of Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan.
Matsubara
Takuma
T
Tsurukai
Taro
T
Hata
Kenji
K
Nishimura
Riko
R
Yoneda
Toshiyuki
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016755:Proto-Oncogene Proteins c-jun / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D012995:Solubility; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D013997:Time Factors
D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1