Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation
Fumiyo Ikeda, … , Anjana Rao, Toshiyuki Yoneda
Fumiyo Ikeda, … , Anjana Rao, Toshiyuki Yoneda
Published August 16, 2004
Citation Information: J Clin Invest. 2004;114(4):475-484. https://doi.org/10.1172/JCI19657.
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Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation

Abstract

Receptor activator of NF-κB ligand (RANKL) plays an essential role in osteoclast formation and bone resorption. Although genetic and biochemical studies indicate that RANKL regulates osteoclast differentiation by activating receptor activator of NF-κB and associated signaling molecules, the molecular mechanisms of RANKL-regulated osteoclast differentiation have not yet been fully established. We investigated the role of the transcription factor c-Jun, which is activated by RANKL, in osteoclastogenesis using transgenic mice expressing dominant-negative c-Jun specifically in the osteoclast lineage. We found that the transgenic mice manifested severe osteopetrosis due to impaired osteoclastogenesis. Blockade of c-Jun signaling also markedly inhibited soluble RANKL-induced osteoclast differentiation in vitro. Overexpression of nuclear factor of activated T cells 1 (NFAT1) (NFATc2/NFATp) or NFAT2 (NFATc1/NFATc) promoted differentiation of osteoclast precursor cells into tartrate-resistant acid phosphatase–positive (TRAP–positive) multinucleated osteoclast-like cells even in the absence of RANKL. Overexpression of NFAT1 also markedly transactivated the TRAP gene promoter. These osteoclastogenic activities of NFAT were abrogated by overexpression of dominant-negative c-Jun. Importantly, osteoclast differentiation and induction of NFAT2 expression by NFAT1 overexpression or soluble RANKL treatment were profoundly diminished in spleen cells of the transgenic mice. Collectively, these results indicate that c-Jun signaling in cooperation with NFAT is crucial for RANKL-regulated osteoclast differentiation.

Authors

Fumiyo Ikeda, Riko Nishimura, Takuma Matsubara, Sakae Tanaka, Jun-ichiro Inoue, Sakamuri V. Reddy, Kenji Hata, Kenji Yamashita, Toru Hiraga, Toshiyuki Watanabe, Toshio Kukita, Katsuji Yoshioka, Anjana Rao, Toshiyuki Yoneda

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Figure 2

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Inhibition of osteoclast differentiation by blockade of JNK activation. ...
Inhibition of osteoclast differentiation by blockade of JNK activation. (A) Osteoclast differentiation of BMMΦ and RAW264 cells was determined by TRAP staining. (B) Lysates of BMMΦ or RAW264 cells were analyzed by immunoblotting using anti–phospho-JNK (anti–p-JNK), JNK1, phospho–c-Jun (p–c-Jun), or c-Jun antibody. (C) Lysates of BMMΦ cells treated with sRANKL and SP600125 (5 μM) as indicated, were analyzed by immunoblotting using anti–phospho-JNK, JNK1, phospho–c-Jun, or c-Jun antibody. (D) Suppression of osteoclastogenesis by JNK inhibitor. Mouse bone marrow cells were incubated with M-CSF and sRANKL in the presence of DMSO or SP600125 (5 μM), and TRAP+ multinucleated osteoclast-like cells were counted. Cont, control (E) The effect of SP600125 on osteoclastogenesis is reversible after 1 day. BMMΦ cells were incubated with M-CSF and sRANKL in the absence (left bar) or presence (middle and right bars) of SP600125 (5 μM). SP600125 was removed after 24 hours by washing carefully (right bar). After 6 days of incubation, TRAP+ multinucleated osteoclast-like cells were counted. (F) Lysates of RAW264 cells infected with control or dominant-negative–JNK1 (DN-JNK1) adenovirus were analyzed by immunoblotting using phospho–c-Jun, c-Jun, or anti-JNK1 antibody. (G) Suppression of osteoclastogenesis by DN-JNK1. RAW264 cells infected with control or DN-JNK1 adenovirus were incubated with sRANKL, and TRAP+ multinucleated osteoclast-like cells were counted. (H) Inhibition of osteoclast differentiation by suppression of c-Jun activation. Mouse spleen cells infected with retroviruses carrying either luciferase siRNA (siLuc) or MKK7 siRNA (siMKK7) were incubated with M-CSF and sRANKL, and TRAP+ multinucleated osteoclast-like cells were counted. The cell lysates were analyzed by immunoblotting as indicated.