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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Article Bone biology

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 3

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Requirement of c-Jun and c-Fos for RANKL-induced osteoclastogenesis. (A)...
Requirement of c-Jun and c-Fos for RANKL-induced osteoclastogenesis. (A) Inhibition of AP-1 transcriptional activity by DN–c-Jun or DN–c-Fos. RAW264 cells were transfected AP-1 reporter construct together with DN–c-Jun or DN–c-Fos, and incubated with sRANKL. Luciferase activity in cell lysates was measured. (B) Suppression of osteoclast differentiation by DN–c-Jun. RAW264 cells infected with control or DN–c-Jun adenovirus were incubated with sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted. (C) Inhibition of osteoclast differentiation by suppression of c-Jun expression. Mouse spleen cells infected with retroviruses carrying either luciferase siRNA or c-Jun siRNA (siJun) were incubated with M-CSF and sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted. The cell lysates were analyzed by immunoblotting using anti–c-Jun or anti–β-actin antibody. (D) Activation of ATF-2 by sRANKL. The lysates of RAW264 cells stimulated with sRANKL were analyzed by immunoblotting using anti–phospho–ATF-2 (anti–p-ATF-2) or anti–ATF-2 antibody. (E) No effects of DN–ATF-2 on osteoclast formation. RAW264 cells infected with control or DN–ATF-2 adenovirus were incubated with sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted. (F) Inhibition of osteoclastogenesis by DN–c-Fos. RAW264 cells infected with control or DN–c-Fos adenovirus were incubated with sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted. (G) No induction of osteoclastogenesis by overexpression of c-Jun and c-Fos. RAW264 cells infected with control, TRAF6, or c-Jun/c-Fos adenovirus were incubated with or without sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted.