Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation
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
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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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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Activation of NFAT1 by RANKL signals and requirement of NFAT for osteocl...
Activation of NFAT1 by RANKL signals and requirement of NFAT for osteoclastogenesis. (A) Activation of NFAT transcriptional activity during osteoclast differentiation. RAW264 cells incubated with or without sRANKL were transfected with a luciferase reporter construct containing three copies of NFAT-responsive elements. Luciferase activity in cell lysates was measured. TRAP (+), TRAP+ osteoclast-like cells treated with sRANKL. RLU, relative light units. (B) Expression of NFAT1 in BMMΦ cells. The lysates of BMMΦ cells were analyzed by immunoblotting using anti-NFAT1 antibody. (C) Nuclear translocation of NFAT1 by sRANKL. RAW264 cells were stimulated with or without sRANKL, and cytoplasmic and nuclear extracts were analyzed by immunoblotting with anti-NFAT1 antibody. (D) Expression of NFAT1 in osteoclasts. Mouse spleen cells were incubated with M-CSF and sRANKL for 6 days and analyzed by immunostaining (upper panel) or immunoblotting (lower panel) using NFAT1 antibody. (E) Transcriptional activation of NFAT1 by TRAF6. COS-7 cells were transfected with a luciferase reporter construct containing three copies of NFAT-responsive elements together with either NFAT1 or TRAF6, or both. Luciferase activity in cell lysates was measured. (F) Suppression of osteoclastogenesis by cyclosporin A. Mouse bone marrow cells were incubated with M-CSF and sRANKL for 6 days in the presence or absence of cyclosporin A (CsA) as indicated. TRAP+ multinucleated osteoclast-like cells were counted under a microscope. (G) Inhibition of osteoclastogenesis by NFAT inhibitor, VIVIT peptide. RAW264 cells infected with control or GFP-VIVIT adenovirus at 20 or 50 MOI were incubated with sRANKL for 6 days, and TRAP+ multinucleated osteoclast-like cells were counted.