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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Induction of osteoclast differentiation by NFAT1 overexpression. (A) Cyt...
Induction of osteoclast differentiation by NFAT1 overexpression. (A) Cytoplasmic and nuclear extracts of RAW264 cells infected with NFAT1 adenovirus were analyzed by immunoblotting with anti-NFAT1 antibody. (B) RAW264 cells were infected with NFAT1 adenovirus at 10, 20, or 50 MOI, and TRAP+ multinucleated osteoclast-like cells were counted. (C) Mouse spleen cells infected with control or constitutively active NFAT1 retrovirus were cultured with M-CSF, and TRAP+ multinucleated osteoclast-like cells were counted. (D) RAW264 cells were transfected with TRAP gene promoter fused to a luciferase reporter construct together with wild-type, constitutively active (CA) or δNC mutant of NFAT1. Luciferase activity in cell lysates was measured. (E) Requirement of putative NFAT-binding element present in the TRAP gene promoter (–523 to –507) for transactivation by NFAT1. RAW264 cells were transfected with serial-deletion mutants of the TRAP gene promoter together with or without NFAT1, and luciferase activities were measured. The sequence of the putative NFAT-binding element in the TRAP gene promoter is indicated. (F) Binding of NFAT1 to NFAT-binding element present in TRAP gene promoter (–523 to –507). Lysates of COS-7 cells transfected with or without NFAT1 were precipitated (Ppt) with the oligonucleotide containing NFAT-binding element (NFAT-BE) from the TRAP gene promoter, and analyzed by immunoblotting using anti-NFAT1 antibody. (G) The lysates of RAW264 cells infected with NFAT1 adenovirus or incubated with sRANKL were determined by immunoblotting with anti-NFAT2 antibody. (H) Induction of osteoclast differentiation by NFAT2 overexpression. RAW264 cells were infected with control or NFAT1 or NFAT2 adenovirus and determined by TRAP staining.