Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation
J. Clin. Invest. Fumiyo Ikeda, et al. 114:475 doi:10.1172/JCI19657 [Go to this article.]

Figure 4
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.