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 5
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.