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 3
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.