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 2
Inhibition of osteoclast differentiation by blockade of JNK activation. (A) Osteoclast differentiation of BMMΦ and RAW264 cells was determined by TRAP staining. (B) Lysates of BMMΦ or RAW264 cells were analyzed by immunoblotting using anti–phospho-JNK (anti–p-JNK), JNK1, phospho–c-Jun (p–c-Jun), or c-Jun antibody. (C) Lysates of BMMΦ cells treated with sRANKL and SP600125 (5 μM) as indicated, were analyzed by immunoblotting using anti–phospho-JNK, JNK1, phospho–c-Jun, or c-Jun antibody. (D) Suppression of osteoclastogenesis by JNK inhibitor. Mouse bone marrow cells were incubated with M-CSF and sRANKL in the presence of DMSO or SP600125 (5 μM), and TRAP⁺ multinucleated osteoclast-like cells were counted. Cont, control (E) The effect of SP600125 on osteoclastogenesis is reversible after 1 day. BMMΦ cells were incubated with M-CSF and sRANKL in the absence (left bar) or presence (middle and right bars) of SP600125 (5 μM). SP600125 was removed after 24 hours by washing carefully (right bar). After 6 days of incubation, TRAP⁺ multinucleated osteoclast-like cells were counted. (F) Lysates of RAW264 cells infected with control or dominant-negative–JNK1 (DN-JNK1) adenovirus were analyzed by immunoblotting using phospho–c-Jun, c-Jun, or anti-JNK1 antibody. (G) Suppression of osteoclastogenesis by DN-JNK1. RAW264 cells infected with control or DN-JNK1 adenovirus were incubated with sRANKL, and TRAP⁺ multinucleated osteoclast-like cells were counted. (H) Inhibition of osteoclast differentiation by suppression of c-Jun activation. Mouse spleen cells infected with retroviruses carrying either luciferase siRNA (siLuc) or MKK7 siRNA (siMKK7) were incubated with M-CSF and sRANKL, and TRAP⁺ multinucleated osteoclast-like cells were counted. The cell lysates were analyzed by immunoblotting as indicated.