Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

Figure 2

Induction of osteoclast differentiation through activation of TRAF-6 and JNK. (a) The protein expression of cytoplasmic molecules of the RANKL-RANK signaling pathway was assessed in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 1 h. (b) Phospho-c-Jun and NFATc1 production was measured in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. (c) The levels of phospho-c-Jun and NFATc1 protein were measured using a JNK inhibitor (SP600125), p38 inhibitor (SB203580), and ERK inhibitor (U0126) at dosages of 10 and 20 M. (d) TRAF-6 mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative siRNA and TRAF-6 siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence and absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). and compared to cells transfected with 0 ng/mL of TRAF-6 siRNA. (e) TRAF-6, phospho-JNK, phospho-c-Jun, and NFATc1 protein levels were measured from RAW 264.7 cells transfected with negative siRNA and TRAF-6 siRNA (50 ng/mL) for 72 h. (f) The mRNA expression of osteoclast-specific genes was measured in controls and negative and TRAF-6 siRNA (50 ng/mL) RAW 264.7 cells. and compared to cells treated with both MSU crystals and RANKL. (g) TRAP and actin ring staining assays for osteoclast-like cells were performed in control RAW 264.7 cells and TRAF-6 siRNA transfected cells under stimulation with both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). compared to control cells treated with both MSU crystals and RANKL. The experimental data were determined by at least three independent tests.

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