The effect of atorvastatin on the phosphorylations of ERK, JNK, Akt, and p38 MARK, induced by TNF-α in FLSs from RA patients (patients 1, 4, and 5). (A) FLSs were cultured in the presence of TNF-α (20 ng/ml) with or without atorvastatin (50 to 100 μM) for 24 hours to determine the effect of atorvastatin on the activations of signaling molecules. Phosphorylated ERK, JNK, Akt, and p38 MARK were analyzed by using Western blotting. Representative Western blots from three independent experiments are shown. (B) The effect of atorvastatin on the phosphorylation of p38 MARK induced by TNF-α in FLSs from an RA patient. After serum deprivation for 24 hours, FLSs were cultured with SB203580 (10 μM) for 2 hours. TNF-α (20 ng/ml) and atorvastatin (50 to 100 μM) were then added, and incubation was continued for another 24 hours. The expressions of RANKL mRNA relative to GAPDH were determined with RT-PCR. (C) RANKL secretion was determined with ELISA. Both SB203580 and atorvastatin inhibited RANKL production by TNF-α.