HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB
Figure 4
Involvement of PKCα-dependent p38 MAPK and JNK1/2 in TNF-α-mediated ROS generation and cPLA2 expression. (a) Cells were pretreated with U0126, SB202190, or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF-α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF-α for 16 h. The levels of p38, JNK1, and cPLA2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF-α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μM) or SP600125 (1 μM) for 1 h before exposure to TNF-α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μM) or SP600125 (1 μM) for 1 h before exposure to TNF-α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti- antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. ; versus TNF-α alone.
