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 5
TNF-α-mediated ROS-dependent activation of NF-κB is required for TNF-α-induced cPLA2 expression. (a) RASFs were pretreated with helenalin for 1 h and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (b) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF-α for 16 h. The levels of p65 and cPLA2 protein were determined by Western blotting. (c) Cells were pretreated with or without Gö6976 (1 μM), SB202190 (1 μM), SP600125 (1 μM), or NAC (10 mM) for 1 h before exposure to TNF-α for the indicated time intervals. The levels of phospho-IKKα/β and phospho-p65 were determined by Western blotting. Cells were pretreated with Gö6976 (1 μM), SB201290 (1 μM), SP600125 (1 μM), NAC (10 mM), or helenalin (1 μM) for 1 h and then incubated with TNF-α for 30 min. (d) Cells were fixed and then labeled using an anti-p65 antibody and FITC-conjugated secondary antibody. (e) The nuclear extract (NE) was prepared and subjected to Western blotting using the indicated antibodies and lamin A used as an internal control. (f) Cells were transiently transfected with NF-κB-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF-α for 4 h. The promoter activity was determined. (g) Cells were pretreated with Gö6976, SB201290, SP600125, DPI, or NAC for 1 h and incubated with TNF-α for 2 h. Chromatin was immunoprecipitated using an anti-p65 antibody. The associated cPLA2 promoter DNA was amplified by polymerase chain reaction (PCR). Input represents PCR products from chromatin pellets before immunoprecipitation. (h) Cells were transfected without or with a cPLA2-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF-α for 6 h. The mRNA levels of cPLA2 (open bars) and cPLA2-luc promoter (shaded bars) were measured. (i) The PGE2 levels in the media were analyzed using a PGE2 EIA kit. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (b), (e), (f), and (g) are the mean ± SEM. ; versus TNF-α alone.
