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 2
TNF-α-induced cPLA2 expression is mediated via the formation of a TNFR1/PKCα complex. RASFs were pretreated with (a) Gö6976 for 1 h or (d) transfected with PKCα or scrambled (scrb) siRNA and then incubated with TNF-α for 16 h. (b) Cells were pretreated without or with Gö6976 (1 μM) or an anti-TNFR1 neutralizing Ab (1 μg/mL) for 1 h and then incubated with TNF-α (30 ng/mL) for the indicated time intervals. (c) RASFs were stimulated with TNF-α for the indicated time intervals. The membrane (ME) and cytosolic extracts (CE) were prepared. The cell fractions were analyzed by Western blot using the indicated antibodies and anti-Gsα and β-actin used as internal controls for ME and CE, respectively. ((e) and (f)) RASFs were pretreated with or without (f) Gö6976 (1 μM) for 1 h and stimulated with TNF-α for the indicated time intervals (e) or for 5 min (f). RASFs were immunoprecipitated (IP) using an anti-PKCα (top) or anti-TNFR1 (bottom) antibody. The cell lysates were immunoprecipitated and analyzed by Western blotting using an anti-PKCα or anti-TNFR1 antibody. Input represents whole cell extracts before immunoprecipitation. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values are the mean ± SEM. In (b) (left, control part), (c), and (e), ; versus vehicle alone. In (a), (b) (right, experimental part), and (d), ; versus TNF-α alone.
