Thrombin Induces COX-2 and PGE₂ Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells

Figure 6

Thrombin induces NF-κB activation via PKCα/MAPKs. (a) Cells were pretreated with helenalin (3 μM), U0126 (10 μM), SB202190 (10 μM), or SP600125 (10 μM) for 1 h and then incubated with thrombin for 30 min. Cells were fixed and then labeled with an anti-phospho-p65 antibody and then FITC-conjugated secondary antibody. Individual cells were imaged. Scale bar: 50 μm. (b) Cells were pretreated with Gö6976 (3 μM), U0126 (10 μM), SB202190 (10 μM), or SP600125 (10 μM) for 1 h and then incubated with thrombin for 4 h. The promoter activity of NF-κB was determined. (c) Cells were pretreated with Gö6976 (3 μM), U0126 (10 μM), SB202190 (10 μM), or SP600125 (10 μM) for 1 h and then incubated with thrombin for 30 min. The NF-κB p65 binding activities were analyzed by a ChIP assay. (d) Cells were pretreated without or with SP600125 (10 μM), SB202190 (10 μM), or U0126 (10 μM) for 1 h, and then incubated with thrombin for the indicated time intervals. The levels of phospho-p65 were determined by Western blot. Data are expressed as of three independent experiments. ^#, as compared with the control or pretreatment with inhibitor indicated in the figure.

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