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

Figure 5

Thrombin induces COX-2 expression via NF-κB. (a) Cells were pretreated with helenalin for 1 h and then incubated with thrombin for 6 h. The levels of COX-2 protein were determined by Western blot. (b) Cells were pretreated with helenalin (3 μM) for 1 h and then incubated with thrombin for 4 h. The mRNA levels and promoter activity of COX-2 were determined. (c) The media saved from (a) were used to determine the levels of PGE2 generation. (d) Cells were transfected with either scrambled or p65 siRNA and then incubated with thrombin for 6 h. The levels of p65 and COX-2 protein were determined. (e) Cells were transfected with wild-type COX-2 promoter or mt-NF-κB COX-2 promoter, and then incubated with thrombin for 4 h. The promoter activity of COX-2 was determined. (f) Cells were treated with thrombin for the indicated time intervals. The nuclear and cytosolic fractions were prepared and subjected to Western blot using an anti-p65 antibody. GAPDH and lamin A were used as a marker protein for cytosolic and nuclear fractions, respectively. (g) Cells were treated with thrombin for the indicated time intervals. The promoter activity of NF-κB was determined. (h) Cells were pretreated without or with helenalin (3 μM) for 1 h and then treated with thrombin for the indicated time intervals. The levels of phospho-p65 were determined by Western blot. (i) Cells were treated with thrombin for the indicated time intervals. The NF-κB p65 binding activities were analyzed by a ChIP assay. 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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