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

Figure 1

Thrombin induces COX-2 expression via PAR1. (a) Cells were treated with thrombin (1, 3, or 10 U/ml) for the indicated time intervals. The protein expression of COX-2 was determined by Western blot. (b, c) Cells were treated with 3 U/ml thrombin for the indicated time intervals. The COX-2 (b) mRNA levels and (c) promoter activity were determined by real-time PCR and promoter assay, respectively. (d) Cells were treated with 3 U/ml thrombin for the indicated time intervals or pretreated with 10 μM CLC or 10 μM NS-398 for 1 h and then treated with thrombin for 8 h. The PGE₂ generation was measured. (e) The levels of PAR1, PAR2, PAR3, PAR4, and β-actin (as an internal control) mRNA on HTSMCs were determined by RT-PCR. (f) Cells were pretreated with PPACK (3 μM) or SCH79797 (10 μM) for 1 h and then incubated with thrombin for 4 h. The mRNA levels and promoter activity of COX-2 were determined. (g) Cells were transfected with either scrambled or PAR1 siRNA and then incubated with thrombin for 6 h. The protein levels of PAR1 and COX-2 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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