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

Figure 3

Thrombin induces COX-2 expression via PKCα. (a) Cells were pretreated with Gö6976 for 1 h and then incubated with thrombin for 6 h. The levels of COX-2 protein were determined. (b) Cells were pretreated with Gö6976 (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) Cells were transfected with either scrambled or PKCα siRNA and then incubated with thrombin for 6 h. The levels of PKCα and COX-2 protein were determined. (d) The media saved from (c) were used to determine the levels of PGE₂ generation. (e) Cells were treated with 3 U/ml thrombin for the indicated time intervals. The membrane and cytosolic fractions were prepared and subjected to Western blot using an anti-PKCα antibody. GAPDH and Gαs were used as a marker protein for cytosolic and membrane fractions, respectively. (f) Cells were pretreated with SCH79797 (10 μM), GPA2 (1 μM), or GPA2A (1 μM) for 1 h and then treated with thrombin for 10 min. The membrane fractions were prepared and subjected to Western blot using an anti-PKCα antibody. Data are expressed as of three independent experiments. ^#, as compared with the control or pretreatment with inhibitor indicated in the figure.

(a)

(b)

(c)

(d)

(e)

(f)