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

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E ₂ (PGE ₂ ) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE ₂ is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE ₂ synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF- κ B was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE ₂ secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), G _i/o protein (GPA2), G _q protein (GPA2A), PKC α (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF- κ B (helenalin) and transfection with siRNA of PAR1, G _q α , G _i α , PKC α , JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKC α phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKC α pathway. Thrombin promoted phosphorylation of NF- κ B p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE ₂ expression triggered by thrombin was engaged in PAR1/G _q or G _i/o /PKC α /MAPK-dependent NF- κ B activation in HTSMCs.
Competing Interests: The authors declare no conflict of interest.
(Copyright © 2022 Chien-Chung Yang et al.)