PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes

Background and Purpose

Different protease-activated receptors (PARs) activated by thrombin are involved in cardiovascular disease, via up-regulation of inflammatory proteins including COX-2. However, the mechanisms underlying thrombin-regulated COX-2 expression in human cardiomyocytes remain unclear.

Experimental Approach

Human cardiomyocytes were used in the study. Thrombin-induced COX-2 protein and mRNA expression, and signalling pathways were determined by Western blot, real-time PCR and COX-2 promoter luciferase reporter assays, and pharmacological inhibitors or siRNAs. PGE₂ generation and cell proliferation were also determined.

Key Results

Thrombin-induced COX-2 protein and mRNA expression, promoter activity and PGE₂ release was attenuated by the PAR1 antagonist ({"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797) or the inhibitors of proteinase activity (PPACK), MEK1/2 (U0126), p38 MAPK (SB202190) or JNK1/2 (SP600125), and transfection with small interfering RNA (siRNA) of PAR1, p38, p42 or JNK2. These results suggested that PAR1-dependent MAPKs participate in thrombin-induced COX-2 expression in human cardiomyocytes. Moreover, thrombin stimulated phosphorylation of MAPKs, which was attenuated by PPACK and {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797. Furthermore, thrombin-induced COX-2 expression was blocked by the inhibitors of AP-1 (tanshinone IIA) and NF-κB (helenalin). Moreover, thrombin-stimulated phosphorylation of c-Jun/AP-1 and p65/NF-κB was attenuated by tanshinone IIA and helenalin, respectively, suggesting that thrombin induces COX-2 expression via PAR1/MAPKs/AP-1 or the NF-κB pathway. Functionally, thrombin increased human cardiomyocyte proliferation through the COX-2/PGE₂ system linking to EP₂ receptors, as determined by proliferating cell nuclear antigen and cyclin D1 expression.

Conclusions and Implications

These findings demonstrate that MAPKs-mediated activation of AP-1/NF-κB pathways is, at least in part, required for COX-2/PGE₂/EP₂-triggered cell proliferation in human cardiomyocytes.