title: PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  Thrombin-induced COX-2 protein and mRNA expression, promoter activity and PGE2 release was attenuated by the PAR1 antagonist (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 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
  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.
authors.affiliation: Graduate Institute of Biomedical Science, Chang Gung University, Tao-Yuan, Taiwan; Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Peter Tzu-Yu
authors.initials: PT
authors.lastname: Chien
authors.forename: Hsi-Lung
authors.initials: HL
authors.lastname: Hsieh
authors.forename: Pei-Ling
authors.initials: PL
authors.lastname: Chi
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D049109:Cell Proliferation; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D044004:Xanthones / Q000494:pharmacology
chemical_list: D016328:NF-kappa B; C545644:PTGER2 protein, human; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D058307:Receptors, Prostaglandin E, EP2 Subtype; D018808:Transcription Factor AP-1; D044004:Xanthones; C053876:6-isopropoxy-9-oxoxanthene-2-carboxylic acid; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; D015232:Dinoprostone