Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes.

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes.

Thrombin/Matrix Metalloproteinase-9-Dependent SK-N-SH Cell Migration is Mediated Through a PLC/PKC/MAPKs/NF-κB Cascade.

Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH3), nonselective protien kinase C (PKC, GF109203X), PKCα/ßII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCß, PKCδ, p38, JNK1/2, IKKα, IKKß, or p65. Moreover, thrombin-stimulated PKCα/ßII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH3, Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

c-Src/Pyk2/EGFR/PI3K/Akt/CREB-activated pathway contributes to human cardiomyocyte hypertrophy: Role of COX-2 induction.

Thrombin and COX-2 regulating cardiac hypertrophy are via various signaling cascades. Several transcriptional factors including CREB involve in COX-2 expression. However, the interplay among thrombin, CREB, and COX-2 in primary human neonatal ventricular cardiomyocytes remains unclear. In this study, thrombin-induced COX-2 promoter activity, mRNA and protein expression, and PGE2 synthesis were attenuated by pretreatment with the inhibitors of c-Src (PP1), Pyk2 (PF431396), EGFR (AG1478), PI3K/Akt (LY294002/SH-5), and p300 (GR343), or transfection with siRNAs of c-Src, Pyk2, EGFR, p110, Akt, CREB, and p300. Moreover, thrombin-stimulated phosphorylation of c-Src, Pyk2, EGFR, Akt, CREB and p300 was attenuated by their respective inhibitors. These results indicate that thrombin-induced COX-2 expression is mediated through PAR-1/c-Src/Pyk2/EGFR/PI3K/Akt linking to CREB and p300 cascades. Functionally, thrombin-induced hypertrophy and ANF/BNP release were, at least in part, mediated through a PAR-1/COX-2-dependent pathway. We uncover the importance of COX-2 regarding human cardiomyocyte hypertrophy that will provide a therapeutic intervention in cardiovascular diseases.

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 (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 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.