u-0126 and phenylalanyl-prolyl-arginine-chloromethyl-ketone

In order to investigate the regulation of chemokines [interleukin-8 (IL-8), growth-regulated oncogene (GRO)α, monocyte chemoattractant protein-1 (MCP-1)) induced by thrombin in endometrial stromal cells (ESCs), the effects of thrombin, a protease activated receptor (PAR)-1 antagonist (PPACK), mitogen-activated protein kinase kinase inhibitor (U0126), phospholipase C inhibitor (U-73122), an antagonist of the intracellular InsP3 receptor (2-aminoethoxy-diphenylborate (2-APB)] and a protein kinase C inhibitor (GF-109203X) on the production of chemokines by ESCs were evaluated.. ESCs from eight endometrial specimens in the secretory phase were cultured and incubated for 24h with thrombin and PPACK, U0126, U-73122, 2-APB or GF-109203X. The levels of IL-8, GROα and MCP-1 in the culture medium were measured by means of ELISA. The activation of MAP kinase was detected by western blot analysis using anti-phosphorylated MAP kinase (ERK1/2) antibody.. Following stimulation by thrombin, the production of IL-8, GROα and MCP-1 increased significantly in a dose-dependent manner. PPACK, U0126, U-73122, 2-APB or GF-109203X suppressed the increases in production of IL-8, GROα and MCP-1 induced by thrombin (P < 0.001, P <0.001 and P <0.001, respectively). MAP kinase activities were induced by treatment with thrombin, and were suppressed by PPACK, U0126, U-73122, 2-APB or GF-109203X.. Our results suggest that thrombin stimulates the production of IL-8, GROα and MCP-1 via PAR-1 by a mechanism involving the MAP kinase system. The increases in IL-8, GROα and MCP-1 may contribute to the maintenance of implantation involving leukocyte chemotaxis.

Topics: Adult; Amino Acid Chloromethyl Ketones; Boron Compounds; Butadienes; Cells, Cultured; Chemokine CCL2; Chemokine CXCL1; Endometrium; Estrenes; Female; Humans; Indoles; Interleukin-8; Maleimides; Nitriles; Pyrrolidinones; Receptor, PAR-1; Stromal Cells; Thrombin

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Topics: Amino Acid Chloromethyl Ketones; Anthracenes; Antithrombins; Blotting, Western; Butadienes; Cell Line; Cells, Cultured; Cyclooxygenase 2 Inhibitors; Enzyme Inhibitors; Flavonoids; Gene Expression; Glycoproteins; Hirudins; Humans; JNK Mitogen-Activated Protein Kinases; Lipoproteins, VLDL; Macrophages; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Nitrobenzenes; Reverse Transcriptase Polymerase Chain Reaction; Sesquiterpenes; Signal Transduction; Sulfonamides; Thrombin; Time Factors

To measure the levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) induced by thrombin in endometrial stromal cells (ESC).. Evaluation of the effects of thrombin, thrombin receptor activator peptide 6 (TRAP-6), and D-phenylalanyl-1-propyl-L arginine chloromethyl ketone (PPACK) on the production of VEGF and MMPs by ESC.. Research laboratory at the Oita University Medical School.. Eight endometrial specimens in the secretory phase.. ESC were incubated for 24 hours with thrombin, TRAP-6, and PPACK.. The levels of VEGF, MMP-1, and active MMP-2 were measured by enzyme-linked immunosorbent assay (ELISA). The presence of protease-activated receptor-1 (PAR-1) and activation of mitogen-activated protein (MAP) kinase were detected by Western blot analysis.. Following stimulation by thrombin and TRP-6, the production of VEGF, MMP-1, and active MMP-2 statistically significantly increased; U0126 and PPACK statistically significantly suppressed the increases in the production of VEGF, MMP-1, and active MMP-2 induced by thrombin and TRAP-6. Activity by MAP kinase was induced by treatment with thrombin and TRAP-6 and was suppressed by PPACK.. The results suggest that thrombin stimulates the production of VEGF and MMPs by a mechanism involving the MAP kinase system. The increases in VEGF and MMPs may contribute to neovascularization, which promotes the proliferation of endometrium and placentation.

Topics: Adult; Amino Acid Chloromethyl Ketones; Blotting, Western; Butadienes; Cells, Cultured; Dose-Response Relationship, Drug; Endometrium; Enzyme-Linked Immunosorbent Assay; Female; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinases; Nitriles; Peptide Fragments; Phosphorylation; Protein Kinase Inhibitors; Receptor, PAR-1; Signal Transduction; Stromal Cells; Thrombin; Up-Regulation; Vascular Endothelial Growth Factor A