interleukin-8 and 2-aminoethoxydiphenyl-borate

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

This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

Topics: Active Transport, Cell Nucleus; AMP-Activated Protein Kinases; Aryl Hydrocarbon Hydroxylases; Benzimidazoles; Benzoflavones; Benzylamines; Boron Compounds; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 1; Cell Line, Tumor; Chelating Agents; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Egtazic Acid; Enzyme Inhibitors; Gene Expression; Humans; Integrin beta Chains; Interleukin-8; Ionomycin; Macrophages; Naphthalimides; Phosphorylation; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; RNA, Small Interfering; Sulfonamides

Using human osteoblastic SaM-1 cells, we investigated the effects of lysophosphatidic acid (LPA) on the production of interleukin (IL)-6 and IL-8, molecules which are capable of stimulating the development of osteoclasts from their haematopoietic precursors, and examined the signal transduction systems involved in their effect on these cells. These human osteoblasts constitutively expressed endothelial differentiation genes (Edg)-2 and Edg-4, which are LPA receptors. LPA increased gene and protein expression of IL-6 and IL-8 in SaM-1 cells. The expression of IL-6 and IL-8 mRNAs was maximal at 1-3h, and the increase in IL-6 and IL-8 synthesis in response to lysophosphatidic acid (1-10 microM) occurred in a concentration-dependent manner. These increases were blocked by Ki16425, an Edg-2/7 antagonist. In addition, LPA caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which was inhibited by pretreatment with Ki16425 or 2-aminoethoxy-diphenylborate (2-APB), an inositol 1,4,5-triphosphate (IP(3)) receptor (IP(3)R) blocker. The pretreatment of SaM-1 cells with U-73122, a phospholipase C (PLC) inhibitor, and 2-APB also inhibited the increase in IL-6 and IL-8 synthesis in response to LPA. These findings suggest that extracellular LPA-induced IL-6 and IL-8 synthesis occurred through Edg-2 (LPA(1) receptor) and the activation of PLC and IP(3)-mediated intracellular calcium release in SaM-1 cells.

Topics: Boron Compounds; Calcium; Dose-Response Relationship, Drug; Estrenes; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate Receptors; Interleukin-6; Interleukin-8; Isoxazoles; Lysophospholipids; Osteoblasts; Propionates; Pyrrolidinones; Receptors, Lysophosphatidic Acid; RNA, Messenger; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured; Type C Phospholipases

We have shown previously that activation of endogenously expressed, Galphaq/11-coupled P2Y2 nucleotide receptors with UTP reveals an intracellular Ca2+ response to activation of recombinant, Galphai-coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+ release underlies this potentiation. The putative Ins(1,4,5)P3 receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li+ block of inositol monophosphatase activity, costimulation of P2Y2 nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y2 nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca2+ release, was enhanced. In these cells, agonist-mediated Ca2+ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P3 generation in the presence of coactivation of P2Y2 nucleotide receptors and CXCR2 would be sufficient for additional Ca2+ release. Potentiated Ca2+ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the Gbetagamma-scavenger Galphat1 (transducin alpha subunit), indicating the involvement of Galphaq and Gbetagamma subunits, respectively.

Topics: 1-Phosphatidylinositol 4-Kinase; Boron Compounds; Calcium; Calcium Signaling; Cells, Cultured; GTP-Binding Proteins; Humans; Inositol Phosphates; Interleukin-8; Kidney; Phosphoric Monoester Hydrolases; Protein Kinase C; Protein-Tyrosine Kinases; Receptor Cross-Talk; Receptors, Interleukin-8B; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; RGS Proteins; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Thapsigargin; Type C Phospholipases; Uridine Triphosphate