h-89 and pyrrolidine-dithiocarbamic-acid

Our previous study showed that human periodontal ligament cells responded to mechanical stress by increasing adenosine triphosphate (ATP) release, accompanied by the increased expression of RANKL and osteopontin. We found that the signaling pathway of mechanical stress-induced osteopontin was mediated through ATP/P2Y(1) receptor and Rho kinase activation but that of mechanical stress-induced RANKL was different. In this study, we further investigated the effect of extracellular ATP on the expression of RANKL and the mechanism involved.. Human periodontal ligament cells were treated with ATP (10-40 microm). The expressions of RANKL and cyclo-oxygenase 2 (COX-2) were examined by RT-PCR and western blot analysis. The level of prostaglandin E(2) was determined using ELISA. Signaling pathways were investigated by using inhibitors and antagonist.. Adenosine triphosphate induced the expression of RANKL. Indomethacin, an inhibitor of COX, could abolish the induction of RANKL expression, suggesting a COX-dependent mechanism. A cAMP-dependent protein kinase inhibitor, H89, and a nuclear factor kappaB (NF kappaB) inhibitor, pyrrolidine dithiocarbamate, inhibited RANKL expression, prostaglandin E(2) production and NF kappaB translocation. In addition, a specific P2Y(1) receptor antagonist, MRS2179, and P2Y(1) small interfering RNA diminished the effect of ATP.. Extracellular ATP stimulates RANKL expression in human periodontal ligament cells through a pathway dependent on the P2Y(1) receptor, cAMP-dependent protein kinase, NF kappaB and COX. Our results suggest that, among the molecules responsible for the effect of mechanical stress, ATP participates in bone resorption or bone homeostasis by mediating its signal through the P2Y(1) receptor and the NF kappaB-COX-RANKL axis in periodontal tissue.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Biomechanical Phenomena; Bone Remodeling; Bone Resorption; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Enzyme Inhibitors; Estrenes; Humans; Indomethacin; Isoquinolines; NF-kappa B; Periodontal Ligament; Phosphodiesterase Inhibitors; Purinergic P2 Receptor Antagonists; Pyrrolidines; Pyrrolidinones; RANK Ligand; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; RNA, Small Interfering; Sulfonamides; Thiocarbamates; Type C Phospholipases

The macrophage-like cell line, J774, was found to respond to immobilized mouse monoclonal IgG2a proteins, but not to soluble forms of IgG2a or IgG2b or to immobilized F(ab')2 of IgG2a, by the increase in the nuclear proteins of two different types of NF-kappa B proteins which differed in their electrophoretic mobilities. Fc gamma 2a receptor-mediated activation of NF-kappa B was blocked by the presence of pyrrolidinedithiocarbamate, neutralizing anti-tumor necrosis factor (TNF)-alpha antibodies, various protein kinase inhibitors (H-89, genistein, or heparin) or intracellular calcium chelator (1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, BAPTA/AM) during stimulation. J774 cells were also found to respond to immobilized IgG2a, but not IgG2b, by the increased production of superoxide, H2O2, and TNF-alpha. Fc gamma 2aR-induced production of H2O2 was inhibited by pretreatment of the cells with pyrrolidinedithiocarbamate, H-89, genistein, heparin, or BAPTA/AM, but not with anti-TNF-alpha antibody. Fc gamma 2aR-induced production of TNF-alpha was, on the other hand, not inhibited by pretreatment of the cells with BAPTA/AM. Although J774 cells responded to exogenously added rTNF-alpha, but not to H2O2, by activation of NF-kappa B, the recombinant TNF-alpha-mediated NF-kappa B activation was enhanced by simultaneous presence of H2O2. These results thus suggest that macrophages respond to the stimulation of Fc gamma 2aR by the production of both reactive oxygen intermediates and TNF-alpha and that endogenous TNF-alpha activates NF-kappa B via the pathway involving reactive oxygen intermediates.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antioxidants; Base Sequence; Binding Sites; Cell Line; Cell Nucleus; Egtazic Acid; Enhancer Elements, Genetic; Female; Genistein; Heparin; HIV-1; Hydrogen Peroxide; Immunoglobulin G; Isoflavones; Isoquinolines; Kinetics; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; NF-kappa B; Oligodeoxyribonucleotides; Protein Binding; Protein Kinase Inhibitors; Pyrrolidines; Reactive Oxygen Species; Receptors, IgG; Recombinant Proteins; Sulfonamides; Thiocarbamates; Tumor Necrosis Factor-alpha