herbimycin and prolinedithiocarbamate

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.

Topics: Antioxidants; Benzoquinones; Cell Line; Cytokines; Enzyme Induction; Enzyme Inhibitors; Flavonoids; Imidazoles; Immunoblotting; Intracellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Lipopolysaccharides; Membrane Proteins; Microglia; Myristoylated Alanine-Rich C Kinase Substrate; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Pyridines; Quinones; Rifabutin; Signal Transduction; src-Family Kinases; Thiocarbamates

Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzoquinones; Cells, Cultured; Chemokine CCL2; Chemotaxis, Leukocyte; Curcumin; Enzyme Inhibitors; Fibroblasts; Gene Expression; Gingiva; Humans; Lactams, Macrocyclic; Lipopolysaccharide Receptors; Lipopolysaccharides; Monocytes; NF-kappa B; Porphyromonas gingivalis; Proline; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Staurosporine; Thiocarbamates; Transcription Factor AP-1