herbimycin and Leukemia-P388

Treatment of a mouse macrophage cell line, P388D1, for 1 h with bacterial LPS caused a transient increase in the level of junB mRNA expression. These cells became refractory in terms of the junB gene response to exposure to a second round of LPS or lipid A, but not to PMA. The LPS-induced desensitized state was not due to the shortening of the half-life of junB mRNA, but was suggested, by nuclear run-on analysis, to be caused by reduction of junB gene transcription. Pretreating cells with herbimycin A, a tyrosine kinase inhibitor, substantially inhibited LPS-induced expression of junB mRNA and decreased tyrosine phosphorylation of 38- to 42-kDa proteins, which comigrated with p38 and p42 mitogen-activated protein (MAP) kinases. Parallel to down-regulation of junB mRNA expression, activation of the p38 MAP kinase was markedly reduced in LPS-tolerant cells, whereas activation of p42 MAP kinase was relatively constant. The specific p38 MAP kinase inhibitor, SB202190, potently inhibited LPS-induced junB mRNA expression. These results suggest that the LPS-induced desensitization of junB gene expression occurs at or upstream of the level of gene transcription and may be involved in a defective LPS-induced p38 MAP kinase pathway.

Topics: Animals; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Dose-Response Relationship, Immunologic; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, jun; Imidazoles; Lactams, Macrocyclic; Leukemia P388; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred DBA; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-jun; Pyridines; Quinones; Rifabutin; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Replication of Ehrlichia risticii was inhibited in P388D1 cells when a protein tyrosine kinase inhibitor (genistein or herbimycin A) was added after internalization of the organism at 3 h postinfection. Upon addition of genistein at day 1, 2, 3, or 4 postinfection, further proliferation of E. risticii was prevented. The inhibition was reversible, since regrowth of E. risticii occurred upon the removal of genistein. Genistein prevented spreading of E. risticii from P388D1 cells to THP-1 cells. Genistein did not prevent binding of [35S]methionine-labeled E. risticii to P388D1 cells but did prevent internalization of [35S]methionine-labeled E. risticii. 14CO2 production from L-[14C]glutamine in Percoll density gradient-purified E. risticii was not inhibited by genistein or herbimycin A, which suggests that these reagents did not directly inhibit ehrlichial energy metabolism. Double indirect immunofluorescence labeling with antiphosphotyrosine antibody and anti-E. risticii antibody revealed colocalization of tyrosine phosphoproteins with ehrlichial inclusions. There was, however, no colocalization of phosphotyrosine with phagosomes containing 0.5-microm-diameter fluorescent beads. Western immunoblot analysis revealed that 52- and 54-kDa proteins were tyrosine phosphorylated only in infected cells and that phosphorylation of these two proteins was reduced when infected cells were treated with genistein for 6 h. These results suggest that protein tyrosine phosphorylation is specific and essential for ehrlichial internalization, replication, and spreading in macrophages but not for binding.

Topics: Animals; Benzoquinones; Ehrlichia; Genistein; Glutamine; Isoflavones; Lactams, Macrocyclic; Leukemia P388; Macrophages; Methionine; Mice; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tyrosine