herbimycin and Inflammation

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time- and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-beta1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.

Topics: Apoptosis; Benzoquinones; Cell Line; Cell Proliferation; Cell Survival; DNA; DNA, Complementary; Dose-Response Relationship, Drug; Epithelial Cells; Fibrinolysin; Humans; Inflammation; Lactams, Macrocyclic; Lung; Nuclear Proteins; Phosphorylation; Plasmids; Protein Binding; Protein Structure, Tertiary; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Quinones; Rifabutin; Time Factors; Transfection; Tumor Suppressor Protein p53; Urokinase-Type Plasminogen Activator

The activating properties of IL-2 and the structure of the IL-2R on human monocytes are well characterized. However, relatively little is known about the biochemical mechanisms involved in IL-2 signal transduction in these cells. We investigated the role of protein tyrosine kinases (PTKs) in the activation of monocytes by IL-2. Incubation of monocytes with the PTK inhibitor herbimycin A (HA) resulted in the dose-dependent suppression of IL-2-induced monocyte tumoricidal activity. This inhibition was rather potent, as a concentration of HA as low as 0.5 microM caused a complete abrogation of cytolytic activity. Furthermore, HA markedly suppressed the ability of IL-2 to induce IL-1 beta, TNF-alpha, IL-6, and IL-8 mRNA expression and protein secretion by monocytes. Anti-phosphotyrosine immunoblotting demonstrated that IL-2 induced a rapid and time-dependent increase in tyrosine phosphorylation of several cellular proteins of molecular masses ranging from 35 to 180 kDa. Interestingly, IL-2 caused a significant up-regulation of the constitutive levels of hck PTK mRNA and protein relative to medium-treated cells as well as an increase in p59hck tyrosine phosphorylation. Finally, we demonstrated by in vitro kinase assay that the specific activity of p59hck PTK was also induced by IL-2 in monocytes. Thus, these data show that the activation of PTKs is required for the triggering of monocyte effector and secretory functions by IL-2 and strongly suggest that p59hck is a key participant in IL-2 signaling in human monocytes.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Cells, Cultured; Cytokines; HT29 Cells; Humans; Inflammation; Interleukin-2; Interphase; Janus Kinase 1; Janus Kinase 3; Lactams, Macrocyclic; Lymphocyte Activation; Macrophage Activation; Monocytes; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-hck; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; src-Family Kinases; Up-Regulation

Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages.

Topics: Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Enzyme Activation; Enzyme Precursors; Extracellular Matrix Proteins; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genistein; Humans; Inflammation; Integrin beta1; Integrins; Interleukin-1; Intracellular Signaling Peptides and Proteins; Isoflavones; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Monocytes; NF-kappa B; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Syk Kinase; Tumor Cells, Cultured; Tyrosine