anisomycin and sodium-arsenite

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCI), or anisomycin, an activator of p38 mitogen-activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)-induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase-activated protein (MAPKAP) kinase-2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Topics: Anisomycin; Arsenites; Butadienes; Cadmium Chloride; Carcinogens; Dithiothreitol; Enzyme Inhibitors; Flavonoids; Glioma; Heat-Shock Proteins; Humans; Hydrogen Peroxide; Imidazoles; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Osmotic Pressure; Oxidants; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Synthesis Inhibitors; Pyridines; Sodium Compounds; Sorbitol; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice null for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

Topics: Animals; Anisomycin; Anti-Inflammatory Agents; Apoptosis; Arsenites; Calcium-Calmodulin-Dependent Protein Kinases; Cytokines; Embryo, Mammalian; Enzyme Activation; Fibroblasts; Genes, Lethal; Interleukin-1; Interleukin-6; Intracellular Signaling Peptides and Proteins; Isoenzymes; Mice; Mice, Inbred DBA; Mice, Mutant Strains; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Receptors, Interleukin-1; Signal Transduction; Sodium Compounds; Stem Cells

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.

Topics: Animals; Anisomycin; Arsenites; Bacterial Proteins; Cell Line; Crosses, Genetic; Cytokines; eIF-2 Kinase; Electrophoresis, Polyacrylamide Gel; Endotoxins; Enzyme Activation; Enzyme Inhibitors; Female; Fibroblasts; Genotype; Hot Temperature; Humans; Hydrogen Peroxide; Interleukin-12; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Double-Stranded; Signal Transduction; Sodium Chloride; Sodium Compounds; Time Factors; Ultraviolet Rays

The effect of the toxic chemical Na-arsenite and the protein synthesis inhibitor anisomycin on glucose transport in primary cultures of bovine chromaffin cells was compared using the effect of insulin-like growth factor I (IGF-I) as a reference. The enhanced uptake of glucose obtained in response to arsenite and anisomycin reached maximum after 60 min, with the response to anisomycin being delayed in onset relative to that of arsenite. At maximal doses the arsenite effect was consistently higher than that of anisomycin and comparable to the approximately 2-fold effect produced by IGF-I. The selective inhibitor of stress-activated protein kinase 2 (SAPK2), SB 203580, inhibited completely anisomycin-induced glucose uptake but only partly suppressed uptake stimulated by arsenite. Both substances, in concentrations producing maximal effects on glucose transport, led to a strong phosphorylation of SAPK2. In contrast to the effect on glucose transport, the arsenite-induced phosphorylation of SAPK2 was relatively slow compared to the anisomycin-induced activation. The results indicate that glucose uptake induced by the two types of cellular stress are mediated by at least two different signaling pathways, which also differ from that activated by IGF-I.

Topics: Animals; Anisomycin; Arsenites; Biological Transport; Cattle; Chromaffin Cells; Deoxyglucose; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Glucose; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Osmotic Pressure; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Sodium Compounds; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation

The Ras/Raf/MAP kinase (ERK) pathway is a major signaling pathway induced by growth factors in mammalian cells. Two other types of mammalian MAP kinases, JNK (SAPK) and p38 (RK, CSBP), are induced by environmental stress. Although the immediate-early gene, egr-1, is induced by growth factors, cytokines, differentiation signals and DNA damaging agents, less is known about its induction by environmental stress and the mechanism involved. Here we report that in NIH3T3 cells, egr-1 is induced by various stress treatments such as heat shock, sodium arsenite, ultraviolet (U.V.) radiation, and anisomycin. p38 and JNK1, but not ERK2, were activated by these stress treatments. Induction of egr-1 by anisomycin is inhibited by a specific inhibitor of p38, SB 203580. We also show that p38 and JNK1 activated by their upstream kinases induce egr-1 promoter activity through activation of the ternary complex factor, Elk-1. The stress treatments also lead to an increase in Egr-1 protein phosphorylation and its DNA binding activity. Together, our data suggest that induction of egr-1 gene by growth factors and stress are mediated through different subgroups of MAP kinases which may also differentially affect egr-1 function on its target genes.

Topics: 3T3 Cells; Animals; Anisomycin; Arsenites; Calcium-Calmodulin-Dependent Protein Kinases; DNA; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Activation; Enzyme Inhibitors; ets-Domain Protein Elk-1; Gene Expression Regulation; Heat-Shock Response; Humans; Imidazoles; Immediate-Early Proteins; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Platelet-Derived Growth Factor; Promoter Regions, Genetic; Proto-Oncogene Proteins; Pyridines; RNA, Messenger; Sodium Compounds; Transcription Factors; Ultraviolet Rays

We have previously shown that osmotic stress activates both the mitogen-activated protein kinase (MAPK) cascade and the stress-activated protein kinase (SAPK, also known as JNK) cascade in rat fibroblastic 3Y1 cells and rat PC12 cells. Here, we show that treatment of these cells with sodium arsenite, a chemical compound that mimics the effects of heat shock, or anisomycin, a protein synthesis inhibitor, induces activation of SAPKs potently. These chemical compounds also stimulated the activity of SEK1/MKK4/JNKK, SAPK activator, and the activity of MEKK, SEK1 activator. Expression of a dominant negative mutant of Ras blocked the anisomycin-induced activation of SAPK and SEK1, but did not affect markedly the arsenite-induced or heat shock-induced activation in PC12 cells. The osmotic-stress-induced activation of SAPK was insensitive to the expression of a dominant negative Ras, but was partly sensitive to down-regulation of protein kinase C. These results suggest the existence of Ras-dependent and Ras-independent activation pathways for the SAPK cascade triggered by environmental stresses including chemical stress in PC12 cells. Cell staining with a specific anti-SAPK serum showed that SAPKs were present in both the cytoplasm and the nucleus under normal conditions, and became located mainly in the nucleus after osmotic stress or ultraviolet treatment, suggesting the nuclear translocation of SAPKs.

Topics: Animals; Anisomycin; Arsenites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Nucleus; Cytoplasm; Enzyme Activation; Genes, ras; Humans; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Osmotic Pressure; PC12 Cells; Protein Kinase C; Protein Kinases; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Rats; Sodium Compounds; Ultraviolet Rays

Rapid, transient expression of the c-fos proto-oncogene is induced by the beta-adrenergic agonist, isoproterenol, in vivo and by numerous factors promoting growth and differentiation in cultured cells. We wanted to determine whether cellular stressors, which are known to induce expression of the gene encoding the major heat shock protein, hsp 70, also induced expression of the c-fos gene. Our findings demonstrate that c-fos mRNA levels increase transiently under conditions of heat stress or sodium arsenite treatment which induce expression of hsp70 mRNA in cultured cell lines. When 3T3 cells are heat shocked in the presence of amiloride, an inhibitor of Na+/H+ exchange, the induction of c-fos mRNA is partially inhibited, whereas that of hsp70 is somewhat enhanced. However, neither response requires ongoing protein synthesis. In fact, dramatic superinduction of c-fos mRNA is observed in cells which are heat shocked in the presence of the protein synthesis inhibitor, anisomycin. A comparison of relative rates of protein synthesis and c-fos mRNA levels during either heat shock or sodium arsenite treatment suggests that the transient suppression of protein synthesis accompanying these treatments may be one factor responsible for the observed c-fos mRNA induction.

Topics: Actins; Animals; Anisomycin; Arsenic; Arsenites; Cell Line; Heat-Shock Proteins; Hot Temperature; Mice; Mice, Inbred BALB C; Protein Biosynthesis; Proto-Oncogenes; RNA, Messenger; Sodium Compounds; Stress, Physiological