Ribotoxic stress response to the trichothecene deoxynivalenol in the macrophage involves the SRC family kinase Hck.
Trichothecene mycotoxins and other translational inhibitors activate mitogen-activated protein kinase (MAPKs) by a mechanism called the "ribotoxic stress response," which drives both cytokine gene expression and apoptosis in macrophages. The purpose of this study was to identify upstream kinases involved in the ribotoxic stress response using the trichothecene deoxynivalenol (DON) and the RAW 264.7 macrophage as models. DON (100 to 1000 ng/ml) dose-dependently induced phosphorylation of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs. MAPK phosphorylation in response to DON exposure occurred as early as 5 min, was maximal from 15 to 30 min, and lasted up to 8 h. Preincubation with inhibitors of protein kinase C, protein kinase A, or phospholipase C had no effect on DON-induced MAPK phosphorylation. In contrast, the Src family tyrosine kinase inhibitors, PP1 (4-amino-5-[4-methylphenyl)]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) and, PP2 (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) concentration-dependently impaired phosphorylation of all three MAPK families. PP1 suppressed DON-induced phosphorylation of the MAPK substrates c-jun, ATF-2, and p90(Rsk). MAPK phosphorylation by two other translational inhibitors, anisomycin and emetine, were similarly Src-dependent. PP1 reduced DON-induced increases in nuclear levels and binding activities of several transcription factors (NF-kappaB, AP-1, and C/EBP), which corresponded to decreases in TNF-alpha production, caspase-3 activation, and apoptosis. Tyrosine phosphorylation of hematopoeitic cell kinase (Hck), a Src found in macrophages, was detectable within 1 to 5 min after DON addition, and this was suppressed by PP1. Knockdown of Hck expression with siRNAs confirmed involvement of this Src in DON-induced TNF-alpha production and caspase activation. Taken together, activation of Hck and possibly other Src family tyrosine kinases are likely to be critical signals that precede both MAPK activation and induction of resultant downstream sequelae by DON and other ribotoxic stressors.
Zhou
Hui-Ren
HR
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824-1224, USA.
Jia
Qunshan
Q
Pestka
James J
JJ
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051572:Proto-Oncogene Proteins c-hck; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013312:Stress, Physiological / Q000139:chemically induced* / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism; D014255:Trichothecenes / Q000633:toxicity*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D014255:Trichothecenes; D014409:Tumor Necrosis Factor-alpha; D011505:Protein-Tyrosine Kinases; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; C007262:deoxynivalenol