ABSTRACT
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.
Subject(s)
Endotoxins/pharmacology , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , eIF-2 Kinase/physiology , Animals , Anisomycin/pharmacology , Arsenites/pharmacology , Bacterial Proteins/metabolism , Cell Line , Crosses, Genetic , Cytokines/metabolism , Electrophoresis, Polyacrylamide Gel , Endotoxins/immunology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/immunology , Fibroblasts/metabolism , Genotype , Hot Temperature , Humans , Hydrogen Peroxide/pharmacology , Interleukin-12/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/metabolism , MAP Kinase Kinase 4 , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Biological , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA, Double-Stranded/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Sodium Chloride/pharmacology , Sodium Compounds/pharmacology , Time Factors , Ultraviolet Rays , eIF-2 Kinase/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
Expression of transfected genes is shown to be suppressed by two intracellular enzymes, RNase L and protein kinase PKR, which function in interferon-treated cells to restrict viral replication. RNase L(-/-) or PKR(-/-) murine embryonic fibroblasts produced enhanced levels of protein from transfected genes compared with wild-type cells. Increased expression of exogenous genes in RNase L(-/-) cells correlated with elevated levels of mRNA and thus appeared to be due to enhanced mRNA stability. Plasmid encoding adenovirus VA RNAs was able to further enhance accumulation of the exogenous gene transcript and protein, even in cells lacking PKR. In contrast to the increased expression of transfected genes in cells lacking RNase L or PKR, expression of endogenous host genes was unaffected by the absence of these enzymes. In addition, a dominant-negative PKR mutant improved expression from a conventional plasmid vector and from a Semliki Forest virus derived, self-replicating vector. These results indicate that viral infections and transfections produce similar stress responses in mammalian cells and suggest strategies for selectively increasing expression of exogenous genes.
Subject(s)
Adenoviridae/genetics , Endoribonucleases/metabolism , Genetic Vectors/genetics , eIF-2 Kinase/metabolism , Adenoviridae/physiology , Animals , Antiviral Agents/metabolism , Cells, Cultured , Endoribonucleases/genetics , Gene Expression Regulation , Mice , Plasmids/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Virus Replication , eIF-2 Kinase/geneticsABSTRACT
Rabbit peritoneal polymorphonuclear neutrophils reduced inorganic [35S]sulfate to [35S]sulfite in vitro, concomitant with incorporation of 35S into a 10.68-kDa cytosolic protein as a S-[35S]sulfo-derivative. Amino-terminal sequencing of the purified protein identified calgranulin C, a member of the S100 protein family. cDNA clones of calgranulins B and C were isolated using oligonucleotide primers based on the established amino acid sequences of other mammalian calgranulins. The complete amino acid sequence of rabbit calgranulin C was deduced from the nucleotide sequence of the corresponding cDNA. It comprises 91 amino acid residues, has a calculated molecular mass of 10.52 kDa, has 74% identity with porcine calgranulin C, and shows high homology with other S100 calcium-binding proteins. Rabbit calgranulin C has a single cysteine residue at position 30, which we believe to be modified to S-[35S]sulfo-cysteine as a consequence of sulfate reduction by neutrophils. The formation of S-[35S]sulfo-calgranulin C appears to be a reaction specific to neutrophils. The specific radioactivity of calgranulin C from the neutrophil culture medium was 50-fold greater than that of the calgranulin C within the cells, suggesting that S-sulfation of calgranulin C might be associated with its secretion.
