mRNA Superinduction Research Articles

Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen.

The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen.

Cell-mediated suppression of human interleukin-2 gene expression at splicing of mRNA

Human IL-2 gene expression is regulated by cell-mediated suppression. Mitogenic stimulation of PBMC induces transient activation of CD8 cells that inhibit expression of this gene. Depletion of CD8 cells elicits marked superinduction of IL-2 mRNA; reintroduction of CD8 cells causes severe inhibition. Moreover, during IL-2 gene induction, splicing of IL-2 precursor transcripts becomes inhibited, resulting in a transient mRNA wave. This block in IL-2 mRNA splicing is relieved by the translation inhibitor, cycloheximide (CHX), which does not stimulate transcription [Gerez et al., J. Biol. Chem. 270, 15569 (1995)]. We show that suppression of IL-2 mRNA expression, whether by CD8 cells, soluble mediators derived from them, or IL-10, is relieved completely by CHX. Hence, suppression involves a CHX-sensitive step. Response to CHX, manifested in superinduction of IL-2 mRNA, is enhanced 10-fold during suppression. Suppression by CD8 cells or soluble mediators leads to rapid degradation of precursor transcripts while relief from suppression leads to a significant rise in precursor RNA. These changes precede effects at the mRNA level. We conclude that suppression induces a block in mRNA splicing and degradation of blocked precursor transcripts. The near-complete absence of IL-2 mRNA superinduction by CHX in Jurkat Th cells, lacking cells with suppressive capacity, supports this interpretation.

Superinduction of Interleukin-6 mRNA in lung epithelial H292 cells depends on transiently increased C/EBP activity and durable increased mRNA stability

Restriction of eukaryotic protein synthesis affects the regulation of some transiently expressed gene transcripts resulting in their superinduction. We determined the transcriptional and post-transcriptional processes implicated in IL-6 mRNA superinduction in a human lung-derived epithelial cell line H292, and their kinetics in the absence and presence of an exogenous stimulus, tumor necrosis factor-α (TNF-α). Cycloheximide (CHI) at 10 μg/ml, which inhibited protein synthesis for 80%, caused a 80-fold induction of IL-6 mRNA level which was due predominantly to a stabilization of IL-6 mRNA (20-fold) early on. Employing transient transfection protocols we noted a small positive effect of CHI on transcription, mediated by the proximal and the distal C/EBP sites of the IL-6 promoter and paralleled by an increased C/EBP DNA-binding activity, similar to that found for exposure to TNF-α alone. TNF-α and CHI synergized on IL-6 mRNA expression (200-fold increase) which was due to an increased transcription, corresponding to a further increased C/EBP DNA-binding activity. However, the effect of CHI on IL-6 gene transcription was transient, in support of the need for ongoing protein synthesis for C/EBP activity. These findings indicate that IL-6 mRNA superinduction, at least in H292 cells, is regulated predominantly by modulating the repressive system that ensures a rapid degradation of IL-6 mRNA.

Enhanced AP-1 and NF-kappaB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells.

Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 microg/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor alpha (TNF-alpha), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4 h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-kappaB and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-kappaB and prolonged AP-1 DNA-binding activities and that the synergism of TNF-alpha and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4 h elapsed between the addition of CHI and that of TNF-alpha. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.

Superinduction of COX-2 mRNA by cycloheximide and interleukin-1β involves increased transcription and correlates with increased NF-κB and JNK activation

Many primary response genes, including cyclooxygenase-2 (COX-2), exhibit mRNA superinduction following agonist stimulation in the presence of translational blockers such as cycloheximide. This is widely assumed to result from mRNA stabilisation. However, superinduction of IL-1β-induced COX-2 mRNA levels by cycloheximide in pulmonary type II A549 cells occurred by increased transcription and not by mRNA stabilisation. Furthermore, equivalent effects were observed on NF-κB binding to COX-2 promoter κB sites and activation of the Jun N-terminal kinases (JNK), p54 and p46. These signalling pathways play important roles in COX-2 induction and may therefore account for the observed increases in COX-2 transcription. These data are consistent with negative feed-back involving down-regulation of NF-κB by de novo IκBα synthesis and suggest that JNK activation may also be down-regulated by a cycloheximide sensitive process.

Increased NF-kB and AP-1 activity and increased stability of IL-8 transcripts underlie IL-8 mRNA superinduction in airway epithelial H292 cells

Increased NF-kB and AP-1 activity and increased stability of IL-8 transcripts underlie IL-8 mRNA superinduction in airway epithelial H292 cells

Increased NF-kB and AP-1 activity and increased stability of IL-8 transcripts underlie IL-8 mRNA superinduction in airway epithelial H292 cells

Increased NF-kB and AP-1 activity and increased stability of IL-8 transcripts underlie IL-8 mRNA superinduction in airway epithelial H292 cells

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Superinduction of NF-κB by Actinomycin D and Cycloheximide in Epithelial Cells

Epithelial cells are actively involved in inflammation and play a role in inflammatory diseases such as asthma. Numerous proinflammatory genes, expressed in the airway epithelium, are regulated by the transcription factor NF-κB. We show that the proinflammatory cytokines, IL-1β and TNFα, as well as a protein kinase C activator cause NF-κB activation in A549 epithelial cells. This observation is consistent with a major role for NF-κB in inflammation. We also demonstrate that IL-1β costimulation with a protein synthesis inhibitor, cycloheximide, or a transcription blocker, actinomycin D, results in superinduction of NF-κB but not the transcription factors Oct 1, AP-1, and Sp-1. We speculate that this may be due to lack ofde novosynthesis of the NF-κB inhibitor, IκBα, and suggest that this phenomena may help explain the widely observed effect of mRNA superinduction of primary response genes in response to translational blockers.

Elevated Gene Expression and Production of Interleukins 2, 4, 5, and 6 during Exposure to Vomitoxin (Deoxynivalenol) and Cycloheximide in the EL-4 Thymoma

Aberrant elevation of serum IgA and induction of murine IgA nephropathy following dietary exposure to the naturally occurring trichothecene vomitoxin (VT or deoxynivalenol) may involve dysregulation of cytokine production at the T cell level. EL4.IL-2 (EL-4), a cloned thymoma that produces interleukins (IL)-2, 4, 5, and 6, was used as a T cell model to investigate the in vitro effects of VT on interleukin production and gene expression. When supernatants of cells stimulated with phorbol 12-myristate 13-acetate (PMA) were assessed by enzyme-linked immunosorbent assay, IL-2, 4, and 5 were increased in the presence of 50 and/or 100 ng/ml VT for 2 and/or 8 days of culture. IL-2, 5, and 6 were also significantly elevated in the presence of 10-100 ng/ml of cycloheximide (CHX), another protein synthesis inhibitor, after 8 days of culture. As demonstrated by Northern analysis, VT at the levels between 50 and 100 ng/ml superinduced IL-2, 4, 5, and 6 mRNAs in PMA-stimulated EL-4 cells during a 24 hr culture period. Similar effects in PMA-treated samples were observed for CHX at 50, 100, 250, 1000, and 10000 ng/ml. mRNA levels for both IL-4 and IL-5, but not IL-2 and IL-6, were increased in unstimulated EL-4 cultures exposed to 50 and 100 ng/ml VT for 48 hr when analyzed by reverse transcriptase-polymerase chain reaction. Using [3H]leucine incorporation as a measurement of protein synthesis, IC50s for VT and CHX were estimated to be 280 and 55 ng/ml, respectively. This study indicates that VT as well as CHX could increase production of several interleukins in the EL-4 model even when present at concentrations that partially inhibited protein synthesis, whereas IL mRNA superinduction occurred across a broader range of concentrations that included maximal protein synthesis inhibition.

Down regulation of IL 2 mRNA by antibody to the 50-kd protein associated with E receptors on human T lymphocyte.

Recent studies have shown that antibodies to certain epitopes on the 50-kd molecule associated with sheep erythrocyte receptors on human T cells can suppress T cell proliferation and interleukin 2 (IL 2) elaboration. We used a human IL 2 cDNA clone to investigate the effect of antibody 9.6 and cyclosporin A (CsA) on the regulation of IL 2 mRNA levels in the cloned human leukemic T cell line Jurkat, J32. Maximal levels of IL 2 mRNA were reached 6 hr after induction of Jurkat cells with a combination of mitogen phytohemagglutinin (PHA) and phorbol ester (TPA). Antibody 9.6, added during the first 4 hr after lymphocyte stimulation, markedly inhibited IL 2 mRNA accumulation induced by low but synergistic combination of PHA (5 micrograms/ml) and TPA (1.0 ng/ml). The inhibition by 9.6 was not demonstrable as the concentration of PHA or TPA was increased. In contrast, the ability of CsA to suppress IL 2 mRNA accumulation appeared to be independent of PHA or TPA concentration and was minimal if CsA was added 4 hr after stimulation. IL 2 mRNA could be superinduced several folds by addition of cycloheximide 3 hr after induction of J32 with mitogens. Antibody 9.6 did not prevent IL 2 mRNA superinduction induced by cycloheximide, whereas CsA, as well as transcription inhibitor DRB, completely blocked this phenomenon. These findings indicate that signals induced by antibody 9.6 regulate IL 2 production at a pre-translational level, are operative for an extended period of time overlapping with the early phase of IL 2 mRNA accumulation, suppress IL 2 gene expression induced by PHA as well as TPA, and that antibody 9.6 and CsA exert their inhibitory effect by distinct mechanism(s).