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The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory moleculesRESULTSTemporal gene-induction patterns in fibroblastsResponse to inducer removalFigure 1 TNF-activated genes can be categorized into three kinetically different groups.Figure 2 Gene expression after TNF withdrawal.Figure 3 Stability of mRNA encoded by genes in groups I, II and III.Table 1 Half-life of mRNA in mouse cellsThe stability of mRNA influences induction patternsGene-induction patterns in primary macrophagesLipopolysaccharide-induced mRNA stabilizationFigure 4 Kinetic patterns of gene expression in macrophages.Relative mRNA stability is ’encoded’ in the genomeDISCUSSIONFigure 5 Gene-expression patterns are determined by the 3prime UTR of a gene.METHODSReagentsCell culture and miceGFP transgenesAnalysis of mRNAStability of mRNAELISAMicroarrayAccession codesACKNOWLEDGMENTSAUTHOR CONTRIBUTIONSReferencesThe stability of mRNA influences the temporalorder of the induction of genes encodinginflammatory moleculesShengli Hao & David BaltimoreThe inflammatory response plays out over time in a reproducible and organized way after an initiating stimulus. Here we showthat genes activated in cultured mouse fibroblasts in response to the cytokine tumor necrosis factor could be categorized intoroughly three groups, each with different induction kinetics. Although differences in transcription were important in determiningthe grouping of these genes, differences in mRNA stability also exerted a strong influence on the temporal order of geneexpression, in some cases overriding that of transcriptional control elements. Transcripts of mRNA expressed early hadabundant AU-rich elements in their 3¢ untranslated regions, whereas those expressed later had fewer. Thus, mRNA stabilityand transcriptional control, two intrinsic characteristics of genes, control the kinetics of gene expression induced byproinflammatory cytokines.As long ago as the first century BC, the inflammatory process wasrecognized as evolving in stereotyped stages1. No matter what invad-ing organism or tissue injury incites the response, the temporal orderof inflammatory events is very similar. From a cellular point of view,neutrophils are the first to arrive at a site of inflammation, and theystay for only a short time (the first 24 h). Monocytes arrive somewhatlater but stay for days. Tissue-remodeling processes take over duringthe late stages of inflammation2.There are two possibilities for how this stereotypical process couldbe orchestrated: each stage could induce the next, or the temporalorder of events could be ‘encoded’ intrinsically in the genes involved inthe inflammatory process. By analyzing events in cultured cells, wehave come to realize that much of the time course of the responseprogram is ‘encoded’ in the genes themselves. The sequential order ofgene expression and the relative duration of the various inflammatoryevents seem to be pre-set as a part of the gene activation program. Theprocess is probably the result of interplay among elements thatregulate transcriptional induction, transcriptional repression andmRNA stability. Here we focus on the function of mRNA stability.Tumor necrosis factor (TNF; A002291) is an important inflamma-tory cytokine involved in both acute and chronic inflammation3.Theeffects of TNF are mediated by a group of transcription factors,including NF-kB, AP-1 and interferon-regulatory factors. Amongthose, NF-kB is a key factor critically involved in most of the effectsof TNF4–6. Global gene activation induced by TNF and other proin-flammatory cytokines6–9has been studied extensively. Such studieshave focused mainly on identifying genes induced in response to aparticular stimulus8, in a particular cell type7or during a particulardisease9. The genes identified have been grouped according to theirfunctions10and transcriptional regulation modes5–9,11. However, thosestudies did not concentrate on examining properties common togenes induced in a particular temporal order.To understand the molecular processes underlying the temporalorder of gene expression after TNF stimulation, we studied culturedfibroblasts, which are representative of cells at the site of an injury. At asite of injury, resident as well as incoming cells produce TNF3; thus, allcells in the vicinity are bathed in TNF throughout the response. Wetherefore examined cells continuously exposed to TNF. We found thatthe activated genes were in three kinetic categories. Although tran-scriptional control was certainly a determinant of the kinetics of geneexpression, the stability of the mRNA encoded by the three groups ofgenes had an unexpectedly key function.RESULTSTemporal gene-induction patterns in fibroblastsWe started our study by examining the timing of gene activationinduced by TNF. For this, we did a microarray analysis of geneexpression in mouse embryonic 3T3 fibroblasts at three times (0.5,2 and 12 h) after the addition of TNF. Using the criterion of a greater-than-twofold induction with a P value of less than 0.01, we found thatapproximately 250 genes were upregulated or downregulated at someor all of these time points (Fig. 1a). We clustered the induced geneswith the 2D Clustering Wizard and agglomerative clustering algo-rithm12provided with the Rosetta Resolver 7.2 software. We thenfurther investigated only those 180 genes that were upregulated afterstimulation with TNF.Received 18 November 2008; accepted 31 December 2008; published online 8 February 2009; doi:10.1038/ni.1699Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. Correspondence should be addressed to D.B. ([email protected]).NATURE IMMUNOLOGY VOLUME 10 NUMBER 3 MARCH 2009 281ARTICLES © 2009 Nature America, Inc. All rights reserved.As reported in an earlier, more limited study, the kinetics of geneinduction by TNF vary considerably from gene to gene5. However, inthis larger set of upregulated genes, we discerned three broad classes ofinduction kinetics, each most directly characterized by the time of peakmRNA expression: group I peaked at 0.5 h, group II peaked at 2 h andgroupIIIpeakedat12h(Fig. 1b). By examining more time points, wewere able to find individual variation in the groups5, but the repre-sentative kinetics of individual genes analyzed by quantitative RT-PCRshowed the strength of the generalization (Fig. 2 and SupplementaryFig. 1 online). These three groups could be called early,


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