The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules

Abstract

The inflammatory response plays out over time in a reproducible and organized way after an initiating stimulus. Here we show that genes activated in cultured mouse fibroblasts in response to the cytokine tumor necrosis factor could be categorized into roughly three groups, each with different induction kinetics. Although differences in transcription were important in determining the grouping of these genes, differences in mRNA stability also exerted a strong influence on the temporal order of gene expression, in some cases overriding that of transcriptional control elements. Transcripts of mRNA expressed early had abundant AU-rich elements in their 3' untranslated regions, whereas those expressed later had fewer. Thus, mRNA stability and transcriptional control, two intrinsic characteristics of genes, control the kinetics of gene expression induced by proinflammatory cytokines.

Figure 1

TNF-activated genes fall into three kinetically different groups. (a) Microarray analysis of gene expression in 3T3 fibroblasts stimulated with recombinant mouse TNF (10 ng/ml) for 0.5, 2, and 12 hours. Activated genes, defined by an induction ratio equal to or greater than 2-fold (P < 0.01), are shown in green and were clustered with an Agglomerative Clustering Algorithm into three groups (I, II, III). Genes suppressed by TNF treatment are shown in red. The number of genes in each group is shown in parentheses. (b). Representative kinetics of expression of genes in each group.

Figure 2

Gene expression after TNF withdrawal (a) 3T3 fibroblasts were exposed to TNF (10 ng/ml) continuously for 10 hours (TNF; red line), or were exposed to TNF for 6 hours, after which TNF was washed away (TNF withdrawal; blue line). Expression of genes in each group was measured by qRT-PCR at indicated time points. The data are mean ± s.d. of triplicate samples and are representative of three to six independent experiments with similar results. (b). 3T3 fibroblasts were stimulated with medium alone (Medium) or with TNF for 6 hours (TNF), or were exposed to TNF for 6 hours after which TNF was washed away and cells were incubated in medium for an additional 18 hours (TNF withdrawal). CCL2 and CCL5 protein in supernatants were measured by ELISA. Note that the production of CCL2 and CCL5 in medium alone was too low (approximately 20 pg/ml) to be shown and CCL5 production is delayed in kinetics and thus lower comparing with CCL2. The data are mean ± s.d. of triplicate samples and representative of three independent experiments with similar results.

Figure 3

mRNA stability of genes in each group. mRNA transcripts encoding the indicated genes were measured by qRT-PCR at indicated times after addition of the transcription inhibitor actinomycin D (ActD) to 3T3 fibroblasts. The results are shown as a ratio to the base line value measured just before ActD addition (in log base 2 scale). Group I, II, and III genes are shown in red, green and blue, respectively. All values were normalized to Rpl32. The data are an average of triplicate samples with variations less than 20% and representative of three independent experiments with similar results.

Figure 4

Kinetic patterns of gene expression in macrophages. Bone marrow-derived macrophages were stimulated with TNF (left) or LPS (right) for the indicated times and gene expression was measured by qRT-PCR. (a) The expression of one representative gene in each group is shown; additional genes are shown in Table 1. (b) Altered expression patterns of Ccl2 and Il1b induced by LPS compared to TNF. The data are mean ± s.d. of triplicate samples and representative of three independent experiments with similar results.

Figure 5

Gene expression patterns are determined by the 3′UTR of a gene. (a) 3T3 fibroblasts were infected with lentiviruses encoding GFP transgenes linked to the promoter of Cxcl10 and the 3′UTR of Ccl5 or Fos. Cells were pre-treated with TNF for 30 minutes before the addition of actinomycin D (ActD). mRNA transcripts encoding GFP, and endogenous Fos and Rpl32, were measured by qRT-PCR at indicated times after addition of ActD. The data are an average of triplicate samples with variations less than 20% and representative of three independent experiments. (b) 3T3 fibroblasts infected with the indicated lentiviruses were stimulated with TNF (20ng/ml). GFP expression was measured by qRT-PCR at indicated time points after the addition of TNF. The data are mean ± s.d. of triplicate samples and representative of three independent experiments with similar results.