Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock - PubMed (original) (raw)

Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock

Michael Hammer et al. J Exp Med. 2006.

Abstract

Activation of the mitogen-activated protein kinase (MAPK) cascade after Toll-like receptor stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase dual specificity phosphatase 1 (DUSP1) as an essential endogenous regulator of the inflammatory response to lipopolysaccharide (LPS). DUSP1-deficient (DUSP1-/-) bone marrow-derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1-/- mice with LPS caused increased lethality and overshooting production of interleukin (IL)-6 and tumor necrosis factor alpha. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, which includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4, and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, interferon gamma and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock.

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Figures

Figure 1.

Figure 1.

In vitro analysis of macrophages from DUSP1−/− mice. (A) Production of IL-6 from LPS-treated macrophages was analyzed by ELISA in supernatants harvested 8 h after stimulation. The mean and standard deviations of triplicate measurements from a representative experiment are shown. (B) Kinetics of MAPK activation by 100 ng/ml LPS in macrophages generated from bone marrow cells of DUSP1+/− or DUSP1−/− mice. 1.5 × 106 cells were stimulated for the indicated times, and whole cell lysates were analyzed by Western blot using antibodies to phosphorylated p38, ERK1/2, JNK, and to γ-tubulin at a 1:1,000 dilution. Data shown are from one representative experiment out of three performed.

Figure 2.

Figure 2.

DUSP1 is induced after injection of LPS and controls survival and cytokine production. (A) Expression levels of DUSP1 in the spleens of WT mice were determined at the indicated times after injection of 10 mg/kg LPS i.p. by Northern blot analysis of 10 μg total RNA. Data shown are from individual mice (n = 3–4) from one representative experiment. (B) Survival of WT (n = 15) and DUSP1−/− mice (n = 13) after i.p. injection of 25 mg/kg LPS. Data are pooled from two experiments. (C and D) Serum levels of cytokines after LPS injection (10 mg/kg). Data shown are the mean and standard deviation (n = 3–4 mice per data point) from representative experiments. Two data points marked by asterisks were beyond the maximum of the standard curve, and the upper limits of the dynamic range of the assay are shown. (C) TNF-α and IL-6 in WT and DUSP1−/− mice on a mixed 129Sv × Bl/6 (left) or pure 129Sv and C3H (right) background (6 h after LPS). (D) Comparison of the effect of deficiency in DUSP1 or IL-10 on serum levels of TNF-α and IL-12p40.

Figure 3.

Figure 3.

Genome-wide analysis of LPS-induced gene expression in the spleens of WT and DUSP1−/− mice. (A) Total splenic RNA was prepared from control WT and DUSP1−/− mice 6 h after i.p. injection of 10 mg/kg LPS (n = 3 per group) and was processed for Affymetrix GeneChip analysis. Venn diagram comparing the numbers of genes significantly up-regulated in WT and DUSP1−/− mice by LPS (for criteria see Materials and methods). Z-score–normalized expression values of LPS-induced genes were subjected to hierarchical clustering analysis. Cluster A contains a group of genes expressed more strongly in the WT spleen. In contrast, cluster C represents a larger subset of LPS-induced genes up-regulated in the absence of DUSP1. Expression data of these 608 genes can be found in Table S1. (B) Differential control of the chemokines CCL3, CCL4, CXCl2, and CXCL10 by DUSP1. Confirmation of the microarray expression data (left: average and standard deviation of signal intensity values) by ELISA determination of serum levels (right: mean and standard deviation, n = 3–4). (C) Increased expression of IL-10 at the mRNA and protein level in DUSP1−/− mice. Northern data from two and three individual mice per condition and organ are shown, and ELISA data are the mean and standard deviation (n = 3–4) from a representative experiment.

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