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The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation

Nature Immunology volume 15, pages 239–247 (2014)Cite this article

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Abstract

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

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Figure 1: Deletion of Dream prevents endotoxin-induced lung inflammatory injury and sepsis-induced mortality.
Figure 2: DREAM and USF1 coordinate TNFAIP3 transcription.
Figure 3: Regulation of Tnfaip3 transcription by DREAM and USF1 in Dream−/− mice.
Figure 4: DREAM deficiency in mice augments basal and induced A20 expression.
Figure 5: DREAM deletion attenuates the TNF-induced activation of TAK1 and IKK in LECs.
Figure 6: DREAM deletion attenuates TNF-induced Jnk and p38 MAPK activation in LECs.
Figure 7: DREAM regulates the expression of NF-κB signaling components and target genes differently.
Figure 8: Expression of wild-type DREAM restores NF-κB signaling components in DREAM-deficient LECs.

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Acknowledgements

We thank J.M. Penninger (Institute of Molecular Biotechnology of the Austrian Academy of Sciences) for DREAM-deficient mice, and Y.B. Wu for help with the isolation and culture of LECs. Supported by the US National Institutes of Health (P01 HL077806).

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Authors and Affiliations

  1. Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois, Chicago, Illinois, USA

    Chinnaswamy Tiruppathi, Dheeraj Soni, Dong-Mei Wang, Jiaping Xue, Vandana Singh, Prabhakar B Thippegowda, Bopaiah P Cheppudira, Rakesh K Mishra, Auditi DebRoy, Kurt Bachmaier, You-Yang Zhao, John W Christman, Stephen M Vogel & Asrar B Malik

  2. Department of Hematology/Oncology, College of Medicine, University of Illinois, Chicago, Illinois, USA

    Zhijian Qian

  3. Department of Medicine, School of Medicine, University of California at San Francisco, San Francisco, California, USA

    Averil Ma

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Contributions

C.T., D.S., J.W.C., A.M. and A.B.M. designed the research; C.T., D.S., D.-M.W., J.X., V.S., P.B.T., B.P.C., R.K.M., A.D., Z.Q., K.B., Y.-Y.Z. and S.M.V. did the experiments; C.T. analyzed data; and C.T. and A.B.M. wrote the manuscript.

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Correspondence to Chinnaswamy Tiruppathi.

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Supplementary Figure 1 Wild type (WT) bone marrow cells fail to induce lung vascular injury in Dream–/– mice following LPS challenge.

a, At 3 weeks following bone marrow cells transplantation, recipient mouse blood was collected from by tail nick. DNA was isolated using blood cells from a recipient female or male mouse. Polymerase chain reaction (PCR) for Sry gene (male specific) and autosomal gene Nme1 was performed to determine the reconstitution of bone marrow cells35. Representative PCR results are shown. Male-WT bone marrow (BM) cells transplanted recipient female Dream–/– mice were positive for Sry gene whereas female-WT bone marrow cells transplanted recipient male Dream–/– mice were negative for Sry gene. Positive and negative controls were included. b and c, WT, Dream–/–, or chimeric (WT-BM→ Dream–/–) mice (6 weeks after transplantation) were challenged with LPS (10 mg/kg, i.p.) and lungs were removed at the indicated times. Lung tissue homogenates were used for immunoblot to determine ICAM-1, A20, DREAM, and β-actin. Representative blots are shown in b. Immunoblots quantified with values normalized to β-actin are shown in c. N = 6 in each group. **P< 0.01; ***p<0.001, difference between Dream+/+ and WT-BM→ Dream–/– group (unpaired two-tailed Student's t-test). d, Cytokines (MCP-1, TNF, and IL-6) in BALF were measured after LPS challenge. N = 6 in each group; *p< 0.05, **p<0.01, ***p<0.001, difference between LPS-injected WT and Dream–/–- mice or WT and WT-BM→ Dream–/– mice (unpaired two-tailed Student's t-test). e, Cytokines (MCP-1, TNF, and IL-6) in serum were measured after LPS challenge. N = 6 in each group; *p< 0.05, **p<0.001, n.s. not significant, difference between LPS-injected WT and Dream–/– mice (unpaired two-tailed Student's t-test). f, MPO activity in BALF was measured after LPS challenge. N = 6 in each group; **p< 0.01, ***p<0.001, difference between LPS-injected WT and Dream–/– mice or WT and WT-BM→ Dream–/– mice (unpaired two-tailed Student's t-test). g, Survival of Dream+/+, Dream-/-, WT-BM→ Dream–/– mice after LPS challenge (10 mg/kg, i.p.). Age and weight matched mice were followed for 6 days after LPS administration. n = 8 in each group. **p< 0.01, vs. Dream+/+ mice using the log-rank test.

Supplementary Figure 2 LPS induces the mobilization of DREAM in BMDMs and analysis of the regulation of A20 transcription coordinated by DREAM and USF1.

a, WT-mice BMDMs were challenged with LPS (1 μg/ml) for different time intervals. After this treatment, cytosolic and nuclear fractions were prepared12 and used for immunoblot analysis to determine DREAM, β-actin (cytosolic marker), or MCM3 (nuclear marker). Data are from representative of two experiments. Values normalized to β-actin or MCM3 are shown in right panels. b, In basal condition, DREAM binding to DREs localized on A20 promoter represses A20 transcription. Inflammatory stimuli (e.g., LPS and TNF)-induced activation of protein kinase A (PKA) results in phosphorylation of nuclear CREM (cAMP-response element modulator)2,3. Interaction of phospho-CREM with DREAM uncouples DREAM from DREs resulting in USF1 binding to DRE-E-box motif, which activates A20 transcription. A20 transcription can be restored by the dephosphoryation of phospho-CREM.

Supplementary Figure 3 LPS induces c-Fos expression.

a, Dream+/+ and Dream–/– mice were challenged with LPS (10 mg/kg, i.p.) and lungs were removed at the indicated times as described in Fig. 1a. Lung tissue was used for Western blot analysis of c-Fos protein expression. n = 5 per time point in each group. **p< 0.01 (unpaired two-tailed Student's t-test), different from Dream+/+ mice. b, BMDMs in culture from Dream+/+ and Dream–/– mice exposed to LPS (100 ng/ml) for different time intervals were used for IB to determine expression of c-Fos. Data shown are representative of three or more experiments. Immunoblots were quantified (right panels). *p<0.05, **p<0.01(unpaired two-tailed Student's t-test); different from Dream+/+.

Supplementary Figure 4 DREAM regulates TAK1 and IKK signaling in BMDMs.

BMDMs in culture from Dream+/+ and Dream–/– mice were exposed to LPS (100 ng/ml) for different time intervals and then cell lysates were used for IB to determine activation of TAK1 (a) and IKK (b). Data shown are representative of three or more experiments. Immunoblots were quantified (right panels). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test);different from Dream+/+.

Supplementary Figure 5 DREAM regulates the transcription of NF-κB signaling components differently.

Dream+/+ or Dream–/– mice lung tissue (LT) mRNA levels of NF-κB signaling components (TRAF2, TRAF6, RIP1, RIP2, IKKγ (Ikbkg), NF-κB1, NF-κB2, RelB, and c-Rel) were determined by qPCR. n = 4 mice per group. Values reported are mean ± S.D. **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test, significantly different from Dream+/+ mice.

Supplementary Figure 6 Model for DREAM regulation of NF-κB signaling and lung inflammatory injury.

IκB kinase (IKKα/IKKβ/IKKγ) activation downstream of TNFR or TLR4 requires K63-linked polyubiquitylation of TRAFs, RIPs, and IKKγ proteins (polyubiquitylation not indicated in the model)19,20. K63-linked polyubiquitylation of these proteins recruits TAK1 to form functional signaling complex which activates IκB kinase and p38 MAPK19,20. Both IκB kinase and p38 MAPK activation amplifies inflammatory responses by inducing the production of inflammatory cytokines and expression of ICAM-1 in lungs. A20 de-ubiquitylates, K63-linked polyubiquitin chains on TRAFs, RIPs, and IKKγ proteins to inhibit IκB kinase and p38 MAPK. This mechanism prevented lung inflammation and injury. DREAM functions by binding to the A20 gene DRE elements thereby represses A20 transcription, and thereby prevents activation of NF-κB and p38 MAPK to prevent lung inflammatory injury.

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Tiruppathi, C., Soni, D., Wang, DM. et al. The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation. Nat Immunol 15, 239–247 (2014). https://doi.org/10.1038/ni.2823

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