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Transcriptional regulation of tristetraprolin by NF-κB signaling in LPS-stimulated macrophages

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Abstract

Lipopolysaccharide (LPS) treatment causes the marked changes of gene expression in macrophages. Tristetraprolin (TTP), which is an mRNA-destabilizing protein that negatively regulates the expression of pro-inflammatory mediators, is induced by LPS. To delineate the molecular mechanism of LPS-stimulated TTP expression, several inhibitors blocking different signaling pathways were used initially. We observed that inhibitors of the NF-κB signaling pathway could down-regulate the TTP expression during LPS-induction. Consistently, TTP expression was increased upon recombinant TNFα stimulation which activates NF-κB signaling. The 5′ regulatory region of zfp36 gene spanning from −2 k to +50 was isolated, which contained a putative NF-κB–binding site located in −1859 to −1850. Analysis of luciferase reporter activity driven by a serial 5′-deletion of TTP promoter showed that NF-κB inhibitor-mediated suppression of LPS or TNFα induced activity was through the predicted κB-binding sites. When the NF-κB-binding site was mutated, the TTP promoter decreased its response to the ectopic expression of NF-κB. Physical interaction analysis including oligonucleotides competition, gel shift and chromatin immunoprecipitation assays demonstrated that NF-κB activated by LPS or TNFα bound to the TTP promoter specifically. These results suggested that during LPS stimulation, NF-κB signaling were activated to regulate the transcription of TTP mRNA.

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Abbreviations

ARE:

AU-rich element

ChIP:

Chromatin-immunoprecipitation

EMSA:

Electrophoretic mobility shift assay

LPS:

Lipopolysaccharide

MAPK:

Mitogen-activated protein kinase

NF-κB:

Nuclear factor-kappa B

RT-PCR:

Reverse transcription-polymerase chain reaction

STAT:

Signal transduction and activator of transcription

TLR:

Toll-like receptor

TNFα:

Tumor necrosis factor-alpha

TTP:

Tristetraprolin

UTR:

Untranslated region

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Acknowledgments

We thank Dr. Bertrant C.M. Tan for the critical reading the manuscript; Ms Yi-Li Liu and I-Ching Huang for technical support on DNA sequencing (ABI 3730) supported in part by Department of Medical Research in NTUH. This study was supported by Academia Sinica and a grant from National Taiwan University (96R0066-33 to M.-S. Chang) and a grant from the National Science Council (NSC97-2311-B-001-019-MY3 to C.-J. Chang).

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Authors and Affiliations

  1. Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan

    Yu-Ling Chen, Yu-Wun Jiang, Yu-Lun Su, Mau-Sun Chang & Ching-Jin Chang

  2. Institute of Molecular Medicine, School of Medicine, National Taiwan University, Taipei, Taiwan

    Sheng-Chung Lee

  3. Institute of Biological Chemistry, Academia Sinica, No. 1 Sec 4 Roosevelt Rd, Taipei, 106, Taiwan

    Sheng-Chung Lee, Mau-Sun Chang & Ching-Jin Chang

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  1. Yu-Ling Chen
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Correspondence to Ching-Jin Chang.

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Chen, YL., Jiang, YW., Su, YL. et al. Transcriptional regulation of tristetraprolin by NF-κB signaling in LPS-stimulated macrophages. Mol Biol Rep 40, 2867–2877 (2013). https://doi.org/10.1007/s11033-012-2302-8

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