Elsevier

Cellular Signalling

Volume 34, June 2017, Pages 1-10
Cellular Signalling

MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression

https://doi.org/10.1016/j.cellsig.2017.02.018Get rights and content

Highlights

  • Background: MKP-1 plays a critical role in regulating inflammation in innate immunity.

  • Results: MKP-1 deficient macrophages responded to LPS challenge with increased IL-1β production.

  • Conclusion: MKP-1 regulates LPS mediated IL-1β production through HIF-1α and ROS.

  • Significance: Modification of MKP-1 provides a new target in HIF-1α regulated inflammation and cancer.

Abstract

Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that plays a major role in inflammatory diseases as well as cancer. The inflammatory response after Toll-like receptor (TLR) 4 activation is tightly regulated through phosphorylation of MAP kinases, including p38 and JNK pathways. The activation of MAP kinases is negatively regulated by MAPK phosphatases (MKPs). MKP-1 preferentially dephosphorylates p38 and JNK. IL-1β is regulated through the activation of MAPK, including p38 as well as several transcription factors. The oxygen-sensitive transcription factor HIF-1α is a known transcription factor for several inflammatory cytokines including IL-1β and IL-6. Here, we report that MKP-1 regulates HIF-1α expression in response to LPS. MKP-1 deficient bone marrow derived macrophages (BMDMs) exhibited increased reactive oxygen species (ROS) production and higher HIF-1α expression. In contrast, the expression of all three isoforms of prolyl hydroxylases (PHDs), which are important in destabilizing HIF-1α through hydroxylation, were significantly decreased in MKP-1 deficient BMDMs. LPS challenge of MKP-1 deficient BMDMs led to a substantial increase in IL-1β production. An inhibitor of HIF-1α significantly decreased LPS mediated IL-1β production both at the transcript and protein levels. Similarly, inhibition of p38 MAP kinase reduced LPS mediated pro-IL-1β and HIF-1α protein levels as well as ROS production in MKP-1 deficient BMDMs. These findings demonstrate a regulatory function for MKP-1 in modulating IL-1β expression through p38 activation, ROS production and HIF-1α expression.

Introduction

In response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) multiple kinase cascades are activated including, mitogen activated protein kinase(s) (MAPKs) [1]. TLRs recognize microbial infections and initiate innate immune responses [2]. The response to LPS is mainly orchestrated by the TLR4 receptor. Docking of LPS to TLR4 recruits the adaptor protein MyD88, which initiates downstream signaling pathways, such as the nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 [2]. This activates several cytokine including, IL-1β production. MAPK phosphatases (MKPs) dephosphorylate TXY motifs on MAPKs and negatively regulate inflammatory responses. MKP-1, also known as dual specificity phosphatase (DUSP)-1, preferentially dephosphorylates phospho p38 and phospho JNK [3]. MKP-1 controls the expression of numerous inflammatory genes and transcription factors through regulation of p38 and JNK.

IL-1β production in response to TLR activation is tightly regulated through several transcription factors. The oxygen-sensitive hypoxia-inducible factor (HIF)-1α is a critical transcriptional regulator for several inflammatory cytokines, including IL-1β and IL-6 [4], [5], [6]. During hypoxia, cytosolic HIF-1α is hydroxylated by prolyl-hydroxylases (PHD) providing a target for polyubiquitination and degradation via the von Hippel-Lindau (VHL) dependent pathway [7]. In addition to hypoxia, a variety of pathogen-derived molecules and inflammatory mediators are able to induce HIF-1α expression under normoxic conditions [6]. Several lines of evidence indicate that TLR4-mediated HIF-1α transcriptional activation is regulated through reactive oxygen species (ROS) [8], [9], [10]. Transactivation of HIF-1α is mainly controlled by its post-translational modifications, such as hydroxylation, ubiquitination, acetylation, and phosphorylation. However, it appears that bacterial endotoxins such as LPS also induce HIF-1α at the transcriptional level [5], [6], [11], [12], [13]. The LPS-mediated signaling cascade leading to accumulation of HIF-1α and IL-1β production is poorly understood. Activation of several pathways, including PI3 kinase, p38 and GSK3 β has been proposed to regulate HIF-1α [10], [11], [14], [15], [16].

In this report, we investigate the role of MKP-1 in LPS-mediated IL-1β production. Using BMDMs derived from wild type (WT) and MKP-1−/− mice, we show that MKP-1 deficient BMDMs exhibit an increased production of IL-1β. This is mechanistically regulated through lower PHDs expression and higher HIF-1α induction in response to LPS. Furthermore, MKP-1 deficient BMDMs exhibit significantly increased mitochondrial and cytoplasmic ROS production.

Section snippets

Chemicals and antibodies

LPS was purchased from Invivogen (San Diego, CA). Phospho-specific antibodies against the phosphorylated form of ERK1/2, p38, JNK, as well as total ERK1/2, JNK, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Total p38 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The IL-1 β antibody was purchased from R&D Systems (Minneapolis, MN). The HIF-1α antibody was purchased from Bioss Inc. (Woburn, MA). Horseradish peroxidase (HRP)-conjugated anti-mouse

MKP-1 deficient BMDMs exhibit higher IL-1β production in response to LPS challenge

IL-1β production is tightly regulated at various levels. Here we studied the effect of MKP-1 deficiency on pro-IL-1β expression, IL-1β transcript and released IL-1β. First, BMDMs derived from WT and MKP-1−/− mice were cultured side by side under equal conditions and challenged with LPS (100 ng/mL) for various time points as indicated. Whole cell lysates were immunoblotted using an antibody against pro IL-1β and equal loading was determined using β-actin antibody (Fig. 1A). The mean densitometric

Discussion

HIF-1α is recognized as the master regulator of the hypoxic response, which activates the transcription of > 100 genes. Under normoxic conditions, activation of TLR and several cytokine receptors can lead to the induction of HIF-1α [5], [31]. There are several mechanisms underlying the HIF-1α induction in response to LPS. TLR4 mediated induction of HIF-1α reflects a combination of increased HIF-1α transcription and decreased HIF-1α degradation (5).

MAP kinases including ERK, JNK, and p38 modulate

Abbreviations

    TLR

    Toll like receptor

    MKP

    MAP kinase phosphatase

    MAPK

    mitogen activated protein kinase

    BMDM

    bone marrow derived macrophage

    ROS

    reactive oxygen species

    ANOVA

    analysis of variance

    HIF-1α

    hypoxia inducible factor 1 alpha

    DUSP

    dual specificity phosphatase

Conflict of interest

The authors declare that no conflict of interest exists.

Author contributions

H. T. and M. B. carried out the experiments. L. S and Y. L. conceived and coordinated the study. C. B and L. S. wrote the manuscript. All authors reviewed the results and approved the final version.

Acknowledgements

This work was supported by the Department of Medicine and the Center for Molecular Medicine and Genetics, Wayne State University School of Medicine (L.S.) and R01 HL 113508 (L.S.).

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