MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation 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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