Elsevier

International Immunopharmacology

Volume 41, December 2016, Pages 98-105
International Immunopharmacology

Ethyl pyruvate inhibits the acetylation and release of HMGB1 via effects on SIRT1/STAT signaling in LPS-activated RAW264.7 cells and peritoneal macrophages

https://doi.org/10.1016/j.intimp.2016.11.002Get rights and content

Highlights

  • Ethyl pyruvate (EP) induces SIRT1 protein expression in RAW264.7 cells.

  • 2 EP significantly reduced the LPS-induced phosphorylation of STAT1 (p-STAT1).

  • Reduction of STAT1 phosphorylation by EP was reversed in RAW264.7 cells transfected with siSIRT1.

  • EP promotes the deacetylation of HMGB1 via the inhibition of p-STAT1 through the upregulation of SIRT1.

Abstract

High mobility group box 1 (HMGB1), a cytokine present in the late phase of sepsis, may be a potential target for the treatment of sepsis. For HMGB1 to be actively secreted from macrophages during infections, it must be post-translationally modified. Although ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has been shown to inhibit the release of HMGB1 in lipopolysaccharide (LPS)-treated RAW 264.7 cells, the underlying mechanism(s) are not yet clear. We investigated the hypothesis that the upregulation of SIRT1 by EP might promote the deacetylation of HMGB1, which reduces HMGB1 release in LPS-activated macrophages. Our results show that EP induced the expression of the SIRT1 protein in RAW264.7 cells and that it significantly inhibited the LPS-induced acetylation of HMGB1. Transfection with a SIRT1-overexpressing vector resulted in a significant decrease in the acetylation of HMGB1 in LPS-activated RAW264.7 cells relative to control cells. The genetic ablation or the pharmacological inhibition of SIRT1 by sirtinol increased LPS-induced HMGB1 acetylation. Moreover, EP inhibited the acetylation of HMGB1 in peritoneal macrophages treated with LPS. Interestingly, EP significantly reduced the LPS-induced phosphorylation of STAT1, which was significantly reversed by siSIRT1 transfection in RAW264.7 cells, indicating that SIRT1 negatively regulates the phosphorylation of STAT1. Overall, the results show that EP promotes the deacetylation of HMGB1 via the inhibition of STAT1 phosphorylation through the upregulation of SIRT1, which reduces HMGB1 release in LPS-activated RAW264.7 cells. In conclusion, EP might be useful in the treatment of diseases that target HMGB1, such as sepsis.

Introduction

Research has demonstrated that the persistent increase in plasma levels of the cytokine high mobility group box 1 (HMGB1) in septic patients is correlated with the degree of organ dysfunction and eventual patient outcomes [1], [2]. Sepsis is a systemic inflammatory response syndrome resulting from the excessive stimulation of the host immune system by pathogenic components and promotes the generation of various proinflammatory cytokines. The overproduction of these cytokines causes systemic inflammation that can lead to lethal damage to multiple organs [3]. Because HMGB1 is a late-phase mediator of sepsis, this molecule has been increasingly recognized as a target for the treatment of sepsis [4]. HMGB1 is actively secreted by stimulated monocytes and macrophages and is passively released by necrotic or damaged cells, stimulating inflammation. Although HMGB1 is localized in the nucleus in almost all cells under noninflammatory conditions, during inflammation, it can be rapidly mobilized to other sites within the cell, such as the cytoplasm and extracellular space, via post-translational modification [5], [6], [7], [8]. For example, hyperacetylation of HMGB1 affects its DNA binding and redirects it toward the cytoplasm [5]. And hyperphosphorylation on serine residues within two nuclear localization signals (NLS) of HMGB1 in monocytes blocks its nuclear import and facilitates cytoplasmic translocation [7], [9]. On the other hand, post-translational methylation [10] and oxidation of HMGB1 [11] are also reported to promote the secretion of HMGB1. Rabadi et al. [12] recently showed a functional link between the deacetylase Sirtuin 1 (SIRT1) and posttranslational modification of HMGB1. In addition, inhibition of HMGB1 release by SIRT1-mediated HMGB1 deacetylation has also been reported to protect non-alcoholic fatty liver disease in rats [13]. These findings strongly suggest that SIRT1 activation plays an important role in the deacetylation of HMGB1 in various inflammatory disorders. Hence, HMGB1 is a novel deacetylation target of SIRT1 [12]. In addition to posttranslational modification of HMGB1 for its secretion, it has been reported that the JAK/STAT1 pathway also plays a critical role in mediating the cytoplasmic accumulation of HMGB1 prior to its subsequent release [14]. Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate, and research has demonstrated that it is a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems, including those of sepsis [15], [16], [17], [18]. Importantly, the anti-inflammatory effects of EP may be related to the inhibition of HMGB1 secretion [15]. Although EP has been shown to inhibit HMGB1 release under septic conditions [18], the molecular mechanism by which EP has this effect is still unclear. In this report, we report that EP is able to induce SIRT1, which inhibits the acetylation of HMGB1 by preventing LPS-induced STAT phosphorylation. In this way, EP, at least in part, reduces HMGB1 release in LPS-activated RAW264.7 cells.

Section snippets

Materials

HyClone Dulbecco's High Glucose Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and antibiotics (penicillin/streptomycin) were acquired from Thermo Fisher Scientific (Waltham, MA). Primary antibodies for HMGB1, β-actin and appropriate secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p-STAT1, t-STAT1, SIRT1 and HA-Tag were purchased from Cell Signaling Technology (Beverly, MA). Anti-acetyl lysine was purchased from Abcam (Cambridge,

Effect of LPS on the acetylation of HMGB1 in RAW264.7 cells

Because acetylation is known to cause the release of HMGB1 in LPS-stimulated RAW264.7 cells [5], the time-dependent kinetic patterns for the release and acetylation of HMGB1 were investigated in these cells following LPS stimulation. Fig. 1A shows that stimulation with LPS initiates the acetylation of HMGB1 as early as 2 h after treatment and that the effect continues over 24 h. Fig. 1B shows time-dependent changes of ratio of acetylated HMGB1 to total HMGB1 (B). As shown in Fig. 1C, the release

Discussion

Several post-translational modifications, including phosphorylation, methylation and acetylation, are involved in the translocation of HMGB1 to the cytoplasm and its subsequent secretion [5], [9], [10]. It is well known that acetylation plays a vital role in regulating HMGB1 release [5], [14], [20]. The hyperacetylation of HMGB1 during inflammation influences its DNA binding and redirects it toward the cytoplasm for secretion [5], [14]. An NAD-dependent class III histone deacetylase, Sirtuin 1,

Conflict of interest

Authors have no conflict of interest to declare.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2016R1A2B4008471).

References (33)

  • A. Oberholzer et al.

    Sepsis syndromes: understanding the role of innate and acquired immunity

    Shock

    (2001)
  • L.F. Gentile et al.

    HMGB1 as a therapeutic target for sepsis: it's all in the timing!

    Expert Opin. Ther. Targets

    (2014)
  • T. Bonaldi et al.

    Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion

    EMBO J.

    (2003)
  • I. Ugrinova et al.

    The effect of PKC phosphorylation on the “architectural” properties of HMGB1 protein

    Mol. Biol. Rep.

    (2012)
  • Y.H. Kim et al.

    N-linked glycosylation plays a crucial role in the secretion of HMGB1

    J. Cell Sci.

    (2016)
  • J.H. Youn et al.

    Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion

    J. Immunol.

    (2006)
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