Ethyl pyruvate inhibits the acetylation and release of HMGB1 via effects on SIRT1/STAT signaling in LPS-activated RAW264.7 cells and peritoneal macrophages
Graphical abstract
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).
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2021, Journal of EthnopharmacologyCitation Excerpt :HMGB1 is a novel deacetylation target of SIRT1. SIRT1 promotes the deacetylation of HMGB1, inhibiting the transcription and exocrine activity of HMGB1 in LPS-activated macrophages and reducing the expression of proinflammatory cytokines (Kim et al., 2016). The expression of HMGB1 is elevated in RA patients and animal models and is mainly concentrated in synovial tissue, synovial fluid and serum (Kaur et al., 2020).
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2021, Pharmacological ResearchCitation Excerpt :PA can increase SIRT1 activity in HepG2 cells [96]. EP can also up-regulate the expression of SIRT1, inhibit LPS-mediated HMGB1 acetylation and STAT1 phosphorylation, finally alleviating inflammation in RAW264.7 cells [97], suggesting that EP can activate SIRT1 activity and play an important role by regulating upstream and downstream molecules. Forkhead box O transcription factor (FOXO) is one of the important downstream molecules of SIRT1 and is involved in various pathological processes, such as aging, oxidative stress, and inflammation [98,99].
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Y.M. Kim and E.J. Park are equally contributed.