Up-down Regulation of HO-1 and iNOS Gene Expressions by Ethyl Pyruvate via Recruiting p300 to Nrf2 and Depriving It from p65

Highlights

• EP induces nuclear translocation of Nrf2 and enhanced the expression of hemeoxygenase 1 in BV2 cells.

• LPS-induces iNOS induction is suppressed by EP-pretreatment and it is reverted by Nrf2 knockdown.

• EP-induced-Nrf2 accumulation in the nucleus recruits p300, which inhibits p65-p300 interaction.

• Nrf2 and p65 competes for p300 binding in GST pull down assay and reporter gene analysis.

• EP enhances protective effect by converging anti-oxidative and anti-inflammatory effects.

Abstract

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to exert robust neuroprotection in various neuropathological conditions, such as, cerebral ischemia and KA-induced seizure animal models. The neuroprotective effect of EP is attributable to the anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated convergence of anti-inflammatory and anti-oxidative functions of EP and present a novel molecular mechanism underlying anti-inflammatory effects of EP, which is conveyed by p300, a transcriptional co-activator for both Nuclear factor E2-related factor 2 (Nrf2) and p65. In BV2 cells, a microglia cell line, EP induced translocation of Nrf2 from the cytosol to the nucleus and enhanced the expression of hemeoxygenase 1 (HO-1) in a dose-dependent manner and 1 h incubation with 10 mM EP increased HO-1 to 4.9-fold. Nrf2 was found to translocate from the cytosol to the nucleus beginning 30 min after EP-treatment and binds to the antioxidant response element (ARE) located on HO-1 promoter. Interestingly, LPS-induced inducible NO synthase (iNOS) induction was substantially suppressed in EP-pre-treated BV2 cells and it was reverted by Nrf2 knockdown. We found that EP-induced Nrf2 accumulation in the nucleus recruits p300, a transcriptional co-activator of both Nrf2 and p65, inhibiting p65-p300 interaction. Competition between Nrf2 and p65 for p300 binding was confirmed by glutathione S-transferase (GST) pull down assay and reporter gene analysis. These results demonstrate that EP induced nuclear translocation of Nrf2 which binds to ARE along with p300 and hampers iNOS expression via depleting p300 from p65. This is a novel anti-inflammatory mechanism conveyed by EP, which enhances protective effect by converging anti-inflammatory and anti-oxidative effects and might be applicable to various Nrf2-activating agents, such as phytochemicals.

Introduction

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been reported to act as an anti-inflammatory molecule and to prevent mortality in mice with sepsis [1]. In a previous report, we demonstrated that EP treatment significantly reduced infarct volumes and alleviated neurological deficits in the postischemic brain [2]. We also showed that EP attenuated kainic acid-induced neuronal cell death in the CA1 and CA3 regions of the mouse hippocampus [3]. The neuroprotective effect of EP in the postischemic brain and in an animal seizure model seemed to be related to the anti-inflammatory effects of EP, which might be mediated by modulation of the NF-κB and p38 MAPK signaling pathways [2], [4]. Recently, we showed that combination treatment of EP and aspirin affords synergistic neuroprotection in the postischemic brain with a wide therapeutic window, in part via differential modulation of the NF-κB signaling pathway [4]. Furthermore, we reported a robust neuroprotective effect of a novel multi-functional compound oxopropanoyloxy benzoic acid (OBA-09), which is a simple ester of pyruvate and salicylic acid, in the postischemic brain [5].

Among various pharmacological effects of EP, ant-inflammatory effect is one of the well-established protective functions, which has been reported in a wide range of pathological conditions, including hemorrhagic shock [6] lethal sepsis and systemic inflammation [7], brain ischemia [2], acute pancreatic injury [8], and thermal injury [9]. Suppression of NF-κB activity has been proposed as a molecular mechanism responsible for the anti-inflammatory effects of EP. It has been reported that EP inhibits DNA binding of p65, a NF-κB subunit, by changing intracellular redox condition to favorable condition for oxidation of key cysteine residue in p65 via decreasing intracellular GSH concentration [10]. EP was also suggested to inhibit DNA binding of p65 by alkylating it at cysteine 38 [11]. Recently, Kim et al. (2008) [12] reported that anti-inflammatory effect of EP is attributable to the inhibition of ROS-dependent STAT signaling and also to the deacetylation of histones H3 and H4 by recruiting HDAC1 to iNOS and TNF-α gene promoters.

In addition to the anti-inflammatory effect, suppression of oxidative damage, inhibition of apoptosis, and metabolic modulation by EP have also been reported. It has been reported that EP effectively scavenges H₂O₂ in non-enzymatic way [13] and also scavenges other reactive intermediates, for example, O₂^.− [13], [14]. ROS scavenging potency of EP seems to be greater than that of pyruvate [15] and these functions can contribute to a robust anti-oxidative effect of EP. Regarding anti-apoptotic function, EP has been shown to decrease apoptosis in various conditions, including dopamine-induced neuronal cell death in PC12 cells [13], a rodent model of hepatic I/R injury [16], and a rodent model of brain ischemic injury [17]. Furthermore, it is conceivable that in aqueous condition, EP is gradually hydrolyzed to form pyruvate and it can influence intracellular metabolism.

In the presence study, we report a novel anti-inflammatory mechanism conveyed by EP, which is obtained by a convergence of anti-inflammatory and anti-oxidative effects, i.e., up-regulation of HO-1 and simultaneous down-regulation of iNOS expression. We present a molecular mechanism underlying the up-down regulation of HO-1 and iNOS gene expression, which is EP-mediated Nrf2 nuclear translocation and binding to p300 and subsequent depletion of p300 from p65.