Molecular and Cellular PharmacologyInteraction of ethyl pyruvate in vitro with NF-κB subunits, RelA and p50
Introduction
Nuclear factor-kappa B (NF-κB) is a central mediator of human immune responses. In mammals, there are at least five NF-κB proteins, RelA, p50, RelB, p52 and RelC, and they form a variety of homodimers and heterodimers (Ghosh et al., 1998, Siebenlist et al., 2005). The dimers exist in the cytoplasm in inactive forms due to the binding of an inhibitory protein called IκB (Ghosh et al., 1998, Siebenlist et al., 2005). Tumor necrosis factor α (TNFα), a cytokine especially important in inducing inflammatory responses, activates the NF-κB signaling pathway. TNFα treatment induces the degradation of IκB, and then, NF-κB dimers translocate to the nucleus where they stimulate the transcription of hundreds of genes that participate in inflammatory responses. Therefore, pharmacological inhibition of NF-κB should substantially attenuate inflammatory responses.
Ethyl pyruvate has been shown to be an effective anti-inflammatory agent in a variety of in vivo models (Fink, 2007), ameliorating damage following endotoxemia (Ulloa et al., 2002), alcoholic hepatitis (Yang et al., 2003), and acute pancreatitis (Yang et al., 2004). These anti-inflammatory effects may be due to inhibition of the NF-κB signaling pathway (Han et al., 2005, Johansson et al., 2008). Each NF-κB protein contains a conserved N-terminal 300-amino acid region known as the rel homology domain (RHD) (Ghosh et al., 1998, Siebenlist et al., 2005). This region is responsible for DNA-binding, nuclear localization, and subunit dimerization (Ghosh et al., 1998, Siebenlist et al., 2005). Ethyl pyruvate was suggested to inhibit NF-κB/DNA-binding activity by alkylating RelA at cysteine 38 (Han et al., 2005). However, the direct interaction of ethyl pyruvate with RelA in vitro has not been reported. In addition, there are no reports that ethyl pyruvate interacts with p50.
Acute lung injury is characterized by alveolar damage with a disruption of the alveolar barrier and inflammatory cells. It has been suggested that alveolar epithelial type II cells prevent injury and promote repair in the lungs (for review, see Sugahara et al., 2006). TNF α has been reported to affect the functions of these cells (Bachurski et al., 1995). A549 cells are immortalized cells, which retain many of the characteristics of alveolar epithelial type II cells. This cell line has been used to study the responses of alveolar epithelial type II cells to several treatments (Burvall et al., 2002, Kuwahara et al., 2006, Kwong et al., 2004, Lu et al., 2003). Recently, utilizing biochemical methods, we have demonstrated that ethyl pyruvate inhibited the nuclear translocation of RelA in A549 cells (Mizutani et al., 2010).
In the present study, we found that the binding of a RelA and p50 dimer to the κB–DNA motif was inhibited by preincubation of the nuclear extract with ethyl pyruvate; therefore, we closely examined the interaction of ethyl pyruvate with RelA and p50. In addition, the inhibitory effects of ethyl pyruvate on the nuclear association of RelA in A549 cells were examined by immunostaining methods.
Section snippets
Materials
The following chemicals and reagents were obtained as indicated: [γ-32P]ATP, Perkin Elmer, Inc. (Boston, MA); Dulbecco's modified Eagle's medium (DMEM), ethyl pyruvate, and phosphate-buffered saline (PBS), Sigma Chemical Co. (St Louis, MO); fetal calf serum (FCS), HyClone (Logan, UT); protease inhibitor cocktail and protein phosphatase inhibitor cocktail (EDTA free), Nacalai Tesque (Kyoto, Japan); SDS-PAGE molecular weight standards, Bio-Rad (Richmond, CA); anti-RelA antibody and anti-p50
Effects of ethyl pyruvate on TNFα-induced increase in NF-κB/DNA-binding activity
We first examined whether or not preincubation of the nuclear extract with ethyl pyruvate inhibited the NF-κB/DNA-binding activity (Fig. 1). We previously examined the effects of ethyl pyruvate on NF-κB-mediated transcription using the NF-κB luciferase reporter gene assay (Mizutani et al., 2010). We found that pretreatment of A549 cells with 10 mM ethyl pyruvate inhibited almost completely the TNFα-induced activation of NF-κB (Mizutani et al., 2010). Therefore, we used 10 mM ethyl pyruvate in the
Discussion
The anti-inflammatory actions of ethyl pyruvate are reportedly due to inhibition of the NF-κB pathway (Han et al., 2005, Johansson et al., 2008). However, it's still not clear whether or not ethyl pyruvate interacts with NF-κB proteins to inhibit their functions. In the present study, we found that the preincubation of nuclear extract from A549 cells with ethyl pyruvate inhibited the NF-κB/DNA-binding activity induced by TNFα treatment. We previously reported that the NF-κB/DNA complex after
Acknowledgments
We gratefully acknowledge the help of Dr. S. Kina (University of the Ryukyus) with the manuscript. This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (K.S. and H.Y.).
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