Interaction of ethyl pyruvate in vitro with NF-κB subunits, RelA and p50.
Ethyl pyruvate, an aliphatic ester derived from pyruvate, reportedly has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-κB). It was suggested that ethyl pyruvate inhibited NF-κB/DNA-binding activity through the covalent modification of RelA. However, the interaction of ethyl pyruvate with RelA in vitro has not been reported. In the present study, we confirmed that treatment of cultured alveolar epithelial cells, A549 cells, with tumor necrosis factor α (TNFα) increased the NF-κB/DNA-binding activity. When the nuclear extract of the cells was incubated with ethyl pyruvate, the NF-κB/DNA-binding activity was strongly inhibited. Because we previously found that the NF-κB/DNA complex included RelA and p50, we bacterially expressed a deletion mutant of RelA, RelA (1-220), and a full-length form of p50. Incubation of RelA (1-220) or p50 with ethyl pyruvate induced dramatic changes in mobility in two types of nondenaturing gel electrophoresis. Electrophoretic mobility shift assays revealed that incubation of RelA (1-220) or p50 with ethyl pyruvate inhibited the DNA-binding activity. Furthermore, immunostaining of A549 cells revealed that ethyl pyruvate inhibited the nuclear association of RelA after TNFα treatment. These results suggest that ethyl pyruvate interacts with RelA and p50 to inhibit their functions at multiple points.
Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
Ayako
A
Mizutani
Noriko
N
Maeda
Seikichi
S
Toku
Sayomi
S
Higa-Nakamine
Yoichiro
Y
Isohama
Hajime
H
Sunakawa
Kazuhiro
K
Sugahara
Hideyuki
H
Yamamoto
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011773:Pyruvates / Q000378:metabolism* / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000737:chemistry / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D052002:NF-kappa B p50 Subunit; D011773:Pyruvates; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; C046522:ethyl pyruvate; D004247:DNA