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Originally published In Press as doi:10.1074/jbc.M101266200 on March 16, 2001

J. Biol. Chem., Vol. 276, Issue 21, 18223-18228, May 25, 2001
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Ikappa B Kinase, a Molecular Target for Inhibition by 4-Hydroxy-2-nonenal*

Chuan Ji, Kevin R. Kozak, and Lawrence J. MarnettDagger

From the Vanderbilt-Ingram Cancer Center and Center in Molecular Toxicology, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 73232-0146

In unstimulated cells, transcription factor NF-kappa B is retained in the cytoplasm by interaction with the inhibitory protein, Ikappa Balpha . Appropriate cellular stimuli inactivate Ikappa Balpha by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-kappa B to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-kappa B in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dose-dependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-kappa B DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IM-induced AP-1 DNA binding activity. HNE inhibits TPA/IM-induced degradation of Ikappa Balpha in both H1299 and Jurkat T cells. The accumulation of Ikappa Balpha parallels the inhibition of its phosphorylation. At doses that inhibit Ikappa Balpha degradation, HNE inhibits Ikappa B kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-kappa B activation that acts by preventing IKK activation and subsequent Ikappa Balpha degradation.

* This work was supported by Research Grant CA47479 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and requests for reprints should be addressed. Tel.: 615-343-7329; Fax: 615-343-7534; E-mail: marnett@toxicology.mc.vanderbilt.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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