4-Hydroxynonenal Prevents NF-κB Activation and Tumor Necrosis Factor Expression by Inhibiting IκB Phosphorylation and Subsequent Proteolysis

Abstract

Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-B. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-B activation by stimuli such as LPS, interleukin (IL)-1␤, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced B-and TNF or IL-8 promoterdependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IB-␣, IB-␤, and, at a later time point, IB-⑀ was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IB-␣. This is the first report which demonstrates that HNE specifically inhibits the NF-B/Rel system. Downmodulation of NF-B-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases.

Diverse agents act at multiple levels to inhibit the Rel/NF-κB signal transduction pathway

“…One class of IkB metabolism blockers includes signaling molecules such as nitric oxide (NO) (Matthews et al, 1996), Extensively Oxidized Low Density Lipoprotein (ox-LDL) (Brand et al, 1997), 4-hydroxynonenal (HNE) (Page et al, 1999), estrogen (E2) (Sun et al, 1998), and prostaglandin A (Rossi et al, 1997). At least one of these inhibitors shows pathway-speci®c inhibition of NF-kB; HNE can inhibit LPS-, IL-1-and phorbol ester-induced activation of NF-kB, but not that induced by TNFa (Page et al, 1999).…”

“…At least one of these inhibitors shows pathway-speci®c inhibition of NF-kB; HNE can inhibit LPS-, IL-1-and phorbol ester-induced activation of NF-kB, but not that induced by TNFa (Page et al, 1999).…”

Transcriptional Inhibition of Interleukin-8 Expression in Tumor Necrosis Factor-tolerant Cells

“…Transfection of THP-1 Cells-In transfection studies pGL2-IL-8 (420 bp of the IL-8 promoter region), a firefly luciferase reporter plasmid, was utilized (30,31). This plasmid (1 g) was transiently co-transfected with 0.2 g of a constitutively active Renilla luciferase control plasmid, pRLtk (Promega, Mannheim, Germany), into THP-1 cells using a DEAE-dextran-based protocol (30,31).…”

Detection of IKKβ-IKKγ Subcomplexes in Monocytic Cells and Characterization of Associated Signaling

“…PAGE and Western Blot Analysis-Cytosolic and nuclear extracts were isolated as described (4,39), and electrophoresis was performed with 12.5% polyacrylamide gels. The proteins were transferred to nitrocellulose membranes using the wet blot technique.…”

“…Immunoprecipitation-Cytosolic extracts were subjected to immunoprecipitation in TNT buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM dithiothreitol, 0.5 M 4-(2-aminoethyl)-benzenesulfonyl fluoride, leupeptin, antipain, aprotinin, pepstatin A, chymostatin 0.75 g/ml each; Sigma) (4,39). Immunoprecipitation was carried out using 35 l of 6% protein A-or G-agarose (Roche Applied Science) for 2 h at 4°C with 1 g of anti-IKK␣ (BD Biosciences), IKK␤ (QED Bioscience), IKK␥ (Santa Cruz Biotechnology), or anti-FLAG antibody.…”