NF-κB-dependent Transcriptional Activation in Lung Carcinoma Cells by Farnesol Involves p65/RelA(Ser276) Phosphorylation via the MEK-MSK1 Signaling Pathway

Joung Hyuck Joo,Anton M Jetten

doi:10.1074/jbc.m800945200

Abstract

In this study, we demonstrate that treatment of human lung adenocarcinoma H460 cells with farnesol induces the expression of a number of immune response and inflammatory genes, including IL-6, CXCL3, IL-1alpha, and COX-2. This response was dependent on the activation of the NF-kappaB signaling pathway. Farnesol treatment reduces the level of IkappaBalpha and induces translocation of p65/RelA to the nucleus, its phosphorylation at Ser(276), and transactivation of NF-kappaB-dependent transcription. Moreover, overexpression of IkappaBalpha or treatment with the NF-kappaB inhibitor caffeic acid phenethyl ester greatly diminishes the induction of inflammatory gene expression by farnesol. We provide evidence indicating that the farnesol-induced phosphorylation of p65/RelA at Ser(276) is important for optimal transcriptional activity of NF-kappaB. The MEK1/2 inhibitor U0126 and knockdown of MEK1/2 expression with small interfering RNAs effectively blocked the phosphorylation of p65/RelA(Ser(276)) but not that of Ser(536), suggesting that this phosphorylation is dependent on the activation of the MEK1/2-ERK1/2 pathway. We further show that inhibition of MSK1, a kinase acting downstream of MEK1/2-ERK1/2, by H89 or knockdown of MSK1 expression also inhibited phosphorylation of p65/RelA(Ser(276)), suggesting that this phosphorylation is dependent on MSK1. Knockdown of MEK1/2 or MSK1 expression inhibits farnesol-induced expression of CXCL3, IL-1alpha, and COX-2 mRNA. Our results indicate that the induction of inflammatory genes by farnesol is mediated by the activation of the NF-kappaB pathway and involves MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA(Ser(276)). The activation of the NF-kappaB pathway by farnesol might be part of a prosurvival response during farnesol-induced ER stress.

Highlights

Isoprenoids are intermediates of the cholesterol/sterol biosynthetic pathway and are formed from mevalonate, which is synthesized from acetyl-CoA by the rate-limiting enzyme 3-hy
We reported that a large number of genes associated with the endoplasmic reticulum (ER)2 stress response are rapidly induced by farnesol treatment, suggesting that farnesolinduced apoptosis is coupled to ER stress [26]
We demonstrate that treatment of human lung adenocarcinoma H460 cells with farnesol induces the expression of a number of immune response and inflammatory-related genes, including COX-2 and several chemo/cytokines, and

Summary

EXPERIMENTAL PROCEDURES

Western Blot Analysis—Cells were harvested and lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS, supplemented with protease and phosphatase inhibitor mixtures I and II (Sigma). The DNA-protein binding reactions were performed in 10 ␮l of binding buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 12.5% glycerol, 0.1% Triton X-100, and 0.5 ␮g/ml bovine serum albumin) with 5 ␮g of nuclear extract, 105 cpm of the radiolabeled oligonucleotide, and 1 ␮g of poly(dI-dC) for 30 min at room temperature. Nuclear proteins were incubated with antip antibody (catalog number sc-109; Santa Cruz Biotechnology) for 20 min at room temperature prior to the addition of labeled oligonucleotide. Changes in the expression of several immune response and inflammatory genes by farnesol

Gene name

RESULTS

DISCUSSION

Signaling Pathway