Leukotriene D4 induces AP-1 but not NFκB signaling in intestinal epithelial cells

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Abstract

We have previously shown that leukotriene D4 (LTD4), a known pro-inflammatory mediator, induces increased survival and proliferation of intestinal epithelial cells. In this study we examined whether LTD4 functions via activation of the transcription factors NFκB and AP-1, which are potent inducers of mitogenesis. We found that the NFκB inhibitory protein IκBα was not degraded upon LTD4 stimulation. Furthermore, nuclear translocation of the classical p65 or alternative p52 subunits of NFκB was not observed. Accordingly, LTD4 stimulation failed to induce NFκB transcriptional activity. Instead we found that LTD4 induced phosphorylation of c-Jun-N-terminal kinase (JNK) and transcriptional activity of AP-1, which could be reduced by a JNK inhibitor. Moreover, LTD4 induced cell proliferation, and this effect was also blocked upon addition of a JNK inhibitor. Our findings show for the first time that JNK/AP-1 but not NFκB is a downstream target of LTD4 in intestinal epithelial cells, suggesting that AP-1 is an important mediator of LTD4-induced mitogenic effects.

Introduction

Leukotrienes are metabolites of arachidonic acid that are released and synthesized from membrane phospholipids via the action of phospholipase A2 and 5-lipoxygenase. Leukotriene D4 (LTD4) is a potent mediator of inflammation [1] and activates the G-protein coupled high-affinity receptor CysLT1 [2]. During the last decade, selective CysLT1 receptor antagonists have proven successful in the treatment of asthma [3], [4].

In chronic inflammation such as inflammatory bowel diseases (IBD), high levels of leukotrienes are found [5]. In addition to increased leukotriene production in the colon, individuals with IBD have a 30–50% increased risk of developing colorectal cancer [6]. The tumor microenvironment contains inflammatory components and growth factors which together promote cell proliferation, survival and migration [7], suggesting that leukotrienes are likely to take part in the neoplastic process. In accordance with this, we have previously found that LTD4 induces proliferation, survival and migration of intestinal epithelial cells [8], [9]. Moreover, we found that high expression of its receptor, CysLT1, in colorectal tumors correlates with poor prognosis [10]. However, the signaling mechanisms of LTD4 in intestinal epithelial cells remain to be elucidated.

Several studies point to the transcription factor NFκB as a link between inflammation and cancer, including inflammation-induced colon cancer (for review see [11]). NFκB mediates a robust response to inflammatory stimuli such as lipopolysaccharides (LPS) and the cytokine TNFα [12]. There are five isoforms of NFκB, namely RelA (p65), RelB, c-Rel, NFκB1 (p105/p50) and NFκB2 (p100/p52) [13]. In dimer formations, these pleiotropic transcription factors orchestrate a number of cell survival as well as mitogenic responses in many different cell types. The classical pathway of NFκB activation is initiated by IκB kinase complex (IKK) phosphorylation of the NFκB inhibitory protein IκBα. As a consequence, IκBα is ubiquitinated and subsequently degraded. This allows the NFκB subunits p50 and p65 to enter the nucleus to enable transcription of target genes [14]. Induction of an alternative pathway, e.g. by LPS, causes cleavage of the p100 precursor of NFκB2 to generate p52 and release of RelB, which after translocation to the nucleus, bind to response elements of target genes [15].

Inflammation-driven carcinogenesis can also be promoted through the activator protein (AP)-1 transcription factor family [16]. The c-Jun N-terminal kinase (JNK) is a MAP kinase that is able to activate AP-1. AP-1 is composed of homo- or heterodimers of Jun, Fos, Maf or ATF subunits, of which c-Jun and c-Fos were originally described as proto-oncogenes [17], [18]. JNK is activated in cells exposed to cellular stress such as ultraviolet light but also to mitogenic stimuli and pro-inflammatory cytokines, including TNFα [19]. The JNK/AP-1 pathway in turn regulates a variety of cellular processes including cell proliferation, differentiation and survival, and the outcome seems to be highly context dependent [20].

The aim of this study was to elucidate whether LTD4 signaling can induce activation of the NFκB or AP-1 transcription factor pathways, which are known to mediate mitogenic signals, in intestinal epithelial cells.

Section snippets

Reagents

LTD4 was obtained from Cayman Chemicals Co. (Ann Arbor, MI), and human recombinant TNFα was purchased from R&D systems (Minneapolis, MN). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA). The cell-permeable JNK inhibitor I, (L)-form (JNKI1), was purchased from Calbiochem (La Jolla, CA). The firefly luciferase pNFκB-Luc construct and the Renilla vector were from BD Biosciences (San Jose, CA). The pGL2Coll73-luc firefly luciferase construct was a kind gift from Dr. Härkönen [21]. Dual

LTD4 does not activate the classical NFκB pathway

One of the important steps in activation of the classical NFκB signaling pathway is degradation of IκBα, which releases the p50/p65 NFκB subunits for translocation into the nucleus [11]. Treatment with LTD4 did not change the protein level of IκBα while the pro-inflammatory cytokine TNFα reduced the IκBα protein level in non-transformed intestinal epithelial Int 407 cells (Fig. 1A). Treatment with the ribosomal inhibitor cycloheximide (10 μg/ml) confirmed that the level of IκBα after LTD4

Discussion

LTD4 is known to play a role in the pathogenesis of several chronic inflammatory diseases, and the secretion of leukotrienes in IBD patients has been previously documented [5]. Accordingly, LTD4 is a potent inflammatory mediator that is able to promote transformation of intestinal epithelial cells [8], [9]. Many studies have focused on transcription factors and their potential as therapeutic targets. Based on previous studies that LTD4 may promote mitogenic signals in intestinal epithelial

Acknowledgments

We thank Maria Juhas for technical assistance. This work was supported by grants awarded to the author AS from the Swedish Cancer Foundation, the Swedish Medical Research Council, the Foundations at Malmö University Hospital, the Ruth and Richard Julin Foundation, Gunnar Nilsson Foundation and the Österlund Foundation, and to AM-L B from the Royal Physiographic Society in Lund, Sweden.

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