IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways

Abstract

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK–STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (−583/−574-bp). However, inhibition of the JAK–STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (−1604/−1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (−1551/−1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK–STAT6 and IRS1/2 and NFATc1.

Introduction

Cytochrome P450 2E1 (CYP2E1), the ethanol-inducible form of cytochrome P450 (CYP), is an important phase I drug metabolizing enzyme since it plays a dual physiological role [1], [2], [3], [4]. It contributes to the defense against the penetration of xenobiotics through its broad substrate specificity, allowing it to detoxify a wide variety of exogenous and endogenous compounds and also plays a role in gluconeogenesis. Paradoxically, it also metabolically activates biologically inert procarcinogens and converts nontoxic substrates to more toxic forms [5]. Furthermore, CYP2E1 is an effective generator of reactive oxygen species such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, rendering it an important component in the process of oxidative stress [2], [3], [6], [7]. As an important component of the adaptive system in the body, the expression of CYP2E1 is under strict regulation at different levels including transcriptional, post-transcriptional, translational, and post-translational [7], [8], [9]. CYP2E1 is induced by both acute and chronic alcohol treatment, as well as under a variety of physiological and pathophysiological conditions. For example, CYP2E1 expression is decreased during inflammation [10] and in alcoholic or non-alcoholic liver diseases [11], [12], and instead increased in diabetic patients [13], [14]. Cytokines, a group of secreted peptides and glycoproteins essential for cellular signaling, are shown to be key mediators in the regulation of CYPs during inflammation and infection [15]. Multiple cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF) and interferon-α/γ (IFN-α/γ) were found to function in the regulation of specific CYPs during lipopolysaccharide (LPS)-induced inflammation. CYP2E1 is also regulated by cytokines both in human and rodent models [16], [17]. Similar to most other CYPs, CYP2E1 is down-regulated by pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF. Interestingly, Abdel-Razzak et al. demonstrated that in primary human hepatocytes interleukin-4 (IL-4), an anti-inflammatory cytokine, significantly up-regulates the expression of CYP2E1 as well as glutathione-S-transferases (GST), although it down-regulates most other P450s [16].

IL-4, mainly produced by T helper (T_H) 2 lymphocytes, is a key cytokine in the regulation of humoral immune response. It contributes to the activation, proliferation, and differentiation of B cells, the secretion of IgE by B lymphocytes, and the prevention of apoptosis in T lymphocytes. An essential biological activity of IL-4 in the development of allergic inflammation is the ability to drive the differentiation of naive T helper type 0 (T_H0) lymphocytes into T_H2 lymphocytes [18]. Its effect relies on signaling through a receptor complex consisting of the specific IL-4 receptor alpha chain (IL-4Rα) and the common gamma chain (IL-4Rγc), resulting in a series of phosphorylation events mediated by receptor-associated kinases [18], [19], [20], [21]. The IL-4R heterodimerization promotes the activation of janus protein kinases (JAKs) that are constitutively associated with IL-4Rα (JAK1) and the IL-4Rγc (JAK3). Phosphorylation of a cassette of three closely spaced tyrosine residues in the cytoplasmic domain of IL-4Rα initiated by activated JAKs enables the recruitment of the transcription factor signal transducer and activator of transcription 6 (STAT6). The tyrosine phosphorylation of STAT6 by JAKs leads to its dimerization and translocation to the nucleus, where it binds to specific cis-acting elements and activates the transcription of IL-4 responsive genes. In addition to the JAK–STAT pathway, the insulin receptor substrate (IRS) family of proteins is also involved in IL-4 signaling. Activation of IRS1/2 results in the activation of mitogen-activated protein kinase (MAPK) signaling cascades as well as the phosphoinositide-3-kinase (PI3K) pathway [21]. Furthermore IL-4-mediated induction of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase as well as IL-4-induced proliferation and differentiation were shown to require simultaneous signaling via both STAT6 and IRS-2 [22], [23].

Previous studies showed that IL-4-induced expression of CYP2E1 is transcriptionally regulated and results in increased mRNA and protein levels in primary human hepatocytes and human hepatoma cells [16], [24]. In the present study using human CYP2E1 promoter constructs transfected into human B16A2 hepatoma cells, we identified two IL-4 responsive elements in the human CYP2E1 promoter, a proximal region (−519/−669-bp) and a more distal one (−1604/−1428-bp). Both regions were required for maximal IL-4-mediated induction of CYP2E1. Moreover siRNA experiments and mutagenesis of the CYP2E1promoter indicate that two well-known IL-4 signaling pathways are responsible for the IL-4 induction of CYP2E1, namely JAK1–STAT6 and IRS1/2 involving the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1).