Elsevier

Biochemical Pharmacology

Volume 66, Issue 5, 1 September 2003, Pages 697-710
Biochemical Pharmacology

Interferon gamma enhances proteasome activity in recombinant Hep G2 cells that express cytochrome P4502E1: modulation by ethanol

https://doi.org/10.1016/S0006-2952(03)00252-1Get rights and content

Abstract

We tested the influence of IFNγ on proteasome activity in parental Hep G2 cells that do not metabolize ethanol, as well as in recombinant Hep G2-derived cells that express either or both alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). IFNγ treatment increased proteasome activity in VL-17A (ADH+, CYP2E1+) and E-47 (CYP2E1+) cells, but not in Hep G2, VI-R2 (parental cells with empty vectors) or in VA-13 (ADH+) cells. Proteasome activation by IFNγ correlated positively with the level of CYP2E1 activity. Treatment of VL-17A cells with agents that inhibit CYP2E1 or the inducible nitric oxide synthase (iNOS) or that prevent the formation of peroxynitrite also blocked proteasome activation by IFNγ, indicating that the proteasome may be directly activated by products of CYP2E1 and iNOS catalysis. While IFNγ treatment increased proteasome activity, it also decreased CYP2E1 activity. Both effects were mediated via the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) pathway, as both were blocked by the JAK2 inhibitor, tyrphostin AG 490. Ethanol treatment of VL-17A cells also caused a similar blockage of these same IFNγ-mediated effects, by inhibiting STAT1 phosphorylation. This inhibition was largely due to ethanol metabolism, as 4-methylpyrazole, an ethanol metabolism inhibitor, restored IFNγ-mediated STAT1 phosphorylation in ethanol-treated cells. Our results lead us to propose that IFNγ initiates signal transduction, which alters the activities of CYP2E1 and iNOS, thereby producing reactive oxygen species. One of these oxidants, possibly peroxynitrite, may be directly involved in proteasome activation. Ethanol metabolism by VL-17A cells suppresses IFNγ-mediated induction of proteasome activity, in part, by preventing STAT1 phosphorylation.

Introduction

The proteasome is a large proteolytic particle that contains multiple catalytic activities for protein degradation. The proteasome participates in the regulation of normal physiological processes in cells, by degrading short-lived regulatory proteins that have roles in vital cellular functions including proliferation and apoptosis, as well as degrading long-lived constitutive enzymes. The proteasome also has an important role in cellular defense by degrading damaged, potentially cytotoxic proteins [1], [2], [3], [4]. Thus, the proteasome is vital for many important cell functions [5], [6]. The enzyme particle is also involved in antigen presentation [7]. The specific subunits of the immunoproteasome, cleave proteins to distinct peptides, which form complexes with major histocompatibility complex (MHC) class I molecules on the cell surface. These complexes can be recognized by cytotoxic T-lymphocytes (CTLs) that eliminate target cells with foreign peptide repertoires [8]. Any MHC class I+ host cell, including the hepatocyte, may be targeted by CTLs [9], [10].

The liver is the primary site of ethanol oxidation, and this organ sustains significant damage from chronic ethanol consumption [11]. In vitro studies using Hep G2-derived cells have demonstrated that ethanol-induced cytotoxicity depends on the levels of expression of the ethanol-metabolizing enzymes, CYP2E1 or ADH [12], [13] and therefore, on the levels of ethanol oxidation. There is evidence that metabolites generated from ethanol oxidation can exert inhibitory effects on a number of cellular pathways including the activity of the proteasome. In hepatocytes of patients with alcohol-induced liver disease, Mallory bodies are typical histological hallmarks [14]. These are believed to form as a result of compromised proteasome function or even proteasome depletion [15]. Ethanol consumption can cause reduced hepatic proteasome activity. This effect is associated with high blood ethanol levels in vivo when ethanol is administered by continuous intragastric feeding, which, also results in increased hepatic lipid peroxidation (oxidative stress) [16], [17]. Impaired proteasome function appears to result from ethanol metabolism [18]. Pro-inflammatory cytokines, including IFNγ, are natural modulators of proteasome activity [19]. IFNγ acts on a cell by binding to surface IFNγ receptors, which initiate the transduction of signals to nuclear genes via the JAK-STAT1 pathway. This occurs by phosphorylation of STAT1, which is then translocated to the nucleus and binds to DNA, initiating downstream effects, which include the modulation of proteasome function in antigen-presenting cells, such as macrophages and dendritic cells [20], [21]. However, the effect of IFNγ on proteasome activity in liver cells has not been characterized.

The aim of this work was to study the response of the proteasome to IFNγ and the transduction of IFNγ signals in recombinant Hep G2 cells incubated in the presence or absence of ethanol. We tested the influence of IFNγ on proteasome chymotrypsin-like activity in parental Hep G2 cells that do not metabolize ethanol, as well as in recombinant Hep G2-derived cells that express either or both ADH and CYP2E1. We also determined whether ethanol itself or the products of its metabolism altered the responsiveness of the proteasome to IFNγ. We found that IFNγ treatment enhanced proteasome activity via the JAK-STAT1 pathway and that this enhancement occurred only in CYP2E1-expressing cells. Here, we provide evidence that this effect may be mediated by peroxynitrite. We further show that prior exposure of cells to ethanol prevented the IFNγ-induced increase in proteasome activity by blocking STAT1 phosphorylation. Portions of this work have been described in recent abstracts [22], [23].

Section snippets

Reagents and media

High glucose Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. The membrane-permeable flourogenic proteasome substrate, methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF-AFC) was obtained from Enzyme Systems Products. Human recombinant IFNγ was from PeproTech, Inc. Anti-IFNγRα was from PharMingen. The antibody preparations to phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and STAT1 were from Cell

Results

To determine whether IFNγ affects the activity of the proteasome, we treated VL-17A and VI-R2 and/or Hep G2 cells with various doses of IFNγ (2–50 ng/mL) for 1–24 hr. Chymotrypsin-like activity of the proteasome was subsequently tested in situ by FLF-AFC hydrolysis. Exposure to IFNγ increased FLF-AFC hydrolysis by 20–50% in VL-17A cells compared with their untreated controls. This increase was first detected after 4 hr of treatment with IFNγ and remained elevated after 24 hr (Fig. 1A). Proteasome

Discussion

The liver is routinely exposed to toxic agents, microbes and viruses arriving via the portal vein from the gastrointestinal tract. Such exposure can activate liver immune cells. Activated intrahepatic lymphocytes secrete IFNγ and other cytokines in response to these stimuli, thus putting hepatocytes under continuous cytokine exposure. Hepatocytes abundantly express proteasome activity and previous studies in vivo have shown that IFNγ can regulate this activity in liver tissue [36]. However, it

Acknowledgements

We thank Dr. Charles Kuszynski and Linda Wilkie for performing flow cytometry for detection of IFNγRα. We are pleased to acknowledge the valuable technical assistance of Jimmie L. Nelson. We thank Dr. Julie Stoner, of the Dept of Preventive and Societal Medicine of the UNMC, for performing some of the statistical analyses. This study was supported by Medical Research Funds from the Department of Veterans Affairs and VA Alcohol Research Center.

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