Articles in PresS. Am J Physiol Gastrointest Liver Physiol (September 1, 2017). doi:10.1152/ajpgi.00027.2017

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Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially

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impairs hepatic protein trafficking and growth hormone signaling

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Erin E. Doody1*, Jennifer L. Groebner1*, Jetta R. Walker2*, Brittnee M. Frizol1, Dean J.

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Tuma3, David J. Fernandez2 and Pamela L. Tuma1†

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Department of Biology, The Catholic University of America, Washington DC 20064 2

Present address: Northern Virginia Community College, Alexandria, VA 22311 3

Department of Internal Medicine, University of Nebraska, Omaha, NE 68105

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*These authors contributed equally †

Corresponding author

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Author contributions: PLT and DJT are responsible for the experimental design. EED, JLG,

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JRW, BMF and DJF performed the experiments. PLT, DJT, JLG and DJF analyzed and

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interpreted data. Figures were compiled by PLT and JLG. PLT wrote the manuscript which all

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authors critically analyzed.

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Running Head: CYP2E1-mediated ethanol metabolism impairs Jak2/STAT5 activation

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Corresponding Author: Pamela L. Tuma, PhD, Department of Biology, The Catholic University

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of America, 620 Michigan Avenue, NE, Washington, DC 20064,

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Tel. 202-319-6681, Fax. 202-319-5721, Email: [email protected]

Copyright © 2017 by the American Physiological Society.

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ABSTRACT

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The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1

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(CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also

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results in the production of reactive oxygen species (ROS) that promote oxidative stress. We

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have previously shown that microtubules are hyperacetylated in ethanol-treated polarized,

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hepatic WIF-B cells and livers from ethanol fed rats. We have also shown that enhanced

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protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion

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and nuclear translocation and that the defects are likely mediated by acetaldehyde. However,

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the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these

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alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated

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alcohol metabolism is required for enhanced acetylation and the trafficking defects, we co-

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incubated cells with ethanol and diallyl sulfide (a CYP2E1 inhibitor) or N-acetyl cysteine (an anti-

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oxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and

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also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat

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surprisingly, both NAS and DAC prevented impaired STAT5B nuclear translocation. Further

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examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B

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activation by growth hormone (GH) was prevented by DAS and NAC. These results were

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confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative

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RT-PCR, we further determined that ethanol exposure led to blunted GH-mediated gene

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expression. In conclusion, we determined that alcohol-induced microtubule acetylation and

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associated defects in microtubule-dependent trafficking are mediated by ADH metabolism

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whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1

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activity.

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New and Noteworthy: Impaired growth hormone-mediated signaling is observed in ethanol

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exposed hepatocytes and is explained by differential effects of ADH and CYP2E1-mediated

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ethanol metabolism on the Jak2/STAT5B pathway.

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Keywords: WIF-B cells, ethanol, hepatotoxicity, CYP2E1

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Abbreviations: 4-MP, 4-methyl pyrazole; ADH, alcohol dehydrogenase; ASGP-R,

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asialoglycoprotein receptor; CYP2E1, cytochrome P450 2E1; DAS, diallyl sulfide; GH, growth

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hormone; GH-R, growth hormone receptor; HDAC6, histone deacetylase 6; NAC, N-acetyl

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cysteine; ROS, reactive oxygen species

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INTRODUCTION

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The liver is the major site of ethanol metabolism, and accordingly, is most susceptible to injury

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from chronic alcohol consumption. Although the progression of alcoholic liver disease is well

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described clinically, little is known about the molecular mechanisms that promote alcohol-

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induced hepatotoxicity. However, alcohol metabolites and enhanced oxidative stress have been

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linked to promoting liver injury. Alcohol is metabolized by two enzymes, alcohol dehydrogenase

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(ADH) and cytochrome P450 2E1 (CYP2E1). ADH-mediated metabolism results in the production

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of acetaldehyde, a highly reactive intermediate that can form stable, covalent modifications on

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other macromolecules (mainly proteins) (reviewed in (39)). CYP2E1-mediated metabolism not

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only produces acetaldehyde, but also highly reactive oxygen and hydroxyethyl radicals (39).

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These oxygen radicals contribute to enhanced oxidative stress and can covalently modify lipids

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(i.e., lipid peroxidation), which in turn promotes their degradation and the production of two other

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highly reactive intermediates, malondialdehyde and 4-hydroxy-2-nonenal. Like acetaldehyde,

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all of these CYP2E1-generated metabolites can from stable, covalent modifications on proteins,

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lipids, DNA and other macromolecules (5, 10, 19, 20, 29, 39, 41). Thus, one hypothesis for

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alcohol-induced hepatotoxicity is that the accumulated covalent modifications during chronic

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alcohol consumption disrupt the normal function of hepatic proteins, lipids and DNA leading to

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hepatic cell dysfunction and injury. However, the targets for modification and how the

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modifications alter proper cell function are not well understood.

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Our ultimate goal is to identify the molecular basis of alcoholic liver injury by identifying and

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defining the mechanisms that lead to alcohol-induced defects in hepatic protein trafficking

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described decades ago in livers from ethanol-fed rats or mice and inferred in patients with

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alcoholic liver disease (reviewed in (35)). Over a dozen years ago, we discovered that

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microtubules are more highly acetylated and more stable in ethanol-treated polarized hepatic

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WIF-B cells, liver slices and in livers from ethanol fed rats (18). In subsequent years, we have

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shown that the defects in microtubule-dependent protein trafficking, including post-Golgi delivery

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of secretory cargo to the cell surface (16), basolateral-to-apical transcytosis (11) and the nuclear

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translocation of a subset of transcription factors (9) can be explained by increased microtubule

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acetylation. We have further shown that increased microtubule acetylation may likely lead to

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impaired vesicle motility by impeding microtubule-based motor translocation and processivity

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along the filamentous tracks (11). We have also correlated alcohol-induced global protein

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acetylation with specific impairment of clathrin-mediated internalization (37, 38). In this case,

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enhanced protein acetylation leads to impaired dynamin recruitment to the necks of invaginating

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coated pits resulting in decreased vesicle fission and impaired endocytosis (38).

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Our early characterization of microtubule modifications in ethanol-treated cells revealed that

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hyperacetylation displayed saturable ethanol dose-dependent and time-dependent kinetics (18)

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implying that this post-translational modification was ultimately dependent on ethanol

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metabolism. More direct evidence for the metabolism-dependence of microtubule acetylation

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and increased stability came from experiments using 4-methyl pyrazole (4-MP) (an ADH

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inhibitor that leads to decreased acetaldehyde levels (30, 31)) and cyanamide (an aldehyde

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dehydrogenase inhibitor that leads to increased acetaldehyde levels (23)). 4-MP co-incubation

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prevented tubulin acetylation whereas cyanamide potentiated the effect indicating that ADH-

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mediated ethanol metabolism is indeed required for increased microtubule acetylation and

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stability and that these effects are likely mediated by acetaldehyde (18). Since then, similar

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studies with 4-MP have also shown that impaired secretion, nuclear translocation and clathrin-

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mediated internalization are also dependent on ADH-mediated ethanol metabolism (9, 16, 38).

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To date, the role of CYP2E1-mediated ethanol metabolites or ROS on microtubule acetylation

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or on these alcohol-induced protein trafficking defects has not been examined.

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The purpose of these studies was to determine if CYP2E1-mediated alcohol metabolism is

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required for alcohol-induced microtubule acetylation and associated defects in microtubule-

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dependent protein trafficking in fully differentiated, polarized, hepatic WIF-B cells (34).

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Importantly, we determined that the WIF-B cells express endogenous ADH and CYP2E1 and

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exhibit ADH, ALDH and CYP2E1 activities comparable to intact hepatocytes (24, 33). They

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produce a time and dose-dependent increase in acetaldehyde, ROS and oxidized proteins upon

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ethanol exposure, also comparable to that observed in intact hepatocytes (24, 33). The WIF-B

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cells display other aspects of alcohol-induced liver injury including increased triglyceride

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accumulation (e.g., fatty liver), an increased lactate/pyruvate redox ratio (24, 33) and exhibit

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alcohol-induced impairments in protein trafficking as described in situ or in isolated hepatocytes

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(8, 16, 18, 38) such that they serve as an excellent model system for the studies described

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here. The approach was straightforward; we co-incubated cells with ethanol and with two well-

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described and long-used pharmacological agents: diallyl sulfide (a CYP2E1 inhibitor in use for

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almost 30 years (3, 4)) or N-acetyl cysteine (a glutathione precursor used as an anti-oxidant first

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described in 1960 (28, 40, 45)). For these studies, we used the same experimental design we

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first described and characterized in 2009 (24) and assayed for selected protein trafficking steps.

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Confirmatory experiments were performed in ethanol-exposed HepG2 cells expressing only

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ADH or only CYP2E1. Finally, quantitative RT-PCR was used to determine if impaired STAT5B

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activation and nuclear translocation blunted GH-mediated gene expression.

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MATERIALS AND METHODS

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Reagents and antibodies. DAS, NAC and monoclonal antibodies against -tubulin or

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acetylated -tubulin were purchased from Sigma–Aldrich (St. Louis, MO). Hepes was

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purchased from HyClone (Logan, UT). Alexa-conjugated secondary antibodies were purchased

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from Life Technologies (Carlsbad, CA). Antibodies against STAT5B (C-17) were purchased

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from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pSTAT5B (C7E5), Jak2 (D2e12) and

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pJak2 (C80) antibodies were purchased from Cell Signaling Technologies (Beverly, MA).

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Recombinant growth hormone (GH) was purchased from Shenandoah Biotechnology (Warwick,

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PA). Polyclonal antibodies against asialoglycoprotein receptor (ASGP-R) and rat serum

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albumin were generously provided by Dr. A. Hubbard (Johns Hopkins University School of

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Medicine, Baltimore, MD).

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Cell culture. WIF-B cells were grown in a humidified 7% CO2 incubator at 37°C as described

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(34). Briefly, cells were grown in F12 medium (Sigma-Aldrich), pH 7.0, supplemented with 5%

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fetal bovine serum (Gemini, Woodland, CA), 10 M hypoxanthine, 40 nM aminopterin and 1.6

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M thymidine. Cells were seeded onto glass coverslips at 1.3 X 104 cells/cm2 and grown for 8–

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12 days until they reached maximum polarity. Cells were treated on day 7 with 50 mM ethanol

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in the absence or presence of 100 M DAS or 5 mM NAS in medium buffered with 10 mM

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Hepes, pH 7.0 for 72 h as described (32). On day 9, cells were reincubated in fresh buffered

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medium in the continued absence or presence of alcohol and/or the pharmacological agents.

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VA-13 and E47 cells (provided by Dr. D. Clemens, University of Nebraska Medical Center,

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Omaha, NE) were grown in a 5% CO2 incubator at 37°C in DMEM containing 10% FBS and 400

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g/ml of zeocin or G418 as described (7). In general, cells were treated on day 2 with 50 mM

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ethanol for 24 h in medium buffered with 10 mM Hepes, pH 7.0. For some experiments, cells

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were additionally treated with 50 nM (1.11 ng/ml) GH for up to 4 h at 37°C as indicated (9).

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Immunofluorescence microscopy. In general, WIF-B, E47 or VA-13 cells were fixed on ice

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with PBS containing 4% paraformaldehyde for 1 min and permeabilized with methanol for

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10 min as described (14). To detect acetylated tubulin, cells were fixed and permeabilized with

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methanol at −20°C for 5 min. Cells were processed for indirect immunofluorescence using anti-

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α-tubulin (1:400), anti-acetylated-α-tubulin (1:250), anti-STAT5B (1:300), anti-pSTAT5B (1:100)

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monoclonal antibodies (Sigma-Aldrich) or anti-ASGP-R (1:1000) polyclonal antibodies. Alexa-

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488 or 568-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used at

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5 μg/ml. Labeled cells were visualized at RT by epifluorescence with an Olympus BX60

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Fluorescence Microscope (OPELCO, Dulles, VA) using an UPlanFl 60x/NA 1.3, phase 3, oil

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immersion objective. Images were taken with an HQ2 CoolSnap digital camera (Roper

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Scientific, Germany) and Metamorph Imaging software (Molecular Devices, Sunny Vale, CA).

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Adobe Photoshop (Adobe Systems Inc, Mountain View, CA) was used to process images and to

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compile figures. To quantitate the relative distributions of STAT5B or phospho-STATB, random

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fields were visualized by epifluorescence and digitized. From micrographs, the average pixel

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intensity of selected regions of interest (ROI) placed in the nucleus or cytoplasm of the same

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cell was measured using the Measure ROI tool of the ImageJ software (National Institutes of

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Health) as described (25, 27). The ratio of nuclear vs. cytoplasmic fluorescence intensities was

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determined. Typically, values were determined from at least 3 independent experiments where

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5-10 random fields were acquired for each condition that contained 15-30 polarized cells each.

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To monitor oxidative stress, WIF-B, E47 or VA-13 cells were incubated with CellROX® Green

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Reagent (Invitrogen) according to the manufacturer’s instructions and fixed with 3.7%

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paraformaldehyde at RT. Labeled cells were visualized as described above.

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Immunoblotting. Proteins were separated using SDS-PAGE, transferred to nitrocellulose and

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immunoblotted with antibodies against total STAT5B (1:5,000-10,000) or Jak2 (1:1000) diluted

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in PBS containing 5% (w/v) milk and 0.1% (v/v) Tween-20. Antibodies specific to phospho-

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STAT5B or phospho-Jak2 were diluted in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-

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20 (both 1:1000). Incubations were performed overnight at 4oC. Immunoreactivity was detected

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using enhanced chemiluminescence (PerkinElmer, Crofton, MD). Relative expression levels

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were determined by densitometric analysis of immunoreactive bands.

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Secretion Assays. Control or treated cells were rinsed five times with prewarmed serum-free

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medium and then reincubated in serum-free medium. At 0, 5, 15 or 30 min after reincubation,

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aliquots of media were collected and analyzed for albumin secretion by immunoblotting as

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described (16). The cell lysates were collected by solubilization directly into SDS-PAGE sample

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buffer. Samples were processed for Western blotting and densitometric analysis of

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immunoreactive bands. The albumin levels secreted for control, DAS or NAC treated cells at 15

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min were set to 100% to which the other corresponding values were normalized.

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Quantitative RT-Jak/STAT signaling pathway PCR arrays. The quantitative RT-PCR

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analysis was performed by SABiosciences (Frederick, MD). Briefly, mature RNA was extracted

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from control or ethanol-treated WIF-B cells additionally treated with GH for 0, 30 or 240 min.

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RNA quality was spectrophotometrically determined. Only RNA that met with the appropriate

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quality standards was further reverse transcribed. The resultant cDNA was used to probe RT2

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Profiler™ Rat Jak/STAT signaling PCR Arrays (96 well-format) purchased from Qiagen

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(Ventura, CA) along with RT2 Sybr™ Green qPCR Mastermix. Fold-changes in gene

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expression were calculated using the double delta cycle threshold (CT) method. The delta CT

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was calculated between the gene of interest and an average of reference house-keeping genes.

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The double delta was calculated by taking the difference of the delta CT for the test group and

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the delta CT for the control group. Fold-changes were calculated using the 2^ (delta CT)

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formula. The CT cut-off was set to 35. Three independent experiments were performed for

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each comparison. Values were considered only if similarities in fold-change were observed in at

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least two out of the three experiments.

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RESULTS

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Ethanol-induced protein acetylation and corresponding defects in protein trafficking are

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not mediated by ROS or CYP2E1 ethanol metabolites.

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Microtubules emanate from centrosomal structures located near the canalicular surfaces in

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WIF-B cells as they do in hepatocytes in situ (Fig. 1A, panel a). Although this orientation is

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maintained in ethanol-treated cells, the microtubules undergo a large morphological change and

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become much shorter, more discrete and more gnarled (Fig. 1A, panel b). Because this gnarled

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phenotype is a hallmark of stable microtubules (22, 26, 42), we stained ethanol-treated cells for

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acetylated -tubulin, a post-translational modification present on stable microtubules. As shown

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in Figure 1B and as we have shown previously (18), the specific anti-acetylated--tubulin

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antibodies recognized microtubules at or near the bile canaliculi in both control and ethanol-

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treated cells. However, the staining was more intense in ethanol-treated cells and was

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observed on microtubules more distal from the bile canaliculi (Fig.1B, panel b marked with

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arrows).

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These morphological changes persisted in ethanol-treated cells that were co-incubated with

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DAS or NAC. Similarly shorter, more discrete and gnarled microtubules were observed as in

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cells treated with ethanol alone (Fig. 1A, panels c and d). Also as in ethanol-treated cells,

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increased levels of acetylated tubulin staining were observed in cells treated with either DAS or

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NAC that were more distal from the canalicular surface (Fig. 1B, panels c and d, marked with

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arrows). These results were confirmed biochemically by immunoblotting cell lysates with

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specific antibodies to detect the total -tubulin pool or the acetylated population (Fig. 1C).

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Importantly, no change in the total amount of -tubulin was detected in any of the treated

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samples. Also importantly, DAS or NAC treatment alone had no significant effect on acetylated

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tubulin levels. Consistent with the morphological analysis and our previous results (18), ethanol

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treatment induced -tubulin acetylation > 2-fold over control levels (Fig. 1C). Also consistent

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with the morphological analysis, additional treatment with DAS or NAC did not prevent the

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ethanol-induced tubulin acetylation. In both cases, approximately 2-fold greater acetylation

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levels were observed. Together, these results indicate that ethanol-induced microtubule

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acetylation is not mediated by ROS or CYP2E1-mediated metabolites and further implicates the

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reactive ethanol metabolite, acetaldehyde, as the important mediator of enhanced acetylation

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(see Discussion).

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To confirm the effects of DAS and NAC on ethanol-treated WIF-B cells as we have previously

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described (24), we monitored oxidative stress by labeling cells with CellROX® Green reagent.

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As expected, addition of ethanol led to a significant increase in labeling indicative of enhanced

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oxidative stress (Fig. 1D, b). Also as expected, virtually no oxidative stress was observed in

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cells additionally treated with DAS (Fig. 1D, c) or NAC (Fig. 1D, d) indicating they effectively

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prevented ROS production thereby confirming our system.

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An ethanol-induced defect in hepatic secretion has been well established in animal models and

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inferred in patients with alcoholic liver disease (reviewed in (35)). Our more recent studies have

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provided strong evidence that ethanol-induced microtubule acetylation is at the heart of the

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impairment likely by interfering with vesicle-mediated surface delivery along microtubule tracks

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(11, 16). If enhanced microtubule acetylation can indeed explain impaired secretion, the

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prediction is that DAS or NAC treatment should not prevent the ethanol-induced defect. To

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measure secretion, we monitored albumin release into the medium by immunoblotting with an

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assay we developed (16). As shown in Figure 2A and as we have previously described,

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decreased basolateral albumin release was observed in ethanol-treated cells (Fig. 2A, left

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panel). When quantitated, we found that secretion was decreased to only 60% of control levels

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(62.9 ± 11.7%) in ethanol-treated cells consistent with our earlier findings (16). In ethanol-

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treated cells co-incubated with DAS (Fig. 2B) or NAC (Fig. 2C), similar levels of impaired

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albumin secretion were observed (68.2 ± 7.2 and 60.4 ± 12.3 of control, respectively). These

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results indicate that impaired secretion is not mediated by ROS or CYP2E1-mediated

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metabolites and further confirm that enhanced microtubule acetylation can explain the defect.

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From a proteomics screen performed in livers from ethanol-fed rats, we determined that ethanol

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led to global increases in protein acetylation (including cortactin, actin and tubulin) (37) and that

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this enhanced acetylation strongly correlated with impaired clathrin-mediated internalization

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(38). If enhanced protein acetylation can explain impaired internalization, we further predicted

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that DAS and NAC should not prevent the ethanol-induced defect in endocytosis. To monitor

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changes in internalization, we immunolabeled control and treated cells for asialoglycoprotein

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receptor (ASGP-R), a receptor known to be internalized by clathrin-meditated endocytosis.

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Consistent with our earlier reports (38), ethanol treatment led to the redistribution of ASGP-R

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from intracellular early endosomes to the cell surface into discrete puncta that we have

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identified as invaginated coated pits (38) (Fig. 3b). Similar redistributions were observed in cells

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co-incubated with DAS (Fig. 3c) or NAC (Fig. 3d) supporting our hypothesis thereby further

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indicating that impaired clathrin-mediated internalization is also not mediated by ROS or

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CYP2E1-mediated metabolites.

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Decreased Jak2/STAT5B activation requires CYP2E1-mediated ethanol metabolism

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We have also strongly correlated ethanol-induced microtubule acetylation with impaired nuclear

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translocation of a subset of transcription factors (9), so the next prediction was that DAS and

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NAC addition should also not prevent this defect. As we have shown previously, ethanol

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treatment led to the cytosolic redistribution of STAT5B at steady state (Fig. 4A, panel c) implying

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impaired nuclear translocation. Semi-quantitation of STAT5B nuclear vs. cytoplasmic

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fluorescence intensities confirmed these observations. At steady state (0 min), the ratio of

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nuclear to cytoplasmic STAT5B staining was significantly decreased in ethanol-treated cells

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(Fig. 4B, left panel). Upon addition of GH for only 5 min, STAT5B nearly completely

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redistributed to the nucleus in control cells, but this translocation was blunted in ethanol-treated

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cells (Fig. 4A, compare panels b and d). Although less pronounced than at steady state, the

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ratio of nuclear to cytoplasmic fluorescence intensity was also decreased in ethanol-treated

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cells after GH addition (Fig. 4B. right panel).

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Somewhat surprisingly, in ethanol-treated cells co-incubated with DAS or NAC, the distributions

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of STAT5B were indistinguishable from control cells with little cytosolic labeling observed at

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steady state (Fig. 4A, panels e and g) and with nearly identical ratios of nuclear to cytoplasmic

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labeling to that of control (Fig. 4B, left panel). Similar results were observed for STAT5B

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distributions after GH addition. Within 5 min, near complete nuclear translocation was observed

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in DAS and NAC treated cells (Fig. 4A, panels f and h) and ratios were restored to control levels

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(Fig. 4B, right panel). Similar results were obtained when labeling for activated and

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phosphorylated STAT5B (pSTAT5B) distributions after GH addition. When quantitated, all

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ratios of nuclear to cytosolic staining were similar to control levels except for cells treated with

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ethanol only, which was decreased to 75% of control (Table 1).

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From our previous studies using pharmacological agents that promote global protein acetylation

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(trichostatin A) or tubulin specific acetylation (taxol at low concentrations) in the absence of

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ethanol, we determined that microtubule acetylation impairs the microtubule-dependent

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translocation of STAT5B to the nucleus after its activation by Jak2 (9). However, these agents

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did not impair Jak2 or STAT5B activation/phosphorylation after GH addition as was observed in

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ethanol-treated cells (9). Thus, to reconcile the disparate results described above, we

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examined the events in STAT5B signaling that are independent of microtubules, yet impaired by

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alcohol administration.

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Upon GH addition, the receptor-associated tyrosine kinase, Jak2, is rapidly activated by cross-

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phosphorylation and in turn phosphorylates the GH receptor (GH-R). The receptor/Jak2

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complex then recruits and phosphorylates STAT5B thereby activating it allowing for subsequent

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microtubule-mediated nuclear translocation. To first examine Jak2 activation, we

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immunoblotted cell lysates with antibodies specific for total or phospho-Jak2 (pJak2) in control

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or treated cells. Importantly, addition of DAS or NAC either alone or in combination with ethanol

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did not change total levels of Jak2 expression (Fig. 5A). In control cells, Jak2 was rapidly

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phosphorylated reaching peak activation within 2 - 5 min after GH addition returning to basal

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levels by ~15 min (Fig. 5B, left side, top panel). As we have reported earlier (9), ethanol

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treatment significantly blunted peak Jak2 activation at 2 min (Fig. 5B, left side, bottom panel) to

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half that seen in control cells (Fig. 5B, right panel). In ethanol-treated cells with added DAS, the

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impairment is nearly completely prevented (Fig. 5C, left panels) with activation kinetics mirroring

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those observed in control cells (Fig. 5C, right panel) whereas NAC addition partially prevented

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the Jak2 phosphorylation defect (Fig. 5D).

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To examine subsequent STAT5B activation by Jak2, we immunoblotted control and treated

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lysates with antibodies specific for total or pSTAT5B. Importantly, addition of DAS or NAC

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alone or in combination with ethanol did not change total levels of STAT5B expression (Fig. 6A).

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In control cells and as expected, STAT5B activation lags behind that of Jak2 with peak

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phosphorylation observed between 5 and 15 min (Fig. 6B, left side, top panel). As we have

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previously shown (9), ethanol treatment significantly blunts peak STAT5B activation at 5 min

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(Fig. 6B, left side, bottom panel) to ~60% of that seen in control cells (Fig. 6B, right panel). In

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contrast, in ethanol-treated cells with added DAS or NAC, the impairment was nearly completely

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prevented (Fig. 6C and D, left panels) with activation kinetics mirroring those observed in control

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cells (Fig. 6C and D, right panels). Together these results indicate that the microtubule-

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independent activation of Jak2 and STAT5B is ultimately impaired by a CYP2E1-mediated

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mechanism.

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To confirm the metabolite-specific responses to alcohol exposure, we continued our studies in

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the well-characterized HepG2-derived E47 and VA-13 cell lines that stably express either

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CYP2E1 or ADH, respectively (reviewed in (6)). As predicted and as previously shown,

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oxidative stress was observed in ethanol-treated E47 cells (Figure 7A, b) confirming CYP2E1-

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mediated ethanol metabolism was occurring. In contrast, but as also predicted, oxidative stress

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was not observed in VA-13 cells in the absence (Fig. 7B, a) or presence of ethanol (Fig. 7B, b)

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consistent with only ADH expression in these cells. To further confirm our inhibitor studies, we

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also labeled cells for acetylated tubulin. In these cell lines, microtubules display the

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characteristic arrangement found in non-polarized cells emanating from a juxta-nuclear MTOC.

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As predicted, only the VA-13 cells displayed enhanced microtubule acetylation morphologically

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(Fig. 7B, d) and biochemically (Fig. 7C). Although the morphological changes were not as

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dramatic as observed for WIF-B cells, when examined closely, enhanced labeling is observed

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for each cell both with respect to overall intensity and to numbers of labeled filaments (compare

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Figs. 7B, c and d). When quantitated, tubulin acetylation was enhanced ~1.5-fold in ethanol-

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treated VA-13 cells. Although more modest than in WIF-B cells, the increase was reproducible

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and expected as these cells are less differentiated than the fully polarized WIF-B cells. In

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contrast, but also as predicted, no increased tubulin acetylation was observed in E47 cells

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morphologically (compare Figs. 7A, c and d) or biochemically (Fig. 7C).

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To confirm that CYP2E1 ethanol metabolism specifically impairs Jak2 and STAT5B activation,

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we immunoblotted lysates from E47 or VA-13 cells treated with GH for total and phospho-Jak2

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or STAT5B. Importantly, addition of ethanol did not change total levels of Jak2 or STAT5B

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expression in either cell type (Fig. 8A). In control E47 cells, Jak2 was rapidly phosphorylated

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reaching peak activation within 2 min after GH addition returning to basal levels by ~15 min (Fig.

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8B). Ethanol treatment significantly blunted peak Jak2 activation at 2 min to nearly half that

379

seen in control cells (Fig. 8C). In control E47 cells and as expected, STAT5B activation lagged

380

behind that of Jak2 with peak phosphorylation varying between 5 or 15 min that was blunted in

381

ethanol-treated cells (Fig. 8C). Although a more modest impairment was observed in E47 cells

382

than in WIF-B cells, when quantitated, Jak2 phosphorylation was reproducibly decreased to

383

~80% of control (82.0 ± 4.0%, p ≤ 0.004) after 15 min of GH exposure. In control VA-13 cells,

384

Jak2 activation was also observed after GH addition, but was somewhat delayed peaking at 5

385

min (Fig. 8D). No change in Jak2 phosphorylation was observed in ethanol-treated VA-13 cells

386

and the activation kinetics mirrored those observed in control cells (Fig. 8D). Similarly, STAT5B

387

phosphorylation displayed delayed activation in both control and ethanol-treated VA-13 cells,

388

but no apparent difference in activation kinetics was observed (Fig. 8E). Together these results

389

confirm our findings that that the microtubule-independent activation of Jak2 and STAT5B are

390

ultimately impaired by a CYP2E1-mediated mechanism.

391 392

Jak/STAT signaling is blunted and not sustained in ethanol-treated hepatic cells.

393

These results indicate that ethanol metabolism leads to impaired Jak2/STAT5B signaling at two

394

steps: Jak2/STAT5B activation upon GH binding to its receptor and subsequent microtubule-

395

dependent STAT5B nuclear translocation. To determine if these impairments impact

396

hepatocyte-specific GH-mediated signaling, we performed quantitative RT-PCR on 96-well

397

arrays of 84 known Jak/STAT signaling pathway genes and 4 house-keeping genes (for

398

normalization) using cDNA prepared from control or ethanol-treated WIF-B cells. We first

399

assayed for changes in steady state gene expression in ethanol-treated cells relative to control.

400

As shown in Table 2, 29 genes showed reproducibly altered expression in ethanol-treated cells

401

with over 70% (21 of 29) of these genes showing decreased expression (indicated in red)

402

suggesting GH/Jak2/STAT5-mediated signaling is blunted in ethanol-treated cells. Of these

403

down-regulated genes, over 70% (15 of 21) are known STAT5 target genes or code for proteins

404

known to interact with/activate GH-R, Jak2 or STAT5 (2, 12, 13, 17) confirming the validity of

405

our screen. Interestingly, the changes in gene expression associated with the cell cycle are all

406

consistent with decreased cell division. Also, the identification of the many cytokines, cytokine-

407

receptors and other immune response modulators are consistent with known alterations in

408

inflammation associated with alcoholic liver disease.

409 410

We next identified the genes that are differentially expressed in control or ethanol-treated cells

411

after acute (30 min) or prolonged (4 h) GH addition. We first identified those genes with

412

differential expression at both time points relative to 0 min. As shown in Table 3, addition of GH

413

for 30 min led to similar changes in Jak/STAT-mediated gene expression in ethanol and control

414

cells (highlighted in gray). Of the 11 genes identified with altered expression in ethanol-treated

415

cells, almost half shared similar changes as those observed for control cells. However, the GH-

416

mediated response in ethanol-treated cells was not sustained. After 4 h, only 2 genes showed

417

sustained altered expression (Egfr and Ptpn1) in ethanol-treated cells with only one value

418

shared with control cells (Epor). This trend is also apparent when changes in gene expression

419

in ethanol-treated cells are compared with those in control for each time point. As shown in

420

Table 4, GH addition for 30 min transiently restored expression to control levels in the majority

421

of genes (15 of 21) with impaired expression in ethanol-treated cells (Table 4). However, after 4

422

h, expression levels returned to steady state levels (compare values for 0 min and 4 h).

423

Interestingly, 4 genes showed altered expression levels independent of GH addition in ethanol-

424

treated cells (Prlr, Cebpb, Cebpd and Irf9) which may reflect other STAT-specific signaling

425

defects. Nonetheless, these results indicate that overall GH/Jak2/STAT5/-mediated signaling is

426

blunted and not sustained in ethanol-treated cells consistent with the defects in activation and

427

translocation we observed.

428 429 430

DISCUSSION

431

To date, the role of CYP2E1-mediated ethanol metabolites or ROS on microtubule acetylation

432

or on alcohol-induced protein trafficking defects has not yet been examined. To determine if

433

CYP2E1-mediated alcohol metabolism is required for the observed impairments, we took a

434

straight-forward approach. To inhibit CYP2E1-mediated ethanol metabolism, we co-incubated

435

cells with ethanol and DAS, and to decrease ROS production, we co-incubated cells with NAC.

436

Addition of either agent failed to prevent microtubule hyperacetylation or prevent acetylation-

437

dependent defects in protein secretion or clathrin-mediated internalization. However, somewhat

438

surprisingly, both agents prevented the alcohol-induced defect in Jak2/STAT5B activation prior

439

to STAT5B microtubule-dependent translocation. These results were confirmed in HepG2 cells

440

expressing only ADH or only CYP2E1. Importantly, quantitative RT-PCR using Jak/STAT

441

signaling arrays revealed that GH mediated hepatic signaling was blunted and attenuated in

442

ethanol-treated cells such that the hepato-protective effects of GH are decreased thereby

443

contributing to hepatic injury.

444 445

Alcohol-induced microtubule acetylation and associated defects in microtubule-

446

dependent protein trafficking are mediated by acetaldehyde

447

Our earlier studies determined that increased microtubule acetylation and stability displayed

448

both ethanol time- and dose-dependence (18). Furthermore, microtubule hyperacetylation and

449

stability was prevented by 4-MP and potentiated by cyanamide indicating that ADH-mediated

450

ethanol metabolism to acetaldehyde was required (18). Since then, similar studies with 4-MP

451

have also shown that impaired secretion, nuclear translocation and clathrin-mediated

452

internalization are also dependent on ADH-mediated ethanol metabolism (9, 16, 38). The

453

studies reported here confirm those conclusions and further establish that ethanol metabolism

454

by ADH to acetaldehyde only (not via other CYP2E1-generated metabolites) is responsible for

455

enhanced microtubule acetylation and associated defects in protein trafficking.

456 457

How acetaldehyde promotes microtubule acetylation is not completely understood, but a

458

possible answer might come from our studies on the distributions, levels and biochemical

459

properties of histone deacetylase 6 (HDAC6), the major tubulin deacetylase. Although ethanol

460

treatment did not alter HDAC6 subcellular distributions in polarized WIF-B cells, it led to a 25%

461

decrease in its protein levels (36). We also determined that HDAC6 binding to endogenous

462

microtubules was impaired in ethanol-treated cells while its ability to bind or deacetylate

463

exogenous tubulin did not change suggesting that tubulin from ethanol-treated cells was

464

modified thereby preventing HDAC6 binding (36). Because both impaired HDAC6 microtubule

465

binding and tubulin hyperacetylation require ethanol metabolism by ADH (but not CYP2E1) (18,

466

36), and because tubulin can be acetaldehyde-adducted (15, 44), we propose that tubulin-

467

acetaldehyde adducts impede HDAC6-tubulin binding thereby preventing deacetylation.

468 469

Decreased Jak2/STAT5B activation requires CYP2E1-mediated ethanol metabolism ROS

470

Unlike for STAT5B translocation, ethanol metabolism by CYP2E1 is responsible for impaired

471

Jak2/STAT5B activation. The mechanism whereby the CYP2E1 generated metabolites impair

472

signaling is not yet known, but clues may come from earlier studies on GH-mediated signaling in

473

liver slices from animals after acute or chronic ethanol administration (1, 21, 43). Despite

474

decreased levels of GH target hepatic gene expression in slices from treated animals, no

475

changes in GH-R numbers, receptor binding or receptor affinity for GH were observed (1, 21,

476

43). Our studies have further shown that total STAT5B or Jak2 protein levels are not changed

477

by ethanol administration in WIF-B, VL-17A, VA-13 or E47 cells or in livers from ethanol-fed rats

478

(9). Thus, blunted gene expression results from impairments in the signal transduction pathway

479

itself, which is fully consistent with our findings that decreased Jak2/STAT5B activation and

480

STA5B nuclear translocation is observed in ethanol-treated cells. These results further suggest

481

that accumulated CYP2E1-generated reactive ethanol metabolites form detrimental adducts on

482

critical residues that alter Jak2 tyrosine kinase activity directly or disrupt Jak2/STAT5B

483

recruitment to GH-R upon ligand binding. Future studies are needed to sort out these and other

484

details.

485 486

Hepatocyte-specific responses to GH-mediated signaling are blunted and attenuated.

487

The primary target organ for GH-mediated signaling is the liver. In general, GH binds its

488

cognate receptor, GH-R, and signaling is mediated by sequential phosphorylation/activation of

489

the tyrosine kinase, Jak2, then the transcription factor, STAT5A/B. Phosphorylated STAT5A/B

490

translocates to the nucleus and binds gamma-interferon-activation sites (GAS) sites thereby

491

promoting expression of specific targets. Because STAT5B protein is present at levels 20-fold

492

greater than STAT5A in liver, the majority of the hepatocyte target genes are mediated by

493

STAT5B signaling (13). The STAT5 target genes regulate a host of hepatic responses including

494

cell growth, cell cycle progression and the metabolism of lipids, steroids, bile acids and drugs

495

(2). Thus it comes as no surprise that defects in STAT5B signaling have been associated with a

496

number of liver diseases including hepatocellular carcinoma, steatosis and fibrosis (2).

497 498

As expected, many of the genes with altered expression in ethanol treated cells at steady state

499

are known STAT5B targets (Prlr, Socs2, Osm, Ifnar, Irf1, Irf9 and Cdkn1) (2, 12, 13, 17). We

500

also noticed that a number of the down-regulated genes encode proteins that interact with the

501

GH-R/Jak2/STAT5 signaling machinery (Csfr, Epor and Egfr), are known tyrosine-binding

502

adaptor proteins (Grb2, CRK and SH2B) or coordinate with STAT5B to regulate gene

503

expression (Nr3c1, Cebpb and Cebpd) (2, 12, 13, 17). Whether these represent direct STAT5B

504

target genes is not yet known, but their decreased expression is consistent with the observed

505

decrease in GH-mediated signaling. Interestingly, one of the few upregulated genes identified

506

at steady state is Ptpn1, a known tyrosine phosphatase. This finding is also consistent with

507

decreased Jak2/STAT5 phosphorylation in ethanol-treated cells. Although we have previously

508

confirmed decreased EGF-R protein levels in livers from ethanol-fed rats (9), our efforts are now

509

aimed at confirming other targets at the protein level and examine their possible roles in

510

impaired GH-mediated signaling.

511 512

Financial support: This work was supported by the National Institute of Alcohol Abuse and

513

Alcoholism grants R01 AA17626 awarded to PLT

514 515

Acknowledgements: We thank Dr. Ann Hubbard (Johns Hopkins University School of

516

Medicine) for providing for the anti-albumin and -ASGP-R antibodies used in these studies. We

517

also thank Dr. Dahn Clemens (University of Nebraska Medical School) for providing the E47

518

and VA-13 cells.

519 520

Disclosures: The authors have nothing to disclose.

521 522

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32. Schaffert CS, Todero SL, Casey CA, Thiele GM, Sorrell MF, and Tuma DJ. Chronic

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33. Schaffert CS, Todero SL, McVicker BL, Tuma PL, Sorrell MF, and Tuma DJ. WIF-B

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34. Shanks MR, Cassio D, Lecoq O, and Hubbard AL. An improved polarized rat hepatoma

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37. Shepard BD, Tuma DJ, and Tuma PL. Chronic Ethanol Consumption Induces Global

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Hepatic Protein Hyperacetylation. Alcohol Clin Exp Res 34, 2010. 38. Shepard BD, Tuma DJ, and Tuma PL. Lysine acetylation induced by chronic ethanol

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39. Tuma DJ and Casey CA. Dangerous byproducts of alcohol breakdown--focus on adducts. Alcohol Res Health 27: 285-290, 2003.

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40. Webb WR. Clinical evaluaton of a new mucolytic agent, acetyl-cysteine. J Thorac Cardiovasc Surg 44: 330-343, 1962. 41. Wehr H, Rodo M, Lieber CS, and Baraona E. Acetaldehyde adducts and autoantibodies against VLDL and LDL in alcoholics. J Lipid Res 34: 1237-1244, 1993. 42. Westermann S and Weber K. Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol 4: 938-947, 2003. 43. Xu X, Ingram RL, and Sonntag WE. Ethanol suppresses growth hormone-mediated cellular responses in liver slices. Alcohol Clin Exp Res 19: 1246-1251, 1995. 44. Yoon Y, Torok N, Krueger E, Oswald B, and McNiven MA. Ethanol-induced alterations of the microtubule cytoskeleton in hepatocytes. Am J Physiol 274: G757-766, 1998. 45. Ziment I. Acetylcysteine: a drug that is much more than a mucokinetic. Biomed Pharmacother 42: 513-519, 1988.

640 641 642 643 644

FIGURE LEGENDS

645

acetylation. A and B, WIF-B cells were treated in the absence (a) or presence of 50 mM

646

ethanol (EtOH) for 72 h (b-d) in the additional presence of 100 M DAS (c) or 5 mM NAC (d) as

647

indicated. In A, cells were labeled for total -tubulin and in B, for acetylated -tubulin.

648

Asterisks indicate selected bile canaliculi. Arrows in B (b-d) indicate the enhanced acetylated

649

microtubule labeling present in long filaments emanating from the bile canaliculi. Bar, 10 m.

650

C, WIF-B cells were treated in the absence (C) or presence of 50 mM ethanol (E) in the

651

additional presence of 100 M DAS or 5 mM NAC as indicated. Cells were immunoblotted for

652

acetylated or total -tubulin as indicated. The fold-increase in tubulin acetylation was

653

determined by densitometric analysis of immunoreactive bands normalized to total tubulin

654

levels. The untreated control values were set to 1.0. Values represent the mean ± SEM from at

655

least three independent experiments. D, WIF-B cells were treated in the absence (a) or

656

presence of 50 mM ethanol (EtOH) (b) for 72 h. Ethanol-treated cells were co-incubated with

657

100 M DAS (c) or 5 mM NAC (d) and labeled with CellROX® Green reagent to monitor

658

oxidative stress by epifluorescence. Only ethanol-exposed cells (b) exhibited ROS.

Figure 1. Addition of DAS or NAC does not prevent ethanol-induced microtubule

659 660

Figure 2. Addition of DAS or NAC does not prevent the ethanol-induced impairment of

661

secretion. A, WIF-B cells were treated in the absence or presence of 50 mM ethanol (EtOH)

662

for 72 h as indicated. In B, cells treated with 100 M DAS were treated in the absence or

663

presence of ethanol, and in C, cells treated with 5 mM NAC were incubated in the absence or

664

presence of ethanol. Cells were rinsed five times with prewarmed serum-free medium and then

665

reincubated in serum-free medium. At 0, 5, 15 and 30 min after reincubation, aliquots of media

666

were collected and analyzed for albumin secretion by immunoblotting (panels on the left). The

667

percent albumin secreted was calculated from densitometric analysis of immunoreactive bands

668

and plotted in the graphs shown on the right. Values are expressed as the mean ± SEM from

669

three independent experiments.

670 671

Figure 3. Addition of DAS or NAC does not prevent the ethanol-induced impairment of

672

clathrin-mediated endocytosis. WIF-B cells were treated in the absence (a) or presence of

673

50 mM ethanol (EtOH) for 72 h (b-d) in the additional presence of 100 M DAS (c) or 5 mM

674

NAC (d) as indicated. Cells were labeled for ASGP-R. Asterisks indicate selected bile

675

canaliculi. Arrows in panels b-d indicate the enhanced ASGP-R basolateral staining observed

676

in treated cells. Bar, 10 m.

677 678

Figure 4. Addition of DAS or NAC prevents the ethanol-induced impairment of STAT5B

679

nuclear translocation. A, WIF-B cells were treated in the absence (a and b) or presence of 50

680

mM ethanol (EtOH) for 72 h (c – h) in the additional presence of 100 mM DAS (e and f) or 5 mM

681

NAC (g and h) as indicated. Cells were incubated for 0 or 5 min with GH and labeled for total

682

STAT5B. Note the enhanced cytosolic staining in cells treated only with ethanol (c and d). Bar,

683

10 m. B, WIF-B cells were treated in the absence (C) or presence of 50 mM ethanol (E) in the

684

additional presence of 100 M DAS or 5 mM NAC as indicated. The ratio of nuclear-to-

685

cytoplasmic fluorescence intensities was calculated for STAT5B at steady state (0 min; left hand

686

panel) or after 5 min of GH addition (right). Values represent the mean ± SEM from at least

687

three independent experiments.

688 689

Figure 5. Addition of DAS or NAC prevents the ethanol-induced impairment of Jak2

690

phosphorylation. A, WIF-B cells were treated in the absence or presence of 50 mM ethanol in

691

the additional presence of 100 M DAS or 5 mM NAC as indicated. Samples were

692

immunoblotted for total Jak2 expression. B, WIF-B cells were treated in the absence or

693

presence of 50 mM ethanol (EtOH) for 72 h as indicated. In C, cells treated with 100 M DAS

694

were treated in the absence or presence of ethanol, and in D, cells treated with 5 mM NAC were

695

incubated in the absence or presence of ethanol. Cells were incubated for up to 15 min with GH

696

and lysates immunoblotted for phospho-Jak2 (pJak2) (panels on the left). Immunoreactivity was

697

measured by densitometry, normalized to total Jak2 levels and plotted as the percent of

698

maximal phosphorylation (panels on the right). Values represent the mean ± SEM from three

699

independent experiments.

700 701

Figure 6. Addition of DAS or NAC prevents the ethanol-induced impairment of STAT5B

702

phosphorylation. A, WIF-B cells were treated in the absence or presence of 50 mM ethanol in

703

the additional presence of 100 M DAS or 5 mM NAC as indicated. Samples were

704

immunoblotted for total STAT5B expression. B, WIF-B cells were treated in the absence or

705

presence of 50 mM ethanol (EtOH) for 72 h as indicated. In C, cells treated with 100 M DAS

706

were treated in the absence or presence of ethanol, and in D, cells treated with 5 mM NAC were

707

incubated in the absence or presence of ethanol. Cells were incubated for up to 15 min with GH

708

and lysates immunoblotted for phospho-STAT5B (pSTAT5B) (panels on the left).

709

Immunoreactivity was measured by densitometry, normalized to total STAT5B levels and plotted

710

as the percent of maximal phosphorylation (panels on the right). Results from a representative

711

experiment from at least three independent experiments are shown for each.

712 713

Figure 7. Only CYP2E1-expressing cells display ethanol-induced ROS while only ADH-

714

expressing cells display alcohol-induced microtubule acetylation. A, E47 or B, VA-13

715

cells were treated in the absence (a and c) or presence of 50 mM ethanol (b and d) for 24 h.

716

Cells were labeled for CellROX® Green to monitor oxidative stress (a-b) or immunolabeled for

717

acetylated tubulin (c-d). C, Cells were immunoblotted for acetylated or total -tubulin as

718

indicated. The fold-increase in tubulin acetylation was determined by densitometric analysis of

719

immunoreactive bands normalized to total tubulin levels. The untreated control values were set

720

to 1.0. Values represent the mean ± SEM from at least three independent experiments.

721 722

Figure 8. Only CYP2E1-expressing cells display ethanol-induced impairments in Jak2

723

and STAT5B activation. A, E47 or VA-13 cells were treated in the absence or presence of 50

724

mM ethanol for 24 h. Cells were incubated for up to 15 min with GH and lysates immunoblotted

725

for total Jak2 and STAT5B as indicated. A representative immunoblot from at least three

726

independent experiments is shown for each. B and C, E47 or D and E, VA-13 cells were

727

treated in the absence or presence of 50 mM ethanol (EtOH) for 24 h as indicated. Cells were

728

incubated for up to 15 min with GH and lysates immunoblotted for phospho-Jak2 (A and D) or

729

phospho-STAT5B as indicated C and E). Immunoreactivity was measured by densitometry,

730

values normalized to total Jak2 or STAT5B levels and maximum values set to 100%. Values

731

represent the mean ± SEM from at least three independent experiments in B, D and E. Due to

732

variability in peak activation time, one representative immunoblot and graph out of four

733

independent experiments are shown in C.

734 735 736 737 738 739

Table 1. Addition of DAS or NAC prevents the ethanol-induced impairment of pSTAT5B nuclear translocation.

nuclear/cytoplasmic Treatment

fluorescence intensity (% of control)

control

100.0 ± 0.0

DAS

96.0 ± 3.6

NAC

103.7 ± 8.2

EtOH

76.3 ± 1.3

EtOH + DAS

111.0 ± 16.5

EtOH + NAC

98.0 ± 6.1

WIF-B cells were treated in the absence or presence of 50 mM ethanol (EtOH) for 72 h in the additional presence of 100 PM DAS or 5 mM NAC as indicated. Cells were incubated for 5 min with GH and labeled for phospho-STAT5B (pSTAT5B). The ratio of nuclear-to-cytoplasmic fluorescence intensities was calculated for pSTAT5B and the untreated control value set to 100%. Values represent the mean ± SEM from at least three independent experiments.

Table 2. Steady state Jak/STAT signaling is blunted in ethanol-treated cells Gene n Fold-change Jak kinases Jak1 3 -1.50 ± 0.09 Jak2 3 -1.57 ± 0.19 Jak3 3 -1.40 ± 0.18 Tyk2 2 -1.57 STATs Stat3 3 -1.42 ± 0.25 Stat5 3 -1.72 ± 0.35 Jak/STAT Receptors Csfr1 3 1.80 ± 0.53 Epor 3 5.44 ± 2.92 Prlr

3

-2.93 ± 0.91

Nr3c1

2

-1.60

Comments

Activated by growth hormone

Activated by growth hormone Colony stimulating factor receptor; ligand binding activates Jak2/STAT5 Erythropoietin-receptor; hepatoprotective; ligand binding activates Jak2/STAT5 Prolactin-receptor; ligand binding activates Jak2/STAT5; can be activated by GH; known STAT5 target gene Glucocorticoid-receptor; co-activator of GH-mediated responses via STAT5 binding

Jak/STAT Modulators Ptpn1 2 1.49 Protein tyrosine phosphatase; can interact with GH-R, STAT5 and Jak2 Socs2 2 -1.47 STAT signaling inhibitor molecule; known STAT5 target gene Cytokines, cytokine receptors and other secreted proteins Crp 2 2.39 C-reactive protein; made in liver is response to inflammation; acute phase response to IL6 Cxcl9 3 2.22 ± 0.64 Chemokine (CXC motif) ligand 9; T-cell attractant induced by IFNJ A2M 3 -2.58 ± 0.67 D2 macroglobulin; inactivates circulating proteases Il20 3 -2.58 ± 0.69 Interleukin 20 Osm 2 -1.89 Oncostatin M; inhibits proliferation and production of other cytokines; known STAT5 target Ifnar 2 -1.50 Interferon D-receptor; known pan-STAT target Il10ra 3 -4.74 ± 2.88 Interleukin 10 receptor A Other immune response modulators Isg15 2 1.84 Interferon-induced 15-kDa Protein; Ub-like protein that attaches to lysine B2M 2 1.52 E2 microglobulin; MHC class 1 component Cebpb 3 -1.53 ± 0.25 CCAAT binding protein E transcription factor; promotes cytokine expression; can interact with STAT5 in liver Cebpd 2 -1.79 CCAAT binding protein G transcription factor; promotes cytokine expression; can interact with STAT5 in liver Irf1 3 -1.48 ± 0.17 Inteferon regulatory factor 1; activates interferon transcription; known STAT5 target gene Irf9 3 -1.69 ± 0.21 Interferon regulatory factor 9; activates interferon transcription; known pan-STAT target Nfkb 3 -1.62 ± 0.23 NFNB; pro-inflammatory transcription factor Cell cycle regulators Cdkn1 2 1.71 Cyclin dependent kinase Inhibitor 1A/p21; blocks cell cycle progression; known STAT5 target Akt 2 -1.70 Akt/protein kinase B; ser/thr kinase; stimulates proliferation Myc 3 -1.45 ± 0.22 Transcription factor that promotes cell cycle entry

Jak/STAT signaling pathway PCR arrays were probed with cDNAs prepared from WIF-B cells treated in the presence or absence of ethanol (see Materials and Methods). The fold-change in gene expression for the 84 genes on the array in ethanol-treated cells relative to control was determined. The values in blue represent the fold-change in genes with increased expression in ethanol-treated cells while the values in red indicate those with decreased expression. Three independent experiments were performed. Values represent the mean ± SEM from three independent experiments (n = 3) or the average from two (n = 2) as indicated. Brief descriptions of gene functions are listed. Known associations between the gene and GH/STAT5-mediated signaling are also provided.

Table 3. Long-term GH-induced Jak/STAT signaling is attenuated in ethanol-treated cells 30 min Control Gene n Fold-Change A2M -1.72 Cdkn1 2 2.82 ± 0.61 Csfr1 3 2.75 Fas 2 Il2ra -1.85 Mcl1a 2 2.06 Socs3 2 Socs5 -Egfrb --2.76 ± 1.04 Gbp1c 3 -3.20 Il10ra 2 -5.5 Il20 2 -3.41 Il2rg 2 Pias1d -Ptpn1 --

30 min Ethanol n Fold-Change 3 1.49 ± 0.30 2 1.74 3 1.93 ± 0.47 -2 2.17 -2 1.97 2 2.19 3 -1.48 ± 0.41 3 1.74 ± 0.36 2 --2 -3.72 3 -1.57 ± 0.29 3 -1.48 ± 0.39

4 h Control Gene n Cdkn1 3 Csfr1 3 Irf1 2 Junb 2 Egfr Epor 2 Il10ra 2 Myc 2 Ptpn1 Spi1e

4 h Ethanol n Fold-Change no sustained changes in up-regulated genes 2 -2.06 3 -2.82 ± 0.46 --2 -2.13 2 -1.73

Fold-Change 2.24 ± 0.14 3.41 ± 1.06 1.7 2.12 --3.10 -4.33 -1.98 ---

Jak/STAT signaling pathway PCR arrays were probed with cDNAs prepared from control or ethanol-treated WIF-B cells after 0, 30 or 240 min of additional treatment with GH (see Materials and Methods). The fold-change in gene expression for the 84 genes on the array in cells treated for 0 min relative to those treated for 30 min (top table) or 4 h (bottom table) was determined for both control and ethanol-treated cells. The values in blue represent the foldchange in genes with increased expression in GH-treated cells while the values in red indicate those with decreased expression. Three independent experiments were performed. Values represent the mean ± SEM from three independent experiments (n = 3) or the average from two (n = 2) as indicated. The shaded rows highlight gene expression changes shared in both control and ethanol-treated cells. aMcl1, myeloid cell leukemia 1 (a BH-3 family member antiapoptotic protein); bEgfr, epidermal growth factor receptor (a known STAT5 target); cGbp1, interferon-induced guanylate binding protein; dPias1, protein inhibitor of activated STAT; eSpi1, Spi-1 proto-oncogene

Table 4. GH transiently restores gene expression in ethanol-treated cells 0 min 30 min 240 min Gene n Fold-change n Fold-change n Fold-change Jak kinases Jak1 3 -1.50 ± 0.09 Jak2 3 -1.57 ± 0.19 3 -1.82 ± 0.45 Jak3 3 -1.40 ± 0.18 3 -1.48 ± 0.29 Tyk2 2 -1.57 2 -2.00 Usf1 3 -1.84 ± 0.26 STATs Stat3 3 -1.42 ± 0.25 Stat5a 3 -1.72 ± 0.35 Jak/STAT receptors Egfr 3 -1.79 ± 0.06 3 -1.90 ± 0.43 3 -1.82 ± 0.28 Ghra Prlr 3 -2.93 ± 0.91 3 -1.59 ± 0.29 3 -2.98 ± 0.58 Nr3c1 2 -1.60 Jak/STAT modulators Socs2 2 -1.47 Socs3 3 -1.83 ± 0.22 Signaling adaptor proteins 3 -1.65 ± 0.36 Grb2b c 2 -1.95 Sh2b1 3 -1.67 ± 0.43 Crkd Cytokines, cytokine receptors and other secreted proteins A2M 3 -2.58 ± 0.67 3 -2.21 ± 0.31 Ifng 3 -1.47 ± 0.17 Il20 3 -2.58 ± 0.69 3 -3.21 ± 0.50 Osm 2 -1.89 Ifnar 2 -1.50 Il2rg 2 -4.45 2 -3.02 Il10ra 3 -4.74 ± 2.88 Other immune modulators Cebpb 3 -1.53 ± 0.25 2 -2.18 2 -1.80 Cebpd 2 -1.79 3 -2.11 ± 0.27 2 -3.72 Gbp1 3 -2.40 ± 0.78 Irf1 3 -1.48 ± 0.17 2 -1.77 Irf9 3 -1.69 ± 0.21 3 -1.54 ± 0.24 3 -1.56 ± 0.25 Nfkb 3 -1.62 ± 0.23 Cell cycle regulators Akt 2 -1.70 Junb 2 -2.85 Myc 3 -1.45 ± 0.22 Src 3 -1.91 ± 0.43

Jak/STAT signaling pathway PCR arrays were probed with cDNAs prepared from control or ethanol-treated WIF-B cells after 0, 30 or 240 min of additional treatment with GH (see Materials and Methods). The fold-change in gene expression in ethanol-treated cells relative to control for each time point was determined. Only genes showing decreased expression levels are shown. Three independent experiments were performed. Values represent the mean ± SEM from three independent experiments (n = 3) or the average from two (n = 2) as indicated. The rows shaded in gray highlight changes in gene expression that are independent of GH addition. aGhr, growth hormone receptor (known Jak2/STAT5 receptor); bGrb2, Growth factor receptor-bound protein 2 (a tyr kinase signaling adaptor protein); cSH2B, SH2 domain-containing protein 2B (a known Jak2/GH-R adaptor protein); dCRK, CRK proto-oncogene (a tyr kinase signaling adaptor protein)

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, b...
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