Research Article

TGF-b enhances alcohol dependent hepatocyte damage viadown-regulation of alcohol dehydrogenase I

Loredana Ciuclan1, Sabrina Ehnert2, Iryna Ilkavets1, Hong-Lei Weng1, Haristi Gaitantzi1,Hidekazu Tsukamoto3, Elke Ueberham4, Nadja M. Meindl-Beinker1, Manfred V. Singer1,

Katja Breitkopf1,�, Steven Dooley1,*,�

1Molecular Hepatology – Alcohol Dependent Diseases, II. Medical Clinic, Faculty of Medicine at Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany; 2Department of Traumatology, TH Munich, Klinikum rechts der Isar, Munich, Germany;

3Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 4Department of Neuroanatomy,Paul-Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany

Background & Aims: Adverse alcohol effects in the liver involve Conclusion: In the presence of ethanol, TGF-b displays pro-stea-

oxidative metabolism, fat deposition and release of fibrogenicmediators, including TGF-b. The work presents an assessmentof liver damaging cross-talk between ethanol and TGF-b inhepatocytes.Methods: To investigate TGF-b effects on hepatocytes, micro-array analyses were performed and validated by qRT-PCR, Wes-tern blot analysis and immunohistochemistry. The cellular statewas determined by assessing lactate dehydrogenase, cellular glu-tathione, reactive oxygen species, lipid peroxidation and neutrallipid deposition. RNA interference was used for gene silencingin vitro.Results: TGF-b is induced in mouse livers after chronic ethanolinsult, enhances ethanol induced oxidative stress and toxicitytowards cultured hepatocytes plus induces lipid-, oxidative stressmetabolism- and fibrogenesis-gene expression signatures. Inter-estingly, TGF-b down-regulates alcohol metabolizing enzymeAdh1 mRNA in cultured hepatocytes and liver tissue from TGF-b transgenic mice via the ALK5/Smad2/3 signalling branch, withSmad7 as a potent negative regulator. ADH1 deficiency is a deter-mining factor for the increased lipid accumulation and Cyp2E1dependent toxicity in liver cells upon alcohol challenge. Further,ADH1 expression was decreased during liver damage in an intra-gastric ethanol infusion mouse model.

Journal of Hepatology 20

Keywords: TGF-b; ALKs; Smad7; Hepatocytes; Liver fibrosis; Alcohol metabolism;Steatosis; Mouse model.Received 9 July 2009; received in revised form 29 September 2009; accepted 30September 2009; available online 6 January 2010* Corresponding author. Tel.: +49 621 3833768; fax: +49 621 3831467.E-mail address: steven.dooley@medma.uni-heidelberg.de (S. Dooley).

� These authors contributed equally to this work.Abbreviations: ALD, alcoholic liver disease; CLD, chronic liver disease; TGF-b1,transforming growth factor-b1; 4-MP, 4-methylpyrazole; HSC, hepatic stellatecell; ECM, extracellular matrix; ADH, alcohol dehydrogenase; MEOS, microsomalethanol oxidizing system; FAEE, fatty acid ethyl esters; ALDH, aldehydehydro-genase; LDH, lactate dehydrogenase; ROS, reactive oxygen species; PCR, poly-merase chain reaction; AOX, aldehyde oxidase; ALK, activin like kinase; Smad,small mother against decapentaplegic; R-Smad, receptor Smad; Cyp2E1, cyto-chrome P450 2E1; CCl4, carbon tetrachloride.

totic action in hepatocytes via decreasing ADH1 expression. LowADH1 levels are correlated with enhanced hepatocyte damageupon chronic alcohol consumption by favoring secondary meta-bolic pathways.� 2009 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Alcohol abuse can lead to alcoholic cirrhosis and end stage liverdisease [1]. Multiple factors mediate toxic effects of alcohol andthe development of alcoholic liver disease (ALD) [2]. TGF-b isthe major fibrogenic cytokine that is elevated during chronic liverdisease (CLD) progression, including ALD [3]. In fibrogenic stagesof ALD, TGF-b accounts for the activation of hepatic stellate cells(HSC) and excessive extracellular matrix (ECM) production [4]. Inaddition, TGF-b was shown to mediate hepatocyte plasticity andmesenchymal transition, thus contributing to (myo)fibroblastpopulations [5]. There is currently a lack of knowledge aboutTGF-b effects in early stages of alcohol dependent liver damage.Signal transducers of TGF-b are the Smads. Upon ligand bindingto cell surface receptors, receptor (R-)Smads are phosphorylated,associate with Smad4, translocate into the nucleus and regulatetarget gene expression. R-Smad signalling is limited by inhibitorySmad7 [6].

Alcohol is metabolized by oxidative and non-oxidative path-ways. The oxidative pathway comprises alcohol dehydrogenases(ADH) and members of the microsomal ethanol oxidizing system(MEOS) as cytochrome P450. The non-oxidative pathway involvesesterification of ethanol to fatty acid ethyl esters (FAEE) [7]. Mam-malian ADHs constitute a complex family of dimeric, zinc-depen-dent enzymes involved in metabolism of alcohol, aldehydes, lipidperoxidation products or steroids. Known adverse products fromethanol metabolism providing hepatocellular toxicity are acetal-dehyde, reactive oxygen species and FAEEs [8]. A cross-talk of cyto-kine signalling pathways with alcohol effects in the liver is widelyunknown. Expression of the ADH1 gene is induced by growth

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Research Article

hormone, insulin-like growth factor I, glucagon and cyclic AMP [9].Several transcriptional regulatory units, including members of theC/EBP family, were identified in its promoter [10].

Surprisingly, hepatic ADH1 and ALDH (aldehyde dehydroge-nase) are inhibited during chronic alcohol abuse in experimentalanimals [11,12], resulting in an increased susceptibility for thedamaging effects of alcohol.

In the present report, we show that TGF-b signalling isinduced in hepatocytes during the early stages of alcohol inducedliver injury, down-regulates ADH1 and enhances cell damage.

Materials and methods

Further details are provided as Supplementary Materials and Methods.

Cell preparation and treatment

In vitro studies were performed with mouse hepatocytes from male C57/BL-6mice [5]. Recombinant TGF-b1 (1–10 ng/ml) and ethanol (100 mM) were addedto serum free culture medium. Alcohol treated plates were sealed with parafilmto prevent evaporation.

Immunoblotting

Western blot analyses were performed as described [13,14]. Antibodies are listedin Supplementary Table 2.

Microarray analysis

Affymetrix (Santa Clara, CA, USA) cDNA microarray analyses were performedwith RNA from three independent hepatocyte preparations stimulated or notfor 1, 6 and 24 h with 5 ng/ml TGF-b [5].

Real-time PCR

One microgram of total RNA was reverse transcribed using Transcriptor FirstStrand cDNA synthesis kit (Roche, Mannheim, Germany). Real-time qRT-PCRwas performed on the Sequence Detection System ABI Prism 7700 (Applied Bio-systems) using TaqMan Universal PCR Master Mix, No AmpErase UNG (Part No.4324018). Levels of gene expression were determined with the comparative DDCt

method using the following assays: ADH1 (No. Mm00507711_m1) and PPIA(house keeping gene; No. Mm02342429g1). Samples were run in triplicate withthe following conditions: initial denaturation, 2 min at 50 �C, 10 min at 95 �C,40 cycles at 15 s at 95 �C and 1 min 60 �C.

Knock-down of ADH1

Adh1-specific siRNAs (Mm_ADH1_2_HPsiRNA; SI00166019) and silencer negativecontrol (AllStars siRNA; 1027287) were purchased from Qiagen. Briefly, cells wereseeded at a density of 1 � 105/cm2 in 24-well plates. Chemically synthesized siR-NAs (10 nmol) were transfected with Lipofectamine™ RNAiMAX (No. 13778-075,Invitrogen) according to the manufacturer’s instructions. Twenty-four hours aftertransfection, the medium was changed and TGF-b was added. Changes in proteinexpression were analysed, 72 h post-transfection with Adh1-specific siRNAs(Fig. 6A).

Alcohol dehydrogenase activity

ADH1 activity was measured as described [12]. Briefly, hepatocytes were culturedat a density of 1 � 105/cm2 in 24-well plates and incubated up to 48 h with 5 ng/ml TGF-b, 0.5 mM Aspirin and 0.5 mM 4-MP (4-methylpyrazole). At the indicatedtime points, the medium was removed and cells were lysed with 1% Triton X-100.ADH activity was measured in the supernatant at 340 nm in 0.4 M Tris buffer (pH7.4) containing 3 mM NAD, 10 mM ethanol and 0.1 mM DTT using a GENios spec-trophotometer (Tecan, Crailsheim, Germany). One enzyme unit catalyses forma-tion of 1 nmol NADH/h and is normalized to protein concentration. Activity isexpressed as units/mg protein.

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Animals

A double-transgenic mouse with doxycycline hydrochloride inducible TGF-bexpression was used to investigate TGF-b dependent Adh1 expression in vivo.In induced mice, TGF-b plasma levels reach values ranging from 250 to1200 ng/ml, being 10–30 times above normal [15]. C57/BL-6 mice intoxicatedfor 6 weeks with ethanol (intragastric feeding) were analyzed: the alcohol dosein these mice peaks at about 25 g/kg/day, and circulating levels reach a daily aver-age of 150 mg/dl [16,17].

ROS production, glutathione depletion, cytochrome P450 activity and lipidperoxidation were investigated as described (Supplementary Material) [18,19].

Immunohistochemistry

Four micrometer sections from paraffin embedded mouse liver was used forimmunohistochemistry [6]. Quantification of histological stainings was per-formed by morphometric analysis with LEICA QWIN software (Solms, Germany)or by calculating the relative number of positive cells. Each analysis was donewith four animals at least. Presented values are mean of 10 fields (magnification,200�) taken from five liver sections per mouse.

Statistics

Statistical significance was determined with Student’s t test for paired data; a pvalue of less than 0.05 was considered significant. In vivo results are expressedas means ± standard error of the mean (SEM) and were analyzed either byMann–Whitney test or by analysis of variance (ANOVA) followed by paired com-parison, when appropriate. All presented data are representative for three differ-ent preparations, investigated in triplicates each.

Results

TGF-b1 signalling is induced in alcoholic liver disease (ALD)

Expression of TGF-b isoforms is increased in liver of ALD patientsand in rodent models of alcohol dependent steatohepatitis [3,20].We assessed R-Smad phosphorylation in liver sections of micechallenged with intragastric ethanol feeding for 6 weeks, whichnormally leads to serious steatosis, focal necrosis and mononu-clear inflammation, but not HSC activation and fibrosis [21]. Wefound that most hepatocytes were immunopositive for nuclearphospho-Smad2, whereas only a few were weakly stained inhealthy controls (Fig. 1A).

TGF-b enhances ethanol induced toxicity and oxidative stress inmouse hepatocytes

Alcohol metabolism, cytokine signalling and oxidative stresscontribute to alcohol-mediated liver injury [22]. Cultured mousehepatocytes are TGF-b sensitive, as determined by R-Smadphosphorylation (Fig. 1B). Challenging hepatocytes with100 mM ethanol is cytotoxic and induces apoptosis, as assessedby Annexin-V staining. Combined treatment with ethanol andTGF-b further increased the number of dead hepatocytes indi-cating synergistic downstream effects. (Fig. 1C and Supplemen-tary Fig. 1). TGF-b also enhances LDH secretion and lipidperoxidation 48 h after ethanol treatment (Fig. 2A, B and Sup-plementary Fig. 2A, B). A 24 h incubation of hepatocytes withTGF-b and ethanol dramatically increased intracellular ROS pro-duction and glutathione depletion, compared to single treat-ments and controls (Fig. 2C and D). These results support arole of TGF-b as an accelerator for adverse ethanol effectstowards hepatocytes.

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Fig. 1. TGF-b signalling is induced in ALD. (A) Representative phospho-Smad2 IHC (brown nuclear staining; 200�) in liver tissue of ethanol-fed mice and controls. Forquantification, positive cells were counted (five observation fields from four different animals, **p <0.01) (B) Immunoblot analysis of Smad1/2/3 phosphorylation in mousehepatocytes, stimulated or not with TGF-b (5 ng/ml). Total Smad2 and b-actin served as loading controls. (C) Annexin-V/Cy3 staining of hepatocytes for apoptosis detection,48 h after stimulation with 5 ng/ml TGF-b and/or 100 mM EtOH (Supplementary Fig. 1).

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TGF-b decreases ADH1 expression in hepatocytes

To determine the mechanistic link of TGF-b and ethanol duringhepatocyte damage, a TGF-b dependent microarray expressionprofile (Affymetrix) was established from mouse hepatocytes(http://www.ebi.ac.uk/arrayexpress/) [5]. Most genes were sig-nificantly regulated at intermediate and late time points (6 and24 h). These identified TGF-b target genes were also validatedby real-time PCR [5]. TGF-b modulates expression of genesinvolved in a variety of biological processes, including alcohol-,lipid- and glucose metabolism as well as oxidative stress (Fig. 3and Supplementary Fig. 3). Interestingly, enzymes directly partic-ipating in alcohol metabolism were down-regulated by TGF-b, i.e.Adh1, aldehyde dehydrogenases (Aldh1a7) and aldehyde oxidase3 (Aox3). Our attention was especially attracted by Adh1 becauseit represents one of the most highly regulated TGF-b target genesin the array and the damaging effects of ethanol on hepatocyteshave previously been reported to be related to reduced ADH1expression [24,25]. Hepatocytes constantly express significantlevels of ADH1 at least until day four in primary culture. (Supple-

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mentary Fig. 4) [23,24]. TGF-b decreases ADH1 expression inmouse and human hepatocytes as measured by semi-quantita-tive real-time PCR (Fig. 4A and Supplementary Fig. 4B). In adose-dependent response, Adh1 mRNA expression was decreasedup to 70%. In line with this, TGF-b reduced enzymatic activity andprotein levels of ADH1 after 24 h of stimulation (Fig. 4B and C). Toconfirm these findings in vivo, ADH1 expression was examined intransgenic mice inducibly over-expressing active TGF-b, and thatdisplay plasma TGF-b levels about 10 times above controls (Sup-plementary Table 4). Consistent with in vitro data, TGF-b express-ing mice exhibit significantly decreased Adh1 mRNA compared tocontrols (Fig. 4D and Supplementary Table 3).

TGF-b down-regulates ADH1 expression in hepatocytes viaactivation of the ALK5 pathway

TGF-b basically signals via two different type I receptor/Smadpathways: ALK1/Smads1, 5, 8 and ALK5/Smads2, 3 [25]. To delin-eate the signalling pathway regulating ADH1, we inhibited theALK5 pathway using SB431542. Accordingly ALK1 expression

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Fig. 2. TGF-b enhances ethanol induced toxicity, oxidative stress and anti-oxidant depression in mouse hepatocytes. Cellular damage after TGF-b and/or alcoholtreatment was determined by (A) percentage of LDH released into the culture medium in relation to total LDH, (B) lipid peroxidation, (C) glutathione (GSH) levels and (D)intracellular oxidative stress (ROS) after 24–72h of TGF-b and/or ethanol treatment; (**p <0.01, *p <0.05 vs control).

Research Article

was knocked-down by adenovirus delivered siRNA prior to TGF-btreatment. SB431542 treatment reduced phosphorylation ofSmad2, but also phosphorylation of Smad1, indicating thatALK5 activity is required for TGF-b dependent ALK1 activation(Fig. 5A). SB431542-dependent inhibition of ALK5 led to a rescueof the TGF-b mediated down-regulation of ADH1 mRNA and pro-tein expression, whereas efficiently knocking-down ALK1, mea-sured as reduced Smad1 phosphorylation, had no effect.(Fig. 5A and B). This indicates that ALK5 and not ALK1, mediatesTGF-b dependent inhibition of ADH1 expression. This assumptionwas confirmed by expression of constitutively active (ca) recep-tors in hepatocytes using adenoviral infection with AdcaALK5or AdcaALK1. The AdcaALK5, but not AdcaALK1 was able toreconstitute the TGF-b effect and decrease Adh1 mRNA expres-sion (Fig. 5C). Furthermore, adenoviral over-expression of TGF-bantagonist Smad7 which inhibits all R-Smad pathways conse-quently reduces Adh1 expression (Fig. 5D). Theoretical analysisof transcriptional regulatory elements within the mouse Adh1promoter with Genomatix Software indicated a Smad binding sitewithin the distal promoter region at �1319 bp, representing apotential TGF-b responsive element (Fig. 5E). Moreover, TIEG(�382 bp) and TCF/LEF-1 (�882 bp) transcription factor bindingelements were also found. Although not yet functionally proven,in silico analysis supports our experimental findings and indicatesa regulatory role of TGF-b towards the Adh1 promoter region viadirect interaction of R-Smads.

Down-regulation of ADH1 participates in TGF-b mediated increase oflipid deposition and oxidative stress level

To further prove that ADH1 expression reduction is a pro-stea-totic effect of TGF-b, we knocked-down ADH1 in cultured hepa-

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tocytes using Adh1 siRNA oligonucleotides transfected 24 hbefore TGF-b stimulation and/or challenged with alcohol.Results were verified by Western blot analysis (Fig. 6A). Tomonitor ethanol metabolism, the residual ethanol level wasdetermined for the culture medium of alcohol treated mousehepatocytes. Ethanol concentration was �80% for ADH1 defi-cient hepatocytes compared to �50% for control cells (Supple-mentary Fig. 5B). With Oil Red O staining we visualized a slightincrease in lipid deposition by ethanol alone, while it wasstrongly enhanced by ethanol plus TGF-b. Interestingly, knock-ing-down ADH1 increased the effect of ethanol as observedwith TGF-b stimulation (Fig. 6A). In contrast, over-expressionof ADH1 by transient transfection rescued, to some extent,the damaging effects of TGF-b and alcohol, and reduced neutrallipid deposition (Fig. 6B). The same tendency was obtainedmeasuring intracellular ROS production. Knocking-down Adh1sufficed to increase oxidative stress and significantly enhancedthe effect of ethanol (Fig. 6C). Inhibition of ADH1 activity by 4-methylpyrazole treatment enhanced cell toxicity followingcombined treatment with ethanol and TGF-b as determinedby LDH leakage assays (Fig. 6D). Alcohol treatment for 8 h ele-vated Cyp2E1 activity in hepatocytes as quantified by HFC for-mation. This effect was enhanced by 4-MP dependentinhibition of ADH (Fig. 6E), thus connecting Cyp2E1 inductionwith Adh1 expression.

To prove that there is a correlation of ADH1 down-regula-tion with liver damage in vivo, ADH1 expression was examinedby immunohistochemistry in liver sections from chronic etha-nol-fed mice as well as in corresponding controls (Fig. 6F).Our results demonstrate a statistically significant reduction ofADH1 in hepatic cytoplasm in sections from ethanol-fed mice(Fig. 6F).

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Fig. 3. TGF-b expression signature. An Affymetrix GeneChip analysis of mouse hepatocytes stimulated or not with 5 ng/ml TGF-b for 24 h. Genes with significantlychanged expression levels (fold change relative to untreated control higher than 1.7) are shown. Negative values indicate repression, while positive ones represent up-regulation. The DAVID tool was used to elucidate biological functions. TGF-b regulates expression of alcohol metabolism related genes (Adh1). Microarray transcript levelsare presented as log2-fold change in 24 h TGF-b stimulated cells vs controls.

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Discussion

Hepatic fibrosis from all aetiologies including advanced ALDcomprises cell activation, fibroblastoid transdifferentiation, ECMdeposition, cytokine release and tissue remodeling [26]. In latedisease stage, TGF-b signalling contributes to stellate cell activa-tion and (myo)fibroblast generation, the latter being responsiblefor ECM deposition and scar formation [1,5,27]. Early after onsetof alcohol dependent liver damage, TGF-b is up-regulated in theliver in animal models and in the human disease [28,29]. Bothare characterized by hepatocyte damage and steatosis, but lackserious inflammation and stellate cell activation. Here, wedescribe nuclear phospho-Smad2 staining in steatotic liver ofmice after intragastric ethanol feeding for 6 weeks.

To characterize the function of TGF-b in early ALD, we inves-tigated its effects on cultured hepatocytes. TGF-b increases theintracellular content of ROS and down-regulates antioxidantgenes in hepatocytes as well as in other cell types [30]. Whencombined with ethanol treatment, TGF-b strongly promotes alco-hol dependent oxidative stress and anti-oxidant depression lead-

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ing to enhanced cellular toxicity. This ‘‘alcohol damagepromoting” effect of TGF-b is further supported by the findingthat ethanol induced lipid peroxidation is increased upon parallelactivation of its signalling pathway.

Leading hypotheses on the mechanism of alcohol dependentcell injury relate to its metabolism and include redox statechanges resulting from ethanol oxidation to acetaldehyde byalcohol and aldehyde dehydrogenases. Metabolism via CYP2E1releases free radicals which diminishes defense systems againstoxidative stress [34]. Furthermore, non-oxidative metabolism ofethanol to FAEEs, a marker of fat accumulation and organ damagemay contribute to pathophysiology of ALD [23].

TGF-b dependent microarray data from hepatocytes implicateregulation of genes involved in glucose, lipid and oxidative stressmetabolism [5]. Adh1, the major enzyme for alcohol metabolismin the acute damaged liver [10], is one of the most strongly reg-ulated TGF-b target genes. TGF-b down-regulates Adh1 mRNAproduction, as well as Adh1 protein and enzymatic activity in cul-tured hepatocytes. A direct link was verified with liver tissuefrom TGF-b transgenic animals displaying TGF-b serum levels

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50

Fig. 4. TGF-b down-regulates ADH1 expression in cultured hepatocytes and in liver tissue from transgenic mice. Mouse hepatocytes in culture were treated or not withTGF-b as indicated. (A) Real-time RT-PCR for Adh1. Gene expression changes were measured in triplicate from three hepatocyte preparations. Relative differences werecalculated using the comparative threshold cycle method (DDCt) by normalization to PPIA. Values are shown as fold change in expression (mean ± SD) relative to control.(B) Western blot and densitometric quantification of ADH1 expression. b-Actin served as the internal control. (C) Enzymatic activity of ADH1 measured as an NADH/NADratio (using ethanol as a substrate). For controls, 0.5 mM Aspirin (inducer of ADH1 activity) and 0.5 mM 4-MP (inhibitor of ADH1) were used. Specific activity is expressed asunits per mg of protein. (D) Adh1 mRNA expression, measured with qRT-PCR in samples from livers of transgenic mice induced to over-express active TGF-b vs controls;**p < 0.01, *p < 0.05.

Research Article

10–30 times above normal [15] and reduced ADH1 expressionupon induction of transgene expression.

The regulation of ADH1 expression has not yet been intenselyinvestigated. However, a number of potential transcription factorbinding sites and regulatory elements were identified within theAdh1 promoter [23], e.g. members of the C/EBP family [10].

In agreement with a proposed regulatory role for TGF-b onAdh1 expression, we identified a TGF-b/Smad3-binding elementwithin the ADH1 promoter in silico. In contrast to most cell types,in which TGF-b exclusively uses the TbRII/ALK5/Smad2/3 path-way, TGF-b may additionally signal via activation of ALK1 andSmad1, 5, 8 in HSCs and HCs [33]. Using functional experimentsthat included constitutively active mutants of ALK5 and ALK1,receptor specific inhibitors as well as the over-expression ofinhibitory Smad7 and siRNA knock-down, we identified ALK5-Smad2/3 signalling, but not ALK1-Smad1/5/8 signalling asrequired for ADH1 down-regulation by TGF-b. However, evidencefor Smad-mediated inhibition of transcription via direct bindingto the identified recognition site, e.g. in concert with other tran-scription factors (for example, via TIEG or TCF/LEF-1) remains tobe provided.

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Physiologically blunting TGF-b signalling occurs on severallevels, including a feedback mechanism via up-regulation ofinhibitory Smad7 [6,32]. Blocking TGF-b by ectopic expressionof Smad7 in hepatocytes suffices to inhibit CCl4-dependent liverdamage and fibrogenesis in mice [5]. Given the observed Smad7inhibition of TGF-b effects on ADH1 expression and ethanolinduced damage in the present study, we hypothesize that abeneficial effect of Smad7 up-regulation also applies for alcoholdependent liver injuries. However, it is not clear how TGF-bpotentiates alcohol damage by down-regulating ADH1. HepaticADH1 activity is decreased in chronic alcohol abuse or after alarge acute ingestion [23], and microsomal cytochrome P450(CYP2E1) is induced to compensate for the lack of ethanolmetabolism to acetaldehyde [31]. We hypothesize that TGF-binhibition of ADH1 primes hepatocytes to metabolize ethanolvia the hepatic microsomal system or the non-oxidative path-way using a yet unidentified link. The increased burden onthe body that ethanol creates potentiates oxidative stress pro-duction. Furthermore, lipid deposition was increased slightlyin ethanol treated hepatocytes compared to controls, whereasit was strongly enhanced by a combined treatment with ethanol

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Fig. 5. TGF-b down-regulates ADH1 via the ALK5 pathway. Cultured hepatocytes were pre-treated with SB431542 (5 lmol/ml) or infected with adenoviruses beforestimulation with TGF-b, as indicated. (A), Western blot and qRT-PCR (B–D) data show that SB431542 and Smad7 inhibit Smad2/3 phosphorylation rescuing TGF-b mediateddown-regulation of ADH1, whereas knocking-down ALK1, functionally proven by blunting PSmad1, had no effect; AdsiALK1, AdcaALK5, AdcaALK1, AdSmad7, adenovirusesencoding siRNA to knock-down ALK1, constitutively active (ca) receptors ALK1/ALK5 and Smad7; AdLacZ, control virus; b-actin was used as loading control. DMSO was thesolvent for the chemical inhibitor; (**p < 0.01, *p < 0.05). (E) The Adh1 mouse promoter, 1680 bp upstream from the transcription start was analyzed using GenomatixSoftware. Among others, a Smad3 binding site (GTCT) within the distal Adh1 promoter region at �1319 bp, was identified.

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and TGF-b. This implies that TGF-b has a major impact on hepa-tic lipid metabolism, possibly involving enhanced down-regula-tion of ADH1. This is supported by recent findings in HepG2cells where decreased ADH1 activity was required for the non-oxidative metabolism of ethanol, resulting in enhanced fat accu-mulation and cytotoxicity compared to cells over-expressingADH1 [12]. Moreover, our own data show that TGF-b and/oralcohol dependent fat deposition in primary mouse hepatocytes

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is abrogated by transient overexpression of ADH1. Furthermore,ADH deficient mice subjected to the Lieber–DeCarli alcohol dietfor 60 days display increased hepatic steatosis and apoptosiscompared to controls [23]. Finally, healthy patients drinkingalcohol with 4-MP dependent inhibition of oxidative metabo-lism display increased FAEE production from non-oxidative eth-anol metabolism, when compared to placebo treated controls[35,36].

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- - + +EtOH (100mM)

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*

*

*

BA

OD

= 5

00 n

m(fo

ld o

f con

trol)

[72h]0

0.5

1.5

2.5

3.5

4.5

5.5

siADH1

% Oil Red O

- - + -EtOH (100mM)

TGF-β1

*

**

OD

= 5

00 n

m(fo

ld o

f con

trol)

[72h]

0

1

2

3

4

5

6

siADH1

7-MFC

- - + +- - + + EtOH (100mM)

4-MP (0.5mM)

4-MP (0.5mM)

*

*

HFC

(pm

ol/m

in/m

g pr

ot.)

[8h]

0

15

30

45

60ROS

- - + +- - + + EtOH (100mM)

*

*

*

*

[ex/

em=4

85/5

27]

[24h] 0

10

20

30

40 LDH

- + + + +- - -+ +- - - + + EtOH (100mM)

*

% L

DH

rele

ase/

tota

l LD

H

*

[48h]TGF-β1 (5ng/ml)

Fig. 6. TGF-b dependent increase of lipid deposition and oxidative stress are mediated via its ADH1 effect. (A) Hepatocytes were transfected with 10 nmol siRNAs forADH1 and scrambled siRNA controls or (B) with 1 lg ADH1 expression plasmid and the empty vector pcDNA3.1 as a control (A and B). ADH1 protein expression knock-down and over-expression was examined by Western blot analysis 48 h later. Microscopic images and quantification of Oil Red O-stained transfected hepatocytes, treatedwith 100 mM EtOH (72h) and/or 5 ng/ml TGF-b (48 h) (magnification, 200�) is shown. Bound Oil Red O was quantified at 500 nm using an ELISA reader. (C) Intracellularoxidative stress (ROS) was assayed using DCFH-DA as substrate. (D) Cellular damage after 4-MP, TGF-b and/or alcohol treatment was determined by measuring thepercentage of LDH release into the culture medium in relation to total LDH. (E) Cyp2E1 activity was assessed 8 h after 100 mM ethanol challenge using 7-MFC as substrate.(F) IHC and quantification of ADH1 positive staining in livers of ethanol-fed mice and controls.

Research Article

414 Journal of Hepatology 2010 vol. 52 j 407–416

Ethanol

ADH

Cytosol

Liver fat accumulation Enhanced oxidative stress

MEOS:Cyp2E1

Microsomes

FAEE synthesis

Endoplasmic Reticulum

Chronic acoholabuse (non/ oxidative

metabolism)

Acute alcohol abuse (oxidative

metabolism)

MEOS:Cyp2E1ADH

As described (Wu H., 2006)

Efficient alcohol metabolite excretion

TGF-β1

Fig. 7. Proposed model of liver damage upon alcohol intoxication. TGF-b enhances alcohol induced hepatocyte damage via down-regulation of ADH1.

JOURNAL OF HEPATOLOGY

Based on the above citations and the present study, we sug-gest the following model: under physiological conditions, ethanolmetabolism via ADH represents the main route of acetaldehydeproduction and alcohol metabolite excretion (Fig. 7). In a chronicsituation, secondary metabolic routes via MEOS with Cyp2E1 up-regulation or a non-oxidative pathway are induced. e.g., Km forethanol oxidation by MEOS is about one order of magnitude fas-ter than ADH1, indicating that the metabolic switch is required tohandle steady state high levels of ethanol. It is expected thatthere must be some selective pressure to metabolize alcohol effi-ciently (it must be assumed to avoid damage), although it isknown that on the other hand MEOS metabolism of alcohol dis-plays several adverse effects, including superoxide radical pro-duction and lipid peroxidation, both associated with negativeoutcome of alcoholic liver injury. This controversy has not yetbeen clarified.

We and others have identified increased levels of TGF-b in tis-sue and serum as well as activated TGF-b signalling in hepato-cytes from livers chronically challenged with alcohol. Here, weprovide evidence that TGF-b is favoring MEOS/non-oxidativealcohol metabolism by decreasing ADH1 expression via theALK5/Smad2/3 signalling pathway, and thus increasing theadverse effects of ethanol.

In conclusion, we show for the first time that TGF-b has damag-ing effects towards hepatocytes when acting in concert with etha-nol. This may occur similarly in vivo. TGF-b-mediated low levels ofADH1 are correlated with enhanced hepatocyte damage uponchronic alcohol consumption by favoring secondary metabolicpathways. It still remains to be shown whether TGF-b participationin steatosis and oxidative stress are required for the progression ofhuman liver injury to HSC activation and fibrogenesis.

Since the ectopic expression of Smad7 inhibits TGF-b depen-dent ADH1 reduction and thus its enhancer role for ALD, the activa-tion of this TGF-b negative regulatory pathway may be beneficial inthe very early stages of alcohol dependent liver damage.

Acknowledgements

The authors who have taken part in this study declared that theydo not have anything to declare regarding funding from industry

Journal of Hepatology 201

or conflict of interest with respect to this manuscript. The studywas supported by grants from the European Research AdvisoryBoard (ERAB), the Dietmar Hopp Foundation, Walldorf, and theBMBF program HepatoSys. We appreciate excellent technicalassistance of C. Stump and A. Müller.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jhep.2009.12.003.

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