Supplementary Materials and Methods
Materials
Antibodies: rabbit polyclonal anti ADH1 (sc-22750,Santa Cruz Biotechnology,
California, USA); rabbit polyclonal phospho-Smad2 (Ser465/467) and phosphoSmad1/3 (9514, Cell Signaling Technology, Frankfurt, Germany); rabbit polyclonal
Smad2 and Smad3 (51-1300 and 51-1500, Zymed Laboratory, Berlin, Germany);
rabbit polyclonal Smad7 (24477, Abcam Ltd., Cambridge, UK); mouse monoclonal actin (A5441, Sigma-Aldrich Missouri, USA). siRNA for ADH1 was from Qiagen
(Hilden, Germany). Chemicals: Human recombinant TGF-1, Peprotech (London,
UK); β-Nicotinamide adenin dinucleotide hydrate from yeast, Oil red and 7-Methoxy4(trifluoromethyl)coumarin substrate (7-MFC), Williams medium E, SB431542,
Insulin, Dexamethasone, Triton-X100 and acetylsalicylic acid, Sigma (Seelze,
Germany ); Protease inhibitor cocktail, phosphatase inhibitor cocktail and rat tail
collagen I, Roche (Mannheim, Germany); Penicillin/Streptomycin, L-glutamin,
Cambrex (Taufkirchen, Germany); CellTrackerTM-Carboximethylfluorescein diacetate
(CMFDA), Molecular Probes (Karlsruhe, Germany); Foetal Bovine Serum and
Lipofectamin2000, Invitrogen (Karlsruhe, Germany); ethanol absolute, J.T. Baker
(City, Holland) and 4-Methylpyrazol from Fluka, (Steinheim am Albuch, Germany).
Cell preparation and treatment
Mouse hepatocytes from male C57/BL-6 mice (100-150g) isolated by collagenase
perfusion were plated on collagen coated 6-well-plates at a density of 5 x105
cells/well in Williams' medium E supplemented with 10 % FBS, 2 mmol L-glutamine,
1 % penicillin/streptomycin and 100 nmol dexamethasone. After 4 hrs of incubation in
5 % CO2 at 37 oC to facilitate attachment, medium was replaced with serum-free
Williams' medium E supplemented with 1 % penicillin/streptomycin, 2 mmol Lglutamine and 100 nmol dexamethasone. The next day, medium was changed to
serum-free Williams' medium E supplemented with 2 mMol L-glutamine and 1%
penicillin/streptomycin [1].
Preparation of cell lysates and immunoblotting
Cell lysates were prepared using RIPA buffer (1X Tris-buffered saline, 1 % Nonidet
P-40, 0.5 % sodium deoxycholate, 0.1 % SDS). Protease inhibitor Mix and
Phosphatase Inhibitor Cocktail II (Roche, Mannheim, Germany) were included before
use. Protein concentration was determined with the DC Protein Assay (Biorad,
München, Germany). Western blot analyses were performed as described.[5]
Individual protein bands were quantified by densitometry using AIDA analysis
software and normalized for -actin. All expression data were confirmed in three
independent experiments. Antibodies are listed in Suppl.Table2.
Isolation of Primary Human Hepatocytes. Human isolated primary
hepatocyteswere obtained according to the institutional guidelines from liver
resections of patients with primary or secondary liver tumors.
Adenovirus infections and transient plasmid transfections
Recombinant adenoviruses expressing constitutively active (ca) mutants for TGF-β
type I receptors (Ad-caALK1 and Ad-caALK5), Smad7 (Ad-Smad7), or encoding
ALK1 siRNA ) were prepared and used as previously described [5]. Briefly, mouse
hepatocytes were infected (day 0) with 50-100 ifu/cell (infectious units ) adenovirus
for 1 hr in William’s medium E containing 5 % FBS. Then, medium was changed to
2
serum free William’s medium E. After 36-48 hrs, target gene expression was
investigated by Westernblot. Infectivity (ifu, infectious units) was determined using
the Rapid Titre Kit from BD Bioscience and 50-100 ifu/cell were used. Generally,
more than 90 % of hepatocytes were infected.
Hepatocytes were transfected with 1µg ADH1 expression plasmids and empty vector
pcDNA3.1 as a control (purchased from Qiagen). Briefly, cells were seeded at a
density of 1x105/cm2 in 24 well plates. Expression plasmids were transfected with
Lipofectamine™ 2000 (Cat. No. 11668-027 Invitrogen, Karlsruhe, Germany)
according to the manufacturer’s instructions. 24 hrs after transfection, medium was
changed and TGF- and or ethanol stimulations proceeded conform with the
standard operating procedure. ADH1 protein overexpression was examined by
Western blot analysis 48 hrs later.
Ethanol metabolism
Hepatocytes were transfected with ADH1 overexpression plasmids and control
vectors, or ADH1 siRNAs and the corresponding scrambled siRNAs, followed by
ethanol stimulation for 12 hrs. Residual ethanol in the medium was measured with an
enzymatic method (Diasys, Germany), which photometrically determines NADH+
formation from NAD by alcohol dehydrogenase dependent oxidation at a wavelength
of 340 nm. A standard curve was generated by immediate measurement of different
ethanol concentrations in medium. Results are expressed as percentage from the
initial ethanol concentration.
Accumulation of lipids - Oil Red O staining
Mouse hepatocytes were plated in 24- or 96-well-plates and stimulated for 3 days
with 170 nM insulin in William’s Medium E (with 1% penicillin/streptomycin and 2 mM
L-glutamine). Following stimulation, cells were fixed with 4% PFA and washed briefly
with 60% isopropanol before staining for 10 min with 0.2% Oil Red O (Sigma-Aldrich
Missouri, USA) in 60% isopropanol. Unbound Oil Red O was removed by washing
with ddH2O. To quantify signals, bound Oil Red O was resolved in 100% isopropanol.
After incubation for 10 min at RT, supernatant was transferred to a transparent 96well-plate and OD was measured at 500 nm with a RAINBOW Thermo ELISA reader
(SLT Labinstruments, Achterwehr, Deutschland).
Annexin-V staining
Apoptosis in cultured mouse hepatocytes was assessed by Annexin-V staining as
previously described [4]. Briefly, 6x104 hepatocytes per well were cultured on glass 8well-chamber slides (BD Biosciences, NJ, USA). After overnight serum starvation,
cells were treated with 5 ng/ml TGF-, 100 mM ethanol or a combination of both for
48 hrs. Residual culture medium was washed off the cells with PBS (2 mM CaCl 2).
Cells were stained for 5 min with 0.25 µg/ml Annexin-V-Cy3 (Abcam Ltd., Cambridge,
UK) and 5 µg/ml Hoechst (Sigma) in PBS (2 mM CaCl2). Unbound stain was washed
off the cells with PBS (2 mM CaCl2) and fluorescent signal is detected immediately.
Lactate dehydrogenase assay
The toxicity of TGF-β and ethanol was determined with a lactate dehydrogenase
(LDH) leakage assay using a cytotoxicity detection kit (Roche, Mannheim, Germany).
Cells (2×104/cm2) were seeded onto 12-well-plates and treated with 5 ng/ml TGF-β
and 100 mM ethanol for 48 h. Cytotoxicity was expressed as percentage of LDH
released into the culture medium in relation to total LDH.
3
Cytochrome P450 assay
Fluorescence-based Cytochrome P450 assays were performed by incubation of
intact cells (in 96-well plates) with selected substrates as previously reported [21].
Briefly, 100 µl reaction buffer (1 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, 0.5 mM
MgCl2, 2 mM CaCl2, 10 mM O-(+)-glucose, and 10 mM HEPES, pH 7.4) containing
the
appropriate
amount
of
the
fluorogenic
substrate
7-Methoxy4(trifluoromethyl)coumarin (7-MFC) were added to each well. After 8 hrs iat 37°C,
supernatants were transferred to white/black 96-well plates and cells were fixed for
protein quantification by sulforhodamine B (SRB) staining. Potential metabolite
conjugates formed were hydrolyzed by incubating supernatants with βglucuronidase/arylsulfatase (150 Fishman units/ml and 1,200 Roy units/ml for 2 h at
37.0°C. Samples were diluted (1:4) with the appropriate quenching solution.
Formation of fluorescent metabolite was quantified with a Fluoroscan Ascent
Fluorescence Microplate Reader. Results are given as picomoles of metabolite per
minute, normalized to total protein content. Methanol-fixed cells were used for
background subtraction.
SRB Staining was performed as reported [21]. In brief, cells were covered with icecold fixation buffer (95% ethanol, 5% acetic acid) and kept at –20°C for 1 h. Fixed
cells were stained with 0.4% SRB (w/v) in 1% acetic acid for 30 min. Unbound dye
was removed by washing with 1% acetic acid. Bound SRB was resolved in 10 mM
unbuffered Tris solution and optical densities were determined at 565 nm. From
optical densities, total protein content was calculated with a standard curve obtained
by plotting the optical density from SRB staining versus total protein contents
measured with the DC Protein Assay Kit (Biorad, Muenchen, Germany).
ROS production, glutathione depletion and lipid peroxidation
Mouse hepatocytes were plated in 96-well-plates. After stimulation with 100 mM
ethanol and/or 5 ng/ml TGF-, cells were washed 3 times with HBSS followed by
incubation with 10 µM DCFH-DA (ROS measurement) or 10 µM monochlorobimane
(MCB; GSH mesurement) in medium for 30 min at 37 °C. Thereafter, cells were
washed three times with HBSS and lysed in 0.1 % Triton-X-100 in HBSS for 10 min
at 37 °C. Cell lysates were transferred to a white 96-well plate (Nunc, Wiesbaden,
Germany) and fluorescence was determined (ex/em 485/527; ROS or 355/460 nm;
GSH) with a Fluoroskan Ascent fluorescence microplate reader (ThermoLabsystems,
Engelsbach, Germany). For measuring lipid peroxidation, cells were treated in the
same way for 48 hrs. Cell lysates were collected in ice-cold HBSS supplemented with
1/50 volume butylated alcohol (0.2 % 2,6-di-tert-butyl-4-methylphenol in ethanol) by
sonication. 1 volume thiobarbituric acid (TBA) (5 mM in acetic acid) solution was
added before boiling the reaction mixture for 60 minutes. Samples were chilled on ice
and 0.5 volumes methanol were added. Samples were centrifuged (13,000 g, 5 min,
RT) and supernatant was transferred to a white 96-well-plate to measure
fluorescence (ex/em 530/590 nm). All in vitro data are presented as mean ± standard
deviation of three measurements for each out of three separate experiments.
In silico promoter analysis
1,500 bp upstream promoter region relative to the mouse Adh1 gene start codon
were analyzed with Genomatix Software to identify putative binding elements
(http://www.genomatix.de).
4
RNA based expression analyses
Total RNA was purified from liver tissue after homogenization in TRIZOL reagent
(GIBCO BRL, Eggenstein, Germany; 1 ml/50mg tissue) and from cultured
hepatocytes using the High Pure RNA isolation kit (Roche, Mannheim, Germany)
according to the manufacturer’s protocol. 1µg total RNA was transcribed to cDNA
using Transcriptor First Strand cDNA synthesis kit (Roche). PCR amplifications were
done for ADH1, Smad7 and rS6 (Suppl. Table 1).
PCR products were obtained after 26 amplification cycles at annealing temperatures
of 60 oC. Samples were run on 1.5 % agarose gels and visualized by
ethidiumbromide staining (0.5 µg/ml) under UV light. The results were analysed by
digital image analysis.
Suppl. Table 1
Gene
Up-stream Primer
Down-stream Primer
Product
size (bp)
rS6
5´-GTG CCT CGT CGG TTG GGA C-3´
5´-GAC AGC CTA CGT CTC TTG GC-3´
320
Adh1
5´-AGG AAG TTC TCC AGG AGA T-3´
5´-TCT TAG CCA TGA AGT CAG CC-3´
264
Microarray analysis
Total RNA extraction and cDNA probe labelling was as described.[5] Hybridization to
microarrays of type moe430_2 from Affymetrix (Santa Clara, CA, USA) and array
scanning were performed according to manufacturers recommendations. Differential
expression
analysis
was
performed
with
ANOVA
in
BioConductor
(http://www.bioconductor.org) using R V2.4.1.
Raw fluorescence intensity values were normalized applying quantile normalization,
and gene expression values were converted to a log2 scale. Significant differences in
gene expression were analysed with limma package for R Multitest. Genes with
mean expression changes greater than 1.7 fold (log2 greater than 0.7655 and lower
than -0.7655) and which were significant at a false-discovery rate of q < 0.05 were
selected as regulated by TGF-. CustomCDF with Entrez based gene/transcript
definitions R package was used for gene annotation. Gene Ontology analysis was
done with DAVID (Database for Annotation, Visualization, and Interpreted Discovery;
http://apps1.niaid.nih.gov/david/. GeneChips microarray study followed MIAME
guidelines issued by the Microarray Gene Expression Data group. We have
deposited all files containing the raw data of the microarray analysis to ArrayExpress
(http://www.ebi.ac.uk/arrayexpress/) at the European Bioinformatics Institute
(Hinxton). The data are available under accession number E-MEXP-1176.
Suppl. Table 2
Antibodies and reagents used in this study:
Antibodies
Cat. No
Clonality
ab5825
RabbitPoly
#3101
RabbitPoly
#9514
RabbitPoly
A-5441
Mouse-
Supplied by
Abcam
Antibody, epitope, location
MADH7
Species*
h, m, r
Dilution
1:500 (WB)
Cell
Signaling
Cell
Signaling
P-Smad2, synthetic phosphopeptide, serine465/467, hSmad2
P-Smad1/3, synthetic phosphopeptide, serine 423/425,
hSmad3
β-actin, N-terminal peptide
h, m, r
h, m, r
1:1,000 (WB)
1:100 (IHC)
1:1,000 (WB)
h, m, r
1:10000 (WB)
Sigma
5
51-1300
sc-22750
Mono
RabbitPoly
RabbitPoly
Zymed
Smad2
h, m, r
1:1,000 (WB)
Santa Cruz
ADH1
h, m, r
1:1,000 (WB)
References
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Klingmuller U, Bauer A, Bohl S, et al. Primary mouse hepatocytes for systems
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2
Goumans MJ, Valdimarsdottir G, Itoh S, et al. Activin receptor-like kinase
(ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol Cell
2003;12:817-28.
3
Dooley S, Delvoux B, Streckert M, et al. Transforming growth factor beta
signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway
that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal
transduction during transdifferentiation of hepatic stellate cells. FEBS Lett
2001;502:4-10.
4
Dooley S, Hamzavi J, Ciuclan L, et al. Hepatocyte-specific Smad7 expression
attenuates TGF-beta-mediated fibrogenesis and protects against liver damage.
Gastroenterology 2008;135:642-59.
5
Ehnert S, Nussler AK, Lehmann A, Dooley S: Blood monocyte-derived
neohepatocytes as in vitro test system for drug metabolism. Drug Metab Dispos
2008, 36(9):1922-1929.
6
Glynn Dennis Jr, Brad T Sherman, Douglas A Hosack, Jun Yang, Wei Gao, H
Clifford Lane and Richard A Lempicki. DAVID: Database for Annotation,
Visualization, and Integrated Discovery Genome Biology 2003, 4:R60
6
Supplementary Results
TGF-β enhances ethanol induced toxicity and oxidative stress in mouse
hepatocytes
To determine appropriate concentrations for TGF-β and ethanol for measurable
effects in hepatocytes, we established dose response curves for cellular damage as
determined by the percentage of LDH release into the culture medium during 48 hrs
treatment. The results indicate 1- 5ng/ml TGF-β1 and 100mM ethanol as lowest
doses inducing a significant response (Suppl.Fig.2A,B).
After the addition of ethanol, plates were sealed with parafilm to prevent evaporation.
With this setting, time dependent variations in the ethanol concentration were due to
its metabolism and not to different evaporation rates. For example, the concentration
of ethanol in the culture medium 12 hours following ethanol addition was reproducibly
reduced to ~ 50% in case of parafilm sealed plates, compared to ~ 33% for unsealed
plates, when related to the initial ethanol concentration (Suppl.Fig.5A). Ethanol was
added in fresh medium every 24 hours, to study long term effects. Control cultures
were treated similarly.
TGF-β decreases ADH1 expression in hepatocytes
Affymetrix oligonucleotide microarray analysis covering the whole mouse genome
was performed to obtain a comprehensive view on TGF-β effects in hepatocytes.
We identified functional groups associated with signal transduction, fibrogenesis and
cell death [5], as well as with transport, metabolism and oxidative stress
(Suppl.Fig.3).
In the metabolism related group, 57 genes belonging to lipid- and 11 to glucosemetabolism. Among them such involved in triacylglycerol metabolism, among others,
the triacylglycerol degradation pathway, lipoprotein lipase (Lpl), hepatic lipase (Lipc),
monoglyceride lipase (Mgll) and liver specific fatty acid binding protein 1 (Fabp1)
were down-regulated. In contrast to this, one of the enzymes synthesizing acyl CoA,
acyl-CoA synthetase bubblegum family member 1 (Acsbg1), and one lipoprotein
transporter, low density lipoprotein receptor-related protein 11 (Lrp11) were
upregulated. Several detoxifying and electron transporting cytochromes were
downregulated (e.g. CYP27A1, CYP2B10, CYP2C70, CYP2D9, CYP39A1,
CYP3A13, CYP4F14 and CYP7B1), whereas the expression of CYP21A1, an
enzyme involved in glucocorticoid production and fat accumulation, was increased.
Moreover, mRNA abundance of several genes involved in the anti-oxidant defense
mechanism was markedly decreased, for example glutathione S-transferases (Gsta2,
Gstt1, Gsta4, Gstt2, Gstm4, Gstm7 and Mgst3), glutathione synthetase (Gss),
whereas glutathione peroxidase 3 (Gpx3) was up-egulated. Consistently with this,
expression of monoamine oxidase A (Maoa), a repressor of the ROS producing
system was down-regulated. Furthermore, some genes involved in glucose
metabolism were changed by TGF- treatment. These include up-regulated
hexokinase 2 (Hk2), insulin-like growth factor 1 (Igf1) and phosphoglucomutase 1
(Pgm1). Other enzymes involved in glycolysis, gluconeogenesis and their regulation
were down-regulated (Aldob, Onecut1, Pklr, Gpd1, Pdk1, Pdk2, Pdk4, Tpi1 and Atf3)
(Fig.3A and Suppl.Fig.3).
In culture, mouse and human hepatocytes share similar phenotypes as seen by light
microscopy photographs (Suppl.Fig.4B).
TGF-β down-regulates ADH1 expression in hepatocytes via activation of the
ALK5 pathway
7
As Smad independent TGF-β signaling occurs in hepatocytes, but inhibition of MAPK
pathway, showed no significant effect on Adh1 expression (data not shown), we
assume that TGF- dependent ADH1 expression reduction in hepatocytes solely
relies on activation of the ALK5/Smad2/3 signaling pathway.
The effect of ADH1 deficiency/ADH1 overexpression on ethanol metabolism
To determine the effect of ADH1 deficiency/ADH1 overexpression on ethanol
metabolism, the residual ethanol concentration was determined in the culture
medium of alcohol treated mouse hepatocytes after 12 hrs of incubation.
Hepatocytes were transfected with ADH1 overexpression plasmids and control
vectors, or ADH1 siRNAs and the corresponding scrambled siRNAs, followed by
ethanol stimulation for 12 hrs. In this setting, the medium ethanol concentration is
reproducibly decreased to ~ 20% in ADH1 overexpressing hepatocytes as compared
to ~ 50% in controls. On the other hand, reducing ADH1 expression with RNAi,
increased ethanol levels to ~ 85% (Suppl.Fig.5B), suggesting significantly
diminished oxidation of ethanol to acetaldehyde. Furthermore, these data confirm
that the measured changes in ethanol levels are due to metabolic effects and not to
evaporation.
We also investigated the combined effect of ethanol and TGF-β. Challenge with
5ng/ml TGF-β for 24 hrs reduced Adh1 mRNA expression with and without ethanol
stimulation (48 hrs, 100mM), although with ethanol the effect was weakened
(Suppl.Fig.5C), which might be due to a stimulatory impact of ethanol for Adh1 [12].
Supplementary figure legends
Suppl.Fig.1. Quantitative analysis of Annexin V staining before and 48 hrs after
treatment with 5 ng/ml TGF-β and/or 100 mM ethanol . Results are presented as
arbitrary units of the measured fluorescence.
Suppl.Fig.2. Cell induced by dose response curves of TGF-β and alcohol
dependent toxicity in hepatocytes.
(A,B) Hepatocytes were stimulated or not with 1, 5, 10 ng/ml TGF- and 50, 100,
200mM ethanol for 48 hrs. Cellular damage was determined by measuring the
percentage of LDH released into the culture medium in relation to total LDH. (three
different mouse hepatocyte preparations were investigated in triplicate
measurements; **p<0.01, *p<0.05 vs control).
Suppl.Fig.3. TGF-β regulates expression of genes involved in its signal
transduction, fibrogenesis, apoptosis, intracellular transport and metabolic
processes in hepatocytes.
(A) Mouse hepatocytes were stimulated or not with 5 ng/ml TGF- for 24 hrs and
total RNA was analyzed using Affymetrix GeneChips. Genes whose expression
levels were significant at a false-discovery rate of q < 0.05 and whose fold change
relative to untreated controls was higher than 1.7 are shown. The DAVID tool was
used to elucidate biological functions that are enriched. The lengths of the bars
represents the number of genes in each modul. Detailed descriptions of the terms
can be found at the homepage of the Gene Ontology project
(http://www.geneontology.org/index.shtml).
8
Suppl.Fig.4. Adh1 mRNA expression remains unchanged during culture of
mouse hepatocytes and TGF-β down-regulates ADH1 expression in the same
manner in human hepatocytes.
(A,B) Mouse and human hepatocytes were cultured in serum-free medium on
collagen-coated plates. Human liver cells were stimulated or not with 5 ng/ml TGF-
for 24 hrs. Adh1 mRNA expression was examined by PCR and rS6 was used as a
loading control. Primers are listed in Suppl.Table 1. (B) Light microscopy
photographs of mouse and human hepatocytes cultured in serum-free medium on
collagen-coated plates for two and five days, respectively. (magnification: 200X).
Suppl.Fig.5. Adh1 dependent ethanol metabolism in cultured hepatocyes
(A) Hepatocytes were stimulated with 100 mM ethanol for 6-24 hrs as indicated.
Alcohol treated plates were sealed or not with parafilm, concentration was measured
in medium at 340 nm and related to a standard curve generated from known ethanol
concentrationsmixed with medium.
(B) Hepatocytes were transfected with control vectors and the corresponding ADH1
siRNAs or ADH1 overexpression plasmids, followed by ethanol (100 mM) stimulation
for 12 hrs (as presented in Supplementary Material and methods). (C) Real-time RTPCR of Adh1 mRNA expression in cultured mouse hepatocytes treated with ethanol
(48hrs) and/or TGF-β (24 hrs) as indicated in supplementary results.
Suppl.Table 3
Serum TGF- concentration in control and TGF- transgenic mice that were
used to analyse Adh1 mRNA expression.
Number
1
2
3
4
5
6
7
8
9
10
wt
wt
wt
wt
wt
TGF- tg
TGF- tg
TGF- tg
TGF-tg
TGF- tg
Serum TGF- concentration
(ng/ml)
50
51
30
40
57
466
240
1017
223
250