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Trichostatin A prevents the accumulation of extracellular matrix in a mouse model of bleomycin-induced skin fibrosis.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 8, August 2007, pp 2755–2764
DOI 10.1002/art.22759
© 2007, American College of Rheumatology
Trichostatin A Prevents the Accumulation of Extracellular
Matrix in a Mouse Model of Bleomycin-Induced Skin Fibrosis
Lars C. Huber,1 Jörg H. W. Distler,2 Falk Moritz,1 Hossein Hemmatazad,1 Thomas Hauser,3
Beat A. Michel,1 Renate E. Gay,1 Marco Matucci-Cerinic,4 Steffen Gay,1
Oliver Distler,1 and Astrid Jüngel1
Objective. Tissue fibrosis is a hallmark compromising feature of many disorders. In this study, we
investigated the antifibrogenic effects of the histone
deacetylase inhibitor trichostatin A (TSA) on cytokinedriven fibrotic responses in vitro and in vivo.
Methods. Skin fibroblasts from patients with systemic sclerosis (SSc) and normal healthy control subjects were stimulated with profibrotic cytokines in combination with TSA. Human Col␣1(I) and fibronectin
were measured using real-time polymerase chain reaction, and levels of soluble collagen were estimated using
the SirCol collagen assay. Electromobilty shift assay
and confocal fluorescence microscopy were used to
investigate the intracellular distribution of Smad transcription factors. For in vivo analysis, skin fibrosis was
quantified by histologic assessment of mouse skin tissue
in a model of bleomycin-induced fibrosis.
Results. Reductions in the cytokine-induced transcription of Col␣1(I) and fibronectin were observed in
both normal and SSc skin fibroblasts following the
addition of TSA. Similarly, the expression of total
collagen protein in TSA-stimulated SSc skin fibroblasts
was reduced to basal levels. The mechanism of action of
TSA included inhibition of the nuclear translocation
and DNA binding of profibrotic Smad transcription
factors. Western blot analysis revealed an up-regulation
of the cell cycle inhibitor p21 by TSA, leading to reduced
proliferation of fibroblasts. In addition, in bleomycininduced fibrosis in mice, TSA prevented dermal accumulation of extracellular matrix in vivo.
Conclusion. These findings provide novel insights
into the epigenetic regulation of fibrosis. TSA and
similar inhibitory compounds appear to represent early
therapeutic strategies for achieving reversal of the
cytokine-driven induction of matrix synthesis that leads
to fibrosis.
Systemic sclerosis (SSc) is characterized by severe
fibrosis of the skin and various internal organs. SSc skin
fibroblasts show distinct features of a dysregulated phenotype in vitro and in vivo, leading ultimately to the
production of high levels of collagen and other proteins
of the extracellular matrix as well as a reduced expression of matrix-degrading enzymes. However, the mechanisms responsible for the activation of fibroblasts, and
thus the development of chronic and progressive fibrotic
disease, remain unclear (for review, see ref. 1).
Several profibrotic cytokines have been shown to
be strongly associated with the pathogenesis of SSc,
including transforming growth factor ␤ (TGF␤) (2),
interleukin-4 (IL-4) (3), and platelet-derived growth
factor (PDGF) (4). Moreover, alterations in the downstream signaling pathways of TGF␤ have been observed,
in particular, an increase in the expression of phosphorylated Smad transcription factors (5,6). These cellular
alterations have been reported to be stable over multiple
generations of SSc fibroblasts in vitro. Genetic studies,
in contrast, could not identify clear associations between specific mutations of the genome and the
Supported in part by the Swiss National Science Foundation
(SNF 3200B0-103691).
1
Lars C. Huber, MD, Falk Moritz, MD, Hossein Hemmatazad, MD, Beat A. Michel, MD, Renate E. Gay, MD, Steffen Gay,
MD, Oliver Distler, MD, Astrid Jüngel, PhD: University Hospital
Zurich, and Zurich Center for Integrative Human Physiology, Zurich,
Switzerland; 2Jörg H. W. Distler, MD: University of ErlangenNuremberg, Erlangen, Germany, and University Hospital Zurich,
Zurich, Switzerland; 3Thomas Hauser, MD, University Hospital Zurich, Zurich, Switzerland; 4Marco Matucci-Cerinic, MD, PhD: University of Florence, Florence, Italy.
Address correspondence and reprint requests to Lars C.
Huber, MD, Center of Experimental Rheumatism, University Hospital
Zurich, Gloriastrasse 25, Zurich 8091, Switzerland. E-mail:
Lars.Huber@usz.ch.
Submitted for publication December 21, 2006; accepted in
revised form April 20, 2007.
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excessive accumulation of extracellular matrix (7). The
profibrotic phenotype of SSc fibroblasts could therefore
be imprinted by a combination of epigenetic alterations,
such as methylation and acetylation, as has been recently
suggested (8).
Epigenetic modifications comprise heritable alterations in the DNA itself without any changes in the
nucleotide sequence. Unlike alterations of the genome,
epigenetic changes are reversible and offer the potential
opportunity to reverse the epigenetic pattern through
therapeutic strategies (9). Modifications of histones, in
particular, histone (de)acetylation, are among the principal mechanisms that have been described as epigenetic
changes.
In normal resting cells, DNA is highly organized
within nucleosomes, in an octomeric core unit of chromatin and in DNA-binding nucleoproteins (histones)
(10). Thereby, the dynamic packaging of chromatin
regulates the extent of gene transcription. Histone hyperacetylation is generally associated with a loosened
state of chromatin and increased rates of gene transcription. Conversely, deacetylation of histones causes tighter
wrapping of the DNA around the nucleosome and
prevents transcription factors and RNA polymerase II
from binding (11). Targeted deacetylation is induced by
histone deacetylases (HDACs), which are multisubunit
enzyme complexes that remove the acetyl group from
the histones via a charge-relay system, using zinc ions
as prosthetic groups (for review, see ref. 12). HDAC
inhibitors function by dislodging the zinc ion, thus
turning off the charge-relay system. The most potent
reversible HDAC inhibitor known is trichostatin A
(TSA), which fits exactly into the catalytic site of the
HDAC enzymes (13).
During recent years, TSA has been used in
clinical trials as a novel therapeutic strategy for various
cancers, and these studies have shown that TSA induces
cell cycle arrest, cell differentiation, and apoptotic cell
death (14–16). Of interest, TSA was also postulated as a
lead compound in the development of antifibrogenic
drugs (17), and was tested as a promising therapeutic
agent in hepatic fibrosis (18) and in the prevention of
cutaneous radiation syndrome (19).
Fibrotic diseases comprise a wide array of clinical
entities. In this regard, novel molecules blocking the excessive accumulation of extracellular matrix are of great
interest in the development of therapeutic strategies. In
the present study, we investigated the antifibrogenic
effects of TSA, both in human SSc skin fibroblasts in
vitro and in an animal model of skin fibrosis in vivo.
HUBER ET AL
PATIENTS AND METHODS
Patients and fibroblast cultures. Normal and SSc
fibroblast cultures were prepared from biopsy specimens that
were obtained from the skin of patients with SSc (n ⫽ 6) and
healthy control subjects (n ⫽ 3) at the University of Florence.
Biopsy samples from the SSc patients were from areas of
affected skin. All patients fulfilled the criteria for SSc as
suggested by LeRoy et al (20), and all subjects signed a consent
form that was approved by the institutional review board of the
University of Florence.
After enzymatic digestion of the skin biopsy specimens
with dispase II (Boehringer-Mannheim, Rotkreuz, Switzerland), cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% heat-inactivated fetal calf
serum (FCS), 25 mM HEPES, 100 units/ml penicillin, 100
␮g/ml streptomycin, 2 mM L-glutamine, and 2.5 ␮g/ml amphotericin B (all from Gibco BRL, Basel, Switzerland). Fibroblasts
from passages 4–8 were used for these experiments. Cells were
treated with TSA (Sigma, St. Louis, MO) in a concentration of
2 ␮M (21). A stock solution of TSA was prepared in ethanol
and stored at ⫺80°C. The final concentration of ethanol in the
medium was 0.06%.
Real-time reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was isolated from SSc fibroblasts
using the RNeasy kit (Qiagen, Basel, Switzerland) according to
the manufacturer’s instructions, which included DNase treatment. To generate complementary DNA (cDNA), total RNA
(300–500 ng) was subjected to RT using Moloney murine
leukemia virus reverse transcriptase (2.5 units/␮l), random
hexamers (2.5 ␮M), dNTPs (2 mM each), and RNase inhibitor
(1 unit/␮l) (all from Applied Biosystems, Rotkreuz, Switzerland). The RT reaction was performed in a total volume of
20 ␮l in a GeneAmp PCR cycler (Applied Biosystems) at 25°C
for 10 minutes, followed by 30 minutes at 48°C and then 5
minutes at 95°C. The cDNA was stored at ⫺20°C until
analyzed further. Samples without enzyme were used as negative controls in the RT reaction, to exclude genomic contamination. Quantification of specific messenger RNA (mRNA)
was performed by TaqMan single-reporter and SYBR Green
real-time PCRs, using the ABI Prism 7700 Sequence Detection
System (Applied Biosystems) as described previously (22).
SYBR Green real-time PCR was performed for human
Col␣1(I) (forward primer 5⬘-TCAAGAGAAGGCTCACGATGG-3⬘, reverse primer 5⬘-TCACGGTCACGAACCACATT3⬘) and human fibronectin 1 (forward primer 5⬘-TTCTAAGATTTGGTTTGGGATCAAT-3⬘, reverse primer 5⬘-TCTTGGTTGGCTGCATATGC-3⬘). To confirm specific amplification by the SYBR Green PCR, a dissociation curve analysis
was performed for each primer pair, and both non-RT negative
controls and water controls were used for these analyses. The
amounts of loaded cDNA were normalized using a predeveloped 18S ribosomal RNA control kit (Applied Biosystems) as
an endogenous control.
Differential gene expression was calculated with the
threshold cycle (Ct), and relative quantification was calculated
with the comparative Ct method. Only samples with a difference of at least 4 cycles between the signals in cDNA samples
and negative controls (corresponding to a 24 [16-fold] difference in expression) were considered for the calculations. All
experiments were performed at least in duplicate.
TRICHOSTATIN A IN SKIN FIBROSIS
Measurement of collagen. Total soluble collagen in cell
culture supernatants was quantified using the SirCol collagen
assay (Biocolor, Belfast, Northern Ireland). For these experiments, confluent cells were incubated for 24 hours with 40 ␮l
DMEM/5% FCS per cm2 of culture dish surface. One milliliter
of Sirius red dye, an anionic dye that reacts specifically with
basic side-chain groups of collagens under assay conditions,
was added to 400 ␮l supernatant, followed by incubation under
gentle rotation for 30 minutes at room temperature. After
centrifugation at 12,000g for 10 minutes, the collagen-bound
dye was redissolved with 1 ml of 0.5M NaOH, and the
absorbance was measured at 540 nm in an MRX enzymelinked immunosorbent assay reader (Dynex Technologies,
Chantilly, VA).
Microtiter tetrazolium (MTS) assay. SSc and normal
dermal fibroblasts were incubated with TSA (2 ␮M) in 96-well
plates for 48–72 hours. Cell proliferation was analyzed using an
MTS assay (CellTiter96 AQueous Cell Proliferation Assay;
Promega, Wallisellen, Switzerland) according to the manufacturer’s instructions. For this analysis, an MRX microplate
reader (Dynex Technologies) was used, at a test wavelength of
490 nm. Untreated fibroblasts were used as controls.
Western blot analysis for p21. Induction of the cell
cycle inhibitor p21WAF1/Cip1 by TSA was analyzed by Western
blotting. SSc skin fibroblasts (2 ⫻ 106/well) were incubated in
the absence or presence of TSA (2 ␮M) for 48 hours and lysed
in Laemmli buffer. Protein extracts from SSc fibroblasts were
analyzed by electrophoresis on 10% sodium dodecyl sulfate–
polyacrylamide gels, and transferred onto a nitrocellulose
membrane. The membrane was blocked for 1 hour with Tris
buffered saline (TBS) containing 0.1% Tween 20 (TBST) and
5% dehydrated skim milk. Blots were then incubated overnight
at 4°C in the presence of monoclonal antibodies against
p21WAF1/Cip1 (Cell Signaling Technology, Beverly, MA) or
␣-tubulin (Sigma). The blots were washed 3 times with TBST
for 15 minutes each, and incubated with horseradish
peroxidase–conjugated goat anti-mouse IgG secondary antibodies at room temperature for 1 hour. Signals were detected
with enhanced chemiluminescence Western blot detection
reagents (Amersham Bioscience, Freiburg, Germany) and
exposed to radiographic films (SuperRX; Fujifilm Medical
Systems, Stamford, CT).
Electrophoretic mobility shift assay (EMSA). SSc skin
fibroblasts were cultured to confluence in cell culture flasks
(225 cm2) and incubated with medium only or with TSA
(2 ␮M) for 48 hours. TGF␤ (5 ng/ml; R&D Systems, Abingdon, UK) was then added for 60 minutes. Cells were collected
by scratching in ice-cold phosphate buffered saline (PBS).
Nuclear extracts were prepared according to the protocol
described by Andrews and Faller (23). The concentration of
nuclear protein was determined using the bicinchoninic acid
protein assay reagent kit (Pierce, Rockford, IL), with normalization for the amount of protein within each experiment.
Nonradioactive EMSA was performed using an EMSA
kit (Panomics, Redwood City, CA) according to the manufacturer’s instructions. Three micrograms of nuclear protein was
incubated for 30 minutes at 21°C with biotinylated oligonucleotides containing the Smad3/4 binding site (Panomics). As a
negative control, the binding reaction was performed in the
presence of an excess of unlabeled double-stranded oligonucleotide. The samples were electrophoretically separated
2757
(120V for 1.5–2 hours) in a nondenaturing polyacrylamide gel
(6% with 2.5% glycerol) and blotted (300 mA for 30–40
minutes) on a Biodyne B (0.45 ␮m) positively charged nylon
membrane (Pall, Basel, Switzerland). The transfer buffer contained 20% methanol, 0.27M Tris, and 2M glycine.
After transfer, the membrane was ultraviolet (UV)–
crosslinked at 254 nm for 3 minutes, using a Stratalinker UV
crosslinker (Stratagene, La Jolla, CA). Biotin was labeled
with alkaline phosphatase–conjugated streptavidin (1:1,000;
Dako, Glostrup, Denmark), and streptavidin was detected
with CDP-Star substrate (Applied Biosystems) according to
the manufacturer’s instructions. Chemiluminescence signals
were visualized by exposing the membrane to an Agfa Curix
Ortho HT-A film (Agfa-Gevaert, Kontich, Belgium) for 2
minutes (24).
Confocal microscopy for Smad3. For confocal fluorescence microscopy, SSc skin fibroblasts (5,000/well) were grown
overnight in 24-well plates containing round coverslips (Hecht
Assistant, Altnau, Switzerland). Fresh medium with 10% FCS,
containing TGF␤ (5 ng/ml) and/or TSA (2 ␮M), was then
added. After incubation at 37°C for 1–2 hours, SSc skin
fibroblasts were fixed with methanol and the nuclei were
stained with DAPI.
The intracellular localization of Smad transcription
factors was investigated by confocal fluorescence microscopy.
Cells were incubated with polyclonal goat antibodies against
Smad3 (Santa Cruz Biotechnology, Santa Cruz, CA) or with
control antibodies, followed by incubation with fluorescein
isothiocyanate (FITC)–labeled secondary rabbit anti-goat antibodies (Dako). Confocal fluorescence microscopy was performed using a Leica SP2 inverted microscope (Leica, Wetzlar,
Germany).
Bleomycin-induced dermal fibrosis. Skin fibrosis was
induced in 6–8-week-old, pathogen-free female C3H/HeJ mice
(Sankyo, Tokyo, Japan) by local injection of bleomycin for
24–28 days (25,26). One hundred microliters of bleomycin
dissolved in PBS at a concentration of 0.5 mg/ml was administered every other day by subcutaneous (SC) injection in
defined areas of the upper back. SC injection of 100 ␮l PBS
was used as the control. Two subgroups of mice were treated
with bleomycin followed by TSA in different concentrations.
TSA was dissolved in PBS/1% DMSO and administered every
other day by intraperitoneal (IP) injection in a total volume of
0.1 ml. The concentrations of TSA were 0.5 ␮g/gm/day for
low-dose therapy and 1 ␮g/gm/day for high-dose therapy. After
4 weeks, the animals were killed by CO2 asphyxiation. The
injected skin was removed and processed for histologic analysis. Experiments were performed in 2 independent series.
Histologic analysis. The injected sections of skin were
fixed in 4% formalin and embedded in paraffin. Fivemicrometer sections were stained with hematoxylin and eosin
for the determination of dermal thickness. For analysis of
connective tissue, Masson’s trichrome staining was performed.
Dermal thickness of the injected sections was analyzed
with an Imager1 microscope (Carl-Zeiss, Jena, Germany) at
200-fold magnification, by measuring the distance between
the epidermal–dermal junction and the dermal–subcutaneous
fat junction at sites of induration in 3 consecutive skin sections
from each animal. In each series of experiments, the dermal
thickness was calculated as the fold increase compared with
that in controls. The analysis was performed by 2 independent
2758
HUBER ET AL
examiners (AJ and LCH) who were blinded to the treatment
groups.
Statistical analysis. Results are expressed as the
mean ⫾ SEM. Statistical analysis was performed using GraphPad Prism software, version 4.03 (GraphPad Software, San
Diego, CA). For analysis between different groups, Wilcoxon’s
test was used. P values less than or equal to 0.05 were
considered significant.
RESULTS
Effects of TSA on the expression of profibrotic
molecules at the mRNA and protein levels. TSA was
described as a lead compound in the development of
antifibrogenic drugs for hepatic fibrosis (17). In this
context, we investigated the antifibrogenic effects of
TSA by exposing human SSc skin fibroblasts to TGF␤ (5
ng/ml) alone or in combination with different concentrations of TSA. As shown in Figure 1, levels of mRNA
for both Col␣1(I) and fibronectin were strongly induced
by TGF␤ in SSc skin fibroblasts, as expected. In particular, following treatment with TGF␤, mRNA for
Col␣1(I) was induced a mean ⫾ SEM 2.7 ⫾ 0.5 fold and
mRNA for fibronectin was induced 5.7 ⫾ 1.3 fold.
These effects were abolished when TSA (2 ␮M)
was added simultaneously with TGF␤ to cultures of SSc
skin fibroblasts. In this regard, addition of TSA reduced
the TGF␤-stimulated production of Col␣1(I) mRNA
from a mean ⫾ SEM 2.7 ⫾ 0.5 fold to 0.9 ⫾ 0.1 fold (P ⬍
Figure 1. Effects of trichostatin A (TSA) on the transcription of
extracellular matrix genes. Systemic sclerosis (SSc) and normal dermal
fibroblasts were stimulated with transforming growth factor ␤ (TGF␤)
(5 ng/ml) (solid bars) or with TGF␤ (5 ng/ml) plus TSA (2 ␮M)
(shaded bars), and compared with unstimulated fibroblasts (open bar).
Results are the mean and SEM fold change in levels of mRNA for
Col␣1(I) and fibronectin relative to that in unstimulated cells, from 4
independent experiments; values are normalized to the expression of
18S ribosomal RNA. ⴱ ⫽ P ⬍ 0.05 versus SSc cells treated with TGF␤
alone.
Figure 2. Inhibitory effects of TSA on the release of collagen protein
in SSc dermal fibroblasts. TGF␤, interleukin-4 (IL-4), and plateletderived growth factor (PDGF) up-regulated the de novo synthesis of
collagen by SSc fibroblasts. When TSA (2 ␮M) was added to the
cultures, both the basal and cytokine-induced production of collagen
were inhibited. Results are the mean and SEM from 4 independent
experiments. See Figure 1 for other definitions.
0.05) and the TGF␤-stimulated induction of fibronectin
mRNA from 5.7 ⫾ 1.3 fold to 2.4 ⫾ 1.3 fold (P ⬍ 0.05)
(n ⫽ 4 for each) (Figure 1). In experiments with skin
fibroblasts from healthy controls, TGF␤ induced the
production of Col␣1(I) mRNA a mean ⫾ SEM 2.3 ⫾ 0.8
fold and the production of fibronectin mRNA 3.5 ⫾ 0.6
fold, whereas the addition of TSA reduced the amount
of TGF␤-induced mRNA production to 1.1 ⫾ 0.5 fold
for Col␣1(I) and to 2.4 ⫾ 1.3 fold for fibronectin
(Figure 1).
We next tested the effects of TSA on skin fibroblasts at the protein level, by analyzing the supernatants
of TGF ␤ - and TSA-treated SSc skin fibroblasts
(Figure 2). Similar to the data obtained from the assessments of mRNA, TGF␤ (5 ng/ml) strongly increased the
de novo synthesis of collagen from a mean ⫾ SEM
190 ⫾ 29 pg/ml to 455 ⫾ 43 pg/ml. The combination of
TGF␤ and TSA down-regulated the synthesis of collagen to 170 ⫾ 55 pg/ml (P ⬍ 0.05), which was below the
basal levels found in cultures of unstimulated cells.
Addition of TSA to unstimulated SSc skin fibroblasts
reduced the synthesis of collagen from 190 ⫾ 29 pg/ml to
115 ⫾ 49 pg/ml. These results clearly indicate that TSA
inhibits the synthesis of collagen by SSc skin fibroblasts.
To investigate whether TSA also blocks the action of other profibrotic cytokines, we exposed SSc skin
fibroblasts to IL-4 (5 ng/ml) or PDGF (20 ng/ml) alone
TRICHOSTATIN A IN SKIN FIBROSIS
or in combination with TSA (2 ␮M) for 48 hours. The
stimulatory effects of IL-4 and PDGF on SSc skin
fibroblasts were similar to the effects observed with
TGF␤. The collagen production by SSc fibroblasts was
increased to 288 ⫾ 73 pg/ml following stimulation with
IL-4, and increased to 235 ⫾ 16 pg/ml following stimulation with PDGF. Strikingly, addition of TSA (2 ␮M)
reversed both the IL-4–induced collagen synthesis (to
103 ⫾ 25 pg/ml) and the PDGF-stimulated collagen
synthesis (to 103 ⫾ 22 pg/ml), representing a return to
normal levels, suggesting that TSA functions as an
inhibitor of the final common pathway in the production
of extracellular matrix proteins.
Kinetics of TSA. We found that the observed
effects of TSA were both dose- and time-dependent.
Untreated cells and TGF␤-stimulated cells were exposed to TSA in concentrations of 100 nM, 500 nM,
1 ␮M, and 2 ␮M. Quantification of collagen protein
within 48 hours after incubation showed that TSA acted
in a clear dose-dependent manner, with the highest
reduction in collagen synthesis observed following addition of 2 ␮M TSA and the lowest reduction observed
with 100 nM TSA. These doses are within the physiologic range of concentrations of TSA that have been
applied in vitro in other studies (18,27).
Similarly, TSA acted in a time-dependent manner. Protein levels were measured following a single
addition of TSA and TGF␤ to the cultures, with the
levels determined at 48 hours, 96 hours, and 144 hours
after incubation. The maximal effects of TSA were
observed after incubation for 48 hours (results not
shown). These results confirm that TSA functions as a
reversible HDAC inhibitor.
Previous studies have shown that TSA might lead
to inhibition of the cell cycle as well as apoptotic cell
death (28,29). With the doses of TSA used in the present
study, no increase in the rate of apoptosis could be
observed, as determined by annexin V/propidium iodide
double-staining and fluorescence-activated cell sorter
analysis (results not shown). However, an effect of TSA
was observed on the cell cycle. When SSc skin fibroblasts
were exposed to TSA (2 ␮M), proliferation of fibroblasts
was reduced by 29 ⫾ 3% after 72 hours, as measured by
MTS assay. To investigate the molecular mechanisms of
this TSA-induced cell cycle inhibition, Western blotting
for the cell cycle inhibitor p21 was performed. When
SSc skin fibroblasts were analyzed after exposure to
TSA for 48 hours, p21 was clearly induced (results not
shown).
Levels of Smad transcription factors. The downstream signaling action of TGF␤ is mainly mediated by
2759
Figure 3. Representative results from electrophoretic mobility shift
assay, demonstrating the inhibition of nuclear Smad3/4 by TSA in SSc
dermal fibroblasts. Lane 1, Positive control; lane 2, free probe without
nuclear extracts; lane 3, nuclear extract from unstimulated SSc fibroblasts; lane 4, nuclear extract from TGF␤-stimulated SSc fibroblasts;
lane 5, nuclear extract from unstimulated SSc fibroblasts treated with
TSA; lane 6, nuclear extract from TGF␤-stimulated SSc fibroblasts
treated with TSA; lane 7, nuclear extract from unstimulated SSc
fibroblasts with an excess of unlabeled cold probe; control lanes were
ran on a separate gel. Signals for Smad3/4–DNA complexes provide
evidence of a strong activation of Smad3/4 by TGF␤ (5 ng/ml) (lane 4).
This activation could be inhibited when TSA (2 ␮M) was added (lane
6). See Figure 1 for definitions.
Smad transcription factors. To determine whether TSA
alters the expression pattern of these molecules, EMSA
was performed. SSc skin fibroblasts were exposed to
TGF␤, alone or in combination with TSA, for 2 hours.
As shown in Figure 3, TGF␤ strongly induced the
DNA-binding fraction of the profibrotic Smad3/4. This
effect, however, was almost completely abolished when
the cells were coincubated with TSA.
These findings were also confirmed on the morphologic level, by confocal fluorescence microscopy of
skin fibroblasts using FITC-labeled anti-Smad3 antibodies (Figure 4). In untreated control SSc fibroblasts
(Figure 4a), green fluorescence within the nuclei, representing the presence of Smad3 molecules, could be
observed only sporadically. After exposure of SSc skin
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HUBER ET AL
Figure 4. Representative images from confocal fluorescence microscopy of human SSc dermal fibroblasts. SSc
fibroblasts were cultured on chamber slides with TSA and/or TGF␤ and stained with DAPI for localization of nuclei
(red) and fluorescein isothiocyanate–labeled anti-Smad3 antibodies (green). Intranuclear Smad3 molecules could be
observed only sporadically in untreated SSc fibroblasts (a) and in TSA-treated SSc fibroblasts (b). Stimulation of the
fibroblasts with TGF␤ resulted in cytoplasmic and nuclear distribution of Smad3 molecules (c). Simultaneous
application of TGF␤ in conjunction with TSA completely abolished this TGF␤-induced nuclear translocation in SSc
fibroblasts (d). See Figure 1 for definitions.
fibroblasts to TSA alone (Figure 4b), a similar pattern of
scant Smad3 distribution was seen. When TGF␤ was
added, however, Smad3 molecules were observed to be
distributed over the cytoplasm and within the nucleus
(Figure 4c). The addition of TSA completely abolished
this TGF␤-induced nuclear translocation (Figure 4d).
Effects of TSA on bleomycin-induced dermal
fibrosis in vivo. Following our in vitro studies, we tested
the effects of TSA in vivo using a previously described
animal model of bleomycin-induced skin fibrosis (25).
No obvious toxic effects were observed in TSA-treated
mice.
Repeated SC application of bleomycin caused
marked thickening of the mouse skin at the sites of
injection. When analyzed histologically, the skin from
bleomycin-exposed animals showed clear signs of tissue
fibrosis, including the accumulation of extracellular matrix within the dermis and the adjacent subcutis, thus
leading to replacement of subcutaneous fat tissue with
fibrillar collagen bundles (Figures 5a and b).
When TSA was administered by IP injection after
SC injections of bleomycin, the fibrotic effects of bleomycin were clearly reduced, as shown in Figures 5c and
d. These observations were quantified by the measurement of dermal thickness. In particular, injection of
bleomycin induced an increase in dermal thickness by a
mean ⫾ SEM 71 ⫾ 14% as compared with that in
untreated control mice (P ⱕ 0.001). Injections of TSA
TRICHOSTATIN A IN SKIN FIBROSIS
2761
Figure 5. Prevention of the accumulation of extracellular matrix by trichostatin A (TSA) in experimental dermal fibrosis. Skin fibrosis was induced
in mice by repeated subcutaneous injections of bleomycin (b–d). Groups of mice were either left untreated (b) or treated with intraperitoneal
injections of TSA (c and d) at a concentration of 0.5 ␮g/gm/day (c) or 1.0 ␮g/gm/day (d). Phosphate buffered saline–treated mice served as controls
(a). Injections of TSA prevented an increase in dermal thickness, as analyzed by computer-assisted measurement of the dermo–epidermal distance
(red bars), and also prevented the accumulation of dense collagen bundles (arrows in b) that would replace subcutaneous fat tissue (asterisks in a
and b versus c and d). Representative tissue sections in a–d were examined at the same magnification (original magnification ⫻ 200).
prevented thickening of the skin. At a TSA dosage of
0.5 ␮g/gm/day, the level of bleomycin-induced dermal
fibrosis was significantly reduced (P ⬍ 0.003), to 16 ⫾
4% of that in PBS-treated control animals. Similar levels
of dermal thickness were observed when TSA was
applied IP at a higher dosage (Figure 6), indicating
that therapeutic effects could be achieved even with
submicromolar doses of TSA.
Moreover, morphologic analysis of the mouse
skin with Masson’s trichrome staining, used for detection of collagen fibers on paraffin-embedded tissue
sections, further revealed the dense accumulation of
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HUBER ET AL
Figure 6. Change in dermal thickness in groups of mice with
bleomycin-induced skin fibrosis treated with TSA at either 1 ␮g/gm/
day (TSA high) or 0.5 ␮g/gm/day (TSA low) or left untreated, as
compared with a control group of phosphate buffered saline (PBS)–
treated mice. Results are the mean and SEM fold change in dermal
thickness relative to controls. ⴱ ⫽ P ⬍ 0.05. See Figure 5 for other
definitions.
collagen fibers induced by repeated bleomycin injections, whereas TSA partly prevented this process. In
particular, dermal collagen bundles were less densely
packed, and the subcutaneous fat was still present.
These data are consistent with the results obtained in
vitro and confirm the role of TSA as a promising lead
compound in the development of antifibrogenic drugs.
DISCUSSION
Fibrosis is a clinical hallmark of many disorders,
and excessive accumulation of extracellular matrix
within internal organs ultimately leads to severe complications, such as respiratory distress, liver failure, and
death. At the moment, no effective treatment is known
to inhibit fibrotic processes. In the context of SSc,
numerous studies have identified a profibrotic phenotype of fibroblasts in which the cells are stimulated by
elevated levels of various cytokines at the early stages of
the disease (4,30), ultimately leading to the development
and progression of tissue fibrosis.
Of interest, the profibrotic cellular features characterizing this phenotype remain unchanged during cell
division (8). In addition to genetic mutations and clonal
selection, which have been suggested as possible mechanisms, there has been growing interest in epigenetic
modifications. Most of the evidence regarding the role
of epigenetic alterations in disease has been gained from
the use of epigenetic drugs, such as HDAC inhibitors.
In the present study, we tested the effect of TSA,
the strongest HDAC inhibitor currently known (12), on
collagen production by skin fibroblasts, using in vitro
and in vivo experiments with SSc skin fibroblasts and a
mouse model of bleomycin-induced skin fibrosis. The
addition of TSA normalized the levels of mRNA transcripts for Col␣1(I) (procollagen) and fibronectin, as
well as the levels of collagen protein in cytokinestimulated SSc skin fibroblasts. Consistent with the data
from other studies, our findings indicate the involvement
of epigenetic histone modifications in the expression of
fibrogenic molecules. The use of HDAC inhibitors prevents the removal of acetyl groups from core histones,
and a state of histone hyperacteylation is usually associated with increased rates of gene transcription.
Since TSA inhibited the growth factor–induced
expression of profibrotic molecules in our experiments, several possible mechanisms of action can be
discussed. One possible mechanism is the induction of
genes essential for the suppression of collagen expression. Conversely, TSA or gene products induced by TSA
might interfere with the signaling cascade involved in
tissue fibrosis. Furthermore, TSA might affect the production of cellular products indirectly by, for example,
inducing cell cycle arrest, apoptosis, or cell death. Binding of TGF␤ to the serine/theronine kinase receptor
(TGF␤RII) activates TGF␤RI and initiates a downstream signaling cascade through Smad transcription
factors. In particular, Smad proteins become activated
by phosphorylation. Phosphorylated Smad2 and Smad3
then form a complex with Smad4, resulting in a heteromeric structure that translocates to the nucleus (6,31).
Together with other DNA-binding factors, the Smad
complexes regulate the expression of several profibrotic
genes. Smad6 and Smad7, in contrast, have been linked
to antifibrotic activities.
Of the factors investigated in the present study,
the DNA-binding activities of Smad3 and Smad4 were
clearly affected, and nuclear translocation was inhibited
by TSA. Whether this effect is attributable to epigenetic
modifications of histones or whether TSA directly inhibits the phosphorylation of Smads has to be addressed in
further studies. Moreover, TSA appears to have other
functional properties in addition to the inhibition of
histone deacetylation. Recent reports have described a
novel, histone acetylation–independent mechanism by
which HDAC inhibitors cause dephosphorylation of
intracellular proteins, which challenges the view of the
role of TSA as a sole HDAC inhibitor (32).
Collagen repressors, such FLI1 and p53, were not
investigated in the present study, but previous findings
suggest that FLI1 is affected by the use of epigenetic
TRICHOSTATIN A IN SKIN FIBROSIS
drugs (8). Alternatively, the fact that TSA provokes cell
cycle arrest could be another explanation for the reduced production of collagen proteins in SSc skin fibroblasts. Several studies performed in cancer cells have
shown that TSA induces the expression of
the cyclin-dependent kinase inhibitor p21, thus leading
to cell cycle inhibition in melanoma and glioma cells
(28,33). Consistent with these findings, TSA strongly
induced the expression of p21 in SSc skin fibroblasts,
and the population of proliferating cells was clearly
reduced. Taken together, the findings from our experiments indicate that TSA prevents the accumulation of
extracellular matrix by several mechanisms, including
epigenetic alterations of the expression pattern of Smad
transcription factors, and interference with regulation of
the cell cycle.
Our data are consistent with the results from
previous studies in which the antifibrotic properties of
TSA have been demonstrated (18,27). However, our
study amends the data obtained in vitro, with experiments performed in vivo using a common animal model
of skin fibrosis. In the bleomycin-induced fibrosis mouse
model, micromolecular concentrations of TSA prevented the development of skin fibrosis without the
occurrence of obvious toxic side effects. After application of 0.5 ␮g/gm of TSA per day, the bleomycin-induced
collagen bundles within the dermis and the replacement
of subcutaneous fat tissue were reduced to levels comparable with those in the skin of untreated animals.
However, increasing the applied concentration of TSA
to 1.0 ␮g/gm/day did not result in further improvement.
These findings indicate that the therapeutic effects of
TSA could be achieved even with doses in the submicromolar range.
In conclusion, we have shown that the HDAC
inhibitor TSA has potent antifibrogenic effects on SSc
skin fibroblasts in vitro. With respect to the pivotal role
of profibrotic cytokines such as TGF␤, PDGF, and IL-4
in the pathogenesis of fibrotic diseases, we were able to
show that TSA abrogates the stimulating effects of these
factors on extracellular matrix production. Moreover, in
a mouse model of bleomycin-induced fibrosis, TSA
prevented the development of skin fibrosis as quantified
by changes to the dermal thickness within the bleomycin
injection sites. With regard to the strong antifibrogenic
activities of TSA, we propose that TSA and related
inhibitory compounds should be considered for use as
early pharmacologic strategies to conquer the development and progression of fibrosis in SSc and other
cytokine-driven fibrotic entities.
2763
ACKNOWLEDGMENTS
We thank all staff members of the laboratory of
electron microscopy at the University of Zurich for their
excellent support, in particular, Dr. M. Hoechli for providing
assistance with the confocal microscopy.
AUTHOR CONTRIBUTIONS
Dr. Huber had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Huber, Distler, Hauser, R. E. Gay, Matucci-Cerinic, S.
Gay, Distler, Jüngel.
Acquisition of data. Huber, Moritz, Hemmatazad, Hauser, Jüngel.
Analysis and interpretation of data. Huber, Distler, S. Gay, Jüngel.
Manuscript preparation. Huber, Moritz, Michel, R. E. Gay, S. Gay,
Distler, Jüngel.
Statistical analysis. Huber.
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