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Histone deacetylase 7 a potential target for the antifibrotic treatment of systemic sclerosis.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 5, May 2009, pp 1519–1529
DOI 10.1002/art.24494
© 2009, American College of Rheumatology
Histone Deacetylase 7, a Potential Target for the Antifibrotic
Treatment of Systemic Sclerosis
Hossein Hemmatazad,1 Hanna Maciejewska Rodrigues,1 Britta Maurer,1 Fabia Brentano,1
Margarita Pileckyte,2 Jörg H. W. Distler,3 Renate E. Gay,1 Beat A. Michel,1 Steffen Gay,1
Lars C. Huber,1 Oliver Distler,1 and Astrid Jüngel1
Objective. We have recently shown a significant
reduction in cytokine-induced transcription of type I
collagen and fibronectin in systemic sclerosis (SSc) skin
fibroblasts upon treatment with trichostatin A (TSA).
Moreover, in a mouse model of fibrosis, TSA prevented
the dermal accumulation of extracellular matrix. The
purpose of this study was to analyze the silencing of
histone deacetylase 7 (HDAC-7) as a possible mechanism by which TSA exerts its antifibrotic function.
Methods. Skin fibroblasts from patients with SSc
were treated with TSA and/or transforming growth
factor ␤. Expression of HDACs 1–11, extracellular
matrix proteins, connective tissue growth factor
(CTGF), and intercellular adhesion molecule 1
(ICAM-1) was analyzed by real-time polymerase chain
reaction, Western blotting, and the Sircol collagen assay. HDAC-7 was silenced using small interfering RNA.
Results. SSc fibroblasts did not show a specific
pattern of expression of HDACs. TSA significantly
inhibited the expression of HDAC-7, whereas HDAC-3
was up-regulated. Silencing of HDAC-7 decreased the
constitutive and cytokine-induced production of type I
and type III collagen, but not fibronectin, as TSA had
done. Most interestingly, TSA induced the expression of
CTGF and ICAM-1, while silencing of HDAC-7 had no
effect on their expression.
Conclusion. Silencing of HDAC-7 appears to be
not only as effective as TSA, but also a more specific
target for the treatment of SSc, because it does not
up-regulate the expression of profibrotic molecules such
as ICAM-1 and CTGF. This observation may lead to the
development of more specific and less toxic targeted
therapies for SSc.
Systemic sclerosis (SSc) is an autoimmune disease characterized by widespread vascular changes and
progressive fibrosis of the skin and internal organs. The
etiology and pathogenesis of SSc are still unknown.
Currently, no effective treatment is available to inhibit
the progression of SSc.
The term “epigenetics” refers to changes in gene
activity that are stable and inherited over rounds of cell
division, but do not involve changes in the DNA sequence of the organism. Several epigenetic modifications have been described, such as DNA methylation
and histone acetylation. Under normal conditions, these
modifications are balanced and reversible, but they may
be altered in disease states. Epigenetic modifications
have been shown to play a role in the pathogenesis of
cancer as well as autoimmune and inflammatory disorders (1,2). Several recent publications have reported
epigenetic modifications in gene transcription in SSc
(3–6). Therefore, the field of epigenetics may provide
new therapeutic targets for treatment strategies.
Dr. Hemmatazad’s work was supported by Schwyzer Stiftung
and by the European Union Sixth Framework Programme (project
AutoCure). Ms Maciejewska Rodrigues’ work was supported by a
Marie Curie grant from the European Union and by the Zurich Center
of Integrative Human Physiology. Dr. Maurer’s work was supported by
the Olga-Mayenfisch Foundation. Dr. Jüngel’s work was supported by
the Swiss National Science Foundation (grant 320000-11684) and by
the European Union Sixth Framework Programme (project AutoCure).
1
Hossein Hemmatazad, MD, Hanna Maciejewska Rodrigues,
Britta Maurer, MD, Fabia Brentano, PhD, Renate E. Gay, MD, Beat
A. Michel, MD, Steffen Gay, MD, Lars C. Huber, MD, Oliver Distler,
MD, Astrid Jüngel, PhD: University Hospital Zurich, and Zurich
Center of Integrative Human Physiology, Zurich, Switzerland; 2Margarita Pileckyte, MD: Kaunas Medical University Hospital, Kaunas,
Lithuania; 3Jörg H. W. Distler, MD: Center of Experimental Rheumatology, University Hospital Zurich, and Zurich Center of Integrative Human Physiology, Zurich, Switzerland, and University of
Erlangen–Nuremberg, Erlangen, Germany.
Address correspondence and reprint requests to Hossein
Hemmatazad, MD, Center of Experimental Rheumatology, University
Hospital Zurich, Gloriastrasse 25, 8091 Zurich, Switzerland. E-mail:
hossein.hemmatazad@usz.ch.
Submitted for publication June 25, 2008; accepted in revised
form February 9, 2009.
1519
1520
The DNA in eukaryotic cells is tightly wrapped
around octamers of histone proteins, restricting its accessibility to factors involved in DNA replication and
transcription. Posttranslational modifications of histone
proteins induce changes in the structure of chromatin
and, therefore, modify gene expression. Hyperacetylated
histones are generally found in transcriptionally active
regions and hypoacetylated histones in transcriptionally
silent regions. The acetylation state of chromatin proteins depends on the balance between the activities of
histone deacetylases (HDACs) and histone acetyltransferases (HATs). Based on structure and biologic activities, mammalian HDACs can be classified into 4 different classes: class I (HDACs 1, 2, 3, and 8), class II
(HDACs 4, 5, 6, 7, 9, and 10), class III (sirtuins 1–7), and
class IV (HDAC-11) (7). HDACs have been shown to be
promising therapeutic targets for cancer therapy, as well
as for inflammatory or fibrotic diseases (5,8,9).
HDAC inhibitors have emerged as a new class of
agents for anticancer therapy. HDAC inhibitors alter the
balance of acetylation and affect many aspects of cellular function, including cell growth, differentiation, cell
death, cell–cell and cell–matrix interactions, and inflammatory responses (10). One of the first discovered
HDAC inhibitors with antitumor activity was trichostatin A (TSA). However, the toxicity of TSA is still a
subject of controversy (11). Even though individual
HDAC isoforms have distinctive physiologic functions,
most known HDAC inhibitors target class I, class II, and
class IV HDACs rather nonselectively. The production
of inhibitors for specific HDAC isoforms is now the
focus of the pharmaceutical industry (12). Therefore,
there is increasing interest in unraveling the properties
and functions of each isoform and exploring their individual roles in the pathogenesis of certain diseases.
We have recently shown a significant reduction in
cytokine-induced transcription of type I collagen and
fibronectin in SSc skin fibroblasts upon treatment with
TSA. In addition, we have demonstrated that the expression of total collagen protein in stimulated SSc skin
fibroblasts was reduced by treatment with TSA. We have
also shown that TSA prevents the dermal accumulation
of extracellular matrix in vivo in the mouse model of
bleomycin-induced fibrosis (6). In the present study, we
analyzed the molecular mechanisms of TSA-mediated
reduction of extracellular matrix in SSc. In order to
define new target molecules, we measured the transcription levels of individual HDACs (HDACs 1–11) in SSc
fibroblasts treated with TSA.
We showed that TSA has different effects on
HEMMATAZAD ET AL
individual HDACs. Most interestingly, TSA almost completely inhibited the transcription of HDAC-7, whereas
the transcripts for HDAC-3 were up-regulated. Specific
gene knockdown of HDAC-7 in SSc fibroblasts resulted
in reduced production of type I and type III collagen on
both the messenger RNA (mRNA) and protein levels.
Therapies that specifically target HDAC-7 could be
safer than the nonselective HDAC inhibitor TSA in the
treatment of SSc, because silencing of HDAC-7 did not
increase the expression of the profibrotic molecules
intercellular adhesion molecule 1 (ICAM-1) and connective tissue growth factor (CTGF). Our results
strongly support the targeting of HDAC-7 to generate a
more specific and less toxic antifibrotic agent for the
treatment of SSc.
MATERIALS AND METHODS
Patients and fibroblast cultures. Normal and SSc
fibroblasts were obtained from the skin of healthy subjects and
patients with scleroderma. All patients fulfilled the criteria for
SSc as described by LeRoy et al (13). All patients signed a
consent form approved by the Institutional Review Board of
Kaunas University.
Primary cultures of human dermal fibroblasts were
established by outgrowth and were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% heatinactivated fetal calf serum (FCS), 25 mM HEPES, 100
units/ml of penicillin, 100 ␮g/ml of streptomycin, 2 mM
L-glutamine, and 2.5 ␮g/ml of amphotericin B (all from Gibco
BRL, Basel, Switzerland). Fibroblasts from passages 3–8 in
monolayer culture were used for the experiments. Cells were
treated with 10 nM to 2 ␮M TSA (Sigma, Buchs, Switzerland).
TaqMan reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was isolated from cultured cells
using an RNeasy kit, including DNase treatment (Qiagen,
Basel, Switzerland) according to the instructions of the manufacturer. To generate complementary DNA (cDNA), total
RNA (300–500 ng) was reverse transcribed using 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 reverse transcriptase 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 by 5 minutes at 95°C. Samples without enzyme in the
RT reaction were used as negative controls to exclude contamination with genomic DNA.
Quantification of specific mRNA was performed by
single-reporter and SYBR Green real-time PCR using an ABI
Prism 7700 Sequence Detection System (Applied Biosystems)
as described previously (14). Predeveloped primer probes were
used for HDACs 1, 2, and 8, platelet-derived growth factor B
(PDGF-B), and PDGF receptor ␤ (PDGFR␤) (all from Applied Biosystems). SYBR Green real-time PCR was performed
for human COL1A1 and fibronectin as described elsewhere
(6).
HDAC-7 AS A TARGET FOR ANTIFIBROTIC THERAPY FOR SSc
The following primers were designed and used: for
ICAM-1, 5⬘-CCT-ATG-GCA-ACG-ACT-CCT-TC-3⬘ (forward) and 5⬘-TGC-GGT-CAC-ACT-GAC-TGA-G-3⬘ (reverse); for COL3A1, 5⬘-GGC-ATG-CCA-CAG-GGA-TTCT-3⬘ (forward) and 5⬘-GCA-GCC-CCA-TAA-TTT-GGTTTT-3⬘ (reverse); for HDAC-3, 5⬘-ATG-CAA-GGC-TTCACC-AAG-AG-3⬘ (forward) and 5⬘-CAG-TCA-TCG-CCTACG-TTG-AA-3⬘ (reverse); for HDAC-4, 5⬘-TGT-ACGACG-CCA-AAG-ATG-AC-3⬘ (forward) and 5⬘-CGG-TTCAGA-AGC-TGT-TTT-CC-3⬘ (reverse); for HDAC-5, 5⬘CAG-CAG-GCG-TTC-TAC-AAT-GA-3⬘ (forward) and 5⬘CGA-TGC-AGA-GAG-ATG-TAG-AGC-A-3⬘ (reverse); for
HDAC-6, 5⬘-GAA-AGT-CAC-CTC-GGC-ATC-AT-3⬘ (forward) and 5⬘-TAG-TCT-GGC-CTG-GAG-TGG-AC-3⬘ (reverse); for HDAC-7, 5⬘-ATG-GGG-GAT-CCT-GAG-TACCT-3⬘ (forward) and 5⬘-GAT-GGG-CAT-CAC-GAC-TATCC-3⬘ (reverse); for HDAC-9, 5⬘-CTG-GAG-CCC-ATCTCA-CCT-T-3⬘ (forward) and 5⬘-TCA-TCA-TCC-TGAGGT-CTG-TCC-3⬘ (reverse); for HDAC-10, 5⬘-GCC-GGATAT-CAC-ATT-GGT-TC-3⬘ (forward) and 5⬘-GAC-GCTTCC-TGT-TGG-ATG-A-3⬘ (reverse); and for HDAC-11, 5⬘GGT-CAG-GAA-GGG-GTA-CAG-GT-3⬘ (forward) and 5⬘ATT-GAG-GGG-GAA-CTC-CAG-AT-3⬘ (reverse).
To confirm specific amplification by SYBR Green
PCR, dissociation curve analysis was performed for each
primer pair, and negative RT controls and water controls were
analyzed for all samples. The amounts of loaded cDNA were
normalized using a predeveloped 18S assay (Applied Biosystems) as an endogenous control. Differential gene expression
was calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification. Only samples with
a difference of at least 4 cycles between the signals in cDNA
samples and the negative RT controls (corresponding to a
16-fold difference in expression) were considered in the calculations. All samples were analyzed in duplicate.
Collagen measurements. Total soluble collagen in cell
culture supernatants was quantified using the Sircol collagen
assay (Biocolor, Belfast, UK). For these experiments, confluent cells were incubated for 24 hours with 40 ␮l of DMEM/
10% FCS per cm2 of culture dish surface. Sirius Red (1 ml), an
anionic dye that reacts specifically with (Gly-X-Y)n tripeptide
in the triple-helix sequence of mammalian collagens under
assay conditions, was added to 100 ␮l of supernatant and
incubated under gentle rotation for 30 minutes at room
temperature. After centrifugation for 10 minutes at 12,000g,
the collagen-bound dye was redissolved with 1 ml of 0.5M
NaOH, and the absorbance was measured at 540 nm in an
MRX enzyme-linked immunosorbent assay reader (Dynex,
Alexandria, VA). All samples were measured in duplicate.
Western blot analysis. For Western blot analysis, SSc
skin fibroblasts (2 ⫻ 105 cells/well) were incubated for 48 hours
in the presence or absence of TSA. Whole cell lysates were
prepared by lysing cells in 2⫻ concentrated Laemmli buffer
(100 mM Tris HCl [pH 6.8], 40% glycerol, 10% sodium dodecyl
sulfate [SDS], 0.7M ␤-mercaptoethanol, and 0.0005% bromphenol blue). Proteins were separated on an SDS–
polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour at room
temperature in 5% nonfat dry milk with 0.05% Tween 20 in
TBS (pH 7.4) and were probed overnight at 4°C with antibodies against HDAC-7, type I collagen, type III collagen,
1521
ICAM-1, PDGF-B, PDGFR␤, and CTGF (Santa Cruz Biotechnology, Santa Cruz, CA) or ␣-tubulin (Sigma). The blots
were washed and incubated for 1 hour at room temperature
with horseradish peroxidase–conjugated secondary antibodies
(Jackson ImmunoResearch, Magden, Switzerland) in 5% nonfat dry milk with 0.05% Tween 20 in TBS (pH 7.4). Bound
antibodies were visualized using enhanced chemiluminescence
(Amersham Pharmacia Biotech, Little Chalfont, UK). Evaluation of the expression of specific proteins was performed with
the Alpha Imager Software system (Alpha Innotech, San
Leandro, CA) via pixel quantification of the electronic image.
Small interfering RNA (siRNA). Fibroblasts from patients with SSc were transfected using an Amaxa Basic Nucleofector kit (Amaxa, Gaithersburg, MD) as described previously
(14). For silencing of HDAC-7, we used HDAC-7 siRNA
(Santa Cruz Biotechnology/Qiagen) and scrambled siRNA
representing an irrelevant coding siRNA (Applied Biosystems)
as a control, which does not target any gene product. The
protocol we used has been optimized and recommended by the
manufacturer. Briefly, proper amounts of cells (5 ⫻ 105 cells)
were resuspended in 100 ␮l of Nucleofector solution, mixed
with 5 ␮l of siRNA (from a 10 ␮M solution), and transfection
was performed using an Amaxa Nucleofector device (Program
U23). After incubation for 24 hours, fresh medium was added,
and the cells were incubated for another 24 hours before RNA
extraction.
Statistical analysis. All data are expressed as the
mean ⫾ SD. Statistical analysis was performed with the
Mann-Whitney U test, using SPSS software (SPSS, Chicago,
IL). P values less than 0.05 were considered significant.
RESULTS
No disease-specific pattern of HDAC mRNA expression in SSc skin fibroblasts. Skin fibroblasts from
SSc patients (n ⫽ 5) and healthy controls (n ⫽ 4) were
analyzed for the expression of all known HDACs
(HDACs 1–11) by TaqMan real-time PCR. Comparing
the ⌬Ct levels for each HDAC, there was no significant
difference in the pattern of constitutive expression of
HDACs in fibroblasts from patients with SSc and those
from healthy controls (Figure 1A). Since the present
study focused on HDAC-7, the expression of this particular HDAC was also measured at the protein level.
The expression of HDAC-7 in SSc fibroblasts was similar to that in healthy control fibroblasts (Figure 1B).
Thus, fibroblasts from patients with SSc show no
disease-specific pattern of HDACs at the transcriptional
level.
Different effects of TSA on the expression of
HDAC-3 and HDAC-7 in SSc fibroblasts. TSA is considered to be a nonspecific HDAC inhibitor. Nevertheless, the direct effects of TSA on the expression of
individual HDACs 1–11 in SSc fibroblasts have not
previously been investigated. Therefore, we treated SSc
fibroblasts with TSA (2 ␮M) (6) for 24 hours and
1522
HEMMATAZAD ET AL
Figure 1. Expression of histone deacetylases (HDACs) in skin fibroblasts from systemic sclerosis (SSc) patients and normal controls
and effects of trichostatin A (TSA) on the expression levels of HDAC-3 and HDAC-7 in SSc fibroblasts. A, Expression of mRNA for
HDACs 1–11 in SSc (n ⫽ 5) and normal (n ⫽ 4) fibroblasts was analyzed by real-time polymerase chain reaction (PCR). Values are
the mean ⫾ SD fold change relative to normal cells, which was set at 1. B, Total cellular extracts of normal and SSc fibroblasts were
analyzed by Western blotting with anti–HDAC-7 antibodies. Blots were stripped and reprobed with anti–␣-tubulin antibodies as a
loading control for normalization. C, Expression of mRNA for HDAC-3 (n ⫽ 6) and HDAC-7 (n ⫽ 7) in untreated (control) and
TSA-treated (2 ␮M for 24 hours) SSc fibroblasts was analyzed by TaqMan real-time PCR. Values are the mean ⫾ SD fold change
relative to control, which was set at 1. D, Protein levels of HDAC-3 and HDAC-7 in untreated (control) and TSA-treated SSc
fibroblasts were analyzed by Western blotting with anti–HDAC-3 and anti–HDAC-7 antibodies. Blots were stripped and reprobed with
anti–␣-tubulin antibodies as a loading control for normalization. Results from 3 independent experiments are shown.
Figure 2. Effects of trichostatin A (TSA) on the expression of different types of collagen in fibroblasts from patients with
systemic sclerosis (SSc). A, Expression of COL1A1 and COL3A1 in untreated (control) and TSA-treated SSc fibroblasts (n ⫽
5) was analyzed by real-time polymerase chain reaction. Values are the mean ⫾ SD change in percentage relative to control,
which was set at 100%. The expression of 18S was used for normalization. B, Protein levels of type I and type III collagen in
untreated (control) and TSA-treated (48 hours) SSc fibroblasts were analyzed by Western blotting with anti–type I collagen and
anti–type III collagen antibodies. Blots were stripped and reprobed with anti–␣-tubulin antibodies as a loading control for
normalization.
HDAC-7 AS A TARGET FOR ANTIFIBROTIC THERAPY FOR SSc
1523
Figure 3. Transfection efficiency of histone deacetylase 7 (HDAC-7) small interfering RNA (siRNA) and expression of HDAC-3
after specific gene knockdown of HDAC-7 in fibroblasts from patients with systemic sclerosis (SSc). A, Expression of mRNA for
HDAC-7 in SSc fibroblasts after specific gene knockdown using siRNA compared with control siRNA (scrambled siRNA representing
an irrelevant coding siRNA) was analyzed by TaqMan real-time polymerase chain reaction (PCR). Values are the mean ⫾ SD change
in percentage relative to control, which was set at 100% (n ⫽ 10). B, Expression of HDAC-7 protein in SSc fibroblasts after inhibition
with siRNA as compared with control siRNA was analyzed by Western blotting with anti–HDAC-7 antibodies. Blots were stripped and
reprobed with anti–␣-tubulin antibodies as a loading control for normalization. C, Expression of mRNA for HDAC-3 in SSc fibroblasts
(n ⫽ 6) treated with HDAC-7 siRNA was analyzed by TaqMan real-time PCR. Values are the mean ⫾ SD fold change relative to
control, which was set at 1. D, The effect of HDAC-7 gene knockdown on the levels of HDAC-3 protein in SSc fibroblasts (n ⫽ 3) was
analyzed by Western blotting with anti–HDAC-3 antibodies. Blots were stripped and reprobed with anti–␣-tubulin antibodies as a
loading control for normalization.
analyzed the mRNA expression of HDACs 1–11 by
TaqMan real-time PCR.
HDAC-7 mRNA was strongly reduced to a
mean ⫾ SD of 0.06 ⫾ 0.01–fold (P ⱕ 0.001) after
treatment with TSA. HDAC-3 mRNA was increased to
5.13 ⫾ 1.46–fold (P ⱕ 0.002) after TSA treatment
(Figure 1C). The transcription levels of all other
HDACs were down-regulated by TSA: to 0.75 ⫾ 0.46–
fold for HDAC-1, 0.59 ⫾ 0.19–fold for HDAC-2, 0.75 ⫾
0.14–fold for HDAC-4, 0.85 ⫾ 0.23–fold for HDAC-5,
0.53 ⫾ 0.15–fold for HDAC-6, 0.87 ⫾ 0.06–fold for
HDAC-8, 0.35 ⫾ 0.06–fold for HDAC-9, 0.67 ⫾ 0.20–
fold for HDAC-10, and 0.67 ⫾ 0.10–fold for HDAC-11
(n ⫽ 3 each).
The mRNA results for HDAC-3 and HDAC-7
were confirmed at the protein level by Western blotting (Figure 1D). Densitometric analysis of the Western blots showed that TSA reduced the expression of
HDAC-7 to 0.037 ⫾ 0.05–fold (P ⬍ 0.05) and increased the expression of HDAC-3 to 2.19 ⫾ 0.76–
fold (P ⬍ 0.05) (n ⫽ 3 each). In conclusion, we found
the most pronounced effects of TSA on the expression
of 2 individual HDACs, namely, HDAC-3 and
HDAC-7.
1524
HEMMATAZAD ET AL
Figure 4. Expression of different types of collagen in histone deacetylase 7 (HDAC-7) small interfering RNA (siRNA)–treated
fibroblasts from patients with systemic sclerosis (SSc). A, Constitutive (unstimulated) and transforming growth factor ␤ (TGF␤)–
induced levels of mRNA for COL1A1 and COL3A1 in SSc fibroblasts (n ⫽ 6) after HDAC-7 knockdown using siRNA compared with
control siRNA (scrambled siRNA representing an irrelevant coding siRNA) were analyzed by quantitative TaqMan real-time
polymerase chain reaction. Values are the mean ⫾ SD change in percentage relative to control, which was set at 100%. B, Expression
of type I and type III collagen in total cellular extracts from HDAC-7 siRNA and control siRNA fibroblasts was analyzed by Western
blotting with anti–type I collagen and anti–type III collagen antibodies. Blots were stripped and reprobed with anti–␣-tubulin
antibodies as a loading control for normalization. C, Type I collagen and type III collagen in whole cell lysates of TGF␤-stimulated
SSc fibroblasts transfected with HDAC-7 siRNA or control siRNA were analyzed by Western blotting. Blots were stripped and
reprobed with anti–␣-tubulin antibodies as a loading control for normalization. D, Total soluble collagen production in supernatants
of HDAC-7 siRNA and control siRNA fibroblasts was measured by the Sircol collagen assay. Values are the mean ⫾ SD change in
percentage relative to control, which was set at 100% (n ⫽ 5 experiments).
Expression of different types of collagen after
TSA treatment. Patients with SSc have increased levels
of type I and type III collagen, with type I being the most
abundant (15). In order to examine whether TSA regulates the expression of collagen, we performed a TaqMan real-time PCR analysis for COL1A1 and COL3A1
in SSc fibroblasts (n ⫽ 5). TSA down-regulated the
expression of mRNA for COL1A1 and COL3A1 by a
mean ⫾ SD of 48 ⫾ 6% and 67 ⫾ 9%, respectively
(Figure 2A). Western blotting was performed to confirm
the results at the protein level (Figure 2B). Densitometric analysis of the Western blots revealed a reduction in
type I and type III collagen by TSA treatment to 0.23 ⫾
0.09–fold and 0.13 ⫾ 0.02–fold, respectively (P ⬍ 0.05)
(n ⫽ 3). The expression of type I and type III collagen
was reduced by TSA to the same extent after stimulation
with transforming growth factor ␤ (TGF␤) (data not
shown). Therefore, both constitutive and induced expression of different types of collagen was downregulated after treatment with TSA.
Specific gene knockdown of HDAC-7. As shown
above, HDAC-7 was considerably down-regulated after
treatment with TSA. To test whether the effects of TSA
on SSc fibroblasts were mainly mediated through the
down-regulation of HDAC-7, HDAC-7 was knocked
down using an RNA interference (RNAi) approach. As
shown in Figure 3A, gene expression of HDAC-7 was
suppressed by 76.20 ⫾ 8.48% (n ⫽ 10; P ⫽ 0.001) in
HDAC-7 AS A TARGET FOR ANTIFIBROTIC THERAPY FOR SSc
1525
Figure 5. Expression of platelet-derived growth factor B (PDGF-B) and PDGF receptor ␤ (PDGFR␤) in histone deacetylase
7 (HDAC-7) small interfering RNA (siRNA)–treated fibroblasts from patients with systemic sclerosis (SSc). A and B,
Expression of mRNA for PDGF-B (A) and PDGFR␤ (B) (n ⫽ 6 each) in SSc fibroblasts transfected with HDAC-7 siRNA as
compared with control siRNA (scrambled siRNA representing an irrelevant coding siRNA) was analyzed by real-time
polymerase chain reaction. Values are the mean ⫾ SD fold change relative to control, which was set at 1. C and D, PDGF-B
(n ⫽ 2) (C) and PDGFR␤ (D) (n ⫽ 3) in whole cell lysates of SSc fibroblasts transfected with HDAC-7 siRNA or control siRNA
were analyzed by Western blotting. Blots were stripped and reprobed with anti–␣-tubulin antibodies as a loading control for
normalization.
HDAC-7 siRNA–treated cells after 48 hours of transfection, as compared with the RNAi control. HDAC-7
was also down-regulated at the protein level 72 hours
after transfection (Figure 3B), demonstrating the successful knockdown of HDAC-7 by siRNA.
Effect of specific gene knockdown of HDAC-7 on
the expression of HDAC-3. Since we observed a significant down-regulation of HDAC-7 after TSA treatment,
while the expression of HDAC-3 was up-regulated, we
wanted to evaluate whether the expression of HDAC-3
was affected by HDAC-7 silencing. Therefore, we measured the expression level of HDAC-3 after knockdown
of HDAC-7 in SSc fibroblasts (n ⫽ 6). Using TaqMan
real-time PCR and Western blot analysis, no considerable changes in HDAC-3 expression at either the
mRNA or the protein level were observed (Figures 3C
and D).
Expression of extracellular matrix proteins after
HDAC-7 silencing. To address the question of whether
the effects of TSA on extracellular matrix components
Figure 6. Expression of connective tissue growth factor (CTGF) and
intercellular adhesion molecule 1 (ICAM-1) in trichostatin A (TSA)–
treated and histone deacetylase 7 (HDAC-7) small interfering RNA
(siRNA)–treated fibroblasts from patients with systemic sclerosis
(SSc). A, Expression of CTGF and ICAM-1 in untreated (control) and
TSA-treated (48 hours) SSc fibroblasts (n ⫽ 2) was analyzed by
Western blotting. Blots were stripped and reprobed with anti–␣tubulin antibodies as a loading control for normalization. B, Expression of CTGF and ICAM-1 in SSc fibroblasts (n ⫽ 2) after HDAC-7
knockdown using siRNA compared with control siRNA (scrambled
siRNA representing an irrelevant coding siRNA) was analyzed by
Western blotting. Blots were stripped and reprobed with anti–␣tubulin antibodies as a loading control for normalization.
1526
are mediated through HDAC-7, we investigated the
expression of extracellular matrix proteins after HDAC7–specific gene knockdown. Whereas the expression of
fibronectin remained unchanged (data not shown), both
constitutive and cytokine-induced gene expression of
COL1A1 and COL3A1 were significantly reduced by
silencing of HDAC-7. The constitutive and TGF␤induced down-regulation of mRNA levels of COL1A1
were 27.0 ⫾ 2.4% (n ⫽ 6; P ⬍ 0.05) and 36.0 ⫾ 14%
(n ⫽ 7; P ⬍ 0.05), respectively (mean ⫾ SD), and for
COL3A1, they were 23.0 ⫾ 4.0% (n ⫽ 5; P ⬍ 0.05) and
43.0 ⫾ 9.0% (n ⫽ 5; P ⬍ 0.05), respectively (Figure 4A).
Western blot analysis was performed to confirm the
results at the protein level (Figures 4B and C). In
addition, using the Sircol collagen assay, the production
of total soluble collagen was found to be reduced by
26 ⫾ 5.7% (n ⫽ 5; P ⬍ 0.05), as shown in Figure 4D. In
conclusion, we found a significant down-regulation of
the expression of type I and type III collagen after
specific gene knockdown of HDAC-7.
Effects of HDAC-7 silencing on the expression of
PDGF-B and PDGFR␤ in SSc fibroblasts. PDGF plays
an important role in the pathogenesis of SSc. Even
though it is almost undetectable in healthy skin, studies
have revealed the increased presence of PDGF and
PDGFRs in biopsy samples of SSc skin (16). According
to Mottet et al (17), HDAC-7 silencing up-regulated the
expression of PDGF-B and its receptor PDGFR␤ in
endothelial cells. In order to examine whether gene
knockdown of HDAC-7 alters the expression of
PDGF-B and PDGFR␤ in SSc fibroblasts, we analyzed
changes in the transcript expression levels of these genes
in cells treated with HDAC-7 siRNA (n ⫽ 6 each). As
shown in Figures 5A and B, there was no significant
change in the levels of PDGF-B or PDGFR␤ in
HDAC-7 siRNA–treated cells versus cells treated with
the RNAi control. Western blotting was performed to
confirm the results at the protein level (Figures 5C and
D). In conclusion, silencing of HDAC-7 had no significant effect on the expression of PDGF-B or PDGFR␤ in
SSc fibroblasts.
Up-regulation of CTGF and ICAM-1 expression
in SSc fibroblasts by treatment with TSA, but not with
silencing of HDAC-7. CTGF is induced by TGF␤ and
modulates fibroblast cell growth, but it also mediates
many of the profibrotic actions of TGF␤ (18). ICAM-1,
an inducible surface glycoprotein that promotes adhesion in immunologic and inflammatory reactions, plays a
role in a variety of inflammatory and neoplastic diseases.
ICAM-1 also contributes significantly to the develop-
HEMMATAZAD ET AL
ment of skin fibrosis, especially via ICAM-1 expression
in skin fibroblasts (19).
To investigate whether acetylation induced by
TSA and specific gene knockdown of HDAC-7 might
play a role in the regulation of CTGF and ICAM-1
proteins, we analyzed by Western blotting the expression
of CTGF and ICAM-1 after TSA treatment as well as
after silencing of HDAC-7. As shown in Figure 6A,
CTGF and ICAM-1 were up-regulated after 48 hours of
treatment with TSA, to 2.55 ⫾ 0.19–fold (n ⫽ 3; P ⬍
0.05) and 21.34 ⫾ 16.71–fold (n ⫽ 3; P ⱕ 0.05),
respectively. Of interest, the expression of CTGF and
ICAM-1 in HDAC-7 siRNA–treated cells remained
unchanged as compared with cells treated with the
siRNA control (Figure 6B). It can be concluded that
silencing of HDAC-7, in contrast to TSA treatment,
does not induce the expression of the profibrotic molecules CTGF and ICAM-1 in SSc fibroblasts, and
HDAC-7 silencing might therefore be a more specific
antifibrotic therapeutic strategy than TSA treatment.
DISCUSSION
The results of this study demonstrate that silencing of HDAC-7, a class II HDAC, might be a more
specific and effective antifibrotic therapeutic approach
in SSc than the use of TSA. Silencing of HDAC-7
significantly reduced the excessive production of extracellular matrix proteins, a characteristic feature of SSc
fibroblasts, without increasing other known profibrotic
molecules, such as ICAM-1 and CTGF.
Recently, we have shown that the nonselective
HDAC inhibitor TSA blocks the cytokine-induced production of type I collagen and fibronectin in fibroblasts
from patients with SSc. In addition, in the bleomycininduced mouse model of skin fibrosis, TSA prevented
the dermal accumulation of extracellular matrix in vivo
(6). The present study is the first to show that TSA not
only blocks the enzymatic activity of HDAC, but it
regulates the protein levels of selective targets by influencing their transcription. We found that almost all
transcripts of the HDACs were reduced by TSA. However, the most interesting finding was that although TSA
almost completely inhibited the transcription of the class
II HDAC member HDAC-7, HDAC-3, a class I HDAC,
was significantly up-regulated after TSA treatment. This
dual function of regulating protein activity and protein
expression by TSA in fibroblasts from patients with SSc
has not previously been described.
Based on these observations, we believe that the
down-regulation of HDAC-7 represents the antifibrotic
HDAC-7 AS A TARGET FOR ANTIFIBROTIC THERAPY FOR SSc
mechanism of TSA, and we suggest that silencing of
HDAC-7 may be a more specific and safer treatment for
SSc than TSA.
TSA is one of the first natural HDAC inhibitors
to be discovered that could target the zinc-dependent
HDAC classes I, II, and IV, but not the NAD-dependent
class III HDACs (11). Despite the ubiquitous distribution of HDACs in the cells, HDAC inhibitors such as
TSA selectively alter only a relative small proportion
(2–10%) of the expressed genes (20). Moreover, in one
study using human lymphoid cell lines, TSA altered only
2% of the expressed genes (8 of ⬃340 genes examined)
(21). It is remarkable that in all these studies, roughly
similar numbers of genes were down-regulated and
up-regulated. For example, in a study using a T cell
leukemia cell line, 22% of expressed genes were altered
by HDAC inhibitors, with approximately similar numbers being up-regulated and repressed (22).
TSA is still considered to be the reference compound for hydroxamic acid–containing HDAC inhibitors, although its costly and highly inefficient production
has encouraged the search for alternative drugs (23).
TSA is one of the most potent HDAC inhibitors that
exerts its effects at very low concentrations (nanomolar),
but its poor bioavailability in vivo, due to an extensive
biotransformation or instability, makes its use a continuing topic of controversy (20,24–26). Unfortunately, none
of the numerous HDAC inhibitors is specific for single
isoforms of HDAC, but few drugs show preferences for
groups of HDACs or for single HDACs. FK228, for
example, shows some preference for class I HDACs, and
tubacin specifically targets HDAC-6 (27,28). Therefore,
the challenge is to develop a new generation of HDAC
inhibitors with improved specificity for certain HDAC
isoforms and an increased efficacy compared with the
pan–inhibitors such as TSA and suberoylanilide hydroxamic acid (SAHA) (29).
We recently reported evidence that TSA in vitro
predominantly abrogates the cytokine-induced production of excessive extracellular matrix and that it prevents
fibrosis in a mouse model of bleomycin-induced fibrosis
(6). We therefore favored the hypothesis that TSA might
serve as an early strategy for the treatment of fibrosis.
However, in the present study, we demonstrated that
TSA also up-regulates the expression of CTGF and
ICAM-1, both of which are characteristically involved in
the pathogenesis of SSc. The production of extracellular
matrix proteins is induced in fibroblasts by TGF␤ during
the early stage of disease and is subsequently maintained
by CTGF (30,31). Additionally, it has been shown that
the expression of CTGF correlates with the severity of
1527
fibrosis (32). Matsushita et al (19) demonstrated that
ICAM-1 deficiency attenuates the development of skin
fibrosis in the tight-skin mouse model. Therefore, the
up-regulation of CTGF and ICAM-1 might counteract
the value of TSA as an antifibrotic drug.
Of interest, in the present study, we demonstrated that TSA significantly blocked the transcription
of HDAC-7. Our results are supported by the recent
study by Dokmanovic et al (33), who reported that the
hydroxamic acid–based HDAC inhibitors vorinostat
(SAHA), which is similar to TSA, selectively downregulated HDAC-7 in several cancer cell lines and, to a
lesser extent, in normal foreskin fibroblasts, with little or
no effect on the expression of other class II HDACs.
Vorinostat has been approved for clinical treatment, and
the authors suggest that the reduced expression of
HDAC-7 might serve as a biomarker for a response to
treatment (33).
HDAC-7 belongs to the class II HDACs that
show a tissue-specific or cell-specific expression and
shuttle between the nucleus and the cytoplasm in response to certain cellular signals. Nucleocytoplasmic
shuttling has been observed for all class II HDACs and
reflects a putatively important regulatory mechanism.
However, it should be stressed that HDAC-7 is expressed in heart and lung tissues, placenta, pancreas,
and skeletal muscle as well as in CD4/CD8 doublepositive thymocytes (34–37). HDAC-7–/– mouse embryos display defects in the development and integrity of
blood vessels (38). Moreover, HDAC-7 protein has been
implicated in several biologic processes, including regulation of gene expression, either as coactivator or corepressor (39,40), and it plays a role in T cell differentiation by inducing Nur-related protein 77 (35).
The enzymatic activity of HDAC-7 maps to the
carboxyl-terminal domain and seems to be dependent on
interaction with the class I HDAC, HDAC-3. The
binding of these 2 HDACs might be mediated by the
transcriptional corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT) and
nuclear receptor corepressor (N-CoR), which simultaneously bind class II HDACs and HDAC-3 (41). Consistent with this, it has recently been reported that
silencing of HDAC-7 also has profound effects on
endothelial cells. Mottet et al (17) reported evidence of
an altered migration of human umbilical vein endothelial cells (HUVECs) upon silencing of HDAC-7. They
showed that this disturbance was at least partly due to an
up-regulation of PDGF-B and PDGFR␤. However, in
our study, silencing of HDAC-7 in SSc dermal fibro-
1528
HEMMATAZAD ET AL
blasts did not affect the expression of PDGF-B or
PDGFR␤.
The impact of HDAC-7 on vascularization is
underlined by another study performed by Chang et al
(38), who showed that HDAC-7 plays a key role in the
maintenance of vascular integrity by repressing matrix
metalloproteinase 10 in HUVECs. Whether this influence of the silencing of HDAC-7 on angiogenesis, as
shown in HUVECs, also occurs in the skin of patients
with SSc is not yet clear.
In our study, we showed that TSA inhibited the
expression of HDAC-7 and up-regulated the expression
of HDAC-3 in fibroblasts from patients with SSc. However, silencing of HDAC-7 by siRNA did not affect the
level of expression of HDAC-3 at either the mRNA or
the protein level. Therefore, we conclude that the antifibrotic effects of TSA are mediated by HDAC-7 independently of HDAC-3. We demonstrated that similar to
TSA, the specific gene knockdown of HDAC-7 significantly reduces both the cytokine (TGF␤)–induced and
the constitutive production of the extracellular matrix
proteins type I and type III collagen. As compared with
TSA, silencing of HDAC-7 does not affect the expression of fibronectin. However, the most pronounced
advantage of silencing HDAC-7 is the specific antifibrotic effect. In contrast to TSA, silencing of HDAC-7
did not influence the expression levels of the profibrotic
molecules CTGF and ICAM-1 in SSc fibroblasts. Therefore, silencing of HDAC-7 might be a new and promising approach to the antifibrotic treatment of SSc.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Drs. Hemmatazad and Jüngel had
full access to all of the data in the study and take responsibility for the
integrity of the data and the accuracy of the data analysis.
Study conception and design. Hemmatazad, Maciejewska Rodrigues,
Maurer, Brentano, Pileckyte, J. H. W. Distler, R. E. Gay, Michel, S.
Gay, Huber, O. Distler, Jüngel.
Acquisition of data. Hemmatazad, Maciejewska Rodrigues, Maurer,
Brentano, Pileckyte, J. H. W. Distler, R. E. Gay, Michel, S. Gay,
Huber, O. Distler, Jüngel.
Analysis and interpretation of data. Hemmatazad, Maciejewska Rodrigues, Maurer, Brentano, Pileckyte, J. H. W. Distler, R. E. Gay,
Michel, S. Gay, Huber, O. Distler, Jüngel.
17.
18.
19.
20.
21.
22.
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