Histone deacetylase 7 a potential target for the antifibrotic treatment of systemic sclerosis.код для вставкиСкачать
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: firstname.lastname@example.org. 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. 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