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ARTHRITIS & RHEUMATISM Vol. 62, No. 1, January 2010, pp 280–290 DOI 10.1002/art.25056 © 2010, American College of Rheumatology

The Transcription Factor Fra-2 Regulates the Production of Extracellular Matrix in Systemic Sclerosis Nicole Reich,1 Britta Maurer,2 Alfiya Akhmetshina,1 Paulius Venalis,1 Clara Dees,1 Pawel Zerr,1 Katrin Palumbo,1 Jochen Zwerina,1 Tatiana Nevskaya,3 Steffen Gay,2 Oliver Distler,2 Georg Schett,1 and Jo ¨rg H. W. Distler1 Objective. Fra-2 belongs to the activator protein 1 family of transcription factors. Mice transgenic for Fra-2 develop a systemic fibrotic disease with vascular manifestations similar to those of systemic sclerosis (SSc). The aim of the present study was to investigate whether Fra-2 plays a role in the pathogenesis of SSc and to identify the molecular mechanisms by which Fra-2 induces fibrosis. Methods. Dermal thickness and the number of myofibroblasts were determined in skin sections from Fra-2–transgenic and wild-type mice. The expression of Fra-2 in SSc patients and in animal models of SSc was analyzed by real-time polymerase chain reaction and immunohistochemistry. Fra-2, transforming growth factor ␤ (TGF␤), and ERK signaling in SSc fibroblasts

were inhibited using small interfering RNA, neutralizing antibodies, and small-molecule inhibitors. Results. Fra-2–transgenic mice developed a skin fibrosis with increases in dermal thickness and increased myofibroblast differentiation starting at age 12 weeks. The expression of Fra-2 was up-regulated in SSc patients and in different mouse models of SSc. Stimulation with TGF␤ and platelet-derived growth factor (PDGF) significantly increased the expression of Fra-2 in SSc fibroblasts and induced DNA binding of Fra-2 in an ERK-dependent manner. Knockdown of Fra-2 potently reduced the stimulatory effects of TGF␤ and PDGF and decreased the release of collagen from SSc fibroblasts. Conclusion. We demonstrate that Fra-2 is overexpressed in SSc and acts as a novel downstream mediator of the profibrotic effects of TGF␤ and PDGF. Since transgenic overexpression of Fra-2 causes not only fibrosis but also vascular disease, Fra-2 might be an interesting novel candidate for molecular-targeted therapies for SSc.

Supported by the University of Erlangen–Nuremberg, Erlangen, Germany (ELAN grant 53410022) and the Interdisciplinary Center of Clinical Research (IZKF), Erlangen, Germany (grant A20). Dr. Maurer’s work was supported by Encysive. Dr. J. H. W. Distler’s work was supported by a Career Support Award of Medicine from the Ernst Jung Foundation. 1 Nicole Reich, DiplBiol, Alfiya Akhmetshina, PhD, Paulius Venalis, MD, Clara Dees, DiplBiol, Pawel Zerr, MSc, Katrin Palumbo, MSc, Jochen Zwerina, MD, Georg Schett, MD, Jo ¨rg H. W. Distler, MD: University of Erlangen–Nuremberg, Erlangen, Germany; 2Britta Maurer, MD, Steffen Gay, MD, Oliver Distler, MD: University Hospital Zurich, Zurich, Switzerland; 3Tatiana Nevskaya, MD, PhD: Russian Academy of Medical Sciences, Moscow, Russia. Dr. O. Distler has received consulting fees, speaking fees, and/or honoraria from Encysive, Actelion, Array BioPharma, Biovitrum, Ergonex, Fibrogen, Nicox, Pfizer, and Bristol-Myers Squibb (less than $10,000 each). Dr. J. H. W. Distler has received consulting fees, speaking fees, and/or honoraria from Encysive/Pfizer and Actelion (less than $10,000 each). Address correspondence and reprint requests to Jo ¨rg H. W. Distler, MD, Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen–Nuremberg, Universita¨tsstrasse 29, 91054 Erlangen, Germany. E-mail: [email protected]. Submitted for publication February 9, 2009; accepted in revised form September 25, 2009.

Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology that affects the skin and a variety of internal organs including the lungs, heart, and gastrointestinal tract. The first manifestations of SSc are apoptosis of microvascular endothelial cells and perivascular inflammatory infiltrates. Later stages of SSc are characterized by massive accumulation of extracellular matrix components such as collagens, glycosaminoglycans, and fibronectin, leading to progressive tissue fibrosis (1,2). The accumulating extracellular matrix disrupts the physiologic tissue structure and frequently results in dysfunction of the affected organs. Tissue fibrosis significantly contributes to the morbidity of SSc patients and is a major cause of death (3). The accumulation of extracellular matrix components in SSc is caused by an 280

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increased production of extracellular matrix by activated fibroblasts (4). Profibrotic cytokines such as transforming growth factor ␤ (TGF␤) and platelet-derived growth factor (PDGF) have been identified as central mediators of fibroblast activation in SSc (5). However, the intracellular signaling cascades by which these cytokines stimulate the production of extracellular matrix are incompletely understood. The activator protein 1 (AP-1) family of transcription factors consists of Jun proteins (c-Jun, JunB, and JunD) and Fos proteins (c-Fos, FosB, Fra-1, and Fra-2) (6,7). AP-1 proteins are expressed as immediate early genes and have been shown to control stress responses including inflammation and wound healing. Two members of the AP-1 family, c-Jun and Fra-2, have recently been implicated in TGF␤ signaling (8). Stimulation of monocytes by interleukin-13 (IL-13) induced binding of Jun and Fra-2, but not other members of the AP-1 family, to the TGF␤ promoter. Furthermore, inhibition of AP-1 signaling by decoy oligonucleotides prevented the activation of the TGF␤ promoter by IL-13, suggesting that AP-1 signaling might be involved in the regulation of TGF␤. Striking evidence for a prominent role of AP-1 signaling in fibrosis came from a recent report describing the generation and the phenotype of mice transgenic for Fra-2. Eferl and coworkers demonstrated that ectopic overexpression of Fra-2 in mice caused systemic fibrotic disease (9). Mice transgenic for Fra-2 are characterized by a prominent dermal fibrosis with massive accumulation of collagen in the dermis. In addition, Fra-2–transgenic mice develop sequentially features of pulmonary arterial hypertension with proliferation of vascular smooth muscle cells and excessive fibrosis of the lung parenchyma. Due to progressive pulmonary disease, Fra-2–transgenic mice die at age 16 weeks from respiratory distress. Bone marrow reconstitution experiments demonstrated that the phenotype of Fra-2–transgenic mice is not primarily mediated by T or B cells, but depends on the overexpression of Fra-2 in mesenchymal cells (9). However, the role of Fra-2 in the pathophysiology of dermal fibrosis in SSc has not been investigated. In the present study, we provide evidence that Fra-2 might play an important role in the pathogenesis of SSc. Fra-2 is overexpressed in patients with SSc, and its expression is induced by TGF␤ and PDGF in an ERK-dependent manner. We also demonstrate that Fra-2 is crucial for the release of collagen by SSc fibroblasts. Together, these data identify Fra-2 as a novel mediator of the profibrotic effects of TGF␤ and

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PDGF and suggest that Fra-2 might be an interesting candidate for antifibrotic therapy in SSc. MATERIALS AND METHODS Patients, Fra-2–transgenic mice, and fibroblast cultures. Fibroblast cultures were obtained from skin biopsy specimens from SSc patients. All patients fulfilled the criteria for SSc as suggested by LeRoy et al (10). Biopsy specimens from SSc patients (n ⫽ 10) were taken from involved skin. Control fibroblasts (n ⫽ 5) were obtained from skin biopsy specimens from healthy age-and sex-matched volunteers. Fra2–transgenic mice have been described previously (9). Wildtype (WT) C57BL/6 mice from the same breedings were used as controls. Human fibroblasts were prepared as outgrowth cultures from skin biopsy specimens and cultured in Dulbecco’s modified Eagle’s medium (DMEM)–Ham’s F-12 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 Invitrogen, Karlsruhe, Germany). In selected experiments, fibroblasts were stimulated with recombinant TGF␤ (10 ng/ml) or PDGF-BB (40 ng/ml) (both from R&D Systems, Abingdon, UK). These concentrations represent standard concentrations used for the stimulation of dermal fibroblasts and are based on the serum levels in SSc patients (11,12). Fibroblasts from passages 4–8 were used for the experiments. All SSc patients and controls signed a consent form approved by the local institutional review boards. Histologic analysis. Skin sections from Fra-2– transgenic mice and controls were fixed in 4% formalin and embedded in paraffin. Two-micrometer–thick sections were stained with hematoxylin and eosin for determination of the dermal thickness. The dermal thickness was analyzed with an Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands) at 100-fold magnification by measuring the maximal distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction at sites of induration in 3 consecutive skin sections from each animal, as described (13,14). The analysis was performed by 2 independent examiners who were blinded to the different groups. Immunohistochemistry for Fra-2 and ␣-smooth muscle actin (␣-SMA). For immunohistochemistry, skin sections were deparaffinized, followed by incubation with 5% serum in phosphate buffered saline for 1 hour to block nonspecific binding and incubation with 3% H202 for 10 minutes to block endogenous peroxidase activity. Cells positive for ␣-SMA in mouse skin sections were detected by incubation with monoclonal anti–␣-SMA antibody (clone 1A4; Sigma-Aldrich, Steinheim, Germany) for 3 hours at room temperature. The expression of Fra-2 in SSc patients and controls was detected by staining with polyclonal mouse anti-human Fra-2 antibody (ab15296; Abcam, Cambridge, UK) overnight at 4°C. Irrelevant isotype-matched antibodies were used as controls. Polyclonal goat anti-mouse antibodies (Jackson ImmunoResearch, Soham, UK) labeled with horseradish peroxidase (HRP) were used as secondary antibodies for 45 minutes at room temperature. Staining was visualized with aminoethylcarbazole, using a peroxidase substrate kit (Vector, Burlingame, CA).

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To determine whether Fra-2 accumulates in the nuclei of activated fibroblasts, skin sections from SSc patients and healthy controls were double stained for Fra-2 and the myofibroblast marker ␣-SMA using polyclonal rabbit anti-human Fra-2 antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) and monoclonal anti–␣-SMA antibodies. Fra-2 staining was visualized with alkaline phosphatase (AP)–conjugated goat anti-rabbit IgG antibodies (Jackson ImmunoResearch, Cambridge, UK) and the Alkaline Phosphatase Substrate Kit IV (Jackson ImmunoResearch). Staining of ␣-SMA was visualized with polyclonal rabbit anti-mouse antibody labeled with HRP and 3,3⬘-diaminobenzidine (Merck, Darmstadt, Germany). Treatment of tight skin 1 mice with imatinib mesylate. Treatment of tight skin 1 mice with imatinib mesylate was performed as described (15). Briefly, imatinib mesylate was dissolved in 0.9% NaCl and injected intraperitoneally in a total volume of 100 ␮l. Three groups with a total of 18 mice were analyzed. One group of TSK-1 mice was treated with imatinib mesylate at a dose of 150 mg/kg/day, and another group of TSK-1 mice was injected with the solvent NaCl. The last group, consisting of control mice not carrying the TSK-1 mutation, also received intraperitoneal injections of NaCl. The treatment was started at age 5 weeks. After 5 weeks of treatment, mice were killed by cervical dislocation and the skin was processed further for histologic analysis. Selective inhibition of ERK signaling. To analyze the role of ERK signaling in the induction of Fra-2 by profibrotic cytokines and in the production of extracellular matrix proteins in SSc fibroblasts, we used PD98059 (New England BioLabs, Frankfurt, Germany), a selective inhibitor of ERK. Fibroblasts were incubated with PD98059 at a concentration of 10 ␮M as previously described (16). In a subset of experiments, fibroblasts were stimulated with TGF␤ in the presence of PD98059. Quantitative real-time polymerase chain reaction (PCR). Total RNA was isolated with the NucleoSpin RNA II extraction system (Machery-Nagel, Du ¨ren, Germany) according to the instructions of the manufacturer. Reverse transcription (RT) into complementary DNA (cDNA) was performed as described using random hexamers (17,18). Gene expression was quantified by TaqMan or by SYBR Green real-time PCR using the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA). Specific primer pairs for each gene were designed with the Primer 3 software. The following primer pairs were used for the analyses: for human ␣1(I) procollagen, 5⬘-TCAAGAGAAGGCTCACGATGG-3⬘ (forward) and 5⬘-TCACGGTCACGAACCACATT-3⬘ (reverse); for human ␣2(I) procollagen, 5⬘-GGTCAGCACCACCGATGTC-3⬘ (forward) and 5⬘-CACGCCTGCCCTTCCTT-3⬘ (reverse); for human ␣1(V) procollagen, 5⬘-CATCCCACCATCACCAAAG-3⬘ (forward) and 5⬘-GGATCGTGTTGGAGGTTGTT-3⬘ (reverse); for human Fra-2, 5⬘-AGCTGGAGGAGGAGAAGTCA-3⬘ (forward) and 5⬘-TGCAGCTCAGCAATCTCC-3⬘ (reverse). Samples without enzyme in the RT reaction (non-RT controls) were used as negative controls. Nonspecific signals caused by primer dimers were excluded by no-template controls and by dissociation curve analysis. A predeveloped 18S assay (Applied Biosystems) was used to normalize for the amounts of cDNA within each sample. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification.

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Western blot analysis. Nuclear extracts were prepared according to the protocol of Andrews and Faller (19). The concentration of nuclear protein was measured with the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL) to normalize for the amounts of loaded protein. Eight micrograms of protein from each sample was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes (Carl Roth, Karlsruhe, Germany) according to standard protocols (20). After blocking with 2% bovine serum albumin for 1 hour, membranes were incubated with polyclonal rabbit anti-human Fra-2 antibodies at a dilution of 1:200 overnight at 4°C. HRP-conjugated polyclonal goat anti-rabbit antibodies (Dako, Hamburg, Germany) at a dilution of 1:2,000 were used as secondary antibodies. Proteins were visualized with the ECL plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK) and exposed to highperformance chemiluminescence film (Amersham Biosciences). Equal loading of proteins was confirmed by visualization of the nuclear protein H1 histone (Invitrogen) or Lamin A and C (New England BioLabs), and the amounts of protein were quantified using ImageJ software, version 1.41 (NIH Image, National Institutes of Health, Bethesda, MD; online at: http://rsbweb.nih.gov/ij/). Western blot for ERK and phosphorylated ERK was performed as described (16). Electrophoretic mobility shift assay (EMSA). Nonradioactive EMSA was performed using an EMSA kit (Panomics, Redwood City, CA), as described (21). Four micrograms of nuclear protein were incubated for 30 minutes at 15°C with biotinylated double-stranded oligonucleotides containing the AP-1 binding site (5⬘-ACACCGCTTGATGAGTCAGCCGGAA-3⬘). To demonstrate the specificity of the binding, the reaction was performed in the presence of an excess of unlabeled double-stranded oligonucleotide (cold probe). The samples were electrophoretically separated (120V for 1–1.5 hours) in a nondenaturing polyacrylamide gel and blotted on a Hybond-N (0.45-␮m) nylon membrane (Amersham Biosciences). After transfer, the membrane was ultraviolet-crosslinked using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). The biotin was labeled with AP-conjugated streptavidin (1:1,000; Panomics), and streptavidin was detected with the ECL plus Western Blotting Detection System and exposed to highperformance chemiluminescence film. Transfection with small interfering RNA (siRNA) against Fra-2. SSc and healthy dermal fibroblasts were transfected using the human dermal fibroblast Nucleofector Kit (Amaxa, Cologne, Germany) with 2-␮M predesigned siRNA duplexes against Fra-2, as described (22,23). Two different siRNA duplexes against Fra-2 were used in combination for nucleofection. The sequences were as follows: for Fra-2 siRNA duplex 1, 5⬘-CCUCCAUGUCCAACCCAUA-3⬘ (sense) and 5⬘-UAUGGGUUGGACAUGGAGG-3⬘ (antisense); for Fra-2 siRNA duplex 2, 5⬘-CCUCGUCUUCACCUAUCCU-3⬘ (sense) and 5⬘-AGGAUAGGUGAAGACGACG-3⬘ (antisense). Fibroblasts transfected with mock siRNA (Ambion, Darmstadt, Germany) were used as controls. Twenty-four hours after siRNA transfection, fibroblasts and cell culture supernatants were collected for further analyses. Collagen measurements. Total soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay (Biocolor, Belfast, UK), as described (17). Confluent

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fibroblasts were incubated for 24 hours with 700 ␮ l DMEM/5% FCS containing ascorbic acid. Three hundred microliters of Sirius red dye, an anionic dye that reacts specifically with basic side chain groups of collagens under assay conditions, was added to 75 ␮l culture supernatant or tissue extract 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 300 ␮l of alkali reagent. The absorbance, which is directly proportional to the amount of newly formed collagen in the sample, was determined at 540 nm with a Spectra MAX 190 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Statistical analysis. Data are expressed as the mean ⫾ SEM. The Wilcoxon signed rank test for related samples and the Mann-Whitney U test were used for statistical analyses. P values less than 0.05 were considered significant.

RESULTS Progressive dermal fibrosis in Fra-2–transgenic mice. Eferl et al described previously an increased SirCol staining in the skin of Fra-2–transgenic mice at age 16 weeks (9). To analyze the time course of Fra-2– mediated dermal fibrosis and to quantify the fibrotic changes, the dermal thickness of Fra-2–transgenic mice and controls was analyzed at ages 9, 12, and 16 weeks. A significant time-dependent increase of the dermal thickness was observed in Fra-2–transgenic mice compared with WT mice, suggesting a progressive dermal fibrosis as observed in SSc patients (Figure 1A). The dermal thickness increased progressively in Fra-2–transgenic mice, but not in control mice. At age 9 weeks, dermal thickness was not different between Fra-2–transgenic mice and controls (mean ⫾ SEM 334 ⫾ 32 ␮m versus 374 ⫾ 10 ␮m). However, the dermal thickness in Fra2–transgenic mice was significantly greater at age 12 weeks (mean ⫾ SEM 446 ⫾ 22 ␮m versus 318 ⫾ 20 ␮m; P ⫽ 0.04) and increased further at age 16 weeks (mean ⫾ SEM 521 ⫾ 21 ␮m versus 363 ⫾ 16 ␮m; P ⫽ 0.003) (results are summarized in Figure 1B). Myofibroblasts are thought to play a key role in tissue fibrosis in SSc. To determine whether increased differentiation of resting fibroblasts into myofibroblasts also occurs in Fra-2–transgenic mice, we stained for the myofibroblast marker ␣-SMA (further information is available from the corresponding author). The numbers of myofibroblasts increased in Fra-2–transgenic mice in parallel to the increase of the dermal thickness. In Fra-2–transgenic mice, the myofibroblast count increased by 60 ⫾ 20% at age 12 weeks and by 138 ⫾ 17% at age 16 weeks compared with that in mice at age 9 weeks. In contrast, the numbers of myofibroblasts re-

Figure 1. Progressive increase of dermal fibrosis in mice overexpressing Fra-2. A, Skin sections from wild-type and Fra-2–transgenic (Fra-2tg) mice were stained with hematoxylin and eosin (H&E) and trichrome. Mice overexpressing Fra-2 showed increased dermal thickness compared with age-matched control mice. Shown are representative sections (original magnification ⫻ 100). B, Dermal thickness was measured in Fra-2–transgenic mice and control mice at ages 9, 12, and 16 weeks. A progressive increase in dermal thickness occurred in Fra-2–transgenic mice over time, and dermal thickness differed significantly from that in controls at age 12 weeks and later. C, In parallel to the increasing dermal thickness, the numbers of myofibroblasts increased steadily and were significantly higher in Fra-2–transgenic mice than in control mice at age 16 weeks. Values in B and C are the mean ⫾ SEM.

mained stable in WT mice over time (Figure 1C). Myofibroblast counts were significantly increased in Fra-2–transgenic mice at age 16 weeks compared with the counts in age-matched control mice (P ⫽ 0.03). Increased expression of Fra-2 in SSc patients and in mouse models of SSc. To investigate whether the expression of Fra-2 is increased in fibrotic disorders, we analyzed the expression of Fra-2 in bleomycin-induced

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Figure 2. Increased expression of Fra-2 in fibrotic disorders. A, In tight skin 1 (TSK-1) mice the expression of Fra-2 was up-regulated by 562% compared with that in control mice. Following treatment of TSK-1 mice with imatinib mesylate, a potent small-molecule inhibitor of the transforming growth factor ␤ and platelet-derived growth factor pathways, the expression of Fra-2 mRNA was completely normalized. The level of Fra-2 mRNA in 9-week-old wild-type (pa/pa) mice was set to 1; the other results are normalized to this value. B–E, Fra-2 was overexpressed in systemic sclerosis (SSc) patients compared with healthy controls (B–D), and increased nuclear accumulation of Fra-2 was detected in SSc patients (E). Expression of Fra-2 was detectable by immunohistochemistry in 64% of SSc patients (B) but in only 27% of healthy volunteers (C). Prominent expression of Fra-2 was seen in SSc fibroblasts (arrows indicate Fra-2–positive fibroblasts) (D). Nuclear staining for Fra-2 was detected in 71% of cells in skin sections from SSc patients, but in only 36% of cells in skin sections from healthy individuals (E). Values in A and E are the mean ⫾ SEM.

dermal fibrosis and in TSK-1 mice, 2 widely used experimental models of SSc (11,13–15,24,25). Expression of messenger RNA (mRNA) for Fra-2 was increased by 50 ⫾ 15% in lesional skin upon bleomycin challenge compared with that in mock-treated mice (P ⫽ 0.03) (data not shown). In the tight skin 1 mouse model, the expression of Fra-2 was up-regulated by 562 ⫾ 248% compared with that in control mice (P ⫽ 0.006) (Figure 2A). We also analyzed the expression of other members of the AP-1 family, JunB, c-Jun, JunD, FosB, and c-Fos, in the mouse model of bleomycin-induced dermal fibrosis and in the TSK-1 mouse model. In the mouse model of bleomycin-induced fibrosis, the mRNA levels of c-Jun and c-Fos were also increased in addition to the mRNA level of Fra-2 (further information is available from the corresponding author). However, in the TSK-1 mouse model, only Fra-2 was induced and no significant upregulations of c-Jun and c-Fos or other members of the AP-1 family were observed (further information is available from the corresponding author). Thus, Fra-2 is the only member of the AP-1 family of transcription factors that is consistently up-regulated in different models of SSc. Next, we determined whether the expression of Fra-2 is also up-regulated in patients with SSc. The expression of Fra-2 protein was detectable in 7 of 11 SSc

patients ex vivo by immunohistochemistry, but in only 3 of 11 controls (Figures 2B and C). Positive staining for Fra-2 was observed in fibroblasts, endothelial cells, and keratinocytes (Figure 2D). In addition to up-regulated expression, an increased nuclear accumulation of Fra-2 was detected in SSc patients. Nuclear staining for Fra-2 was detected in 71 ⫾ 7% of cells in skin sections from SSc patients compared with 36 ⫾ 5% of cells in skin sections from healthy individuals (P ⫽ 0.003) (Figure 2E). Moreover, double staining for Fra-2 and the myofibroblast marker ␣-SMA revealed that Fra-2 was strongly expressed and accumulated in the nucleus of activated SSc fibroblasts in lesional skin. Together, these findings suggest that Fra-2 signaling is overactivated in SSc patients. TGF␤ and PDGF stimulate the expression of Fra-2 in SSc fibroblasts. To investigate whether the overexpression of Fra-2 in the skin of SSc patients might be mediated by profibrotic cytokines, SSc fibroblasts were stimulated with TGF␤ and PDGF. TGF␤ and PDGF are overexpressed in SSc and are thought to be key players in the pathogenesis of SSc (26). Incubation of SSc fibroblasts with TGF␤ and PDGF stimulated significantly the expression of Fra-2 mRNA and protein (Figures 3A–C). TGF␤ and PDGF significantly increased the mRNA level of Fra-2 by 93 ⫾ 37% and

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Figure 3. Transforming growth factor ␤ (TGF␤) and platelet-derived growth factor (PDGF) induce the expression of Fra-2 in systemic sclerosis (SSc) fibroblasts. The gene expression of Fra-2 was analyzed by SYBR Green real-time polymerase chain reaction on the mRNA level, and the expression of Fra-2 protein was analyzed by Western blot. The expression levels of untreated fibroblasts were set to 100%; the other results are normalized to this value. A and B, TGF␤ (A) and PDGF (B) both stimulated the expression of Fra-2 mRNA in SSc fibroblasts. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus control. C, Consistent with the induction of Fra-2 transcripts, a prominent induction of Fra-2 was also observed on the protein level by Western blot upon stimulation with TGF␤ and PDGF. The amounts of Fra-2 protein were increased by 123 ⫾ 24% upon stimulation with TGF␤ and by 134 ⫾ 29% upon stimulation with PDGF.

173 ⫾ 47%, respectively, compared with controls (both P ⬍ 0.05) (Figures 3A and B). Consistent with the induction of Fra-2 transcripts, a strong increase of Fra-2 was also observed on the protein level upon stimulation of SSc fibroblasts with TGF␤ and PDGF (Figure 3C). TGF␤ and PDGF also stimulated the expression of Fra-2 in healthy dermal fibroblasts. There was a trend toward stronger induction of Fra-2 mRNA in SSc fibroblasts compared with control fibroblasts. However, this trend failed to reach statistical significance (further information is available from the corresponding author). The time kinetics of Fra-2 induction differed between TGF␤ and PDGF. The maximal effects of TGF␤ were observed within 6 hours, whereas the induction of Fra-2 by PDGF was more pronounced after 24 hours (further information is available from the corresponding author). Since PDGF induces TGF␤ in SSc fibroblasts, we determined whether the induction of Fra-2 by PDGF is mediated indirectly via TGF␤. Inhibition of TGF␤ signaling with neutralizing antibodies against TGF␤ completely prevented the up-regulation of Fra-2 by PDGF (further information is available from the corresponding author). In contrast, neutralizing antibodies against TGF␤ did not reduce the basal expression of Fra-2, suggesting that the inhibitory effect was operative only upon stimulation with PDGF, but not in unstimulated cells. Together, these findings indicate that PDGF does not exert direct effects on the expression of

Fra-2, but that the induction of Fra-2 by PDGF is mediated indirectly via TGF␤. To determine whether TGF␤ signaling and Fra-2 are activated in the same cells, we performed double staining for Fra-2 and Smad3, a common mediator of the profibrotic effects of TGF␤ in fibroblasts. We found a colocalization of the stainings for Fra-2 and Smad3 in a significant proportion of fibroblast-like cells in the dermis. However, Fra-2 was also detected in Smad3negative cells, suggesting that other, non-Smad3– dependent pathways might additionally stimulate the expression of Fra-2 in SSc (further information is available from the corresponding author). To confirm the stimulatory effects of TGF␤ and PDGF on Fra-2 in vivo, TSK-1 mice were treated with imatinib mesylate, a potent small-molecule inhibitor of TGF␤ and PDGF pathways with potent antifibrotic effects (27). Treatment with imatinib mesylate completely normalized the expression of Fra-2 and decreased the mRNA levels of Fra-2 in TSK-1 mice to those in control mice (Figure 2A). The expression of Fra-2 is regulated by ERK. The mitogen-activated kinase ERK has been identified as a key component of TGF␤ signaling (16). To determine whether ERK signaling is activated in dermal fibroblasts upon stimulation with TGF␤, Western blot for ERK and phosphorylated ERK was performed. A significant increase in phosphorylated ERK was de-

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Figure 4. Regulation of Fra-2 expression by ERK. A, Inhibition of ERK by the specific inhibitor PD98059 significantly reduced the expression of Fra-2 mRNA upon stimulation with transforming growth factor ␤ (TGF␤). Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus stimulation with TGF␤ alone. B, Consistent with the mRNA data, a 73 ⫾ 4% decrease was also observed on the protein level in systemic sclerosis fibroblasts incubated with PD98059.

tected within 30 minutes after stimulation with TGF␤ (further information is available from the corresponding author). ERK has been shown to be an important upstream regulator of AP-1 signaling (28–30) and is also activated by TGF␤ in SSc fibroblasts. To determine whether ERK regulates the expression of Fra-2 in SSc fibroblasts, selective inhibition of ERK by the smallmolecule inhibitor PD98059 was performed. Preincubation with PD98059 decreased the stimulatory effects of TGF␤ on Fra-2 mRNA by 63 ⫾ 7% (Figure 4A), indicating that ERK regulates the expression of Fra-2 in SSc fibroblasts upon TGF␤ stimulation. Consistent with the results on the mRNA level, inhibition of ERK strongly reduced the levels of Fra-2 protein (Figure 4B). Fra-2 signaling is regulated by TGF␤-and ERKdependent pathways in SSc fibroblasts. To directly demonstrate that Fra-2 signaling is active in fibroblasts from SSc patients and that Fra-2–containing AP-1 complexes bind to the respective DNA binding sites, EMSA with supershift analysis was performed. AP-1–DNA complexes were already detectable in unstimulated SSc fibroblasts. Addition of antibodies against Fra-2 led to supershift of the AP-1–DNA complexes, thereby demonstrating that these complexes contained significant amounts of Fra-2 protein. Addition of an excess of unlabeled DNA probes containing the AP-1 binding site strongly reduced the signal intensity of AP-1–DNA

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complexes and supershift complexes, underlining the specificity of the signals (data not shown). To determine whether inhibition of ERK modifies the DNA binding of Fra-2, EMSA was performed with nuclear extracts from SSc fibroblasts in the presence or absence of the ERK inhibitor PD98059 (further information is available from the corresponding author). When SSc fibroblasts were coincubated with PD98059, a strongly decreased signal for AP-1–DNA complexes was found (further information is available from the corresponding author). Together, these data suggest that inhibition of ERK potently blocks nuclear translocation and DNA binding of Fra-2–containing AP-1 complexes. Fra-2 stimulates the synthesis of collagen in dermal fibroblasts. After demonstrating that Fra-2 is overexpressed in SSc and that profibrotic cytokines stimulate its expression, we next determined whether Fra-2 regulates the synthesis of extracellular matrix in SSc fibroblasts using an siRNA approach. After transfection with siRNA against Fra-2, a down-regulation of the mRNA levels of Fra-2 by 59 ⫾ 6% was observed, thereby confirming the effective inhibition of Fra-2 signaling by this approach. Knockdown of Fra-2 decreased the basal expression of mRNA for Col1a1 to 56 ⫾ 10% of that in mock-transfected cells (P ⬍ 0.05). Furthermore, the levels of Col1a2 and Col5a1, which also accumulate in SSc, were down-regulated to 59 ⫾ 9% and 67 ⫾ 10%, respectively (both P ⬍ 0.05) (Figure 5A). Inhibition of Fra-2 also significantly decreased the release of collagen protein from SSc fibroblasts. Transfection with siRNA against Fra-2 reduced the amount of collagen protein in the supernatant to 54 ⫾ 6% of that in mock-transfected cells (P ⬍ 0.05) (Figure 5B). In contrast to its effects on the expression of collagen, knockdown of Fra-2 did not alter the expression of major matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). The mRNA levels of MMP-1, MMP-3, or TIMP-1 were not altered in SSc fibroblasts transfected with siRNA against Fra-2 compared with those in mocktransfected cells (further information is available from the corresponding author). In addition to the regulation of the basal production of collagen by SSc fibroblasts, Fra-2 plays an important role in the induction of extracellular matrix proteins upon stimulation with TGF␤ and PDGF. In SSc fibroblasts transfected with siRNA against Fra-2, the increase of Col1a1 by TGF␤ was reduced by 57 ⫾ 17% compared with that in SSc fibroblasts transfected with mock siRNA (further information is available from the corresponding author). In addition to Col1a1, knock-

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Figure 5. Knockdown of Fra-2 reduces the basal expression of extracellular matrix proteins in systemic sclerosis (SSc) fibroblasts. A, After transfection of SSc fibroblasts with small interfering RNA (siRNA) against Fra-2, the basal expression of mRNA for Col1a1, Col1a2, and Col5a1 decreased significantly in SSc fibroblasts. B, Inhibition of Fra-2 signaling also decreased the amount of collagen protein in the supernatant. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus mock siRNA–transfected cells.

down of Fra-2 reduced the induction of Col1a2 and Col5a1 by 55 ⫾ 23% and 60 ⫾ 4%, respectively (further information is available from the corresponding author). Similar results were obtained for PDGF. Reduction of Fra-2 decreased the stimulatory effects of PDGF on the levels of Col1a1, Col1a2, and Col5a1 by 74 ⫾ 9%, 76 ⫾ 15%, and 69 ⫾ 10%, respectively (further information is available from the corresponding author). We have previously shown that inhibition of ERK

reduces the release of extracellular matrix proteins from SSc fibroblasts (16). To investigate whether Fra-2 acts as a downstream mediator of ERK for the synthesis of collagen, Fra-2 and ERK signaling were simultaneously inhibited. Inhibition of ERK by PD98059 after knockdown of Fra-2 by siRNA did not reduce further the levels of Col1a1 in SSc fibroblasts compared with those in mock-transfected controls (to 60 ⫾ 13% with PD98059 and to 57 ⫾ 10% without PD98059) (Figure

Figure 6. Fra-2 acts as a downstream mediator of ERK for the synthesis of collagen. Inhibition of ERK by PD98059 after silencing of Fra-2 by small interfering RNA (siRNA) did not further reduce the synthesis of Col1a1 (A), Col1a2 (B), and Col5a1 (C) in systemic sclerosis fibroblasts compared with that resulting from inhibition of Fra-2 alone, suggesting that Fra-2 is a crucial downstream mediator of ERK-induced matrix production. Values are the mean ⫾ SEM.

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6A). Similarly, simultaneous inhibition of ERK and Fra-2 signaling did not reduce Col1a2 and Col5a1 levels to a greater extent than inhibition of Fra-2 alone (for Col1a2, to 56 ⫾ 17% with PD98059 and to 59 ⫾ 9% without PD98059; for Col5a1, to 56 ⫾ 7% with PD98059 and to 67 ⫾ 10% without PD98059) (Figures 6B and C). These data suggest that Fra-2 is a crucial downstream mediator of ERK-induced matrix production in fibroblasts. DISCUSSION We demonstrate overexpression of Fra-2 in patients with SSc and in animal models of SSc with prominent expression in fibroblasts. The up-regulation of Fra-2 in SSc might be mediated by PDGF and TGF␤, which induce Fra-2 in SSc fibroblasts. Activation of ERK is critical for the induction of Fra-2, since inhibition of ERK prevents the stimulatory effects of TGF␤ and PDGF. Furthermore, we provide evidence that Fra-2 regulates the production of extracellular matrix in SSc fibroblasts, since knockdown of Fra-2 decreases the synthesis of collagen at baseline and upon stimulation. Together, our findings suggest that Fra-2 plays an important role in the activation of fibroblasts in SSc by mediating the profibrotic effects of PDGF and TGF␤. However, further studies are needed to dissect the molecular mechanisms by which overexpression of Fra-2 induces fibrosis and to determine whether activation of other pathways augments the profibrotic effects of Fra-2. Although it was beyond the scope of this study to investigate the role of other members of the AP-1 family in the pathogenesis of fibrotic diseases, the profibrotic effects observed in our study might be specific for Fra-2. Mice overexpressing different members of the AP-1 family under ubiquitous promoters have been described (6,31–34). However, an SSc-like disease with fibrosis of the skin and the lungs has not been reported in mice transgenic for members of the AP-1 family other than Fra-2. The AP-1 family members c-Fos and Fra-1 target primarily the bone, and mice overexpressing c-Fos or Fra-1 develop osteosarcoma and osteopetrosis, respectively (31,32). Overexpression of JunD causes lymphopenia (33). Mice transgenic for FosB, c-Jun, and JunB have no overt phenotype (6,31,34). Thus, Fra-2 might be the only member of the AP-1 family that regulates the production of extracellular matrix and plays a role in tissue fibrosis. Moreover, we demonstrate that Fra-2 is upregulated in animal models of dermal fibrosis and in SSc patients and that TGF␤ and PDGF stimulate the expres-

sion of Fra-2. TGF␤ and PDGF signaling pathways are activated in SSc and might thus directly drive the overexpression of Fra-2 in vivo. Consistent with our findings, TGF␤ and PDGF have been shown to activate AP-1 signaling, although the effect on Fra-2 has not yet been investigated. TGF␤-mediated activation of AP-1 transcription factors has been shown to regulate the expression of MMP-1 and TIMP-1 (35). Similarly, PDGF stimulated the activity of c-Fos and the DNA binding of AP-1 in NIH3T3 cells (36). Here we show that inhibition of ERK decreases the expression of Fra-2 in SSc fibroblasts. Our findings are supported by previous reports identifying ERK as a critical activator of AP-1 in the context of neoplastic transformation (28,29). A subset of JB6 cells undergoes neoplastic transformation in an AP-1–dependent manner upon exposure to tumor promoters, whereas another subset of JB6 cells is protected from transformation. Subsequent analyses demonstrated that the levels of ERK are reduced in resistant cells. Transfection of resistant cells with WT ERK rendered resistant JB6 cells susceptible to tumor promoters, whereas transfection with dominant-negative mutants of ERK prevented transformation (28,29). ERK has previously been implicated in activation of fibroblasts and fibrosis. ERK is phosphorylated and activated upon stimulation with TGF␤, and inhibition of ERK reduced the production of extracellular matrix proteins by SSc fibroblasts (16). Furthermore, increased phosphorylation of ERK was observed in fibrotic lesions of asbestosis (37). ERK activation thus appears to be an important step in activating Fra-2 and AP-1, and the profibrotic effects of ERK might be mediated in part by induction of Fra-2. MatInspector analyses indicated regulatory binding sites for AP-1 in the promoters of Col1a1 and Col1a2. Consistent with these predictions, AP-1 complexes bound to the putative AP-1 binding sites between the residues –265 to –241 in the Col1a2 promoter, and deletion of the AP-1 binding site reduced the induction of Col1a2 by TGF␤ (38). Our findings suggest that Fra-2 might be the central mediator of the regulatory effects described for AP-1. Fra-2 regulates the expression of Col1a1, Col1a2, and Col5a1, all of which have been shown to accumulate in fibrotic lesions of patients with SSc. Overexpression of Fra-2 induced tissue fibrosis (9), whereas knockdown of Fra-2 reduced the basal production of Col1a1, Col1a2, and Col5a1 as well as the induction of collagens upon stimulation with profibrotic cytokines. In conclusion, the present study demonstrates that TGF␤ and PDGF up-regulate the expression of

Fra-2 IN THE PATHOGENESIS OF SSc

Fra-2 in SSc. We identified Fra-2 as a novel downstream mediator of the profibrotic effects of TGF␤ and PDGF that stimulates the release of collagen in an ERKdependent manner. Together with the observation that transgenic overexpression of Fra-2 causes dermal and pulmonary fibrosis in mice, these data indicate that Fra-2 might play an important role in the pathogenesis of SSc as a downstream mediator of the stimulatory effects of TGF␤ and PDGF on the synthesis of collagen. ACKNOWLEDGMENTS We thank Erwin F. Wagner (Centro Nacional de Investigaciones Oncologicas, Madrid, Spain) and Peter Hasselblatt (Freiburg University Hospital, Freiburg, Germany) for helpful discussions and ideas and for providing Fra-2– transgenic mice, and we also thank Maria Halter for excellent technical support.

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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. Dr. J. H. W. Distler 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 conception and design. Reich, Gay, O. Distler, Schett, J. H. W. Distler. Acquisition of data. Reich, Maurer, Akhmetshina, Venalis, Dees, Zerr, Palumbo, Zwerina, Nevskaya, J. H. W. Distler. Analysis and interpretation of data. Reich, Gay, O. Distler, J. H. W. Distler.

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DOI 10.1002/art.27185

Clinical Images: Gastric antral vascular ectasia in systemic sclerosis

The patient, a 63-year-old woman diagnosed as having limited cutaneous systemic sclerosis (1), presented with transfusiondependent anemia. Esophagogastroduodenoscopy revealed typical features of gastric antral vascular ectasia (GAVE), also known as watermelon stomach because of the long red streaks alternating with normal gastric mucosa that resemble the markings on a watermelon (left) (2). GAVE is rare in scleroderma but is often responsible for both acute and chronic gastrointestinal hemorrhage. Because of recurrent bleeding in the patient, vascular ectasias were treated using endoscopic argon plasma coagulation (right) (3). Six sessions were required to achieve near-total elimination of the lesions. Now, 18 months after GAVE was diagnosed, the patient’s condition has improved substantially, and no additional transfusions have been required. 1. Lonzetti LS, Joyal F, Raynauld JP, Roussin A, Goulet JR, Rich E, et al. Updating the American College of Rheumatology preliminary classification criteria for systemic sclerosis: addition of severe nailfold capillaroscopy abnormalities markedly increases the sensitivity for limited scleroderma. Arthritis Rheum 2001;44:735–6. 2. Selinger CP, Ang YS. Gastric antral vascular ectasia (GAVE): an update on clinical presentation, pathophysiology and treatment. Digestion 2008;77: 131–7. 3. Marie I, Ducrotte P, Antonietti M, Herve S, Levesque H. Watermelon stomach in systemic sclerosis: its incidence and management. Aliment Pharmacol Ther 2008 15;28:412–21.

Rene Thonhofer, MD Cornelia Siegel, MD Markus Trummer, MD Alexander Gugl, MD State Hospital Mu ¨rzzuschlag Mu ¨rzzuschlag, Austria