ARTHRITIS & RHEUMATISMVol. 65, No. 3, March 2013, pp 792–804DOI 10.1002/art.37809© 2013, American College of Rheumatology

Tropisetron Suppresses Collagen Synthesis inSkin Fibroblasts via �7 Nicotinic Acetylcholine Receptorand Attenuates Fibrosis in a Scleroderma Mouse Model

Agatha Stegemann,1 Anca Sindrilaru,2 Beate Eckes,3 Adriana del Rey,4 Alexander Heinick,1

Jan S. Schulte,1 Frank U. Muller,1 Sergei A. Grando,5 Bernd L. Fiebich,6

Karin Scharffetter-Kochanek,2 Thomas A. Luger,1 and Markus Bohm1

Objective. There is increasing evidence thatserotonin (5-hydroxytryptamine [5-HT]) and distinct5-HT receptors are involved in the pathogenesis ofsystemic sclerosis. The aim of this study was to test thehypothesis that tropisetron, a routinely used antiemeticagent previously characterized as a 5-HT3/4 receptor–modulating agent, can directly affect collagen synthesisin vitro and attenuate experimentally induced fibrosisin vivo.

Methods. Functional in vitro studies were per-formed using human dermal fibroblasts (HDFs). Signaltransduction studies included immunofluorescenceanalysis, Western immunoblotting, promoter reporterassays, cAMP/Ca2� measurements, and use of phar-macologic activators and inhibitors. Gene silencingwas performed using small interfering RNA. Putativereceptors of tropisetron were detected by semiquantita-tive reverse transcription–polymerase chain reaction(RT-PCR) and immunofluorescence. The murine modelof bleomycin-induced scleroderma was used to assess

the antifibrogenic and antifibrotic effects of tropisetronin vivo. Collagen expression in vitro, ex vivo, andin situ was determined by real-time RT-PCR analy-sis, Western immunoblotting, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunohisto-chemical analysis.

Results. Tropisetron suppressed collagen synthe-sis induced by transforming growth factor �1 (TGF�1).This effect was independent of 5-HT3/4 receptor but wasmediated via �7 nicotinic acetylcholine receptor(�7nAChR). Suppression of TGF�1-induced collagensynthesis occurred via an unknown molecular mecha-nism not involving modulation of the Smad, cAMP, Akt,c-Jun, or MAPK pathway. In vivo, tropisetron not onlyprevented skin fibrosis but also reduced the collagencontent in established dermal fibrosis induced by bleo-mycin.

Conclusion. Tropisetron directly reduces collagensynthesis in HDFs via an �7nAChR-dependent mecha-nism. The antifibrogenic and antifibrotic effects ofthis agent observed in a mouse model of bleomycin-induced scleroderma indicate the future potential oftropisetron in the treatment of fibrotic diseases such asscleroderma.

Systemic sclerosis (SSc) is a chronic inflamma-tory connective tissue disease that affects the skin andinternal organs. Key pathogenetic features are vasculardamage and excessive tissue fibrosis/sclerosis due toincreased expression of profibrotic cytokines such astransforming growth factor �1 (TGF�1) or constitutiveactivation of TGF�1-mediated signaling pathways (1,2).This results in aberrant production of extracellular ma-trix (ECM). The pathogenesis of SSc is incompletelyunderstood (3,4), and new effective antifibrotic thera-pies are needed.

Supported by the German Scleroderma Foundation and theDFG (grants KO 4110/1-1 to Drs. Stegemann and Bohm, SFB 497 andKFO 142 to Drs. Sindrilaru and Scharffetter-Kochanek, and KR 558 toDr. Eckes).

1Agatha Stegemann, PhD, Alexander Heinick, PhD,Jan S. Schulte, MD, Frank U. Muller, MD, Thomas A. Luger, MD,Markus Bohm, MD: University of Munster, Munster, Germany; 2AncaSindrilaru, MD, Karin Scharffetter-Kochanek, MD: University of Ulm,Ulm, Germany; 3Beate Eckes, PhD: University of Cologne, Cologne,Germany; 4Adriana del Rey, PhD: University of Marburg, Marburg,Germany; 5Sergei A. Grando, MD, PhD: University of California,Irvine; 6Bernd L. Fiebich, PhD: University of Freiburg, Freiburg,Germany.

Address correspondence to Markus Bohm, MD, Departmentof Dermatology, Laboratory for Neuroendocrinology of the Skin andInterdisciplinary Endocrinology, University of Munster, Von EsmarchStrasse 58, 48149 Munster, Germany. E-mail: bohmm@uni-muenster.de.

Submitted for publication March 9, 2012; accepted in revisedform November 20, 2012.

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Targeting neuroendocrine receptors that modu-late collagen metabolism may be a novel approach forthe treatment of SSc. Our group recently observed that�-melanocyte–stimulating hormone suppresses collagensynthesis in human dermal fibroblasts (HDFs) and hasantifibrogenic activity in a mouse model of SSc (5,6).Perhaps the earliest neuroendocrine mediator impli-cated in the pathogenesis of SSc was serotonin (5-hydroxytryptamine [5-HT]). Serotonin is a vasoactivesubstance that may mediate the initial phase of vaso-constriction in Raynaud’s phenomenon in patients withSSc. Antiserotoninergic strategies have therefore beenused in SSc (7). Interestingly, 5-HT stored in plateletsinduced ECM synthesis, and mice lacking the 5-HT2

receptor were protected against experimentally inducedfibrosis (8). Further, cyproheptadine and other 5-HTreceptor antagonists are used for the treatment of car-cinoid syndrome, in which tumor cells secrete 5-HT andSSc-like skin changes occur (9–11).

Interestingly, 5-HT receptors are expressed invarious nonneuronal cells, including those in skin (12).The biology of 5-HT receptors is complex. At least 17different receptor subtypes have been described (13).Antagonists selective for 5-HT receptor have been de-veloped, and some of them are used in daily medicalpractice. Tropisetron was characterized as a 5-HT3receptor antagonist that binds with high affinity to5-HT3 receptor and with lower affinity to 5-HT4 recep-tor (14). It has antiemetic activity by antagonizing 5-HT3receptor in the gut and is approved for the treatmentof nausea and vomiting during chemotherapy in patientswith cancer.

In this study, we investigated whether tropisetronsuppresses experimentally induced skin fibrosis in a mousemodel of SSc. We also investigated the mechanismbehind this action of tropisetron.

MATERIALS AND METHODS

Cell culture and reagents. Normal neonatal and adultHDFs (Tebu-Bio) were cultured as previously described (5).Fibroblasts (3T3) (PromoCell) were cultured in RPMI 1640medium, as previously described (6). Human colon tissue wasprovided by D. Bettenworth, MD (University of Munster,Germany), and synovial fibroblasts were provided by S. Gras-sel, MD (University Hospital Regensburg, Germany). Tropise-tron was obtained from Novartis Pharma, and 5-HT andbleomycin were purchased from Sigma. TGF�1 was obtainedfrom PeproTech, forskolin (FSK) and a PhosphodiesteraseInhibitor Set I were from Calbiochem-Novabiochem, and�-bungarotoxin was from Tocris. The specific �7 nicotinicacetylcholine receptor (�7nAChR) agonist AR-R17779 wassynthesized at AstraZeneca Pharmaceuticals.

Establishment of HDFs from patients with SSc. HDFsfrom the lesional skin of 6 patients with SSc (mean age 54.2years, range 31–72 years) were established as previously de-scribed (15). All patients fulfilled the American College ofRheumatology criteria for a diagnosis of SSc (16). The estab-lishment of HDFs from patients with SSc was approved by thelocal ethics committees of the Universities of Munster andCologne.

Cell viability test. The cytotoxicity of tropisetron wasassessed by XTT assay (Roche Diagnostics), as previously de-scribed (17).

RNA extraction, semiquantitative reverse transcription–polymerase chain reaction (RT-PCR), and real-time RT-PCR.Total RNA was prepared from cells using an RNeasy Mini Kit(Qiagen) or from skin using TRIzol (Invitrogen). Comple-mentary DNA synthesis was performed with a RevertAidcDNA Kit (Fermentas). Established primers and conditionswere used for semiquantitative and real-time RT-PCRs of5-HT receptors (18), �7nAChR (19,20), type I collagen (5,6),plasminogen activator inhibitor 1 (PAI-1), and tissue inhibitorof metalloproteinases 1 (TIMP-1) (21). Primers for �-smoothmuscle actin (�-SMA) were as follows: forward 5�-GACCGA-ATGCAGAAGGAGAT-3� and reverse 5�-CCACCGATCC-AGACAGAGTA-3�. Quantification of the gene expressionof each sample was performed using the 2���Ct method (22),using GAPDH as an internal standard with the ground condi-tion set at 1.

Plasmid constructs and promoter reporter assays. Theluciferase plasmid construct (CAGA)9-Lux, which is specificfor Smad3, was a gift from S. Dennler and J. M. Gauthier(Glaxo Wellcome) (23). The 3T3 fibroblasts were transfectedwith DOTAP (Roth), treated with TGF�1 and tropisetron,followed by promoter reporter assay. Luciferase activity wasmeasured with a kit from Promega.

Determination of C-terminal type I procollagen pro-peptide (CPI). We used a commercially available enzyme-linked immunosorbent assay (ELISA) (Takara) to measuretype I collagen secretion. HDFs were stimulated with TGF�1alone or with tropisetron. Cell culture supernatants wereharvested after 24 hours.

Gene silencing of �7nAChR. Small interfering RNA(siRNA) oligonucleotides with the target sequence GAUAA-CAGUCUUACUCUCU and the nontargeting control siRNA#2 were designed by Thermo Scientific Dharmacon. HDFswere transfected with siRNA for 24 hours according to themanufacturer’s protocol.

Determination of cAMP levels. HDFs were stimulatedwith tropisetron, alone or with TGF�1, for 30 minutes in thepresence of a Phosphodiesterase Inhibitor Set I. FSK served asa positive control. Intracellular cAMP levels were measured bya commercially available enzyme immunoassay (AmershamPharmacia Biotech).

Determination of 5-HT by high-performance liquidchromatography (HPLC). HDFs were stimulated with TGF�1(10 ng/ml) or were left untreated. Supernatants were harvestedafter 24 hours. Ten microliters of cold 0.4M perchloric acid wasadded to 50 �l of the cell supernatants or 10�7M 5-HTstandards. After centrifugation, supernatants were subjected toisocractic electrochemical detection–HPLC separation using a25-cm Supelcosil LC-18 column and 50 mM citrate/phosphatebuffer solution (pH 3.9) containing 10% acetonitril as eluent.

ANTIFIBROGENIC AND ANTIFIBROTIC EFFECT OF TROPISETRON 793

The detection limit was 10 pg 5-HT in 40 �l injection volume(0.25 ng/ml).

Western immunoblotting. Cells were either lysed withboiling Laemmli sample buffer or digested with pepsin in500 �l 0.5M acetic acid for 24 hours at 4°C for the detection oftype I collagen protein. Lysates were separated by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis followed byimmunoblotting, as previously described (6). The followingantibodies were used: anti–type I collagen (DPC Biermann),anti-Smad2/3 (BD Biosciences), anti–phospho-Smad3 (Ser433/435)(Cell Signaling Technology), anti–5-HT3 receptor (Imgenex),anti–5-HT4 receptor (Millipore), anti-�7nAChR (R&D Sys-tems), anti–phospho-p38, anti–phospho–ERK-1/2, anti-ERK-1/2, anti–phospho–c-Jun, and anti–phospho-Akt (all from CellSignaling Technology). Equal protein loading was assured bymembrane stripping and reprobing with an antibody against�-actin (Santa Cruz Biotechnology) or �-tubulin (Calbiochem-Novabiochem). Densitometric analysis was performed usingImageJ software. All Western immunoblots were repeated atleast 3 times.

Immunofluorescence analysis in vitro. Cells wereseeded in 8-well chamber slides, stimulated as indicated, fixedwith methanol at �20°C or with 4% paraformaldehyde. Slideswere blocked with 5% rabbit or donkey serum followed byincubation for 1.5 hours with a mouse polyclonal antibodyagainst Smad2/3 (1 �g/ml; BD Biosciences) or a rabbit�7nAChR antibody (R&D Systems). Bound primary anti-bodies were detected with rabbit anti-mouse or donkey anti-rabbit antibodies coupled to Texas Red (Dianova). DAPI(Fluka) was used for nuclear labeling. Cells were imaged byfluorescence microscopy (Carl Zeiss).

Ca2� imaging. HDFs were seeded on round 20-mm–diameter glass slides at 25,000 cells per slide. After 24 hours offetal calf serum (FCS) deprivation, cells were incubated with4 �M Fluo-4 AM (Invitrogen) in Tyrode’s solution (2 mMCaCl, 140 mM NaCl2, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES,10 mM glucose, pH 7.4) for 15 minutes, and then the dye waswashed out for 10 minutes with Tyrode’s solution. Agents weredirectly applied into the incubation chamber, and fluorescencewas recorded using a Zeiss LSM 710 confocal fluorescencemicroscope (excitation 488 nm, Fluo-4 emission 493–622 nm).

Mouse model of scleroderma. The bleomycin-inducedskin fibrosis model initially described by Yamamoto et al (24)was used. All animal experiments were performed with per-mission from the local veterinary authorities of the Universityof Ulm, Germany. C3H mice (4 groups of 5 mice each) weretreated with bleomycin (10 �g), tropisetron (10 �g), bleomycinplus tropisetron, or NaCl (negative control). The mice werekilled after 21 days. To test whether tropisetron has effects onestablished skin fibrosis, a post-bleomycin approach was cho-sen (25). Accordingly, C3H mice were injected with bleomycin(10 �g) for 21 days followed by injections with NaCl (negativecontrol) or tropisetron (10 �g) for a further 14 days. Skinbiopsy specimens were obtained for RNA extraction, collagenprotein determination, and immunohistochemical analysis.

Histochemistry and immunostaining. Masson’s tri-chrome staining of mouse skin cryosections was performedaccording to the manufacturer’s protocol (Sigma). Type Icollagen immunostaining was performed using rabbit anti-mouse type I collagen or rabbit IgG as an isotype control asprimary antibodies (Chemicon). An Alexa Fluor 555–labeled

goat anti-rabbit antibody (Caltag) was used as secondaryantibody, followed by nuclear staining with DAPI (Fluka).Digital pictures were acquired with a Zeiss Axiophot micro-scope.

Assessment of dermal thickness. Skin sections frommice treated with NaCl, bleomycin, tropisetron, or bleomycinplus tropisetron were stained with hematoxylin and eosin.Sections were viewed under a Zeiss Axiophot microscope.Three measurements were performed for each microscopicfield, and 5 different fields were analyzed for each mouse(n � 3). Densitometric analysis was performed using ImageJsoftware.

Determination of collagen protein content ex vivo.Mouse skin samples were weighed and incubated in 0.5Macetic acid in the presence of protease inhibitor cocktail(Roche Diagnostics). One milligram of pepsin (Sigma) per500 �l 0.5M acetic acid was added to each sample, followed byincubation for 24 hours at 4°C. After centrifugation, super-natants were precipitated with methanol/chloroform, afterwhich 2� Laemmli sample buffer was added to dissolve theprotein pellets. After normalization for wet weight, sampleswere subjected to SDS-PAGE and stained with 0.5% Coomas-sie brilliant blue (Bio-Rad).

Statistical analysis. All experiments were performedat least 3 times. Expression and activity levels were calculatedas the mean � SD, and deviations from normality wereassessed by the Shapiro-Wilk test. Differences between 2 meanvalues were assessed by Student’s t-test. One-way analysis ofvariance and Tukey’s test were used for the analysis of Ca2�

fluorescence intensity. P values less than 0.05 were consideredsignificant.

RESULTS

Suppressive effect of tropisetron on TGF�1-induced collagen synthesis in HDFs. We first investi-gated the effect of tropisetron in neonatal HDFs stim-ulated with TGF�1. TGF�1 is a key profibrotic factorthat up-regulates the synthesis of collagen and otherECM genes implicated in fibrosis (26,27). First, theamount of secreted type I collagen was measured by CPIELISA. In HDFs treated with TGF�1, CPI secretionwas significantly increased compared with control. Tro-pisetron dose-dependently suppressed TGF�1-inducedCPI secretion by 21–48% (Figure 1A, lower panel). Toclarify whether this effect of tropisetron is limited onlyto deposited type I collagen or involves a reduction inintracellular collagen synthesis, Western immunoblot-ting was performed. As expected, treatment of HDFswith TGF�1 (10 ng/ml) increased the expression oftype I collagen protein. Tropisetron reduced the intra-cellular protein expression of type I collagen whencostimulated with TGF�1 compared with stimulationwith TGF�1 alone (Figure 1A, upper panel).

Next, real-time PCR analysis of Col�1(I) andCol�2(I) was performed to investigate whether tropise-

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tron reduces collagen synthesis at the RNA level.TGF�1 (10 ng/ml) increased the expression of type Icollagen messenger RNA (mRNA). Coincubation withtropisetron at a dose of 10 �g/ml suppressed this effectof TGF�1 by �40% for Col�1(I) and by �30% forCol�2(I) compared with incubation with TGF�1 alone(Figure 1B). Tropisetron per se did not alter type Icollagen gene expression (additional information isavailable from the corresponding author). Moreover, thetropisetron doses used in the above experiments werenoncytotoxic (additional information is available fromthe corresponding author).

The suppressive effect of tropisetron on collagensynthesis was confirmed using neonatal HDFs from 2different donors (Figure 1C). Furthermore, tropisetronattenuated TGF�1-induced type I collagen mRNA ex-pression in HDFs established from the normal skin of3 adult donors (Figure 1D). Interestingly, tropisetronalso reduced TGF�1-mediated induction of �-smoothmuscle actin (�-SMA), a myofibroblast marker (addi-tional information is available from the correspondingauthor). In summary, tropisetron suppressed TGF�1-induced type I collagen synthesis as well as �-SMAmRNA expression in HDFs.

Figure 1. Tropisetron (TRO) suppresses transforming growth factor �1 (TGF�1)–induced collagen synthesis in human dermal fibroblasts (HDFs).A, Bottom, Tropisetron dose-dependently attenuates TGF�1-mediated secretion of C-terminal type I procollagen propeptide (CPI). HDFs weretreated with TGF�1 (10 ng/ml) and tropisetron at the indicated concentrations for 24 hours, and the supernatants were subjected to enzyme-linkedimmunosorbent assay (n � 3). Top, Costimulation with tropisetron plus TGF�1 reduces the intracellular protein expression of type I collagen (COL)compared with stimulation with TGF�1 alone. HDFs were exposed to TGF�1 (10 ng/ml) or TGF�1 plus tropisetron (10 �g/ml) for 24 hours,followed by Western immunoblotting of type I collagen. Pepsin-digested lysates were subjected to sodium dodecyl sulfate–polyacrylamide gelelectrophoresis and analyzed by Western immunoblotting using an anti–type I collagen antibody. The amounts of type I collagen were assessed bydensitometric evaluation of Col�1(I) and Col�2(I). B, Treatment with TGF�1 plus tropisetron (10 �g/ml) suppresses the effect of TGF�1 onCol�1(I) and Col�2(I). HDFs were exposed to TGF�1 (10 ng/ml) plus several doses of tropisetron, as indicated, for 24 hours, followed by real-timereverse transcription–polymerase chain reaction (RT-PCR) of Col�1(I) and Col�2(I) transcripts. C and D, Tropisetron suppresses TGF�1-inducedcollagen expression in neonatal HDFs (C) and adult HDFs (D) derived from 3 different donors. Neonatal HDFs and adult HDFs were stimulatedwith TGF�1 (10 ng/ml), tropisetron (10 �g/ml), or TGF�1 plus tropisetron for 24 hours. Type I collagen mRNA expression was determined byreal-time RT-PCR. Bars show the mean � SD. � � P 0.05; �� � P 0.01 versus TGF�1 alone. Rel. � relative.

ANTIFIBROGENIC AND ANTIFIBROTIC EFFECT OF TROPISETRON 795

No effect of tropisetron on several canonicalsignal transduction pathways activated by TGF�1. Togain insight into the mechanism by which tropisetronsuppresses TGF�1-mediated ECM synthesis, we ana-lyzed the Smad pathway, which is activated by TGF�(28,29). Treatment of HDFs with TGF�1 led to nucleartranslocation of Smad2/3 in HDFs within 30 minutes, asshown by immunofluorescence analysis (Figure 2A).Simultaneous stimulation of cells with tropisetron andTGF�1, however, did not alter TGF�1-mediatedSmad2/3 translocation (Figure 2A). Moreover, preincu-bation with tropisetron for 24 hours also did not affectTGF�1-induced Smad2/3 nuclear accumulation (datanot shown). In accordance with this finding, tropisetrondid not reduce TGF�1-induced Ser433/435 phosphoryla-tion of Smad3 in HDFs (Figure 2B).

Next, we performed promoter reporter assayswith (CAGA)9-Lux, a Smad3/4-specific luciferase con-struct (21,30). In these experiments, 3T3 fibroblastswere used, because transfection of HDFs with thisplasmid was not efficient. TGF�1 treatment increased(CAGA)9-Lux promoter activity compared with notreatment. However, tropisetron failed to alter thisTGF�1 effect, and the tropisetron alone also did notchange basal Smad promoter activity (Figure 2C). Insupport of the lack of an effect of tropisetron on Smadsignaling, expression of 2 TGF�1-regulated Smad targetgenes, PAI-1 and TIMP-1 (31), was unaffected by thedrug (additional information is available from the cor-responding author). These data showed that tropisetrondoes not abrogate TGF�1-mediated collagen synthesisin HDFs via Smad interference.

Figure 2. Tropisetron does not affect Smad signaling in HDFs in vitro. A, Tropisetron does not inhibit TGF�1-induced nuclear translocation ofSmad2/3 in HDFs. Cells were treated with TGF�1 (10 ng/ml) as a positive control or with TGF�1 plus tropisetron (10 �g/ml) for 30 minutes,followed by immunofluorescence analysis using an anti-Smad2/3 antibody. Bar � 10 �m. B, Tropisetron does not alter Smad3 phosphorylation inHDFs. Cells were stimulated for 30 minutes with TGF�1 and TGF�1 plus tropisetron. Cell lysates were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis and analyzed by Western immunoblotting, using an anti–phospho-Smad3 (Ser433/435) antibody. The sameblot was stripped and reprobed with an anti-Smad2/3 antibody. Results in A and B are from a representative experiment of 3 independentexperiments performed. C, Tropisetron does not modulate Smad3/4 promoter activity. Transient transfection of 3T3 cells (n � 3) was performedwith a Smad3/4-specific luciferase construct, (CAGA)9-Lux. Cells were incubated with TGF�1 (positive control) and TGF�1 plus tropisetron for 6hours and assayed for luciferase activity. Bars show the mean � SD. � � P 0.001 versus control. See Figure 1 for definitions.

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We also questioned whether tropisetron affectsTGF�1-mediated activation of additional signal trans-duction pathways involved in collagen expression (32).TGF�1 treatment of HDFs resulted in weak phosphor-ylation of p38, c-Jun, and Akt, but this response was notaltered by treatment with tropisetron (additional infor-mation is available from the corresponding author).Moreover, neither TGF�1 nor tropisetron, alone or incombination, altered the phosphorylated state of ERK-1/2 under the given experimental conditions (additionalinformation is available from the corresponding author).

Mediation of the suppressive effect of tropisetronon collagen synthesis by HDFs. Previously, we demon-strated that cAMP plays an important role in modu-lating the TGF�-mediated responses of several ECMgenes (21,30). In order to investigate whether the ef-fect of tropisetron is mediated by activation of thecAMP pathway, we used the adenylate cyclase inhibitorSQ22536. SQ22536 did not affect the suppressive effectof tropisetron on TGF�1-induced collagen synthesis, asshown by real-time RT-PCR analysis (Figure 3A), sug-gesting that tropisetron acts in a cAMP-independent

Figure 3. Tropisetron attenuates TGF�1-induced collagen synthesis in HDFs via a cAMP- and 5-hydroxytryptamine (5-HT)–independentmechanism. A, Inhibition of adenylate cyclase does not abrogate the suppressive effect of tropisetron on TGF�1-induced collagen expression. HDFs(n � 3) were pretreated with the adenylate cyclase inhibitor SQ22536 (100 �M) for 30 minutes and stimulated for 24 hours with TGF�1 (10 ng/ml),tropisetron (10 �g/ml), or TGF�1 plus tropisetron in the presence and absence of SQ22536. Type I collagen mRNA expression was determined byreal-time RT-PCR. Bars show the mean � SD. � � P 0.05 versus TGF�1 alone. B and C, Tropisetron does not elevate cAMP expression in HDFs.Intracellular cAMP expression in HDFs treated for 30 minutes with several doses of tropisetron alone (B) and in combination with TGF�1(10 ng/ml) (C) was measured by enzyme-linked immunosorbent assay (n � 3). Bars show the mean � SD. � � P 0.001 versus control. D, Left,Messenger RNA expression of 5-HT3 receptor (5-HT3-R) and 5-HT4 receptor is undetectable in HDFs, as determined by semiquantitative RT-PCRanalysis. �-actin served as a housekeeping gene product control. Lane 1, Colon cDNA (positive control). Lane 2, HDF cDNA. Lane 3, HDF RNA.Right, Neither 5-HT3 receptor nor 5-HT4 receptor was expressed in HDFs, as shown by Western immunoblotting. Protein lysates were subjectedto sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Human colon lysate served as positive control. Equal protein amounts were confirmedusing �-actin antibody as a loading control. One representative Western blot of 3 performed is shown. FSK � forskolin (see Figure 1 for otherdefinitions).

ANTIFIBROGENIC AND ANTIFIBROTIC EFFECT OF TROPISETRON 797

manner in HDFs. In accordance with this, tropisetronalone at several doses failed to increase intracellularcAMP levels over control, as shown by ELISA (Figure3B). In contrast, FSK, an activator of adenylate cyclase,strongly elevated cAMP expression (Figure 3B). Fur-thermore, neither TGF�1 alone nor TGF�1 plus tro-pisetron at different doses affected intracellular cAMPlevels (Figure 3C). Therefore, tropisetron did not elicitits suppressive effect on TGF�1-induced collagen syn-thesis in HDFs via activation of the cAMP pathway.

Because both 5-HT3 receptor and 5-HT4 receptorwere previously identified as binding sites for tropisetron(14), we next investigated whether HDFs express thesereceptors. Surprisingly, mRNA expression of both5-HT3 receptor and 5-HT4 receptor was undetectable inHDFs, as shown by semiquantitative RT-PCR, whereasthese receptors were present in the human colon tissueused as positive control (Figure 3D, left panel). Inaccordance with this, both receptors were not expressedin HDFs at the protein level, as demonstrated byWestern blotting (Figure 3D, right panel). In contrast,single protein bands of the expected sizes of 5-HT3

receptor (60 kd) and 5-HT4 receptor (45 kd) wereobserved in cell lysates of human colon (33,34) (Figure3D, right panel). The data showed that 5HT3/4 receptorscannot mediate the suppressive effect of tropisetron onTGF�1-induced collagen synthesis, because HDFs lackthese receptors.

Because the suppressive effect of tropisetron onTGF�1-mediated collagen synthesis occurred in theabsence of exogenous 5-HT, we further sought to deter-mine whether the drug antagonizes endogenously pro-duced 5-HT by HDFs. Conditioned medium from cellswas analyzed by HPLC (detection limit 0.25 ng/ml) forthe presence of 5-HT, which was detectable in neitherthe experimental culture medium (0% FCS) nor inconditioned medium from HDFs. In addition, no detect-able amounts of 5-HT were observed in supernatantsof HDFs stimulated with TGF�1 for 24 hours (data notshown). These results showed that tropisetron acts in thecomplete absence of 5-HT.

Effect of �7nAChR on the anti-TGF�1 effect oftropisetron in HDFs. Because both putative tropisetronreceptors, 5-HT3 receptor and 5-HT4 receptor, are notpresent in HDFs, we investigated whether �7nAChRcould mediate the effect of tropisetron on TGF�1-induced collagen synthesis. It has been reported that�7nAChR binds tropisetron due to its high homology to5-HT3 receptor (35,36). However, the functional signif-icance of this observation is unknown. Expression of

�7nAChR in neonatal HDFs was detected via semi-quantitative RT-PCR and immunofluorescence analysis(additional information is available from the corre-sponding author), in accordance with earlier studies(37). Indeed, Ca2� signaling after treatment of HDFswith the specific �7nAChR agonist AR-R17779 wasdetected. This effect was blocked by �-bungarotoxin, an�7nAChR antagonist (38). Tropisetron also led to Ca2�

signaling, but it was less potent than that induced byAR-R17779 (see Supplementary videos 1–3, availableon the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/doi/10.1002/art.37809/abstract;additional information is available from the correspond-ing author).

Next, we performed functional readouts withthe above agents. The suppressive effect of tropisetronon TGF�1-induced type I collagen gene expression inHDFs was completely neutralized by �-bungarotoxin(Figure 4A). Like tropisetron, AR-R17779 attenuatedthe inductive effect of TGF�1 on type I collagen tran-scripts (Figure 4B).

Finally, we confirmed the critical role of�7nAChR in the context of TGF�1-mediated collagensynthesis by gene silencing. Transfection of HDFs with�7nAChR siRNA markedly decreased mRNA expres-sion of this receptor by �90%, whereas nontargetingsiRNA had no effect (Figure 4C, inset). Cells were thentreated with �7nAChR siRNA or nontargeting siRNAand stimulated with TGF�1 or TGF�1 plus tropisetron,respectively. Gene silencing of �7nAChR abolished thesuppressive effect of tropisetron on TGF�1-mediatedCol�1(I) and Col�2(I) mRNA expression, as shown byreal-time RT-PCR (Figure 4C). Moreover, �7nAChRsiRNA but not nontargeting siRNA neutralized thesuppressive effect of tropisetron on TGF�1-mediatedCPI secretion (Figure 4D). In summary, these findingsidentified �7nAChR as the essential and functionaltropisetron receptor that mediates the suppressive effecton TGF�1-induced collagen synthesis in HDFs.

Effect of tropisetron on experimentally inducedfibrosis and established skin fibrosis induced by bleo-mycin. To assess the in vivo significance of the observedin vitro effects of tropisetron in HDFs, we used anestablished mouse model of SSc in which dermal fibrosisis induced by subcutaneous injections of bleomycin (24).First, the possible preventive effect of tropisetron onbleomycin-induced skin fibrosis was assessed by deter-mining the amount of collagen at both the RNA andprotein levels in skin samples, using real-time RT-PCR.Subcutaneous injection of bleomycin led to significantlyincreased mRNA expression levels of Col�1(I) and

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Col�1(III) compared with control. Tropisetron stronglyreduced the mRNA expression levels of Col�1(I)(mean � SD �79 � 21%) and Col�1(III) (�79 � 11%)in skin samples from bleomycin-treated mice (Figure5A). At the protein level, bleomycin treatment likewiseresulted in an increase in the amount of extractablecollagen in skin, as shown by SDS-PAGE after pepsindigestion. This effect of bleomycin was suppressed bytropisetron (Figure 5B).

The extent of dermal fibrosis was also assessed byhistologic and immunohistochemical analysis. Masson’strichrome staining revealed intense dermal fibrosis inbleomycin-treated mice, as visualized by blue staining(Figure 5C). Cotreatment with tropisetron suppressedthis effect of bleomycin, while tropisetron alone did nothave any effect. The beneficial effect of tropisetron onbleomycin-induced skin fibrosis was confirmed by type Icollagen immunostaining. Bleomycin injection increased

Figure 4. The �7 nicotinic acetylcholine receptor (�7nAChR) mediates the effect of tropisetron on TGF�1-induced collagen synthesis in HDFs.A, The suppressive effect of tropisetron on TGF�1-induced collagen synthesis is neutralized by �-bungarotoxin (�-BTX). HDFs (n � 3) werepretreated with the specific �7nAChR antagonist �-BTX (5 nM) for 30 minutes and stimulated for 24 hours with TGF�1 (10 ng/ml) alone or TGF�1plus tropisetron (10 �g/ml) in the presence of the inhibitor. Collagen mRNA expression was determined by real-time RT-PCR. � � P 0.01 versuscontrol; E � P 0.05 versus TGF�1 plus tropisetron. B, The specific �7nAChR agonist AR-R17779 mimics the action of tropisetron onTGF�1-induced collagen expression. Cells (n � 3) were stimulated with TGF�1 (10 ng/ml) alone, AR-R17779 alone (50 �M), or both agents for24 hours. Collagen mRNA expression was determined by real-time RT-PCR. � � P 0.05 versus TGF�1 alone. C, Small interfering RNA (siRNA)knockdown of �7nAChR abolishes the suppressive effect of tropisetron on TGF�1-induced type I collagen synthesis in HDFs, as determined byreal-time RT-PCR. Inset, Gene ablation of �7nAChR was confirmed by real-time RT-PCR. � � P 0.05 versus TGF�1; �� � P 0.001 versuscontrol. D, The suppressive effect of tropisetron on TGF�1-mediated CPI secretion is neutralized by �7nAChR siRNA but not nontargeting siRNA,as determined by enzyme-linked immunosorbent assay of secreted CPI. Both RNA and protein expression analyses were performed 3 times. � � P 0.05 versus TGF�1. Bars show the mean � SD. See Figure 1 for other definitions.

ANTIFIBROGENIC AND ANTIFIBROTIC EFFECT OF TROPISETRON 799

the extent of deposited dermal collagen compared withthe negative control as well as tropisetron alone, whilecotreatment with bleomycin and tropisetron led to astrong decline in the extent of collagen staining (Figure5C). In accordance with these readouts, treatment ofmice with bleomycin resulted in an increase of dermalthickness, whereas tropisetron significantly reduced thiseffect (additional information is available from thecorresponding author).

To evaluate whether tropisetron is also operativeon established skin fibrosis, we used a post-bleomycin

approach described initially by other investigators (25).Accordingly, cutaneous fibrosis was first induced bybleomycin injections for 21 days followed by daily injec-tions with either NaCl (negative control) or tropisetron(10 �g) for a further 14 days. Tropisetron reducedbleomycin-induced collagen induction after the onset ofskin fibrosis, as shown by mRNA (Figure 5D, left panel)and protein readouts (Figure 5D, right panel). Thesefindings demonstrated that tropisetron had both preven-tive (antifibrogenic) and therapeutic (antifibrotic) activ-ity in the given mouse model of bleomycin-induced SSc.

Figure 5. Tropisetron has antifibrogenic and antifibrotic effects in a mouse model of bleomycin (BLM)–induced systemic sclerosis. A, Mice wereinjected with NaCl (0.9%), bleomycin (10 �g), tropisetron (10 �g/ml), or both reagents daily for 21 days. Cotreatment with tropisetron de-creased mRNA expression of type I and type III collagen in mouse skin, as determined by real-time RT-PCR. Bars show the mean � SD (n � 3).� � P 0.05; �� � P 0.01 versus bleomycin alone. B, Tropisetron reduces type I collagen deposition in bleomycin-treated mouse skin. Mice weretreated as indicated followed by protein extraction from skin samples. Results are representative of 3 independent experiments with identical results.C, Bleomycin induces intense dermal fibrosis in mouse skin sections, and cotreatment with tropisetron suppresses this effect. Mouse skin sectionswere stained with Masson’s trichrome or van Gieson’s stain or were immunoprobed with an anti–type I collagen antibody. Results are representativeof those in each of the 4 treatment groups. Bar � 100 �m. D, Tropisetron reduces established skin fibrosis. Mice were injected subcutaneously withbleomycin (10 �g) for 21 days followed by daily injections with either NaCl (0.9%) or tropisetron (10 �g) for a further 14 days. Post-bleomycintreatment with tropisetron decreases type I and type III collagen mRNA expression, as determined by real-time RT PCR (left panel) and type Icollagen deposition, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (right panel) in mouse skin. Bars show the mean �SD (n � 4). � � P 0.05 versus bleomycin plus NaCl. See Figure 1 for other definitions.

800 STEGEMANN ET AL

Effect of �7nAChR on dermal fibroblasts frompatients with SSc. In a final set of experiments, wedetermined the expression of �7nAChR in skin andfibroblasts from patients with SSc. As shown by semi-quantitative RT-PCR, transcripts for �7nAChR weredetectable in both normal and diseased adult humanskin (Figure 6A, top panels). Moreover, �7nAChRtranscripts were present in normal fibroblasts and fibro-blasts from patients with SSc (n � 3 each) (Figure 6A,bottom panels). To check whether the detected�7nAChR is functionally active in the latter cells, wetreated them with TGF�1 and tropisetron. Tropisetronreduced TGF�1-induced type I collagen mRNA expres-sion as well as CPI secretion in these cells (Figures 6Band C). In addition, tropisetron reduced TGF�1-

mediated �-SMA induction, as shown by real-time RT-PCR (Figure 6D).

DISCUSSION

Here, we provide evidence for a modulatingeffect of tropisetron on collagen synthesis in vitro andin vivo. In vitro, tropisetron dose-dependently sup-pressed type I collagen synthesis, and this effect wasmediated by �7nAChR. Validation of �7nAChR as thecrucial mediator of the anticollagenic effects of tropise-tron was shown by pharmacologic approaches usingspecific antagonists and agonists of �7nAChR as wellas by gene silencing of this receptor. The receptor�7nAChR belongs to a family of neurotransmitter-gated

Figure 6. The �7 nicotinic acetylcholine receptor (�7nAChR) is present in the skin and fibroblasts of patients with systemic sclerosis (SSc), andtropisetron (TRO) has modulatory effects on transforming growth factor �1 (TGF�1)–induced type I collagen and �-smooth muscle actin (�-SMA)expression in SSc fibroblasts. A, Transcripts for �7nAChR are expressed in both normal and SSc skin (top panels) and in both normal fibroblastsand fibroblasts from patients with SSc (bottom panels). Gene expression was determined by semiquantitative reverse transcription–polymerase chainreaction (RT-PCR). Lane 1, Synovial fibroblasts (positive control). Lane 2, H2O (negative control). Lanes 3–5, Human dermal fibroblasts (HDFs)from the skin of patients with SSc (n � 3). Lanes 6–8, HDFs from the skin of normal controls (n � 3). B and C, Tropisetron suppressesTGF�1-induced C-terminal type I procollagen propeptide (CPI) secretion, as shown by enzyme-linked immunosorbent assay (B) and collagenmRNA expression (C). D, Tropisetron reduces TGF�1-mediated �-SMA induction, as demonstrated by real-time RT-PCR. Bars show the mean �SD (n � 3). � � P 0.01 versus TGF�1 in B; 0.05 versus TGF�1 in C and D. To check whether the detected �7nAChR is functionally active,the cells were treated with TGF�1 and tropisetron. Tropisetron reduced TGF�1-induced type I collagen mRNA expression as well as CPI secretionin these cells (B and C). Rel. � relative.

ANTIFIBROGENIC AND ANTIFIBROTIC EFFECT OF TROPISETRON 801

ion channel proteins, mediating the exchange of ionssuch as Na�, Ca2�, and K� (39). Notably, expression ofthese receptors is not confined to neuronal cells (40)but also occurs in non-neuronal tissue, including skin(41). The �7nAChR detected in HDFs was shown to befunctionally active and to mediate the biologic effects oftropisetron in the context of TGF�1-mediated expres-sion of several ECM target genes.

In order to elucidate the molecular mechanism ofthe anti-TGF�1 effect of tropisetron, we investigatedseveral canonical signal transduction pathways of TGF�that are known to regulate collagen metabolism (30,32).Tropisetron did not affect the activation of canonicalSmad, c-Jun, Akt, or MAPK signal transduction path-ways in response to TGF�1. Moreover, the drug did notalter intracellular levels of cAMP. Previous studiesrevealed that �7nAChR agonists can activate the JAK/STAT pathway (42). However, inhibition of STAT-3suppressed collagen production in keloid fibroblasts (43).Moreover, STAT-4 was recently described to exert pro-fibrotic effects by controlling cytokine release in a mousemodel of SSc (44). Based on these findings, involvementof the STAT-3/4 pathways in the anticollagenic effect oftropisetron in HDFs is unlikely. Notably, the �7nAChRagonist nicotine was shown to inhibit the productionof inflammatory mediators via modulation of the NF-�Bpathway in macrophages (45). Further studies areneeded to clarify the signaling pathways downstream of�7nAChR that lead to the anticollagenic effect of tro-pisetron in HDFs.

To assess the in vivo relevance of our in vitrofindings, we tested tropisetron in a mouse model ofestablished bleomycin-induced SSc. Most importantly,tropisetron not only prevented bleomycin-induced skinfibrosis but also had antifibrotic effects in establishedfibrosis; the latter finding suggests a therapeutic po-tential of this drug in SSc patients with advanced skin(and organ) fibrosis. Notably, it has been reported thatmice with targeted disruption of �7nAChR have de-creased amounts of type I collagen protein and elastin(46). The discrepancy between the suppressive effect oftropisetron acting via �7nAChR in our in vitro experi-ments and these in vivo observations in which no inflam-matory stimulus was included needs to be further inves-tigated. Indeed, the bleomycin-induced fibrosis modelis regarded as an inflammation-driven model for SSc.Therefore, tropisetron, via �7nAChR, may also elicitantiinflammatory effects that could contribute to theattenuating effect on bleomycin-induced skin fibrosis.In animal models of inflammation, selective �7nAChR

agonists have been effective in suppressing macrophagecytokine production and inflammatory responses (47–51).

Regarding the future use of tropisetron in SSc,we evaluated the expression of �7nAChRs in the skinand fibroblasts of patients with SSc. The presence ofthese receptors in the affected skin and fibroblasts ofthese patients in particular indicated that this target sitefor tropisetron is preserved. Most importantly, tro-pisetron was shown to be operative even in HDFs frompatients with SSc. As in normal HDFs, tropisetronsuppressed TGF�1-mediated type I collagen synthesisand also attenuated expression of �-SMA, a myofibro-blast marker.

In summary, we demonstrated a novel effect oftropisetron, namely the inhibition of collagen synthesis,and provided insight into the molecular mechanism ofthis effect in HDFs. We identified �7nAChR as theessential receptor that mediates the anti-TGF�1 effectof tropisetron in HDFs and confirmed the relevance ofthese observations in vivo. Tropisetron not only pre-vented experimentally induced skin fibrosis but alsohad antifibrotic effects on established fibrosis inducedby bleomycin. Our findings encourage not only a de-tailed investigation of the cutaneous cholinergic systemin SSc but also the exploitation of tropisetron and other�7nAChR agonists as a future treatment option forfibrotic diseases.

ACKNOWLEDGMENTS

We are grateful to Mara Apel, Nicole Gross, and AnneErpenbeck-Leuer (Munster), and Gabi Scherr (Cologne) forexpert technical assistance.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Bohm had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Stegemann, Sindrilaru, Grando, Fiebich,Bohm.Acquisition of data. Stegemann, Sindrilaru, Heinick, Schulte.Analysis and interpretation of data. Stegemann, Eckes, del Rey,Muller, Scharffetter-Kochanek, Luger, Bohm.

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