CC-930

Jun N-terminal kinase as a potential molecular target for prevention and treatment of dermal ﬁbrosis

Nicole Reich, Michal Tomcik, Pawel Zerr, Veronika Lang, Clara Dees,

Jérome Avouac, Katrin Palumbo, Angelika Horn, Alﬁya Akhmetshina,
1

Christian Beyer, Weilin Xie, Brydon L Bennett, Oliver Distler, Georg Schett, Jörg H W Distler

Department for Internal

ABSTRACT

extracellular matrix components in SSc is caused by

Medicine 3 and Institute for Clinical Immunology, University
of Erlangen-Nuremberg,
Erlangen, Germany
Objectives The hallmark of systemic sclerosis (SSc) is the accumulation of extracellular matrix proteins by pathologically activated ﬁbroblasts. This study analysed
an increased production of extracellular matrix by activated ﬁbroblasts. Proﬁbrotic cytokines such as transforming growth factor beta (TGFβ) and plate-

2
Department of Clinical and
the antiﬁbrotic effects of the selective c-Jun N-terminal
let-derived growth factor (PDGF) are important

⦁ xperimental Rheumatology,
⦁ irst Faculty of Medicine,
Institute of Rheumatology,
Charles University in Prague,
Prague, Czech Republic
kinase (JNK) inhibitor, CC-930, which recently entered ﬁrst clinical trials as a novel antiﬁbrotic approach. Methods Phosphorylated c-Jun was detected by western blot and immunohistochemistry. The model of
players in the pathogenesis of SSc as they potently activate ﬁbroblasts and are sufﬁcient to induce ﬁbrosis in vivo. However, the intracellular signal- ling cascades, by which these cytokines stimulate

3
Paris Descartes University,
bleomycin-induced dermal ﬁbrosis and the tight skin 1
the production of extracellular matrix and induce

Rheumatology A department,
Cochin Hospital and INSERM
U781, Necker Hospital, Paris,
France
(TSK1) mouse model were used to investigate the effects of CC-930 on the prevention of experimental ﬁbrosis. The potential of CC-930 to induce regression of ﬁbrosis was
ﬁbrosis are incompletely understood.
Jun N-terminal kinases (JNK) belong to the superfamily of mitogen-activated protein kinases

4
Celgene, San Diego, California,
assessed in a modiﬁed model of established ﬁbrosis.
(MAPK). JNK are phosphorylated and thereby acti-

USA
Center of Experimental
Rheumatology and Zurich
Center of Integrative Human
Physiology, University Hospital
Zurich, Zurich, Switzerland
Correspondence to
Jörg H W Distler, Department of Internal Medicine 3 and Institute for Clinical Immunology,
Universitätsstr, University of
Erlangen-Nuremberg, 91054,
Erlangen, Germany;
[email protected]

Received 10 October 2011
Accepted 29 November 2011
Published Online First
18 January 2012
Results Transforming growth factor beta (TGFβ) and platelet-derived growth factor (PDGF) activate JNK
and stimulate the phosphorylation of its downstream target c-Jun. Incubation with CC-930 prevented the phosphorylation of c-Jun and reduced the stimulatory levels of these cytokines on the release of collagen. Inhibition of JNK prevented dermal thickening,
myoﬁbroblast differentiation and the accumulation of collagen in a dose-dependent manner in mice challenged with bleomycin and in TSK1 mice. In addition to the prevention of ﬁbrosis, treatment with pharmacologically relevant doses of CC-930 also induced regression of established experimental ﬁbrosis.
Conclusions These data identify JNK as a downstream mediator of the pro-ﬁbrotic effects of of TGFβ and PDGF in SSc ﬁbroblasts. Selective inhibition of JNK by CC-930 exerted potent antiﬁbrotic effects in vitro and in different models in vivo. JNK might thus be a novel molecular target for the treatment of ﬁbrosis in SSc.

Systemic sclerosis (SSc) is a connective tissue dis- ease that affects the skin and a variety of internal organs including lungs, gastrointestinal tract and heart. The predominant changes in the early stages of SSc are perivascular inﬂammatory inﬁltrates and apoptosis of microvascular endothelial cells. Later stages of SSc are characterised by massive accumu- lation of extracellular matrix components such as different types of collagen and ﬁbronectin leading to progressive tissue ﬁbrosis. The accumulating extracellular matrix disrupts the physiological tis- sue structure and frequently results in dysfunction of the affected organs. Tissue ﬁbrosis is a major cause of morbidity and a major contributor to the increased mortality in SSc. The accumulation of
vated in response to extracellular stimuli such as cytokines. JNK convert these extracellular stimuli into intracellular responses by activating down- stream target genes such as members of the activa- tor protein 1 (AP-1) family of transcription factors. JNK control cellular activation, differentiation and proliferation and thereby play key roles in the reg- ulation of tissue homeostasis and inﬂammation. Indirect evidence suggests that JNK might also play a role in ﬁbrotic diseases: JNK might be activated by pro-ﬁbrotic cytokines. TGFβ, which potently stimulates the activation of resting ﬁbroblasts and induces the release of extracellular matrix, activates JNK in epithelial cells. Target genes of JNK, such as the AP-1 family members c-Jun and other mem- bers of the AP-1 family have been implicated in the pathogenesis of ﬁbrosis in SSc. However, the roles of JNK in SSc and potential therapeutic effects of JNK inhibitors have not been evaluated. Antiﬁbrotic effects of JNK inhibitors in animal mod- els of SSc would have direct translational implica- tions because CC-930 is currently under evaluation in the clinic (www.clinicaltrials.gov; trial identiﬁer NCT01203943). The ﬁrst results indicate that JNK can be selectively targeted in humans by CC-930, and that inhibition of JNK is well tolerated with a low rate of severe adverse events.
The aim of the present study was to deﬁne the role of JNK in ﬁbroblast activation and tissue ﬁbro- sis in SSc and to evaluate the therapeutic potential of the selective inhibition of JNK. We demonstrate that JNK is activated by pro-ﬁbrotic cytokines in SSc ﬁbroblasts. Inhibition of JNK by the selective inhibitor CC-930 inhibits the release of extracellu- lar matrix proteins in cultured ﬁbroblasts, prevents ﬁbrosis in different models of SSc, and induces

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Figure 1 JNK is activated by TGFβ and PDGFin SSc. (A) Increased levels of phosphorylated c-Jun, the primary substrate of JNK, in SSc patients. Representative images are shown at 200-fold or 1000-fold magniﬁcation as indicated. (B) The JNK pathway is activated by pro-ﬁbrotic cytokines, and this activation is blocked by CC-930. To analyse JNK signalling in dermal SSc ﬁbroblasts, the phosphorylation of its target c-Jun was analysed by western blot. Incubation with TGFβ (lane 2) or PDGF(lane 4) increased the protein level of phosphorylated cJun compared with unstimulated ﬁbroblasts (lane 1). Pretreatment of ﬁbroblasts with CC-930 reduced the stimulatory effects of TGFβ (lane 3) and in particular of PDGF(lane 5). Equal loading of proteins was conﬁrmed by quanti ﬁcation of β-actin. JNK, c-Jun N-terminal kinase; p-cJun, phosphorylated cJun; PDGF,platelet-derived growth factor; SSc, systemic sclerosis; TGFβ, transforming growth factor beta.

regression of pre-established ﬁbrosis without toxic side effects. JNK might thus be a novel molecular target for the treatment of SSc and other ﬁbrotic diseases.

MATERIAL AND METHODS
Patients and ﬁbroblast cultures
Fibroblast cultures were obtained from skin biopsies of clini- cally involved skin from SSc patients (n=9). All patients ful-

with immunosuppressive or other potentially disease-mod- ifying drugs at the time of biopsy. Control ﬁbroblasts were obtained from skin biopsies of healthy age and sex-matched volunteers. Fibroblasts were prepared as outgrowth cultures from skin biopsies and cultured as described. Fibroblasts from passages 4 –8 were used for the experiments. All patients and controls signed a consent form approved by the local insti- tutional review boards.

ﬁlled the criteria for SSc as suggested by LeRoy and Megsger.
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The median age of SSc patients was 50 years (range 24 –75 years) and their median disease duration was 7 years (range 1.5–12 years). Five patients had limited cutaneous SSc and four had diffuse cutaneous SSc. All patients were positive for antinuclear antibodies; three of the patients were positive for anticentromere antibodies and two were positive for anti- topoisomerase-1 antibodies. None of the patients was treated

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Selective inhibition of JNK by CC-930
CC-930 was kindly provided by Celgene (Summit, New Jersey, USA). The speci ﬁcity of CC-930 was proved by exten- sive biochemical pro ﬁling against a panel of 209 protein kinases. At 3 μM, only three human kinases other than JNK showed inhibition that was greater than 80% in the screen- ing panel; MAPK2, epidermal growth factor receptor (EGFR)

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Figure 2 JNK regulates the release of collagen from ﬁbroblasts. (A) Inhibition of JNK signalling by CC-930 reduced the basal production of extracellular matrix components. Incubation of SSc ﬁbroblasts with CC-930 for 24 h reduced the expression of col 1a1, col 1a2, and ﬁbronectin-1 mRNA and the release of protein into supernatants. The levels of mock-treated ﬁbroblasts were deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. *p<0.05; **p<0.01. Stimulation of dermal SSc ﬁbroblasts with TGFβ and PDGFincreased the release of collagen into the supernatants. CC-930 completely blocked these stimulatory effects. The collagen levels of untreated ﬁbroblasts were deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. *p<0.05; **p<0.01. (B) Inhibition of JNK signalling by CC-930 reduced the stimulatory effects of pro-ﬁbrotic cytokines on collagen production. Stimulation of ﬁbroblasts with TGFβ and PDGFincreased the release of collagen into the supernatants. CC-930 completely blocked these stimulatory effects. The collagen levels of untreated ﬁbroblasts were deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. *p<0.05; **p<0.01. (C) CC-930 does not exert toxic side effects on dermal ﬁbroblasts. The proliferation rate of cultured dermal SSc ﬁbroblasts was analysed 24 h after incubation with CC-930. No differences could be measured, suggesting CC-930 was not toxic to ﬁbroblasts. The levels of mock-treated ﬁbroblasts were deﬁned as 100%; other results were normalised to this value. JNK, c-Jun N-terminal kinase; PDGF,platelet-derived growth factor; SSc, systemic sclerosis; TGFβ, transforming growth factor beta.

and mutated EGFR L861Q. Follow-up studies determined IC values of 480 nM (MAPK2) and 379 nM (EGFR). The IC values of JNK 1, 2, and 3 isomers were 52 nM, 5 nM and 5 nM, respectively. Dermal ﬁbroblasts were incubated with CC-930 at concentrations from 0.1 to 10 μM for 24 h. For con- trols, ﬁbroblasts were incubated with the solvent dimethyl sulphoxide in the same concentrations. In subsets of experi- ments, ﬁbroblasts were stimulated with recombinant TGF β at 10 ng/ml or PDGF-BB at 40 ng/ml (both R&D Systems, Abingdon, UK). For stimulation experiments ﬁbroblasts were cultured in Dulbecco ’s modiﬁed essential medium/F12 con- taining 0.1% fetal bovine serum and ascorbic acid (50 μg/ml) for 3 days before adding cytokines.

MTT assay
The microtitre tetrazolium (MTT) assay was used to assess the metabolic activity of cells incubated with CC-930, as described. SSc ﬁbroblasts were incubated with 1 μM CC-930 in 96-well plates for 20 h. Then MTT was added at a ﬁnal concentration of 1 mg/ml, and the cells were further incubated at 37°C for 4 h. Mock-treated ﬁbroblasts were used as controls, and all other results were normalised to untreated cells.

Western blot analysis
After rinsing twice with phosphate-buffered saline, cells were trypsinised and lysed with Laemmli buffer. Five micrograms of protein from each sample were separated by 10% sodium dodecylsulphate–polyacrylamide gel electrophoresis and elec- trotransferred onto polyvinylidene diﬂuoride membranes according to standard protocols. Immunoblots were incu- bated with monoclonal antibodies against c-Jun and phospho- c-Jun (Santa Cruz, Heidelberg, Germany). After incubation with horseradish peroxidase-conjugated rabbit anti-mouse antibodies (Dako, Hamburg, Germany), signals were detected with ECL western blotting detection reagents (Amersham Bioscience, Freiburg, Germany). For con ﬁrmation of the equal loading of proteins, the amount of β-actin was determined using mouse anti-human β -actin antibodies (Sigma-Aldrich, Munich, Germany).

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Immunohistochemistry
Skin biopsies were obtained from clinically involved skin of patients with SSc (n=10). The median age of SSc patients was 45 years (range 21–56 years) and their median disease duration was 4.5 years (range 0.5–6 years). Five patients had limited cutaneous SSc and ﬁve patients had diffuse cutaneous SSc. None of the patients was treated with immunosuppressive or other potentially disease-mod- ifying drugs at the time of biopsy. Control biopsies were obtained from age and sex-matched healthy volunteers. Formalin-ﬁxed, par- afﬁn-embedded skin sections were stained with antibodies against phosphorylated c-Jun (Santa Cruz Biotechnology Inc, Heidelberg, Germany). Peroxidase labelled mouse immunoglobulins (Dako, Glostrup, Denmark) were used as secondary antibodies. Irrelevant isotype-matched antibodies were used as controls. Staining was visualised with 3,3-diaminobenzidine tetrahydrochloride (DAB) peroxidase substrate solution (Sigma-Aldrich). Irrelevant isotype- matched antibodies were used as controls. Staining was visualised with DAB peroxidase substrate solution (Sigma-Aldrich).

Quantitative real-time PCR
Total RNA was isolated with the NucleoSpin RNA II extraction system (Machery-Nagel, Düren, Germany) and reverse transcribed into complementary DNA with random hexamers, as described. Gene expression was quanti ﬁed by TaqMan or by SYBR green real-time PCR using the ABI Prism 7700 sequence detection sys- tem (PE Applied Biosystems, Rotkreuz, Switzerland) using prim- ers described previously. Samples without enzyme in the reverse transcription reaction were used as negative controls to exclude genomic contamination. Unspeciﬁc signals caused by primer dim- ers were excluded by dissociation curve analysis and by no tem- plate controls. A predeveloped 18S assay (Applied Biosystems) was used to normalise for the amounts of loaded cDNA. Differences were calculated with the threshold cycle and the comparative threshold cycle method for relative quanti ﬁcation.

Collagen measurements
Total soluble collagen in cell culture supernatants was quanti- ﬁed using the SirCol collagen assay (Biocolor, Belfast, Northern Ireland) as described.

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Figure 3 Bleomycin-induced dermal ﬁbrosis was prevented by treatment with CC-930. (A) Dermal ﬁbrosis was strongly reduced by treatment with CC-930. Representative skin sections stained with haematoxylin and eosin are shown at 100-fold magniﬁcation. The dermal thickness (B), the hydroxyproline content (C) and the numbers of myoﬁbroblasts (D) were reduced dose-dependently by treatment with CC-930. The efﬁcacy of CC-930 was comparable to that of imatinib, which served as a positive control. The levels of control mice were deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. *p<0.05; **p<0.01. NaCl, sodium chloride.

Bleomycin-induced dermal ﬁbrosis
Dermal ﬁbrosis was induced in 6-week-old DBA/2J mice by injections of bleomycin in deﬁned areas of the upper back. One hundred microlitres of bleomycin dissolved in 0.9% sodium chloride (NaCl) at a concentration of 0.5 mg/ml were injected subcutaneously every other day. Subcutaneous injections of 100 μl NaCl were used as controls for treatment with bleomycin. Five different groups of mice with a total number of 36 mice were analysed. One group received injections of NaCl and the other four groups were challenged with bleomycin. Two of the bleomycin-treated groups also received CC-930 in doses of 50 mg/kg and 150 mg/kg by oral gavage twice a day. Another group was also treated intraperitoneally with 50 mg/kg imatinib as a positive control for response to antiﬁbrotic treatment. CC-930 and imatinib were initiated together with the ﬁrst challenge of bleomycin over 3 weeks. After treatment, animals were killed by cervical dislocation.

TSK 1 mouse model
To investigate the role of JNK in a model of SSc that is less depen- dent on inﬂammatory changes than bleomycin-induced dermal

of TSK1 mice that were treated with CC-930 at doses of 50 mg/kg or 150 mg/kg, with imatinib at a dose of 50 mg/kg or that did not receive any antiﬁbrotic treatment. The ﬁfth group consisted of control mice of the same genetic background not carrying the TSK1 mutation. The treatment was started at an age of 5 weeks and the outcome was evaluated at an age of 10 weeks.

Mouse model of established dermal ﬁbrosis
To evaluate the regression of ﬁbrosis on inhibition of JNK, a mod- iﬁed model of bleomycin-induced dermal ﬁbrosis was used. In this model, treatment was initiated 3 weeks after the beginning of the challenge with bleomycin, when signiﬁcant dermal ﬁbro- sis was already established. The outcome of six different groups with a total number of 40 mice was analysed. The ﬁrst group of mice received subcutaneous injections of NaCl for 6 weeks. The second group was injected for 3 weeks with bleomycin fol- lowed by injections of NaCl for another 3 weeks to analyse the degree of ﬁbrosis before treatment, and to control the spontane- ous regression of ﬁbrosis. The third group of mice was killed after 6 weeks of injections with bleomycin. The fourth and the ﬁfth group were treated with CC-930 at doses of 50 mg/kg and

ﬁbrosis,
the antiﬁbrotic potential of CC-930 was evaluated in
150 mg/kg for the last 3 weeks of continuous challenge with

the tight skin 1 (TSK1) mouse model of SSc. Five groups with a total number of 51 mice were analysed. Four groups consisted

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bleomycin for 6 weeks. The sixth group was a positive control group consisting of mice challenged with bleomycin for 6 weeks

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Figure 4 Antiﬁbrotic effects of CC-930 in TSK1 mouse model. (A) Treatment with CC-930 dose-dependently reduced ﬁbrosis in TSK1 mice. Representative skin sections stained with haematoxylin and eosin are shown at 40-fold magniﬁcation. Treatment of TSK1 mice with CC-930 reduced hypodermal thickening (B) and completely prevented myoﬁbroblast differentiation (C). The antiﬁbrotic effects of 150 mg/kg CC-930 were comparable with that observed with 50 mg/kg imatinib. The levels of pa/pa mice were deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. **p<0.01. TSK, tight skin 1.

and treated in parallel with imatinib at doses of 50 mg/kg for the last 3 weeks.

Histological analysis
Skin sections were stained with haematoxylin and eosin for better visualisation of the tissue structure. The dermal thickness was anal- ysed with a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands) by measuring the distance between the epider- mal–dermal junction and the dermal –subcutaneous fat junction at four different skin sections of each mouse. The hypodermal thickness was determined by measuring the thickness of the sub- cutaneous connective tissue beneath the panniculus carnosus at four different sites at the upper back in each mouse. The evalu- ation was performed by two independent examiners.

Detection of myoﬁbroblasts
Myoﬁbroblasts are characterised by the expression of α-smooth muscle actin (αSMA). Fibroblasts positive for αSMA were detected by incubation with monoclonal anti-αSMA antibod- ies (clone 1A4; Sigma-Aldrich, Steinheim, Germany). The expression was visualised with horseradish peroxidase-labelled secondary antibodies and DAB (Sigma-Aldrich). Monoclonal mouse immunoglobulin G antibodies (Calbiochem, San Diego, California, USA) were used for controls.

Hydroxyproline assay
The collagen content of 3 mm punch biopsies from lesional skin samples was determined by hydroxyproline assays as described.

Statistics
Data are expressed as mean±SEM. The Wilcoxon signed rank tests for related samples and the Mann–Whitney U test were

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used for statistical analyses. A p value of less than 0.05 was con- sidered statistically signiﬁcant.

RESULTS
The JNK pathway is activated by pro-ﬁbrotic cytokines and blocked by CC-930
Active JNK phosphorylates the AP-1 transcription factor c-Jun and the phosphorylation of c-Jun is commonly used as a func- tional read-out for the activity of JNK. To assess the activ- ity of JNK in SSc, we performed immunohistochemistry for phosphorylated c-Jun. p-cJun was detected in ﬁbroblasts, ves- sels and in the epidermis. Increased p-cJun was observed in samples from SSc patients compared with healthy controls. Increased staining was particularly observed in ﬁbroblasts and small vessels of SSc, whereas the differences in the epidermis were less pronounced. ( ﬁgure 1A). To analyse whether TGF β and PDGF induce the activation of JNK in SSc ﬁbroblasts, western blot analysis of cultured dermal ﬁbroblasts for c-Jun was performed. Furthermore, we analysed whether the JNK inhibitor CC-930 decreased activation and phosphorylation of c-Jun upon stimulation by TGF β and PDGF. Stimulation with TGF β and PDGF potently increased the phosphorylation of c-Jun ( ﬁgure 1B). In contrast, preincubation with CC-930 at a clinically relevant concentration of 1 μM reduced the stimu- latory effects of TGF β and PDGF on the phosphorylation of c-Jun. In particular, CC-930 completely prevented the stimu- latory effect of PDGF on c-Jun phosphorylation in ﬁbroblasts ( ﬁgure 1B).

Inhibition of JNK reduced the synthesis of extracellular matrix in SSc ﬁbroblasts
We next investigated whether inhibition of JNK might inhibit the basal release of extracellular matrix proteins from SSc

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Figure 5 CC-930 induced regression of experimental ﬁbrosis. (A) Treatment with CC-930 did not only stop further progression, but also induced regression of bleomycin-induced dermal ﬁbrosis. Representative skin sections stained with haematoxylin and eosin are shown at 100-fold
magniﬁcation. Treatment with CC-930 reduced dermal thickening (B), hydroxyproline content (C) and myoﬁbroblast counts (D) to below pretreatment levels despite ongoing challenge with bleomycin. The level of mice receiving sodium chloride injections for 6 weeks was deﬁned as 100%; other results were normalised to this value. Data represent the mean±SEM. *p<0.05; **p<0.01. NaCl, sodium chloride.

ﬁbroblasts. CC-930 dose-dependently reduced the synthesis of extracellular matrix proteins in SSc ﬁbroblasts. No dif- ferences were observed between ﬁbroblasts isolated from patients with limited cutaneous SSc and patients with dif- fuse cutaneous SSc. Maximal effects were observed at con- centrations of 1 μM. At this concentration, CC-930 reduced the messenger RNA levels of col 1a1 and col 1a2 to 71±11% and 61±13%, respectively, and the levels of ﬁbronectin 1 to 55±14% (p<0.02 compared with mock-treated ﬁbroblasts for all) ( ﬁgure 2A). Consistent with the decreased mRNA levels, incubation with 1 μM CC-930 also reduced the amount of collagen protein in the supernatant to 79±7% (p=0.001) com- pared with control ( ﬁgure 2A). In contrast to SSc ﬁbroblasts, no signiﬁcant effects on the basal collagen synthesis were

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observed in unstimulated healthy dermal ﬁbroblasts (data not shown).
We next analysed whether CC-930 can prevent the pro- ﬁbrotic effects of TGFβ and PDGF in healthy dermal ﬁbroblasts. Incubation with CC-930 at a concentration of 1 μM decreased the stimulatory effects of TGFβ by 230±13% (p=0.005) and of that of PDGF by 73±23% (p=0.04) (ﬁgure 2B).
To exclude the possibility that the inhibitory effects on the release of extracellular matrix proteins are due to the toxicity of CC-930 with a general reduction of metabolic activity, MTT assays were performed. CC-930 did not reduce metabolic activ- ity in antiﬁbrotic concentrations, suggesting that inhibition of JNK speciﬁcally prevents ﬁbroblast activation and matrix syn- thesis without toxic side effects (ﬁgure 2C).

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Figure 6 Proposed mechanism of action of novel anti ﬁbrotic therapies and potential interactions. JNK, c-Jun N-terminal kinase; NCID, Notch intracellular domain; PDGFR,platelet-derived growth factor receptor; PPARγ, peroxisome proliferator-activated receptor gamma; RTK, receptor tyrosine kinase; TCF,; TGFR, transforming growth factor β receptor.

Inhibition of JNK prevents bleomycin-induced ﬁbrosis
To investigate the role of JNK in inﬂammation-driven models resembling early inﬂammatory stages of SSc, the mouse model of bleomycin-induced dermal ﬁbrosis was used. Treatment with CC-930 was well tolerated. No signs of toxicity such as weight loss, reduced activity, or rufﬂed fur were observed.
Dermal ﬁbrosis with dense accumulation of collagen bundles, inﬂammatory inﬁltrates and replacement of the subcutaneous fat tissue by connective tissue was observed in sham-treated bleomycin-challenged mice. In mice treated with CC-930, the development of ﬁbrosis by bleomycin was signiﬁcantly amelio- rated (ﬁgure 3A). Treatment with CC-930 reduced dermal thick- ening in a dose-dependent manner by up to 45±2% at doses of 150 mg/kg (p=0.001). This reduction in dermal thickening was comparable to that observed with imatinib at doses of 50 mg/ kg (ﬁgure 3A,B). Inhibition of JNK by CC-930 also prevented the accumulation of collagen in lesional skin. The hydroxy- proline content of the skin area injected with bleomycin was dose-dependently reduced by up to 53±3% in mice treated with 150 mg/kg CC-930 compared with bleomycin-treated animals (p=0.043) (ﬁgure 3C). Consistent with reduced dermal thicken- ing and the decreased accumulation of collagen, inhibition of JNK also inhibited the differentiation of resting ﬁbroblasts into metabolically active myoﬁbroblasts. At doses of 150 mg/kg, the myoﬁbroblast differentiation induced by bleomycin challenge was completely prevented (p=0.001) (ﬁgure 3D).

Inhibition of JNK prevents ﬁbrosis in the TSK1 model
To conﬁrm the role of JNK in a less inﬂammation-dependent model of ﬁbrosis that better resembles the late stages of SSc, we assessed the antiﬁbrotic effects of CC-930 in the TSK1 mouse model. Treatment with CC-930 also exerted potent antiﬁbrotic effects in the TSK1 model, and reduced hypodermal thickening in a dose-dependent manner by up to 85±4% (p<0.001) com- pared with sham-treated TSK1 mice (ﬁgure 4A,B). Consistent

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with previous reports, imatinib prevented histological changes in TSK1 mice. Of interest, the reduction in hypodermal thick- ening at doses of 150 mg/kg CC-930 was more pronounced than with 50 mg/kg imatinib (ﬁgure 4B). Inhibition of JNK by CC-930 also reduced myoﬁbroblast counts in the TSK1 model in a dose-dependent manner. At doses of 150 mg/kg, myoﬁbro- blast differentiation was completely prevented, and the myoﬁ- broblast counts did not differ from control mice not carrying the TSK1 mutation (p=0.004 compared with untreated TSK1 mice) (ﬁgure 4C).

Inhibition of JNK can induce regression of established ﬁbrosis To evaluate whether inhibition of JNK might also be effective for the treatment of established ﬁbrosis, a modiﬁed model of bleomycin-induced dermal ﬁbrosis was used. Prolonged injec- tions of bleomycin for 6 weeks resulted in progressive ﬁbrosis with increased dermal thickening to 226±6% compared with a dermal thickening of 175±8% after 3 weeks of bleomycin chal- lenge (p=0.021 compared with NaCl injections for 6 weeks). The dermal thickness in mice treated with CC-930 for the last 3 weeks was signiﬁcantly reduced to 127±4% at doses of 150 mg/ kg (p=0.008 compared with bleomycin for 6 weeks) (ﬁgure 5A,B). CC-930 thus decreased the level of dermal thickness under the pretreatment level (127±4% vs 175±8% compared with NaCl injections for 6 weeks, p=0.008 and p=0.021). Besides dermal thickness, the number of myoﬁbroblasts was strongly reduced on treatment with 150 mg/kg CC-930 under the pretreatment level (171±31% vs 443±15% compared with NaCl injections for 6 weeks, p=0.012 and p=0.014) (ﬁgure 5C). In addition, the accumulation of collagen assessed by hydroxyproline assay was signiﬁcantly reduced in mice treated with 150 mg/kg CC-930 for the last 3 weeks of continuous bleomycin challenge for 6 weeks (p=0.025) (Figure 5D). The inhibition of JNK thus not only pre- vents the development of ﬁbrosis in different models, but can also induce the regression of pre-existing ﬁbrosis.

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DISCUSSION
In this paper we highlight an important role of JNK in experi-

cannabinoid receptor 2 agonists primarily target ﬁbroblast activation indirectly by inhibiting the release of pro- ﬁbrotic

mental ﬁbrosis and demonstrate that JNK signalling is activated
mediators from inﬂammatory cells.
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by pro-ﬁbrotic cytokines. TGFβ and PDGF, two key players in ﬁbrotic disease activate JNK in SSc ﬁbroblasts, as demonstrated by phosporylation of its downstream target c-Jun in vitro and in vivo. Consistent with our results in ﬁbroblasts, an increased phosphorylation of JNK on stimulation with TGFβ has also been demonstrated in tracheal epithelial cells and in breast cancer cells. These ﬁndings demonstrate that JNK is a common target of pro-ﬁbrotic mediators and indicate a role of JNK as intracel- lular mediators of the pro-ﬁbrotic effects of TGFβ and PDGF.
Indeed, selective inhibition of JNK by CC-930 potently
We did not observe toxic effects of CC-930 in antiﬁbrotic concentrations. Inhibition of JNK did not reduce the metabolic activity of cultured ﬁbroblasts. Mice treated with CC-930 did not show any signs of toxicity such as weight loss, reduced activity, or rufﬂed fur. In an extension of our preclinical data, phase III clinical trials with different inhibitors of JNK for the treatment of rheumatoid arthritis and different types of cancer showed that inhibition of JNK is well tolerated in humans. The ﬁrst data on CC-930 in humans are also available. Results from dosing studies in healthy volunteers have indicated that CC-930

reduced the activation of ﬁbroblasts. CC-930 inhibited the
is well tolerated and exposure is dose-proportional.
Moreover,

release of extracellular matrix proteins. Inhibition of JNK also prevented experimental ﬁbrosis in the bleomycin model of early inﬂammatory stages of SSc. Moreover, inhibition of JNK also induced the regression of pre-established experimental ﬁbro- sis. The mouse model of bleomycin-induced ﬁbrosis is highly dependent on inﬂammation and this results in hypersensitiv- ity to anti-inﬂammatory therapies. CC-930 also exerted potent anti ﬁbrotic effects in the TSK1 model of later, non-in ﬂamma- tory, stages of SSc. In contrast to bleomycin-induced ﬁbrosis, inﬂammatory inﬁltrates are not observed in the TSK1 model and TSK1 mice are not considered to be hyperresponsive to anti-inﬂammatory therapies in general. However, B cells seem to play a very central role in the development of ﬁbrosis in TSK1 mice, and the TSK1 model may overestimate the effects of B-cell targeting therapies. These limitations should be con- sidered when interpreting the results obtained in individual models. However, a prudent combination of different models may overcome many of the limitations of individual animal models. In the case of targeting JNK, additional evidence of anti ﬁbrotic effects is provided from models of other ﬁbrotic disorders. Consistent with our data obtained with CC-930, JNK mice are protected from liver inﬂammation and sub- sequent ﬁbrosis induced by a choline-deﬁcient L -amino acid- de ﬁned diet. Deﬁciency of JNK also prevented subepithelial collagen deposition in an ovalbumin-driven model of allergic airways disease. Together, these ﬁndings suggest that JNK plays crucial roles in the pathogenesis of ﬁbrotic diseases.
⦁ everal potential targets for translational therapies of SSc have recently been identiﬁed ( ﬁgure 6). Some of them target similar pathways at different levels. Tyrosine kinase inhibi- tors such as imatinib, inhibitors of Src kinases, Jak-2 inhibi- tors and also JNK inhibitors are all growth factor signalling at different levels and may exert their antiﬁbrotic effects by the inhibition of intracellular cascades that are activated by TGFβ
⦁ he antiﬁbrotic effects of peroxisome
proliferator-activated receptor gamma agonists may also pri- marily be mediated by the inhibition of TGFβ signalling. Binding of serotonin to 5-HT2B receptors stimulates the release of TGFβ. The inhibition of 5-HT2B may thus exert its antiﬁ- brotic effects indirectly by decreased TGF β signalling. The morphogenic pathways such as Wnt, Hedgehog and Notch, which have recently been implicated in ﬁbrosis, interact with each other and with TGFβ signalling during development.
Although direct links between these pathways and TGFβ signalling in ﬁbrosis have not yet been established, altered TGFβ signalling may contribute to the pro-ﬁbrotic effects of Wnt, Hedgehog and Notch pathways. Antibodies against interleukin (IL)-4 and IL-13 inhibit other pro- ﬁbrotic cytokines, but also directly target ﬁbroblast activation. In contrast, IL6 receptor antagonists, cannabinoid receptor 1 antagonists and

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no unexpected drug-dependent effects were observed from phase I clinical assessment. These data suggest that inhibition of JNK by inhibitors such as CC-930 is well tolerated, and that a potential application in patients with ﬁbrotic diseases might not be hindered by toxicity.
In summary, we demonstrate that JNK is activated by pro- ﬁbrotic cytokines and regulates the synthesis of extracellular matrix proteins. Selective inhibition of JNK by CC-930 prevented skin ﬁbrosis in different models resembling different stages of SSc, and induced regression of pre-established experimental ﬁbrosis. As inhibition of JNK is well tolerated and inhibitors are currently being evaluated in clinical trials for other indications, JNK might be an interesting molecular target in SSc and other ﬁbrotic diseases.
Acknowledgments The authors would like to thank Maria Halter and Anna-Maria Herrmann for excellent technical assistance.
Funding This study was funded by grants DI 1537/1-1 and DI 1537/2-1 of the Deutsche Forschungsgesellschaft, the Career Support Award of Medicine of the Ernst Jung Foundation, grants A20 and A40 of the Interdisciplinary Center of Clinical Research in Erlangen, a grant from Celgene and the CMH research project no 00000023728.
Ethics approval Ethics approval was obtained from the local institutional review boards. Patient consent Obtained.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed. REFERENCES
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PAFR Signal

PAFR has won the GFOA

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