Src kinases in systemic sclerosis: Central roles in fibroblast activation and in skin fibrosis

ARTHRITIS & RHEUMATISMVol. 58, No. 5, May 2008, pp 1475–1484DOI 10.1002/art.23436© 2008, American College of Rheumatology

Src Kinases in Systemic Sclerosis

Central Roles in Fibroblast Activation and in Skin Fibrosis

Catherine Skhirtladze,1 Oliver Distler,2 Clara Dees,1 Alfiya Akhmetshina,1 Nicole Busch,1

Paulius Venalis,1 Jochen Zwerina,1 Bernd Spriewald,1 Margarita Pileckyte,3

Georg Schett,1 and Jorg H. W. Distler1

Objective. Src kinases are nonreceptor tyrosinekinases, which have been implicated in cytoskeletalorganization and cell mobility. This study was under-taken to evaluate the potential of Src kinases as noveltargets of antifibrotic therapies.

Methods. Fibroblast cultures were obtained from10 patients with systemic sclerosis (SSc) and 5 healthysubjects. Src signaling was inhibited using small-molecule inhibitors and overexpression of a dominant-negative mutant of Src and of the endogenous inhibitorCsk. The expression of extracellular matrix proteins wasanalyzed by real-time polymerase chain reaction and bySirCol collagen assay. Toxic effects were excluded byMTT assay and staining for annexin V and propidiumiodide. The mouse model of bleomycin-induced dermal

fibrosis was used to assess the role of Src kinases indermal fibrosis in vivo.

Results. Stimulation with transforming growthfactor � and platelet-derived growth factor activated Srcsignaling in dermal fibroblasts from patients with SScand healthy donors. Incubation with the Src kinaseinhibitors or overexpressed mutant Src or Csk reducedthe synthesis of messenger RNA for COL1A1, COL1A2,and fibronectin 1. A dose-dependent reduction in colla-gen release was also observed at the protein level. Noinhibitory effects on proliferation and no increase in thenumber of apoptotic or necrotic fibroblasts were ob-served. Consistent with the in vitro data, inhibition ofSrc kinases prevented experimental dermal fibrosis.Dermal thickness, the amount of collagen protein, andthe number of myofibroblasts were reduced in a dose-dependent manner.

Conclusion. These findings indicate that Src ki-nases play important roles in the activation of fibro-blasts and in the development of experimental fibrosis.Thus, Src kinases might be interesting targets for novelantifibrotic therapies in SSc.

Systemic sclerosis (SSc) is characterized in itslater stages by massive fibrosis with accumulation ofextracellular matrix (ECM) components (1,2). Tissuefibrosis is a major cause of death and contributessignificantly to morbidity in SSc patients (3). The accu-mulation of ECM components in SSc is due to increasedproduction by activated fibroblasts (4). The mechanismsof the pathologic activation of fibroblasts in SSc are notcompletely understood, although profibrotic cytokinessuch as transforming growth factor � (TGF�) andplatelet-derived growth factor (PDGF) have been iden-tified as playing a key role in fibrosis in SSc (5).

Supported by the University of Erlangen-Nuremberg (ELANgrant 53410022) and the Interdisciplinary Center of Clinical Research,Erlangen (grant A20). Dr. Jorg H. W. Distler’s work was supported bythe Ernst Jung Foundation Career Support Award of Medicine.

1Catherine Skhirtladze, DiplBiol, Clara Dees, DiplBiol, AlfiyaAkhmetshina, DiplBiol, Nicole Busch, DiplBiol, Paulius Venalis, MD,Jochen Zwerina, MD, Bernd Spriewald, MD, Georg Schett, MD, JorgH. W. Distler, MD: Department of Internal Medicine 3 and Institutefor Clinical Immunology, University of Erlangen-Nuremberg, Erlan-gen, Germany; 2Oliver Distler, MD: Zurich Center of IntegrativeHuman Physiology, and University Hospital Zurich, Switzerland;3Margarita Pileckyte, MD: Kaunas Medical University Hospital, Kau-nas, Lithuania.

Drs. Skhirtladze and Oliver Distler contributed equally to thiswork.

Dr. Oliver Distler has received consulting fees, speaking fees,and/or honoraria (less than $10,000) from Array BioPharma. Dr. JorgH. W. Distler has received consulting fees, speaking fees, and/orhonoraria (less than $10,000 each) from Encysive and Actelion.

Address correspondence and reprint requests to Jorg H. W.Distler, MD, Department of Internal Medicine 3, University ofErlangen-Nuremberg, Universitatsstrasse 29, Erlangen 91054, Ger-many. E-mail: [email protected].

Submitted for publication October 18, 2007; accepted inrevised form January 18, 2008.

1475

However, therapeutic approaches to selectively inhibitthe pathologic activation of fibroblasts and target fibro-sis in SSc, e.g., by tyrosine kinase inhibitors, are begin-ning to enter clinical development.

Src kinases are a family of 9 nonreceptor tyrosinekinases (6). Like most tyrosine kinases, Src kinases havelow basal activity and become transiently activated uponappropriate stimulation (7). The activation of Src ki-nases is tightly regulated. Cellular stimulation with cer-tain cytokines, including PDGF, and activation of inte-grin signaling stimulate phosphorylation of a tyrosineresidue in their catalytic region and thereby activate Srcsignaling (7,8). Another major profibrotic mediator,TGF�, has also been shown to activate Src signaling inhepatoma cells, ovarian cancer cell lines, and mesangialcells (9–11), and to inhibit Src signaling in prostatecancer cells and endothelial cells (12,13). In contrast,phosphorylation of a tyrosine residue at the C-terminusby the C-terminal kinase (Csk) induces intramolecularbinding of the C-terminus to the SH2 domain of Srckinases, which places Src kinases into an inactive con-formation. Thus, Csk is a major endogenous inhibitor ofSrc kinases (6,7).

Src kinases are critically involved in adhesion viaregulation of the activation of focal adhesion kinase(FAK) (7,14). FAK is crucial for transmission of integrinsignaling upon adhesion of fibroblasts to the ECM. Inter-estingly, FAK has been linked to fibrosis, since it promotesdifferentiation from resting fibroblasts into myofibroblasts(15). Furthermore, inhibition of FAK has been shown toameliorate experimental pulmonary fibrosis (14).

Since Src kinases have been shown to be acti-vated by PDGF and Src is a major regulator of theprofibrotic activity of FAK, we hypothesized that Srckinases might play a role in fibrotic disorders such asSSc. The evaluation of Src kinases as potential noveltargets for the treatment of SSc is particularly relevant,because the first combined inhibitor of Src and Ablkinases, dasatinib, was recently approved for treatmentof chronic myelogenous leukemia resistant to imatinib(16). We investigated the role of Src kinases in SSc andevaluated the efficacy of Src inhibition as a novel,antifibrotic therapeutic approach, using toxicity assaysand in vitro and in vivo models of dermal fibrosis.

MATERIALS AND METHODS

Patients and fibroblast cultures. Fibroblast cultureswere obtained from biopsy specimens from lesional skin ofpatients with SSc (n � 10) and from healthy volunteers (n � 5),as previously described (17). All patients fulfilled the criteria

for SSc as described by LeRoy et al (18). All patients andcontrols signed a consent form approved by the local institu-tional review boards.

Incubation with small-molecule inhibitors of Src.Stimulation experiments were performed in Dulbecco’s mod-ified Eagle’s medium/1% fetal calf serum. Dermal fibroblastswere incubated with 2 highly specific small-molecule inhibitorsof Src kinases, 4-(4�-phenoxyanilino)-6,7-dimethoxyquinazo-line (PADMQ) at concentrations ranging from 10 nM to 1,000nM, and 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid amide (SU6656) at con-centrations ranging from 0.1 �M to 10 �M, for 24 hours (19).Both inhibitors were obtained from Calbiochem (Darmstadt,Germany). The concentrations of PADMQ and SU6656 usedrepresent therapeutic ranges and cover both the 50% inhibi-tion concentration (IC50) and the IC90 for Src. SU6656 inhibitsthe Src family kinases Src, Fyn, Yes, and Lyn, with IC50 valuesof 0.02–0.28 �M. In contrast, the IC50 values for the inhibitionof PDGF receptor �, insulin-like growth factor receptor I,cABL, and Csk were �10 �M, �20 �M, 1.74 �M, and 7.63 �M,respectively, and therefore significantly higher (19). Similarly,PADMQ inhibits Src with an IC50 of 15 nM and is selectiveover other tyrosine kinases, such as vascular endothelialgrowth factor (VEGF) receptor 2 (by 88-fold) and c-FMS (by190-fold) (20). Thus, both inhibitors are very specific for Srckinases. Control fibroblasts were incubated with the samevolumes of the solvent DMSO. In a subset of experiments,recombinant TGF� (10 ng/ml) or PDGF-BB (40 ng/ml) (bothfrom R&D Systems, Abingdon, UK) was added 60 minutesafter the Src inhibitor.

Western blot analysis. Protein isolation and Westernblotting were performed according to standard protocols (21).

Real-time quantitative polymerase chain reaction(PCR). Total RNA was reverse-transcribed, and quantificationof gene expression by TaqMan or by SYBR Green real-timePCR was performed according to standard protocols (17,22).

Transfection with Src, kinase-deficient Src, and Csk.SSc fibroblasts were transfected with wild-type Src, wild-typeCsk, and a kinase-deficient mutant of Src (Src �k) (kindlyprovided by Dr. Kari Alitalo, University of Kuopio, Kuopio,Finland and Dr. Sally Parsons, University of Virginia, Char-lottesville, VA, respectively [23,24]) using the human dermalfibroblast Nucleofector Kit (Amaxa, Cologne, Germany), aspreviously described (25). As controls, transfections with theempty vector were performed according to the same protocol.Overexpression of Src and Csk after transfection was con-firmed by real-time PCR analysis.

Collagen measurements. Total soluble collagen in cellculture supernatants and skin samples was quantified by SirColcollagen assay (Biocolor, Belfast, UK), as previously described(26).

MTT assay. The metabolic activity of cells incubatedwith PADMQ was assessed by MTT assay, as previouslydescribed (27).

Quantification of apoptotic and necrotic cells. Theinfluence of Src inhibition on the vitality of SSc and healthydermal fibroblasts was analyzed by staining for annexin V andpropidium iodide (PI) after incubation with PADMQ for 7days.

Bleomycin-induced dermal fibrosis. Skin fibrosis wasinduced in 7-week-old, pathogen-free, female DBA/2 mice

1476 SKHIRTLADZE ET AL

(n � 10) (Charles River, Sulzfeld, Germany) by local injectionsof bleomycin over 21 days (28,29). Bleomycin (100 �l) wasdissolved in 0.9% NaCl at a concentration of 0.5 mg/ml andadministered every other day by subcutaneous injection indefined areas of the upper back. Control mice (n � 10) wereinjected subcutaneously with 100 �l 0.9% NaCl. Two sub-groups were treated with bleomycin and different concentra-tions of the Src inhibitor SU6656. SU6656 was dissolved in20% DMSO/80% NaCl and administered twice daily by intra-peritoneal injection in a total volume of 100 �l. One group ofmice (n � 8) received 2 mg/kg SU6656 twice daily, and asecond group of mice (n � 5) received 6 mg/kg SU6656 twicedaily. Mice in the control group and the bleomycin groupreceived intraperitoneal injections of 100 �l 20% DMSO/80%NaCl in order to exclude any antifibrotic effect of this solvent.The doses of SU6656 used in this study have previously beenshown to effectively inhibit Src kinases in vivo (30). After 21days, animals were killed by cervical dislocation.

Histologic analysis. Injection sites were examined witha Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, TheNetherlands). Dermal thickness was determined by measuringthe maximal distance between the epidermal–dermal junctionand the dermal–subcutaneous fat junction at sites of indura-tion in 3 consecutive skin sections from each animal, aspreviously described (28). The analysis was performed by 2independent examiners who were blinded with regard totreatment group.

Detection of myofibroblasts. Myofibroblasts were iden-tified by staining for �-smooth muscle actin (�-SMA).

Statistical analysis. Data are expressed as the mean �SEM. Wilcoxon’s signed rank test for related samples and theMann-Whitney U test were used for statistical analyses. Pvalues less than 0.05 were considered significant.

RESULTS

Activation of Src signaling in dermal fibroblastsby profibrotic cytokines. TGF�, PDGF, and endothelin1 (ET-1) are overexpressed in SSc and are major stimuli

of the increased production of ECM by SSc fibroblasts.To test whether these profibrotic cytokines activate Srcsignaling, dermal fibroblasts were stimulated with re-combinant TGF�, PDGF, and ET-1. Equal loading ofprotein was confirmed by detection of the housekeepingprotein �-tubulin (results not shown). All of thesecytokines strongly stimulated the phosphorylation of Srcat the tyrosine residue 416 in SSc dermal fibroblasts,demonstrating that Src signaling cascades are activatedupon stimulation with TGF�, PDGF, and ET-1. Themaximal effect was observed after 10 minutes (Figure 1).After 60 minutes, Src signaling returned to baselinelevels. Similar results were obtained in healthy dermalfibroblasts (results not shown).

Reduction in basal production of ECM proteinsby inhibition of Src kinases. After demonstrating thatprofibrotic cytokines activate Src signaling in dermalfibroblasts, we investigated whether Src signaling plays arole in the production of ECM proteins. To assess theeffects of small-molecule inhibitors of Src kinases on theproduction of ECM proteins under basal conditions,dermal fibroblasts were treated with the Src inhibitorsPADMQ and SU6656. A dose-dependent down-regulation of the basal synthesis of messenger RNA(mRNA) for COL1A1, COL1A2, and fibronectin 1 wasobserved in SSc and healthy dermal fibroblasts treatedwith Src kinase inhibitors (data available upon requestfrom the corresponding author).

At concentrations of 10 nM, PADMQ reducedthe production of COL1A1 by SSc fibroblasts by amean � SEM of 19 � 8%. A significant decrease in thelevel of COL1A1 mRNA was detected in fibroblaststreated with 50 nM PADMQ, compared with untreated

Figure 1. Activation of Src signaling in dermal fibroblasts from patients with systemic sclerosis, before stimulation and after 10 minutes (10�) and60 minutes of stimulation with transforming growth factor � (TGF�), platelet-derived growth factor BB (PDGF-BB), or endothelin 1. Theseprofibrotic cytokines induced potent phosphorylation, and thereby activation of Src signaling, within 10 minutes (upper lanes). After 60 minutes, Srcsignaling returned to baseline levels. Equal loading was confirmed with the bicinchoninic acid protein assay, by quantification of total Src (lowerlanes) and �-tubulin (results not shown).

Src KINASES IN SYSTEMIC SCLEROSIS 1477

Figure 2. Reduction in the synthesis of extracellular matrix proteins upon inhibition of Src kinases by the small-molecule inhibitorPADMQ, and by overexpression of a dominant-negative mutant of Src (Src �k) and the endogenous inhibitor Csk. a–c, Levels ofmRNA for a, COL1A1, b, COL1A2, and c, fibronectin 1 in untreated control fibroblasts, fibroblasts stimulated with transforminggrowth factor � (TGF�), and fibroblasts stimulated with TGF� and treated with 50 nM or 250 nM PADMQ. PADMQ down-regulatedthe TGF�-induced synthesis of all 3 proteins in dermal fibroblasts from patients with systemic sclerosis (SSc) and healthy donors, ina dose-dependent manner, as determined by real-time polymerase chain reaction with SYBR Green. Bars show the mean and SEMprotein level, normalized to the level in control fibroblasts (set at 100). � � P � 0.05 versus fibroblasts stimulated with TGF�. d–f, Levelsof mRNA for d, COL1A1, e, COL1A2, and f, fibronectin 1 in mock-transfected SSc fibroblasts and SSc fibroblasts transfected withwild-type (WT) Src, Src �k, or Csk. Src signaling was stimulated by overexpression of WT Src. Inhibition of Src signaling byoverexpression of either Src �k or the endogenous inhibitor Csk strongly reduced the expression of all 3 proteins. Bars show the meanand SEM protein level, normalized to the level in mock-transfected cells (set at 100).


fibroblasts (mean � SEM decrease of 34 � 21%; P �0.05). The inhibitory effects of PADMQ were maximalat concentrations of 250 nM, with a mean � SEMreduction in the COL1A1 level of 52 � 11% (P � 0.05).PADMQ also reduced the levels of mRNA for COL1A2and fibronectin 1 in a dose-dependent manner. Treat-ment with 250 nM PADMQ reduced the synthesis ofmRNA for COL1A2 and fibronectin 1 in SSc fibroblastsby 57 � 10% and 62 � 8%, respectively (P � 0.05 forboth). No apparent differences in the effect of Srcinhibition on the synthesis of ECM proteins were ob-served between male and female patients or betweenpatients with limited cutaneous SSc and those withdiffuse cutaneous SSc. Similar results were obtained inhealthy dermal fibroblasts.

Reduction in TGF�-induced production of ECMproteins by inhibition of Src kinases. Next, we analyzedthe role of Src kinases in the production of ECMproteins in SSc and healthy dermal fibroblasts stimu-lated with TGF�. Treatment with PADMQ reduced theTGF�-mediated induction of ECM proteins to belowbaseline levels. The inhibitory effects of PADMQ on theTGF�-stimulated production of ECM proteins weremore pronounced than the effects on basal production.Dose-dependent reductions in COL1A1, COL1A2, andfibronectin 1 levels were observed. Treatment of SScfibroblasts with 250 nM PADMQ reduced COL1A1levels by a mean � SEM of 76 � 17%, COL1A2 levelsby 80 � 2%, and fibronectin 1 levels by 74 � 10%,compared with the levels observed in TGF�-stimulatedfibroblasts (P � 0.05 for all) (Figures 2a–c).

Reduction in PDGF-induced production of ECMproteins by inhibition of Src kinases. PDGF stimulatedthe synthesis of COL1A1 to 184 � 28% of control levelsin SSc fibroblasts (data available upon request from thecorresponding author). Pretreatment with the Src kinaseinhibitor blocked the stimulatory effects of PDGF onCOL1A1 in a dose-dependent manner, with a mean �SEM reduction of 72 � 5% in the COL1A1 level in SScfibroblasts treated with 250 ng/ml (P � 0.05). Inhibitionof Src kinases also reduced the synthesis of mRNA forCOL1A2 and fibronectin 1, with maximal reductions of77 � 16% and 79 � 9%, respectively, in SSc fibroblasts(P � 0.05 for both). Comparable inhibitory effects wereobserved in healthy dermal fibroblasts stimulated withPDGF.

To confirm the role of Src kinase in ECM pro-duction, SU6656, another inhibitor of Src kinases, whichhas previously been tested in vivo (30), was evaluated forits effects on the production of ECM proteins by dermal

fibroblasts. SU6656 significantly reduced the basal aswell as the cytokine-stimulated production of ECMproteins in a dose-dependent manner (data not shown).

Reduction in production of ECM proteins byoverexpression of mutant Src and overexpression of theendogenous inhibitor Csk. To confirm the role of Srcsignaling in the production of ECM proteins on anadditional experimental level, SSc fibroblasts were trans-fected with expression vectors for Src, Src �k, and Csk(Figures 2d–f). The efficacy of the transfections wasconfirmed on the mRNA level by real-time PCR (datanot shown). Stimulation of Src kinase signaling bytransfection with wild-type Src increased the levels ofCOL1A1, COL1A2, and fibronectin 1 to a mean � SEMof 162 � 7%, 180 � 25%, and 183 � 17%, respectively,compared with mock-transfected cells. Furthermore,inhibition of Src signaling by transfection with adominant-negative Src �k mutant or the endogenousinhibitor Csk reduced the expression of ECM proteins.Overexpression of Src �k decreased the expression ofCOL1A1 to 52 � 4% of the levels in controls, and Cskdecreased the expression of COL1A1 to 57 � 8% of thelevels in controls (Figure 2d). Similarly, the levels ofCOL1A2 and fibronectin 1 were strongly reduced in SScfibroblasts overexpressing Src �k and Csk (Figures2d–f).

Reduction in the release of collagen protein byinhibition of Src signaling. The reduced production ofECM proteins in SSc and healthy dermal fibroblastscaused by inhibition of Src signaling by small-moleculeinhibitors, by overexpression of a dominant-negative Srcmutant, and by the endogenous inhibitor Csk was con-firmed on the protein level using the SirCol collagenassay. Consistent with the results obtained on themRNA level, collagen synthesis was found to be stronglyreduced in SSc and healthy fibroblasts upon inhibition ofSrc signaling (Figures 3a and b). PADMQ reduced thesynthesis of collagen in a dose-dependent manner.Treatment with 10 nM PADMQ reduced the release ofcollagen by 10 � 5%, and treatment with 50 nM PADMQreduced the release of collagen by 38 � 8% (Figure 3a).The maximal reduction, 68 � 4% of the collagen produc-tion level in untreated SSc fibroblasts, was observed with250 nM of PADMQ (P � 0.05). Similar results wereobtained in healthy dermal fibroblasts.

Consistent with the findings obtained using thesmall-molecule inhibitor, inhibition of Src signaling byoverexpression of Src �k and Csk reduced the release ofcollagen to 52 � 4% and 57 � 8%, respectively, of thelevels in mock-transfected cells (Figure 3b). Overexpres-sion of wild-type Src, however, increased the level of


collagen to 183 � 17% (Figure 3b), thereby confirmingthe important role of Src signaling in the synthesis ofECM proteins in SSc fibroblasts.

Lack of effect of blockade of Src signaling on theexpression of matrix-degrading enzymes or their inhib-itors. To exclude the possibility that the reduced pro-duction of ECM proteins was counterbalanced by aconcomitant increase in the expression of matrix-degrading enzymes or their inhibitors, the expression ofmajor matrix metalloproteinases (MMPs) and tissueinhibitors of metalloproteinases (TIMPs) was analyzed.Incubation with chemical inhibitors of Src did not alterthe expression of MMP-1, MMP-2, MMP-3, or MMP-13in dermal fibroblasts (data available upon request fromthe corresponding author). Levels of TIMP-1, TIMP-2,and TIMP-3 were also unaffected. No changes in themRNA levels of these MMPs and TIMPs were observedin SSc fibroblasts transfected with wild-type Src, Src �k,or Csk (data not shown).

Lack of effect of inhibition of Src signaling on theviability of cultured dermal fibroblasts. To determinewhether inhibition of Src kinases causes toxic side effectsin fibroblasts, the effects of inhibition of Src on meta-bolic activity and cell viability were analyzed. Treatmentwith PADMQ did not reduce metabolic activity asassessed by MTT assay. No significant differences wereobserved between SSc and healthy dermal fibroblaststreated with 250 nM or 1,000 nM PADMQ and un-treated controls. In contrast, a significant reduction inmetabolic activity was observed in cells treated withDMSO (data not shown).

Treatment with PADMQ in concentrations up to1,000 nM for 7 days did not increase the number ofannexin V–positive cells, suggesting that treatment-related inhibition of Src signaling does not induceapoptosis under basal conditions in dermal fibroblastsfrom SSc patients and controls (data available uponrequest from the corresponding author). Furthermore,

Figure 3. a, Levels of collagen protein released in untreated control fibroblasts and fibroblasts treated with 10,50, or 250 nM PADMQ. PADMQ inhibited the basal synthesis of collagen in dermal fibroblasts from patientswith SSc and healthy donors, in a dose-dependent manner, as determined by SirCol collagen assay. Bars show themean and SEM collagen level, normalized to the level in control fibroblasts (set at 100). � � P � 0.05 versuscontrol fibroblasts. b, Levels of collagen protein released in mock-transfected SSc fibroblasts and SSc fibroblaststransfected with WT Src, Src �k, or Csk. Overexpression of Src �k and of the endogenous Src inhibitor Cskstrongly reduced the release of collagen by SSc fibroblasts, whereas overexpression of WT Src increased thesynthesis of collagen protein. Bars show the mean and SEM collagen level, normalized to the level inmock-transfected cells (set at 100). See Figure 2 for definitions.


no differences were observed between the SU6656-treated group and the untreated group with regard tothe numbers of PI-positive, necrotic fibroblasts uponinhibition of Src kinases.

Prevention of experimental dermal fibrosis byinhibition of Src signaling. To evaluate the potential ofSrc kinases as novel targets for antifibrotic therapies invivo, the efficacy of SU6656 was tested in a mouse modelof bleomycin-induced dermal fibrosis. Hematoxylin andeosin staining revealed a dense accumulation of thick-ened collagen bundles in the dermis of skin sectionsfrom bleomycin-injected mice that were not treated withSU6656. In mice treated with bleomycin, the dermalthickness increased significantly compared with controls(169 � 11%; P � 0.001) (Figures 4a, b, and e).

Intraperitoneal injections of the Src kinase inhib-itor SU6656 prevented the development of skin fibrosisin a dose-dependent manner. In mice treated with 2mg/kg SU6656 twice daily, the mean � SEM increase indermal thickness was reduced to 134 � 9% of thethickness in controls (P � 0.001 versus mice injectedwith bleomycin) (Figures 4c and e). In mice treated with6 mg/kg SU6656 twice daily, no increased accumulation

of collagen bundles was detectable. Consistent with thisobservation, the increase in dermal thickness upon in-jection of bleomycin was completely prevented in micetreated with 6 mg/kg SU6656 twice daily (109 � 12%;P � 0.002 versus mice injected with bleomycin) and didnot differ significantly from the dermal thickness incontrol mice (P � 0.23) (Figures 4d and e).

To further confirm the antifibrotic effects of Srcinhibition in vivo, the accumulation of collagen proteinin lesional skin was quantified by SirCol collagen assay.In mice treated with bleomycin alone, the amount ofcollagen in the skin was increased to 231 � 11%compared with controls (P � 0.05). In mice treated with2 mg/kg SU6656 twice daily, the increase in the amountof collagen protein in lesional skin samples was 59 �16% of the increase in mice treated with bleomycinalone. In mice treated with 6 mg/kg SU6656 twice daily,the increase in the level of collagen protein was 85 �19% less than the increase in mice treated with bleomy-cin alone (P � 0.05) (Figure 5).

Myofibroblasts possess a high capacity for theproduction of ECM proteins and are thought to play akey role in the pathogenesis of fibrotic disease. Immu-

Figure 4. Prevention of bleomycin-induced dermal fibrosis in a mouse model by the Src kinase inhibitor SU6656. SU6656 reduced the accumulationof extracellular matrix proteins in a dose-dependent manner. a–d, Representative tissue sections from a, an untreated control mouse, b, a mousetreated with bleomycin alone, c, a mouse treated with bleomycin and 2 mg/kg SU6656 twice daily (bid), and d, a mouse treated with bleomycin and6 mg/kg SU6656 twice daily (original magnification � 100). e, Dermal thickness in untreated control mice, mice treated with bleomycin alone, andmice treated with bleomycin and 2 mg/kg or 6 mg/kg SU6656. Treatment with SU6656 reduced the dermal thickness in a dose-dependent manner.Bars show the mean and SEM thickness normalized to dermal thickness in control mice (set at 100). � � P � 0.05 versus mice treated with bleomycinalone.


nohistochemical analysis of �-SMA demonstrated thattreatment with SU6656 significantly decreased the num-ber of myofibroblasts in lesional skin from mice in adose-dependent manner. In bleomycin-treated mice, thenumber of myofibroblasts per high-power field increasedto 326 � 7% compared with control mice (P � 0.001)(Figure 6). In mice treated with 2 mg/kg SU6656 twicedaily, the increase in the number of myofibroblasts was60 � 13% less than the increase in mice treated withbleomycin alone (P � 0.002). In mice treated with 6mg/kg SU6656 twice daily, the increase in the number ofmyofibroblasts was 85 � 19% less than the increase inmice treated with bleomycin alone (P � 0.003) (Figure6). Of note, the number of myofibroblasts in micetreated with bleomycin and 6 mg/kg SU6656 twice dailydid not differ significantly from that observed in controlmice (P � 0.38).

Treatment with 2 mg/kg or 6 mg/kg SU6656 twicedaily was well tolerated. Body weight did not differbetween the group of mice treated with bleomycin aloneand the groups treated with bleomycin and SU6656.

DISCUSSION

We demonstrated that Src kinases are activatedby profibrotic cytokines in fibroblasts from SSc patients.Inhibition of Src signaling strongly reduced the synthesisof COL1A1, COL1A2, and fibronectin 1 withoutcounter-regulatory changes in TIMPs and MMPs. Inhi-bition of Src did not reduce metabolic activity or cellviability under basal conditions. Inhibition of Src kinasesefficiently prevented dermal fibrosis in mice in vivo, withreduced dermal thickness and collagen content in the

Figure 5. Level of collagen protein in lesional skin from untreatedcontrol mice, mice treated with bleomycin alone, and mice treated withbleomycin and 2 mg/kg or 6 mg/kg SU6656 twice daily (bid), determinedby SirCol collagen assay. SU6656 reduced the accumulation of extracel-lular matrix proteins in mice with bleomycin-induced dermal fibrosis. Barsshow the mean and SEM collagen level normalized to the level in controlmice (set at 100). � � P � 0.05 versus mice treated with bleomycin alone.

Figure 6. Number of myofibroblasts in untreated control mice, micetreated with bleomycin alone, and mice treated with bleomycin and 2mg/kg or 6 mg/kg SU6656 twice daily (bid). Myofibroblasts in lesionalskin were identified by immunohistochemical analysis of �-smoothmuscle actin. Treatment with SU6656 decreased the number of myofibro-blasts in a dose-dependent manner. Bars show the mean and SEMnumber of myofibroblasts, normalized to the number in control mice(set at 100). � � P � 0.05 versus mice treated with bleomycin alone.


skin. Thus, the data presented herein indicate that Srckinases might be an interesting novel target for antifi-brotic approaches to the treatment of SSc.

We have shown that PDGF, ET-1, and TGF�, 3key players in the pathogenesis of SSc, potently stimu-late Src signaling in SSc and healthy dermal fibroblasts.The activation of Src kinases by PDGF in human dermalfibroblasts observed in our study is consistent withprevious findings in other cell types (8,31). In contrast tothe effects of PDGF, the effects of TGF� on Src kinaseshave been demonstrated to differ substantially amongcell types. TGF� stimulates Src signaling in hepatomacells, ovarian cancer cell lines, and mesangial cells,whereas it inhibits Src in prostate cancer cells andendothelial cells (9–13). In the present study, Src signal-ing was strongly activated by TGF� in dermal fibro-blasts. Thus, the effect of Src on the synthesis of ECMproteins seems to be specific to cell type and mightdepend on the cell type–specific expression of transcrip-tion factors and coactivators.

The profibrotic effects of Src kinases are of directclinical interest, because an Src kinase inhibitor, dasat-inib, was recently approved for the treatment of chronicmyelogenous leukemia resistant to imatinib. Dasatinib isa highly selective small-molecule tyrosine kinase inhibi-tor that selectively targets Src kinases, Abl kinases, andPDGF receptors (32). We and other investigators havedemonstrated that the combined inhibition of Abl ki-nases and PDGF receptor by imatinib exerts very potentantifibrotic effects in vitro and in vivo (28,33–35). Da-satinib inhibits the activity of Abl kinases 300-fold to1,300-fold more potently than does imatinib, and dasat-inib is active in most cell lines resistant to imatinib(32,36). Based on the potent antifibrotic effects of se-lective inhibitors of Src, combined inhibition of Src kinasesin addition to Abl and PDGF receptor by dasatinibmight have even more potent antifibrotic effects thaninhibition of Abl and PDGF receptor alone. However,further studies are needed to confirm this hypothesis.

Of note, the antifibrotic effects of Src kinaseinhibitors were not accompanied by toxic effects. Inhi-bition of Src kinases did not reduce the metabolicactivity of cultured SSc and healthy dermal fibroblastseven in high concentrations. Induction of apoptosis ornecrosis was not observed. Furthermore, mice toleratedSU6656 well, with no reduction in body weight or otherobvious signs of toxicity. The lack of obvious side effectsin our study is indirectly supported by studies of thecombined Src/Abl inhibitor dasatinib, which was welltolerated in clinical trials (36). Thus, inhibition of Srckinases, either alone or in combination with Abl kinases

and PDGF receptor, might represent a novel, nontoxicantifibrotic approach to the treatment of SSc.

Since treatment of established fibrosis is a majortherapeutic aim, preclinical studies of the activity ofnovel therapeutics are of major interest. Treatment ofestablished dermal fibrosis with Src kinase inhibitors hasnot yet been evaluated, but we are currently performingexperiments addressing the role of different tyrosinekinase inhibitors in the treatment of dermal fibrosis.

Although no toxic effects were observed in fibro-blasts or mice, potential antiangiogenic side effects ofantifibrotic drugs are an important concern in SSc, sincevascular disease is a major feature of SSc that signifi-cantly contributes to the morbidity of patients. Srckinases have indeed been implicated in endothelial cellfunction and angiogenesis. VEGF activates Src in avianendothelial cells, and overexpression of a dominant-negative Src mutant has been shown to inhibit VEGF-induced vascular permeabilization and angiogenesis(37). Thus, Src inhibits VEGF-induced angiogenesisunder normal circumstances. However, in SSc patientsan uncontrolled overexpression of VEGF has beendemonstrated (22). This overexpression has deleteriousrather than beneficial effects on angiogenesis that mightfurther aggravate vascular disease in SSc. Inhibition ofSrc signaling might therefore help to control the dys-regulated activation of VEGF signaling and its negativeeffects on angiogenesis in SSc. The net effect of Srcinhibitors on angiogenesis in SSc is unclear and needsfurther investigation.

In summary, our results demonstrate that Srckinases can be activated in SSc fibroblasts upon stimu-lation with profibrotic cytokines and regulate the syn-thesis of ECM proteins. Inhibition of Src kinases re-duced the production of ECM in vitro and inexperimental dermal fibrosis in vivo without toxic sideeffects. These data suggest that Src kinases might bepromising molecular targets for therapy in fibrotic dis-eases such as SSc.

AUTHOR CONTRIBUTIONS

Dr. Jorg H. W. Distler had full access to all of the data in thestudy and takes responsibility for the integrity of the data and theaccuracy of the data analysis.Study design. Oliver Distler, Schett, Jorg H. W. Distler.Acquisition of data. Skhirtladze, Dees, Akhmetshina, Busch, Venalis,Jorg H. W. Distler.Analysis and interpretation of data. Oliver Distler, Akhmetshina,Pileckyte, Schett, Jorg H. W. Distler.Manuscript preparation. Oliver Distler, Zwerina, Schett, Jorg H. W.Distler.Statistical analysis. Jorg H. W. Distler.Performance of in vivo experiments. Spriewald.


REFERENCES

1. D’Angelo WA, Fries JF, Masi AT, Shulman LE. Pathologicobservations in systemic sclerosis (scleroderma): a study of fifty-eight autopsy cases and fifty-eight matched controls. Am J Med1969;46:428–40.

2. Scheja A, Hellmer G, Wollheim FA, Akesson A. Carboxyterminaltype I procollagen peptide concentrations in systemic sclerosis:higher levels in early diffuse disease. Br J Rheumatol 1993;32:59–62.

3. Hesselstrand R, Scheja A, Akesson A. Mortality and causes ofdeath in a Swedish series of systemic sclerosis patients. AnnRheum Dis 1998;57:682–6.

4. Varga J, Bashey RI. Regulation of connective tissue synthesis insystemic sclerosis [review]. Int Rev Immunol 1995;12:187–99.

5. Gay S, Jones RE Jr, Huang GQ, Gay RE. Immunohistologicdemonstration of platelet-derived growth factor (PDGF) andsis-oncogene expression in scleroderma. J Invest Dermatol 1989;92:301–3.

6. Lowell CA. Src-family kinases: rheostats of immune cell signaling.Mol Immunol 2004;41:631–43.

7. Okutani D, Lodyga M, Han B, Liu M. Src protein tyrosine kinasefamily and acute inflammatory responses. Am J Physiol Lung CellMol Physiol 2006;291:L129–41.

8. Catarzi S, Biagioni C, Giannoni E, Favilli F, Marcucci T, Ianto-masi T, et al. Redox regulation of platelet-derived-growth-factor-receptor: role of NADPH-oxidase and c-Src tyrosine kinase.Biochim Biophys Acta 2005;1745:166–75.

9. Kim HP, Kim TY, Lee MS, Jong HS, Kim TY, Lee JW, et al.TGF-�1-mediated activations of c-Src and Rac1 modulate levels ofcyclins and p27(Kip1) CDK inhibitor in hepatoma cells replatedon fibronectin. Biochim Biophys Acta 2005;1743:151–61.

10. Mishra R, Zhu L, Eckert RL, Simonson MS. TGF-�-regulatedcollagen type I accumulation: role of Src-based signals. Am JPhysiol Cell Physiol 2007;292:C1361–9.

11. Tanaka Y, Kobayashi H, Suzuki M, Kanayama N, Terao T.Transforming growth factor-�1-dependent urokinase up-regula-tion and promotion of invasion are involved in Src-MAPK-dependent signaling in human ovarian cancer cells. J Biol Chem2004;279:8567–76.

12. Atfi A, Drobetsky E, Boissonneault M, Chapdelaine A, ChevalierS. Transforming growth factor � down-regulates Src family proteintyrosine kinase signaling pathways. J Biol Chem 1994;269:30688–93.

13. Manganini M, Maier JA. Transforming growth factor �2 inhibitionof hepatocyte growth factor-induced endothelial proliferation andmigration. Oncogene 2000;19:124–33.

14. Vittal R, Horowitz JC, Moore BB, Zhang H, Martinez FJ, ToewsGB, et al. Modulation of prosurvival signaling in fibroblasts by aprotein kinase inhibitor protects against fibrotic tissue injury. Am JPathol 2005;166:367–75.

15. Thannickal VJ, Lee DY, White ES, Cui Z, Larios JM, Chacon R,et al. Myofibroblast differentiation by transforming growth fac-tor-�1 is dependent on cell adhesion and integrin signaling viafocal adhesion kinase. J Biol Chem 2003;278:12384–9.

16. Jabbour E, Cortes J, Kantarjian H. Dasatinib for the treatment ofPhiladelphia chromosome-positive leukaemias [published erratumappears in Expert Opin Investig Drugs 2007;16:1135]. Expert OpinInvestig Drugs 2007;16:679–87.

17. Distler JH, Hagen C, Hirth A, Muller-Ladner U, Lorenz HM, delRosso A, et al. Bucillamine induces the synthesis of vascularendothelial growth factor dose-dependently in systemic sclerosisfibroblasts via nuclear factor-�B and simian virus 40 promoterfactor 1 pathways. Mol Pharmacol 2004;65:389–99.

18. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T,Medsger TA Jr, et al. Scleroderma (systemic sclerosis): classifica-tion, subsets and pathogenesis. J Rheumatol 1988;15:202–5.

19. Blake RA, Broome MA, Liu X, Wu J, Gishizky M, Sun L, et al.SU6656, a selective src family kinase inhibitor, used to probegrowth factor signaling. Mol Cell Biol 2000;20:9018–27.

20. Tian G, Cory M, Smith AA, Knight WB. Structural determinantsfor potent, selective dual site inhibition of human pp60c-src by4-anilinoquinazolines. Biochemistry 2001;40:7084–91.

21. Distler JH, Jungel A, Kowal-Bielecka O, Michel BA, Gay RE,Sprott H, et al. Expression of interleukin-21 receptor in epidermisfrom patients with systemic sclerosis. Arthritis Rheum 2005;52:856–64.

22. Distler O, Distler JH, Scheid A, Acker T, Hirth A, Rethage J, etal. Uncontrolled expression of vascular endothelial growth factorand its receptors leads to insufficient skin angiogenesis in patientswith systemic sclerosis. Circ Res 2004;95:109–16.

23. Bergman M, Joukov V, Virtanen I, Alitalo K. Overexpressed Csktyrosine kinase is localized in focal adhesions, causes reorganiza-tion of �v�5 integrin, and interferes with HeLa cell spreading. MolCell Biol 1995;15:711–22.

24. Ishizawar RC, Tice DA, Karaoli T, Parsons SJ. The C terminus ofc-Src inhibits breast tumor cell growth by a kinase-independentmechanism. J Biol Chem 2004;279:23773–81.

25. Distler JH, Jungel A, Huber LC, Seemayer CA, Reich CF III, GayRE, et al. The induction of matrix metalloproteinase and cytokineexpression in synovial fibroblasts stimulated with immune cellmicroparticles. Proc Natl Acad Sci U S A 2005;102:2892–7.

26. Distler JH, Jungel A, Caretto D, Schulze-Horsel U, Kowal-Bielecka O, Gay RE, et al. Monocyte chemoattractant protein 1released from glycosaminoglycans mediates its profibrotic effectsin systemic sclerosis via the release of interleukin-4 from T cells.Arthritis Rheum 2006;54:214–25.

27. Distler JH, Huber LC, Hueber AJ, Reich CF III, Gay S, Distler O,et al. The release of microparticles by apoptotic cells and theireffects on macrophages. Apoptosis 2005;10:731–41.

28. Distler JH, Jungel A, Huber LC, Schulze-Horsel U, Zwerina J,Gay RE, et al. Imatinib mesylate reduces production of extracel-lular matrix and prevents development of experimental dermalfibrosis. Arthritis Rheum 2007;56:311–22.

29. Yamamoto T, Takagawa S, Katayama I, Yamazaki K, HamazakiY, Shinkai H, et al. Animal model of sclerotic skin. I. Localinjections of bleomycin induce sclerotic skin mimicking sclero-derma. J Invest Dermatol 1999;112:456–62.

30. Severgnini M, Takahashi S, Tu P, Perides G, Homer RJ, JhungJW, et al. Inhibition of the Src and Jak kinases protects againstlipopolysaccharide-induced acute lung injury. Am J Respir CritCare Med 2005;171:858–67.

31. Chen KC, Zhou Y, Zhang W, Lou MF. Control of PDGF-inducedreactive oxygen species (ROS) generation and signal transductionin human lens epithelial cells. Mol Vis 2007;13:374–87.

32. Kantarjian H, Jabbour E, Grimley J, Kirkpatrick P. Dasatinib[review]. Nat Rev Drug Discov 2006;5:717–8.

33. Abdollahi A, Li M, Ping G, Plathow C, Domhan S, Kiessling F, etal. Inhibition of platelet-derived growth factor signaling attenuatespulmonary fibrosis. J Exp Med 2005;201:925–35.

34. Daniels CE, Wilkes MC, Edens M, Kottom TJ, Murphy SJ,Limper AH, et al. Imatinib mesylate inhibits the profibrogenicactivity of TGF-� and prevents bleomycin-mediated lung fibrosis.J Clin Invest 2004;114:1308–16.

35. Wang S, Wilkes MC, Leof EB, Hirschberg R. Imatinib mesylateblocks a non-Smad TGF-� pathway and reduces renal fibrogenesisin vivo. FASEB J 2005;19:1–11.

36. Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, PaquetteR, et al. Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med 2006;354:2531–41.

37. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A,et al. Src blockade stabilizes a Flk/cadherin complex, reducingedema and tissue injury following myocardial infarction. J ClinInvest 2004;113:885–94.


Src kinases in systemic sclerosis: Central roles in fibroblast activation and in skin fibrosis

Documents

Transcript of Src kinases in systemic sclerosis: Central roles in fibroblast activation and in skin fibrosis