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(The FASEB Journal. 2001;15:797-806.)
© 2001 FASEB

Molecular mechanisms of TGF-ß antagonism by interferon {gamma} and cyclosporine A in lung fibroblasts

OLIVER EICKELBERG1, ANDREAS PANSKY*, ELEONORE KOEHLER*, MICHEL BIHL*, MICHAEL TAMM*, PIUS HILDEBRAND*, ANDRE P. PERRUCHOUD*, MICHAEL KASHGARIAN and MICHAEL ROTH*

Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA; and
* Department of Research and Internal Medicine, University Hospital, 4031 Basel, Switzerland

1Correspondence: Yale University School of Medicine, Department of Pathology, 310 Cedar St. LB20, New Haven, CT 06520-8023, USA. E-mail: oliver.eickelberg{at}yale.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung fibrosis is a fatal condition of excess extracellular matrix (ECM) deposition associated with increased transforming growth factor ß (TGF-ß) activity. Although much is known about its pathological features, our understanding of the signal transduction pathways resulting in increased ECM and collagen deposition in response to TGF-ß is still incompletely defined. We have previously reported that a JunD homodimer of the transcription factor AP-1 is specifically activated by TGF-ß in lung fibroblasts. Here we demonstrate that JunD is also specifically required for TGF-ß-induced effects. Antisense against JunD, but not c-fos or c-jun, significantly inhibited collagen deposition in response to TGF-ß in primary human lung fibroblasts. We then investigated the ability of pharmacological agents to inhibit TGF-ß-induced signaling and collagen deposition. Cs-A and IFN-{gamma}, but not glucocorticoids, cyclophosphamide, or azathioprine, inhibited TGF-ß-induced signaling, as assessed by luciferase reporter gene assays, and collagen deposition. TGF-ß antagonism by Cs-A was associated with direct inhibition of JunD activation, as demonstrated by electrophoretic mobility shift analyses. In contrast, the effects of IFN-{gamma} required signal transducer and activator of transcription (STAT)-1. We thus identify the JunD isoform of AP-1 as an essential mediator of TGF-ß-induced effects in lung fibroblasts. TGF-ß-induced signaling and collagen deposition are efficiently antagonized by Cs-A and IFN-{gamma} treatment, both of which exhibit distinct molecular mechanisms of action. These observations therefore offer novel targets for future therapy of fibrotic lung disease.—Eickelberg, O., Pansky, A., Koehler, E., Bihl, M., Tamm, M., Hildebrand, P., Perruchoud, A. P., Kashgarian, M., Roth, M. Molecular mechanisms of TGF-ß antagonism by interferon {gamma} and cyclosporine A in lung fibroblasts.


Key Words: lung fibrosis • tumor growth factor ß • AP-1 • IFN-{gamma} • Cs-A


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LUNG FIBROSIS IS a fatal clinical entity with poor prognosis. It is presumably triggered by an initial inflammatory process that results in a fibroproliferative disorder of the interstitium (1 2 3) . Our insight into the pathogenesis of lung fibrosis is still very incomplete. However, the key feature of lung fibrosis is the accumulation of extracellular matrix (ECM) molecules, mainly collagens, in the intra-alveolar and interstitial space of the lung. This compromises tissue homeostasis and ultimately leads to the loss of lung function characteristic to lung fibrosis (4 5 6) . Current therapy of lung fibrosis includes corticosteroids with or without cytotoxic agents. However, this approach is ineffective and limited in that it lacks the support of large multicenter trials (7) . In addition, severe side effects often require cessation of therapy (1 , 7 8 9 10) .

Lung fibrogenesis is determined by a delicate balance between ECM synthesis and degradation, both of which are tightly regulated in the physiological state. During the development of lung fibrosis, this balance is shifted toward increased ECM accumulation (5 , 6) . Mechanisms that have been described to increase lung fibrogenesis in vivo include increased synthesis of collagens, decreased lung proteinase activity, as well as recruitment of profibrogenic cell populations to fibrotic sites (4 , 11 , 12) . Moreover, discrete cell-to-cell interactions between the alveolar epithelium and the mesenchyme exert antifibrotic functions that seem to be lost in lung fibrosis due to increased alveolar epithelial apoptosis (13 , 14) .

With respect to increased ECM accumulation, transforming growth factor ß (TGF-ß) has evolved as a key molecular mediator stimulating ECM accumulation (4 , 11 , 12 , 15) . In animal models of lung fibrosis, an increase in TGF-ß expression precedes collagen accumulation (6 , 16 17 18 19) . Experimental blockade of TGF-ß activity through neutralizing antibodies or receptor competition has been found to inhibit fibrosis (20 21 22) . Accordingly, antagonism of TGF-ß activity in the lung seems to be a promising approach for the treatment of lung fibrosis or other diseases associated with increased ECM accumulation. Such an approach, however, faces significant obstacles. In addition to its stimulatory effects on ECM deposition, TGF-ß is one of the major anti-inflammatory mediators (23) . It is a major modulator of the immune response in the lung, preventing persistent inflammation in this highly antigen-exposed compartment (23 , 24) . Hence, blockade of TGF-ß activity in mouse lung leads to severe perivascular inflammation and death (25) , thereby limiting the prospect of TGF-ß antagonism as a therapeutic approach in fibrosis.

The lung fibroblast is the key cell responsible for synthesizing the major components of the pulmonary ECM (5) . Cultures of primary human lung fibroblasts have provided valuable insight into the cellular events and signaling pathways that trigger ECM deposition in this tissue (26 27 28 29) . In this study, we chose primary lung fibroblasts as a model system to evaluate the contribution of specific signaling pathways activated by TGF-ß. We demonstrate that JunD is a key mediator of TGF-ß-induced effects in primary human lung fibroblasts. Cyclosporine A (Cs-A) and interferon {gamma} (IFN-{gamma}) are identified as potent antagonists of TGF-ß activity in vitro, both of which exhibit discrete molecular mechanisms of action. Thus, we identify two potent drugs that may be of promise for inhibition of TGF-ß-induced fibrogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Cultures of primary human lung fibroblasts were established from tissue obtained during lobectomy or pneumectomy from patients assigned to surgery due to lung cancer (26) . Lung tissue was taken from radiological normal lung distant from the tumor and kept overnight at 4°C in phosphate-buffered saline (PBS). These methods have been approved by the ethical committee of the Faculty of Medicine, University Basel (# M75/97). The next day, tissue was cut into small pieces (5x5 mm) and placed into cell culture flasks (Falcon, Basel, Switzerland) precoated with 10% fetal bovine serum (FBS) (Life Technologies, Inc./BRL Life Technologies, Gaithersburg, Md.). Outgrowing fibroblasts were allowed to reach confluence and passaged by trypsinization. Fibroblasts were cultured in RPMI medium 1640 supplemented with 20 mM HEPES and 10% FBS. All experiments were performed with cells between passages 2 and 5. All drugs were used at the indicated concentrations. Wild-type and STAT-1 knockout mouse fibroblasts were cultured in DMEM supplemented with 20 mM HEPES and 10% FBS. No antibiotics or antimycotics were added to culture media at any time.

Antisense oligonucleotides
Antisense oligonucleotides were synthesized as phosphorothioate-modified oligos corresponding to the 5'-ends of mRNAs for JunD, c-jun, c-fos, or STAT-1 (MWG Biotech, Munich, Germany). The oligonucleotides were JunD, 5'-TTC GCG TAG ACA GG-3'; c-jun, 5'-CGT TTC CAT CTT TGC AGT-3'; c-fos, 5'-GC GTT GAA GCC CGA GAA-3'; STAT-1, 5'-TAC CAC TGA GAC ATC CTG-3'; random control, 5'-ACC GTT CGC TGT TAT CTT-3'. For antisense experiments, cells were washed twice in PBS and then incubated with the indicated oligonucleotides for 24 h before treatment in regular growth medium. Transfection was performed with Tfx-50 (Promega Corp., Madison, Wis.) at a molar ratio of 1:3 (oligos:lipid).

Electrophoretic mobility shift assays
Nuclear extracts for gel shift analyses were prepared as described previously (30) . Cells were washed twice in ice-cold PBS and harvested in 1 ml of PBS with a rubber policeman. Samples were centrifuged for 1 min at 4000 g (4°C) and cell pellets were resuspended in 100 µl low-salt buffer [20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10% glycerol, and a set of proteinase inhibitors, CompleteTM]. After 10 min of incubation on ice, the samples were centrifuged at 10,000 g for 1 min (4°C) and the supernatants (cytosolic extracts) immediately frozen in a dry ice/ethanol bath. Pelleted nuclei were resuspended in 60 µl of high salt buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, supplemented with CompleteTM) and nuclear proteins were extracted by shaking on ice for 30 min. Samples were centrifuged at 13,000 g for 10 min (4°C) and the supernatants taken as nuclear extracts.

DNA mobility shift assays were performed using oligonucleotides comprising the consensus sequences for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') (27) and STAT-1 (5'-CAT TTC CCG TAA ATC AT-3') (31) . Oligos were endlabeled with [{gamma}-32P]-ATP using T4-polynucleotide kinase. Aliquots of nuclear extracts (2 µg) were incubated with 5 ng labeled oligonucleotides under binding conditions [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 µg/ml poly (dI-dC)] in a total volume of 10 µl. Incubations were carried out at room temperature for 30 min. Protein–DNA complexes were applied to 4% polyacrylamide gels, electrophoresed, and analyzed by autoradiography.

Determination of total ECM and collagen deposition
Total deposited ECM and collagen deposition into ECM were assessed by proline incorporation assays, as described earlier (26) . Primary human lung fibroblasts were seeded into 12-well plates (Falcon), allowed to reach 80% confluence, and placed into media containing 0.5% FBS for 24 h. Fibroblasts were treated with the indicated concentrations of TGF-ß 1 with or without drugs in the presence of 0.5 µCi/ml [3H]-proline (Amersham Life Science, Buckinghamshire, U.K.) and 10 µg/ml ascorbic acid. Determination of de novo deposition of total proteins and collagens was assayed in deposited ECM after 2 washes in PBS and cell lysis in 25 mM NH4OH for 10 min at room temperature. ECM was fixed twice with 70% ethanol and washed twice with 50 mM Tris HCI/1 mM CaCl2/1 mM proline, pH 7.5. Fixed ECM was incubated in 750 µl collagenase assay buffer [50 mM Tris-HCI (pH 7.5), 5 mM CaCI2, and 2.5 mM N-ethylmaleimide containing 120 units/ml collagenase (C0773 from Clostridium histolyticum, Sigma, St. Louis, Mo.)] for 4 h at 37°C. In parallel, an identical set of experiments was incubated in assay buffer without collagenase. After 4 h of incubations, supernatants containing collagenase digestible proteins were removed and residual ECM was solubilized by overnight incubation in 0.3 M NaOH/1% sodium dodecyl sulfate. Equal aliquots of supernatants and solubilized residual ECM were then subjected to liquid scintillation counting.

Calculations were made as follows: 1) disintegrations per minute (dpm) of solubilized ECM without collagenase digestion = total deposited proteinaceous ECM; 2) (dpm of supernatant without collagenase digestion x 100)/(dpm of supernatant without collagenase digestion + dpm of solubilized ECM without collagenase digestion) = % background; and 3) dpm of supernatant with collagenase digestion - % background = dpm deposited collagen, as published earlier (26 , 32 , 33) .

Luciferase reporter gene assays
Luciferase reporter gene assays were carried out in cells transiently transfected with p3TP-Lux, a TGF-ß-responsive reporter (34) . In brief, cells were seeded onto 48-well plates (1x104 cells/well) and serum-deprived for 24 h. Cells were subjected to transient transfection using LT-1 (Panvera, Madison, Wis.) at a DNA to reagent ratio of 1:3 (using 0.3 µg of plasmid per well). Transfections were carried out in the presence of FBS for 6 h at 37°C in humidified atmosphere. After transfection, cells were overlaid with media containing the various agents, as indicated. After 16 h, cells were washed twice with ice-cold PBS, lysed, and equal amounts of lysates were analyzed for firefly luciferase expression. In brief, 10 µl aliquots of cell lysates were mixed with 50 µl of luciferase reagent buffer and luminescence of the samples was integrated over a time period of 10 s in a luminometer. To control for unspecific effects, identical experiments were carried out in parallel using an empty reporter gene vector (27) .

Statistical analysis
Data were obtained from multiple cell lines of primary human lung fibroblasts, unless otherwise noted. Total ECM and collagen measurements were performed in triplicate using at least two independent sets of experiments per culture. Luciferase assays were performed in quadruplicate with three independent sets of experiments. For statistical analysis, Student’s t test and ANOVA analysis were performed. A P value < 0.01 was used to determine significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AP-1 is required for TGF-ß-induced fibrosis
We previously analyzed total ECM and collagen deposition by primary human lung fibroblasts (26) . In this cell type, TGF-ß potently induces both total ECM and collagens. AP-1 was found to be the major transcription factor activated in response to TGF-ß. Specifically, TGF-ß induced a JunD homodimer of the AP-1 complex, which was required for interleukin 6 induction (27) .

We investigated whether the JunD/AP-1 induction was required for TGF-ß-induced collagen deposition, as assessed by analyses of total ECM and collagens. JunD levels in human lung fibroblasts were suppressed by incubation with specific antisense oligonucleotides, which effectively and specifically inhibited TGF-ß-induced AP-1 activation. Figure 1a represents a characteristic gel shift demonstrating inhibition of TGF-ß induced AP-1 activation in the presence of JunD antisense oligonucleotides. In contrast, antisense oligonucleotides against c-jun, c-fos, or STAT-1 had no effect on AP-1 activation in response to TGF-ß (Fig. 1a ).



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  		Figure 1. JunD antisense oligonucleotides inhibit TGF-ß-induced AP-1 activation and collagen deposition. Primary human lung fibroblasts were pretreated with 5 µM antisense oligonucleotides (AS) for 24 h. Media were aspirated and fresh media containing 2 ng/ml TGF-ß1 were added. a) Cells were harvested after 2 h of TGF-ß1 treatment with or without the indicated antisense oligonucleotides. Nuclear proteins were extracted and equal aliquots (2 µg) analyzed for AP-1 activation. n/s = nonspecific. b) Cells were labeled with 0.5 µg/ml [3H]-proline and incubated for an additional 24 h in the presence of the indicated oligonucleotides. Media were aspirated; cells were lysed and deposited ECM was immediately fixed in 70% ethanol. Total ECM and collagen deposition were determined in triplicate as described. *P < 0.001 vs. control, **P < 0.001 vs. TGF-ß.

Specific suppression of JunD through this strategy led to inhibition of total ECM and collagen deposition by lung fibroblasts. As shown in Fig. 1b , TGF-ß stimulated total ECM and collagen deposition by 301 ± 10% and 376 ± 17%, respectively (P<0.001). In the presence of 10 µM JunD antisense oligonucleotides, this effect of TGF-ß was reduced to 163 ± 16% and 208 ± 12%. Thus, blockage of AP-1 activation significantly inhibited total ECM and collagen deposition by 44.5 ± 4% and 45.8 ± 4%, respectively (P<0.001). In contrast, nonspecific scrambled oligonucleotides were ineffective (Fig. 1b ).

Cyclosporine A and interferon {gamma} potently inhibit TGF-ß-induced effects
We next evaluated whether TGF-ß-induced collagen deposition in vitro could be inhibited by pharmacological agents used for therapy of lung fibrosis. We investigated the drugs hydrocortisone (HC), cyclophosphamide (CP), and azathioprine (AZT). We also analyzed the effects of the immunosuppressant cyclosporine A (Cs-A) and the cytokine IFN-{gamma}. Primary human lung fibroblasts were stimulated with TGF-ß and treated with concentrations of the drugs known to be achieved in vivo. Fig. 2a shows the results obtained with each drug over concentration ranges of 3 logs. Neither AZT, CP, nor HC affected ECM or collagen deposition in response to TGF-ß. In addition to HC, we analyzed several modified glucocorticoids (cortisone, dexamethasone, or fluticasone), none of which was able to inhibit ECM or collagen deposition in response to TGF-ß (data not shown).



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  		Figure 2. Cs-A and IFN-{gamma} antagonize TGF-ß activity in human lung fibroblasts. a) Confluent primary human lung fibroblasts were placed in 0.5% FBS for 24 h and then stimulated with TGF-ß1 (2 ng/ml). After 2 h, cells were labeled with 0.5 µg/ml [3H]-proline and incubated for an additional 36 h with or without the listed drugs at the indicated concentrations. Total ECM and collagen deposition were determined as described. *P < 0.001 vs. control, **P < 0.001 vs. TGF-ß. b) antifibrotic efficacy of all five drugs was calculated as % inhibition of TGF-ß-induced ECM and collagen deposition. Data represent the mean ± SD of triplicate measurements in cultures of primary human lung fibroblasts obtained from 10 different individuals. *P < 0.001. c) Primary human lung fibroblasts were seeded into 48-well plates and transfected with the TGF-ß-responsive reporter gene p3TP-Lux (300 ng/well). Cells were then treated with TGF-ß (2 ng/ml) ± the indicated concentrations of IFN-{gamma} or Cs-A. Cell extracts were harvested after 24 h and luciferase activities were counted in a standard Luminometer. Identical sets of cells were transfected in parallel with an empty luciferase vector to control for unspecific effects of the drugs. Normalized luciferase activities were expressed as fold induction compared to control. *P < 0.001 vs. control, **P < 0.001 vs. TGF-ß.

In contrast, the profibrogenic effect of TGF-ß was potently inhibited by Cs-A and IFN-{gamma}. Cs-A was significantly effective at a concentration of 2000 ng/ml, and still showed minor inhibition at 200 ng/ml (Fig. 2a ). At 2000 ng/ml, Cs-A inhibited TGF-ß-induced ECM and collagen deposition by 25 ± 4% and 62 ± 5%, respectively (P<0.001) (Fig. 2b ). By comparison, IFN-{gamma} was an even more potent antifibrotic substance in this model. Its effect was nearly complete at 20 ng/ml. At this concentration, TGF-ß-induced total ECM deposition was inhibited by 66 ± 7% and collagen deposition by 92 ± 3% (P<0.001) (Fig. 2b ). Significant inhibition still occurred with as little as 200 pg/ml of IFN-{gamma}. Notably, all of these observations were repeated in multiple cultures of primary human lung fibroblasts, obtained from at least 10 individuals.

We then sought to determine whether these effects were restricted to ECM deposition, or whether they would also be evident in signal transduction pathways directly activated by TGF-ß. We analyzed activation of the TGF-ß-responsive reporter gene p3TP-Lux (containing three AP-1-responsive elements cloned in front of a portion of the PAI-1 promoter) (34) in response to TGF-ß, as well as its modulation by Cs-A and IFN-{gamma}. As demonstrated in Fig. 2c , p3TP-Lux is significantly activated by TGF-ß (1 ng/ml) in human lung fibroblasts (3.75 ± 0.23-fold induction). IFN-{gamma} almost completely inhibited this induction, both at 20 and 2 ng/ml (Fig. 2c ), similar to its effect on TGF-ß-induced collagen deposition. Cs-A, although clearly less potent than IFN-{gamma}, also significantly inhibited TGF-ß-induced p3TP-Lux activation, both at concentrations of 2000 and 200 ng/ml. The inhibition of TGF-ß-induced effects by IFN-{gamma} and Cs-A therefore occurs in the AP-1 signaling pathway, which is activated by TGF-ß in human lung fibroblasts.

Cs-A directly inhibits AP-1 activation by TGF-ß
We then sought to closer define the molecular mechanisms by which Cs-A and IFN-{gamma} produced their antifibrotic effects. As described above, AP-1 was required for TGF-ß-induced collagen deposition. Typically, AP-1 activation was rapid and pronounced within 4 h of TGF-ß treatment (Fig. 3a ). We investigated whether Cs-A or IFN-{gamma} could directly affect TGF-ß-induced AP-1 activation and DNA binding by gel shift analyses. We observed no effect of IFN-{gamma} (20 ng/ml to 200 pg/ml) on TGF-ß-induced AP-1 activation throughout 16 h of exposure (data not shown). In contrast, Cs-A potently suppressed TGF-ß-induced AP-1 activation over 8 h of cotreatment. As shown in Fig. 3b , Cs-A treatment alone had limited effects on AP-1 binding to DNA, by comparison to TGF-ß. However, TGF-ß-induced AP-1 activation was significantly down-regulated when fibroblasts were cotreated with TGF-ß and Cs-A (compare lanes T+C to lanes T at each time point).



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  		Figure 3. Cyclosporine A inhibits TGF-ß-induced AP-1 activation. a) Confluent human lung fibroblasts were placed in 0.5% FBS for 24 h. Cells were stimulated with TGF-ß1 (2 ng/ml) and nuclear proteins were extracted at the indicated time points. Equal protein aliquots (2 µg) were incubated with [{gamma}-32P]-labeled oligonucleotides containing the consensus sequence for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3'). AP-1 DNA binding activity was analyzed on a 4% nondenaturing polyacrylamide gel. Data is representative for identical experiments performed with cultures of lung fibroblasts from ten different individuals. b) Samples were processed as described for panel a, with the exception that fibroblasts were treated with TGF-ß1 alone (2 ng/ml) (T), cyclosporine A alone; 2000 ng/ml) (C), or cotreated with TGF-ß 1 and cyclosporine A (T+C) for the indicated time points. AP-1 = AP-1 DNA binding activity, n/s = nonspecific binding activity.

IFN-{gamma} exerts antifibrotic activity via STAT-1
Earlier studies showed that IFN-{gamma} activates signal transducer and activator of transcription (STAT)-1, which integrates with signals generated by TGF-ß (35) . We therefore investigated whether STAT-1 accounted for differences in ECM deposition in TGF-ß-, IFN-{gamma}-, or Cs-A-treated human lung fibroblasts. IFN-{gamma} rapidly increased STAT-1 activation and binding to DNA. This was observed within as little as 60 min after IFN-{gamma} exposure, and lasted up to 6 h (Fig. 4a ). As shown, neither TGF-ß nor Cs-A could activate STAT-1 binding by itself. However, when cells were cotreated with IFN-{gamma} and TGF-ß, STAT-1 activation was significantly enhanced as compared to IFN-{gamma} treatment alone (Fig. 4b ). These results suggested that changes in STAT-1 activity could contribute to the antifibrotic effect of IFN-{gamma}.



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  		Figure 4. STAT-1 is rapidly activated by interferon {gamma} (IFN-{gamma}), but not TGF-ß 1 or Cs-A in human lung fibroblasts. a) Confluent human lung fibroblasts were placed in 0.5% FBS for 24 h and stimulated with TGF-ß 1 (2 ng/ml), IFN-{gamma} (20 ng/ml), or Cs-A (2000 ng/ml) for the indicated times. Nuclear proteins were extracted and equal protein aliquots (2 µg) were processed for gel shift analysis with [{gamma}-32P]-labeled oligonucleotides containing the consensus sequence for STAT-1 (5'-CAT TTC CCG TAA ATC AT-3'). Data is representative for experiments performed with cultures of lung fibroblasts from five different individuals. b) Cells were prepared and stimulated as described above with the exception that cells were costimulated with IFN-{gamma} (20 ng/ml) and the indicated concentrations of TGF-ß 1. Nuclear extracts were prepared at the indicated time points and analyzed for STAT-1 activation. STAT-1 = STAT-1 DNA binding activity, n/s = nonspecific binding activity.

Pretreatment of lung fibroblasts with antisense oligonucleotides against STAT-1 prevents STAT-1 activation by IFN-{gamma}, as well as its ability to suppress TGF-ß-induced ECM deposition (data not shown). To confirm STAT-1 involvement, we therefore analyzed TGF-ß-induced signaling and ECM deposition in fibroblasts generated from wild-type and STAT-1 knockout mice. Figure 5 demonstrates induction of collagen deposition in response to TGF-ß in wild-type (150±4%) and STAT-1 knockout cells (126±4%). In wild-type fibroblasts, this was effectively inhibited by IFN-{gamma} and Cs-A at concentrations analogous to those effective in human lung fibroblasts (Fig. 5) . However, in STAT-1 knockout cells the inhibitory effect of IFN-{gamma} on collagen deposition was absent, whereas the inhibitory activity of Cs-A was retained (Fig. 5) .



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  		Figure 5. IFN-{gamma} requires STAT-1 activity for inhibition of TGF-ß-induced collagen deposition. Confluent wild-type and STAT-1 knockout fibroblasts were placed in 0.5% FBS for 24 h and then stimulated with TGF-ß1 (2 ng/ml). After 2 h, cells were labeled with 0.5 µg/ml [3H]-proline and incubated for an additional 24 h in the presence of IFN-{gamma} or Cs-A at the indicated concentrations. Media were aspirated, cells were lysed, and deposited ECM was immediately fixed in 70% ethanol. Total ECM and collagen deposition were determined in triplicate as described. Data is representative for two independent sets of experiments. *P < 0.01 vs. control, **P < 0.005 vs. TGF-ß.

Similar effects were seen in TGF-ß signaling. In both wild-type and STAT-1 knock-out fibroblasts, TGF-ß potently induced p3TP-Lux expression (10.6±3.9 and 9.5±2-fold inductions, respectively, P<0.005) (Fig. 6 ).This induction was almost completely blocked by IFN-{gamma} in wild-type fibroblasts over a concentration range from 2–20 ng/ml. However, in cells with a deletion of STAT-1, IFN-{gamma} was unable to inhibit TGF-ß signaling, indicating that STAT-1 was absolutely required for inhibition of TGF-ß activity in response to IFN-{gamma}. In contrast, Cs-A was able to inhibit TGF-ß signaling in both wild-type and STAT-1 knockout fibroblasts irrespective of the presence of STAT-1 (Fig. 6) .



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  		Figure 6. IFN-{gamma} requires STAT-1 activity for inhibition of TGF-ß-induced signaling. Fibroblasts from wild-type and STAT-1 knockout fibroblasts were seeded into 48-well plates and transfected with the TGF-ß-responsive reporter gene p3TP-Lux at 300 ng/well. Cells were then treated with TGF-ß (2 ng/ml) ± the indicated concentrations of IFN-{gamma} or Cs-A. Cell extracts were harvested after 24 h and luciferase activities were counted in a standard Luminometer. Identical sets of cells were transfected in parallel with an empty luciferase vector to control for unspecific effects of the drugs. Normalized luciferase activities were expressed as fold induction compared to control. *P<0.001 vs. control, **P<0.001 vs. TGF-ß.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lung fibrosis in the form of idiopathic pulmonary fibrosis represents a life-threatening syndrome with a median survival of 3–5 years after diagnosis (1 , 7 , 9) . Current therapeutic options are ineffective and associated with severe morbidity and poor outcome (7 , 8) . The hallmark feature of lung fibrosis is the transformation of the lung interstitium by a destructive fibroproliferative response, characterized by accumulation of ECM in the lung interstitium and disintegration of the physiological mesenchymal/epithelial interaction (3 , 11 , 12 , 36) . The initial trigger of disease onset is controversial, but it is suggested to be an inflammatory stimulus. As the disease progresses, increased fibrogenesis becomes a major pathophysiologic determinant. In this respect, recent research has focused on the role of TGF-ß as a key mediator of ECM and collagen accumulation in the lung. An animal model of transient TGF-ß overexpression in the lung closely resembles the human disease and shows distinct features of accumulated ECM (18) . We therefore chose TGF-ß as a fibrogenic stimulus in our model of primary human lung fibroblasts to assess particular contributions of distinct TGF-ß signaling pathways on ECM accumulation in vitro.

We found that AP-1/JunD plays a central role in TGF-ß-induced signaling and ECM accumulation in lung fibroblasts. JunD is a distinct isoform of the AP-1 transcription factor that can form homodimers or heterodimers with other jun- or fos-isoforms (37) . AP-1 complexes are described to be central to TGF-ß signal transduction in many cell culture models, including human lung fibroblasts (27 , 38 39 40) . The biological effects mediated by AP-1 depend on the isoform specific composition of the AP-1 dimer. We have previously published that in the identical model, TGF-ß-induced AP-1 complexes consist of JunD homodimers. Similar observations have recently been made in untransformed lung and intestinal epithelial cells (41) , human KMST fibroblasts (42) , and mouse keratinocytes (43) , indicating that the JunD isoform is a major contributor to TGF-ß signaling in many cell types. In this study, we have extended these observations in that we show that AP-1/JunD is not only activated, but also required for TGF-ß-induced ECM accumulation in lung fibroblasts.

Transcription factors belonging to the SMAD or Sp1 family have also recently been described to be activated by TGF-ß (45) . It is therefore possible that these factors could also contribute to ECM deposition and be considered possible targets for antifibrotic drugs (44 , 45) . Indeed, SMADs have been found to interact with and potentiate AP-1-dependent gene expression, but the precise mechanism of synergism between SMADs and AP-1 is unknown (40 , 46 , 47) . As such, the prospect of inhibiting distinct pathological effects of TGF-ß (such as ECM accumulation) through selective down-regulation of signaling components while leaving other effects (such as cell cycle regulation) untouched presents an intriguing idea of antagonizing growth factor activity.

The antifibrotic efficiency of Cs-A fit with its ability to block TGF-ß-induced AP-1 activation. The molecular target of Cs-A is the Ca2+- and calmodulin-dependent protein phosphatase calcineurin (48) . Inhibition of calcineurin activity by Cs-A thereby accounts for the drug’s biological effects. Calcineurin has been shown to be involved in AP-1 activation in immune cells (49 , 50) . Furthermore, the immunophilin FKBP12, which mediates the effects of the immunosuppressants FK506 and rapamycin, has been shown to directly interfere with TGF-ß signal transduction (51) , indicating that more immunosuppressive drugs of this class may be valuable for antagonizing TGF-ß activity.

IFN-{gamma}, in contrast, did not directly affect DNA binding activity of AP-1. Moreover, we demonstrated that it required STAT-1 for its antifibrotic effects. STAT-1 is rapidly activated on IFN-{gamma} treatment and immediately translocates to the nucleus (52) . STAT-1-dependent gene expression requires the ubiquitous coactivators CBP/p300, which bind to STATs with high affinity, thereby initiating and potentiating IFN-{gamma}-induced gene expression (53 , 54) . In most cell systems, the availability of CBP/p300 is the rate-limiting step for transcription factor mediated gene expression, including STAT-1 and AP-1 (55 56 57) . In human lung fibroblasts, p300 is expressed at low levels (O. Eickelberg, unpublished observations). Under IFN-{gamma} and TGF-ß cotreatment, STAT-1 and AP-1 thus compete for limiting amounts of p300 in order to be transcriptionally active (Fig. 7) . When IFN-{gamma} treatment increases STAT-1 affinity and binding to p300, AP-1 still demonstrates DNA binding activity but lacks transcriptional activation, as recently shown (56 , 57) . This mechanism therefore explains the higher antifibrotic potency of IFN-{gamma} compared to Cs-A.



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  		Figure 7. A model for the antagonistic effects of Cs-A and IFN-{gamma} on TGF-ß-induced effects. TGF-ß activity is mediated via JunD/AP-1 activation resulting in increased collagen deposition in lung fibroblasts. Inhibition of these effects by Cs-A is achieved via direct inhibition of JunD/AP-1 activation (left axis). In contrast, effects of IFN-{gamma} require STAT-1, which inhibits AP-1 transcriptional activity via competition for CBP/p300 (right axis).

Do these investigations have any significance or clinical benefit for future treatment of lung fibrosis? The antifibrotic effect of IFN-{gamma} has long been suspected from animal models of bleomycin-induced lung fibrosis (58 59 60) , but only recently a pilot study by Ziesche et al. revealed its potency for the treatment of lung fibrosis in humans (61) . This study found that lung function of patients with lung fibrosis was significantly improved under IFN-{gamma} and glucocorticoid cotreatment compared to glucocorticoid treatment alone. In this respect, our study identifies STAT-1 as the immediate effector of IFN-{gamma} required for its inhibitory effect. Compounds that specifically activate or mimic STAT-1 in lung fibroblasts might therefore avoid side effects observed with systemic IFN-{gamma} treatment, and thus increase the therapeutic potential of IFN-{gamma}.

In contrast, the role of Cs-A as an antifibrotic drug in vivo is controversial. Cs-A is primarily used as an immunosuppressant for transplant tolerance. However, earlier observations described its potential for the treatment of interstitial lung diseases presenting with lung fibrosis (62 63 64 65 66) . In these reports, most patients showed a favorable response to Cs-A in terms of lung function. Concerning its narrow therapeutic window with systemic uptake, aerosolized Cs-A may prove to be valuable in future controlled trials of IPF. It would therefore be desirable to investigate the efficacy of Cs-A in animal models of lung fibrosis.

Initially we were surprised by the clear inefficacy of glucocorticoids, azathioprine, or cyclophosphamide to antagonize TGF-ß-induced ECM accumulation in vitro, since these drugs are used as primary therapy in patients with lung fibrosis (7 , 67) . However, response rates to these compounds are known to be poor (10–30%) and their therapeutic efficacy is questioned. On a molecular biological level, it is also unclear as to whether cotreatment of cells with TGF-ß and glucocorticoids has synergistic or antagonistic effects. Depending on the cell type studied, glucocorticoid treatment can either enhance (68) or repress (69) TGF-ß-induced effects.

In summary, we have identified distinct molecular mechanisms by which Cs-A and IFN-{gamma} antagonize TGF-ß effects. The response to injury with fibrosis is commonly found in many species and tissues and is to a great extent associated with increased activity of TGF-ß. Basic pathophysiologic mechanisms of fibrosis demonstrate high degrees of similarity between organ systems. As such, the presented results provide novel ideas for future treatment of fibrosis in diverse organ systems such as kidney, liver, or lung.


   ACKNOWLEDGMENTS
 
O.E. is a recipient of a Feodor-Lynen Fellowship from the Alexander von Humboldt Foundation, and is currently supported by the Juvenile Diabetes Foundation International. We are indebted to Drs. R. Wells, D. Vicencio, B. Robibaro, C. Eickelberg, and T. McCarthy for generous help and invaluable discussion. We thank S. Bertschin and T. Woodtli for their technical expertise, D. Levy for providing wild-type and STAT-1 knockout fibroblasts, J. Massague for 3TP-Lux, and Dr. J. Pohl (ASTA Medica, Frankfurt/Main, Germany) for providing mafosfamide, the active compound of cyclophosphamide. O.E. is grateful to G. Giebisch and M. Centrella for superior guidance and advice.

Received for publication May 4, 2000. Revision received August 21, 2000.
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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