MCB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shi-Wen, X.
Right arrow Articles by Leask, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shi-Wen, X.
Right arrow Articles by Leask, A.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, July 2006, p. 5518-5527, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.00625-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Constitutive ALK5-Independent c-Jun N-Terminal Kinase Activation Contributes to Endothelin-1 Overexpression in Pulmonary Fibrosis: Evidence of an Autocrine Endothelin Loop Operating through the Endothelin A and B Receptors

Xu Shi-Wen,1 Fernando Rodríguez-Pascual,2 Santiago Lamas,2 Alan Holmes,1 Sarah Howat,3 Jeremy D. Pearson,3 Michael R. Dashwood,4 Roland M. du Bois,5 Christopher P. Denton,1 Carol M. Black,1 David J. Abraham,1* and Andrew Leask6*

Centre for Rheumatology, Department of Medicine, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom,1 Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, C.S.I.C., Ramiro de Maeztu 9, E-28040 Madrid, Spain,2 Centre for Cardiovascular Biology & Medicine, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL, United Kingdom,3 Department of Molecular Pathology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom,4 Interstitial Lung Disease Unit, Royal Brompton Hospital, Imperial College of Science, Technology and Medicine, Emmanuel Kaye Building, 1B Manresa Road, London SW3 6LR, United Kingdom,5 CIHR Group in Skeletal Development and Remodeling, Division of Oral Biology, Schulich School of Medicine and Dentistry, Dental Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada6

Received 11 April 2006/ Returned for modification 27 April 2006/ Accepted 8 May 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor ß (TGF-ß) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-ß induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-ß are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proper lung function requires efficient gas exchange through specialized structures, termed alveoli, whose structure ultimately depends on the underlying connective tissue consisting of fibroblasts and extracellular matrix (ECM) (for a review, see reference 19). Damage to the lung can occur in response to environmental insults or inflammation, resulting in the induction of a tissue repair program involving the coordinated de novo synthesis of epithelia, blood vessels, and connective tissue (42). The proper repair of connective tissue requires that fibroblasts synthesize and contract new ECM components, including collagen and fibronectin (5). These activities are conducted by a specialized type of fibroblast termed the myofibroblast, which expresses {alpha}-smooth muscle actin ({alpha}-SMA), a protein which promotes ECM contraction and remodeling (6, 20, 55). In normal wound repair, myofibroblasts disappear and organ function is restored; however, should the tissue repair program not appropriately terminate, myofibroblasts persist in the lesion, resulting in the extensive, exaggerated amount of excessively contracted ECM characteristic of scar tissue (17). Indeed, a characteristic of scar tissue is the presence of {alpha}-SMA-enhanced contraction of the extracellular matrix by lesional fibroblasts (20, 55). In addition, in vitro and in vivo studies have consistently shown that fibroblasts isolated from patients with systemic sclerosis (SSc) directly contribute to the excessive scarring observed in fibrosis by enhancing production of ECM components (22, 27, 28, 49). Excessive scarring can result in organ failure and death (40).

A growing body of evidence implicates the vasoconstrictive peptide endothelin-1 (ET-1) as a mediator of organ-based fibrosis (1, 50-52, 54). There are three known endothelin isoforms (–1, –2, and –3), which arise by proteolytic processing of large precursors (~200 amino acid residues). Intermediates, termed big endothelins, are excised from prepropeptides at sites containing paired basic amino acids and are subsequently cleaved at Trp-21-Val/Ile-22 to produce mature 21-residue, biologically active peptides (3, 44). The enzyme responsible for the specific cleavage at Trp-21 has been termed endothelin-converting enzyme (39, 56); its mRNA is stabilized in response to injury, resulting in the generation of bioactive endothelin (45).

Elevated levels of ET-1 are observed in patients with persistent, chronic fibrosis, suggesting that ET-1 may contribute not only to normal tissue repair but also to the pathogenesis of fibrosis (1, 29, 34). Elevated levels of circulating ET-1 in patients with skin and lung fibrosis correlate with the severity of the fibrotic phenotype (1, 29, 34). This increase in circulating ET-1 is paralleled by an increase in ET-1 synthesis in vivo (1, 29, 34). When added to fibroblasts, ET-1 promotes ECM production and contraction (50-52). ET-1 is overproduced by fibroblasts isolated from patients with fibrotic lung of scleroderma and contributes to the persistent myofibroblast phenotype of these cells (51). Lung fibroblasts from patients with pulmonary fibrosis associated with SSc (fibrosing alveolitis associated with systemic sclerosis [FASSc]) produce elevated levels of ET-1 (51). Endogenous ET-1 activity in lung FASSc fibroblasts directly contributed to the contractile phenotype of the FASSc fibroblasts, as blocking ET-1 signaling by the specific ET-1 dual receptor antagonist bosentan and the ETA antagonist PD156707 greatly reduced the ability of FASSc fibroblasts to contract a collagen gel matrix (51). In addition, FASSc fibroblasts produced elevated levels of {alpha}-SMA, ezrin, moesin, and paxillin, which depended on endogenous ET-1 signaling and phosphatidylinositol 3-kinase (51). Therefore, the enhanced contractile ability of the FASSc fibroblast depends on the elevated levels of endogenous ET-1 expression demonstrated by lung FASSc fibroblasts. Consequently, elucidating the molecular basis for the overproduction of ET-1 in fibroblasts isolated from patients with pulmonary fibrosis is necessary not only to understand the mechanism behind the persistence of the myofibroblast in this disorder but also to develop novel antifibrotic therapies.

In addition to ET-1, other secreted proteins have been demonstrated to play key roles in fibrogenesis. For example, the potent profibrotic transforming growth factor ß (TGF-ß) has long been known to induce fibroblasts to produce and contract ECM in vitro and in vivo, and anti-TGF-ß strategies are effective at alleviating fibrosis in animal models (31). Subcutaneous injection of TGF-ß alone results in transient fibrosis that depends on the continuous application of ligand, whereas sustained fibrotic response to TGF-ß requires an additional stimulus (35, 48). Consistent with this notion, it was recently demonstrated that TGF-ß and ET-1 cooperate to induce myofibroblast formation (46). Recently, it was shown that TGF-ß induces ET-1 production in vascular endothelial cells via Smads and Ap-1 (43). However, whether ET-1 is induced by TGF-ß in lung fibroblasts is unknown. Furthermore, to what extent TGF-ß contributes to the overexpression of ET-1 in pulmonary fibrosis is unclear. There is no therapy for fibrotic disease; consequently, an appreciation of the molecular basis of the origin and persistence of the myofibroblast phenotype will not only influence tissue engineering and regenerative medicine but also have a significant impact on the treatment of pathological fibrosis. Indeed, the signal transduction cascades involved with promoting the expression of profibrogenic proteins in fibrosis, including pulmonary fibrosis, is almost wholly unknown. Such knowledge is necessary for developing an understanding of the origin of fibrosis and in the design of antifibrotic therapies.

In this report we investigate the regulation of ET-1 expression, in the presence or absence of added TGF-ß, in normal and fibrotic pulmonary fibroblasts. Our studies provide new insights into the mechanism underlying ET-1 overproduction in pulmonary fibrosis and hence into the molecular basis for chronic fibrotic disease.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and cell culture. Fibroblasts were grown by explant culture from open lung biopsy specimens from SSc patients taken for histological staging of lung fibrosis, and control samples were taken from normal lungs not used for transplant. The group of eight SSc patients fulfilled the criteria of the American College of Rheumatology for the diagnosis of SSc with lung involvement. The sex ratio was six females to two males. Fibroblasts were used between passages 2 and 5 (49).

Smad knockout and wild-type fibroblasts (a generous gift from Anita B. Roberts, NIH, Bethesda, MD) were isolated from PCR-genotyped Smad3 wild-type and knockout newborn mice by standard methods and were cultured in Dulbecco's modified Eagle's medium (DMEM)-10% fetal bovine serum-1% Pen-Strep. TAK1 wild-type and knockout fibroblasts (a generous gift from Sankar Ghosh, Yale University) (47) were similarly cultured.

Measurement of ET-1 in control and SSc lung fibroblast culture supernatants. Endothelin-1 secretion was measured in supernatants collected from confluent monolayer cultures of normal and SSc lung fibroblasts in serum-free medium by using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's (Biomedica, Vienna, Austria) instructions. This assay uses two antibodies directed against different epitopes of ET-1 and has a sensitivity of <1.0 pg/ml. These data were adjusted in accordance with cell counts at the time of sampling, and values are given as amounts of ET-1 per milliliter per 106 cells.

Western blot analysis. Normal and SSc lung fibroblasts were grown to confluence in DMEM with 10% fetal calf serum and then serum starved in DMEM with 0.5% bovine serum albumin for 24 h. After serum starvation, cells were stimulated with 4 ng TGF-ß1 from 0 to 180 min with 0.5% bovine serum albumin. For blocking experiments, cells were incubated for 24 h in the presence or absence of the JNK inhibitor SP600125 (10 µM; Calbiochem), the ALK5 inhibitor SB 431542 (10 µM; Tocris), the JNK inhibitor SP600125 (10 µM; Calbiochem), the platelet-derived growth factor (PDGF) receptor inhibitor Gleevec (imatinib mesylate, 2 mM; Novartis, Basel, Switzerland), the angiotensin II inhibitor Losartan (100 nM; Merck, Whitehouse Station, NJ), or recombinant human interleukin 1 (IL-1) receptor antagonist (50 ng/ml; R&D Systems, Minneapolis, MN). In addition, the ETA receptor antagonist PD156707, sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimethoxy-benzyl)-but-2-enoate (10 µM); the ETB receptor antagonist BQ788, N-cis-2,6-dimethyl-piperidimocarbonyl-L-gMeLeuD-Nle-ONa (10 µM); and the mixed ETA/B receptor antagonist bosentan (10 µM) (all from M. Clozel, Actelion Pharmaceuticals, Allschwil, Switzerland) were used. TAK1 cells were similarly cultured and treated for 30 min with ET-1 (100 nM; R and D Systems) for phospho-JNK blots and for 24 h for {alpha}-SMA blots. For the experiment in which the ability of bosentan to reduce the TGF-ß induction of {alpha}-SMA was tested, cells isolated from three normal individuals were preincubated for 1 h prior to the incubation with TGF-ß1 for 24 h.

Cell layer lysates were examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 12% polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes at 30 V for 90 min. Membranes were blocked by incubation for 1 h with 5% nonfat milk in phosphate-buffered saline containing 0.2% Tween 20, and antigens were detected using specific antibodies. Cell layer lysates (10 µg/sample) were probed using antibodies directed against JNK1, c-jun, and c-fos (all from Santa Cruz, CA) or antibody against {alpha}-SMA (Sigma, St Louis, MO) followed by incubation with appropriate horseradish peroxidase-conjugated bound secondary antibody (Jackson ImmunoResearch Laboratories, Hornby, Canada). Signal was detected using an enhanced chemiluminescence protocol (Amersham Biosciences, Piscataway, NJ) as described by the manufacturer.

Reverse transcription-PCR. Lung fibroblasts were serum starved for 18 h and treated with 4 ng TGF-ß for 4 h. Total RNA was isolated using Trizol (Invitrogen), and the integrity of the RNA was verified by gel electrophoresis. Total RNA (10 µg) was reverse transcribed in a 20-µl reaction volume containing an oligonucleotide (dT18) and random decamers (dN10) using Moloney murine leukemia virus reverse transcriptase (Promega) for 1 h at 37°C. The cDNA was diluted to 100 µl with diethylpyrocarbonate-treated water, and the target was measured by real-time PCR using FastStart DNA Master SYBR green (Roche Applied Science) according to the manufacturer's instructions. Triplicate samples were run, transcripts were measured in picograms, and expression values were standardized to values obtained with control 28S RNA primers. Primers (Sigma Genosys) used to amplify ET-1 were as follows: 5'-TTC TCT CTG CTG TTT GTG GC3-' (forward) and 5'-CCA AGT CCA TAC GGA ACA AC-3' (reverse).

Construction of reporter plasmids and cell transfection. Luciferase reporter constructs, a 650-bp fragment of the human ET-1 promoter (650-bp ppET-1-prom-luc), and constructs with specific mutations in the Smad or AP-1 binding sites were generated by PCR and cloned into pGL3-basic (43). A luciferase reporter gene driven by multiple copies of a Smad response element (courtesy of Peter ten Dijke) was also used. Transient transfection experiments were performed as described previously (43, 52). Promoter/reporter constructs were transfected into lung fibroblasts using FuGENE6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Promoter/reporter plasmids were cotransfected with pCMV-ßGal (Clontech), which was used to adjust for differences in transfection efficiencies between samples. Following transfection, cells were incubated in DMEM with 0.5% fetal bovine serum for 18 h. Media were changed, and cells were incubated in the presence or absence of inhibitors for 45 min and cultured for an additional 24 h in the presence or absence of TGF-ß1 (4 ng). Fibroblasts were rinsed once with phosphate-buffered saline, and cellular protein was extracted using 200 µl of reporter lysis buffer (Promega Corp, Madison, WI). Reporter gene activity was measured by luminometry (Turner Designs, Sunnyvale, CA) using luciferase and ß-galactosidase assays (Tropix Inc. Bedford, MA) according to the manufacturers' instructions. Values given are means ± standard errors of triplicate assays from three individual experiments.

Fibroblasts were also cotransfected with empty expression vector; expression vectors encoding Smad 7 (P. ten Dijke, Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden) (38); or TAM-67 (7) (M. J. Birrer, National Cancer Institute, Bethesda, Md.), a dominant negative c-jun expression vector which blocks the activity of all endogenous Jun and Fos proteins by forming nonfunctional heterodimers. Alternatively, reporter constructs were transfected with expression vector encoding JNK interacting protein 1 (jip-1), a protein inhibiting JNK/c-jun (A. J. Whitmarsh, University College, London, United Kingdom) (59). For blocking experiments, cells were treated overnight in the presence or absence of the ALK5 inhibitor SB 431542 (10 µM; Tocris).

Nuclear extract preparation and electrophoretic mobility shift assays. Electrophoretic mobility shift assays were performed with nuclear extracts or purified c-Jun and radiolabeled double-stranded oligonucleotides, and anti-c-jun or anti-HuR antibody (Santa Cruz), using standard procedures (43).

Statistical analysis. Data were analyzed by using the unpaired Student t test or nonparametric tests as appropriate. The probability values obtained are indicated in the text and/or in the figure legends when statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-ß induces ET-1 in normal lung fibroblasts through a Smad-independent, Ap-1-dependent pathway. TGF-ß induces ET-1 expression in endothelial cells (43). To determine whether TGF-ß induces ET-1 production in normal human lung fibroblasts, we cultured normal lung fibroblasts to 80% confluence. We then cultured cells in the absence of serum for 18 h and then in the presence or absence of TGF-ß1 (4 ng/ml) for an additional 24 h. ET-1 production was then assessed using a specific ELISA recognizing ET-1 protein. We found that TGF-ß significantly (P < 0.05) increased ET-1 protein production in normal lung fibroblasts (Fig. 1A).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. TGF-ß induces ET-1 production in primary human normal lung fibroblasts. (A) TGF-ß induces ET-1 protein. Normal lung fibroblasts were cultured and treated with or without TGF-ß1 (4 ng/ml) as described in Materials and Methods. Conditioned media were analyzed for ET-1 production using a specific ET-1 ELISA. TGF-ß1 significantly (P < 0.05) induced ET-1 protein. Data for an average of six wells ± standard deviation are shown. (B) TGF-ß induces the ET-1 promoter. Normal lung fibroblasts were transfected with an ET-1 promoter/luciferase reporter construct and treated with or without TGF-ß1 (4 ng/ml) as described in Materials and Methods. Cell layers were analyzed for luciferase expression and adjusted for differences in transfection efficiency between samples revealed by ß-galactosidase expression produced by a cotransfected construct containing a cytomegalovirus promoter-driven ß-galactosidase gene. TGF-ß1 significantly (P < 0.05) induced ET-1 promoter activity. Data for an average of six wells ± standard deviation in three independent experiments are shown.

 
To extend these data, we determined whether TGF-ß increased ET-1 production at least in part through elements in the ET-1 promoter. To perform this analysis, we transfected into normal human lung fibroblasts a construct possessing 805 nucleotides upstream of the transcription initiation start site of the human ET-1 promoter subcloned 5' upstream of the luciferase reporter gene. Following transfection, cells were serum starved and exposed in the presence or absence of TGF-ß1 for 24 h. We found that TGF-ß induced the ET-1 promoter, which mirrored our results examining ET-1 protein (Fig. 1B). Previously, it was shown that TGF-ß induced ET-1 promoter activity in endothelial cells through Smads and Ap-1 (43). To evaluate whether such a mechanism might be operative in lung fibroblasts, we evaluated whether ET-1 promoter/luciferase reporter constructs containing mutations in the Smad or Ap-1 elements were capable of responding to TGF-ß. We found that whereas mutation of the Smad element of the ET-1 promoter did not affect the ability of the ET-1 promoter to respond to TGF-ß in lung fibroblasts, removal of the Ap-1 element significantly attenuated the response of the ET-1 promoter to TGF-ß (Fig. 2A). Extending these results, overexpression of the inhibitory Smad7, achieved by a transfecting a Smad7-encoding expression vector previously shown to reduce induction of Smad-dependent signaling (8, 25, 38), did not affect the TGF-ß induction of the ET-1 promoter but was able to reduce induction of a generic Smad-responsive promoter (Fig. 2B). Similarly, TGF-ß was able to induce the ET-1 promoter even in Smad3–/– fibroblasts (Fig. 2C). These results are in contrast to previous data, showing that Smad3-responsive promoters and genes are not induced by TGF-ß in Smad3–/– fibroblasts (25, 41). Conversely, inhibition of JNK with SP600125 and overexpression of dominant negative c-jun (TAM-67) (18) blocked the TGF-ß induction of the ET-1 promoter (Fig. 2B). Similarly, overexpression of JIP-1, which selectively deactivates the JNK pathway by cytoplasmic retention of JNK (24), inhibited the TGF-ß induction of ET-1 in normal fibroblasts of gene expression mediated by JNK, which occurs in the nucleus. Collectively, these results suggest, in contrast to the situation in endothelial cells, that TGF-ß induces ET-1 production in pulmonary fibroblasts in a Smad-independent Ap-1-dependent fashion.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. TGF-ß induces the ET-1 promoter in primary human normal lung fibroblasts in a Smad-independent JNK/Ap-1-dependent fashion. (A) Effect of mutating the Ap-1 or the Smad element of the ET-1 promoter on TGF-ß induction of ET-1. Normal lung fibroblasts were transfected with a wild-type ET-1 promoter/luciferase reporter construct (WT) or an otherwise identical construct containing mutated Smad (Smad mut) or Ap-1 (Ap-1 mut) elements. These elements were previously shown to be functional in endothelial cells (43). Mutation of the Ap-1 element, but not the Smad element, significantly (P < 0.05) reduces the TGF-ß1 induction of the ET-1 promoter. Data for an average of six wells ± standard deviation in three independent experiments are shown. (B) Effect of inhibition of Smad or c-jun/Ap-1 pathways on TGF-ß1-induced ET promoter activity. A wild-type ET-1 promoter/luciferase reporter construct (WT) was transfected into normal lung fibroblasts with or without empty expression vector, expression vectors encoding Smad 7 or TAM-67 (dominant negative c-jun [dn c-jun]) or 10 µM JNK inhibitor SP-600125. Cells were treated with or without TGF-ß1 (4 ng/ml). Inhibition of JNK/c-jun, but not Smad, signaling significantly (P < 0.05) reduced the TGF-ß1 induction of the ET-1 promoter. As a control, a generic Smad3-responsive promoter (SBE-lux) was transfected into fibroblasts. Induction of this promoter by TGF-ß1 was sensitive to overexpression of Smad7. Data for an average of six wells ± standard deviation in three independent experiments are shown. (C) Effect of loss of Smad3 expression on TGF-ß1-induced ET-promoter activity. The wild-type ET-1 promoter containing a Smad3 recognition sequence functionally operative in endothelial cells (WT) (43) was transfected into Smad3+/+ or Smad3–/– embryonic fibroblasts (Smad3 WT or Smad3 KO, respectively) and treated with or without TGF-ß1 (4 ng/ml). By using these cells, Smad3 has previously been shown to be the Smad operating in driving Smad binding element (SBE)-dependent transcriptional activation in fibroblasts and induction of TGF-ß/Smad-dependent protein expression (25, 41). Loss of Smad3 did not affect the TGF-ß1 induction of the ET-1 promoter, but the induction of SBE-lux was impaired. Data for an average of six wells ± standard deviation in three independent experiments are shown. (D) Effect of bosentan treatment on TGF-ß induction of {alpha}-SMA. Fibroblasts from three normal individuals (N1, N2, and N3) were treated with or without ET-1 (100 nM) or TGF-ß1 (4 ng/ml) as indicated. Twenty-four hours later, cell extracts were prepared and subjected to Western blot analysis with the indicated antibodies.

 
TGF-ß-induced {alpha}-SMA production is inhibited by antagonism of the endothelin A/B receptors. To verify that the ability of TGF-ß to induce ET-1 was of functional relevance to fibroblasts, cells isolated from three normal individuals were pretreated with or without the ETA/ETB receptor antagonist bosentan prior to treatment with or without TGF-ß1 for 24 h. Cell extracts were then prepared and subjected to Western blot analysis with anti-{alpha}-SMA antibody. The ability of TGF-ß to induce {alpha}-SMA depended on ET-1 as bosentan blocked TGF-ß-induced {alpha}-SMA protein expression (Fig. 2D).

Fibrotic lung fibroblasts display increased JNK/Ap-1 activation. ET-1 protein is overexpressed by fibrotic lung fibroblasts isolated from patients with scleroderma (FASSc) and is essential for the persistence of the myofibroblast phenotype in these cells (51). As we were interested in examining the mechanism underlying ET-1 overproduction in fibrotic lung fibroblasts, and since we had demonstrated the central contribution of Ap-1 to the induction of the ET-1 promoter in normal lung fibroblasts, we sought to ascertain whether the elevated expression of ET-1 in fibrotic lung fibroblasts could be due to an increased level of JNK activation in this cell type. To investigate this question, we cultured normal and FASSc fibroblasts to 80% confluence. We then cultured cells in the absence of serum for 18 h, prior to exposure of cells to TGF-ß1 (4 ng/ml) for various lengths of time. Cell layers were harvested and subjected to Western blot analysis with anti-JNK1 and anti-phospho-JNK1 antibodies. We found that, whereas TGF-ß was able of inducing JNK activation in normal and FASSc lung fibroblasts, fibrotic lung fibroblasts displayed constitutively elevated JNK activation as visualized by increased phosphorylation revealed by the anti-phospho-JNK1 antibody and an increase in c-jun phosphorylation revealed by an anti-phopsho-c-jun antibody (Fig. 3).


Figure 3
View larger version (68K):
[in this window]
[in a new window]
  		FIG. 3. Fibrotic lung fibroblasts isolated from patients with lung involvement in scleroderma (FASSc) display elevated, constitutive JNK1 activation that is ALK5 independent. Normal and FASSc fibrotic lung fibroblasts were serum starved and treated with or without TGF-ß1 (4 ng/ml) for the durations indicated. Cell layers were harvested and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with anti-phospho-JNK1 and anti-JNK1 antibodies. Cells were also treated in the presence and absence of the ALK5 TGF-ß type I receptor inhibitor SB 431542 (26). Whereas TGF-ß1 induces JNK1 phosphorylation in an ALK5-dependent fashion, FASSc fibroblasts display elevated, ALK5-independent JNK phosphorylation.

 
To extend these results and to further evaluate whether the Ap-1/JNK cascade was constitutively activated in fibrotic lung fibroblasts, we examined whether nuclear extracts prepared from fibrotic lung fibroblasts displayed levels of Ap-1 DNA binding activity that were elevated compared to those of nuclear extracts isolated from normal lung fibroblasts. To perform this analysis, we radiolabeled an annealed double-stranded oligomer bearing the functional Ap-1 binding element of the ET-1 promoter and used this fragment as a probe in gel shift reactions with extracts prepared from normal and fibrotic lung fibroblasts. Our gel shift analyses revealed that protein binding to the Ap-1 element of the ET-1 promoter in extracts prepared from fibrotic lung fibroblasts was increased compared to that in extracts prepared from control fibroblasts (Fig. 4A). Experiments using an anti-c-jun antibody to block nuclear factor binding to radiolabeled probe confirmed that c-jun recognized the Ap-1 site in the ET-1 promoter (Fig. 4B). Collectively, our results suggested that elevated JNK/Ap-1 activation was a feature of fibrotic lung fibroblasts isolated from FASSc patients.


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 4. Nuclear extracts from FASSc lung fibroblasts display elevated levels of Ap-1 binding activity. (A) FASSc nuclear extracts contain elevated Ap1 binding activity. A radiolabeled probe containing the consensus Ap-1 element of the ET-1 promoter (43) was incubated with nuclear extracts prepared from pulmonary fibroblasts isolated from two normal individuals (N1 and N2) or two individuals with pulmonary fibrosis associated with scleroderma (S1 and S2). Cells were treated in the presence or absence of TGF-ß1 for 30 min prior to harvesting. TGF-ß induces binding to the Ap-1 element of the ET-1 promoter; extracts from FASSc cells show elevated endogenous Ap-1 binding activity. To verify that protein binding was due to Ap-1 binding activity, protein binding to DNA was competed by addition of a 100-fold molar excess of cold oligomer bearing a consensus Ap-1 site, but not by oligomers containing consensus Ap-2 or Sp-1 sites. (B) Transcription factor c-jun binds the Ap1 element in the ET-1 promoter. Preincubation of extracts for 1 h with anti-c-jun, but not anti-HuR (control), reduced factor binding to probe, indicating that c-jun was required for protein/DNA complex formation. Individuals N2 and S2 from panel A were examined.

 
Elevated ET-1 production in fibrotic lung fibroblasts results from c-jun/JNK activation. To determine whether elevated Ap-1/JNK activity in fibrotic lung fibroblasts contributed to the elevated ET-1 expression observed in fibrotic lung fibroblasts, we first found that, consistent with our results examining ET-1 protein (51), fibrotic lung fibroblasts displayed increased levels of ET-1 mRNA compared to those in normal lung fibroblasts as revealed using real-time PCR analysis with primers detecting ET-1 mRNA (Fig. 5A). Extending these data, we found that the ET-1 promoter showed markedly increased activity in fibrotic lung fibroblasts relative to control normal lung fibroblasts (Fig. 5B). Mutation of the Ap-1 element, but not the Smad element, of the ET-1 promoter significantly reduced the elevated level of ET-1 promoter activity in fibrotic lung fibroblasts (Fig. 5B), suggesting that Ap-1 contributed to the overexpression of ET-1 in fibrotic lung fibroblasts. Consistent with this notion, the JNK inhibitor SP600125 did not affect the basal ET-1 promoter activity in normal lung fibroblasts but significantly reduced the elevated ET-1 promoter activation observed in FASSc lung fibroblasts (Fig. 5B). These results suggested that elevated c-jun/JNK activation in fibrotic lung fibroblasts was responsible, at least in part, for the elevated expression of ET-1 in FASSc lung fibroblasts.


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 5. FASSc lung fibroblasts show elevated ET-1 expression in a Smad-independent c-jun/JNK-dependent fashion. (A) FASSc lung fibroblasts (LF) possess elevated levels of ET-1 mRNA. Normal and FASSc lung fibroblasts were cultured to confluence and treated for 18 h in the presence or absence of the dual ETA/ETB receptor antagonist bosentan. Cells were cultured from four normal individuals and four individuals with FASSc. Total RNA was harvested, reverse transcribed into cDNA, and subjected to real-time PCR analysis with primers recognizing human ET-1 and 28S RNA. Ratios of ET-1/28S RNA levels ± standard deviations are shown (n = 3). Note that in the presence of bosentan, the elevated level of ET-1 mRNA in FASSc fibroblasts is markedly reduced. (B) Elevated expression of ET-1 is JNK/c-jun dependent. Normal and FASSc lung fibroblasts were transfected with a wild-type ET-1 promoter/luciferase reporter construct (WT) or an otherwise identical construct containing mutated Smad (Smad mut) or Ap-1 (Ap-1 mut) elements. Mutation of the Ap-1 element, but not the Smad element, significantly (P < 0.05) reduced the TGF-ß1-induction of the ET-1 promoter. Similarly, addition of the JNK inhibitor SP-600125 (SP; 10 µM) reduced ET-1 promoter activity in FASSc, but not normal, lung fibroblasts. Data for an average of six wells ± standard deviation in three independent experiments are shown.

 
An autocrine endothelin loop results in the elevated JNK activation in scleroderma lung fibroblasts. To further investigate the molecular basis for the increased activation of the ET-1 promoter in fibrotic lung fibroblasts, we investigated whether the elevated level of ET-1 promoter activity in fibrotic lung fibroblasts was dependent on TGF-ß signaling via the TGF-ß type I/ALK5 receptor. First, we showed that TGF-ß induced ET-1 in both normal and fibrotic lung fibroblasts in a fashion which depended on the TGF-ß type I/ALK5 receptor, as a specific ALK5 antagonist SB 431542 blocked TGF-ß-induced ET-1 promoter activity in normal and fibrotic lung fibroblasts (Fig. 6). Conversely, ALK5 inhibition was not able to reduce the overexpression of the ET-1 promoter in fibrotic lung fibroblasts (Fig. 6). Collectively, these results suggested that whereas ALK5-dependent JNK activation was required for the TGF-ß induction of the ET-1 promoter in normal and fibrotic lung fibroblasts, ALK5-independent JNK activation contributed to the overexpression of ET-1 in fibrotic lung fibroblasts. To test this hypothesis, we investigated whether ALK5 inhibition using SB 431542 (26) blocked the constitutive hyperactivation of JNK in fibrotic lung fibroblasts. Confirming that ALK5 was required for the TGF-ß induction of JNK1, we used Western blot analysis to show that ALK5 inhibition by SB 431542 blocked the TGF-ß induction of JNK1 activation in normal and fibrotic lung fibroblasts (Fig. 3A). Conversely, we found that ALK5 inhibition had no significant effect on the elevated, constitutive JNK1 activation in fibrotic lung fibroblasts (Fig. 3A).


Figure 6
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6. ALK5 is not required for the overexpression of ET-1 in FASSc lung fibroblasts. Normal and FASSc lung fibroblasts were transfected with an ET-1 promoter/luciferase reporter construct and treated in the presence or absence of TGF-ß1 (4 ng/ml) for 24 h. Cells were treated in the presence or absence of the ALK5 inhibitor SB-431542 or the JNK inhibitor SP600125. Whereas ALK5 inhibition blocked the TGF-ß induction of the ET-1 promoter in FASSc lung fibroblasts (P < 0.05; compared by Student's t test to TGF-ß-treated sample), ALK5 inhibition did not significantly decrease the elevated basal ET-1 promoter activity in FASSc lung fibroblasts (P < 0.05; compared by Student's t test to basal ET-1 promoter activity in FASSc fibroblasts).

 
To test the notion that ET-1 may be responsible for the increased induction of JNK1 in fibroblasts, we first tested whether recombinant ET-1 could induce JNK1 in normal fibroblasts. To perform this experiment, we added ET-1 to normal fibroblasts for 30 min and then subjected cells to Western blot analysis to detect JNK1 and phosphorylated JNK1. Our results demonstrated that ET-1 could indeed induce JNK1 (Fig. 7). Previously, it had been shown that TAK1 could impair the ability of growth factors to induce JNK1 (47). We showed that TAK1 was required for the ability of ET-1 to induce JNK1 as ET-1 was not able to induce phosphorylation of JNK1 in TAK1 knockout fibroblasts (Fig. 7). Demonstrating the ability of TAK1 to mediate ET-1 action, ET-1 was not able to induce {alpha}-SMA protein expression in TAK1-deficient fibroblasts (Fig. 7).


Figure 7
View larger version (53K):
[in this window]
[in a new window]
  		FIG. 7. ET-1 induces JNK1 phosphorylation and {alpha}-SMA production through TAK1. Wild-type fibroblasts (TAK WT) or fibroblasts in which TAK1 was deleted (TAK KO) (47) were treated with or without recombinant ET-1 (100 nM) for 30 min (for phospho-JNK1 blots) or 24 h (for {alpha}-SMA blots). Cell layers were harvested and subjected to Western blot analysis with the antibodies indicated. It is notable that ET-1 did not induce JNK1 phosphorylation or induce {alpha}-SMA expression in TAK1 knockout fibroblasts, and basal {alpha}-SMA expression was lower in TAK1 knockout fibroblasts.

 
Additional secreted proteins believed to contribute to fibrosis include PDGF, IL-1, and angiotensin, yet application of the PDGF inhibitor Gleevec (37), the soluble IL-1 receptor (13), and the angiotensin II receptor inhibitor losartan (14) also had no significant impact on the elevated JNK activation in scleroderma lung fibroblasts (Fig. 8). We then evaluated whether constitutive JNK activation in FASSc lung fibroblasts was under control of an autocrine ET loop. To our surprise, we found that the dual ETA/ETB receptor antagonist bosentan reduced the amount of JNK1 in scleroderma cells to that in normal fibroblasts (Fig. 8). Bosentan did not affect basal JNK1 activation in normal fibroblasts (Fig. 8). Conversely, the single ETA receptor antagonist PD156707 and the ETB receptor antagonist BQ788 were relatively ineffective at reducing JNK1 activation in FASSc fibroblasts (Fig. 8). Extending these results, we showed, using real-time PCR analysis, that Bosentan reduced the elevated levels of ET-1 mRNA in fibrotic lung fibroblasts (Fig. 5A). These results suggest that an autocrine ET loop, acting through the ETA and ETB receptors, is responsible for the elevated JNK activation in fibrotic lung fibroblasts and hence for the elevated ET-1 transcription observed in this cell type.


Figure 8
View larger version (39K):
[in this window]
[in a new window]
  		FIG. 8. An autocrine ET loop results in the elevated JNK1 activation observed in FASSc lung fibroblasts. Scleroderma lung fibroblasts were treated with the PDGF receptor inhibitor Gleevec, the angiotensin II receptor inhibitor losartan, a soluble IL-1 receptor antagonist (IL-1ra), the dual ETA/ETB receptor inhibitor bosentan (Anti-ETAr&Br), the ETA receptor inhibitor PD156707 (Anti-ETAr), or the ETB receptor inhibitor BQ788 (Anti-ETBr). Cell extracts were prepared and subjected to Western blot analyses with the indicated antibodies. Inhibition of autocrine ET signaling reduced the activation of JNK1 in FASSc fibroblasts to that of normal fibroblasts. It is notable that bosentan (Anti-ETAr&Br) treatment did not appreciably affect basal JNK1 activation in normal fibroblasts.

 
JNK inhibition reduces the overexpression of profibrotic markers in fibrotic lung fibroblasts. Given the role of ET-1 in maintaining lung fibrosis (51), these results suggested that inhibition of c-jun/JNK upstream may be of benefit in combating fibrosis. Previously, we showed that the overexpression of {alpha}-SMA, a hallmark of the fibrotic phenotype, by fibrotic scleroderma lung fibroblasts at least partially depended on endogenous ET signaling through the ETA and ETB receptors (51). To extend these previous results and to confirm our hypothesis that JNK inhibition would reduce the overexpression of profibrotic markers in fibrotic lung fibroblasts, we investigated whether incubation of FASSc lung fibroblasts with the JNK inhibitor SP600125 would reduce the overexpression of {alpha}-SMA in this cell type. Fibroblasts isolated from three normal individuals and three individuals with FASSc were examined. In all cases examined, SP600125 did not affect {alpha}-SMA expression in normal fibroblasts but significantly reduced the elevated {alpha}-SMA production in the FASSc fibroblasts (Fig. 9). Similarly, SP600125 reduced ET-1 production by FASSc fibroblasts (Fig. 9). Collectively, our results suggest that a constitutively elevated JNK activation, mediated by an ET-1 autocrine loop, contributes to the activated fibrotic phenotype of fibroblasts isolated from patients with pulmonary fibrosis (Fig. 10).


Figure 9
View larger version (50K):
[in this window]
[in a new window]
  		FIG. 9. JNK inhibition reduces overexpression of ET-1 and {alpha}-SMA by FASSc lung fibroblasts. Scleroderma lung fibroblasts were treated with the JNK inhibitor SP600125 for 24 h. Cell extracts were harvested and subjected to Western blot analyses with the antibodies indicated. Lung fibroblasts from three normal individuals (N1, N2, and N3) and three individuals with FASSc (S1, S2, and S3) were examined. SP600125 inhibits {alpha}-SMA production by FASSc fibroblasts. Similarly, conditioned media from identical experiments were pooled and subjected to ELISA to detect ET-1 levels.

 

Figure 10
View larger version (25K):
[in this window]
[in a new window]
  		FIG. 10. Schematic diagram of autocrine ET signaling, TGF-ß, and JNK contribution to overexpression of ET-1 in fibrotic FASSc lung fibroblasts and hence to fibrosis. Constitutively activated ET-dependent JNK activates the ET-1 promoter in an ALK5-independent fashion. Exogenous TGF-ß induces the ET-1 promoter through JNK, leading to additional production of ET-1. TGF-ß also directly induces profibrotic proteins (31). These events result in enhanced production of profibrotic proteins and ECM production, remodeling, and contraction.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TGF-ß and ET-1 interactions in fibrosis. Many studies have also shown that TGF-ß is a central profibrotic cytokine in scleroderma (16). TGF-ß is a potent inducer of myofibroblast formation (17). Levels of TGF-ß mRNA and protein are increased in the lungs of scleroderma patients with pulmonary fibrosis as visualized by in situ hybridization of lung biopsy samples (12) or measurement of TGF-ß mRNA in mononuclear cells from bronchial lavage fluids (15, 33). Thus, ET-1 overexpression in lung is within a context of elevated TGF-ß expression from immune cells. Our results, showing that TGF-ß can induce ET-1 in normal and fibrotic pulmonary fibroblasts, suggest that ET-1 may be a downstream mediator of TGF-ß. However, we also showed that whereas inhibition of the TGF-ß type I/ALK5 receptor blocked the TGF-ß induction of ET-1 in normal and fibrotic fibroblasts, ALK5 inhibition did not affect the elevated ET-1 expression in fibrotic lung fibroblasts nor the elevated JNK activation observed in this cell type. We then demonstrated that ET-1 could induce JNK1 phosphorylation and {alpha}-SMA expression through TAK1. Finally, we showed that activated JNK1 in fibrotic cells was dependent on an autocrine ET loop and that inhibition of JNK1 reduced the elevated production of type I collagen and {alpha}-smooth muscle actin in fibrotic lung fibroblasts. It is interesting to note that, in this context, we recently found that the overexpression of {alpha}-SMA in fibrotic fibroblasts was independent of signaling through the ALK5 receptor (11). In fibrotic disease, and consistent with the notions that ET-1 and TGF-ß cooperate to cause myofibroblast formation (46) and that factors working with TGF-ß are required to generate sustained fibrotic responses in vivo (35), the constitutive ALK5-independent JNK/Ap-1-dependent ET-1 expression observed in fibrotic pulmonary fibroblasts, caused by an autocrine ET loop, would be expected to cooperate with ALK5-dependent ET-1 produced in response to TGF-ß to generate the fibrotic phenotype. Thus, ALK5/TGF-ß-independent ET-1 production, which would not be expected to be under the controls normally regulating TGF-ß signaling in fibroblasts (31), is likely to be a key feature contributing to pathological fibrotic disease in lung (Fig. 10). Our results suggest that understanding the interactions among ET-1 and TGF-ß is likely to have a major impact in understanding wound healing and the progression to pathological fibrotic responses in vivo. Smads were not required for the TGF-ß induction of ET-1.

These results are consistent with observations that TGF-ß indirectly causes an increase in {alpha}-SMA mRNA and protein expression, which occurs in Smad3 knockout fibroblasts (30). In addition, Smad3 knockout mice are only partially resistant to the bleomycin model of fibrosis, indicating that there is a non-Smad component to the acquisition of the fibrotic phenotype (30). Our observation that TGF-ß-induced {alpha}-SMA requires ET-1 signaling through the ETA and ETB receptors is consistent with these observations.

Dysregulation of signal transduction in pulmonary fibrosis. Collectively, our results are consistent with the notion that signaling pathways normally controlling gene expression of key profibrotic genes are dysregulated in fibrotic disease, resulting in the bypassing of controls that normally suppress profibrotic responses (31). In this regard, it is interesting to note that the JNK activation observed in fibrotic lung fibroblasts would be predicted to generally suppress Smad-dependent signaling as activation of JNK/c-jun represses non-Ap-1-dependent Smad-responsive promoters in favor of combined Smad/Ap-1-dependent transcriptional responses (32, 57, 58). For example, the net result of Smad/JNK activation would be to block matrix metalloproteinase 1 and enhance TIMP-1 expression (21, 33a)—and hence indirectly affect matrix accumulation—yet attenuate Smad-dependent activation of type I collagen and CCN2 (32, 57, 58). In this regard, although "leading edge" dermal scleroderma fibroblasts show increased Smad3 activity (36), dermal scleroderma fibroblasts are less responsive to exogenous TGF-ß, as visualized by the induction of the type I collagen promoter, than their normal counterparts (4). That ET-1 induces CCN2 and type I collagen may be especially important in the context of fibrosis (52). That such dysregulation of signaling exists might explain the divergent results that have been obtained when the effect of Smad signaling pathways have been examined in fibrotic cells (25, 36) and further emphasize the importance of a complex series of interactions involving MAP kinase cascades such as JNK (this report and reference 21) or ERK (8-10, 32, 52, 53) in regulating fibrogenic responses, either dependent or independent of TGF-ß. It is interesting to note, consistent with our observations that the ability of TGF-ß to induce {alpha}-SMA is ET-1 dependent and that ET-1 induced {alpha}-SMA production requires JNK1, that JNK inhibition blocks the ability of TGF-ß to induce {alpha}-SMA expression in lung fibroblasts (23). It is likely that fibrosis results from a dysregulation of signal transduction pathways that renders the fibrotic fibroblast incapable of responding to signals that normally downregulate the wound healing/fibrotic response (2).

In conclusion, we have provided evidence that both TGF-ß/ALK5-dependent and TGF-ß/ALK5-independent induction of ET-1 are likely to play key roles in the expression of ET-1 in pulmonary fibrosis and hence in fibrogenesis. Dysregulated constitutive activation of JNK, due to an autocrine ET loop, contributes to the enhanced basal production of ET-1 in fibrotic lung fibroblasts and hence to the constitutively activated persistent myofibroblast phenotype observed in pulmonary fibrosis.


   ACKNOWLEDGMENTS
 
This work was supported by the Raynaud's and Scleroderma Association Trust, the Arthritis Research Campaign, the Nightingale Trust, the Welton Foundation, the Canadian Institutes of Health Research, and Gap B Funds from the University of Western Ontario. A.L. is a New Investigator of the Arthritis Society (the Scleroderma Society of Ontario).


   FOOTNOTES
 
* Corresponding author. Mailing address for David J. Abraham: Centre for Rheumatology, Department of Medicine, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom. Phone: 44-(0)207-794-0500, ext. 34062. Fax: 44-(0)207-794-0432. E-mail: d.abraham{at}medsch.ucl.ac.uk. Mailing address for Andrew Leask: CIHR Group in Skeletal Development and Remodeling, Division of Oral Biology, Schulich School of Medicine and Dentistry, Dental Sciences Building, University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone: (519) 661-2111, ext. 81102. Fax: (519) 850-2459. E-mail: Andrew.Leask{at}schulich.uwo.ca. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Abraham, D. J., R. Vancheeswaran, M. R. Dashwood, P. Pantelides, W. Shi-Wen, R. M. du Bois, and C. M. Black. 1997. Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease. Am. J. Pathol. 151:831-841.[Abstract]

2. Abraham, D. J., X. Shiwen, C. M. Black, S. Sa, Y. Xu, and A. Leask. 2000. Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts. J. Biol. Chem. 275:15220-15225.[Abstract/Free Full Text]

3. Anggard, E. E., R. M. Botting, and J. R. Vane. 1990. Endothelins. Blood Vessels 27:269-281.[Medline]

4. Asano, Y., H. Ihn, K. Yamane, M. Kubo, and K. Tamaki. 2004. Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts. J. Clin. Investig. 113:253-264.[CrossRef][Medline]

5. Badylak, S. F. 2002. The extracellular matrix as a scaffold for tissue reconstruction. Semin. Cell Dev. Biol. 13:377-383.[CrossRef][Medline]

6. Bell, E., B. Ivarsson, and C. Merrill. 1979. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76:1274-1278.[Abstract/Free Full Text]

7. Brown, P. H., T. K. Chen, and M. J. Birrer. 1994. Mechanism of action of a dominant-negative mutant of c-Jun. Oncogene 9:791-799.[Medline]

8. Chen, Y., I. E. Blom, S. Sa, R. Goldschmeding, D. J. Abraham, and A. Leask. 2002. CTGF expression in mesangial cells: involvement of SMADs, MAP kinase and PKC. Kidney Int. 62:1149-1159.[CrossRef][Medline]

9. Chen, Y., D. J. Abraham, X. Shi-Wen, C. M. Black, K. M. Lyons, and A. Leask. 2004. CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin. Mol. Biol. Cell 15:5635-5646.[Abstract/Free Full Text]

10. Chen, Y., X. Shi-Wen, J. van Beek, L. Kennedy, M. McLeod, E. A. Renzoni, G. Bou-Gharios, S. Wilcox-Adelman, P. F. Goetinck, M. Eastwood, C. M. Black, D. J. Abraham, and A. Leask. 2005. Matrix contraction by dermal fibroblasts requires TGFbeta/ALK5, heparan sulfate containing proteoglycans and MEK/ERK: insights into pathological scarring in chronic fibrotic disease. Am. J. Pathol. 167:1699-1711.[Abstract/Free Full Text]

11. Chen, Y., X. Shi-Wen, M. Eastwood, C. M. Black, C. P. Denton, A. Leask, and D. J. Abraham. 2006. Contribution of activin receptor-like kinase 5 (transforming growth factor beta receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts. Arthr. Rheum. 54:1309-1316.[CrossRef][Medline]

12. Coker, R. K., G. J. Laurent, P. K. Jeffery, R. M. du Bois, C. M. Black, and R. J. McAnulty. 2001. Localisation of transforming growth factor beta1 and beta3 mRNA transcripts in normal and fibrotic human lung. Thorax 56:549-556.[Abstract/Free Full Text]

13. Cominelli, F., C. C. Nast, B. D. Clark, R. Schindler, R. Lierena, V. E. Eysselein, R. C. Thompson, and C. A. Dinarello. 1990. Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis. J. Clin. Investig. 86:972-980.[Medline]

14. Criscione, L., H. Thomann, S. Whitebread, M. de Gasparo, P. Buhlmayer, P. Herold, F. Ostermayer, and B. Kamber. 1990. Binding characteristics and vascular effects of various angiotensin II antagonists. J. Cardiovasc. Pharmacol. 16(Suppl. 4):S56-S59.

15. Deguchi, Y., and S. Kishimoto. 1991. Spontaneous activation of transforming growth factor-beta gene transcription in broncho-alveolar mononuclear cells of individuals with systemic autoimmune diseases with lung involvement. Lupus 1:27-30.[Abstract/Free Full Text]

16. Denton, C. P., and D. J. Abraham. 2001. Transforming growth factor-beta and connective tissue growth factor: key cytokines in scleroderma pathogenesis. Curr. Opin. Rheumatol. 13:505-511.[CrossRef][Medline]

17. Desmouliere, A. 1995. Factors influencing myofibroblast differentiation during wound healing and fibrosis. Cell Biol. Int. 19:471-476.[CrossRef][Medline]

18. Domann, F. E., J. P. Levy, M. J. Birrer, and G. T. Bowden. 1994. Stable expression of a c-JUN deletion mutant in two malignant mouse epidermal cell lines blocks tumor formation in nude mice. Cell Growth Differ. 5:9-16.[Abstract]

19. Gadek, J. E., G. A. Fells, R. L. Zimmerman, and R. G. Crystal. 1984. Role of connective tissue proteases in the pathogenesis of chronic inflammatory lung disease. Environ. Health Perspect. 55:297-306.[Medline]

20. Grinnell, F. 1994. Fibroblasts, myofibroblasts, and wound contraction. J. Cell Biol. 124:401-404.[Free Full Text]

21. Hall, M. C., D. A. Young, J. G. Waters, A. D. Rowan, A. Chantry, D. R. Edwards, and I. M. Clark. 2003. The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1. J. Biol. Chem. 278:10304-10313.[Abstract/Free Full Text]

22. Harrison, N. K., A. C. Argent, R. J. McAnulty, C. M. Black, B. Corrin, and G. J. Laurent. 1991. Collagen synthesis and degradation by systemic sclerosis lung fibroblasts. Responses to transforming growth factor-beta. Chest 99(Suppl. 3):71S-72S.[Medline]

23. Hashimoto, S., Y. Gon, I. Takeshita, K. Matsumoto, S. Maruoka, and T. Horie. 2001. Transforming growth factor-ß1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH 2-terminal kinase-dependent pathway. Am. J. Respir. Crit. Care Med. 163:152-157.[Abstract/Free Full Text]

24. Heo, Y. S., S. K. Kim, C. I. Seo, Y. K. Kim, B. J. Sung, H. S. Lee, J. I. Lee, S. Y. Park, J. H. Kim, K. Y. Hwang, Y. L. Hyun, Y. H. Jeon, S. Ro, J. M. Cho, T. G. Lee, and C. H. Yang. 2004. Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125. EMBO J. 23:2185-2195.[CrossRef][Medline]

25. Holmes, A., D. J. Abraham, S. Sa, X. Shiwen, C. M. Black, and A. Leask. 2001. CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J. Biol. Chem. 276:10594-10601.[Abstract/Free Full Text]

26. Inman, G. J., F. J. Nicolas, J. F. Callahan, J. D. Harling, L. M. Gaster, A. D. Reith, N. J. Laping, and C. S. Hill. 2002. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62:65-74.[Abstract/Free Full Text]

27. Ivarsson, M., A. McWhirter, C. M. Black, and K. Rubin. 1993. Impaired regulation of collagen pro-alpha 1(I) mRNA and change in pattern of collagen-binding integrins on scleroderma fibroblasts. J. Investig. Dermatol. 101:216-221.[CrossRef][Medline]

28. Jimenez, S. A., E. Hitraya, and J. Varga. 1996. Pathogenesis of scleroderma. Collagen Rheum. Dis. Clin. N. Am. 22:647-674.

29. Kawaguchi, Y., K. Suzuki, M. Hara, T. Hidaka, T. Ishizuka, M. Kawagoe, and H. Nakamura. 1994. Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis. Ann. Rheum. Dis. 53:506-510.[Abstract/Free Full Text]

30. Lakos, G., S. Takagawa, S. J. Chen, A. M. Ferreira, G. Han, K. Masuda, X. J. Wang, L. A. DiPietro, and J. Varga. 2004. Targeted disruption of TGF-beta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. Am. J. Pathol. 165:203-217.[Abstract/Free Full Text]

31. Leask, A., and D. J. Abraham. 2004. TGF-beta signaling and the fibrotic response. FASEB J. 18:816-827.[Abstract/Free Full Text]

32. Leask, A., A. Holmes, C. M. Black, and D. J. Abraham. 2003. Connective tissue growth factor gene regulation. Requirements for its induction by transforming growth factor-beta 2 in fibroblasts. J. Biol. Chem. 278:13008-13015.[Abstract/Free Full Text]

33. Ludwicka, A., T. Ohba, M. Trojanowska, A. Yamakage, C. Strange, E. A. Smith, E. C. Leroy, S. Sutherland, and R. M. Silver. 1995. Elevated levels of platelet derived growth factor and transforming growth factor-beta 1 in bronchoalveolar lavage fluid from patients with scleroderma. J. Rheumatol. 22:1876-1883.[Medline]

33. Mauviel, A., K. Y. Chung, A. Agarwal, K. Tamai, and J. Uitto 1996. Cell-specific induction of distinct oncogenes of the Jun family is responsible for diferential regulation of collagenase gene expression by TGFß in fibroblasts and keratinocytes. J. Biol. Chem. 271:10917-10923.[Abstract/Free Full Text]

34. Miyauchi, T., N. Suzuki, T. Yuhara, T. Akama, H. Suzuki, and H. Kashiwagi. 1992. Significance of plasma endothelin-1 levels in patients with systemic sclerosis. J. Rheumatol. 19:1566-1571.[Medline]

35. Mori, T., S. Kawara, M. Shinozaki, N. Hayashi, T. Kakinuma, A. Igarashi, M. Takigawa, T. Nakanishi, and K. Takehara. 1999. Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: a mouse fibrosis model. J. Cell. Physiol. 181:153-159.[CrossRef][Medline]

36. Mori, Y., S. J. Chen, and J. Varga. 2003. Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts. Arthritis Rheum. 48:1964-1978.[CrossRef][Medline]

37. Myllarniemi, M., J. Frosen, L. G. C. Ramirez, E. Buchdunger, K. Lemstrom, and P. Hayry. 1999. Selective tyrosine kinase inhibitor for the platelet-derived growth factor receptor in vitro inhibits smooth muscle cell proliferation after reinjury of arterial intima in vivo. Cardiovasc. Drugs Ther. 13:159-168.[CrossRef][Medline]

38. Nakao, A., M. Afrakhte, A. Moren, T. Nakayama, J. L. Christian, R. Heuchel, S. Itoh, M. Kawabata, N. E. Heldin, C. H. Heldin, and P. ten Dijke. 1997. Identification of Smad7, a TGFß-inducible antagonist of TGF-ß signalling. Nature 389:631-635.[CrossRef][Medline]

39. Ohnaka, K., R. Takayanagi, M. Nishikawa, M. Haji, and H. Nawata. 1993. Purification and characterization of a phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelium. J. Biol. Chem. 268:26759-26766.[Abstract/Free Full Text]

40. Panos, R. J., R. L. Mortenson, S. A. Niccoli, and T. E. King, Jr. 1990. Clinical deterioration in patients with idiopathic pulmonary fibrosis: causes and assessment. Am. J. Med. 88:396-404.[CrossRef][Medline]

41. Piek, E., W. J. Ju, J. Heyer, D. Escalante-Alcalde, C. L. Stewart, M. Weinstein, C. Deng, R. Kucherlapati, E. P. Bottinger, and A. B. Roberts. 2001. Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts. J. Biol. Chem. 276:19945-19953.[Abstract/Free Full Text]

42. Razzaque, M. S., and T. Taguchi. 2003. Pulmonary fibrosis: cellular and molecular events. Pathol. Int. 53:133-145.[CrossRef][Medline]

43. Rodriguez-Pascual, F., M. Redondo-Horcajo, and S. Lamas. 2003. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression. Circ. Res. 92:1288-1295.[Abstract/Free Full Text]

44. Rubanyi, G. M., and L. H. Botelho. 1991. Endothelins. FASEB J. 5:2713-2720.[Abstract]

45. Shao, R., Z. Shi, P. J. Gotwals, V. E. Koteliansky, J. George, and D. C. Rockey. 2003. Cell and molecular regulation of endothelin-1 production during hepatic wound healing. Mol. Biol. Cell 14:2327-2341.[Abstract/Free Full Text]

46. Shephard, P., B. Hinz, S. Smola-Hess, J. J. Meister, T. Krieg, and H. Smola. 2004. Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes. Thromb. Haemost. 92:262-274.[Medline]

47. Shim, J. H., C. Xiao, A. E. Paschal, S. T. Bailey, P. Rao, M. S. Hayden, K. Y. Lee, C. Bussey, M. Steckel, N. Tanaka, G. Yamada, S. Akira, K. Matsumoto, and S. Ghosh. 2005. TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev. 19:2668-2681.[Abstract/Free Full Text]

48. Shinozaki, M., S. Kawara, N. Hayashi, T. Kakinuma, A. Igarashi, and K. Takehara. 1997. Induction of subcutaneous tissue fibrosis in newborn mice by transforming growth factor beta—simultaneous application with basic fibroblast growth factor causes persistent fibrosis. Biochem. Biophys. Res. Commun. 240:292-297.[Medline]

49. Shi-Wen, X., C. P. Denton, A. McWhirter, G. Bou-Gharious, D. J. Abraham, R. M. du Bois, and C. M. Black. 1997. Scleroderma lung fibroblasts exhibit elevated and dysregulated collagen type I biosynthesis. Arthritis Rheum. 40:1237-1244.[Medline]

50. Shi-Wen, X., C. P. Denton, M. R. Dashwood, A. M. Holmes, G. Bou-Gharios, J. D. Pearson, C. M. Black, and D. J. Abraham. 2001. Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1. J. Investig. Dermatol. 116:417-425.[CrossRef][Medline]

51. Shi-Wen, X., Y. Chen, C. P. Denton, M. Eastwood, E. A. Renzoni, G. Bou-Gharios, J. D. Pearson, M. Dashwood, R. M. du Bois, C. M. Black, A. Leask, and D. J. Abraham. 2004. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. Mol. Biol Cell. 15:2707-2719.[Abstract/Free Full Text]

52. Shi-Wen, X., S. L. Howat, E. A. Renzoni, A. Holmes, J. D. Pearson, M. R. Dashwood, G. Bou-Gharios, C. P. Denton, R. M. du Bois, C. M. Black, A. Leask, and D. J. Abraham. 2004b. Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK. J. Biol. Chem. 279:23098-23103.[Abstract/Free Full Text]

53. Stratton, R., V. Rajkumar, M. Ponticos, B. Nichols, X. Shiwen, C. M. Black, D. J. Abraham, and A. Leask. 2002. Prostacyclin derivatives prevent the fibrotic response to TGF-beta by inhibiting the Ras/MEK/ERK pathway. FASEB J. 16:1949-1951.[Abstract/Free Full Text]

54. Teder, P., and P. W. Noble. 2000. A cytokine reborn? Endothelin-1 in pulmonary inflammation and fibrosis. Am. J. Respir. Cell Mol. Biol. 23:7-10.[Free Full Text]

55. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier, and R. A. Brown. 2002. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3:349-363.[CrossRef][Medline]

56. Turner, A. J., and L. J. Murphy. 1996. Molecular pharmacology of endothelin converting enzymes. Biochem. Pharmacol. 51:91-102.[CrossRef][Medline]

57. Verrecchia, F., L. Vindevoghel, R. J. Lechleider, J. Uitto, A. B. Roberts, and A. Mauviel. 2001. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner. Oncogene 20:3332-3340.[CrossRef][Medline]

58. Verrecchia, F., C. Tacheau, E. F. Wagner, and A. Mauviel. 2003. A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor-beta-driven SMAD3/4-specific gene expression. J. Biol. Chem. 278:1585-1593.[Abstract/Free Full Text]

59. Willoughby, E. A., G. R. Perkins, M. K. Collins, and A. J. Whitmarsh. 2003. The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK. J. Biol. Chem. 278:10731-10736.[Abstract/Free Full Text]

Molecular and Cellular Biology, July 2006, p. 5518-5527, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.00625-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles: