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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 225-231, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0140OC

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Enhances Collagen Production by Human Lung Fibroblasts

Vladimir V. Yurovsky

Research Service, Veterans Affairs Maryland Health Care System and Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland

Address correspondence to: Vladimir V. Yurovsky, University of Maryland School of Medicine, MSTF Room 8-19, 10 South Pine Street, Baltimore, MD 21201. E-mail: vyurovsk{at}umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the tumor necrosis factor family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes and brain cells. Soluble TRAIL at high concentrations was found to induce apoptotic death in normal human lung fibroblasts, whereas at low concentrations it was found to stimulate collagen production by these cells. Collagen {alpha}2(I) mRNA expression was assessed by semiquantitative reverse transcriptase/polymerase chain reaction; total soluble collagen was measured in culture supernatants by the Sircol assay. Both {alpha}2(I) collagen mRNA level and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (> 4-fold increase in mRNA level) at 1 ng/ml TRAIL. Analysis of the transcriptional response in TRAIL-stimulated fibroblasts, using DNA microarray hybridization, revealed an augmented expression of a number of genes involved in tissue remodeling, including those related to the transforming growth factor-ß (TGF-ß) pathway. DNA microarray results for the increase in TGF-ß1 mRNA level were confirmed by Northern blot analysis and by measurements of total active TGF-ß1 in culture supernatants. In addition, pan-specific TGF-ß antibody was shown to inhibit TRAIL-stimulated collagen mRNA and protein expression. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGF-ß production that acts in an autocrine manner.

Abbreviations: fluorescein isothiocyanate, FITC • c-Jun N-terminal kinase, JNK • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • nuclear factor {kappa}B, NF-{kappa}B • polymerase chain reaction, PCR • transforming growth factor-ß, TGF-ß • tumor necrosis factor–related apoptosis-inducing ligand, TRAIL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to be expressed by activated T lymphocytes (1, 2), raising the possibility that this molecule might be involved in T cell–mediated cytotoxicity and/or immune regulation. A soluble form of TRAIL has also been detected in biologic fluids and implicated particularly in viral infection (3). Soluble TRAIL can induce apoptosis in a variety of transformed cell lines and in normal human hepatocytes and brain cells (1, 4–7). The bioactive form of TRAIL is homotrimer, and the activity of soluble TRAIL is enhanced by crosslinking monomers with an antibody (1). Induction of apoptosis by TRAIL takes place through death-signaling receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind to decoy receptors DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), which do not contain functional death domains, and to a soluble receptor osteoprotegerin (reviewed in Ref. 8). The presence of multiple receptors suggests, first, that TRAIL is involved in multiple processes, and second, that restricted expression of TRAIL receptors may regulate the sensitivity of cells to TRAIL-induced apoptosis. However, it has been shown recently that cell resistance to TRAIL-induced apoptosis is controlled intracellularly rather than at the level of expression of death receptors and decoy receptors (9, 10).

Relatively little is known about possible nonapoptotic pathways induced by TRAIL. The components of different TRAIL signaling pathways have not been well defined, though possible roles of tumor necrosis factor receptor 1-associated death domain protein, Fas-associated death domain factor, tumor necrosis factor receptor-associated factor 2, or receptor-interacting protein in TRAIL signaling have been suggested (11–14). The downstream signaling pathways after TRAIL binding to DR4, DR5, or DcR2 involve c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B) (13–15).

We have found the expression of TRAIL in type 2 CD8+ T cell clones that have undergone oligoclonal expansion in the lungs of patients with systemic sclerosis (scleroderma), and are able to stimulate collagen production in lung fibroblasts in vitro (manuscript in preparation). The presence of CD8+ T cells producing type 2 cytokines in the lungs of patients with systemic sclerosis is associated with greater decline in pulmonary function over time that is indicative of the development of lung fibrosis (16). The objective of this study is to examine the effects of TRAIL on human lung fibroblasts. Among the family members, TRAIL displays highest homology to CD95 (Fas/APO-1) ligand, receptor of which may not only mediate cell apoptosis, but also mediate the proliferation of normal human fibroblasts (17, 18). Considering structural and functional similarities between TRAIL and CD95 ligand, the dual signaling programs of TRAIL might be also anticipated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culture
Three lines of human lung fibroblasts (8F1024, 8F1608, and 9F0892; BioWhittaker, Walkersville, MD) were obtained at autopsy from persons with intracranial trauma. The cells were cultured in FGM-2 medium containing 2% fetal bovine serum, and were drawn to the quiescent state by incubation in serum-free medium for 72 h before experiments. Fibroblasts in early passages were used for experiments.

Flow Cytometry
The cells were trypsinized with Trypsin/EDTA solution (BioWhittaker) for 1 min, washed with ice-cold phosphate-buffered saline, fixed with 2% paraformaldehyde for 10 min, permeabilized with 0.1% saponin for 20 min at room temperature, and stained with mouse monoclonal IgG1 anti-TRAIL-R1, -R2, -R3, or -R4 (all from Alexis Biochemicals, San Diego, CA) for 30 min at 4°C. Secondary antibody was fluorescein isothiocyanate (FITC)-labeled rat anti-mouse IgG1 (BD PharMingen, San Diego, CA). Flow cytometry was performed using FACScan and analyzed with the CellQuest software (BD Immunocytometry Systems, San Jose, CA).

Cell Death and Viability Assays
Fibroblasts were grown either in a 96-well plate at 104 cells/well for colorimetric test or in 6-well plates at 2 x 105 cells/well for flow cytometry analysis. Titrated doses of recombinant human TRAIL (Alexis Biochemicals) were added to the quiescent cells in the presence of crosslinking antibody (2 µg/ml) for 24 h. For colorimetric test of cell viability, CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) was added for the last hour of culture. The absorbance of the medium was determined at 490 nm. The percentage of dead cells was calculated relative to the cells treated with crosslinking antibody alone. For flow cytometry, fibroblasts were trypsinized, washed with ice-cold phosphate-buffered saline, and stained with FITC-labeled annexin V and propidium iodide (both from BD PharMingen). Data were collected (30,000 events) using FACScan and analyzed with the CellQuest software.

Collagen mRNA Measurements
Fibroblasts were grown in 6-well plates to 80–90% confluence, then were drawn to the quiescent state and treated for 24 h with titrated doses of TRAIL in triplicates in the presence of crosslinking antibody. Afterward, the cells were washed and subjected to RNA isolation, using TRIzol reagent (Life Technologies, Rockville, MD) as per the manufacturer's protocol. One microgram of RNA was reverse transcribed to cDNA, using random primers, and amplified in a 25-µl polymerase chain reaction (PCR) mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 0.4 mM dNTPs, 1 µM 5'- and 3'-primers, and 1 unit Taq polymerase. The following primer pairs were used: {alpha}2(I) collagen forward, 5'-GATGGTGAAGATGGTCCCACAGG-3', and reverse, 5'-GGTCGTCCGGGTTTTCCAGGGT-3'; ribosomal protein S9 forward, 5'-GATGAGAAGGACCCACGGCGTCTGTTCG-3', and reverse, 5'-GAGACAATCCAGCAGCCCAGGAGGGACA-3'. PCR amplification was done in a thermal cycler PTC-200 (MJ Research, Waltham, MA), using the following conditions: denaturation at 95°C for 30 s and primer annealing and extension at 72°C for 2 min. For semiquantitative detection of PCR products, 5 µl of each reaction mixture was electrophoresed in a 1.8% agarose and stained with ethidium bromide. The gels were scanned and quantitated using NucleoVision gel imaging and densitometry system (NucleoTech, San Mateo, CA). The amount of {alpha}2(I) collagen transcript was normalized to the amount of S9 transcript in the same sample. In blocking experiments, quiescent fibroblasts were treated with 10 ng/ml TRAIL in the presence of crosslinking antibody (2 µg/ml) and pan-specific transforming growth factor-ß (TGF-ß) rabbit antibody or normal rabbit IgG (both from R&D Systems, Minneapolis, MN) at 20 µg/ml. Quantification of {alpha}2(I) collagen and S9 transcripts was done in 6 h by real-time PCR, using the LightCycler (Roche Molecular Biochemicals, Indianapolis, IN), with the same primer pairs and SYBR Green I detection, according to the manufacturer's instructions.

Collagen Protein Measurement
Total soluble collagen was measured in culture supernatants by the Sircol assay (Biocolor, Belfast, N. Ireland). One milliliter of Sirius Red reagent was added to 150 µl test sample and mixed for 30 min at room temperature. The collagen–dye complex was precipitated by centrifugation at 16,000 x g for 5 min; washed twice with 0.5 ml of methanol-free ethanol and dissolved in 1 ml 0.5 M NaOH; the absorbance was measured at 540 nm. The calibration curve was set up on the basis of collagen standard provided by the manufacturer. The assay was performed in duplicate, and the mean of two data was determined for individual sample.

DNA Microarray Analysis
Five micrograms of RNA from fibroblasts treated for 24 h with 1 ng/ml TRAIL and/or crosslinking antibody were used for making [33P]-labeled cDNA probes to hybridize to the GeneFilters microarrays containing 4,108 named human genes (Research Genetics, Huntsville, AL). cDNA preparation and labeling, hybridization, and membrane washing were done according to the manufacturer's instructions. Detection was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), followed by data analysis using the Pathways software (Research Genetics). The density of each array element was normalized for total spot density. Further analysis was done with the program Significance Analysis of Microarrays (19) that uses repeated permutations of the data to determine whether the differences in the expression of a gene between groups are significant. The results obtained from DNA arrays using this method correlate well with Northern blot analyses (19). This program revealed 2,420 genes whose expression was significantly related to the response. Genes were considered as differentially expressed if their mRNA level in TRAIL-stimulated fibroblasts deviated from that in fibroblasts treated with crosslinking antibody alone by at least a factor of 2.5 in at least three of four experiments. This subset of genes was clustered hierarchically into groups on the basis of the similarity of their expression profiles, using the program Cluster (20), and the results were visualized with the program TreeView (20).

Northern Blot Analysis
Three micrograms of RNA from fibroblasts treated for 24 h with titrated doses of TRAIL plus crosslinking antibody were processed for Northern blot hybridizations on Zeta-Probe membranes (Bio-Rad, Hercules, CA), according to standard protocol (21). Blots were sequentially hybridized with [32P]-labeled probes for TGF-ß1 that was amplified from cDNA clone 136821 (Research Genetics) and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech, Palo Alto, CA) as a loading control. The membranes were scanned with a PhosphorImager, and band densities were analyzed with the ImageQuant software (Molecular Dynamics).

TGF-ß1 Measurement
TGF-ß1 was measured in culture supernatants by an enzyme-linked immunosorbent assay, using the TGF-ß1 Quantikine kit (R&D Systems). The protein quantification was performed according to the manufacturer's recommendations, including the activation of latent TGF-ß1 with 0.167 N HCl. The assay was performed in duplicate, and the mean of two data was determined for individual sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Soluble TRAIL on Fibroblast Viability
Soluble multimerized TRAIL and normal human lung fibroblasts were used as a model of selective TRAIL influence on fibroblasts. A prerequisite for such study is the presence of TRAIL receptors on target cells. The expression of TRAIL receptors in fibroblasts was analyzed by flow cytometry (Figure 1) . All four cellular TRAIL receptors were detected in permeabilized cells, allowing both the cell-surface and intracellular staining. The expression of TRAIL-R2 was the most pronounced. These data were consistent with the pattern of TRAIL receptor expression in human lung fibroblasts shown by others (9), with TRAIL-R1 and -R2 being primarily associated with the Golgi network, whereas TRAIL-R3 and -R4 being localized predominantly in the nucleus. Fibroblast viability in the response to TRAIL was assessed by colorimetric assay and flow cytometry (Figure 2) . Soluble multimerized TRAIL did not induce the proliferation of fibroblasts, but at doses >= 100 ng/ml was found to cause their apoptotic death.



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  		Figure 1. Expression of TRAIL receptors in human lung fibroblasts (line 9F0892). Cells were trypsinized, permeabilized, stained with mouse IgG1 anti–TRAIL-R1, -R2, -R3, or -R4 plus FITC-labeled rat anti-mouse IgG1, and analyzed by flow cytometry. Solid line, TRAIL-R1; thin line, TRAIL-R2; dashed line, TRAIL-R3; dotted line, TRAIL-R4; shaded area, isotype control staining.

 


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  		Figure 2. Soluble multimerized TRAIL triggers apoptosis in human lung fibroblasts. Quiescent fibroblasts were treated for 24 h with titrated doses of recombinant human TRAIL in the presence of cross-linking antibody. (A) Colorimetric test of cell viability (line 9F0892), using CellTiter 96 AQueous One Solution Reagent. The percentage of dead cells is expressed relative to the cells treated with crosslinking antibody alone. (B) Flow cytometric analysis of cells undergoing apoptosis. Fibroblasts (line 8F1608) were trypsinized, stained with FITC-labeled annexin V and propidium iodide, and analyzed by flow cytometry. Early apoptotic cells (open bars) were determined as annexin V–positive, propidium iodide–negative; late apoptotic and dead cells (filled bars) as propidium iodide–positive. The data are presented as mean ± SD of triplicate cultures. *P < 0.05 by unpaired t test, TRAIL plus crosslinking antibody versus antibody alone. Similar results were obtained with three different fibroblast lines.

 
Effects of TRAIL on Collagen Production by Fibroblasts
TRAIL at lower doses was found to stimulate both {alpha}2(I) collagen mRNA expression and total soluble collagen secretion by human lung fibroblasts (Figure 3) . The peak response (> 4-fold increase in collagen mRNA level) was observed at 1 ng/ml TRAIL. The peak collagen protein secretion was also seen at doses 0.1 and 1 ng/ml. This suggests that TRAIL at concentrations below the apoptosis-inducing threshold has fibroblast-stimulating properties.



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  		Figure 3. Effects of TRAIL on collagen production by lung fibroblasts. Quiescent fibroblasts (line 9F0892) were treated with titrated doses of TRAIL in the presence of crosslinking antibody for 24 h. The level of {alpha}2(I) collagen mRNA was determined by reverse transcriptase-PCR and normalized to the level of ribosomal protein S9 mRNA in the same sample. Total soluble collagen was measured in culture supernatants using the Sircol assay. The data are presented as mean ± SD of triplicate cultures. *P < 0.05 by unpaired t test, TRAIL plus crosslinking antibody versus antibody alone. The upregulation of collagen production mediated by soluble TRAIL was observed in six separate experiments with three fibroblast lines. Open bars, mRNA; filled bars, protein.

 
Transcriptional Program in the Response of Lung Fibroblasts to TRAIL
DNA microarray hybridization was used to measure the changes in mRNA levels of 4,108 genes in lung fibroblasts treated with 1 ng/ml TRAIL for 24 h. Genes that were differentially expressed, with a 2.5-fold or greater difference in at least three experiments, could be classified into a number of functional categories. Some of the categories are listed in Figure 4 . The expression of a cohort of genes involved in signal transduction, DNA transcription, and tissue remodeling was found to substantially increase. Those included genes for protein kinase JNK1, transcription factors Sp3 and c-Fos (which have been implicated in control of collagen gene expression [22, 23]), collagen types I through VI and XV, fibronectin, laminin, and elastin.



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  		Figure 4. Differential gene expression patterns in fibroblasts (line 8F1024) treated for 24 h with 1 ng/ml TRAIL plus crosslinking antibody and in those treated with crosslinking antibody alone. Gene expression profiles were generated using GeneFilters microarrays and analyzed using the Pathways software. Seven hundred fifty-three genes were selected whose mRNA levels changed in response to TRAIL stimulation by at least a factor of 2.5 in at least three of four independent hybridizations. The genes were grouped into categories on the basis of their most likely role. Other categories and hierarchical trees are not presented. For each gene, the ratio of mRNA level in fibroblasts treated with TRAIL to its level in fibroblasts treated with crosslinking antibody alone is represented by a color, according to the color scale at the bottom.

 
Surprisingly, TRAIL also enhanced the transcription of potent regulators of extracellular matrix (ECM) synthesis: TGF-ß1, -ß2, and connective tissue growth factor; the latter might be induced by TGF-ß (24). In addition, the levels of latent TGF-ß–binding protein 1 and 2, insulin-like growth factor 2 receptor, and thrombospondins 1 and 2 were increased. Latent TGF-ß–binding proteins are important in the secretion of TGF-ß and in targeting it to ECM, whereas insulin-like growth factor 2 receptor and thrombospondins are involved in activation of latent TGF-ß by its release from latent TGF-ß–binding protein and removal of the latency-associated peptide (25, 26). Also increased were levels of accessory TGF-ß receptors endoglin and betaglycan; the latter may concentrate TGF-ß at the cell surface and stabilize it in a conformation optimal for binding to the signaling receptors (27). In contrast, the transcription of decorin, an ECM protein that binds TGF-ß and presumably inhibits its effects (28), was found to decrease. These data suggested that TRAIL might exert its activity via TGF-ß–mediated autocrine and/or paracrine stimulation of fibroblasts.

Involvement of the TGF-ß1 Pathway in Fibroblast Response to TRAIL
To further address the question of whether the effects of TRAIL on fibroblasts might be mediated by the TGF-ß pathway, TGF-ß1 gene expression was analyzed by Northern blotting (Figure 5) . TRAIL was found to upregulate the level of TGF-ß1 transcript, with the peak response at 0.1 ng/ml TRAIL. The level of total active TGF-ß1 in culture supernatants was also shown to increase upon treatment with TRAIL (Figure 6) . In addition, pan-specific TGF-ß antibody was tested for its ability to neutralize the activity of TRAIL (Figure 7) . The {alpha}2(I) collagen mRNA expression and total soluble collagen secretion were markedly decreased in the presence of anti–TGF-ß, but not control antibody. These observations substantiate the existence of a hitherto unknown molecular crosstalk between the TRAIL and TGF-ß pathways.



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  		Figure 5. Northern analysis of TGF-ß1 gene expression in TRAIL-stimulated fibroblasts. The amount of TGF-ß1 mRNA was compared in fibroblasts (line 9F0892) treated for 24 h with titrated doses of TRAIL plus crosslinking antibody. Blots were hybridized with probes for TGF-ß1 and GAPDH to correct for the loading difference. (A) Representative Northern blot. (B) Quantitative analysis of blot densities. The TGF-ß1 band intensities were normalized to the correspondent GAPDH band intensities and presented as means of triplicates ± SD. *P < 0.05 by unpaired t test, TRAIL plus crosslinking antibody versus antibody alone.

 


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  		Figure 6. TRAIL increases the TGF-ß1 protein level in fibroblast culture supernatants. Quiescent fibroblasts (line 9F0892) were treated for 24 h with titrated doses of TRAIL plus crosslinking antibody. The level of TGF-ß1 in acid-activated supernatants was measured using the TGF-ß1 Quantikine Assay. The data are presented as mean ± SD of triplicate cultures. *P < 0.05 by unpaired t test, TRAIL plus crosslinking antibody versus antibody alone.

 


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  		Figure 7. Anti–TGF-ß antibody inhibits TRAIL-stimulated collagen expression. Quiescent fibroblasts (line 9F0892) were treated with 10 ng/ml TRAIL in the presence of crosslinking antibody and pan-specific TGF-ß or control antibody. The amount of {alpha}2(I) collagen mRNA was determined in 6 h by real-time PCR and normalized to ribosomal protein S9 mRNA in the same sample. Total soluble collagen was measured in another set of fibroblasts in 24 h, using the Sircol assay. The data are presented as mean ± SEM of triplicate cultures. *P < 0.05; **P < 0.01, by unpaired t test. The inhibition of collagen expression by anti–TGF-ß antibody was observed in three independent experiments. Open bars, mRNA; filled bars, protein.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, soluble multimerized TRAIL at concentrations >= 100 ng/ml was found to induce apoptotic death in normal human lung fibroblasts. This observation differs from other papers that reported the resistance of normal human lung fibroblasts to cytotoxic activity of TRAIL (5, 9). The discrepancy may reflect a difference in TRAIL oligomerization or in the assessment of cell viability. Walczak and coworkers (5) used leucine zipper form of TRAIL and crystal violet assay for cell viability; Zhang and colleagues (9) assessed cell viability by propidium iodide staining, but did not apply crosslinking of TRAIL. Furthermore, fibroblasts may represent a heterogeneous population of cells (29, 30) with different sensitivity to TRAIL-induced apoptosis.

However, the estimated level of soluble TRAIL in human plasma is ~ 1 ng/ml (3, 31). At this concentration TRAIL was found to stimulate fibroblasts for ECM production. The dual signaling programs have also been reported for CD95 engagement in normal human fibroblasts, with cellular proliferation being initiated by CD95 aggregation at low levels, whereas apoptosis is initiated at high levels (18). CD95 ligand has also been shown to be a potent chemoattractant for human neutrophils at concentrations incapable of inducing cell apoptosis (32).

A potential link between the anti-apoptotic and collagen expression regulatory pathways may be provided by protein kinase JNK. DNA microarray analysis of fibroblasts treated with TRAIL revealed the augmented transcription of JNK1; it also has been found to be activated during cell protection from TRAIL-induced apoptosis (15). On the other hand, JNK provides a major regulatory step in activation of transcription factor AP-1 (33), which, in turn, has been shown to regulate the mouse and human {alpha}2(I) collagen promoter activity (23, 34). The elevated expression of transcription factor c-Fos, a member of AP-1 family, was also observed in DNA microarrays (Figure 4). Other factors such as NF-{kappa}B may additionally contribute to the TRAIL-induced collagen upregulation, although in comparison with tumor necrosis factor, TRAIL is a very weak NF-{kappa}B activator (35).

However, the microarray data should not be overinterpreted. Some degree of crosshybridization might be assumed among various collagen cDNA spots as well as among zinc finger protein cDNA spots. Although microarray approach may provide a new insight into the complex molecular mechanisms, the results of this analysis should be complemented by alternative methods.

Microarray data suggested that TRAIL might stimulate collagen expression via the TGF-ß regulatory feedback loop. The increase in TGF-ß1 mRNA level was confirmed by Northern blot analysis and by measurements of total active TGF-ß1 in culture supernatants. In addition, pan-specific TGF-ß antibody was shown to inhibit TRAIL-stimulated collagen expression. TGF-ß has been reported to induce the expression of Fas-associated death domain factor-like interleukin-1–converting enzyme-inhibitory protein (36), which is a major cellular inhibitor of apoptosis induced by TRAIL (10, 37). Thus, a possible scenario includes the upregulation of TGF-ß to protect fibroblasts from TRAIL-induced apoptosis, but TGF-ß also confers the increase in ECM synthesis. Although TGF-ß alone is unlikely to maintain the elevated collagen production in normal or scleroderma fibroblasts (38), it might be able to augment the collagen upregulation induced by TRAIL. TRAIL expressed on bronchoalveolar CD8+ T cells also may contribute to lung fibroblast stimulation, leading to pulmonary fibrosis. Further studies are needed to elucidate the molecular details of these interactions, particularly the extent to which collagen expression is under the control of the TRAIL pathway itself. The functional synergy has also been reported between the TRAIL and interferon pathways in breast cancer cells, with TRAIL mediating induction of interferon-ß (39).

Taken together, these data indicate that TRAIL, at doses below the apoptosis-inducing threshold, can upregulate ECM production by fibroblasts. The mechanisms of this stimulation likely involve triggering the TGF-ß pathway of autocrine and paracrine activation of fibroblasts. If this process continues uncontrolled, it may contribute to the development of fibrosis, particularly in the lungs of patients with systemic sclerosis. The molecular crosstalk observed between TRAIL and TGF-ß signaling pathways may be the target for therapeutic intervention.


   Acknowledgments
 
The author wishes to thank Natalya Tsymbalyuk for technical assistance. This work was supported by a Type II Merit Review Award from the Department of Veterans Affairs.

Received in original form July 30, 2002

Received in final form August 27, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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