Published ahead of print on August 24, 2006, doi:10.1165/rcmb.2006-0132OC
American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 206-212, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0132OC
Imbalance of Receptor-Regulated and Inhibitory Smads in Lung Fibroblasts from Bleomycin-Exposed Rats
Anne V. Gonzalez,
Frédérique Le Bellego and
Mara S. Ludwig
Meakins-Christie Laboratories, McGill University Health Center, Montreal, Quebec, Canada
Correspondence and requests for reprints should be addressed to Mara S. Ludwig, Meakins-Christie Laboratories, 3626 St Urbain Street, Montreal, PQ, H2X 2P2 Canada. E-mail: mara.ludwig{at}mcgill.ca
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Abstract
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Transforming growth factor (TGF)- plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF- is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF- stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF- in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-, may contribute to the fibrotic phenotype characteristic of BLF.
Key Words: transforming growth factor • bleomycin • lung fibrosis
Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease, of unknown etiology, and for which there is currently no effective treatment (1, 2). IPF is characterized histologically by the presence of fibroblastic foci, which are sites of fibroblast/myofibroblast proliferation and extracellular matrix deposition (3). The chemotherapeutic agent bleomycin has been used to induce an experimental model of lung fibrosis in rodents. Although imperfect, the model shares several features with IPF, including heterogeneous distribution of fibrosis, accumulation of extracellular matrix molecules, and differentiation of fibroblasts into a myofibroblast phenotype (3, 4).
Transforming growth factor (TGF)- is a multifunctional cytokine, with an essential role in wound healing and tissue repair after injury (5). TGF-, in particular the TGF-1 isoform, has been widely implicated in tissue fibrosis (6). In IPF, TGF-1 is increased at sites of extracellular matrix gene expression (7). Overexpression of TGF-1 along alveolar surfaces in experimental rats results in severe and prolonged pulmonary fibrosis (8). Conversely, inhibition of TGF- by antibodies, soluble receptors, or expression of the proteoglycan decorin markedly diminishes the fibrotic response to bleomycin (9–11).
TGF- isoforms bind to transmembrane serine/threonine kinase receptors and activate intracellular mediators known as Smads. TGF- first binds to the TGF- type II receptor, resulting in phosphorylation of the type I receptor. The TGF- type I receptor then phosphorylates the receptor-regulated (R-Smad) Smad2 or Smad3. Once activated, the R-Smads associate with the "common" Smad (co-Smad) Smad4. This complex translocates to the nucleus, where transcriptional responses are elicited on many target genes, either directly or through interaction with transcription factors. The inhibitory Smad7 prevents R-Smad phosphorylation by binding to the type I receptor, and constitutes an auto-regulatory loop in TGF- signal transduction (12, 13).
We have conducted studies in the bleomycin rat model to investigate the role of TGF- and Smad signaling in the fibrotic process. Myofibroblasts isolated from bleomycin-exposed animals demonstrated increased production of TGF- and enhanced secretion of the extracellular matrix constituents, proteoglycans (14). Experiments in whole lung from animals exposed to bleomycin showed that Smad expression was altered. Using cytosolic and nuclear lung tissue extracts, we found increased nuclear levels of Smad3 at days 7 and 14, increased nuclear levels of phosphorylated Smad2/3 at days 7, 14, and 28 and decreased cytosolic levels of Smad7 at days 7, 14 and 28 after bleomycin exposure (15).
Fibroblasts and myofibroblasts are the putative cells responsible for extracellular matrix production in the fibrotic process (14, 16). Based on our previous findings, we postulated that myofibroblasts were the key cell involved in the altered expression of Smads observed in the bleomycin-exposed animals. We hypothesized that an imbalance between receptor-regulated and inhibitory Smads, and an altered response to TGF-, could explain the enhanced fibrotic reaction typical in this model. Therefore, we isolated fibroblasts from rat lungs 14 d after bleomycin or saline exposure, and measured the expression of Smad signaling molecules at baseline and after TGF-1 stimulation.
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MATERIALS AND METHODS
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Materials
Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco, Invitrogen Corporation (Burlington, ON, Canada). LabTekII chamber glass slides were obtained from Nalge Nunc International (Naperville, IL). Hoechst 33258 came from Molecular Probes (Eugene, OR) and PermaFluor from Thermo Electron Corporation (Pittsburgh, PA). The protease inhibitor cocktail (Complete Mini) was from Roche (Laval, PQ, Canada), and the BCA Protein Assay Kit was obtained from Pierce (Rockford, IL). Bleomycin was obtained from Bristol-Myers Squibb (Princeton, NJ). Recombinant Human TGF-1 was purchased from R&D Systems (Minneapolis, MN). Antibodies were obtained from the following sources: vimentin and  -smooth muscle actin (-SMA) antibodies from Sigma (St. Louis, MO); p-Smad2/3 (sc-11769-r) and Smad7 (sc-7004) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); p-Smad3 (#9514) from Cell Signaling (Beverly, MA); Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG from Molecular Probes (Burlington, ON, Canada). The p-Smad2/3 antibody is a rabbit polyclonal antibody that recognizes both phosphorylated Smad2 and Smad3, with greater affinity for Smad3. The Smad7 antibody is a goat polyclonal antibody, raised against a peptide of Smad7 of human origin, and that also recognizes Smad7 of rat origin. The p-Smad3 antibody is a rabbit polyclonal antibody, raised against a synthetic phospho-peptide corresponding to residues surrounding Ser433 and Ser435 of human origin. PVDF membranes (Hybond-P) and ECL plus were purchased from Amersham Biosciences (Baie d'Urfé, PQ, Canada). RNA isolation kit (RNeasy kit) and Smad7 primers (QuantiTect primer assay QT00188699) were purchased from Qiagen (Mississauga, ON, Canada). SuperScript II reverse transcriptase, RNase inhibitor, DTT, dNTPs, and 5X FS buffer were all from Invitrogen. Actinomycin D was obtained from Sigma.
Experimental Design
Pulmonary fibrosis was induced in male Sprague-Dawley rats by a single intratracheal instillation of 1.5 units of bleomycin in 0.3 ml saline. Control rats received an equal volume of saline only. The animals were killed at 14 d by exsanguination under pentobarbital sodium anesthesia. After perfusion with sterile PBS, the lungs were excised. All animal experiments were performed in accordance with institutional guidelines (McGill University, Montreal, PQ, Canada).
Primary Rat Lung Fibroblast Culture
Cultures of rat lung fibroblasts were established by enzymatic dissociation of finely minced lung tissue as previously described (14). Fibroblasts were cultured at 37°C in a 5% CO2 incubator, in DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin. Experiments were performed with fibroblasts (n = 3 for both BLF and NLF) between the fifth and eighth passages.
Immunofluorescence Imaging
Immunofluorescent staining was used to detect vimentin, -SMA, and expression of p-Smad2/3 in BLF and NLF. Fibroblasts were seeded at 5,000 cells per well and grown to subconfluence on 8-well LabTekII chamber glass slides. Staining was performed in baseline conditions and after overnight serum starvation followed by stimulation with TGF-1 (10 ng/ml) for 30 min. Cells were washed in PBS and fixed in ice-cold methanol for 10 min at –20°C. After blocking with 20% normal goat serum in PBS for 1 h at room temperature, cells were incubated with the antibody against vimentin, -SMA, or p-Smad2/3 (10 µg/ml) overnight at 4°C. The cells were washed in PBS and incubated with fluorescent goat anti-mouse or goat anti-rabbit IgG for 1 h at room temperature. After washing the cells again in PBS, the nuclei were stained with Hoechst, and the slides were mounted with PermaFluor. Negative controls were processed in a similar fashion, except incubation with the primary antibody was omitted. The slides were examined using the BX51 Olympus fluorescence microscope attached to a CoolSNAP-Pro color digital camera (Carsen Group, Markham, ON, Canada).
Western Blot Analysis
Whole cell lysates were prepared at baseline, and after stimulation with TGF-1 (10 ng/ml) at the following time points: 0, 30 min, 1 h, 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h. The lysis buffer consisted of 150 mM NaCl, 10 mM Tris, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate, to which a protease inhibitor cocktail was added. Protein concentration was determined by the Bradford technique. Equal amounts of protein (20–30 µg) were resolved in a 10% polyacrylamide gel and transferred to PVDF membranes overnight at 30 V. Membranes were blocked with 5% dry milk in Tris-buffered saline with 0.1% Tween20 (TBST), or 5% bovine serum albumin (BSA) in TBST before incubation with the p-Smad3 antibody. Membranes were then immunoblotted with primary antibodies (p-Smad3 and Smad7) at a dilution of 1:1,000 in 5% BSA in TBST for 1 h at room temperature. After washing with TBST, the membranes were incubated with a 1:10,000 dilution of horseradish peroxidase–conjugated anti-goat or anti-rabbit antibody. Detection was performed using enhanced chemiluminescence. To confirm equal protein loading and transfer, membranes were stripped (in a buffer containing 62.5 mM Tris HCl pH 6.7, 2% SDS, and 100 mM -mercaptoethanol) and reprobed using anti-actin antibodies. Densitometric analysis was accomplished with an image analyzer software, the Fluorchem FC800 system (Alpha Innotech, San Leandro, CA). Density values for Smad levels were normalized to the actin values.
RNA Extraction and Real-Time RT-PCR
Changes in Smad7 mRNA levels after TGF-1 stimulation were measured by real-time RT-PCR. In addition, Smad7 mRNA stability was assessed. After stimulation with TGF-1 for 1 h, Actinomycin D (5 µg/ml) was added to the lung fibroblast cultures and RNA extraction performed at times 0, 0.5, 1, 1.5, 2, 4, 6, and 8 h. Total RNA was extracted using the Qiagen RNA isolation kit, according to the manufacturer's instructions. Reverse transcription of 500 ng of total RNA was performed in a total volume of 20 µl consisting of SuperScript II reverse transcriptase, RNase inhibitor, 0.1M DTT, dNTPs, 5X FS buffer, and RNase-free water. One microliter of cDNA was PCR-amplified in a reaction volume of 20 µl containing QuantiTect Smad7 primers, SYBR Green Supermix and RNase-free water. One microliter of cDNA was also amplified for ribosomal S9 RNA, using S9 primers at 500 nM and a similar reaction mixture. PCR was performed for 41 cycles, consisting of 94°C for 15 s followed by 60°C for 30 s and 72°C for 23 s, using the LightCycler (Roche). Standard curves for quantification of Smad7 and S9 cDNA were constructed by serial 10-fold dilution of purified cDNA. Quantification of the amplified products was performed by melting curve analysis, with data normalized to S9 RNA. PCR mixtures without cDNA were included in all reactions to exclude possible contamination.
Statistical Analysis
All values are reported as means ± SE of three observations. Student's unpaired t test was used to analyze the differences in p-Smad3 and Smad7 levels in NLF and BLF at baseline. One-way ANOVA was used to analyze changes in p-Smad3 and Smad7 mRNA and protein in response to TGF- in the two groups, followed by Dunnett's multiple comparison test. Differences were considered statistically significant at a P level of 0.05.
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RESULTS
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Characterization of Isolated Lung Fibroblasts
Phenotypic characterization of NLF and BLF by immunofluorescence has been previously described (14). Both NLF and BLF stained positively for vimentin (a fibroblast marker), with no discernable difference in the staining pattern. In contrast, only weak staining was observed for -SMA (a smooth muscle marker) in NLF, while the major proportion of BLF stained positively for -SMA, reflecting differentiation into a myofibroblast phenotype (data not shown).
Immunofluorescence Detection of p-Smad2/3 in BLF versus NLF
Immunofluorescence microscopy was used to detect levels of p-Smad2/3 in BLF and NLF. At baseline, staining for p-Smad2/3 was increased in BLF compared with NLF (Figures 1A and 1B). The immunofluorescence signal was concentrated in the nucleus, as evidenced by counter-staining with Hoechst (Figures 1C and 1D). Stimulation with TGF-1 (10 ng/ml) for 30 min resulted in a similar increase in nuclear staining of p-Smad2/3 in both BLF and NLF (Figures 1E and 1F).
Baseline Smad Expression in BLF versus NLF
Western blot analysis was used for comparison of the levels of p-Smad3 and Smad7 in BLF and NLF. At baseline, levels of p-Smad3 were increased  3-fold in BLF compared with NLF (Figure 2A, P < 0.05). In contrast, baseline levels of the inhibitory Smad7 were comparable in BLF and NLF (Figure 2B). We calculated the ratio of p-Smad3/Smad7 (both values normalized to actin) in BLF and NLF. The ratio was 4-fold greater in BLF compared with NLF (Figure 2C, P < 0.05). We questioned whether direct exposure of fibroblasts to bleomycin could lead to activation of p-Smad3. After incubation of NLF with bleomycin (1, 10, and 100 µg/ml for 24 or 48 h), no significant changes in p-Smad3 levels were observed (data not shown).
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Figure 2. Baseline levels of p-Smad3 (A) and inhibitory Smad7 (B) in BLF and NLF. Ratio of normalized p-Smad3 to Smad7 levels in BLF and NLF (C). Western blot analysis and quantification were performed on whole cell lysates. Equal protein loading was confirmed by normalizing for actin values. Data represent mean ± SE of two separate experiments, n = 3 in each group. *P < 0.05.
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Levels of p-Smad3 in Response to TGF-1
We examined the response of BLF and NLF to stimulation with TGF-1 at various time points. Levels of p-Smad3 were significantly increased in both BLF and NLF, 30 min and 1 h after stimulation with TGF-1 10 ng/ml (Figures 3A and 3B, P < 0.01 and P < 0.05). Comparison of the response in BLF versus NLF revealed no significant difference in p-Smad3 levels after TGF- stimulation. Within 24 h of stimulation, levels of p-Smad3 returned to baseline levels, with BLF returning to their higher initial value.
Regulation of Smad7 in Response to TGF-1
We questioned whether the regulation of Smad7 in response to TGF- would be altered in BLF versus NLF. Levels of Smad7 mRNA were measured at baseline and 1, 2, 6, and 24 h after stimulation with TGF-1 (10 ng/ml). Baseline levels of Smad7 mRNA were comparable in BLF and NLF. After stimulation with TGF-1, levels of Smad7 mRNA were rapidly upregulated in both BLF and NLF, with a maximal increase 1 h after TGF- (Figure 4A, P < 0.01 for BLF and P = 0.05 for NLF). Levels of Smad7 mRNA at 1 h after TGF- were not significantly different between BLF and NLF. The decay of Smad7 mRNA, after TGF- stimulation, was evaluated by blocking further transcription with Actinomycin D. Stability of Smad7 mRNA was comparable in BLF and NLF, with a half-life of 30 min (Figure 4B). Smad7 protein levels were measured after TGF- stimulation at various time points (12, 18, 24, 36, and 48 h). There was a significant increase in Smad7 protein at 24, 36, and 48 h after TGF- stimulation in NLF only (Figures 5A and 5B, P < 0.05, one-way ANOVA).
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DISCUSSION
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Pulmonary fibrosis remains a difficult clinical problem, with limited therapeutic options. Progress has been made, over the past decade, in defining the various clinical entities that constitute the idiopathic interstitial pneumonias (3). A paradigm shift has occurred, from viewing lung fibrosis as an inflammatory process culminating in fibrosis, to recognizing the process as constituting "abnormal wound healing" (17). Much remains to be understood about the molecular mechanisms underlying this exuberant fibrogenesis. TGF- is known to be a key mediator of tissue fibrosis at multiple sites (5). We previously examined changes in Smad expression, critical mediators of TGF- signaling, in whole lung tissue extracts from bleomycin-exposed rats (15). In the current experiment, we sought to elucidate the patterns of Smad expression in isolated lung fibroblasts, the putative effector cells in pulmonary fibrosis, after in vivo exposure to bleomycin.
We found that baseline levels of phosphorylation of the receptor-regulated Smad3 were increased in BLF compared with NLF. Despite this increased activation of TGF- signaling, levels of the inhibitory Smad7 were comparable between the two groups of fibroblasts. This finding suggests an imbalance between positive and negative intracellular mediators of TGF- signaling in lung fibroblasts, after bleomycin exposure. We also examined changes in the levels of p-Smad3 and Smad7 in response to TGF-1 stimulation. While the pSmad3 response was similar in BLF and NLF, changes in Smad7 protein were more substantial in NLF.
Some aspects of the study design warrant further discussion. We isolated lung fibroblasts at a single time point after bleomycin exposure. We chose this time point based on the findings of previous studies done in our laboratory. Ebihara and coworkers reported on the temporal changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis (18). Maximal increases in resistance and elastance were observed at 14 d after bleomycin exposure. Concurrently, increases in the proteoglycans versican, heparin sulfate proteoglycan (HSPG), biglycan, and fibromodulin measured in whole lung extracts were also maximal (18). Venkatesan and colleagues examined the expression of proteoglycans in rat lung fibroblasts 14 d after in vivo bleomycin exposure, and again detected increased production of proteoglycans in BLF compared with NLF. In addition, in the whole lung tissue experiments, Smad signaling was significantly altered 14 d after bleomycin exposure (15). We performed experiments with fibroblasts between the fifth and eighth passages. Phan and coworkers previously reported increased collagen synthesis in lung fibroblasts isolated from bleomycin-treated rats compared with controls; differences in collagen production persisted at the same level until the tenth passage (16). The limitations of the intratracheal bleomycin model of lung fibrosis have been recently emphasized by Borzone and colleagues (19). While the bleomycin model is not equivalent to IPF, it reproduces key morphologic features of pulmonary fibrosis, and remains a critical model both for the elucidation of fibrogenesis mechanisms and assessment of potential therapies (4, 20).
Upon binding of TGF- to its receptor, the receptor-regulated Smad2 and Smad3 become phosphorylated and translocate to the nucleus, where the promoters of various target genes are activated either directly or through interaction with a variety of transcription factors (12, 13). We chose to examine the activation of Smad3 more specifically, because of mounting evidence that Smad3 is a critical mediator of the fibrotic response (21). The nonredundant functions of Smad2 and Smad3 are reflected in significant differences in their respective knockouts: Smad2-null mice do not survive beyond embryogenesis, whereas Smad3-null mice are viable but exhibit defects in mucosal immunity and die at 1–6 mo of age (21). Among the target genes activated by TGF- are a variety of extracellular matrix genes, including COL1A1 and COL1A2 (22), and the dependence of this process on Smad3 has been demonstrated in dermal fibroblasts (22, 23). The high levels of p-Smad3 found at baseline in BLF, in the current experiment, may therefore contribute to their fibrogenic phenotype.
Smad7 is a TGF-–inducible antagonist of TGF- signaling that was initially identified in 1997 by Nakao and coworkers (24). Upon activation of the TGF- receptor, Smad7 mRNA is rapidly up-regulated, and down-regulation of TGF- signaling occurs via two mechanisms. Smad7 binds the activated TGF- receptor and prevents phosphorylation of the receptor-regulated Smads. In addition, Smad7 recruits ubiquitin ligases to the TGF- type I receptor, with subsequent protein degradation (13). In the current experiment, we describe baseline levels of p-Smad3 3 times higher in BLF versus NLF. This is consistent with the increased secretion of TGF-1 by bleomycin-exposed fibroblasts we reported in a previous experiment (14), and suggests that autocrine TGF-1 signaling by BLF may be contributing to the maintenance of abnormally elevated baseline levels of p-Smad3. However, despite this increased activation of TGF- signaling, we found similar levels of Smad7 in BLF compared with NLF. The overall state of activation of the cell, as determined by the ratio of p-Smad3 to Smad7, therefore favored positive intracellular signaling of TGF-.
These results are consistent, in part, with our previous findings in whole lung extracts, where at 14 d after bleomycin exposure we found an 2-fold increase in nuclear phosphorylated Smad2/3, but decreased cytoplasmic expression of Smad7 (15). The increased levels of p-Smad3 are comparable between the two studies; the modest difference likely relates to the fact that the nuclear extracts used in the previous study were derived from all lung cell types, not just fibroblasts. In contrast, we found comparable baseline levels of Smad7 mRNA and protein in BLF and NLF, not the paradoxical decrease in Smad7 we previously observed in cytoplasmic whole lung extracts, at 14 d after bleomycin exposure. We cannot rule out that another cell type involved in the early response to bleomycin contributed to this decrease in Smad7. We chose to examine fibroblasts in the current experiment, as this is the putative cell type responsible for enhanced matrix deposition in lung fibrosis. However, it is likely that other cells (e.g., epithelial cells) also contribute to the disordered expression of Smads seen in the whole lung. We are unaware of data on this question in either IPF or the bleomycin model of lung fibrosis, but there is some information available from airway biopsies in patients with asthma. Nakao and colleagues (25) showed decreased Smad7 immunoreactivity in epithelial cells of bronchial biopsies from remodeled asthmatic airways as compared with control airways. Further, the expression of Smad7 in bronchial epithelial cells was inversely correlated to basement membrane thickness and airway hyperresponsiveness.
The concept of an imbalance between receptor-regulated and inhibitory Smads has been reported in other fibrotic disease processes. Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix, resulting in progressive cutaneous and visceral fibrosis. Dong and coworkers (26) obtained dermal fibroblasts from patients with scleroderma and from control subjects, and found decreased basal Smad7 expression in scleroderma fibroblasts. In contrast, Smad3 expression was increased, as was phosphorylation of Smad2 and 3. In a murine model of scleroderma induced by subcutaneous injections of bleomycin, increased phosphorylation of Smad2/3 with relative Smad7 deficiency was also noted (27). Fukasawa and colleagues (28) noted diminished levels of Smad7 protein in a model of progressive renal fibrosis induced by unilateral ureteral obstruction, while immunoreactivity for nuclear phosphorylated Smad2 and 3 and fibrosis were increased. Decreased Smad7 expression has also been shown to contribute to myocardial fibrosis in the infarcted rat heart (29). In contrast, relatively little is known about changes in Smads in fibrotic pulmonary disease.
We examined changes in p-Smad3 and Smad7 after stimulation with TGF-. We found rapid increases in p-Smad3 in BLF and NLF, peaking at 30 min after TGF- in both groups of fibroblasts. We suspect phenotypic differences between BLF and NLF relate to baseline activation of TGF- signaling in BLF, rather than differential response to TGF-, per se. We also examined changes in Smad7 after TGF- at the mRNA and protein level. Smad7 is known to be rapidly induced in response to TGF- stimulation. Inadequate upregulation of Smad7 in response to TGF- could result in a deficient down-regulation of TGF- signaling and persistent cellular activation. Using real-time PCR, we found rapid increases in Smad7 mRNA in both BLF and NLF, suggesting that the auto-regulatory response to TGF- persisted despite insufficient levels of Smad7 at baseline in BLF. In addition, the stability of the induced Smad7 mRNA was comparable in BLF and NLF. This is in contrast to the results of Dong and associates (26), who showed decreased induction of Smad7 mRNA in response to TGF- in dermal fibroblasts isolated from patients with scleroderma versus control subjects. In the current study, the Smad7 protein response to TGF- was diminished in BLF compared with NLF. This finding suggests that a defect in the post-transcriptional regulation of Smad7 may be present in BLF. Increased ubiquitin-dependent degradation of Smad7, as described by Fukasawa and colleagues (28) in a model of renal fibrosis, could also contribute to the inadequate levels of Smad7 protein. Further investigation of the regulation of Smad7 in response to TGF- in this model is warranted.
Modulation of Smad signaling holds promise as a therapeutic modality for pulmonary fibrosis. It represents an alternative to systemic blockade of TGF-, which while effective in inhibiting fibrosis, would almost certainly result in unacceptable toxicity (30). Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis, making it a potential target for antifibrotic therapy (31). Transient gene transfer and overexpression of Smad7 was shown to prevent bleomycin-induced pulmonary fibrosis in mice (32). Overexpression of Smad7 also inhibits the fibrotic effect of TGF- on renal tubular epithelial cells, and prevents liver fibrosis in rats (33, 34). Halofuginone, a low-molecular-weight plant alkaloid, rapidly induces expression of Smad7 mRNA and reduces TGF-–induced phosphorylation of Smad2 and 3 (35). Halofuginone has been shown to ameliorate radiation-induced fibrosis, and one study has demonstrated reduction in bleomycin-induced pulmonary fibrosis (36). A novel, orally active TGF- receptor I kinase inhibitor was recently shown to inhibit the initiation and progression of lung fibrosis induced by adenovirus-mediated gene transfer of TGF-1 (37). Interestingly, short-term drug administration halted the progression of fibrosis, suggesting that a similar approach could be effective in treating chronic fibrosis while limiting the toxicity associated with long-term systemic inhibition of TGF-.
In conclusion, we have demonstrated that lung fibroblasts, isolated from rats after exposure to bleomycin, demonstrate an imbalance between the level of activation of the receptor-regulated Smad3 and the inhibitory Smad7. Insufficient negative feedback on TGF- signaling by Smad7 is likely to contribute to maintenance of a pro-fibrotic phenotype in these cells. Modulation of intracellular TGF- signaling represents a potential therapeutic target for pulmonary fibrosis.
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Footnotes
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This work was supported by Canadian Institutes of Health Research, the J T Costello Memorial Research Fund, and a CIHR/CLA/Glaxo Fellowship (A.V.G.).
Originally Published in Press as DOI: 10.1164/rccm.2006-0132OC on August 24, 2006
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
Received in original form April 3, 2006
Accepted in final form August 3, 2006
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References
|
|---|
- Gross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J Med 2001;345:517–525.[Free Full Text]
- Idiopathic pulmonary fibrosis: diagnosis and treatment. Am J Respir Crit Care Med 2000;161:646–664.[Free Full Text]
- ATS/ERS international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2002;165:277–304.[Free Full Text]
- Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol 2005;33:9–13.[Abstract/Free Full Text]
- Blobe GC, Schirmann WP, Lodish HF. Role of transforming growth factor in human disease. N Engl J Med 2000;342:1350–1358.[Free Full Text]
- Bartram U, Speer CP. The role of transforming growth factor in lung development and disease. Chest 2004;125:754–765.
- Broekelmann TJ, Limper AH, Colby TV, McDonald JA. Transforming growth factor 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991;88:6642–6646.[Abstract/Free Full Text]
- Sime PJ, Xing Z, Graham FL, Gauldie J. Adenoviral-mediated gene transfer of active TGF-1 induces prolonged aggressive fibrosis in rat lung. J Clin Invest 1997;100:768–776.[Medline]
- Giri SN, Hyde DM, Hollinger MA. Effect of antibody to transforming growth factor-beta on bleomycin-induced accumulation of lung collagen in mice. Thorax 1993;48:956–966.
- Wang Q, Wang Y, Hyde DM, Gotwals PJ, Koteliansky VE, Ryan ST, Giri SN. Reduction of bleomycin-induced lung fibrosis by TGF- soluble receptor in hamsters. Thorax 1999;54:805–812.[Abstract/Free Full Text]
- Kolb M, Margetts PJ, Galt T, Sime PJ, Xing Z, Schmidt M, Gauldie J. Transient transgene expression of decorin in the lung reduces the fibrotic response to bleomycin. Am J Respir Crit Care Med 2001;163:770–777.[Abstract/Free Full Text]
- Heldin CH, Miyazono K, ten Dijke P. TGF- signalling from cell membrane to nucleus through Smad proteins. Nature 1997;390:465–471.[CrossRef][Medline]
- Shi Y, Massagué J. Mechanisms of TGF- signaling from cell membrane to the nucleus. Cell 2003;113:685–700.[CrossRef][Medline]
- Venkatesan N, Roughley PJ, Ludwig MS. Proteoglycan expression in bleomycin lung fibroblasts: role of transforming growth factor-1 and interferon-
 . Am J Physiol Lung Cell Mol Physiol 2002;283:L806–L814.[Abstract/Free Full Text] - Venkatesan N, Pini L, Ludwig MS. Changes in Smad expression and subcellular localization in bleomycin-induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol 2004;287:L1342–L1347.[Abstract/Free Full Text]
- Phan SH, Varani J, Smith D. Rat lung fibroblast collagen metabolism in bleomycin-induced pulmonary fibrosis. J Clin Invest 1985;76:241–247.[Medline]
- Selman M, King TE Jr, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151.[Abstract/Free Full Text]
- Ebihara T, Venkatesan N, Tanaka R, Ludwig MS. Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Am J Respir Crit Care Med 2000;162:1569–1576.[Abstract/Free Full Text]
- Borzone G, Moreno R, Urrea R, Meneses M, Oyarzún M, Lisboa C. Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001;163:1648–1653.[Abstract/Free Full Text]
- Snider GL, Celli BR, Goldstein RH, O'Brien JJ, Ludey EC. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 1978;117:289–297.[Medline]
- Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 2004;85:47–64.[CrossRef][Medline]
- Verrecchia F, Chu ML, Mauviel A. Identification of novel TGF-/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 2001;278:17058–17062.
- Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-: involvement of Smad3. J Invest Dermatol 1999;112:49–57.[CrossRef][Medline]
- Nakao A, Afrakhte M, Morén A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, et al. Identification of Smad7, TGF- inducible antagonist of TGF- signalling. Nature 1997;389:631–635.[CrossRef][Medline]
- Nakao A, Sagara H, Setoguchi Y, Okada T, Okumura K, Ogawa H, Fukuda T. Expression of Smad7 in bronchial epithelial cells is inversely correlated to basement membrane thickness and airway hyperresponsiveness in patients with asthma. J Allergy Clin Immunol 2002;110:873–878.[CrossRef][Medline]
- Dong C, Zhu S, Wand T, Yoon W, Li Z, Alvarez RJ, ten Dijke P, While B, Wigley FM, Goldschmidt-Clermont PJ. Deficient Smad7 expression: a putative molecular defect in scleroderma. Proc Natl Acad Sci USA 2002;99:3908–3913.[Abstract/Free Full Text]
- Takagawa S, Lakos G, Mori Y, Yamamoto T, Nishioka K, Varga J. Sustained activation of fibroblast transforming growth factor-/Smad signaling in a murine model of scleroderma. J Invest Dermatol 2003;121:41–50.[CrossRef][Medline]
- Fukasawa H, Yamamoto T, Togawa A, Ohashi N, Fujigaki Y, Oda T, Uchida C, Kitagawa K, Hattori T, Suzuki S, et al. Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice. Proc Natl Acad Sci USA 2004;101:8687–8692.[Abstract/Free Full Text]
- Wang B, Hao J, Jones SC, Yee MS, Roth JC, Dixon IM. Decreased Smad7 expression contributes to cardiac fibrosis in the infarcted rat heart. Am J Physiol Heart Circ Physiol 2002;282:H1685–H1696.[Abstract/Free Full Text]
- Crystal RG, Bitterman PB, Mossman B, Schwarz MI, Sheppard D, Almasy L, Chapman HA, Frideman SL, King TE Jr, Leinwand LA, et al. Future research directions in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2002;166:236–246.[Abstract/Free Full Text]
- Zhao J, Shi W, Wang YL, Chen H, Bringas P Jr, Datto MB, Frederick JP, Wand XF, Warburton D. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2002;282:L585–L593.[Abstract/Free Full Text]
- Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Mihazono K, Iwamoto I. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999;104:5–11.[Medline]
- Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ, Lan HY. Smad7 inhibits fibrotic effect of TGF-beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol 2002;13:1464–1472.[Abstract/Free Full Text]
- Dooley S, Hamzavi J, Breitkopf K, Wiercinska E, Said HM, Lorenzen J, ten Dijke P, Gressner AM. Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 2003;125:178–191.[CrossRef]
- Xavier S, Piek E, Fujii M, Javelaud D, Mauviel A, Flanders KC, Samuni AM, Felici A, Reiss M, Yarkoni S, et al. Amelioration of radiation-induced fibrosis. Inhibition of transforming growth factor- signaling by halofuginone. J Biol Chem 2004;279:15167–15176.[Abstract/Free Full Text]
- Nagler A, Firman N, Feferman R, Cotev S, Pines M, Shoshan S. Reduction in pulmonary fibrosis in vivo by halofuginone. Am J Respir Crit Care Med 1996;154:1082–1086.[Abstract]
- Bonniaud P, Margetts PJ, Kolb M, Schroeder JA, Kapoun AM, Damm D, Murphy A, Chakravarty S, Dugar S, Higgins L, et al. Progressive Transforming Growth Factor 1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Cell Mol Biol 2004;171:889–898.
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MATERIALS AND METHODS
