Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

Jingsong Zhao1,*, Wei Shi2,*, Yan-Ling Wang1, Hui Chen1, Pablo Bringas Jr.1, Michael B. Datto3, Joshua P. Frederick3, Xiao-Fan Wang3, and David Warburton2

1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles 90033; 2 Departments of Surgery and Pediatrics, and the Cell and Developmental Biology Program, The Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027; and 3 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta ) signaling plays an important regulatory role during lung fibrogenesis. Smad3 was identified in the pathway for transducing TGF-beta signals from the cell membrane to the nucleus. Using mice without Smad3 gene expression, we investigated whether Smad3 could regulate bleomycin-induced pulmonary fibrosis in vivo. Mice deficient in Smad3 demonstrated suppressed type I procollagen mRNA expression and reduced hydroxyproline content in the lungs compared with wild-type mice treated with bleomycin. Furthermore, loss of Smad3 greatly attenuated morphological fibrotic responses to bleomycin in the mouse lungs, suggesting that Smad3 is implicated in the pathogenesis of pulmonary fibrosis. These results show that Smad3 contributes to bleomycin-induced lung injury and that Smad3 may serve as a novel target for potential therapeutic treatment of lung fibrosis.

transforming growth factor-beta ; hydroxyproline


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY FIBROSIS IS A DISEASE characterized by excessive mesenchymal cell proliferation and concomitant collagen accumulation within the alveolar and interstitial compartments of the lung (36). This inflammatory and fibroproliferative response to lung injury occurs via concerted interaction of various cytokines and growth factors within the tissue microenvironment (7). Among them, transforming growth factor-beta (TGF-beta ) is known to play an essential role in the pathogenesis of lung fibrosis (3). In animal models of pulmonary fibrosis, levels of TGF-beta mRNA and protein are increased (12, 15, 28).

TGF-beta is both mitogenic and chemotactic for fibroblasts, monocytes, and macrophages and promotes accumulation of extracellular matrix proteins by increasing their synthesis while inhibiting matrix degradation (3). Treatment of animals with TGF-beta antagonists, in the form of either neutralizing antibodies or the TGF-beta -binding proteoglycan decorin, prevented bleomycin-induced lung fibrosis in vivo, indicating that TGF-beta ligand is required for the development of pulmonary fibrosis (10, 11). Moreover, intratracheal instillation of TGF-beta -soluble type II receptor protein dramatically reduced bleomycin-induced lung fibrosis in hamsters (34), and exogenous overexpression of Smad7, an inhibitor in the TGF-beta signaling pathway, prevented bleomycin-induced lung fibrosis in mice (25).

The identification of the Smad family of signal transducer proteins has unraveled novel mechanisms by which TGF-beta receptors signal from the cell membrane to the nucleus (14). The activated TGF-beta receptor complex induces phosphorylation of Smad3 and its closely related homolog Smad2, which bind with the common mediator Smad4. These multisubunit Smad hetero-oligomers subsequently translocate into the nucleus, where they direct transcriptional activities to affect the cell's response to TGF-beta stimulation (22). Smad3 null mutant mice have been generated in different laboratories by targeted disruption of exon 1, or exon 2 or exon 8 in the Smad3 gene (8, 39, 43). All of these Smad3 knockout mice were viable and fertile although they were smaller than wild-type mice. The Smad3 null mutant mice with targeted disruption in exon 1 or exon 8 display forelimb malformation, impaired immunity, and diminished T cell responsiveness to TGF-beta (8, 39). However, only the Smad3 null mutant mice with target disruption in exon 2 became moribund with colorectal adenocarcinomas at the age of 4-6 mo (43). Therefore, Smad3 seems essential in many TGF-beta -mediated development and cell growth control processes.

Although Smad3 mediates intracellular signaling from TGF-beta ligands, which are potent fibrogenic molecules, the unique biological function of Smad3 during lung fibrogenesis has not been explored in vivo. In the present study, we tested the hypothesis that Smad3 regulates pulmonary fibrogenesis, using a bleomycin-induced model of interstitial lung fibrosis in mice, in which TGF-beta signaling is known to be required for the development of fibrotic injury (11, 18). Mice null mutant for Smad3 were thus chosen to evaluate the contribution of Smad3 protein to pulmonary fibrosis in vivo. We have found that the lack of Smad3 attenuated bleomycin-induced lung fibrosis in mice, suggesting that Smad3 plays a pivotal role during tissue injury that leads to lung fibrosis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Smad3 null mutant mice. Smad3 null mutant (-/-) mice were bred from C57BL/6 mice heterozygous for a targeted disruption of exon 1 of the Smad3 gene (8). The genotypes of both wild-type and Smad3-deficient mice were determined by PCR analysis on tail DNA obtained from 3-wk-old animals, as previously described (8). Mice were kept under specific pathogen-free conditions in the University of Southern California (USC) animal facility until use.

Bleomycin-induced lung injury. Female wild-type and Smad3 null mutant mice, 9 wk of age, were randomly selected for either bleomycin or saline vehicle control treatment. The average body weight for wild-type mice is ~20 g and for Smad3 knockout mice is ~15 g. Administration of bleomycin (Sigma, St. Louis, MO) or saline vehicle was performed by a constant subcutaneous infusion through a micro-osmotic pump (model 1007D; Alza, Palo Alto, CA) from days 0-7 (13). In mice anesthetized with pentobarbital sodium (30-40 µg/g ip), the minipump loaded with bleomycin sulfate (0.1 mg/g mouse body wt dissolved in saline) was implanted subcutaneously on the back of the mice, slightly posterior to the scapulae (38). All mice were monitored regularly and received food and water ad libitum. Mice were killed after 4 wk, and the pumps were examined to ensure that they had delivered the entire dosage in each mouse. The body weight after 4 wk of treatment slightly increased in wild-type mice treated with saline vehicle, and no significant changes of the body weight were observed for the rest of the experimental groups. Lungs were removed for immediate RNA extraction, measurements of hydroxyproline content, or fixation for histological analysis. The bleomycin dose used herein was shown to consistently produce pulmonary fibrosis with a low mortality rate (<10%) in preliminary experiments with mice of the same genetic background. Animal protocols used herein were approved by the USC Institutional Animal Care and Use Committee and were in accordance with the National Institutes of Health (NIH) guidelines for animal welfare.

Histology and immunohistochemistry. Mouse lungs were fixed by perfusion with 4% paraformaldehyde-PBS before routine processing and paraffin embedding. Coronal sections, 5-µm thick, were prepared and stained with hematoxylin and eosin for histological examination. Alternatively, lung sections were processed for Masson's trichrome stain, a specific histochemical stain for collagen and elastin (20).

Lung tissue sections were deparaffinized, and endogenous peroxidase was blocked. Sections to be stained for fibronectin were treated with blocking goat serum for 1 h and incubated overnight with the primary antibody. The rabbit polyclonal anti-fibronectin antibody (Sigma) was used at a dilution of 1:100. Biotinylated proliferating cell nuclear antigen (PCNA) primary antibody, biotinylated secondary antibody, and streptavidin-peroxidase conjugate were purchased from Zymed (South San Francisco, CA). The peroxidase reaction products were red colored with aminoethyl carbazole and dark brown with diaminobenzidine. Normal rabbit IgG, bovine serum albumin, and water were run in parallel slides as negative controls.

Histological scoring of lung fibrosis. For the quantitative morphological analysis of fibrotic changes in adult mouse lungs, a numerical fibrotic scale was used (31). Briefly, hematoxylin-eosin-stained lung sections were viewed under the microscope for identification of lesions. The resultant pulmonary lesions were defined as follows: 0, absence of lesion; 1, occasional small localized subpleural foci; 2, thickening of interalveolar septa and subpleural foci; and 3, thickened continuous subpleural fibrous foci and interalveolar septa. The severity of the fibrotic changes in each lung section was assessed as a mean score of severity from observed microscopic fields. More than 20 fields within each lung section were evaluated under high-power field using the above predetermined scale of severity. To prevent observer's bias, all histological specimens were coded and examined without knowledge of experimental conditions. Each specimen was scored independently by two or three observers.

RNA extraction, reverse transcription, and competitive PCR. Total RNA from adult mouse lungs was extracted by guanidinium thiocyanate following homogenization as we have documented previously (40). Extracted total RNA was immediately reverse transcribed by incubating at 37°C for 1 h in the presence of ribonuclease inhibitor, oligo(dT)12-18, and MMLV reverse transcriptase (GIBCO-BRL). The resultant cDNA products were used for competitive PCR quantification.

Competitive PCR methodology for specific mRNA quantification of pulmonary genes has been described in detail elsewhere (41). Briefly, a set of primers was designed for mouse type I procollagen alpha 1-chain [alpha 1(I)] to amplify a 210-bp cDNA fragment (24). To generate competitor cDNA for alpha 1(I) competitive PCR assay, the above desired primer sequences were engineered into a heterologous DNA fragment using same strategy as we documented earlier (40). Consequently, both alpha 1(I) cDNA and its competitor utilize the same set of primers in the alpha 1(I) competitive PCR. The alpha 1(I) competitor was 268 bp in length. Both alpha 1(I) and its competitor PCR products were subsequently DNA sequenced to verify their identities. Competitive PCR assays for both fibronectin and TGF-beta 1 were developed in a similar manner as that for alpha 1(I) (42).

PCR amplification was carried out using a modification of a previously described assay for TGF-beta type II receptor (40). Reverse-transcribed samples derived from 20-50 ng total RNA were added to a PCR reaction mixture containing a known amount of competitor to achieve a total volume of 50 µl. beta -actin-competitive PCR as an internal control was performed in parallel on the same assayed samples. As a negative control for genomic DNA, non-reverse-transcribed total RNA was also included in the competitive PCR assays.

Hydroxyproline quantification. Total hydroxyproline content of the left lung was measured as an assessment of lung collagen content. A spectrophotometric assay was used, with modification, to quantify lung hydroxyproline (30). Lung tissues were homogenized in ice-cold trichloroacetic acid. Precipitated pellets were hydrolyzed for 16 h at 110°C in 6 N HCL. After NaOH neutralization, hydrolysates were assayed colorimetrically with dimethylaminobenzaldehyde to quantify hydroxyproline. Purified hydroxyproline (Sigma) was used to set a standard. Hydroxyproline content was expressed as micrograms of hydroxyproline per left lung.

Analysis of inflammatory cell profile of bronchoalveolar lavage fluid. Wild-type and Smad3 -/- null mutant mice were treated with bleomycin or saline vehicle as described above. At posttreatment day 7, the mouse was killed and an 18G plastic catheter was cannulated into the trachea. Cold sterile PBS (1 ml) was used to inflate the lung, and the lavage fluid was recovered with ~80% of the original volume. Cells were then pelleted by centrifugation and resuspended in 1 ml of fresh PBS. The concentration of the cells was determined by counting cells in hemocytometer. A drop of cell suspension was used to make a cell smear on a Superfrost-coated glass slide. After Giemsa staining, macrophage/monocyte, lymphocyte, and neutrophil were counted under microscope, and the percentage of each type cell was calculated based on the total number of counted cells.

Data presentation and statistical analysis. All experiments were repeated at least three times, with similar results obtained within repetitive experiments. All data were expressed as means ± SD. A Student's t-test was used for comparison of statistical difference between any two experimental groups, and P values < 0.05 were generally considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Loss of Smad3 attenuates bleomycin-induced lung fibrosis in young adult mice. To determine the novel mechanism of Smad3 protein during lung injury, we used a well-recognized model of lung fibrosis induced by the antineoplastic antibiotic bleomycin (18, 19). Mouse lung fibrogenesis was induced by bleomycin released from an osmotic minipump subcutaneously implanted in both wild-type and Smad3-deficient mice, as described in MATERIALS AND METHODS.

Histological examination of lung specimens demonstrated that continuous subcutaneous bleomycin administration (0.1 mg/g for 7 days) induced multifocal fibrotic lesions in normal wild-type mouse lungs, primarily in the subpleural regions with thickened interalveolar septa (Fig. 1B), whereas a well-alveolized normal histology was seen in both wild-type and Smad3 knockout adult mice treated with saline vehicle (Fig. 1, A and C). However, when Smad3 null mutant mice were given bleomycin treatment, fibrotic lesions were much less severe in the subpleural areas, and only a slight degree of interstitial fibrogenesis was detected (Fig. 1D). Bleomycin-initiated pathological changes in mice were therefore reduced in the absence of Smad3 gene expression.


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Fig. 1.   Histopathological effects induced by bleomycin in wild-type and Smad3 null mutant lung tissues. Histological sections were all stained with hematoxylin and eosin. A: wild-type mice with saline control. B: wild-type mice with subcutaneous infusion of bleomycin. C: Smad3-deficient mice with saline vehicle treatment. D: Smad3-deficient mice with bleomycin administration. WT, Wild-type; KO, knockout; SV, saline vehicle; BL, bleomycin. Bar = 100 µm.

Because bleomycin-induced lung fibrosis could result from either increased lung inflammation or merely increased extracellular collagen production by fibroblast cells, we therefore studied the possible cellular mechanism underlying the phenotype that deficiency of Smad3 attenuated bleomycin-induced lung fibrosis. By examining inflammatory cell profiles of mouse lung bronchoalveolar lavage fluid (BALF) at day 7 post-bleomycin treatment, we found that the number of BALF cells was significantly increased in both wild-type and Smad3-deficient mouse lungs, although more of an increase of BALF cell number in wild-type mouse lungs than Smad3-deficient mouse lung was observed (data not shown). However, the inflammatory cell type profiles are comparable between wild-type and Smad3 deficient mouse BALF (Table 1). In both wild-type and Smad3 null mutant mice treated with saline vehicle control, lymphocyte was the major subpopulation in BALF (89.5 ± 2.1 and 91.5 ± 0.7%). After 7-day bleomycin treatment, macrophage and monocyte counts dramatically increased and became the major subpopulation in BALFs of both wild-type and Smad3 knockout mice (57 ± 5.7 and 59 ± 2.8%). Therefore, our results indicated that bleomycin-induced lung inflammatory responses in Smad3 null mutant mice were not significantly changed compared with that in wild-type mice and that attenuation of bleomycin-induced pulmonary fibrosis in Smad3 deficient mice is therefore unlikely to be due to reduction of inflammatory response in the lungs.

                              
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Table 1.   Inflammatory cell profile of bronchoalveolar lavage fluid

Lung fibrotic changes in both normal and Smad3-defective lungs were quantitatively examined by morphological scoring using the histological criteria detailed in MATERIALS AND METHODS. Histological scoring of lung fibrosis showed a significant suppression of bleomycin-induced fibrosis in Smad3-deficient mice compared with wild-type mice (Table 2): 2.88 ± 0.33 for bleomycin-treated wild-type and 0.57 ± 0.49 for bleomycin-treated Smad3 knockout mice (P < 0.05). In addition, fibrotic lesions were not noted in Smad3 null mutant lungs of mice that received saline vehicle, suggesting loss of Smad3 function alone does not contribute to lung injury (Table 2). These findings further indicate that a lack of Smad3 gene expression attenuates bleomycin-induced fibrotic pathogenesis in mouse lungs.

                              
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Table 2.   Morphological scoring of lung fibrosis in WT and Smad3 KO mice

Changes in whole lung content of collagen, a major extracellular matrix component of fibrosis, were also evaluated by measuring hydroxyproline amount in the lung. As shown in Fig. 2, lung hydroxyproline content was significantly increased in bleomycin-treated wild-type mice vs. saline control mice: at day 28 after bleomycin administration, the lung hydroxyproline content of wild-type mice was 83.7 µg/left lung, compared with 41.1 µg/left lung measured in wild-type mice of saline controls (P < 0.05). Although Smad3 null mutant mice with control saline treatment yielded 39.4 µg hydroxyproline/left lung, the lung hydroxyproline content of bleomycin-treated Smad3-deficient mice was similar: 41.8 µg/left lung (Fig. 2). The above observation, that loss of Smad3 prevented the elevated level of hydroxyproline content seen in normal mice treated with bleomycin, confirmed our conclusion that Smad3 gene expression is necessary for bleomycin-induced lung fibrosis in mice.


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Fig. 2.   Hydroxyproline content in wild-type and Smad3 null mutant mouse lungs. Each bar represents the mean value ± SD of the lung hydroxyproline level of 4-6 mice from each genotype, and assayed condition and is expressed as micrograms per left lung tissue. *Significant difference (P < 0.05) between bleomycin-treated wild-type mice and bleomycin-treated Smad3-deficient mice.

Bleomycin-mediated fibrogenic responses, as shown by interstitial deposition of collagen and fibronectin, are reduced in lungs without Smad3 gene expression. Fibroblast proliferation in both the pleura and interstitium was accompanied by evidence of extensive deposition of extracellular matrix proteins such as collagens, elastin, and fibronectin in adult mouse lung tissues (29). Distribution of collagen was visualized by Masson's trichrome staining, and the extent of collagen deposition in lung sections is shown in Fig. 3A. The lungs of saline-treated animals appeared normal, with only thin bands of collagen immediately adjacent to large vessels and airways. After bleomycin infusion, the lungs of wild-type mice contained dense staining of collagen replacing large areas of lung parenchyma. In contrast, the areas of collagen accumulation in the lung of bleomycin-treated, Smad3-deficient mice were apparently fewer in number and considerably less dense. The lack of Smad3 seems to reduce interstitial collagen deposition in bleomycin-treated mouse lungs.


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Fig. 3.   Histological and immunohistological evaluation of loss of Smad3 on bleomycin-induced lung fibrosis. A: photomicrographs of Masson's trichrome-stained sections of lung tissues. Arrows indicate collagen staining. B: fibronectin immunostaining in adult mouse lungs. Positive fibronectin staining is indicated by arrows. C: proliferating cell nuclear antigen (PCNA) immunostaining of the lung sections. PCNA positive cells in mesenchyme are indicated by arrows. WT+V, Wild-type lungs treated with saline vehicle control; WT+B, wild-type lungs treated with bleomycin; KO+B, Smad3 knockout mouse lungs treated with bleomycin; Neg. Ctrl, negative control for fibronectin immunostaining, in which normal rabbit serum instead of specific rabbit anti-fibronectin antibody was used. Bar = 50 µm.

Furthermore, we examined fibronectin deposition in lung tissues by immunohistochemistry (Fig. 3B). Fibronectin immunostaining was intense in the fibrotic interstitium of bleomycin-treated wild-type mice, compared with control wild-type mice treated with saline alone. However, such an induction of fibronectin protein expression in fibrotic wild-type lung was diminished in bleomycin-treated Smad3-deficient lung, indicating that bleomycin-induced fibronectin accumulation in lung mesenchyme is abolished in the absence of Smad3 gene expression in mouse lungs. Therefore, aside from reduction of fibrogenesis, loss of Smad3 leads to attenuation of bleomycin-induced accumulation of both collagen and fibronectin in adult mouse lungs.

In addition, we also measured cell proliferation by staining the lung tissue section with anti-PCNA antibody (Fig. 3C). PCNA is detected only in proliferating cells. In wild-type mice, bleomycin treatment significantly increased the number of PCNA-positive cells, mainly in mesenchyme. However, treatment with bleomycin in Smad3-deficient mice failed to increase the number of PCNA-positive cells, indicating that Smad3 may be essential for bleomycin-induced fibroblast proliferation during pulmonary fibrosis.

To further evaluate the functional effect of Smad3 during bleomycin-induced lung fibrosis, we assessed gene expression of type I procollagen, fibronectin, and TGF-beta 1 in the adult mouse lungs of both wild and mutant genotypes of Smad3. Using competitive RT-PCR, we were able to quantitate mRNA expression of the above genes in the young adult mice treated with either saline vehicle or bleomycin (Fig. 4). Both alpha 1(I) and fibronectin mRNA were upregulated in wild-type mice after bleomycin administration, compared with uninjured wild-type lungs with saline treatment (Fig. 4B): type I procollagen and fibronectin mRNA were induced in fibrotic lungs by 3.8- and 4.0-fold, respectively (P < 0.05). There was significantly less mRNA expression of both type I procollagen and fibronectin in the lungs of Smad3 knockout mice after bleomycin administration: type I procollagen and fibronectin mRNA amounts showed a similar 1.1- and 1.0-fold change, respectively, in bleomycin-treated Smad3-null lungs compared with saline-treated normal lungs (Fig. 4B). Loss of Smad3 alone without bleomycin administration did not affect mRNA expression of both type I procollagen and fibronectin in the mouse lungs (Fig. 4B). Therefore, overexpression of both type I procollagen and fibronectin, in bleomycin-mediated lung fibrogenesis, is no longer detected in Smad3 -/- lungs after bleomycin administration.


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Fig. 4.   Effect of Smad3 deficiency on bleomycin-induced gene expression in adult mouse lungs. A: competitive RT-PCR electrophoretic patterns for type I procollagen (alpha 1 chain), fibronectin, and transforming growth factor (TGF)-beta 1. B: quantification of mRNA levels of type I procollagen (alpha 1 chain), fibronectin, and TGF-beta 1 after densitometric scanning of competitive PCR electrophoretic patterns. Results are expressed as relative abundance after beta -actin normalization and are means ± SD for at least 6 mice in each experimental group. *Significantly different (P < 0.05) from the mean value of the wild-type treated with bleomycin. alpha 1(I), Type I procollagen alpha 1-chain; FN, fibronectin.

Because Smad3 is an intracellular mediator of TGF-beta signaling, TGF-beta gene expression in response to bleomycin, in both wild-type and Smad3 -/- lungs, was measured. As shown in Fig. 4, we observed that subcutaneous infusion of bleomycin increased the TGF-beta mRNA level in wild-type adult mice: a 5.1-fold induction of TGF-beta mRNA amount was seen in wild-type lungs in response to bleomycin infusion (P < 0.05). However, Smad3 deficiency did not prevent bleomycin-induced upregulation of TGF-beta mRNA expression in mouse lungs (Fig. 4): TGF-beta mRNA level was increased by a significant 4.0-fold with bleomycin treatment in the absence of Smad3 (P < 0.05). The above findings indicate that loss of Smad3 does not affect bleomycin-induced TGF-beta mRNA production in mouse lungs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Progressive fibrosis in lung, kidney, liver, heart, bone marrow, and skin is a major cause of both suffering and death. Pulmonary fibrosis is a devastating disorder with a median survival of 4-5 years after onset of symptoms in affected patients (7). Pulmonary fibrosis is initiated by inflammation followed by a massive production of fibrous connective tissue in the interalveolar septa (4). This fibrotic process results in an excessive number of fibroblasts, an increase in lung collagen content, abnormal spatial distribution of extracellular matrix proteins, and, ultimately, deteriorated lung function (10). However, the molecular mechanisms responsible for the inflammatory process and the ensuing fibrotic alternations in the connective tissue in lung fibrosis are not well understood.

Recent advances in cell and cytokine biology have suggested that TGF-beta is important in the process of bleomycin-induced pulmonary fibrosis in animals, as well as in idiopathic pulmonary fibrosis in humans (21). Pulmonary TGF-beta levels are elevated in mice and rats after intratracheal bleomycin instillation, during the development of pneumonitis and fibrosis (15, 37). Adenovirus-mediated gene transfer of active TGF-beta 1 was sufficient to induce prolonged severe fibrosis in rat lungs (29). It is thought that alveolar macrophages, as stimulated by bleomycin-induced injury, secrete a large quantity of bioactive TGF-beta , thereby inducing lung fibroblasts in the alveolar interstitium to synthesize collagen, resulting in pulmonary fibrosis (18, 19). Although the precise mechanisms of the lung fibrosis process remain enigmatic, TGF-beta signaling appears to be one important mediator of pulmonary fibrosis in both animals and humans (3).

Due to its implication in lung fibrogenesis, blockade of the endogenous TGF-beta pathway has been proposed as a molecular strategy to ameliorate pulmonary fibrosis and is therefore of potential clinical significance. Both TGF-beta -neutralizing antibody and decorin, a TGF-beta -binding proteoglycan, have been shown to reduce bleomycin-induced lung fibrosis (10, 11). Using a soluble version of TGF-beta type II receptor, Wang et al. (34) found that pulmonary fibrosis including excess collagen accumulation was greatly reduced in hamsters with bleomycin administration. Recently, Smad7, an intracellular antagonist of TGF-beta signaling, has been shown to attenuate bleomycin-induced lung fibrosis in mice receiving intratracheal injection of recombinant adenovirus overexpressing Smad7 (25). Taken together, these results could mean that abrogation of TGF-beta signaling is sufficient to offer a rescuable effect against lung fibrogenesis.

Smad3-deficient mice generated by three different research groups survive into adulthood, although they have displayed some impaired immune functions (8, 39, 43). Availability of adult Smad3 null mutant mice made it possible to investigate the function of Smad3 during lung inflammation and fibrogenesis. Using a bleomycin-mediated lung injury model, we have demonstrated that lung fibrotic reactions, including collagen accumulation, were significantly attenuated in Smad3 null mutant mice with targeted disruption of exon 1. Our results suggest that loss of Smad3 in these mutant mice has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where enhanced TGF-beta production is associated with excess collagen synthesis. Therefore, by disrupting Smad3 gene in mice, fibrotic responses in lung cells to TGF-beta are selectively lost, resulting in refractoriness to bleomycin-induced lung fibrogenesis. In addition, loss of Smad3 did not appear to affect bleomycin-induced macrophage/monocyte infiltration and related TGF-beta production, suggesting that TGF-beta synthesis, as induced by bleomycin, is independent of Smad3-mediated molecular pathway. A similar finding of unaltered TGF-beta expression was documented in bleomycin-treated mouse lungs regardless of overexpression of Smad7, a TGF-beta pathway antagonist (25). It has also been recently shown that mice with a deletion of the Smad3 gene by targeted disruption in exon 8 exhibited accelerated wound healing with an increased rate of re-epithelialization and decreased extracellular matrix deposition (1, 2). However, significantly reduced local infiltration of monocytes and reduced local TGF-beta expression were observed in the wound skin of Smad3 null mutant mice, suggesting that part of this phenotype may be secondary to the changes of TGF-beta ligand level. In contrast, monocyte infiltration and TGF-beta induction in bleomycin-treated lung of our Smad3 gene-deficient mice were not changed, which indicated that Smad3 may be directly involved in the production of fibrotic extracellular proteins in bleomycin-treated lung tissue. Therefore, lack of Smad3 may thus modify the different cellular response to TGF-beta in different tissues and suggests a molecular basis to TGF-beta -mediated biological effects (23).

An excess accumulation of collagen in the lung is the hallmark of pulmonary fibrosis. Aside from the reduction of lung fibrosis, we found that loss of Smad3 inhibited bleomycin-induced collagen accumulation in the lung interstitium. Recent evidences have shown that Smad3 is involved in the transcriptional activation of TGF-beta -mediated stimulation of collagen expression (5, 32, 33). Smad3 is necessary for the formation of transcriptional activation complex in the stimulation of type I collagen gene expression by TGF-beta ligands in human skin fibroblasts (6, 9). Smad3 also acts synergistically with transcriptional factor TFE3 to activate TGF-beta -induced transcription of the plasminogen activator inhibitor-1 gene (16). Therefore, one of the possible mechanisms by which abrogation of Smad3 gene expression prevented bleomycin-induced lung fibrosis may be mediated by having a direct negative effect on collagen expression in mouse lungs. However, it is also possible that reduction of collagen expression in bleomycin-treated Smad3 null mutant lungs is mediated by other indirect effects, such as increased expression of the transcriptional coactivator p300/CBP or reduced expression of the TGF-beta pathway inhibitor Smad7 (9, 27). The molecular mechanism of Smad3 action on collagen expression needs to be studied further.

Furthermore, induction of fibronectin expression in bleomycin-treated whole lungs was also attenuated in Smad3 null mutant mice compared with that in wild-type mice, whereas bleomycin-induced TGF-beta expression was not changed in the absence of Smad3 function, as shown in this in vivo study. Interestingly, studies on cultured mouse embryonic fibroblasts that were isolated from exon 8-deleted Smad3 null mutant mice indicate that lack of Smad3 function does not block TGF-beta -induced fibronectin expression, but that TGF-beta autoinduction relies on the expression of Smad3 (27). The discrepancy between these studies and those reported herein suggests that fibroblasts in adult lungs may respond differently to TGF-beta signaling compared with embryonic fibroblasts, or that the TGF-beta -triggered signaling in epithelial cells is necessary for regulation of fibronectin expression in nearby fibroblasts in vivo. The latter effect would have been missing in embryonic fibroblast cultures in vitro.

Smad3 occupies a central position in TGF-beta -mediated signal transduction (22). Smad3 is a direct substrate for the protein serine kinase of the activated TGF-beta receptor complex. Upon phosphorylation by the cognate receptors, Smad3 relocates into the nucleus and binds to specific gene promoters in cooperation with other transcriptional proteins, thus activating transcription of TGF-beta -responsive genes. On the other hand, Smad2, a close homolog of Smad3 due to an extra exon insert, has not yet been shown to interact directly with DNA, although Smad2 can still be brought to DNA by association with other proteins (26). Although Smad2 and Smad3 appear to have redundant functions when overexpressed in vitro, the unique abilities of Smad3 to bind DNA directly indicate that these two Smads may regulate distinct target genes in vivo. This idea is further supported by the striking differences in their respective null phenotypes: Smad3 null mice are both viable and fertile, whereas Smad2-deficient mice do not survive beyond embryogenesis (35). However, although we have now clearly defined a functional importance for Smad3 in participation of bleomycin-induced lung fibrosis, more studies are required to define the action of Smad2 during lung inflammation and fibrogenesis.

We found that loss of Smad3 did not entirely inhibit lung fibrosis induced by bleomycin. Compared with bleomycin-treated wild-type mice, lung phenotype in Smad3 null lungs with bleomycin infusion demonstrated attenuated pulmonary fibrogenesis and overtly normal lung alveolar structures. However, minor lesions such as sporadic thickening of alveolar septa were also observed. Such an incomplete inhibition of lung fibrosis was also found in bleomycin-treated lungs overexpressing adenovector-mediated Smad7, an intracellular TGF-beta signaling antagonist (25). One explanation could be that the existence of Smad2 could account for the incomplete suppression of TGF-beta signal transduction in the absence of Smad3. In agreement with this, earlier data also imply that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs (25). In addition, previous reports have suggested the role of an array of cytokines and growth factors, apart from TGF-beta , in the development of lung fibrosis, including tumor necrosis factor-alpha , platelet-derived growth factor, insulin growth factor-1, and interleukins (17). Thus it is conceivable that abrogation of the action of other cytokines, in addition to TGF-beta , is required to achieve complete inhibition of lung fibrosis.

In summary, we have shown that loss of Smad3 gene expression attenuated bleomycin-induced lung fibrosis, including collagen accumulation, establishing a novel functional significance for Smad3-participation in lung fibrogenesis. Because overproduction of TGF-beta is a chief cause of tissue fibrosis in various human fibrotic disorders including those of the lung, our data suggest that the disruption of the Smad3-mediated TGF-beta signal transduction may be of therapeutic benefit in treating lung injuries due to fibrosis.


    ACKNOWLEDGEMENTS

This study is supported by NIH Grants HL-61286 (J. Zhao), CA-75368 (X.-F. Wang), HL-44977, HL-44060, and HL-60231 (D. Warburton); American Lung Association Research Grants (J. Zhao and W. Shi); and Childrens Hospital Los Angeles Research Institute Career Development Award (W. Shi). J. Zhao is a recipient of a National Heart, Lung, and Blood Institute Independent Scientific Award.


    FOOTNOTES

* J. Zhao and W. Shi contributed equally to this work.

Present address for J. Zhao: SangStat Medical Corp., 6300 Dumbarton Cir., Fremont, CA 94555 (E-mail: jingsongzhao{at}yahoo.com).

Address for reprint requests and other correspondence: W. Shi, Depts. of Surgery and Pediatrics, and Cell & Developmental Bio. Prog., The Childrens Hospital Los Angeles Research Inst., 4650 Sunset Blvd., Los Angeles, CA 90027.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00151.2001

Received 28 April 2001; accepted in final form 26 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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