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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transforming growth factor- (TGF-
)
signaling plays an important regulatory role during lung fibrogenesis.
Smad3 was identified in the pathway for transducing TGF-
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-; hydroxyproline
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TGF-
) is known to play an essential role in the
pathogenesis of lung fibrosis (3). In animal models of
pulmonary fibrosis, levels of TGF-
mRNA and protein are increased
(12, 15, 28).
TGF- 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-
antagonists, in the form of either neutralizing antibodies or the
TGF-
-binding proteoglycan decorin, prevented bleomycin-induced lung
fibrosis in vivo, indicating that TGF-
ligand is required for the
development of pulmonary fibrosis (10, 11). Moreover,
intratracheal instillation of TGF-
-soluble type II receptor protein
dramatically reduced bleomycin-induced lung fibrosis in hamsters
(34), and exogenous overexpression of Smad7, an inhibitor
in the TGF-
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- receptors signal from the cell membrane to the nucleus (14). The
activated TGF-
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-
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-
(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-
-mediated development and cell growth control processes.
Although Smad3 mediates intracellular signaling from TGF- 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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 procollagenHydroxyproline 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
|
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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- is important in the process of bleomycin-induced pulmonary fibrosis in animals, as well as in idiopathic pulmonary fibrosis in
humans (21). Pulmonary TGF-
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-
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-
, 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-
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- pathway has been proposed as a molecular strategy to ameliorate
pulmonary fibrosis and is therefore of potential clinical significance.
Both TGF-
-neutralizing antibody and decorin, a TGF-
-binding
proteoglycan, have been shown to reduce bleomycin-induced lung fibrosis
(10, 11). Using a soluble version of TGF-
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-
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-
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- production is associated with excess
collagen synthesis. Therefore, by disrupting Smad3 gene in mice,
fibrotic responses in lung cells to TGF-
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-
production,
suggesting that TGF-
synthesis, as induced by bleomycin, is
independent of Smad3-mediated molecular pathway. A similar finding of
unaltered TGF-
expression was documented in bleomycin-treated mouse
lungs regardless of overexpression of Smad7, a TGF-
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-
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-
ligand level. In
contrast, monocyte infiltration and TGF-
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-
in different tissues and suggests a molecular basis
to TGF-
-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--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-
ligands
in human skin fibroblasts (6, 9). Smad3 also acts synergistically with transcriptional factor TFE3 to activate
TGF-
-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-
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- 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-
-induced fibronectin expression, but that TGF-
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-
signaling compared with embryonic fibroblasts, or that the TGF-
-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--mediated signal
transduction (22). Smad3 is a direct substrate for the
protein serine kinase of the activated TGF-
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-
-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- signaling
antagonist (25). One explanation could be that the
existence of Smad2 could account for the incomplete suppression of
TGF-
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-
, in the development of lung fibrosis, including tumor necrosis factor-
, 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-
, 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- 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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ashcroft, GS,
and
Roberts AB.
Loss of Smad3 modulates wound healing.
Cytokine Growth Factor Rev
11:
125-131,
2000[ISI][Medline].
2.
Ashcroft, GS,
Yang X,
Glick AB,
Weinstein M,
Letterio JL,
Mizel DE,
Anzano M,
Greenwell-Wild T,
Wahl SM,
Deng C,
and
Roberts AB.
Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.
Nat Cell Biol
1:
260-266,
1999[ISI][Medline].
3.
Border, WA,
and
Noble NA.
Transforming growth factor beta in tissue fibrosis.
N Engl J Med
331:
1286-1292,
1994
4.
Chandler, DB,
Hyde DM,
and
Giri SN.
Morphometric estimates of infiltrative cellular changes during the development of bleomycin-induced pulmonary fibrosis in hamsters.
Am J Pathol
112:
170-177,
1983[Abstract].
5.
Chen, SJ,
Yuan W,
Lo S,
Trojanowska M,
and
Varga J.
Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta.
J Cell Physiol
183:
381-392,
2000[ISI][Medline].
6.
Chen, SJ,
Yuan W,
Mori Y,
Levenson A,
Trojanowska M,
and
Varga J.
Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3.
J Invest Dermatol
112:
49-57,
1999
7.
Cooper, JA, Jr.
Pulmonary fibrosis: pathways are slowly coming into light.
Am J Respir Cell Mol Biol
22:
520-523,
2000
8.
Datto, MB,
Frederick JP,
Pan L,
Borton AJ,
Zhuang Y,
and
Wang XF.
Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction.
Mol Cell Biol
19:
2495-2504,
1999
9.
Ghosh, AK,
Yuan W,
Mori Y,
and
Varga J.
Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-beta involves functional cooperation with p300/CBP transcriptional coactivators.
Oncogene
19:
3546-3655,
2000[ISI][Medline].
10.
Giri, SN,
Hyde DM,
Braun RK,
Gaarde W,
Harper JR,
and
Pierschbacher MD.
Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis.
Biochem Pharmacol
54:
1205-1216,
1997[ISI][Medline].
11.
Giri, SN,
Hyde DM,
and
Hollinger MA.
Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.
Thorax
48:
959-966,
1993[Abstract].
12.
Gurujeyalakshmi, G,
Hollinger MA,
and
Giri SN.
Regulation of transforming growth factor-beta1 mRNA expression by taurine and niacin in the bleomycin hamster model of lung fibrosis.
Am J Respir Cell Mol Biol
18:
334-342,
1998
13.
Harrison, JH, Jr,
and
Lazo JS.
High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis.
J Pharmacol Exp Ther
243:
1185-1194,
1987[Abstract].
14.
Heldin, CH,
Miyazono K,
and
ten Dijke P.
TGF-beta signalling from cell membrane to nucleus through SMAD proteins.
Nature
390:
465-471,
1997[ISI][Medline].
15.
Hoyt, DG,
and
Lazo JS.
Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice.
J Pharmacol Exp Ther
246:
765-771,
1988[Abstract].
16.
Hua, X,
Liu X,
Ansari DO,
and
Lodish HF.
Synergistic cooperation of TFE3 and smad proteins in TGF-beta-induced transcription of the plasminogen activator inhibitor-1 gene.
Genes Dev
12:
3084-3095,
1998
17.
Kelley, J.
Cytokines of the lung.
Am Rev Respir Dis
141:
765-788,
1990[ISI][Medline].
18.
Khalil, N,
Bereznay O,
Sporn M,
and
Greenberg AH.
Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation.
J Exp Med
170:
727-737,
1989[Abstract].
19.
Khalil, N,
Whitman C,
Zuo L,
Danielpour D,
and
Greenberg A.
Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.
J Clin Invest
92:
1812-1818,
1993[ISI][Medline].
20.
Madtes, DK,
Elston AL,
Hackman RC,
Dunn AR,
and
Clark JG.
Transforming growth factor-alpha deficiency reduces pulmonary fibrosis in transgenic mice.
Am J Respir Cell Mol Biol
20:
924-934,
1999
21.
Mason, RJ,
Schwarz MI,
Hunninghake GW,
and
Musson RA.
NHLBI Workshop Summary. Pharmacological therapy for idiopathic pulmonary fibrosis. Past, present, and future.
Am J Respir Crit Care Med
160:
1771-1777,
1999
22.
Massague, J.
TGF-beta signal transduction.
Annu Rev Biochem
67:
753-791,
1998[ISI][Medline].
23.
Massague, J.
Wounding Smad.
Nat Cell Biol
1:
E117-E119,
1999[ISI][Medline].
24.
Monson, JM,
Friedman J,
and
McCarthy BJ.
DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene.
Mol Cell Biol
2:
1362-1371,
1982[ISI][Medline].
25.
Nakao, A,
Fujii M,
Matsumura R,
Kumano K,
Saito Y,
Miyazono K,
and
Iwamoto I.
Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.
J Clin Invest
104:
5-11,
1999
26.
Piek, E,
Heldin CH,
and
Ten Dijke P.
Specificity, diversity, and regulation in TGF-beta superfamily signaling.
FASEB J
13:
2105-2124,
1999
27.
Piek, E,
Ju WJ,
Heyer J,
Escalante-Alcalde D,
Stewart CL,
Weinstein M,
Deng C,
Kucherlapati R,
Bottinger EP,
and
Roberts AB.
Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.
J Biol Chem
276:
19945-19953,
2001
28.
Raghow, R,
Lurie S,
Seyer JM,
and
Kang AH.
Profiles of steady state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis.
J Clin Invest
76:
1733-1739,
1985[ISI][Medline].
29.
Sime, PJ,
Xing Z,
Graham FL,
Csaky KG,
and
Gauldie J.
Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung.
J Clin Invest
100:
768-776,
1997
30.
Stegemann, H,
and
Stalder K.
Determination of hydroxyproline.
Clin Chim Acta
18:
267-273,
1967[ISI][Medline].
31.
Tran, PL,
Weinbach J,
Opolon P,
Linares-Cruz G,
Reynes JP,
Gregoire A,
Kremer E,
Durand H,
and
Perricaudet M.
Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.
J Clin Invest
99:
608-617,
1997
32.
Vindevoghel, L,
Kon A,
Lechleider RJ,
Uitto J,
Roberts AB,
and
Mauviel A.
Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.
J Biol Chem
273:
13053-13057,
1998
33.
Vindevoghel, L,
Lechleider RJ,
Kon A,
de Caestecker MP,
Uitto J,
Roberts AB,
and
Mauviel A.
SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.
Proc Natl Acad Sci USA
95:
14769-14774,
1998
34.
Wang, Q,
Wang Y,
Hyde DM,
Gotwals PJ,
Koteliansky VE,
Ryan ST,
and
Giri SN.
Reduction of bleomycin induced lung fibrosis by transforming growth factor beta soluble receptor in hamsters.
Thorax
54:
805-812,
1999
35.
Weinstein, M,
Yang X,
Li C,
Xu X,
Gotay J,
and
Deng CX.
Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2.
Proc Natl Acad Sci USA
95:
9378-9383,
1998
36.
Weissler, JC.
Idiopathic pulmonary fibrosis: cellular and molecular pathogenesis.
Am J Med Sci
297:
91-104,
1989[ISI][Medline].
37.
Westergren-Thorsson, G,
Hernnas J,
Sarnstrand B,
Oldberg A,
Heinegard D,
and
Malmstrom A.
Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats.
J Clin Invest
92:
632-637,
1993[ISI][Medline].
38.
Yaekashiwa, M,
Nakayama S,
Ohnuma K,
Sakai T,
Abe T,
Satoh K,
Matsumoto K,
Nakamura T,
Takahashi T,
and
Nukiwa T.
Simultaneous or delayed administration of hepatocyte growth factor equally represses the fibrotic changes in murine lung injury induced by bleomycin. A morphologic study.
Am J Respir Crit Care Med
156:
1937-1944,
1997
39.
Yang, X,
Letterio JJ,
Lechleider RJ,
Chen L,
Hayman R,
Gu H,
Roberts AB,
and
Deng C.
Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.
Embo J
18:
1280-1291,
1999
40.
Zhao, J,
Bu D,
Lee M,
Slavkin HC,
Hall FL,
and
Warburton D.
Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.
Dev Biol
180:
242-257,
1996[ISI][Medline].
41.
Zhao, J,
Lee M,
Smith S,
and
Warburton D.
Abrogation of Smad3 and Smad2 or of Smad4 gene expression positively regulates murine embryonic lung branching morphogenesis in culture.
Dev Biol
194:
182-195,
1998[ISI][Medline].
42.
Zhao, J,
Shi W,
Chen H,
and
Warburton D.
Smad7 and Smad6 differentially modulate transforming growth factor beta -induced inhibition of embryonic lung morphogenesis.
J Biol Chem
275:
23992-23997,
2000
43.
Zhu, Y,
Richardson JA,
Parada LF,
and
Graff JM.
Smad3 mutant mice develop metastatic colorectal cancer.
Cell
94:
703-714,
1998[ISI][Medline].