1 Center for Craniofacial Molecular Biology, Departments of Surgery and Pediatrics, and Cell and Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, University of Southern California Schools of Dentistry and Medicine, Los Angeles, California 90033; and 2 Molecular Virology and Immunology Program, Department of Pathology and Biology, Health Science Center, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Excessive transforming growth factor (TGF)-
signaling has been implicated in pulmonary hypoplasia associated with
bronchopulmonary dysplasia, a chronic lung disease of human prematurity
featuring pulmonary fibrosis. This implies that inhibitors of TGF-
could be useful therapeutic agents. Because exogenous TGF-
ligands are known to inhibit lung branching morphogenesis and
cytodifferentiation in mouse embryonic lungs in ex vivo culture, we
examined the capacity of a naturally occurring inhibitor of TGF-
activity, the proteoglycan decorin, to overcome the inhibitory effects
of exogenous TGF-
. Intratracheal microinjection of a recombinant
adenovirus containing decorin cDNA resulted in overexpression of the
exogenous decorin gene in airway epithelium. Although exogenous TGF-
efficiently decreased epithelial lung branching morphogenesis in
control cultures, TGF-
-induced inhibition of lung growth was
abolished after epithelial transfer of the decorin gene. Additionally,
exogenous TGF-
-induced antiproliferative effects as well as the
downregulation of surfactant protein C were abrogated by decorin in
cultured embryonic lungs. Moreover, lung branching inhibition by
TGF-
could be restored by the addition of decorin antisense
oligodeoxynucleotides in culture, indicating that decorin is both
specifically and directly involved in suppressing TGF-
-mediated
negative regulation of lung morphogenesis. Our findings suggest that
decorin can antagonize bioactive TGF-
during lung growth and
differentiation, establishing the rationale for decorin as a candidate
therapeutic approach to ameliorate excessive levels of TGF-
signaling in the developing lung.
transforming growth factor-; intratracheal microinjection; epithelium-specific gene transfer; recombinant adenovirus; competitive
polymerase chain reaction; antisense oligodeoxynucleotide
inhibition
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INTRODUCTION |
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THE PRIMITIVE LUNG EPITHELIUM undergoes cell proliferation, branching morphogenesis, and alveolar saccular formation as well as concomitant cell lineage differentiation to form an organ capable of conducting respiratory gases to a large, diffusable interface with the circulation. Lung development is now known to take place within a complex milieu of peptide growth factors, which may affect many parameters of cell behavior including cell proliferation, cell survival, migration, differentiation, and extracellular matrix deposition (reviewed in Ref. 13). It is therefore likely that soluble factors instruct embryonic, fetal, and neonatal lung development by coordinated temporospatial autocrine/paracrine signaling.
Transforming growth factor (TGF)-1, -2, and -3 are members of a
large family of cytokines that includes activin, bone morphogenetic proteins, and Müllerian inhibiting substance. TGF-
s initiate their cellular action by binding primarily to three cell
surface-receptor proteins termed TGF-
type I, type II, and type III
receptors. On binding to TGF-
s, the TGF-
type III receptor
(betaglycan) presents TGF-
directly to the TGF-
type II receptor,
a serine/threonine kinase subunit (23, 24, 32). The TGF-
type II
receptor kinase trans-phosphorylates
the type I receptor after TGF-
type I-type II receptor
heteromerization, which, in turn, displaces betaglycan from TGF-
ligand binding (25, 27). The TGF-
type I-type II receptor complex
subsequently trans-phosphorylates
intracellular Smad proteins and so eventually propagates a
phosphorylation signal into the nucleus (6, 11, 21, 35).
TGF- signaling is an important negative regulator during embryonic
lung development as demonstrated by gain- and loss-of-function studies
in embryonic mouse lungs in organ culture as well as in transgenic and
null mutant mice. TGF-
1 and TGF-
2 both inhibit pulmonary
branching morphogenesis in culture (30, 39), whereas TGF-
3 null
mutant mice have a specific neonatal lethal lung phenotype (18). On the
other hand, abrogation of TGF-
type II receptor signaling, either
with antisense oligodeoxynucleotides (ODNs) or with pulmonary
epithelium-specific overexpression of a dominant-negative TGF-
type
II receptor, significantly stimulates lung morphogenesis in culture and
prevents TGF-
-induced downregulation of epithelial differentiation
marker genes such as surfactant protein (SP) C (39, 40), indicating
that physiological TGF-
signaling negatively modulates lung
development. In contrast, overexpression of TGF-
1 in the alveolar
epithelium with the human SP-C promoter generates a neonatal lethal,
hypoplastic pulmonary phenotype with reduced saccular formation and
abnormal epithelial differentiation, supporting the conclusion that
excessive TGF-
signaling can inhibit lung morphogenesis in vivo
(42).
Aberrant expression of TGF-, occurring as a result of lung disease
and injury, could therefore perturb finely regulated TGF-
signaling
and so result in abnormalities of lung growth, differentiation, and
development. Pulmonary fibrosis is a prominent feature of bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, and increased concentrations of TGF-
1 have been found
in the bronchoalveolar lavage fluid of human premature infants who
develop a severe form of BPD (20). Excessive TGF-
signaling is known
to induce a chronic fibrotic lung injury in rats as well as in the
bleomycin model of chronic fibrosis (19, 31). Alveolar hypoplasia, a
major sequela of neonatal hyperoxia, is also associated with high
levels of TGF-
activity in premature lungs (3). Taken together,
excessive TGF-
signaling appears to adversely disrupt the orderly
temporospatial molecular cascades that normally govern lung
morphogenesis and cytodifferentiation. Therefore, therapeutic
strategies to antagonize TGF-
signaling could ameliorate lung injury
and augment lung repair in the developing lung.
Decorin belongs to the family of small leucine-rich proteoglycans that
have been implicated as key regulators of both matrix assembly and
cellular growth (15). Decorin consists of leucine-rich repeats of
20-24 amino acids and a single site for glycosaminoglycan side-chain attachment. Decorin is a 100-kDa proteoglycan found in the
interstitial extracellular matrix, with a core protein of 45 kDa.
Decorin specifically binds and neutralizes TGF- ligands via its
protein moiety, thus acting as a TGF-
inhibitor (37). Because
decorin occurs naturally, it may be involved in the physiological regulation of the effects of TGF-
ligands. By sequestering bioactive TGF-
through formation of inactive TGF-
-decorin complexes,
decorin could be useful as an antagonistic agent to downmodulate
TGF-
signaling in the prevention and treatment of lung disease and injury.
In the present report, we used decorin to antagonize exogenous
TGF--mediated inhibition of lung morphogenesis and
cytodifferentiation in a well-characterized model of embryonic mouse
lung morphogenesis ex vivo defined culture. A recombinant adenovirus
expressing decorin was utilized, via an intratracheal microinjection to
introduce the adenovirus, in a gene transfer approach to induce
epithelium-specific decorin overexpression in embryonic mouse lungs in
culture (40). We found that recombinant adenoviral expression of
decorin in the pulmonary epithelium specifically overcame exogenous
TGF-
-mediated negative regulation of lung epithelial branching.
Decorin thus appears to be a candidate rational therapy to negatively
regulate excessive TGF-
signaling during lung morphogenesis, injury,
and repair.
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MATERIALS AND METHODS |
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Recombinant adenovirus constructs. Construction of the recombinant adenovectors has been described in detail elsewhere (2, 31). Briefly, full-length human decorin cDNA was cloned into a shuttle vector containing the human cytomegalovirus promoter and the E1 region of the human type 5 adenovirus genome. The recombinant adenovirus, packaged with the decorin gene, was then generated by homologous recombination after cotransfection with the shuttle vector and a virus-rescuing vector (10). The presence of decorin cDNA in the viral genome was further verified by restrictive digestion and Southern hybridization.
High titers of the resultant replication-deficient recombinant
adenovirus, designated AdDcn, were purified by CsCl gradient centrifugation and PD-10 Sephadex chromatography. Purified viruses were
plaque titered in 293 cells, divided into aliquots in PBS-10% glycerol, and stored at 80°C until used (36) and are
expressed in plaque-forming units (pfu). The control virus was
constructed with the E1 region deleted and contained no exogenous
genes. AdLacZ expressing
-galactosidase was also prepared to
facilitate the localization of exogenous expression in transfected
cells (40).
Administration of recombinant adenoviruses and embryonic lung culture. Timed-pregnant Swiss-Webster female mice were obtained by mating and checking for vaginal plugs on the same day so that the variation in gestational age was <6 h. Embryos on embryonic day 11.5 (ED11.5; 6-7 mm in length, with ~45 somites) were dissected (day of plug is defined as ED0) in sterile Hanks' buffer, and the lung primordia (with trachea attached) were removed by microdissection.
Intratracheal microinjection was performed as we previously described (40). Aided with a microinjection needle and a microscope, desirable concentrations of adenovirus were injected intratracheally into the embryonic respiratory tract until both proximal and distal airways of the lung explants were filled with injected solution. Injected lungs were incubated for 1 h at room temperature for viral transfection before lung culture.
Early mouse embryonic lungs (ED11.5) were cultured routinely under
serum-free, chemically defined conditions as previously reported (33,
40). TGF- ligands, TGF-
neutralizing antibodies (R&D Systems,
Minneapolis, MN), and/or decorin ODNs were added exogenously to the
lung culture medium as needed. The cultures were maintained in 100%
humidity with an atmosphere of 95% air-5% CO2 for 4 days.
Quantification of branching morphogenesis. Because branching morphogenesis per se is the key bioassay readout for testing the functional role of decorin during embryonic lung development, three independent measurements of branching morphogenesis were devised: 1) the number of airway generations from the trachea to the most distal branch of the longest visible airway, 2) the number of air sacs visible around the periphery of the lung explants, and 3) computerized pattern recognition analysis of the number of individual terminal respiratory units in the whole explant. These analyses were performed on whole mounts of lung explants without knowledge of the experimental conditions, with transillumination to visualize the structures and photomicroscopy to record the permanent image, and on paraffin-embedded sections of fixed tissue.
RNA extraction, reverse transcription, and competitive PCR quantification. Total RNA from cultured lung explants was extracted by guanidinium thiocyanate after homogenization as documented by Zhao et al. (38). Total extracted RNA was immediately incubated at 37°C for 1 h in 20 µl of 10 mM Tris (pH 8.4), 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 5 U of ribonuclease inhibitor, 0.5 mM deoxynucleotide triphosphates, 100 pmol of oligo(dT)12-18, and 200 U of Moloney murine leukemia virus reverse transcriptase (US Biochemicals, Cleveland, OH). The resultant cDNA products were used for competitive PCR assays.
Competitive PCR methodology for specific mRNA quantification of
pulmonary genes has been described previously (38). Briefly, a set of
primers ( primers 1 and
2; Fig.
1A)
were designed for murine decorin to amplify a cDNA fragment of 182 bp
in size (29). Two composite primers were synthesized for decorin
competitor construction (Fig. 1A):
each composite primer had the target decorin primer sequence (solid
boxes) incorporated into a stretch of sequence (open boxes) designed to
hybridize to the opposite strand of a heterologous DNA fragment. The
desired primer sequences ( primers 1 and 2) were thus
engineered into a competitor cDNA after PCR amplification, ensuring
that both decorin cDNA and decorin competitor utilize the same set of
primers in the decorin competitive PCR. The decorin competitor was 268 bp in length. Both decorin and its competitor PCR products were DNA
sequenced to ensure their identities. Competitive PCR assay for
biglycan was developed in a manner similar to that for decorin.
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PCR amplification was carried out with a modification of a previously
described assay for the TGF- type II receptor (39). The PCR mixture
containing a known amount of competitor was added to
reverse-transcribed samples derived from 20 ng of total RNA or to
dilutions of standard cDNA templates in a total volume of 50 µl.
-Actin competitive PCR as an internal control was routinely performed on the same samples. As a negative control for genomic or
viral DNA, un-reverse-transcribed total RNA was also included in the
competitive PCR assays.
Immunocytochemistry. Cultured lungs were fixed with paraformaldehyde and embedded into paraffin. Embryonic lung sections (5 µm thick) on HistoGrip-coated slides were subsequently prepared for immunohistochemical study. Decorin antibodies were generous gifts from Dr. Larry Fisher (National Institute of Dental Research, National Institutes of Health, Bethesda, MD) and were used at the recommended concentrations (7). Biotinylated second antibody and streptavidin-peroxidase conjugate were used to detect bound antibody. Subsequent addition of aminoethylcarbazole chromogen generated a reddish precipitate surrounding the decorin antigen (Zymed, South San Francisco, CA). Normal rabbit serum, bovine serum albumin, and water were run in parallel with decorin antibodies to yield negative controls.
Densitometric and statistical analysis. Electrophoresis after PCR amplification was performed on 3% agarose gels (NuSieve 3:1, FMC Bioproducts, Rockland, ME) in a submarine gel unit (CBS Scientific, Del Mar, CA), where target and competitor PCR products were separated by size. DNA bands were visualized by staining with 5 µg/ml of ethidium bromide. Images were both photographed by Polaroid 667 films for a permanent record and captured by a computerized scanner for intensity measurement. The intensity was determined by densitometric analysis in pixels with ImageQuant band-analyzing software (Molecular Dynamics, Sunnyvale, CA). Means ± SD were calculated, and the significance of differences between means was evaluated by t-test (criterion for significance is P < 0.05).
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RESULTS |
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Adenovirus-mediated epithelium-specific overexpression
of human decorin in mouse lung explants in culture. To
assess the effects of decorin on the TGF--mediated inhibition of
embryonic lung development, we initiated the study by establishing an
adenoviral transfer strategy to introduce decorin cDNA into embryonic
lungs undergoing branching morphogenesis in culture. A
replication-defective adenovirus carrying human decorin cDNA (AdDcn)
under the control of a human cytomegalovirus promoter was prepared as
described in MATERIALS AND METHODS.
Because TGF-
ligands are known to inhibit embryonic lung epithelial
branching morphogenesis in culture, intratracheal microinjection of
recombinant adenovirus was administered on ED11.5 lung explants before culture.
To determine that the lung explants microinjected intratracheally with
recombinant adenovirus express the transgene, cultured lungs
microinjected with AdDcn or control virus were extracted for
total RNA. The subsequent reverse-transcribed products from embryonic
lungs were then used for PCR assays. Only lungs microinjected with
AdDcn, not with control virus, expressed exogenous human decorin mRNA
(Fig.
2A) when
human-specific PCR primers to decorin cDNA were used. To measure the
level of decorin transgene overexpression, a set of primers common to
both human and mouse decorin were synthesized to anneal to
identical sequences on their respective cDNAs.
Competitive PCR assays were developed to quantify mouse,
human, or total (mouse plus human) decorin mRNA amounts present in the
microinjected lungs. The construction of mouse decorin competitive PCR
is shown as a paradigm in Fig. 1. Intratracheal microinjection of AdDcn yielded a pfu-dependent transgene overexpression of human decorin mRNA
levels, whereas control virus microinjection at corresponding pfu
titers resulted only in basal levels of murine decorin mRNA as shown by
competitive PCR electrophoretic patterns for both human and mouse
decorin (Fig. 2A). However,
overexpression of the human decorin transgene did not affect endogenous
mouse decorin gene expression in comparison with medium and virus
controls (Fig. 2A) as quantified by
competitive PCR with a gene-specific primer set to amplify
the murine decorin. Thus intratracheal microinjection of AdDcn
induced only a pfu titer-dependent overexpression of the exogenous
human decorin gene but not of the endogenous mouse counterpart gene.
Endogenous levels of other small leucine-rich proteoglycans, including
biglycan and fibromodulin, were also not changed by AdDcn infection
into embryonic lungs in culture (data not shown). Densitometric
analysis of decorin competitive PCR electrophoretic pattern confirmed
the virus dose-dependent induction of transgene expression in
AdDcn-microinjected lungs (Fig.
2B). A 9- and a 23-fold
overexpression of decorin transgene were present, respectively, when
embryonic lungs were microinjected with AdDcn at 2 × 109 and 2 × 1010 pfu/ml
(P < 0.05), whereas infection with
control virus at the above concentrations failed to elevate decorin
gene expression. As a result, using the AdDcn-mediated gene transfer
approach, we were able to achieve decorin gene overexpression during
embryonic lung development in culture.
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Decorin immunohistochemistry on cultured embryonic lungs microinjected with recombinant adenovirus was performed to confirm the overproduction of the transgene and localize the exogenous human decorin protein. A human-specific decorin antibody was used to localize cells with transgene expression in lungs infected intratracheally with AdDcn. Exogenous human decorin protein was detected in both proximal (Fig. 2C, a1 and a2) and distal (Fig. 2Cb) airway epithelial cells with high intensities of immunostaining, whereas mesenchymal cells were not stained (Fig. 2C, arrowheads). Human decorin immunoactivities in both major and terminal bronchial cells indicate that AdDcn microinjected into the tracheal lumen was able to fill the whole respiratory tract, achieving efficient transfection in epithelial cells lining both the proximal and distal airways. However, the above characteristic immunoreactive pattern was no longer observed when mouse-specific decorin antibody replaced human-specific decorin antibody (data not shown), further supporting the notion that exogenous human decorin transgene was overexpressed in lung epithelium when AdDcn was used for microinjection. Additionally, lungs microinjected with control virus yielded only background staining (Fig. 2Cc). Thus we conclude that intratracheal microinjection of AdDcn, not of control virus, resulted in overexpression of the exogenous decorin transgene at both the mRNA and protein levels exclusively in the airway epithelium, not in the mesenchyme, in embryonic lungs in culture.
Intratracheal microinjection of AdDcn prevents
exogenous TGF--mediated inhibition of embryonic lung
branching morphogenesis in culture. Embryonic murine
lungs (ED11.5) underwent extensive morphogenesis in culture to develop
into a characteristic branching pattern (Fig.
3A,
a and
b). The use of the chemically
defined, serumless culture system in the present study enabled us to
separate the effects of decorin overexpression from those of hypoxia,
reduced placental blood flow, or systemic metabolic effects mediated
through the sympathoadrenal axis as well as allowing us to perform the functional blocking and pharmacological experiments in a precisely controlled manner. As Zhao et al. (39) have
previously shown, both TGF-
1 (5 ng/ml; Fig.
3Ac) and TGF-
2 (0.1 ng/ml; Fig.
3Ae) inhibit lung branching
morphogenesis in culture as quantified by the number of terminal
branches, resulting in a hypoplastic phenotype. However,
TGF-
-mediated dose-dependent lung branching inhibition was abolished
when TGF-
neutralizing antibody was added in addition to the TGF-
ligand in lung culture (Fig. 3B, c and
d).
Analogous to the function of TGF-
neutralizing antibody, decorin is known to modulate soluble TGF-
by binding to it with high
affinity, thereby inactivating TGF-
. Decorin was therefore overexpressed in the lung epithelium with intratracheal microinjection of AdDcn in cultured embryonic lungs. As shown in Fig.
3B, decorin transgene expression in
embryonic lungs completely blocked the concentration-dependent
inhibition of lung branching morphogenesis by either TGF-
1 or
TGF-
2 (a and
b, respectively). In lungs
microinjected with AdDcn, 20 ng/ml of TGF-
1 failed to significantly
inhibit lung branching (94.8% of medium control), whereas TGF-
1 of
the same dose significantly decreased lung morphogenesis (64.7% of medium control; P < 0.05). Likewise,
TGF-
2 (0.4 ng/ml) failed to alter lung branching in the presence of
the decorin transgene (98.9% of medium control), whereas the same dose
of TGF-
2 inhibited lung branching morphogenesis when the control
virus was used for microinjection (67.3% of medium control;
P < 0.05). Thus cultured lungs
microinjected with AdDcn were resistant to exogenous TGF-
(Fig.
3A, d
for TGF-
1 and f for TGF-
2) in
comparison to TGF-
-treated lungs without decorin transgene
overexpression in culture (Fig. 3A,
c for TGF-
1 and
e for TGF-
2). Our observation that
administration of either TGF-
neutralizing antibody or decorin
transgene overexpression prevented TGF-
-induced inhibition on
embryonic lung epithelial branching morphogenesis suggests that decorin
acts as an inhibitor of TGF-
ligands in a manner similar to that of
TGF-
neutralizing antibodies.
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To further examine the biological significance of decorin gene transfer
into lung epithelium, various doses of decorin adenovirus were used for
embryonic lung intratracheal microinjection (Fig. 4). Both TGF-1 and TGF-
2 were able to
significantly decrease lung branching in the presence of the control
virus regardless of viral concentrations. However, a dose-dependent
reversal of the TGF-
-mediated negative influence on lung
morphogenesis was obtained in lungs microinjected with AdDcn and
cultured in the presence of either TGF-
1 (5 ng/ml; Fig. 4,
left) or TGF-
2 (0.1 ng/ml; Fig.
4, right). Cultured embryonic lung
explants epithelially transfected with AdDcn at 2 × 1010 pfu/ml showed complete
restoration of lung branching morphogenesis from the TGF-
-mediated
inhibition regardless of the presence of exogenously added TGF-
1
(95.5%) or TGF-
2 (103.4%). In comparison, both TGF-
1 and
TGF-
2 efficiently decreased lung branching morphogenesis in cultured
lungs transfected with control virus of the same dose (66.5% for
TGF-
1 and 70.2% for TGF-
2; P < 0.05). Therefore, the above observation that decorin recombinant
adenovirus pfu dependently release TGF-
-induced inhibition on lung
branching morphogenesis further supports the conclusion that decorin
counteracts TGF-
-mediated negative signaling on epithelial branching
morphogenesis during early embryonic lung development.
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Restoration of TGF--mediated lung
branching inhibition in AdDcn-microinjected embryonic lungs treated
with human decorin antisense ODN. To ascertain the
specificity of the effects reported in Intratracheal
microinjection of AdDcn prevents exogenous
TGF-
-mediated inhibition of embryonic lung branching
morphogenesis in culture, AdDcn-infected
lung explants were treated with an antisense ODN complementary to the
human decorin cDNA sequence (28). The results showed a dose-dependent
suppression of the steady-state level of human decorin-specific
transcripts in AdDcn-treated lungs cultured with human decorin
antisense ODN as measured by human decorin competitive PCR (Fig.
5). Antisense ODN specific to human decorin (30 µM) decreased the exogenous decorin mRNA level to a remnant (4.4%) in comparison with medium control-treated AdDcn-infected lungs
in culture, whereas either scrambled or sense ODN to human ODN at
experimental doses showed no effect on exogenous decorin transgene
expression in cultured embryonic lungs. In support of the above
results, both endogenous decorin and biglycan mRNA amounts were
quantified and confirmed not to be changed in response to human decorin
ODN treatment in cultured lungs (Fig.
5A). Thus the human decorin
antisense ODN markedly and specifically inhibited exogenous transgene
expression in lung explants intratracheally microinjected with AdDcn,
without negatively affecting endogenous gene expression in lungs in
culture.
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In the next step, human decorin antisense ODN was used to test the
specificity of the effects of decorin transgene-mediated reversal on
TGF--induced negative regulation of embryonic lung branching
morphogenesis. Both TGF-
1 (5 ng/ml) and TGF-
2 (0.1 ng/ml) inhibit
lung morphogenesis, as measured by the number of terminal sacs, in
cultured lungs microinjected with the control virus regardless of the
presence of either sense or antisense human decorin ODN in culture
(Fig. 6). Exogenous decorin transgene overexpression in lung epithelium overcame the negative
regulation of lung branching with either TGF-
1 or TGF-
2 added
exogenously to the culture medium in the presence of either human
decorin sense (Fig. 6) or scrambled (data not shown) ODN. In contrast, TGF-
-induced lung epithelial branching inhibition was restored in
AdDcn microinjected lungs coadministered with antisense ODN to human
decorin in culture (Fig. 6), whereas a simultaneous inhibition of
exogenous human decorin mRNA expression was shown (Fig.
5A). Therefore, the observation that
TGF-
inhibited lung branching in AdDcn-microinjected lungs treated
with human decorin antisense ODN indicates that decorin is specifically
and directly involved in TGF-
signaling-mediated growth control of
embryonic lung epithelial branching.
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Suppression of both cyclin A and SP-C gene expression
by exogenous TGF- is abolished in cultured embryonic
lungs overexpressing decorin. TGF-
-induced growth
arrest occurs late in the G1 phase and is accompanied by a reduction in the steady-state level of cyclin A
mRNA in mammalian cells. Our data indicate that AdDcn induces
refractoriness to TGF-
-induced lung branching inhibition and
epithelial growth in lungs in culture. Therefore, cyclin A was used as
a marker gene to evaluate the rate of cellular
proliferation during embryonic lung development in the present study.
As shown in the electrophoretic pattern of cyclin A competitive PCR
(Fig. 7), we found that both TGF-
1 and
TGF-
2 exert inhibitory effects on cyclin A mRNA expression in
cultured lungs regardless of the control virus microinjection and the
presence of sense control human decorin ODN. On the other hand, neither
TGF-
1 nor TGF-
2 repressed cyclin A gene expression in lungs
overexpressing decorin, indicating that decorin transgene
expression prevented the TGF-
-mediated antiproliferative effect on
embryonic lung growth. Furthermore, the TGF-
-mediated growth
inhibition effect on lung development was restored in
AdDcn-microinjected lungs cultured with human decorin antisense ODN.
The above observation that decorin overrides TGF-
-mediated negative
regulation of lung epithelial growth was also confirmed by the
measurement of proliferating cell nuclear antigen, a mitogenic index of
proliferating cells, through proliferating cell nuclear antigen
morphometry on cultured lung sections (data not shown). These findings
further support our conclusion that decorin overexpression specifically
blocks antiproliferative signaling by TGF-
during embryonic lung
development.
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Gene expression of SPs is well characterized to be downregulated by
TGF- signaling during embryonic lung development. Because SPs are
primarily expressed in lung epithelium during embryonic lung
development, SP-C was chosen as a lung epithelium-specific differentiation marker to evaluate the function of decorin on TGF-
signaling during early lung development. When added to the culture
medium, TGF-
1 or TGF-
2 reduced epithelial SP-C gene expression in
medium control lungs (data not shown) as well as in lungs infected with
control virus (Fig. 7) as demonstrated by the competitive PCR
electrophoretic pattern for SP-C mRNA quantification. However,
exogenous TGF-
1 or TGF-
2 no longer inhibited epithelial SP-C mRNA
expression when lungs were epithelially infected with AdDcn.
Additionally, TGF-
-mediated negative regulation of SP-C gene
expression was restored in AdDcn-infected lungs in the presence of
antisense ODN to human decorin.
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DISCUSSION |
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The TGF- signaling pathway has been implicated in the normal
temporospatial pattern of lung morphogenesis and pulmonary-specific gene expression. Signaling molecules including TGF-
ligands, TGF-
receptors, and Smad-2, -3, and -4 have been the focus of studies to elucidate the functional significance of
TGF-
signaling during normal lung development and pathogenesis (30,
39, 40, 42). However, little is known about the biological role of the TGF-
-interacting proteoglycans during lung growth and injury. Therefore, this is the first report to demonstrate that decorin can act
as a TGF-
antagonist to attenuate exogenous TGF-
signaling during
lung morphogenesis in culture, establishing the rationale that decorin,
a naturally occurring TGF-
inhibitor, may be potentially useful in
ameliorating TGF-
overproduction during lung growth, injury, and
repair. Using a gene transfer approach in embryonic mouse lungs in
culture, we discovered that decorin overexpression released the lungs
from exogenous TGF-
-mediated branching inhibition to regain a normal
phenotype of branching morphogenesis. Additionally, decorin transgene
expression in the lung epithelium prevented both the TGF-
-induced
antiproliferative effect and the downregulation of SP gene expression.
The experimental evidence we present herein suggests that decorin might
be a therapeutically useful component of a negative loop that can
attenuate excessive TGF-
signaling during lung growth and development.
Decorin is capable of binding to TGF- ligand with high affinity,
resulting in the formation of inactive TGF-
-decorin complexes (12).
Decorin therefore acts in an analogous manner to TGF-
neutralizing
antibodies. In support of this notion, we showed that both decorin
overexpression and the addition of TGF-
neutralizing antibodies are
effective in blocking TGF-
-mediated inhibition of lung branching
morphogenesis in culture. TGF-
is known to induce
G1/S phase cell cycle arrest in
lung epithelial cells as demonstrated by the reduction of cyclin A gene
expression. Formation of either decorin-TGF-
or TGF-
antigen-antibody complexes apparently inactivates TGF-
and therefore
results in the release of TGF-
-induced pulmonary epithelial cell
cycle arrest. However, unlike TGF-
antibodies, decorin is a
naturally occurring inhibitor of TGF-
bioactivity. Decorin can also
be overproduced at high levels in lung tissue with recombinant
adenovirus-mediated gene transfer as presented in the present study.
Excessive TGF-
signaling has been implicated in lung diseases such
as BPD. A reduction in decorin mRNA levels in the whole lung and
decreased decorin immunoreactivity have been detected in lungs
developing hyperoxic injury (34).
Increased TGF- production appears to be characteristic of several
fibrotic diseases including hepatic cirrhosis, pulmonary fibrosis, and
glomerular sclerosis. Administration of decorin protein inhibits the
increased production of extracellular matrix and attenuates glomerular
sclerosis in a rat model of glomerulonephritis (4). Gene transfer of
decorin cDNA also increased the decorin protein present in the kidney
where it markedly ameliorated kidney fibrosis with a simultaneous
reduction in the expression level of TGF-
1 in the renal glomeruli
(17). In bleomycin-induced lung fibrosis, neutralization of TGF-
by
systemic treatment with its antibodies reduced lung collagen
accumulation (9). Likewise, decorin has been demonstrated to be as
effective as TGF-
neutralizing antibodies in exerting an
antifibrotic effect, including downregulation of matrix protein
expression in a bleomycin hamster model of lung fibrosis (8).
Therefore, decorin offers a novel rationale for therapeutic
intervention that may be useful in treating fibrotic lung diseases
associated with an overproduction of TGF-
.
Decorin is a secreted protein that is deposited in the lung
interstitium as localized by decorin immunohistochemistry in the present study. Decorin, like other leucine-rich proteoglycans, is a
connective tissue organizer, orienting and ordering collagen fibrils by
binding with collagen proteins, thereby establishing the exact topology
of fibrillar collagens in tissue. Besides TGF-, decorin can also
bind to a variety of adhesive and nonadhesive proteins including
fibronectin, thrombospondin, and various types of collagens (16). In
null mutant animals, disruption of the decorin gene leads to skin
fragility and abnormal collagen morphology, characterized by
uncontrolled lateral fusion of fibrils (5). Being capable of
simultaneously binding to both TGF-
ligands and collagen fibrils,
decorin is able to tether TGF-
onto the interstitial tissue matrix,
resulting in sequestration of TGF-
ligands. Therefore, we postulate
that ectopically overexpressed decorin in lung epithelium after
intratracheal microinjection of recombinant decorin adenovirus forms a
steric barrier through formation of bioinactive TGF-
-decorin
heterodimers to prevent soluble TGF-
from accessing TGF-
-receptor
complexes and thereby abolishes the negative effect of exogenous
TGF-
signaling on lung growth and development.
Decorin antisense ODN experiments performed herein were designed to
demonstrate that the observed refractoriness to exogenous TGF--mediated lung growth inhibition after decorin adenovirus injection was due directly and specifically to decorin overexpression in lung tissue. We also showed that antisense ODN to human decorin effectively suppresses exogenous decorin gene expression without affecting endogenous murine decorin gene expression in mouse lung explants transfected with AdDcn (14). Our results that the antisense ODN to human decorin restored exogenous TGF-
-mediated epithelial branching inhibition in AdDcn-infected lung cells or tissue
indicate that decorin exerts specific anti-TGF-
effects during lung
development. In support of the above conclusion, the expression levels
of other related small leucine-rich proteoglycans including biglycan
and fibromodulin were unaltered regardless of the presence of either decorin transgene or decorin antisense ODN. Thus it is decorin, not
other related TGF-
interaction proteoglycans, that is involved in
antagonizing exogenous TGF-
-mediated negative regulation of lung
growth and differentiation.
In addition to decorin, TGF--interacting proteins and proteoglycans
such as betaglycan (TGF-
type III receptor), biglycan, fibromodulin,
and endoglin may also be implicated in the negative regulation of
TGF-
signaling and therefore may affect key aspects of pulmonary
morphogenesis and injury. Betaglycan is a TGF-
ligand presenter that
physically associates with TGF-
type II receptor. Betaglycan
increases the affinity between TGF-
ligand and TGF-
type II
receptor (41). Endoglin is a dimeric TGF-
1 and TGF-
3 binding
protein of endothelial cells that modulates cellular responses to
TGF-
s and can form heteromeric signaling complexes with TGF-
type
II and type I receptors (22). The endoglin gene is mutated in
Osler-Weber-Rendu hereditary telangiectasia type 1, a condition that is
characterized by large intrapulmonary arteriovenous malformations (26).
Although we provide evidence of a functional role for decorin as a
natural TGF-
inhibitor that can counteract increased TGF-
signaling during lung morphogenesis, injury, and repair, the biological
significance of other related TGF-
-associated proteoglycans remains
to be elucidated.
Using an adenovector-mediated gene transfer strategy, we show the
promise of decorin as a potential treatment or preventive measure for
TGF--mediated lung diseases through decorin gene therapy.
Intratracheal microinjection of recombinant adenovirus to introduce
transgene expression may be of medical significance because of its
simplicity, apparent safety, and lack of toxicity (1). The
replication-deficient adenovirus has a wide spectrum of host cell
range, including cells in both normal and pathological states. The
adenovector offers a high efficiency of exogenous gene delivery and
resultant transgene expression. The replication-defective virus is
incapable of integrating into the host genome and thus does not
negatively impact on intrinsic host cell function. Using the decorin
recombinant adenovirus, we have shown the feasibility of achieving high
expression of the decorin transgene in lung epithelial cells during
embryonic lung development. The present report therefore supports the
hypothesis that decorin, as a naturally occurring inhibitor of
excessive TGF-
activity, may be particularly useful in designing
novel strategies to treat lung diseases due to aberrant TGF-
production.
We conclude that decorin can play an important functional role in
regulating TGF- signaling during lung growth, differentiation, and
development through sequestering bioactive TGF-
ligands. Decorin, as
a naturally occurring biological molecule that antagonizes TGF-
bioactivity, may be potentially useful in designing new rational
therapeutic strategies to ameliorate excessive TGF-
signaling in
injured lungs. It is possible that decorin may, together with other
proteoglycans, orchestrate TGF-
-mediated signaling on cellular
growth, differentiation, and gene expression during lung development.
We speculate that finely regulated TGF-
signaling through decorin
may serve to modulate and therefore balance the positive functions of
signaling by other peptide growth factor pathways, including
epidermal growth factor, fibroblast growth factor, platelet-derived
growth factor, and vascular endothelial growth factor, which exert
positive and permissive influences on lung development.
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ACKNOWLEDGEMENTS |
---|
This work is supported by National Heart, Lung, and Blood Institute Grants HL-44060, HL-44977, HL-60231 (to D. Warburton), and HL-61286 (to J. Zhao).
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FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Warburton, USC Center for Craniofacial Molecular Biology, 2250 Alcazar St., CSA 103, Los Angeles, CA 90033 (E-mail: dwarburton{at}chla.usc.edu).
Received 26 January 1999; accepted in final form 8 April 1999.
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