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Am J Physiol Lung Cell Mol Physiol 286: L1179-L1187, 2004; doi:10.1152/ajplung.00188.2003
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Matrix GLA protein modulates branching morphogenesis in fetal rat lung

Kirk A. Gilbert and Stephen R. Rannels

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Submitted 10 December 2003 ; accepted in final form 7 January 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The regulation of matrix {gamma}-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

lung development; vitamin K; sonic hedgehog; patched; bone morphogenetic protein-4; fibroblast growth factor-10; fibronectin; explants; mesenchyme; {gamma}-carboxyglutamic acid

MAMMALIAN LUNG BRANCHING MORPHOGENESIS is a complex growth process involving temporal and spatial expression of numerous gene products from diverse gene families. From the initial formation of the rudimentary lung bud, a continual signaling interaction occurs between the epithelial and mesenchymal compartments to ensure proper growth and differentiation of the developing lung. The end result is a highly branched organ capable of efficient gas exchange over impressively large epithelial and endothelial surface areas. The cellular and molecular regulation of lung growth is an area of intense research, and, although the molecular regulation of lung branching morphogenesis has been the subject of recent reviews (9, 43), the process is still incompletely understood.

Matrix {gamma}-carboxyglutamic acid (GLA) protein (MGP) is a vitamin K-dependent extracellular matrix protein originally isolated from bovine bone and subsequently found in many soft tissues, with highest expression observed in lung (16). Interestingly, MGP accumulates in the extracellular matrix of only cartilage and bone (18, 33), suggesting that its broad tissue distribution warrants important, yet undefined, roles for the protein. MGP belongs to a limited family of proteins that contain posttranslationally modified GLA residues, with the blood-coagulating proteins being the most well-characterized members of this family. The {gamma}-carboxylase responsible for converting glutamate to GLA requires vitamin K hydroquinone as a cofactor, and vitamin K antagonists such as warfarin impair protein function by preventing the recycling of oxidized vitamin K epoxide back to its reduced hydroquinone form (15).

Results from numerous studies suggest that MGP is involved in cell growth and differentiation. MGP is expressed in vitro in a large variety of cells, including vascular smooth muscle cells (14, 30, 42), osteoblasts (1, 7), chondrocytes (1, 7, 44), fibroblasts (7), kidney epithelial cells (6), pulmonary epithelial cells (34), breast epithelial cells (11), and cardiac myocytes (16). In most cell culture models, MGP expression increases with increasing cell density, with the highest rates of expression and secretion occurring in postconfluent, nondividing cells (6). During early development, in situ hybridization reveals that MGP expression is restricted to the epithelial-mesenchymal border in developing lung and limb buds (26). High MGP expression is observed in embryonic kidney and calvaria, with peak mRNA and protein levels occurring during the perinatal period in many developing organs, such as kidney, lung, and spleen (47). Taken together, the cell-tissue profile, location, and time course of expression of MGP underscore the complexity of its function and lend support to its potential regulatory role in cellular processes of growth and differentiation.

Although present in most vertebrate tissues examined, the physiological function of MGP is not completely understood. Recently, it has been demonstrated that mice lacking MGP develop extensive vascular and cartilaginous calcification to such an extreme that null mutant animals die within 2 mo postpartum due to rupture of the thoracic or abdominal aorta (27). The extensive arterial calcification, osteopenia, and growth plate inhibition present in these MGP–/– animals suggest that one of MGP's primary roles may be as an inhibitor of tissue calcification. Indeed, virally driven MGP overexpression in cultured embryonic chick chondrocytes results in blocked mineralization of these cells (44). Similarly, in vivo overexpression of MGP in developing chick limb bud causes inhibition of cartilage mineralization, delayed chondrocyte maturation, and inhibition of endochondral bone ossification (44).

Given the increasing evidence that MGP is an important regulator of growth and differentiation and that MGP is highly expressed in the lung, we sought to further define the role of MGP in the process of lung development. This study provides a detailed description of lung MGP expression over a time course of pre- and postnatal periods of lung growth and development. In addition, fetal lung explants were used as a model of lung branching morphogenesis to specifically address MGP's involvement in this process.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of fetal lung explants. Timed pregnant adult Wistar rats were purchased (Charles River Laboratories) and housed according to institutional policies. Pregnant rats were euthanized 13–14 days postcoitum, and embryonic (E) 13- to 14-day fetal rat pups were aseptically dissected from the uterine decidua into ice-cold, sterile PBS. With the aid of a dissecting stereomicroscope, lung bud rudiments were dissected free from the embryos and placed on cell culture inserts (0.4 µm size; Falcon) in six-well tissue culture plates (Falcon). Lung explants were cultured in BGJb medium (Fitton-Jackson Modification; Life Technologies) supplemented with 5% carbon-stripped fetal calf serum. Typical culture conditions consisted of three to five explants per well. The medium was changed at 24-h intervals, and lung explants were photographed on a daily basis over the culture interval. At specific times in the culture interval, explants were removed from the inserts and flash-frozen in liquid nitrogen, with subsequent storage at –80°C until RNA isolation was performed. For experiments involving immunohistochemistry, explants were carefully removed from the inserts and fixed for 1 h at 4°C in 4% paraformaldehye, followed by dehydration through an alcohol series and storage at –20°C until further use.

Lung RNA isolation. Total RNA was isolated from fetal lung explants based on the method of Chomczynski and Sacchi (12). Briefly, lung explants were homogenized in 0.75 ml of Tri-Reagent (Molecular Research), with manual disruption of the tissue by using conical-shaped pestles designed to fit 1.5-ml microcentrifuge tubes. Samples were homogenized on ice according to the Tri-Reagent protocol. RNA was quantified by spectrophotometry and stored at –80°C for subsequent use in RT-PCR; DNase digestion of total RNA samples was performed before spectrophotometric quantitation.

RT-PCR analysis. Total RNA (0.1–1 µg) was reverse-transcribed for 1 h at 42°C by using poly(dT) primers, 500 µM deoxynucleotides, 10 mM dithiothreitol, 1x RT buffer, and an RNase H– reverse transcriptase (Superscript H–; Invitrogen) in a total volume of 20 µl. After RT, samples were variably diluted for subsequent PCR amplification; dilutions were empirically based on relative abundance of the respective gene product in fetal lung. MGP mRNA expression was quantified by competitive PCR, as described previously (17). Both endogenous MGP and an MGP cDNA mimic amplified with equal efficiency by using the same set of primers that included forward primer 5'-CGGAGAAATGCCAACACCTT-3' and reverse primer 5'-GCAACGAACAATCTGTG-3'. PCR reactions included 200 fg of MGP mimic, 5 ng of reverse-transcribed total RNA containing 200 µM each of deoxynucleotide, 5 µl 10x polymerase buffer, 250 µM each of forward and reverse primers, and one unit of Pfu polymerase (Clontech Laboratories) in a total volume of 50 µl. Products were amplified in a programmable thermocycler under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C (20 s), annealing at 50°C (30 s), extension at 72°C (1 min), with a final extension at 72°C for 8 min. For analysis, amplified products were run on 1.5% agarose gels with ethidium bromide staining. Gels were scanned on a Fluorimager imaging system (Molecular Dynamics) and quantified with the ImageQuant software package. An accurate measure of experimentally induced changes in MGP mRNA expression for any given series of samples could be determined by calculating the ratio of MGP to mimic.

Semiquantitative PCR was used to measure sonic hedgehog (Shh), patched (Ptc), bone morphogenetic protein (BMP)-4, and fibroblast growth factor (FGF)-10 mRNA expression in cultured explants, with the rat homologue to prokaryotic elongation factor Tu (EFTu) or GAPDH used to normalize expression of these genes. Gene-specific primers included the following: Shh forward primer 5'-GGAAAACACTGGAGCAGACC-3' from mouse sequence base pairs 28–47; Shh reverse primer 5'-CCACGGAGTTCTCTGCTTTC-3' from base pairs 335–316 to give a 309-base pair product for Shh amplification; Ptc forward primer 5-TCGCTATGGAGCAGATTTCC-3' from mouse sequence base pairs 136–155; Ptc reverse primer 5'-AGTGCTGAGTCCAGGTGTTG-3' from base pairs 505–486 to give a 371-base pair product for PTC amplification; BMP-4 forward primer 5'-CTTCTCTGAGCCTTTCCAGC-3' from mouse sequence base pairs 266–285; BMP-4 reverse primer 5'-CCTGGGATGTTCTCCAGATG-3' from base pairs 750–731 to give a 486-base pair product for BMP-4 amplification; FGF-10 forward primer 5'-ACTGACACATTGTGCCTCAGCC-3' from rat sequence base pairs 14–35; FGF-10 reverse primer 5'-GCCACATACATTTGCCTGCC-3' from base pairs 556–537 to give a 554-base pair product for FGF-10 amplification; GAPDH forward primer 5'-ACCACAGTCCATGCCATCAC-3'; GAPDH reverse primer 5'-CAAAGGTGGAGGAATGGGAG-3'; EFTu forward primer 5'-CACTTTGGGTGTGAAACAGC-3'; and EFTu reverse primer 5'-GAACGACGATCGATCTTCTC-3'.

Lung protein isolation and Western blot analysis. Proteins (20–25 µg) were separated by SDS-PAGE on 15% SDS-polyacrylamide gels, followed by transfer to nitrocellulose membranes with the use of a Transblot apparatus (Bio-Rad Laboratories). Immunodetection of transferred proteins was performed by standard Western blot procedure. Briefly, nonspecific binding sites were blocked with blotto (5% nonfat dry milk and 0.05% Tween-20 in PBS) for 1 h. Membranes were incubated in primary antibody (1:2,000 dilution in blotto) for 2 h at room temperature and then washed extensively. A subsequent incubation in a solution containing the biotinylated secondary antibody (Vector Laboratories) was performed for 1 h, followed by another round of extensive washing. Immunodetection was visualized by using enhanced chemiluminescence detection reagents (ECL; Amersham) and short exposure to X-ray film.

Immunohistochemistry. For experiments aimed at in situ localization of specific proteins, lungs were fixed and paraffin embedded for histological processing into 4-µm tissue sections. Sections were mounted on glass slides, deparaffinized, and rehydrated. Antigen retrieval was accomplished by microwave heating in 10 mM citric acid (pH 6.0). Sections were then incubated in blocking solution containing 10% goat serum in PBS Tween-20. Primary antibody incubation was performed overnight at 4°C, followed by a series of washes in Tris-buffered saline. Detection of primary antibody was performed by using a Vectastain avidin-biotin complex kit (Vector Laboratories). Vector VIP was the substrate chromogen with methyl green used as a counterstain. Parallel sections were stained with hematoxylin-eosin to visualize general morphology.

Antibody treatment of fetal lung explants. Two synthetic peptides corresponding to specific rat MGP protein sequences were used to generate anti-MGP antibodies. The first sequence was a 12-residue peptide located in the propeptide-like region of rat MGP and contains the sequence NH2-P F T N R R N A N T F I-COOH. This region binds the vitamin K-dependent {gamma}-carboxylase required for conversion of glutamic acid residues to {gamma}-carboxylated GLA residues. The antibodies generated with this peptide have been termed the "midpeptide antibody." The second peptide sequence was a 12-residue peptide located at the extreme C-terminal portion of rat MGP and contains the sequence NH2-Y N R Y F R Q R R G A K-COOH. The antibodies generated with this peptide were termed the "C-terminal antibody." Affinity-purified antibodies isolated from rabbits challenged with these peptide immunogens were routinely used in explant cultures at a 1:100 to 1:200 dilution of 1 mg/ml antibody, for a final concentration of 5–10 ng/µl. Overnight incubation of anti-MGP antibodies with the respective peptides used for generating the antibodies before their use in explant culture or immunohistochemistry was performed to successfully block anti-MGP antibody effects and/or immunostaining.

The fibronectin antibody was a mouse monoclonal antibody (EP5; sc-8422, Santa Cruz Biotechnology; stock concentration 200 µg/ml) raised against a recombinant protein corresponding to fibronectin of human origin; dilution of this antibody for use in explant culture experiments was 1:100, resulting in a final concentration of 2 ng/µl.

Microinjection of antibodies into the lung lumen. A microinjector system (Narishige, MO-202) was mounted to a stereoscope for injection of nanoliter amounts of undiluted MGP antibodies or PBS (control) into the lumen of lung explants positioned on cell culture inserts. Micropipettes were drawn by using a Narishige PC-10 device, and injection pressures were generated with a FemtoJet (Eppendorf) system. Injection volumes were precalibrated, and both control and experimental solutions were initially colorized with Coomassie blue to visualize fluid distribution. Subsequent experiments were performed without the addition of Coomassie blue. Beginning on day 2 of culture, ~50–100 nl of antibody were injected directly into the trachea and distal luminal spaces, resulting in an even distribution of solution throughout the lumen of the whole lung or individual lobes. It was estimated that the local exposure of antibody was as much as four times that used for external, mesenchymal application. After microinjections, cell culture inserts were replaced, and explants were incubated at 37°C for the remainder of the culture interval.

Quantitation of explant morphology. To assess morphological changes in fetal lung explants, terminal lung buds were counted with the aid of a microscope and statistically compared across culture conditions. Terminal lung buds were defined as acinar structures separated by distinct septae located at the periphery of the growing explants.

Data analysis. Quantitation of morphogenesis was as described above. For some experiments, such as immunostaining or in situ analysis of specific tissue sections or specimens, results were assessed by using a qualitative approach. PCR experiments were repeated at least three times for statistical comparisons. Significant differences in gene expression or morphological measurements resulting from various treatment protocols were determined by using Student's t-test. Results were considered significant at values of P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
MGP expression in developing lung. MGP mRNA levels were determined in rat lung from gestational day 13 (E13) through the perinatal period and in the adult by using a competitive PCR assay, as previously described (17) (Fig. 1). MGP mRNA was found in early lung primordia at E15 and continued to rise during gestation, with a small peak occurring just before birth. Postnatally, levels dropped slightly and were steadily maintained until a sharp increase was observed during the alveolarization interval, beginning in the second postnatal week. MGP mRNA was most highly expressed in adult lung. These results demonstrate that MGP message expression is developmentally regulated, with higher levels of expression occurring during periods of lung development traditionally characterized by low-cell proliferation and increased differentiation.



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  		Fig. 1. Embryonic and postnatal expression of matrix {gamma}-carboxyglutamic acid protein (MGP). MGP mRNA expression was detected as early as embryonic day (E) 15, and expression increased to peak fetal levels just before birth. Postnatally, expression remained relatively stable in the first week, followed by a dramatic rise in message levels near the end of the second postnatal week, with highest levels observed in the adult. PN, postnatal day. Inset: a gel is shown from a competitive PCR reaction in which MGP mimic was added to each sample of reverse-transcribed RNA. The ratio of MGP (bottom band) to mimic (top band) gives an accurate quantitation of MGP mRNA present in the RNA sample.

 
In addition to overall changes in MGP expression during lung development, site-specific localization of protein was determined. Fetal lungs were prepared as described for immunohistochemical localization of MGP protein by using the C-terminal anti-MGP antibody (Fig. 2). MGP immunostaining was associated predominantly with the mesenchymal cells surrounding epithelial tips of distal buds (Fig. 2, A and C). This staining pattern was compared with that of pro-surfactant protein (SP)-C in adjacent sections, where pro-SP-C was found in the epithelium of early embryonic lungs (Fig. 2, B and D).



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  		Fig. 2. Immunolocalization of MGP. MGP was immunolocalized in rat lung histological sections from several stages of fetal and postnatal growth. Vector VIP was the substrate chromogen, with positive MGP staining appearing as a dark purple precipitate; methyl green was used as a counterstain. A: E15 fetal lung reveals MGP deposition predominantly in the mesenchyme surrounding the growing epithelial tips, accompanied by light epithelial expression. B: adjacent section processed for pro-surfactant protein-C (SP-C) immunolocalization demonstrated positive epithelial expression of pro-SP-C at E15. C: E17 lung shows similar pattern of MGP expression as that seen at E15, with highest expression in peripheral mesenchyme. D: pro-SP-C protein expression at E17 is localized to epithelial tubules. E: at E20, MGP localization remains throughout the mesenchyme, with more dense epithelial expression in bronchiolar structures. F: boxed region from E at higher magnification shows mesenchymal and epithelial expression of MGP. G: PN5 lung section incubated with MGP primary antibody previously blocked with C-terminal peptide shows no positive staining, demonstrating specificity of the C-terminal MGP antibody. H: adjacent PN5 section to that shown in G shows continued MGP localization to the bronchiolar regions, with maintenance of light expression in the lung parenchyma. I: higher magnification from H (boxed region) shows a more detailed view of MGP expression in PN5 lung parenchyma, some of which is highlighted by arrowheads. J and K: with continued alveolar maturation and thinning of the blood-gas barrier, MGP expression in PN8 lung continues in the bronchiolar airways and lung parenchyma. Arrowheads in K highlight some of the parenchymal staining. L and M: similar to earlier postnatal lung sections, PN14 MGP lung expression is localized to both airway and alveolar epithelial cells (arrowheads). N, O, and P: adult rat lung sections express MGP in the bronchiolar and alveolar epithelial regions. N: no staining was observed with blocked primary antibody. P: close-up of the boxed region in O, with arrowheads highlighting some of the alveolar deposition of MGP in adult lung.

 
Later in gestation, MGP protein became more widely distributed in the lung. By E20, additional staining was observed in the bronchial epithelium, along with continued staining in the mesenchyme (Fig. 2, E and F). As the mesenchyme became more differentiated in early postnatal lung, MGP was localized to airway epithelial cells and vascular walls, as well as light deposition in the surrounding parenchyma (Fig. 2, H and M). In the adult, light distribution throughout the parenchyma and airway cells was still evident, with more intense staining lining the airways (Fig. 2, O and P). This pattern of distribution in adult lung is consistent with other reports (26, 27). No staining was observed in the absence of primary antibody or if anti-MGP antibody was preincubated with a blocking peptide (Fig. 2, G and N).

To further investigate MGP regulation in the developing lung, particularly during early branching morphogenesis, an in vitro explant model was used. Explants remain in the pseudoglandular phase of lung development during culture. This model provides an ideal system in which to examine specific regulators of lung growth and differentiation by using reagent-addition protocols. Increased MGP mRNA expression over the culture interval mimics the early embryonic in vivo time course (17). A stereotypic morphological pattern of explant growth was observed in control cultures, characterized by a high degree of distal epithelial branching (Fig. 3A). Interfering with MGP function by addition of anti-MGP antibodies to the culture medium revealed a completely different explant morphology after 4 days in culture. Compared with control cultures, anti-MGP antibody treatment induced a grossly dilated morphology characterized by elongated epithelial tubules (Fig. 3A). By the end of the culture interval, lung branching, as assessed by terminal lung bud count, was significantly reduced in anti-MGP antibody-treated explants by using either the midpeptide or C-terminal peptide-derived antibodies (Fig. 3C). Both of these antibodies were specific for MGP, as assessed by Western blotting of purified MGP isolated from rat femurs (Fig. 3B). In contrast to the external application of anti-MGP antibody, explant growth was not altered when antibodies were microinjected directly into the lumen of specific lobes of intact explants (Fig. 4), suggesting that endogenous MGP exerts its effects primarily through the mesenchyme.



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  		Fig. 3. Terminal lung bud counts are reduced with MGP antibody treatment. Two different domain-specific antibodies to MGP were added independently to explant culture medium, and explants were harvested after 4 days in culture. A: the photomicrographs shown are representative of the explant morphology normally observed under control conditions or in the presence of peptide-specific anti-MGP antibodies. B: Western blot of purified MGP isolated from rat femur shows specificity of both the C-terminal antibody (C-term Ab) and mid-peptide antibody (Mid-Pep Ab) for MGP. MW, molecular weight. C: 16 explants each were photographed and used for analysis of terminal bud counts. Values are means ± SE. Significant reduction in terminal lung buds was observed in explants treated with either anti-MGP antibody compared with control explants, *P < 0.001. CSFCS, carbon-stripped fetal calf serum.

 


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  		Fig. 4. Luminal microinjection of MGP antibody does not alter explant growth. Intact explants were microinjected after 24 h in culture with 50–100 nl of either anti-MGP antibody (1 mg/ml) or PBS (control) into each luminal space (arrows indicate which spaces were microinjected). Limiting antibody exposure to the epithelium of intact explants did not significantly alter gross lung morphology after 3 days in culture. The solutions that were microinjected into the explants shown above contained 0.1% Coomassie blue to better visualize fluid distribution. Subsequent experiments were performed in the absence of the dye with identical results.

 
A series of experiments demonstrated the dynamic nature of the anti-MGP antibody-induced dilation of fetal lung explants. The presence of antibody throughout the culture interval resulted in marked dilation of distal airways compared with untreated lungs (Fig. 5, A and B). When C-terminal antibody was externally applied after 1 or 2 days of culture, distal bud dilation was evident, whereas morphology of medial structures was comparable to controls (Fig. 5, C and D, respectively). Conversely, when antibody was withdrawn after 1 or 2 days in culture, normal terminal branching and bud formation resumed, with the more medial structures remaining dilated (Fig. 5, E and F, respectively). Blocking peptide was also added to explant cultures following anti-MGP antibody withdrawal to block any residual antibody that may have remained in the tissue (Fig. 5, G and H). The resulting morphology was similar to antibody withdrawal in the absence of blocking peptide, as seen in Fig. 5, E and F. Peptide alone had no effect on gross explant morphology and growth (Fig. 5I). Preincubation of anti-MGP antibody with its immunogenic peptide completely blocked the antibody-induced dilation (Fig. 5J). Thus addition or withdrawal of anti-MGP antibody at different times during the culture interval altered only the most distal morphology. These results suggest that MGP expression and function are both site specific and time dependent, requiring cyclic deposition with each new generation of epithelial branching.



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  		Fig. 5. Effects of MGP antibody addition and withdrawal on explant morphology. C-term Ab (final concentration of 10 ng/l) added to the medium surrounding explants for the entire culture interval (B) resulted in dramatically dilated airways and tubules compared with the highly branched control cultures that terminated in numerous distal buds (A). When antibody was withdrawn after 1 day (D1; E) or 2 days (D2; F) in culture, proper branching and bud formation resumed; the extent of recovery was directly dependent on how long the antibody was present. F and H: for example, explants treated with antibody for 2 days had many dilated proximal structures and a rim of normal distal buds. C and D: applying antibody at different times during the culture interval altered only the most distal morphology. G and H: addition of blocking peptide following antibody withdrawal did not further affect morphology, suggesting that free antibody did not remain in the cultures. Indeed, blocking peptide alone had no effect on explant morphology (I), with resulting growth resembling control conditions (A). J: finally, preincubation of anti-MGP antibodies with blocking peptide before the addition to explant culture completely blocked the antibody-induced inhibition of branching. D3, day 3; D0, day 0.

 
Based on our results demonstrating altered branching with MGP antibody treatment (Figs. 3 and 5), we examined the expression of some genes known to play a role in lung branching morphogenesis in antibody-treated explants (Table 1). Anti-MGP antibody treatment caused a significant downregulation of MGP mRNA expression (Table 1 and Fig. 6). Antibody treatment also significantly downregulated the expression of Shh and Ptc mRNA, whereas the expression of Bmp-4 and Fgf-10 was not significantly different from that of controls (Table 1). Interestingly, antifibronectin antibody treatment of fetal explants caused a similar dilated morphology as that seen with anti-MGP (Fig. 6), reflected by a 21% decrease in terminal bud counts (71.75 ± 4.66 in control vs. 56.75 ± 1.38 in fibronectin antibody-treated explants; Fig. 6B). Combined treatment with both anti-MGP and antifibronectin antibodies resulted in an even greater reduction in terminal bud counts. Additionally, antifibronectin antibody also caused a similar decrease in MGP mRNA expression as that seen with anti-MGP antibody (Fig. 6C); however, the combination of antifibronectin and anti-MGP antibodies did not further reduce MGP mRNA expression. These results suggest that these two proteins coordinately contribute to regulation of branching morphogenesis in the developing lung.


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  		Table 1. Gene expression analysis following anti-MGP antibody treatment of fetal explants

 


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  		Fig. 6. Antifibronectin antibodies reduce fetal lung branching and MGP mRNA expression. Explants treated for 4 days with antibodies to fibronectin (FN; 1:100 dilution of 200 g/ml stock), MGP (1:100 dilution of 1 mg/ml stock), or in combination were analyzed for changes in morphology and for alterations in MGP mRNA expression. A similar dilated morphology was observed with either antibody treatment (A), resulting in a significant reduction in terminal bud counts compared with controls (B). Combined antibody treatment resulted in a further reduction in terminal branching compared with treatment with only 1 of the antibodies. In addition to altered branching, antibody treatments also reduced MGP mRNA expression, as determined by competitive PCR (C). Terminal bud counts were calculated from 4 explants each and are expressed as means ± SE. *P < 0.03; #P < 0.001. RNA expression data are expressed as means ± SE from 3 separate experiments. *P < 0.001. Control RNA expression was normalized to a value of 1 for comparison between experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results identify MGP as a potential regulatory factor in the process of lung growth and development. The time course of expression during the pre- and postnatal periods suggests that MGP is developmentally regulated, with peak expression occurring during periods of relatively low cellular proliferation and increased cellular differentiation. The prenatal rise in mRNA expression occurs at a time when the proximal airway epithelium loses the ability to respond to distal mesenchymal signals, suggestive of a terminally differentiated phenotype (36). Similarly, the increase in MGP expression occurring in the second postnatal week coincides with alveolar differentiation and lung maturation. The time course of expression that we observed is similar to that reported in a limited study in the mouse (26), in which MGP mRNA was expressed peripherally at the epithelial-mesenchymal border early in gestation and became more restricted to proximal airways later in development. The high levels observed in the adult are consistent with a proposed role of the lung in secretion of MGP into the vascular system, where MGP is found in significant concentrations (32). Because of this, it is possible that high-circulating blood levels of MGP could cross the placenta and rescue an MGP–/– fetus (27) during development in a manner similar to that observed in transforming growth factor (TGF)-{beta}1–/– mice (23). The lethal phenotype associated with the absence of MGP would then be acquired postnatally, resulting ultimately in arterial calcification and death (27).

Antibody-directed interference of MGP function with peptide-derived anti-MGP antibody addition to cultured fetal lung explants resulted in grossly dilated epithelial tubules characterized by significantly reduced branching. We did not observe any signs of antibody-induced toxicity on explant growth, as exemplified by the antibody addition and withdrawal experiments (see Fig. 5). Although dilated, antibody-treated explants grew to a similar size as controls, and preincubation of the antibody with blocking peptide abolished the dilatory effects of anti-MGP. Explant growth with peptide treatment alone resembled controls. Additionally, microinjection of undiluted MGP antibody into the lumen of intact explants (see Fig. 4) did not alter growth over the culture interval.

Expression of specific genes known to regulate the process was altered with MGP antibody treatment, suggesting that mesenchymal MGP may interact with or regulate additional proteins involved in lung growth and development. Evidence for this interaction has been demonstrated in other tissues. Attempts to purify MGP from bone extracts by using CaCl2/urea-based protein purification protocols demonstrated that MGP is tightly associated with fibronectin and a member of the BMP protein family, subsequently identified as BMP-2 (39). Indeed, more recent reports have shown that MGP binds strongly with BMP-2 in ligand-binding studies (41) and that BMP-2 co-precipitates with MGP in transfected human embryonic kidney 293 cells (45), suggesting that MGP may regulate or contribute to the functional role of BMP-2 through their physical interaction. BMPs are members of the TGF-{beta} superfamily and function in many diverse cellular processes during development, including cellular differentiation and tissue morphogenesis. Several BMP antagonists, some of which act as BMP binding proteins, limit the activity or availability of BMPs and thus regulate BMP function. In this respect, one of the roles of MGP may be to limit the availability of these proteins by binding and sequestering the protein in specific locations during growth and differentiation.

Although BMP-2 expression has not been reported in lung, a closely related family member, BMP-4, plays an important role in lung branching morphogenesis, where its expression is limited to the epithelial tips of distal lung buds and surrounding mesenchyme during lung development. It is hypothesized that lung BMP-4 functions to inhibit local epithelial proliferation at distal bud tips, forming a suppression zone at the branch point and forcing lateral growth (4). The regulation of BMP-4 expression or function in early lung development is not completely understood. Exogenous addition of BMP-4 to E11.5 mouse lung explant cultures actually enhanced lung branching morphogenesis (5, 37), an effect that was blocked by adenoviral-induced overexpression of the BMP-4 antagonist gremlin (37). Lung branching was not affected in explants, however, when BMP-4 was microinjected into the lung lumen, thereby restricting exposure to the luminal epithelium (5). Targeted epithelial misexpression of Bmp-4 using the SP-C enhancer/promoter produced normal mouse lungs at E11.5 and E12.5, but, by E15.5, transgenic lungs were significantly smaller and less branched than normal (4). These studies underscore the importance of tightly regulated spatial and temporal expression of Bmp-4 for normal lung branching to occur. A recent hypothesis suggests that BMP-4 signaling in regions of high expression (such as at the bud tips) could induce inhibitors of BMP-4 that, in turn, modulate or inhibit local BMP activity, allowing bud induction to occur (13). Thus it is possible that MGP may act as a binding protein to BMP-4, limiting its inhibitory capacity. The reduced branching observed in the presence of anti-MGP antibodies is consistent with this hypothesis.

Interestingly, MGP expression in a mouse chondrocyte cell line (MC615) is reduced in the presence of either BMP-2 or BMP-4 (40). In osteogenic sarcoma cells, however, MGP expression is unaltered by BMP-4, except in the presence of retinoic acid (RA), where the cytokine further enhanced the RA-induced expression of MGP (50). These studies suggest a cell type-specific regulatory interaction with MGP and BMP-4 that likely is dependent on the presence of additional molecular regulators. In our model system, anti-MGP antibody treatment did not significantly alter overall BMP-4 expression, but site-specific or cell type-specific changes in expression remain to be determined. It is of interest to note that RA has been reported to repress endogenous MGP expression in a cell-type-specific manner through RA receptor binding to a response element located on the MGP promoter (22). Other studies, however, demonstrated elevated MGP expression in the presence of RA (7). It is known that RA, an important morphogen in the control of lung branching morphogenesis (10), exerts its effects on gene expression in a concentration-dependent manner. It is also likely that RA-induced changes in gene expression alter both the spatial and temporal balance of specific regulatory proteins, which, in turn, affect the local growth characteristics of the developing lung. Indeed, BMP-4 expression patterns become much more diffuse, with severely attenuated expression at the epithelial tips in explants cultured in the presence of RA (28). We speculate that MGP and BMP-4 may be integral components in a regulatory complex of proteins in the cleft region and that paracrine factors such as RA may contribute to the regulation of expression at specific sites.

The effects of anti-MGP antibody treatment on expression of Shh and Ptc are likely the result of the antibody-induced decrease in branching, as these proteins are important mediators of this process. Several members of the Shh signaling pathway are required for normal pattern formation and morphogenesis in a variety of developing tissues, including the lung. Shh expression begins very early in lung development, where its highest levels are found in the distal epithelium of growing lung bud tips (4, 38). Its receptor, Ptc, is expressed at high levels in the distal mesenchyme, adjacent to the Shh-expressing epithelium (2). Several lines of evidence suggest that Shh plays a role in controlling site-specific FGF-10 expression in the developing lung. Overexpression of Shh in transgenic mice causes downregulation of Fgf-10 and Ptc (3), whereas Shh null mutant mice are characterized by abnormal, diffuse mesenchymal expression of Fgf-10 and severely altered lung development (31). Inhibition of Shh and Ptc mRNA expression in the presence of anti-MGP antibody would prevent the normal local downregulation of Fgf-10 by Shh, ultimately resulting in altered cleft formation and attenuated lung branching. Our data suggest that MGP may normally contribute to the induction of Shh, which, in turn, dampens the growth-stimulatory function of FGF-10. This relationship may be reciprocal in nature, as evidenced by the consistent downregulation of MGP mRNA expression observed in the presence of anti-MGP. We speculate that a critical balance of key gene products is required for normal branch formation to occur and that, if an imbalance arises in one component, expression of other regulatory proteins is affected.

TGF-{beta} is another important signaling molecule in lung development that also has a regulatory influence on MGP expression. Zhou et al. (51) demonstrated arrested lung sacculation and delayed epithelial cell differentiation in transgenic mice with a constitutively activated TGF-{beta}1 gene. At day 16 of development, transgenic mouse lungs contained fewer acinar buds than their control nontransgenic littermates. Perturbation of the TGF-{beta} type II receptor or TGF-{beta}1 antisense oligonucleotides further demonstrated the negative influence of TGF-{beta}1 on embryonic murine lung morphogenesis (46). It is likely that TGF-{beta}1 is an important signal for the local induction of MGP, because this growth factor is known to regulate expression of several other ECM proteins (19, 35). TGF-{beta}1 stimulates MGP expression in a concentration-dependent manner in fetal lung explants (49), a result that we have also observed in our system (data not shown). Mesenchymal targeting of adenoviral-directed TGF-{beta}1 gene upregulation resulted in a spatially specific increase in TGF-{beta}1 expression, accompanied also by increased fibronectin and MGP mRNA expression (48). Thus the growth-regulating effects induced by TGF-{beta}1 are likely mediated, in part, by the stimulation of ECM proteins, including MGP and fibronectin.

Additional studies have demonstrated a colocalization of MGP and fibronectin (8, 25), suggesting that these two matrix proteins work coordinately to regulate complex cell-matrix interactions. The establishment of a suppression zone, or cleft region, during branching morphogenesis is, in part, accomplished by spatially specific deposition of several ECM proteins, resulting in very local decrease in cellular proliferation, morphogenetic stabilization, and a remodeling of the ECM (29). Overexpression of antisense fibronectin RNA in Madin-Darby canine kidney cells severely attenuated branching morphogenesis, decreased cell growth rates, and reduced cell migration (21). Conversely, vascular smooth muscle cells incubated with an active fragment of fibronectin resulted in accelerated phenotypic changes accompanied by increased expression of MGP (20). The decrease in MGP expression and branching observed with antifibronectin antibodies in the present study (see Fig. 6) is consistent with a regulatory interaction between the two proteins. Overall, these data suggest that TGF-{beta}1 activation may be an important initial step in orchestrating cleft formation, and, based on the present results with anti-MGP and antifibronectin antibody treatments and reduced fetal lung branching, it is reasonable to suggest that MGP and fibronectin play important downstream roles in this process. Indeed, it has been suggested that ECM proteins may be able to, in turn, modulate the local activity of TGF-{beta}1 and keep it sequestered in specific regions so that it is available to surrounding cells at the appropriate time (35). It is important to emphasize that normal branching patterns are mediated by very specific spatial and temporal patterns of expression. These protein-protein interactions are presently under investigation to further define the role of MGP in fetal lung growth and differentiation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RFA HL-62869.


   ACKNOWLEDGMENTS
 
We thank Jing Zhou and Dr. Carolyn Lloyd for excellent technical assistance in these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Rannels, Dept. of Cellular and Molecular Physiology, H-166, The Pennsylvania State Univ. College of Medicine, 500 Univ. Drive, Hershey, PA 17033 (E-mail: srannels{at}psu.edu).

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Barone LM, Owen TA, Tassarini MS, Bortell R, Stein GS, and Lian JB. Developmental expression and hormonal regulation of the rat matrix GLA protein (MGP) gene in chondrogenesis and osteogenesis. J Cell Biochem 46: 351–365, 1991.[CrossRef][Web of Science][Medline]
  2. Bellusci S, Furuta Y, Rush MG, Henderson R, Winnier G, and Hogan BL. Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124: 53–63, 1997.[Abstract]
  3. Bellusci S, Grindley J, Emoto H, Ito N, and Hogan BL. Fibroblast growth factor 10 (FGF-10) and branching morphogenesis in the embryonic mouse lung. Development 124: 4867–4878, 1997.[Abstract]
  4. Bellusci S, Henderson R, Winnier G, Oikawa T, and Hogan BL. Evidence from normal expression and targeted misexpression that bone morphogenetic protein-4 (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122: 1693–1702, 1996.[Abstract]
  5. Bragg AD, Moses HL, and Serra R. Signaling to the epithelium is not sufficient to mediate all of the effects of transforming growth factor beta and bone morphogenetic protein 4 on murine embryonic lung development. Mech Dev 109: 13–26, 2001.[CrossRef][Web of Science][Medline]
  6. Cancela ML, Hu B, and Price PA. Effect of cell density and growth factors on matrix GLA protein expression by normal rat kidney cells. J Cell Physiol 171: 125–134, 1997.[CrossRef][Web of Science][Medline]
  7. Cancela ML and Price P. Retinoic acid induces matrix Gla protein gene expression in human cells. Endocrinology 130: 102–108, 1992.[Abstract/Free Full Text]
  8. Cancela ML, Williamson MK, and Price PA. The putative RGD-dependent cell adhesion activity of matrix Gla protein is due to higher molecular weight contaminants. J Biol Chem 269: 12185–12189, 1994.[Abstract/Free Full Text]
  9. Cardoso WV. Molecular regulation of lung development. Annu Rev Physiol 63: 471–494, 2001.[CrossRef][Web of Science][Medline]
  10. Cardoso WV, Williams MC, Mitsialis SA, Joyce-Brady M, Rishi AK, and Brody JS. Retinoic acid induces changes in the pattern of airway branching and alters epithelial cell differentiation in the developing lung in vitro. Am J Respir Cell Mol Biol 12: 464–476, 1995.[Abstract]
  11. Chen L, O'Bryan JP, Smith HS, and Liu E. Overexpression of matrix Gla protein mRNA in malignant human breast cells: isolation by differential cDNA hybridization. Oncogene 5: 1391–1395, 1990.[Web of Science][Medline]
  12. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[Web of Science][Medline]
  13. Chuang PT and McMahon AP. Branching morphogenesis of the lung: new molecular insights into an old problem. Trends Cell Biol 13: 86–91, 2003.[CrossRef][Web of Science][Medline]
  14. Farzaneh-Far A, Proudfoot D, Weissberg PL, and Shanahan CM. Matrix Gla protein is regulated by a mechanism functionally related to the calcium-sensing receptor. Biochem Biophys Res Commun 277: 736–740, 2000.[CrossRef][Web of Science][Medline]
  15. Ferland G. The vitamin K-dependent proteins: an update. Nutr Rev 56: 223–230, 1998.[Web of Science][Medline]
  16. Fraser JD and Price PA. Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase. J Biol Chem 263: 11033–11036, 1988.[Abstract/Free Full Text]
  17. Gilbert KA and Rannels SR. Glucocorticoid effects on vitamin K-dependent carboxylase activity and matrix GLA protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol 285: L596–L597, 2003.
  18. Hale JE, Fraser JD, and Price PA. The identification of matrix Gla protein in cartilage. J Biol Chem 263: 5820–5824, 1988.[Abstract/Free Full Text]
  19. Heine UI, Munoz EF, Flanders KC, Roberts AB, and Sporn MB. Colocalization of TGF-{beta}1 and collagen I and II, fibronectin, and glycosaminoglycans during lung branching morphogenesis. Development 109: 29–36, 1990.[Abstract]
  20. Hu WY, Fukuda N, Satoh C, Jian T, Kubo A, Nakayama M, Kishioka H, and Kanmatsuse K. Phenotypic modulation by fibronectin enhances the angiotensin II-generating system in cultured vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 20: 1500–1505, 2000.[Abstract/Free Full Text]
  21. Jiang ST, Chuang WJ, and Tang MJ. Role of fibronectin deposition in branching morphogenesis of Madin-Darby canine kidney cells. Kidney Int 57: 1860–1867, 2000.[CrossRef][Web of Science][Medline]
  22. Kirfel J, Kelter M, Cancela LM, Price PA, and Schule R. Identification of a novel negative retinoic acid responsive element in the promoter of the human matrix Gla protein gene. Proc Natl Acad Sci USA 94: 2227–2232, 1997.[Abstract/Free Full Text]
  23. Letterio JJ, Geiser AG, Kulkarni NS, Roche S, Sporn MB, and Roberts AB. Maternal rescue of transforming growth factor-beta 1 null mice. Science 264: 1936–1938, 1994.[Abstract/Free Full Text]
  24. Levine RA, Serdy M, Guo L, and Holzschu D. Elongation factor Tu as a control gene for mRNA analysis of lung development and other differentiation and growth regulated systems. Nucleic Acids Res 21: 4426, 1993.[Free Full Text]
  25. Loeser RF and Wallin R. Cell adhesion to matrix Gla protein and its inhibition by an Arg-Gly-Asp-containing peptide. J Biol Chem 267: 9459–9462, 1992.[Abstract/Free Full Text]
  26. Luo G, D'Souza R, Hogue D, and Karsenty G. The matrix Gla protein is a marker of the chondrogenesis cell lineage during mouse development. J Bone Miner Res 10: 325–334, 1995.[Web of Science][Medline]
  27. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, and Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386: 78–81, 1997.[CrossRef][Medline]
  28. Malpel S, Mendelsohn C, and Cardoso WV. Regulation of retinoic acid signaling during lung morphogenesis. Development 127: 3057–3067, 2000.[Abstract]
  29. Mollard R and Dziadek M. A correlation between epithelial proliferation rates, basement membrane component localization patterns, and morphogenetic potential in the embryonic mouse lung. Am J Respir Cell Mol Biol 19: 71–82, 1998.[Abstract/Free Full Text]
  30. Mori K, Shioi A, Jono S, Nishizawa Y, and Morii H. Expression of matrix Gla protein (MGP) in an in vitro model of vascular calcification. FEBS Lett 433: 19–22, 1998.[CrossRef][Web of Science][Medline]
  31. Pepicelli CV, Lewis PM, and McMahon AP. Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 8: 1083–1086, 1998.[CrossRef][Web of Science][Medline]
  32. Price PA, Thomas GR, Pardini AW, Figueira WF, Caputo JM, and Williamson MK. Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats. J Biol Chem 277: 3926–3934, 2002.[Abstract/Free Full Text]
  33. Price PA, Urist MR, and Otawara Y. Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone. Biochem Biophys Res Commun 117: 765–771, 1983.[CrossRef][Web of Science][Medline]
  34. Rannels SR, Cancela ML, Wolpert EB, and Price PA. Matrix Gla protein mRNA expression in cultured type II pneumocytes. Am J Physiol Lung Cell Mol Physiol 265: L270–L278, 1993.[Abstract/Free Full Text]
  35. Roberts AB, McCune BK, and Sporn MB. TGF-{beta}: regulation of the extracellular matrix. Kidney Int 41: 557–559, 1992.[Web of Science][Medline]
  36. Shannon JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev Biol 166: 600–614, 1994.[CrossRef][Web of Science][Medline]
  37. Shi W, Zhao J, Anderson KD, and Warburton D. Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis. Am J Physiol Lung Cell Mol Physiol 280: L1030–L1039, 2001.[Abstract/Free Full Text]
  38. Urase K, Mukasa T, Igarashi H, Ishii Y, Yasugi S, Momoi MY, and Momoi T. Spatial expression of sonic hedgehog in the lung epithelium during branching morphogenesis. Biochem Biophys Res Commun 225: 161–166, 1996.[CrossRef][Web of Science][Medline]
  39. Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J, Lietze A, Tempst P, Hunkapiller M, and DeLange RJ. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci USA 81: 371–375, 1984.[Abstract/Free Full Text]
  40. Valcourt U, Ronziere MC, Winkler P, Rosen V, Herbage D, and Mallein-Gerin F. Different effects of bone morphogenetic proteins 2, 4, 12, and 13 on the expression of cartilage and bone markers in the MC615 chondrocyte cell line. Exp Cell Res 251: 264–274, 1999.[CrossRef][Web of Science][Medline]
  41. Wallin R, Cain D, Hutson SM, Sane DC, and Loeser R. Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2). Thromb Haemost 84: 1039–1044, 2000.[Web of Science][Medline]
  42. Wallin R, Cain D, and Sane DC. Matrix Gla protein synthesis and gamma-carboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells. Thromb Haemost 82: 1764–1767, 1999.[Web of Science][Medline]
  43. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, and Cardoso WV. The molecular basis of lung morphogenesis. Mech Dev 92: 55–81, 2000.[CrossRef][Web of Science][Medline]
  44. Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, Pacifici M, and Iwamoto M. Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb. J Cell Biol 147: 1097–1108, 1999.[Abstract/Free Full Text]
  45. Zebboudj AF, Imura M, and Bostrom K. Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem 277: 4388–4394, 2002.[Abstract/Free Full Text]
  46. 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 181: 242–257, 1996.
  47. Zhao J and Nishimoto SK. Matrix Gla protein gene expression is elevated during postnatal development. Matrix Biol 15: 131–140, 1996.[CrossRef][Web of Science][Medline]
  48. Zhao J, Sime PJ, Bringas JR P, Tefft JD, Buckley S, Bu D, Gauldie J, and Warburton D. Spatial-specific TGF-beta-1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung. Eur J Cell Biol 78: 715–725, 1999.[Web of Science][Medline]
  49. Zhao J and Warburton D. Matrix Gla protein gene expression is induced by transforming growth factor-beta in embryonic lung culture. Am J Physiol Lung Cell Mol Physiol 273: L282–L287, 1997.[Abstract/Free Full Text]
  50. Zhou H, Hammonds RG Jr, Findlay DM, Martin TJ, and Ng KW. Differential effects of transforming growth factor-beta 1 and bone morphogenetic protein 4 on gene expression and differentiated function of preosteoblasts. J Cell Physiol 155: 112–119, 1993.[CrossRef][Web of Science][Medline]
  51. Zhou L, Dey CR, Wert SE, and Whitsett JA. Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-{beta}1 chimeric gene. Dev Biol 175: 227–238, 1996.[CrossRef][Web of Science][Medline]



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