1Department of Pathology, Children's and Brigham and Women's Hospitals and Harvard Medical School, and 2Department of Medicine, Children's Hospital, Boston, Massachusetts 02115; 3Department of Pediatrics, Harbor-UCLA Research and Educational Institute, Torrance, California 90502; 4Department of Degenerative Neurological Diseases, National Institute of Neuroscience, Tokyo 187-8502; and 5Japan Society for the Promotion of Science, Tokyo 102-8471, Japan
Submitted 18 December 2002 ; accepted in final form 31 July 2003
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ABSTRACT |
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gastrin-releasing peptide; neuromedin B; bombesin receptor subtype 3; dexamethasone; cell proliferation; type II cell differentiation
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Other BLPs include the amphibian phyllolitorins (PLs), which are immunologically and functionally similar to BLP (20, 22). Identified as potent smooth muscle constrictor peptides in rodents (10), putative mammalian PLs became of major interest when their potential clinical relevance emerged. BLP immunoreactivity in bronchoalveolar lavage fluid from asymptomatic human smokers was found to have an amino acid content most consistent with a PL-like peptide (1). However, this mammalian homolog has yet to be identified. PLs elicit the same panel of effects as BLP, including cell proliferation (19, 22).
Several major developmental effects of BLP have been defined. In embryonic mouse lung buds, BLP induces branching morphogenesis (19). In mice and baboons, BLP promotes widespread cell proliferation and differentiation of type II pneumocytes and neuroendocrine cells (9, 29), as well as surfactant phospholipid secretion (4). The effects of BLP can be blocked by BLP receptor-specific antagonists, which can inhibit type II cell differentiation in fetal primate lung explants (9, 35).
Three mammalian BLP receptors have been cloned (6, 23): GRP/bombesin-preferring receptors (GRPR) (7, 27), neuromedin B (NMB) receptors (NMBR) (33), and the orphan bombesin receptor subtype 3 (BRS-3) (12, 15). GRPR and BRS-3 are expressed in primate fetal lung (9). These are seven-transmembrane-spanning, G protein-coupled receptors primarily leading to PKC-dependent signal transduction with inositol trisphosphate generation (13). BLP treatment of 3T3 embryonic fibroblasts leads to tyrosine phosphorylation of focal adhesion kinase (p125FAK) preceding MAP kinase activation and cell proliferation (26, 37). By in situ hybridization, GRPR mRNA is detected in the mesenchyme around developing airways and blood vessels in mice and in epithelial cells in rodents, humans, and nonhuman primates (9, 19, 21, 31, 34). The effect of BLP on surfactant phospholipid synthesis by purified type II cells requires coculture with fetal pulmonary fibroblasts (8). GRPR mRNA levels in fetal lung mesenchyme peak during the canalicular phase, preceding type II pneumocyte differentiation (19, 31, 34). Subsequently, GRPR and GRP levels fall to low or undetectable levels during normal alveolarization, especially after birth (19, 29).
BLPs have been demonstrated to play a significant role in normal fetal lung growth and maturation (28). These effects include the promotion of branching morphogenesis in embryonic mouse lung buds, which is optimally induced by an amphibian bombesin-related peptide, Leu8-PL (L8PL). In fetal mouse lung explants, bombesin was shown to promote fetal lung development, but no other BLP was tested in mice. In fetal baboon lung explants, similar effects were observed using bombesin, GRP, or L8PL, but no significant effects occurred with NMB (9).
In the present study, we tested the hypothesis that different BLPs regulate fetal lung development via one or more of the known mammalian BLP receptors: GRPR, NMBR, and/or BRS-3. 1) We analyzed the ontogeny of BLP and BLP receptor gene expression and the localization of these mRNAs in developing murine lung. 2) We assessed the regulation of BLP receptor gene expression by agents known to modulate lung morphogenesis. 3) Using dose-response analyses to begin to address potential receptor specificities, we determined the effects of several previously untested BLPs on type II cell differentiation and cell proliferation in normal fetal mouse lung explants. Cumulatively, our observations support a direct role for multiple BLP receptors in murine fetal lung development.
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MATERIALS AND METHODS |
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Timed-pregnant Swiss-Webster mice were obtained from Taconic Laboratories (Germantown, NY) at gestational day 7. We strictly adhered to the National Research Council Guide for the Care and Use of Laboratory Animals throughout all phases of the study. The Animal Care Committee of the Brigham and Women's Hospital and Children's Hospital reviewed and approved the protocols used in the study.
Preparation of RNA and cDNA
Lungs were harvested from fetal mice at embryonic (E) days 1218 (E12E18) and from neonatal mice on the day of birth [postnatal (P) day 1 (P1)]. Total RNA was prepared from snap-frozen lung tissue using TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA integrity was evaluated by examination of 18S and 28S bands after separation on ethidium bromide-stained 1.5% agarose-formaldehyde gels. Northern blot analyses were carried out according to standard protocols. cDNA was prepared using SuperScript 2 RNase reverse transcriptase (GIBCO-BRL) according to the manufacturer's instructions.
RNA Analysis
RT-PCR. Synthetic oligodeoxynucleotides, designed to span at least one intron, were purchased from Oligos Etc (Wilsonville, OR). Primer pairs and PCR conditions are given in Table 2. PCR were carried out using Taq polymerase (Boehringer Mannheim, Indianapolis, IN) as specified by the manufacturer. Briefly, 1 µl of the reverse-transcribed RNA mixture was added to a 50-µl PCR, each of which was subjected to the number of cycles specified in Table 2. Each cycle included denaturation for 1 min at 94°C and annealing for 1.5 min and extension for 2 min at 72°C with a thermal cycler (MJ Research, Watertown, MA). Negative controls consisted of an equal volume of water substituted for the volume of RNA in the RT reaction.
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PCR products were analyzed on 1.5% agarose gels containing ethidium bromide and blotted onto nitrocellulose according to standard protocols (5). Internal oligonucleotide probes were end labeled using T4 polynucleotide kinase, hybridized, washed, and exposed to Kodak Biomax film, as described elsewhere (36). These RT-PCR assay conditions were strictly defined to yield semiquantitative results, as previously described (36), resulting in linear detection of positive control bands over a 2-log range of input positive control RNA. Relative amounts of specific mRNAs could subsequently be normalized with actin mRNA in the same RT reaction mixture. Densitometry was carried out by three-dimensional integration of the area of whole bands on autoradiograms with a Scanjet-3-P (Hewlett-Packard, Palo Alto, CA) interfaced with Scan Analysis 2.55 software (Biosoft, Ferguson, MO).
In situ hybridization. Nonisotopic in situ hybridization was carried out to localize mRNAs in tissue sections, as described in detail previously (9). Briefly, fetal mouse lungs were fixed for 34 h in 4% paraformaldehyde, transferred into 30% sucrose at 4°C overnight, and then embedded in OCT, frozen, and stored at -70°C. Frozen sections (5 µm) were placed on Fisher "Plus" slides. Murine NMBR and BRS-3 cDNAs have been described previously (11, 33). The cRNA probes were labeled with biotin-14-dCTP according to the manufacturer's specifications (GIBCO-BRL Life Technologies, Frederick, MD). Frozen sections were incubated with proteinase K (10 µg/ml) and then hybridized at 42°C overnight with biotin-14-labeled cRNA (0.25 µg/ml hybridization buffer). After they were washed, the slides were developed with streptavidin-alkaline phosphatase conjugate in blocking buffer (100 mg/ml of blocking reagent) and then with nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate solution (Roche Molecular Biochemical, Mannheim, Germany). Sections were counterstained with 2% aqueous methyl green, and coverslips were applied using aqueous mounting medium.
Lung Organ Cultures
Fetal lung was harvested on either E17 or E18, chopped into 0.5-mm cubes, and cultured in six-well plates for 48 h at 37°C in 5% CO2-95% air on a rocking platform, as described previously (29). Tissue viability was checked by histological analyses after 48 h, satisfactory baseline incorporation of [3H]choline and [3H]thymidine, and satisfactory positive control responses with dexamethasone.
Determination of 3H-Labeled DNA and 3H-Labeled Saturated Phosphatidylcholine
After 44 h of culture with BLPs or dexamethasone, lung organ cultures were incubated with [3H]thymidine (4 µCi/ml) or [3H]choline (16 µCi/ml; NEN-Dupont, Boston, MA) for 4 h at 37°C in 5% CO2-95% air on a rocking platform before being harvested and analyzed for 3H-labeled DNA, DNA content, 3H-labeled saturated phosphatidylcholine, and protein content, as described previously (29). Experimental values were normalized by defining the mean of the control groups as baseline and expressing values as percent changes above or below this level.
Statistical Analyses
Numerical data were analyzed using the unpaired Student's t-test. Values are means ± SE.
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RESULTS |
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To study BLP and BLP receptor gene expression during murine lung ontogeny, tissue was harvested daily from E12E18 and several postnatal days up to 2 wk of age (P1P14). The results of semiquantitative RT-PCR analyses of lung RNA are shown in Fig. 1A. Results of relative densitometric analysis, normalized for -actin, are given in Fig. 1. The GRP gene is expressed throughout embryonic, fetal, and postnatal lung development, with highest levels at E15, E16, and P1 (Fig. 1, A and B). The three BLP receptor mRNAs are expressed in different temporal patterns during ontogeny. GRPR mRNA is detected at E12, peaks at E16, and then falls off to very low levels after birth. NMBR mRNA peaks at E14 but is also present at low to moderate levels from E13 to P1 and at P7. BRS-3 is present at moderate levels at all time points between E12 and P14, with peaks at E15, E16, and P4P7. Similar results were obtained in three separate experiments using RNA from different pools of fetal mouse lung at all time points (data not shown).
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In Situ Hybridization for BLPR mRNAs
In situ hybridization was carried out using antisense cRNA probes to determine localization of GRPR, NMBR, and BRS-3 mRNAs in murine fetal lung at E14, E16, and E18 (Figs. 2 and 3). First, GRPR was localized to airway epithelium and mesenchymal cells at all three time points (Fig. 2). At E14E16, GRPR was expressed moderately to strongly in the mesenchyme, predominantly around developing airways and blood vessels; a strong signal was also present in the epithelium at E14 and E16. Consistent with the results of RT-PCR analysis, the overall signal for GRPR declined by E18 in the epithelium and mesenchyme.
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In contrast, NMBR mRNA was localized predominantly to the diffuse mesenchymal compartment of developing lung at E14 and E18; a moderate NMBR mRNA signal was also present in the airway epithelium at E14 and E16.
Similarly, a strong signal for BRS-3 mRNA was localized to airway epithelium and scattered mesenchymal cells at E14 and E18; at E16, there was a stronger signal for BRS-3 in the airway epithelium. No specific hybridization signal was observed at any time point with any of the sense control probes (Figs. 2 and 3).
Regulation of GRPR, NMBR, and BRS-3 Gene Expression by Dexamethasone and Bombesin
To determine whether agents known to modulate fetal lung development in vivo and in vitro can regulate GRPR, NMBR, or BRS-3 gene expression, semiquantitative RT-PCR analyses were carried out. Again, PCR conditions were determined such that a positive control mRNA was detected within the linear range of the assay and at less than half-maximal signal-to-noise ratio, as previously described (36). We chose dexamethasone as an agent that induces type II cell differentiation and decreased cell proliferation and bombesin as a prototypical BLP that is known to augment cell differentiation and growth. Results of a typical 2-day culture experiment are given in Fig. 4. We carried out densitometric analysis of the RT-PCR bands obtained in four similar experiments. The pooled results of relative densitometric analysis are given in Fig. 4, BD. Dexamethasone treatment led to a dose-dependent increase in GRPR mRNA levels (Fig. 4B), with a twofold increase in relative amounts of GRPR mRNA with 10 nM dexamethasone (P < 0.01). Second, we observed a greater than threefold induction of GRPR gene expression by its specific ligand bombesin at 10 nM (P < 0.0001), again in a dose-dependent fashion (Fig. 4C). Finally, NMBR gene expression was suppressed by bombesin in a dose-dependent manner (Fig. 4D), with about half as much NMBR mRNA in cultures treated with 10 nM bombesin as in the negative control (P < 0.015). There was no change in BRS-3 mRNA levels with any tested dose of dexamethasone or bombesin (data not shown).
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[3H]Thymidine and [3H]Choline Incorporation in Fetal Lung Explants
To determine whether all three groups of BLPs can promote type II cell differentiation and/or generalized cell proliferation in fetal mice, lung explants were cultured with BLPs from each of the three recognized BLP families (Table 1) vs. dexamethasone as a control. Peak [3H]thymidine incorporation occurred on E18 (see Fig. 6). The maximal effect was observed with 0.1 nM GRP (75% increase over baseline). GRP also elicited significant responses of smaller magnitude on E17 (<15% increase at 0.11.0 nM GRP; Fig. 5). In parallel cultures from the same litters of mice, NMB stimulated [3H]thymidine incorporation
20% over baseline at 0.11.0 nM on E18 (Fig. 6), with a peak effect (
70% increase over baseline) observed at 10 nM. NMB also elicited significant responses of smaller magnitude on E17 (
25% increase at 0.11.0 nM NMB; Fig. 5). L8PL had minimally significant effects on cell proliferation (<15% inhibition with 1.010 nM L8PL on E17 and <10% stimulation with 0.11.0 nM L8PL on E18). Low-dose (1.010 nM) dexamethasone stimulated thymidine incorporation (
1020%) on E17 and E18. At higher doses, dexamethasone inhibited [3H]thymidine incorporation on E17 (>50% inhibition with 100 nM) and E18 (>25% inhibition with 10 nM).
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In contrast, maximal effects on stimulating choline incorporation were observed on E17 (Fig. 5) and E18 (Fig. 6). GRP at 0.1 nM induced an 20% increase in choline incorporation on E17 and E18. The magnitude of these GRP effects was similar to that of the 0.1 nM dexamethasone control on E17 (Fig. 5), whereas the response to 10 nM dexamethasone on E18 was of greater magnitude (>30%). NMB inhibited choline incorporation
1525% on E17 at 1.0, 10, and 100 nM; L8PL was inhibitory (
35%) only at 100 nM. In contrast, on E18, 10 nM NMB stimulated choline incorporation to the same extent (
30%) as the 10 nM dexamethasone control. High-dose (100 nM) L8PL inhibited choline incorporation on E17, whereas low- and high-dose (1.0 and 100 nM) L8PL stimulated choline incorporation on E18.
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DISCUSSION |
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The temporal and spatial distribution of gene expression has been reported to differ during development between different members of other gene families (17, 25). The ontogeny of GRP and GRPR genes in fetal mouse lung has been previously analyzed in our laboratory (19, 29). To complete the assessment of BLP receptor gene expression in developing mouse lung, we demonstrate mRNAs encoding NMBR and BRS-3 as well. GRP, GRPR, and BRS-3 mRNAs are detectable as early as E12 and E13; NMBR mRNA is detectable at E13 and reaches peak levels on E14. This timing coincides with the period of branching morphogenesis, suggesting a potential regulatory role for GRP and the three BLP receptors in early organogenesis, as we previously addressed (19). Whereas GRPR mRNA levels in the lung drop off markedly shortly after birth, NMBR and BRS-3 mRNAs are sustained.
Others have shown that glucocorticoids can inhibit GRPR mRNA levels in pancreatic acinar cells (18). Not infrequently, mesenchymal cells and epithelial cells can have opposite responses to the same ligand (2, 14). The well-recognized effect of dexamethasone on promoting fetal lung maturation could be mediated in part by dexamethasone-mediated upregulation of GRPR on fibroblasts and enhanced signaling through the GRPR pathway (9). The opposite regulation of GRPR and NMBR gene expression is interesting, because GRP promotes but NMB inhibits choline incorporation in developing E17 lung. Teleologically, this dichotomy may reflect ligand-specific (bombesin) upregulation of the preferred native receptor (GRPR) and decreased levels of secondary receptors with lower affinity (NMBR).
It should be emphasized that the identity of the native BRS-3 ligand is unknown. Furthermore, bombesin, NMB, and PL have very low affinity for BRS-3; hence, the effects of these three peptides on fetal lung are unlikely to be mediated via BRS-3. This is further supported by our observation that bombesin does not alter BRS-3 gene expression in fetal lung explants. In addition, neither a mammalian PL nor a mammalian PL receptor has been identified. The effects of amphibian PL in the present study could be mediated in part by GRPR, but it is likely that additional novel BLP receptors are involved as well.
Our physiological data suggest the possibility that GRPR and NMBR play different roles in lung development. Low- and high-dose GRP induce a marked increase in cell proliferation on E18 that is likely to be mediated via GRPR and NMBR and, potentially, other novel BLP receptors. In contrast, only low-dose GRP stimulates significant choline incorporation on E17, supporting a unique role for GRPR in type II cell differentiation. This effect on type II cell differentiation is likely to be mediated via GRPR on fetal lung mesenchymal cells, because we previously demonstrated a requirement for these cells to elicit the GRP effect on type II cells in culture (8). In contrast, low-dose NMB promotes only thymidine incorporation, consistent with a major role for NMBR in cell proliferation. Higher-dose (10 nM) NMB stimulates a greater degree of cell proliferation (70%), similar to the effect of GRP at low and high doses, suggesting that 10 nM NMB may function via GRPR and/or other novel BLP receptors. In contrast to GRP, NMB (1.0100 nM) only inhibits choline incorporation on E17, whereas 10 nM NMB is stimulatory on E18. The latter effect is likely to be mediated via GRPR.
The effects of BLPs on E17 lung explants (Fig. 5) differ markedly from their effects on E18 cultures (Fig. 6). These differences are likely to reflect large changes in developing murine lung between E17 and E18. On E17, the lung is in the late canalicular phase, during which there is widespread ingrowth of capillaries into developing alveolar septa as well as the induction of type II cell differentiation. On E18, the lung is entering the saccular phase: type II cell differentiation is more advanced, and structural remodeling predominates, together with intense epithelial cell proliferation. These developmental differences may explain why low-dose GRP promotes cell proliferation most intensely on E18, when GRPR is expressed predominantly in the proliferating epithelium. In contrast, low-dose GRP stimulates type II cell differentiation most strongly on E17, when GRPR mRNA is most strongly expressed in the mesenchyme.
It is unlikely that a PL receptor plays a major role in the observed responses. L8PL has only weak (<15%) effects on thymidine incorporation at any dose. L8PL had more than a marginal effect on choline incorporation at 100 nM, which could be mediated via NMBR (33). Previously, we demonstrated that morphogenesis of embryonic mouse lung bud can be augmented by high-dose bombesin but low-dose L8PL (19). Cumulatively, these data suggest that mammalian PL may function preferentially during early lung embryogenesis, whereas GRP plays a significant role in promoting cell differentiation later in fetal development.
BRS-3 mRNA was detectable in E12 mouse lung, and this expression continued through E18 and after birth. BRS-3 mRNA was localized to epithelium and mesenchymal cells throughout the developing lungs at E14E18. These data suggest that the BRS-3 receptor may modulate branching morphogenesis and/or cell differentiation, analogous to GRPR and/or the putative mammalian PL receptor. However, GRPR knockout mice (32) have several phenotypic features that are the opposite of behavioral and metabolic changes observed in BRS-3 knockout mice (24). Therefore, it is possible that BRS-3 signaling might serve to downregulate effects of GRPR in developing lung.
In conclusion, our investigation supports a significant role for NMBR and GRPR in murine lung development. NMBR promotes cell proliferation in fetal mouse lung, consistent with its expression in airway epithelium of fetal mouse lung. In contrast, low-dose GRP/bombesin appears to act via GRPR to increase cell proliferation and type II cell differentiation, with maturational effects mediated via a mesenchymal cell intermediary (8).
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-50045 and UO1-HL-52638 (M. E. Sunday).
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FOOTNOTES |
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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.
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REFERENCES |
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