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Role of Neuregulin-1ß in the Developing Lung

Department of Pediatrics, Division of Newborn Medicine, Tufts University and Floating Hospital for Children; Department of Cell Biology, Harvard Medical School; and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Christiane E. L. Dammann, M.D., Department of Pediatrics, Division of Newborn Medicine, Floating Hospital for Children at Tufts-New England Medical Center, 750 Washington Street, Boston, MA. E-mail: cdammann{at}tufts-nemc.org

	ABSTRACT

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Neuregulins play a critical role in the developing heart, nervous, and mammary systems. Neuregulin-1–induced cardiac, neuronal, and mammary differentiation is based on a cell–cell communication model, where the ligand neuregulin-1 is produced and secreted by one cell type, which does not express its receptors erbB3 and erbB4 and acts on neighboring cell types that do express these receptors. We proposed that neuregulin-1 affects fetal lung maturation through a similar mechanism. Immunostaining showed neuregulin-1 in fetal lung that increased in fibroblasts at the onset of surfactant synthesis. Neuregulin-1ß was found to be secreted by the fetal lung fibroblast and stimulated type II cell surfactant synthesis. Both fetal lung fibroblast-conditioned media and neuregulin-1 stimulated erbB2 receptor phosphorylation in type II cells. The effects of neuregulin-1 and of fibroblast-conditioned media on both surfactant synthesis and type II cell erbB2 phosphorylation were specifically blocked by antibody to neuregulin-1. Thus, neuregulin-1ß may control fetal lung maturation through mesenchymal–epithelial interactions in a paracrine mechanism similar to that described for the developing heart, brain, and mammary systems.

Key Words: fetal lung development • fibroblast–pneumocyte factor • fibroblast-conditioned medium • erbB receptors • fibroblast type II cell communication

ErbB receptors comprise a family of membrane-spanning tyrosine kinase receptors typified by the best known member, the epidermal growth factor receptor, but also including erbB2, erbB3, and erbB4. The primary ligands for erbB3 and erbB4 are various forms of the neuregulin (NRG) growth factor. It has recently been shown that NRG-1 and the receptors erbB2 and erbB3 are expressed in the midtrimester human fetal lung (1). Knockout animal models for NRG-1, erbB2, erbB3, and erbB4 have not been helpful in determining whether these molecules are important in the regulation of lung development, including late fetal lung surfactant production, because of their early embryonic lethality at about embryonic Day 10.5. Extensive studies show that erbB receptors and their ligands are involved in the regulation of cell proliferation, cell differentiation, and cell survival (2–6). In particular, these activities appear crucial to developing systems. For example, it has been shown that the morphogenesis of mammary tissue is dependent on sequential mesenchyme–epithelial interactions mediated by NRG (7).

Fetal lung surfactant synthesis requires communication between mesenchyme and adjacent type II epithelial cells. The specific nature of the communication is poorly understood. Conditioned media from fetal lung fibroblasts stimulate fetal type II epithelial cells to synthesize surfactant. This activity is ascribed to the presence of an unidentified polypeptide, termed fibroblast–pneumocyte factor (FPF) (8, 9). Here we demonstrate that NRG-1ß, a stromal-derived growth factor active in cell–cell communication in mammary development (7), is secreted by fetal lung fibroblasts. Purified NRG-1ß mimics the stimulatory effect of lung fibroblast-conditioned medium (FCM) on surfactant synthesis. Moreover, a neutralizing antibody to NRG-1 inhibits this stimulatory activity in the FCM. This indicates that NRG-1ß plays a major role in type II cell maturation.

	METHODS

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Complete details of the methods are provided in the online supplement. The animal research protocol was approved by the institutional animal research committee. The recombinant epidermal growth factor–like domain of NRG-1ß was expressed and purified as previously described (10, 11) and was used in all experiments.

Fetal Lung FCM
Fibroblast cultures were prepared as described previously using time-dated pregnant Swiss Webster mice (Taconic Farms, Germantown, NY) killed on Day 17 or Day 18 of gestation (term Day 19) and determination of fetal sex (12). FCM was prepared with serum-free Dulbecco's modified Eagle medium (DMEM) without or, in Day 17 cells only, with 10^-8 M dexamethasone for 24 hours. FCM was stored at 4°C until used for analysis of FPF activity. Only the FCM from female lungs was used to control for known sex differences in the timing of lung maturation, including the stimulating activity in the FCM (13, 14).

Primary Fetal Type II Cell Cultures
Supernatants obtained after the first hour of differential adherence for fibroblast cultures were centrifuged at 650 x g for 10 minutes. Cell pellets were resuspended in collagenase and incubated for 2–3 hours at 37°C and then stored on ice for 45 minutes. After resuspension of the pellet in DMEM with 20% FCS, cells were incubated on ice for 45 minutes. Cells were plated in 100-mm² tissue culture dishes and incubated 60 minutes at 37°C. Supernatants were removed and centrifuged as before, and the cell pellet was resuspended in DMEM with 20% FCS. Cells were replated in 24-well tissue culture plates (5–10 x 10⁵ cells/well) and cultured in 1-ml DMEM with 10% FCS at 37°C until 90–100% confluent. Cell purity was confirmed by staining (15) and was more than 92% as previous published (16).

Immunohistochemistry
Immunostaining was performed on sex-specific Swiss Webster mice fetuses (Day 15 to Day 18 of gestation). Immunostaining was performed as previously described (16, 17) using Vectastain Avidin and biotinylated alkaline phosphatase macromolecular complex (ABC-AP) kits according to manufacturer's instructions.

[³H] Choline Incorporation
The stimulation of surfactant synthesis was determined as incorporation of [³H] choline into disaturated phosphatidylcholine (DSPC) as previously described (14) using both mouse lung epithelial (MLE-12) cells and primary fetal type II cell cultures. Treatment conditions included NRG-1ß (1 and 10 nM), NRG-1ß plus NRG-1 NRG-1ß blocking antibody (NRG-1) (MLE-12 cells), FCM (1:1 with DMEM), FCM plus 10 µg/ml, or NRG-1 alone (MLE-12 cells). Information on the specificity of the NRG-1ß blocking antibody is included in the online supplement. In primary type II cells, nonspecific IgG antibody (IgG) was used throughout as a control for the specific neutralizing activity of the NRG-1. Results were normalized as a percentage of experiment-specific control subjects. All experiments included FCM with known surfactant synthesis stimulatory activity as a positive control.

[³H] Thymidine Uptake
Cell proliferation was measured as [³H] thymidine incorporation into DNA as previously described by Zhou and Young (18), using MLE-12 cells and primary fetal type II cells. Treatment conditions, administered for 20 hours after 24 hours serum starvation, were NRG-1ß (1 and 10 nM), FCM (1:1 with DMEM), NRG-1ß plus NRG-1 (MLE-12 cells), FCM plus NRG-1, NRG-1 alone (MLE-12 cells), or nonspecific IgG (primary type II epithelial cells). Control subjects were kept in serum-free medium. Results were normalized as a percentage of experiment-specific control subjects.

lmmunoprecipitation and lmmunoblotting
Immunoprecipitation and immunoblotting studies were performed using MLE-12 cells treated with various conditions. After immunoprecipitation, proteins were separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blots prepared, and proteins visualized by chemiluminescence. Blots were stripped and reprobed with receptor-specific antibodies. For the NRG immunoprecipitation, fresh FCM was added for a total of five times to concentrate the protein.

	RESULTS

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NRG-1 Expression in the Fetal Lung
Immunolocalization in fetal whole lung tissue from gestational Day 15 to Day 18 showed NRG-1 protein in the columnar epithelia of the small airway earlier in gestation, specifically Day 15 (Figure 1A) . With further progression of the development at Day 17 of gestation, NRG-1 was more diffusely present specifically in the subalveolar mesenchyme underlining the cuboidal epithelia of the terminal saccules (Figure 1B). Double immunostaining of Day 17 lung tissue with Hoxb5, a nuclear mesenchymal marker, showed Hoxb5 staining of the fibroblast nucleus and confirmed pronounced NRG staining of the cytoplasm of the fibroblasts adjacent to the cuboidal epithelial (Figures 1D and 1E).

View larger version (135K): [in this window] [in a new window]   Figure 1. Neuregulin (NRG) immunostaining (stained blue) in female fetal mouse lung at Day 15 (A) and Day 17 (B) of gestation. Day 17 control lung (C) was processed the same way, but not exposed to NRG-1 antibody (all x40 magnification). Double immunostaining in female fetal mouse lung at Day 17 using Hoxb5 as a nuclear marker for the mesenchyme (stained blue) in combination with NRG-1 (stained brown) in x40 (D) and x100 magnification (E). Arrow in (E) indicates representative cluster of NRG-1–positive mesenchymal cells underlying airway epithelium; the arrowhead indicates representative Hoxb5 as blue nuclear staining of mesenchyme near airway epithelium. In Day 17 fetal mouse there is also rare columnar epithelial cell cytoplasmic staining for Hoxb5 as we have previously reported (11).

View larger version (28K): [in this window] [in a new window]   Figure 2. NRG-1 immunoprecipitation (IP). NRG-1 protein was immunoprecipitated from fibroblast-conditioned medium (FCM) from Day 18 female fetal mouse lung fibroblasts (lane 2) and conditioned media from Day 18 type II cells (lane 3) using NRG-1 antibody bound to protein A sepharose beads. Precipitates were blotted with anti–NRG-1 antibody. The 44 kD recombinant NRG-1ß produced by High Five insect cells was used as a positive control (lane 1).

View larger version (48K): [in this window] [in a new window]   Figure 3. NRG-1 stimulation of MLE-12 cell growth and differentiation. Cells were treated for 24 hours with Dulbecco's modified Eagle medium (DMEM) (bar 1), 1-nM (bar 2) or 10-nM NRG-1ß (bar 3), FCM from Day 18 female fetal lung fibroblasts (bar 4), FCM plus neutralizing NRG-1 (bar 5), or NRG-1 alone (bar 6). (A) The extent of NRG-1ß–stimulated surfactant synthesis was determined by [3H] choline incorporation into DSPC. Bars represent mean ± SEM of 14–16 determinations expressed in percent of experiment-specific control subjects. (B) The extent of cell proliferation was determined by [3H] thymidine incorporation into DNA. Bars represent mean ± SEM of 9–31 determinations expressed in the percentage of experiment-specific control subjects. *p < 0.05 compared with DMEM treatment.

NRG-1 Effect on erbB Receptor Tyrosine Phosphorylation in the MLE-12 Cells
As shown in Figure 4A , all four known members of the erbB receptor family are present in MLE-12 cells (Figure 4, lower panel). Tyrosine phosphorylation of epidermal growth factor receptor (erbB1) was stimulated by epidermal growth factor (Figure 4, lane 2, upper panel), whereas NRG-1ß stimulated phosphorylation of erbB2 (Figure 4, lane 5, upper panel). ErbB3 exhibited high constitutive phosphorylation (Figure 4, lane 6, upper panel), whereas erbB4 was not phosphorylated at baseline (Figure 4, lane 8, upper panel) and was not stimulated by NRG-1ß (Figure 4, lane 9, upper panel). FCM from Day 18 female fetal lung fibroblasts, which exhibited marked FPF activity (Figure 3A), stimulated erbB2 tyrosine phosphorylation (Figure 4B, lane 3, left upper panel), which was comparable to the effect of NRG-1ß (Figure 4, lane 2, left upper panel). The effect of FCM on erbB2 phosphorylation was inhibited by neutralizing NRG-1 (Figure 4, lane 4, upper left panel), but not by a nonspecific IgG (Figure 4, lane 5, upper left panel), indicating that NRG-1ß is responsible for erbB2 stimulation. On the other hand, FCM from Day 17 female fetal lung fibroblasts was not able to stimulate erbB2 phosphorylation (Figure 4B, lane 7, right upper panel) unless the fibroblasts had been pretreated with cortisol (Figure 4, lane 8, right upper panel). Again, erbB2 stimulation with FCM from cortisol-treated Day 17 female fibroblasts was inhibited with the neutralizing NRG-1 antibody (Figure 4, lane 9, right upper panel), but not with control IgG antibody (Figure 4, lane 10, right upper panel). Quantification by densitometry of the blots confirmed these effects (Figure 4C).

View larger version (101K): [in this window] [in a new window]   Figure 4. ErbB receptor expression and FCM stimulation of erbB2 tyrosine phosphorylation in mouse lung epithelial (MLE-12) cells. (A) ErbB receptor expression profile by immunoprecipitation (IP). MLE-12 cells were stimulated with the indicated purified growth factors. Cell lysates were immunoprecipitated with the indicated antibodies, and precipitates blotted with antiphosphotyrosine (anti-pY) antibody (upper panel). Membranes were then reprobed with receptor-specific antibodies (lower panel). (B) FCM stimulation of erbB2. FCMs from fetal Day 18 (left panel) and Day 17 (right panel) female fetal lung fibroblasts, untreated or glucocorticoid pretreated, were compared. Cells were treated overnight with various conditions. ErbB2 was immunoprecipitated from lysates and blotted first with antiphosphotyrosine antibody (upper panels) and then with anti-erbB2 antibody (lower panels). Treatment conditions were as follows: DMEM (lane 1), recombinant NRG-1ß positive control (30 nM for 2 minutes; lanes 2 and 6), FCM from Day 18 female fetal fibroblasts (lane 3), FCM (Day 18) with neutralizing NRG-1 (lane 4), or FCM (Day 18) with IgG (lane 5), FCM from Day 17 fibroblasts (lane 7), FCM from cortisol-treated Day 17 fibroblasts (lane 8), cortisol-treated Day 17 FCM with NRG-1ß (lane 9), and cortisol-treated Day 17 FCM with IgG (lane 10). (C) Quantification by densitometry of three different experiments. Treatment conditions were compared with untreated control subjects within the same blot. Bars represent the mean ± SEM of three determinations (upper graph) or three to six determinations (lower graph) expressed in percent of experiment-specific control subjects. FCM from Day 18 fetal lung fibroblasts was used in the experiments presented in the upper graph. The effect of cortisol-stimulated FCM from Day 17 fetal lung fibroblasts on erbB 2 phosphorylation is presented in the lower graph. *p < 0.05 compared with control; p < 0.05 compared with nonspecific IgG treatment. EGF = epidemial growth factor.

View larger version (43K): [in this window] [in a new window]   Figure 5. NRG-1 effect on type II epithelial cell growth and differentiation. Cells were treated for 24 hours with DMEM alone (bar 1), 1 nM (bar 2) or 10 nM NRG-1ß (bar 3), FCM from Day 18 female fetal lung fibroblasts (bar 4), FCM with neutralizing NRG-1 (bar 5), or FCM with nonspecific IgG (bar 6). (A) The extent of NRG-1ß–stimulated surfactant synthesis was determined by [3H] choline incorporation into DSPC. Bars represent mean ± SEM of 5–12 determinations expressed in percent of experiment-specific control subjects. (B) The extent of cell proliferation was determined by [3H] thymidine incorporation into DNA. Bars represent mean ± SEM of 3–16 determinations expressed in percent of experiment-specific control subjects. *p < 0.05 compared with control (DMEM) treatment. p < 0.01 compared with FCM treatment or to FCM plus nonspecific IgG.

	DISCUSSION

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Although over a dozen splice variants of the NRG-1 gene have been described (19, 20), a 44- to 45-kD soluble form of NRG-1ß appears to be the predominant form of the protein found in conditioned media from transformed fibroblasts (21) and cultured mammary tumor cells (19). The presence of NRG-1ß in the fetal lung FCM suggests its potential for a role in lung mesenchyme–epithelia interactions during fetal lung cell maturation.

Surfactant DSPC synthesis is a marker for lung epithelial type II cell differentiation. The stimulation of DSPC synthesis by FCM is the accepted definition of FPF activity (9). We studied two models commonly used to examine the control of type II cell differentiation, MLE-12 cells and fresh isolated primary cultures of fetal lung type II cells. We employed the MLE-12 epithelial cell model system, which provides a stable and reproducible assay system for studying the stimulation of DSPC synthesis (22). The MLE-12 cell line was obtained from a lung adenocarcinoma derived by expressing the simian virus-40 large T antigen under the lung-specific surfactant protein-C (SP-C) promoter in transgenic mice (23). MLE-12 cells exhibit characteristics of type II cells, including the expression of surfactant proteins SP-B and SP-C, formation of microvilli and multivesicular bodies, and a strong response to fetal FCM with increased DSPC synthesis (22). In this study, we showed that purified recombinant NRG-1ß mimicked the effect of FCM on surfactant synthesis, an effect specifically blocked by adding neutralizing NRG-1 antibody to the FCM. As would be expected, the antibody also blocked NRG-1ß stimulation. NRG-1ß also stimulated DSPC synthesis in the primary fetal lung type II cell culture system, similar in effect and magnitude to the FCM. These stimulatory effects of FCM and NRG-1ß also were specifically reversed by neutralizing antibody to NRG-1ß. Thus, these results lead to the suggestion that NRG-1ß may be a component of FPF activity.

NRG-1ß stimulated proliferation of MLE-12 cells, whereas FCM from Day 18 fetal lung fibroblasts had no stimulatory effect on cell proliferation in these cells. This is not surprising, because FCM from Day 18 may contain other factors than NRG-1 that could inhibit cell proliferation in this tumor-derived cell line. A possible candidate for such a factor is transforming growth factor–ß1, which is known to suppress immature fetal lung fibroblast proliferation (18, 24) and is present in FCM (25). In the primary fetal type II cells FCM from Day 18 fetal lung fibroblasts had again no mitogenic effect. The effect of purified NRG-1ß on thymidine incorporation in primary fetal type II epithelial cells differed from its effect on the tumor-derived MLE-12 cells. NRG did not stimulate mitogenesis in the primary fetal type II cells, which followed precisely the response profile of the FCM in these cells. These Day 18 fetal type II cells are almost mature and at the peak of fetal surfactant synthesis, and thus, one could speculate that even substances like NRG-1, which carry mitogenic activity, favor the further promotion of epithelial cell maturation in late fetal lung development. The fact that NRG-1ß–stimulated proliferation in MLE-12 cells but not in the primary fetal type II cell may be related to the differential susceptibility of primary cells and transformed cells toward mitogenic factors.

To compare the effect of NRG-1ß and FCM on erbB receptor phosphorylation in type II epithelial cells, we again used both the MLE-12 cells and the primary fetal type II cells. NRG-1ß stimulated erbB2 phosphorylation in MLE-12 cells. There was no effect on erbB4, and no apparent change in the high constitutive phosphorylation of erbB3; erbB2 is an orphan receptor with no known ligand and is phosphorylated only in dimers with other activated erbB receptors. Hence, these results suggest that NRG-1ß redirected constitutively phosphorylated erbB3 to heterodimerize with and activate erbB2. Because the tyrosine phosphorylation of erbB2 was reproducibly stimulated by NRG-1ß, we compared this effect to the response to FCM. FCM from Day 18 fetal fibroblasts, which expresses FPF activity, stimulated erbB2 phosphorylation similar to NRG-1ß. FCM from immature Day 17 fetal mouse lung fibroblasts, which lacks FPF activity, did not stimulate erbB2 phosphorylation. Only when Day 17 fibroblasts were pretreated with glucocorticoids to stimulate production of FPF (14) did the FCM from these pretreated cells stimulate erbB2 phosphorylation. The stimulatory effect on erbB receptor phosphorylation of both FCMs containing FPF activity was inhibited by NRG-1 antibody.

The immunohistochemistry for NRG-1 in the primary fetal mouse lung supports our hypothesis that NRG-1 plays a role in mesenchymal–epithelial cell communication controlling fetal lung maturation. Before the late gestational onset of surfactant synthesis, NRG-1 protein was observed in both mesenchymal and epithelial cells. At the time of the development of mesenchymal–epithelial cell interaction leading to surfactant production, NRG-1 protein staining appeared and became pronounced at the mesenchymal–epithelial cell border, underlining the suggestion of a paracrine involvement for NRG-1 in lung cell differentiation similar to the role in mammary lobuloalveolar budding during pregnancy (7). We and others have shown by immunohistochemistry that ErbB receptors are expressed in fetal lung epithelial cells in vivo (1, 26, 27).

In summary, NRG-1 localizes to the proper location for mesenchymal–epithelial communication. FCM from cultured Day 18 fetal mouse lung fibroblasts contains NRG-1ß. Purified NRG-1ß mimics the effect of FCM on surfactant synthesis and erbB receptor stimulation, and NRG-1 antibody inhibits FPF activity in the FCM. Surfactant synthesis is crucial to fetal lung development in preparation for a normal transition at birth. An insufficient synthesis of surfactant is primarily responsible for neonatal respiratory distress syndrome. Our results imply that a suppression or immaturity of the NRG-1 I erbB signaling system during lung development might contribute to this disease state.

	Acknowledgments

 
The authors thank Dr. MaryAnn Volpe for her help with the double immunostaining and Lucia D. Pham for the preparation of the fetal lung fibroblast and type II cell cultures.

	FOOTNOTES

 
Supported by National Institutes of Health HL 04436, HL 37930, CA 71702, and IROCA 71702–01; the Charles H. Hood Foundation (Boston, MA); and the Peabody Foundation (Boston, MA).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form May 24, 2002; accepted in final form March 24, 2003

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