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Am J Physiol Lung Cell Mol Physiol 293: L429-L435, 2007. First published June 1, 2007; doi:10.1152/ajplung.00451.2006
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ErbB4 regulates fetal surfactant phospholipid synthesis in primary fetal rat type II cells

Katja Zscheppang,1,2,3 Washa Liu,1 MaryAnn V. Volpe,1 Heber C. Nielsen,1 and Christiane E. L. Dammann1,3

1Department of Pediatrics, Division of Newborn Medicine, Tufts University and Floating Hospital for Children, Boston, Massachusetts; 2University of Applied Sciences Lausitz, Senftenberg, Germany; and 3Department of Pediatric Pulmonology and Neonatology, Hannover Medical School, Hannover, Germany

Submitted 16 November 2006 ; accepted in final form 31 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insufficient fetal surfactant production leads to respiratory distress syndrome among preterm infants. Neuregulin signals the onset of fetal surfactant phospholipid synthesis through formation of erbB receptor dimers. We hypothesized that erbB4 downregulation in fetal type II epithelial cells will downregulate not only fetal surfactant phospholipid synthesis, but also affect proliferation and erbB receptor localization. We tested these hypotheses using small interfering RNA (siRNA) directed against the erbB4 gene to silence erbB4 receptor function in cultures of primary day 19 fetal rat lung type II cells. ErbB4 siRNA treatment inhibited erbB4 receptor protein expression, fibroblast-conditioned medium induced erbB4 phosphorylation, and fetal surfactant phospholipid synthesis. Cell proliferation, measured as thymidine incorporation, was also inhibited by erbB4 siRNA treatment. Downregulation of erbB4 receptor protein changed erbB1 localization at baseline and after stimulation, as determined by confocal microscopy and subcellular fractionation. We conclude that erbB4 is an important receptor in the control of fetal lung type II cell maturation.

small interfering RNA

SURFACTANT SYNTHESIS IN PREPARATION for birth is a crucial component of fetal lung development. Insufficient surfactant production by immature type II cells contributes to the respiratory distress syndrome among preterm infants (1), which still ranks seventh among the causes of infant death in the United States (18). Development of fetal surfactant synthesis requires communication between the pulmonary mesenchyme and type II epithelial cells. Lung fibroblasts secrete substances that stimulate type II cells to synthesize surfactant (24). Today, we know that the growth factor neuregulin (NRG)-1, a ligand of the erbB receptors 3 and 4, is at least in part responsible for this fetal surfactant synthesis initiating effect (7). NRG-1 and ErbB receptors play critical roles in the early development of the heart and nervous system (19). Mouse embryos with a disrupted gene for NRG-1 or for the receptors erbB2, erbB3, or erbB4 die in midgestation from defects in cardiac or neuronal development (10, 14, 19, 23). Because of the midgestation lethality, these animals are not helpful in determining the importance of these receptors or their ligand NRG in late gestation fetal surfactant synthesis.

ErbB receptor activation involves the binding of a specific ligand such as NRG. Ligand binding triggers the formation of erbB homo- and heterodimers (13). Different erbB receptor dimers play different roles in different organ systems during development (15). We have shown that in fetal lung type II epithelial cells, erbB4 and erbB1 appear to be the major communication and dimerization partners (16a, 29). For this study, we hypothesized that erbB4 heterodimers are important for normal type II cell development. We proposed that erbB4 downregulation in fetal type II epithelial cells will downregulate fetal surfactant phospholipid synthesis and affect type II cell growth and erbB1 localization in these cells, implying that erbB4 heterodimers are important for normal type II cell differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plastic tissue culture dishes, flasks, and six-well tissue culture plates were obtained from BD Biosciences (San Jose, CA); mouse monoclonal anti-actin clone AC-40, epidermal growth factor (EGF), Gelvatol, DABCO, and DMEM were from Sigma (St. Louis, MO); fetal calf serum was from Hyclone (Logan, UT); goat anti-rabbit IgG (HRP-labeled), goat anti-mouse IgG (HRP-labeled), [3H]choline (specific activity 86 Ci/mol), and [3H]thymidine (specific activity 20 Ci/mmol) from Perkin Elmer Life Sciences (Boston, MA); 10% nonimmune goat serum from Zymed Laboratories (San Francisco, CA); rabbit polyclonal IgG anti EGFR antibody 1005, rabbit polyclonal IgG anti erbB3 antibody C-17, rabbit polyclonal IgG anti erbB4 antibody C-18, and mouse monoclonal IgG anti erbB4 antibody C-7 were from Santa Cruz (Santa Cruz, CA); rabbit polyclonal anti c-erbB2 antibody Ab-1, mouse monoclonal anti c-erbB2 antibody Ab-15, mouse monoclonal anti c-erbB3 antibody Ab-6, mouse monoclonal anti c-erbB3 antibody Ab-2, mouse monoclonal anti c-erbB3 antibody Ab-11, and mouse monoclonal anti c-erbB3 antibody Ab-5 were from Neo Markers (Fremont, CA); protein A Sepharose CL-4B was from Amersham Biosciences (Uppsala, Sweden); Precision Plus Protein standards (dual color) and Protran nitrocellulose transfer membrane were from Bio-Rad Laboratories (Hercules, CA); recombinant HRP-linked anti-phosphotyrosine antibody RC20 was from Transduction Laboratories (Palo Alto, CA); Alexa Fluor 488 goat anti-mouse IgG (H+L) and Alexa Fluor 568 goat anti-rabbit IgG (H+L) were from Molecular Probes (Eugene, OR); Transit-TKO Transfection Reagent and Label IT small interfering RNA (siRNA) Tracker Intracellular Localization Kit were from Mirus (Madison, WI); erbB4 siRNA, Silencer Negative Control #1 siRNA (scrambled siRNA), and GAPDH (positive control) siRNA were from Ambion (Austin, TX). CellTiter 96 AQueous Nonradioactive Cell Proliferation Assay was from Promega (Madison, WI). Neuregulin 1beta was produced using an expression vector kindly provided by Kermit Carraway III (UC Davis, CA) and purified by the GRASP Center for GI Research at Tufts-New England Medical Center (Tufts Univ., Boston, MA).

Preparation of fibroblast conditioned medium and primary fetal rat type II epithelial cell cultures. An animal research protocol approved by the institutional animal care and use committee at Tufts-New England Medical Center was used. Pregnant Sprague-Dawley rats were killed on day 21 [for fibroblast conditioned medium (FCM) collection] or day 19 (for type II cell isolation) of gestation by CO2 inhalation. Uteri were removed, and fetuses were kept in DMEM on ice. The lungs were removed, washed in sterile Hanks' buffered salt solution, and minced with a razor blade. The minced lungs were incubated with DNase and trypsin for 12 min at 37°C. The reaction was stopped by DMEM containing 10% charcoal-stripped fetal calf serum (FCS–). The cells were filtered, centrifuged, resuspended in DMEM containing 10% FCS–, and plated in culture dishes for 60 min at 37°C (21% O2/5% CO2) to allow differential adherence of lung fibroblasts. Fibroblasts were grown to confluence and then serum starved for 24 h to prepare conditioned media.

For type II cell isolation, the supernatants from the first differential adherence were centrifuged again. The cell pellet was resuspended in DMEM containing 10% FCS– and plated in culture flasks for 60 min at 37°C for a second differential adherence. Supernatants were again removed and centrifuged. Cell pellets were resuspended in DMEM containing 20% FCS–, and 1 or 0.5 x 106 cells were plated in 12-well plates (for Western blot and choline incorporation studies) or in 24-well plates (for thymidine incorporation studies), respectively.

siRNA experiments. In preliminary experiments, a cocktail (12) of three predesigned siRNAs, each at several incremental doses (20 nM, 30 nM, 40 nM, and 80 nM each), was used to determine the optimal concentration of each oligonucleotide for optimal erbB4 downregulation in type II epithelial cells. A concentration of 30 nM for each siRNA oligonucleotide (total 90 nM concentration) produced the maximal decrease in erbB4 protein content. The sequences of siRNA oligonucleotides to target the rat erbB4 mRNA are shown in Table 1. Methodology for transfection of primary cells was adapted from the method of Elbashir et al. (8) and from the manufacturer's guidelines (Mirus, Madison, WI) as previously published (27). Cells were transfected 24 h after plating at ~50% confluence with the erbB4 siRNA cocktail (GC content of 41.3%) using Transit-TKO Transfection Reagent over 48 h. The media were then changed to serum-free DMEM alone or DMEM:FCM (1:1), each condition containing the siRNA treatments and transfection reagent, for an additional 24 h resulting in a total exposure time of 72 h. Control conditions in each experiment included no treatment (control), transfection reagent alone (TKO), scrambled siRNA (90 nM, negative control, siRNA sequence not found in any known gene, guanine cytosine content of 43%), and siRNA directed against GAPDH (50 nM, positive control).


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  		Table 1. Sequences of small interfering RNA oligos used to target the rat erbB4 mRNA

 
Western blotting of the erbB receptors. Cells were plated in 12-well plates in DMEM containing 20% FCS–. Twenty-four hours after being plated, cells were treated for 48 h with siRNA. Cells were switched to free DMEM or DMEM containing FCM (1:1) still containing the siRNA for the next 24 h. Some cells were stimulated with NRG-1beta (33 nM) for 2 min. All cells were washed with ice-cold PBS and lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.1% Triton X-100, 10% glycerol, 1 mM Na3VO4, 1 mM NaF, 1 mM ZnCl2, 10 mM beta-glycerolphosphate, 5 mM tetrasodium pyrophosphate, 1 mM PMSF, and 4 mg/ml each of aprotinin, leupeptin, and pepstatin) as described before (7). Lysates were cleared by microcentrifugation. Aliquots were used for protein determination. Sixty micrograms of the total protein was boiled in Laemmli buffer for 5 min at 100°C. The proteins were separated by 7% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Protein loading was controlled by Ponceau staining. The blots were blocked using 1% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST), incubated with recombinant HRP-linked anti-phosphotyrosine antibody Py20 (1:5,000) overnight at 4°C, washed three times with TBST, and the proteins were visualized by enhanced chemiluminescence. Membranes were stripped in stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 0.8% beta-mercaptoethanol) for 30 min at 50°C, blocked with 1% BSA in TBST, and reprobed with erbB4 (C18), actin (clone AC-40), or GAPDH antibodies overnight at 4°C.

Subcellular fractionation. Cells were treated as described above in the Western blotting protocol and then scraped in PBS. Subcellular fractionation was done as described before (2). Cells were pelleted and suspended in nuclei buffer (pH 7.9, 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1% Triton X-100, 0.5 mM DTT), kept on ice for 10 min, and centrifuged at 10,000 g at 4°C for 10 min. The supernatants containing the cytoplasmic fractions were removed and saved for study. The pellets were suspended in high salt buffer (pH 7.9, 10 mM HEPES, 400 mM NaCl, 0.1 mM EDTA, 5% glycerol, 0.5 mM DTT), kept on ice for 20 min, and centrifuged at 10,000 g at 4°C for 10 min. The supernatants containing the nuclear fractions were used for study. Twenty micrograms of total protein of the cytoplasmic fractions and 10 µg of total protein of the nuclear fractions were used for Western blotting.

[3H]choline incorporation. [3H]choline incorporation into disaturated phosphatidylcholine (DSPC) was determined as previously described (7). Briefly, cells were plated in 12-well plates in DMEM containing 20% FCS–. After an initial 24-h incubation, cells were treated for 48 h with siRNA treatments. Then, cells were treated with siRNA in serum-free DMEM or DMEM containing FCM (1:1) and incubated with 2 µCi [3H]choline over 24 h. Some cells were stimulated with NRG-1beta (33 nM) for 2 min before all cells were harvested. Aliquots were used for protein assay (17). After lipid extraction (9), DSPC was isolated by treatment with osmium tetroxide and separated by thin-layer chromatography on silica gel H chromatography sheets. The resulting spots were scraped, and incorporated [3H]choline was measured by beta scintillation counting.

[3H]thymidine uptake. We used a thymidine uptake assay as previously described (7), which has been shown to correlate well with increased labeling by autoradiography (16) and bromodeoxyuridine uptake (6). Briefly, cells were plated in 24-well plates in DMEM containing 20% FCS– for 24 h and treated similarly in the first 48 h. After the first 48 h of siRNA treatments, cells were treated again with the different siRNAs in serum-free DMEM or DMEM containing FCM (1:1) while simultaneously incubated with 1 µCi [3H]thymidine per milliliter of media. Some control as well as siRNA-treated cells were stimulated with NRG-1beta (33 nM) for 2 min before all cells were washed three times with ice-cold PBS and trypsinized. Aliquots were used for DNA determination (3). Incorporated [3H]thymidine was measured in a beta scintillation counter.

MTS assay. The CellTiter 96 AQueous Nonradioactive Cell Proliferation Assay was used to determine the number of viable cells. Cells were plated in 96-well plates in 100 µl of DMEM containing 20% FCS. After 24 h, 20 µl of MTS/PMS solution was added, and cells were incubated for 3 h at 37°C. The absorbance was than recorded at 490 nm using an ELISA plate reader.

Confocal microscopy. Primary day 19 rat type II cells were cultured on glass coverslips. After 24 h of incubation, cells were treated for 48 h with siRNA treatments. Then, cells were treated with the same siRNA treatments in serum-free DMEM or DMEM containing FCM (1:1) for the final 24 h. Immunofluorescence was done as described before (21). Briefly, cells were rinsed with DMEM, fixed for 20 min in 3% paraformaldehyde, and permeabilized for 2 min with 0.2% Triton X-100 in PBS. After blocking in 10% normal goat serum, cells were incubated with primary erbB antibody (EGFR Ab1005, erbB4 AbC7) for 30 min at room temperature, washed with PBS, and incubated with secondary antibody (Alexa Fluor 488, Alexa Fluor 568) for 30 min at room temperature. Cells were mounted in Gelvatol/DABCO and analyzed using a Leica TCS-SP2 confocal laser scanning microscope.

Fluorescein labeling of siRNA. The transfection efficiency was determined by fluorescent labeling of the siRNA. The labeling reaction was prepared by incubating molecular biology-grade H2O, 10x labeling buffer A, Label IT siRNA Tracker Reagent, and 10 µg of siRNA for 1 h at 37°C. Unreacted Label siRNA Tracker Reagent was removed from the labeled siRNA by ethanol precipitation. The labeled siRNA was resuspended in siRNA dilution buffer, and cells were incubated with the labeled siRNA for 48 h.

Data analysis. The effects of erbB4 siRNA treatment on choline and thymidine incorporation and on erbB4 receptor protein expression were expressed as percentages of scrambled siRNA-treated values. To evaluate the results for their statistical significance, a two- or one-tailed t-test or Mann-Whitney U-test, with post hoc Bonferroni correction for multiple comparisons, where appropriate, were used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ErbB4 downregulation in fetal rat type II epithelial cells. Primary fetal rat type II cells were transfected with three different siRNAs targeting three different sequences in the transcribed erbB4 mRNA, a methodology that has been used by others (12). To identify the most effective dose of siRNA in these primary fetal type II cells, we performed a dose-response curve and found that concentrations of 60 nM and 120 nM showed similar effects with a dose-dependent U-shaped response curve (Fig. 1A). This U-shaped response has been documented for some genes, is not related to the sequence of siRNA used, and starts at ~100 ng of siRNA (Dr. Susan Magdaleno, Ambion, Austin, TX, personal communication). To minimize toxicity while optimizing the siRNA inhibitory effect, we chose the dose for subsequent experiments that was midrange between these doses at 90 nM (30 nM of each sequence). The transfection efficiency using this concentration, observed by fluorescein labeling of the siRNA, was 71%. Densitometry (Fig. 1B) of eight experiments using the combination of three different sequences against erbB4 at a concentration of 30 nM each showed a significant decrease in erbB4 protein to 74 ± 2.5% (means ± SE, n = 8, P < 0.0001) compared with scrambled siRNA.


Figure 1
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  		Fig. 1. Downregulation of erbB4 receptor protein and phosphorylation by erbB4 small interfering RNA (siRNA). A: primary fetal rat type II cell lysates after treatment with different concentrations of scrambled siRNA (left) or erbB4 siRNA (right). The top shows the effect on erbB4 phosphorylation, and the middle shows the effect on erbB4 protein content. Actin reprobing was done to control for protein loading. Densitometry results show the effect of erbB4 siRNA on protein expression (B) and erbB4 phosphorylation in nonstimulated, neuregulin (NRG)-stimulated, and fibroblast-conditioned medium (FCM)-stimulated cells for scrambled siRNA treatment (C, left) and erbB4 siRNA cocktail treatment (C, right). **P < 0.0001, n = 8, *P < 0.029, n = 4. As a positive control, GAPDH siRNA was used to downregulate GAPDH (D, top). Actin reprobing was done as an internal control for protein loading (D, bottom).

 
Endogenous baseline phosphorylation was reduced to 81 ± 8% (means ± SE, n = 4, P = 0.05) with the same concentration of erbB4 siRNA treatment compared with scrambled siRNA treatment. NRG-induced phosphorylation was reduced to 80 ± 15% (means ± SE, n = 4, P = 0.17), and FCM-induced phosphorylation was significantly reduced to 73 ± 12% (means ± SE, n = 4, P = 0.029) by erbB4 siRNA treatment (Fig. 1C).

We used GAPDH siRNA as a positive control to more fully evaluate the possibility of nonspecific effects of siRNA in our experiments. GAPDH protein was reduced to 52% by the GAPDH siRNA (Fig. 1D) without significantly affecting erbB4 protein or erbB4 phosphorylation (Fig. 1A).

Effect of erbB4 protein downregulation on fetal surfactant phospholipid synthesis measured by choline incorporation. siRNA directed against erbB4 reduced baseline DSPC production to 60 ± 5% (means ± SE, n = 14, P < 0.0001) compared with the scrambled siRNA treated controls (100 ± 7%, n = 15) (Fig. 2A). There was an additional inhibitory effect on NRG- or FCM-induced choline incorporation. ErbB4 siRNA treatment decreased NRG-induced choline incorporation to 80 ± 10% (means ± SE, n = 9, P = 0.09) and FCM-induced choline incorporation significantly to 75 ± 9% (means ± SE, n = 10, P = 0.01) compared with NRG- or FCM-stimulated scrambled siRNA-treated cells (Fig. 2B).


Figure 2
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  		Fig. 2. Effects of erbB4 siRNA treatment on surfactant phospholipid synthesis (A and B) and cell proliferation (C and D). Cells were treated with erbB4 siRNA (white bar), and baseline choline incorporation was expressed as percent of scrambled siRNA-treated cells (gray bar) (A). *P < 0.0001, n = 14. Shown is choline incorporation of scrambled siRNA and erbB4 siRNA-treated cells, exposed to NRG and FCM. Results of erbB4 siRNA-treated cells are expressed as percent of scrambled siRNA-treated cells (gray bars) (B). *P = 0.01, n = 10. Cells were treated with erbB4 siRNA (white bar), and baseline thymidine incorporation (*P < 0.05, n = 12) (C) or cell viability (**P < 0.002, n = 24) (D) was expressed as percent of scrambled siRNA-treated cells (gray bar). DPM, disintegrations per minute.

 
Effect of erbB4 protein downregulation on cell proliferation measured by thymidine incorporation and a cell proliferation assay. Neither NRG nor FCM stimulated proliferation in these primary type II cells at day 19 of gestation. Rather, these treatments caused a trend towards inhibition of cell proliferation to 84 ± 7% (means ± SE, n = 15, P = 0.17) for NRG and 82 ± 7% (means ± SE, n = 15, P = 0.14) for FCM compared with untreated control cells (100 ± 9%, n= 14) (data not shown). This is in agreement with our previous finding for this stage of development shown in isolated fetal mouse type II cells (5).

ErbB4 siRNA treatment alone decreased thymidine incorporation to 70 ± 8% (means ± SE, n= 12, P = 0.01) compared with scrambled siRNA-treated control cells (100 ± 7%, n = 15) (Fig. 2C). ErbB4 siRNA treatment further promoted the NRG- (90 ± 15%, means ± SE, n = 15) and FCM- (80 ± 8%, means ± SE, n = 15) induced inhibitory effect seen in scrambled siRNA-treated control cells. These changes were not statistically significant (data not shown).

To verify that these results reflect decreased cell proliferation rather than decreased mitochondrial DNA synthesis, we performed an additional cell proliferation assay as described in MATERIALS AND METHODS. The erbB4 siRNA treatment showed a downregulation of cell proliferation to 89 ± 4% (means ± SE, n = 8, P = 0.02) compared with scrambled siRNA-treated control cells (Fig. 2D). These results confirmed the inhibition of proliferation shown with the thymidine incorporation assay.

Effect of erbB4 siRNA treatment on erbB1 localization. Confocal microscopy was used to measure downregulation of erbB4 protein (green fluorescence) and cellular localization of erbB4 and erbB1 (red fluorescence) in scrambled siRNA-treated control rat type II epithelial cells (Fig. 3) and erbB4 siRNA-treated cells. Cells were either kept unstimulated (top) or treated for 24 h with FCM (bottom). In control cells treated with scrambled siRNA (controls), erbB4 staining was diffuse in the cytoplasm and in the nucleus. FCM treatment led to colocalization of erbB4 and erbB1 and an additional enrichment of both receptors in the cell membrane.


Figure 3
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  		Fig. 3. Effects of erbB4 downregulation on cellular erbB4 and erbB1 receptor localization. Confocal microscopy was used to study the colocalization (orange dye, C and F) of erbB4 (green dye, A and D) and erbB1 (red dye, B and E) in primary day 19 fetal rat type II cells. The left panels show localization signals of erbB4 and erbB1 in type II cells treated with scrambled siRNA alone (top left) or stimulated with FCM (bottom left). The right panels show localization signals of both receptors in type II cells treated with erbB4 siRNA cocktail alone (top right) or also stimulated with FCM (bottom right).

 
In contrast, erbB4 siRNA treatment led to an overall decreased staining with a more diffuse intracellular location of erbB4 receptor protein in the cytoplasm and the nucleus. ErbB4 downregulation also led to a change in erbB1 localization. In erbB4 siRNA-treated cells not stimulated with FCM, erbB1 was localized to the cell membrane, the cytoplasm, and the nucleus. Furthermore, the observed FCM-induced nuclear enrichment of erbB1 in control cells was disturbed in erbB4 siRNA-treated cells, leading to an inhibition of colocalization with erbB4 in FCM-treated cells (Fig. 3).

Subcellular fractionation studies underline the change in ErbB1 localization in the siRNA-treated cells seen by confocal imaging. Western blots demonstrated that in erbB4 siRNA-treated cells, the remaining erbB4 is localized to the cytoplasm and the nucleus, similar to erbB1. FCM stimulation led to enrichment of erbB4 in the nucleus, whereas erbB1 did not relocate to the nuclear fraction (Fig. 4). These subcellular fractionation data underline the observation that erbB4 siRNA treatment interferes with the FCM-induced nuclear localization of erbB1.


Figure 4
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  		Fig. 4. Subcellular localization of erbB4 and erbB1 after erbB4 downregulation. Subcellular fractionation of erbB4 siRNA-treated unstimulated (left) and FCM-stimulated (right) cells. Each left lane contains the cytoplasmic (cyto) and the right lane the nuclear (nucl) fraction. The top panel shows the effect on erbB4 protein content, and the bottom panel the effect on erbB1 protein content.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
NRG plays an important role at the onset of fetal surfactant synthesis (7). Here we focused on the NRG receptor erbB4 in primary fetal rat type II cells at day 19 of gestation, shortly before the onset of fetal surfactant synthesis. We observed that downregulation of erbB4 using siRNA downregulated surfactant phospholipid synthesis in day 19 rat fetal type II epithelial cells, implying an important role of erbB4 ligand and receptor in this element of fetal lung development.

Day 19 fetal rat type II cells were used because at this day in gestation, endogenous fibroblast type II cell communication mediating initiation of fetal surfactant synthesis takes place (4), which includes NRG secretion by the fetal lung (5). This is not surprising since NRG is known as signaling protein mediating cell-cell interactions (30). ErbB4 is the most prominent receptor in late fetal type II cells (27) and the signaling receptor for NRG. We used an in vitro gene silencing method with siRNA to inhibit erbB4 receptor function in these fetal type II cells. Similar to studies in COS cells (12), in a rat type II cell line (L2), the greatest reduction of erbB4 protein was accomplished with a combination of three siRNAs targeting three different sequences of erbB4 mRNA, at a concentration of 20 nM each (unpublished data). In primary rat type II cells, a combination of all three siRNAs, at a concentration of 30 nM each, resulted in maximal reproducible inhibition of erbB4 receptor protein amount and receptor phosphorylation.

The most prominent and functionally important surfactant lipid is DSPC. In the fetal lung, its synthesis is commonly considered a marker of lung type II cell differentiation (24). We found that siRNA directed against erbB4 caused an inhibition of surfactant DSPC synthesis to 60% at a downregulation level of erbB4 receptor protein and phosphorylation to 74%. Since the overall transfection efficiency was 71% and the half-life of erbB4 receptor protein is unknown in these cells, we believe that the level of erbB4 receptor present in our study in the erbB4 siRNA-treated cells may represent residual erbB4 receptor protein produced not only during the culture period, but in vivo levels present before the start of culture. In addition, erbB receptor density is an important factor in receptor dimerization and function (5, 25, 28). Thus this level of erbB4 reduction down to 71% may readily cause significant loss of erbB4 function.

Stimulation of fetal type II cells with either NRG or FCM to induce surfactant synthesis is well documented (7, 24). In this study, NRG or FCM stimulation of type II cells treated with erbB4 siRNA did not overcome the baseline inhibitory effect of erbB4 downregulation on surfactant phospholipid synthesis, implying that their major effect is signaled through erbB4. FCM contains substances that initiate type II cell differentiation (24) and play a crucial role in endogenous mediated lung maturation (22). This mesenchyme-epithelial cell interaction is influenced by hormones and growth factors (11). NRG is responsible, at least in part, for this surfactant synthesis initiating effect (7). Further in vivo animal studies of surfactant synthesis are necessary to resolve mechanisms of fibroblast- type II cell communication stimulating surfactant synthesis in the fetal lung.

In many developing organ systems, including the fetal lung, cell differentiation is accompanied by reduced cell proliferation (20, 26). We therefore studied the effect of erbB4 siRNA on cell proliferation using thymidine incorporation. ErbB4 receptor downregulation appeared to inhibit cell proliferation to 70% in these primary day 19 rat type II cells. Both NRG and FCM further decreased proliferation in siRNA-treated and untreated type II cells, thereby suggesting that NRG and FCM promote maturation at this time point of gestation. This is in agreement with our previously published results in primary day 18 mouse type II cells (7). On the other hand, we would have expected that erbB4 downregulation would overcome the inhibitory effect of these growth factors, whereas in fact the level of thymidine incorporation was very similar among all siRNA treatment types, regardless of the presence of either NRG or FCM. Therefore, we speculate that factors other than erbB4 regulate cell proliferation at this time in gestation.

In type II epithelial cells, erbB4 and erbB1 appear to be major dimerization partners (16a, 29). We used confocal microscopy to test the effect of erbB4 siRNA downregulation on localization and colocalization patterns of erbB1 and erbB4. ErbB1 showed a diffuse staining in the cytoplasm and the nucleus in nontreated and FCM-treated cells. In contrast, erbB4 was localized to the nucleus in both nonstimulated and stimulated cells. FCM treatment led to an additional enrichment in the cell membrane and to a colocalization between erbB1 and erbB4 in the perinuclear region. In contrast, erbB4 siRNA treatment led to an enrichment of erbB1 at the cell membrane. Additionally, the colocalization of erbB1 and erbB4 was inhibited by erbB4 siRNA treatment. Our localization and fractionation studies cannot distinguish between cytoplasm, Golgi, and plasma membrane for the exact cellular localization outside of the nucleus, but the data support the likelihood that the reduced level of erbB4 protein induced by siRNA was sufficient to alter receptor dynamics and function. Further studies are necessary to elucidate the exact intracellular localization and function of erbB receptors in the fetal type II cell.

Our results suggest that erbB4 plays a prominent role in basal fetal surfactant phospholipid synthesis at this gestational age and extend the understanding of the mechanisms by which FCM induce fetal type II cell maturation. The results may also indicate that the role of erbB4 in fetal surfactant phospholipid synthesis is not completely compensated for by the activity of one of the other erbB receptors, all of which are expressed in type II cells (7). In vivo studies are needed to confirm this conclusion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-04436, HL-37930, and HD-044784, the Deutsche Forschungsgemeinschaft (Da 378/ 3-1), and the Peabody Foundation (Boston, MA). Confocal microscopy studies were performed at the Confocal Imaging Center of the Tufts Univ. Center for Neuroscience Research, supported by P30-NS-047243 (R. Jackson). Neuregulin was purified by the GRASP Center, supported by P30-DK-34928.


   ACKNOWLEDGMENTS
 
We thank Drs. Rob Jackson and Lai Ding of the Confocal Imaging Center for consultative support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. E. L. Dammann, Dept. of Pediatrics, Tufts New England Medical Center, 750 Washington St., Boston, MA 02111 (e-mail: cdammann{at}tufts-nemc.org)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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E. Purevdorj, K. Zscheppang, H. G. Hoymann, A. Braun, D. von Mayersbach, M.-J. Brinkhaus, A. Schmiedl, and C. E. L. Dammann
ErbB4 deletion leads to changes in lung function and structure similar to bronchopulmonary dysplasia
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L516 - L522.
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