Neuregulin-1 and Human Epidermal Growth Factor ... - ATS Journals

Neuregulin-1 and Human Epidermal Growth Factor Receptors 2 and 3 Play a Role in Human Lung Development In Vitro Neil V. Patel, Michael J. Acarregui, Jeanne M. Snyder, Jonathan M. Klein, Mark X. Sliwkowski, and Jeffrey A. Kern Departments of Internal Medicine, Pediatrics, and Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa; and Genentech, Inc., South San Francisco, California

The human epidermal growth factor receptor (HER) family consists of four distinct receptors: HER1 (epidermal growth factor receptor), HER2, HER3, and HER4. Their specific activating ligands are collectively known as neuregulins (NRG). We hypothesized that one member of the NRG family, NRG-1, and the HER family would play a role in fetal lung development. To test this hypothesis, we defined NRG-1 and HER gene expression in mid-trimester human fetal lung tissue. HER2 and HER3 messenger RNA and protein were detected in the fetal lung, but HER4 expression was not detected. Immunohistochemical staining of fetal lung tissue localized HER2 and HER3 protein to the developing lung epithelium. NRG-1 expression was not found in freshly isolated human fetal lung, but it was observed in fetal lung explants after 2 d of explant culture. Immunohistochemistry of cultured human fetal lung explants revealed that NRG-1 protein was also expressed in pulmonary epithelial cells. Exposing human fetal lung to recombinant NRG-1 activated the HER receptor complex as measured by ⵑ 4-fold increases in receptor phosphotyrosine content. In addition, NRG-1 increased explant epithelial cell volume density ⵑ 2-fold (P ⬍ 0.03); increased epithelial cell proliferation ⵑ 2-fold, as determined by bromodeoxyuridine labeling (P ⫽ 0.002); and reduced surfactant protein-A (SP-A) levels by 53% (P ⬍ 0.05). These data are consistent with an autocrine regulatory process mediated by NRG-1 activation of HER2/HER3 heterodimers expressed on developing human fetal lung epithelial cells. Receptor activation results in increased lung epithelial cell proliferation and volume density, and decreased SP-A production, a marker of type II pneumocyte differentiation.

Receptor tyrosine kinases (RTKs) and their specific ligands play essential roles in intracellular and intercellular communication. During development, these interactions are a mechanism for coordinating growth and differentiation (1–3). Specific RTKs have already been shown to play a role in lung development. For example, insertional inactivation of the platelet-derived growth factor receptor-A in mice reduced the number of alveolar myofibroblasts, decreased alveolar septation, and resulted in emphysematous lung tissue (4). Inhibition of signaling through the fibroblast growth factor receptor (FGFR) in transgenic (Received in original form July 12, 1999 and in revised form October 19, 1999) Address correspondence to: Jeffrey A. Kern, M.D., C-33A, GH, Dept. of Medicine, University of Iowa, Iowa City, IA 52242. E-mail: jeffrey-kern@ uiowa.edu Abbreviations: bromodeoxyuridine, BrdU; dibutyryl-cyclic adenosine monophosphate, dBt-cAMP; epidermal growth factor, EGF; epidermal growth factor receptor, EGFR; ethylene glycol-bis(␤-aminoethyl ether) N,N,N⬘, N⬘tetraacetic acid, EGTA; fibroblast growth factor receptor, FGFR; human epidermal growth factor receptor, HER; immunoglobulin, Ig; dissociation constant, Kd; neuregulin, NRG; phosphate-buffered saline, PBS; polymerase chain reaction, PCR; reverse transcriptase, RT; receptor tyrosine kinase, RTK; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; surfactant protein-A, SP-A. Am. J. Respir. Cell Mol. Biol. Vol. 22, pp. 432–440, 2000 Internet address: www.atsjournals.org

mice with lung-specific expression of a dominant negative FGFR blocked airway branching and epithelial differentiation without inhibiting outgrowth of the mainstem bronchi (5). This resulted in undifferentiated epithelial tubes extending from the bifurcation of the trachea to the diaphragm and ultimately perinatal death. Antisense oligonucleotides directed against keratinocyte growth factor (KGF), the FGFR-activating ligand, inhibited terminal bud formation in embryonic mouse lung explants (6). Thus, RTKs appear to play several critical roles in lung development. Human epidermal growth factor receptors (HER) are another important mediator of cell growth and differentiation (7, 8). The HER family belongs to the subclass I RTK superfamily and consists of four distinct receptors: epidermal growth factor receptor (EGFR; also called HER1), HER2, HER3, and HER4. In the lung, alveolar type II cell differentiation and maturation are regulated in part through an epidermal growth factor (EGF)-EGFR interaction (9– 11). Loss of the EGF-induced EGFR signal in EGFR knockout mice resulted in lungs with impaired branching, deficient alveolarization, and a marked reduction in alveolar volume (12). In human fetal lung tissue, inhibition of signal transduction by blocking EGFR tyrosine kinase activity resulted in decreased surfactant protein-A (SP-A) expression (13). SP-A is a calcium-dependent lectin associated with lung surfactant, and a marker of type II pneumocyte differentiation (14, 15). All four receptor proteins are transmembrane tyrosine kinases and are structurally similar, with two cysteine-rich extracellular domains, a membrane spanning domain, a tyrosine kinase intracellular domain, and a variable cytoplasmic tail (16, 17). However, HER2, HER3, and HER4 are divergent from EGFR, with their extracellular amino acid sequence no more than 47% homologous to EGFR (16, 18, 19). There are also distinct intracellular sequences within the HER family’s cytoplasmic tails that result in different second messenger interactions and signal pathway activation. The role of these family members in lung development is unknown. Ligands for HER3 and HER4 include a family of three proteins collectively known as neuregulins (20), and individually as neuregulin (NRG)-1, NRG-2, and NRG-3 (21–23). NRG-1 and NRG-2 gene expression has been reported in the human adult and developing lung (21, 22). Our studies have focused on NRG-1 in lung development, although NRG-2 may also play a role. At least 15 different isoforms of NRG-1 are known to result from alternative splicing of a single transcript (17). Some isoforms of NRG-1 are synthesized as a 75-kD membrane-bound proform that can be cleaved in an extracellular juxta-membrane domain to release a 45-kD soluble NRG-1 ligand (21, 24). All active NRG-1 isoforms share

Patel, Acarregui, Snyder, et al.: NRG-1, HER2, and HER3 in Lung Development

a specific sequence in the extracellular domain, an EGF-like domain that is necessary and sufficient for bioactivity. As with many receptors, ligand-mediated dimerization of the HER must occur in order to form a stable binding site for NRG-1 (25). Heterodimers of HER proteins (HER2/HER3, HER2/HER4) appear to form high affinity NRG-1 binding sites (dissociation constant [Kd] ⬇ 100 pM) (26–30), whereas homodimers (HER3/HER3, HER4/HER4) form low affinity sites (Kd ⬇ 5–10 nM) (27, 28, 31, 32). Interaction with NRG-1 activates the receptor’s intrinsic tyrosine kinase, leading to autophosphorylation or transphosphorylation of the receptor dimer, and initiates a transmembrane signaling cascade (33). HER3 and HER2 homodimers are not activated by NRG-1. HER2 homodimers do not bind NRG-1 (17). However, HER2 can be activated in a heterodimer receptor complex. HER3 homodimers can bind NRG-1, but the intracellular kinase catalytic domain of HER3 is inactive due to amino acid substitutions in its tyrosine kinase domain (18, 30). The role of NRG-1 and the HER family in lung growth and development is not known. HER2 has been detected in embryonic rat tissue, including lung epithelium (24, 34, 35), and in human pulmonary epithelium (36, 37). Studies from our laboratory have shown that HER2 is overexpressed in approximately 30% of epithelial lung cancers (36–38) and that the HER2 axis is capable of regulating pulmonary epithelial cell growth because proliferation of lung cancer cell lines can be modulated through HER2 (39) and blocked with specific tyrosine phosphorylation inhibitors. These data suggest that HER2 is involved in pulmonary epithelial cell proliferation. Less is known about HER3; however, it is also present in fetal and adult murine lung tissue (35, 40). To date, no reports exist concerning HER4 expression in human lung tissue. Recent publications have shown the importance of members of the HER/ NRG-1 receptor-ligand system in development. In HER2, HER4, and NRG-1 murine knockout models, all of the animals died of central nervous system and cardiac malformations on or before Day 11 of gestation (41–43). The role of the receptor or ligand knockout in lung development is unclear, as the deaths occurred before lung organogenesis. HER3 knockout mice have gross malformations of the peripheral nervous system and Schwann cells (44, 45). Some of these embryos survive to birth but die shortly thereafter because of respiratory insufficiency (44). We hypothesized that NRG-1 and the HER family would be expressed in the lung during development and may play a role in normal lung organogenesis. To test this hypothesis, we first defined NRG-1 and HER expression in human fetal lung tissue. In addition, we studied the functional effect of NRG-1 receptor activation in fetal lung explants. A role for the receptor in either epithelial cell proliferation or differentiation could be postulated based on results reported in other organ systems (41–44). Therefore, we examined the effect of receptor activation on pulmonary epithelial cell content and SP-A production in a human fetal explant model.

Materials and Methods Human Fetal Lung Explant Culture Human fetal lung tissue was obtained from mid-trimester abortuses (17–21 wk) under a protocol approved by the University of

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Iowa Human Subjects Review Committee. Fetal lung explants were prepared from eight different fetuses. The major airways were removed and the distal lung was minced into 1-mm3 pieces. The explants were placed on sterile lens paper, which rested on a metal grid, in a 35-mm culture dish containing 1 ml of serum-free Waymouth MB 752/1 medium with added penicillin G (100 U/ml), streptomycin (100 ␮g/ml), and amphotericin-B (0.25 ␮g/ml). The explants were incubated at 37⬚C over a four- to six-day period in a humidified atmosphere of 5% CO2 with daily media changes. When used, NRG-1 was added at the initiation of culture (10⫺8 M) and refreshed daily with media changes. Elapsed time from tissue recovery to culture initiation or snap-freezing in liquid nitrogen was 4 to 6 h. The day of harvest was considered Day 0 (D0) for our cultures. Days 1 to 6 (D1, D2, D3, D4, D5, D6) of culture were sequential 24-h intervals after the day of tissue recovery (D0).

Reverse Transcriptase/Polymerase Chain Reaction Total tissue RNA was prepared from human fetal lung explants harvested from the explant culture system on Days 0, 2, 4, and 6. RNA was extracted using RNA-Stat 60 (Tel-test B, Friendswood, TX) according to the manufacturer’s directions. Contaminating DNA was removed by a 15-min incubation with deoxyribonuclease I (DNase I; 1 U) (GIBCO BRL, Grand Island, NY). The reaction was stopped with ethylenediaminetetraacetic acid (2 mM final concentration) and heating (65⬚C for 15 min). DNase I–treated RNA (10 ␮g) was reverse transcribed (42⬚C for 2 h) using 5 ␮g/ml oligo-dT in polymerase chain reaction (PCR) buffer (25 mM MgCl2, 10 mM deoxynucleotide triphosphate, and 0.1 mM dithiothreitol with 200 U reverse transcriptase [RT]). All singlestranded RNA was digested at the end of the reaction period with RNase H (5 U, 37⬚C for 20 min). Aliquots of the reaction mix were then subjected to PCR (94⬚C for 45 s, 55⬚C for 45 s, 72⬚C for 45 s; 40 cycles). PCR was carried out using specific primers for HER2, HER3, HER4, and NRG-1 (HER2: F 5⬘-AGGGAAACCTGGAACTCACC-3⬘, R 5⬘-TGGA-TCAAGAC-CCCTCCTT-3⬘; HER3: F 5⬘-CAGGTGCTGGCCTTGCTTTT-3⬘, R 5⬘-GTGGCTGGAGTTGGTGTTAT-3⬘; HER4: F 5⬘-TCCAGCCCAGCGATTCTCAG-3⬘, R 5⬘-GGCCAGTACAGGACTTATGG-3⬘; NRG-1: F 5⬘-GCGAATTCC-ATCTTGTAAAATGTGCG-3⬘, R 5⬘-CTCGGCCGCTACTCCG-CCTCCATAAATTCAATC-3⬘). PCR products were separated on a 2% agarose/Tris-acetate-EDTA (TAE) gel and visualized with ethidium bromide staining.

Immunoblot Analysis Approximately 0.1 g of tissue was placed in lysis buffer (20 mM Tris, 0.33 M sucrose, 0.5 mM ethylene glycol-bis(␤-aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid [EGTA], 0.5 mM leupeptin, 0.03 mM aprotinin, 100 mM N-ethylmaleimide, 1% Triton-X 100, pH 8.0) and homogenized for 30 s. The protein from the homogenized tissue was quantified by the method of Bradford (Bio-Rad, Hercules, CA). Equal protein amounts were aliquoted into Laemmli buffer and boiled for 5 min. The protein was electrophoresed on 4 to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gels (Bio-Rad) and electroblotted to a nitrocellulose membrane. Nonspecific binding was blocked by a 2-h incubation in 5% nonfat dry milk in phosphatebuffered saline (PBS) at room temperature. The membrane was probed with specific antibodies to HER2, HER3, HER4 (HER2 polyclonal antibody, K-15; ErbB-3 polyclonal antibody, C-17; ErbB-4 polyclonal antibody, C-18; all from Santa Cruz Biotechnology, Santa Cruz, CA), or NRG-1 (3G11 polyclonal antibody; Genentech, Inc., South San Francisco, CA). All HER antibodies were produced in rabbits and raised against synthetic peptides derived from the human sequence. 3G11 is a murine monoclonal antibody directed against the human NRG-1 EGF-like domain (amino

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acids 177–244). All primary antibodies were used at a final concentration of 0.1 ␮g/ml. Positive controls consisted of the K562 human chronic myelogenous leukemia cell line transfected with full-length complementary DNAs for either HER2, HER3, or HER4 expressing the appropriate protein. Specific bands were visualized using a horseradish peroxidase–labeled goat antirabbit immunoglobulin (Ig)G or goat antimouse IgG, both used at 0.02 ␮g/ml, and visualized by enhanced chemoluminescence detection (ECL; Amersham, Piscataway, NJ). The relative amount of HER protein at each time point was quantified by scanning densitometry with the aid of NIH Image software.

Immunohistochemistry Lung explants were flash-frozen in liquid nitrogen at the appropriate time points and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA). Sections were cut to 6 to 8 ␮m thickness at ⫺20⬚C and thawmounted on microscope slides (SuperFrost Plus; Fisher Scientific, Springfield, NJ). Frozen sections were hydrated in PBS, fixed in neutral buffered formalin, endogenous peroxidase activity quenched with 0.3% H2O2, and blocked with horse serum. Sections were incubated with primary antibody (0.1 ␮g/ml in PBS) directed against HER2, HER3, or HER4 (Santa Cruz Biotechnology) or NRG-1 (Neomarkers; Lab Vision, Fremont, CA) for 16 h at 4⬚C. Antibodies are described in the previous section. Secondary antibody was Vectastain Elite universal antibody containing polyclonal goat antirabbit Ig followed by avidin-biotin conjugate reagent (Vector Laboratories, Burlingame, CA) according to manufacturer’s directions. Staining was visualized by incubation with diaminobenzadine substrate followed by counterstaining with Harris hematoxylin (50% solution).

Phosphotyrosine Analysis Human fetal lung explants were placed in serum-free Waymouth media and recombinant NRG-1(177–244) was added to 10⫺8 M final concentration. At various times after NRG-1(177–244) addition, the samples were homogenized in lysis buffer with protease and phosphatase inhibitors (50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, 0.03 mM aprotinin, 100 mM N-ethylmaleimide, 1 mM Na3VO4, 1 mM NaF, pH 7.4). Proteins were separated on a 4 to 15% SDS-PAGE gradient gel (Bio-Rad) and electroblotted to a nitrocellulose membrane. Nonspecific binding was blocked by a 2-h incubation with 5% nonfat dry milk in PBS at room temperature. The membrane was then probed with a specific polyclonal antibody to phosphotyrosine residues (Upstate Biotechnology, Lake Placid, NY). An EGF-stimulated human vulvar cancer cell line, A431, served as the positive control. Specific bands were visualized with a horseradish peroxidase–labeled goat antirabbit antibody and visualized by ECL detection (Amersham).

Morphometric Analysis Frozen sections from human fetal lung explants were sectioned (6–7 ␮m thickness) and mounted as described, hydrated in PBS, stained in Harris hematoxylin solution, dehydrated in ethanol and xylene, and cover slips applied. Morphometric changes were quantified by point counting using a method previously described (46, 47). One slide representing each condition from eight separate experiments, with each experiment using a separate donor, was analyzed. The specimens were viewed with a light microscope using a ⫻40 objective and an eyepiece reticule with a 10 ⫻ 10 grid with 100 intersections. The tissue at each of the grid intersection points was scored as being lumen, connective tissue, or epithelium. Slides were evaluated independently by two observers blinded to treatment conditions. Data were expressed as the volume densities of the lumen and epithelium. The lumen volume density was calcu-

lated by dividing the number of lumen points by the total points from all components (epithelium plus connective tissue plus lumen). The volume density of the epithelium was calculated as the number of epithelial tissue points divided by total points from tissue only (epithelium plus connective tissue). Conditions were compared using the unpaired, two-tailed Student’s t test.

Bromodeoxyuridine Labeling of Explant Culture Explant cultures of human fetal lungs were cultured in the presence of 10⫺8 M NRG-1 for 4 d as described. During the final 24 h of culture, bromodeoxyuridine (BrdU; 2 mM) (Sigma Chemical Co., St. Louis, MO) was added. The explants were harvested, fixed in formalin, processed to paraffin, and 5-␮m sections cut and mounted on glass slides. BrdU immunoreactivity was detected with a commercially available kit (Zymed Laboratories Inc., South San Francisco, CA) according to the manufacturer’s instructions. The proportion of total epithelial cells labeled was determined by counting the total number of epithelial cells in a prealveolar duct and the number of BrdU positive cells in the same duct by light microcopy. In two separate experiments using tissue from two different donors, six representative ducts were randomly selected and counted for a total of twelve determinations per condition. Results were compared using an unpaired, two-tailed Student’s t test.

NRG-1 Effect on SP-A Production In Vitro NRG-1(177–244) (10⫺8 M) was added daily to fetal lung explants maintained in serum-free Waymouth medium over 4 d. Control explant tissue was maintained in serum-free Waymouth medium without NRG-1(177–244), whereas the positive control was exposed to dibutyryl-cyclic adenosine monophosphate (dBt-cAMP) at 10⫺6 M. Tissue from each condition was placed in lysis buffer (20 mM Tris, 0.33 M sucrose, 0.5 mM EGTA, 0.5 mM leupeptin, 0.03 mM aprotinin, 100 mM N-ethylmaleimide, 1% Triton-X 100, pH 8.0) and homogenized for 30 s. The protein from the homogenized tissue was quantified by the method of Bradford (Bio-Rad). Equal protein amounts were aliquoted into Laemmli buffer and boiled for 5 min. The protein was electrophoresed on 4 to 15% SDS-PAGE gradient gels (Bio-Rad) and electroblotted to a nitrocellulose membrane. Nonspecific binding was blocked by a 2-h incubation in 5% nonfat dry milk in PBS at room temperature. The membrane was then probed with a guinea pig, polyclonal human SP-A antisera (1:1,000 dilution) (14) and bound antibody detected with an alkaline phosphatase–conjugated goat antiguinea pig IgG (whole molecule) (Sigma) and 5-bromo-4-chloro-3indoyl phosphate/nitro blue tetrazolium (BCIP/NBT alkaline phosphatase substrate; Sigma). Data were compared using an unpaired, two-tailed Student’s t test.

Results Expression of HER Family Receptors in Developing Human Fetal Lung We first determined the expression of HER2, HER3, and HER4 in the human fetal lung. Human fetal lung tissue was obtained and total RNA extracted. Equal amounts of total RNA were subjected to RT-PCR using specific primers for HER2, HER3, and HER4 (Figure 1A). On D0 (day of tissue recovery), single bands representing the PCR products for HER2 and HER3 were found in the human fetal lung tissue (D0; Figure 1A, lane 1), whereas no HER4 PCR product was identified on D0. Total RNA that was not reverse transcribed (Figure 1A, lanes 5 and 6) had no PCR product for any receptor, confirming that the PCR products were not a result of DNA contamination. Protein analysis


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Figure 1. Identification of HER2 and HER3 in human fetal lung explants. (A) Total RNA was obtained from mid-trimester human fetal lung explants (D0) or explants cultured for the indicated days. The RNA was incubated with reverse transcriptase (RT⫹), whereas control samples (RT⫺) were not. PCR products were separated on a 2% TAE gel, stained with ethidium bromide (1 mg/liter, 1 h), and photographed. Lane 1 ⫽ D0; lane 2 ⫽ D2; lane 3 ⫽ D4; lane 4 ⫽ D6; lane 5 ⫽ D0 (RT⫺); lane 6 ⫽ D4 (RT⫺); lane 7 ⫽ positive complementary DNA (cDNA) control. Expected PCR product sizes were HER2 ⫽ 296 bp, HER3 ⫽ 366 bp, and HER4 ⫽ 495 bp. The gel is representative of two experiments. (B) Equal protein amounts (10 ␮g) from homogenates of mid-trimester human fetal lung explants (D0) or explants cultured for the indicated days were suspended in Laemmli buffer, electrophoresed through a 4–15% SDS-PAGE gel, blotted onto nitrocellulose, and membranes probed for HER2, HER3, and HER4. Bound antibody was detected using chemiluminescence techniques. The respective antibodies are specific for HER2, HER3, and HER4, and do not cross-react with other receptor family members. A positive control consisted of K562 cells transfected with a full-length cDNA to HER2, HER3, or HER4, and expressing the respective full-length protein. The blot is representative of five experiments.

Figure 2. Immunostaining for HER2, HER3, and NRG-1 in mid-trimester human fetal lung and cultured fetal lung explants. Human fetal lung was stained on D0 or explants were cultured for 4 d in control media, harvested, and then stained for HER2, HER3, and NRG1. The lumens of prealveolar ducts are indicated with an “L,” the connective tissue between the ducts is indicated with a “CT,” and the arrows indicate the epithelium of the prealveolar ducts. The primary antibody was directed against HER2 (A) and HER3 (C) using D0 tissue. In B, D, and G, HER2, HER3, and NRG-1 antibodies were used to analyze D4 tissue, respectively. Negative staining controls are included in E (D0) and F (D4). Staining is representative of eight different experiments, with tissue obtained from eight separate donors. Original magnification: ⫻400.

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Figure 3. Identification of NRG-1 in human fetal lung explants. (A) Total RNA was obtained from mid-trimester human fetal lung explants (D0) or explants cultured for the indicated days. The RNA was incubated with reverse transcriptase (RT⫹), whereas control samples (RT⫺) were not. PCR products were separated on a 2% TAE gel, stained with ethidium bromide (1 mg/liter, 1 h), and photographed. Lane 1 ⫽ D0; lane 2 ⫽ D2; lane 3 ⫽ D4; lane 4 ⫽ D0 (RT⫺); lane 5 ⫽ D4 (RT⫺); lane 6 ⫽ positive cDNA control. Expected PCR product size for NRG-1 ⫽ 175 bp. The gel is representative of two experiments. (B) Equal protein amounts (10 ␮g) from homogenates of mid-trimester human fetal lung explants (D0) or explants cultured for the indicated days were suspended in nonreducing Laemmli buffer, electrophoresed through a 4–15% SDS-PAGE gel, blotted onto nitrocellulose, and the membranes probed for NRG-1 (3G11; Genentech, Inc.). Bound antibody was detected using chemiluminescence techniques. The blot is representative of five experiments.

also identified HER2 and HER3 in the D0 fetal lung tissue. Immunoblot analysis of human fetal lung proteins on D0 identified both HER2 and HER3 protein expression but no HER4 protein expression (Figure 1B). The expressed protein was 185 kD for HER2 and 180 kD for HER3, data that agree with the published molecular mass for the full-length receptor protein (18, 26, 48, 49). Immunohistochemical staining of D0 fetal lung tissue further confirmed the presence of HER2 and HER3 proteins (Figure 2). HER2 and HER3 staining was localized only to the lung epithelium. No HER2 or HER3 protein staining was observed in mes-

Figure 4. Effect of NRG-1(177–244) on NRG-1 receptor phosphorylation. Human fetal lung tissue was stimulated in vitro with NRG-1(177–244) (10⫺8 M) and harvested at the indicated time points in buffer containing protease and phosphatase inhibitors. Equivalent protein amounts (10 ␮g) were separated by SDSPAGE, electroblotted to nitrocellulose, and probed with a phosphotyrosine antibody. Increased phosphorylation of HER proteins (p180 band) was identified. Increased phosphorylation of lower molecular weight proteins was also seen (open arrow), possibly representing downstream tyrosine phosphorylated substrates. EGF-stimulated A431 cells were used as a positive control with phosphorylated EGFR represented by the p170 band. The blot is representative of five experiments using tissue obtained from different donors.

enchymal cells. Thus, HER2 and HER3 proteins are specifically expressed in pulmonary epithelial cells of freshly isolated mid-trimester human fetal lung tissue. The levels of HER2 and HER3 were also modulated in human fetal lung explants over time in culture in vitro (Figure 2). Staining intensity for HER2 and HER3 increased during the time in culture. All staining for HER2 and HER3 remained in the lung epithelium during differentiation in vitro. This observation was consistent with our immunoblot analysis where the average relative amount of HER2 in-

Figure 5. The effect of NRG-1(177–224) on the development of human fetal lung explant development in vitro. Fetal lung explants were cultured as described in media supplemented with 10⫺8 M NRG-1(177–244) or no additions for 6 d. Samples were frozen, sectioned, mounted on slides, stained, and point counting performed. (A) NRG-1(177–244) significantly increased explant epithelium volume density (P ⫽ 0.03). (B) NRG-1(177–244) increases lumenal volume density. Results represent the mean and standard deviation of eight experiments. Data were compared using a two-tailed, unpaired Student’s t test.


creased 3.8-fold (P ⫽ 0.03) to a maximum level on D4 of culture (Figure 1B). The mean relative amount of HER3 similarly increased 3.5-fold (P ⫽ 0.03) with time in culture and reached a maximum on D4 of culture (Figure 1B). HER4 was not detected at any time point. These data support the formation of a high affinity NRG-1 receptor (HER2/HER3 heterodimer) in the human lung during midgestation that is modulated during in vitro differentiation. Expression of NRG-1 in Developing Human Lung With the identification of HER proteins capable of forming a high affinity NRG-1 binding site in fetal lung, we next determined if the ligand was also present. Human fe-

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tal lung samples were analyzed for NRG-1 gene expression by RT-PCR. Unlike the HER gene expression, a specific NRG-1 PCR product was not detected in reverse transcribed RNA from freshly isolated human fetal lung tissue (Figure 3A). In addition, immunoblot analysis using an NRG-1–specific antibody, 3G11 (Genentech Inc.), did not reveal NRG-1 protein expression in the starting tissue, D0 (Figure 3B). However, with time in culture, NRG-1 messenger RNA (mRNA) and protein became detectable. As shown in Figure 3A, lanes 2 and 3, a specific NRG-1 PCR product became detectable on D2 of culture and increased on D4. No PCR product was seen in samples not exposed to RT, confirming that the PCR band was not due to DNA contamination. NRG-1 protein expression was also identified by immunoblot analysis on D2 and D4 of culture (Figure 3B). The 75-kD band presumably represents the unprocessed, membrane-bound form of the protein. In addition, a 45-kD band was identified on D2 and increased on D4, which most likely represents the soluble form of NRG-1. Immunohistochemistry of human fetal lung in explant culture also revealed NRG-1 protein specifically expressed in pulmonary epithelial cells of mid-trimester human fetal lung (Figure 2). NRG-1 expression was not identified in mesenchymal cells. Thus, NRG-1 was expressed in the same epithelial cell population as HER2 and HER3 in the human fetal lung explants. A Functional NRG-1 Receptor Is Present in Developing Human Lung To begin to assess the role of HER2 and HER3 in human fetal lung, an analysis of NRG-1–induced changes in human fetal lung receptor phosphotyrosine content was performed. When freshly isolated human fetal lung (D0) in serum-free Waymouth medium was exposed to NRG-1(177–244) for 20 min, receptor activation was readily detected, as shown in Figure 4. The relative intensity of the p180 band representing tyrosine phosphorylated HER2 or HER3 increased ⵑ 4-fold in comparison to the unstimulated control. Receptor phosphorylation returned to baseline levels within 2 h, whereas increased concentrations of lower molecular weight phosphorylated proteins were maintained, possibly representing downstream tyrosine phosphorylated substrates. Low concentrations of phosphorylated receptor were present in the unstimulated samples, suggesting the existence of baseline receptor phosphorylation.

Figure 6. NRG-1 stimulates pulmonary epithelial cell proliferation. Explant cultures of human fetal lungs were cultured in the presence of 10⫺8 M NRG-1 for 4 d (A) or with no stimulation (B) as described (original magnification: ⫻400). During the final 24 h of culture, the explants were incubated with BrdU (2 mM), then fixed, sections cut (5 ␮m), and BrdU immunoreactivity detected. The lumens of prealveolar ducts are indicated with an “L,” the connective tissue between the ducts is indicated with a “CT,” the closed arrows indicate the BrdU-labeled epithelial cells, whereas the open arrows point to unlabeled epithelial cells. Two separate experiments were performed using tissue from different donors.

Modulation of the NRG-1/HER System Results in Increased Epithelial Cell Proliferation and Morphologic Changes during In Vitro Fetal Lung Development We next sought to determine the developmental response to NRG-1 receptor activation in human fetal lung. Human fetal lung explants were cultured over 6 d, as previously described, in the presence or absence of exogenous NRG1(177–244) (10⫺8 M). At the end of 6 d, the samples were harvested, stained, and point counting was performed to determine the volume density of epithelial, mesenchymal, and lumenal components. Human fetal lung explants grown in the presence of exogenous NRG-1(177–244) showed almost a 2-fold increase in pulmonary epithelial cell volume density (Figure 5A) (P ⬍ 0.03). Interestingly, the lumenal volume density also increased 1.7-fold with NRG-1(177–244) stimula-

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tion (Figure 5B), and approached, but did not reach, statistical significance (P ⫽ 0.1). These data suggested that pulmonary epithelial cells were proliferating in response to HER activation. To further define the responding cells, we performed BrdU labeling of NRG-1–stimulated human fetal lung explants. As shown in Figure 6, after 4 d of culture, proliferating cells in the untreated explants were localized to the epithelium. After 4 d of NRG-1 exposure, labeled cells remained confined to the epithelial cell layer. The number of proliferating cells as defined by BrdU labeling in distal airway ducts in NRG-1–exposed explants was directly compared with unstimulated explants. The unstimulated explants had 27.2 ⫾ 14.2% of all epithelial airway cells labeled, whereas the NRG-1–exposed explants had 48.7 ⫾ 16.1% (P ⫽ 0.002). Taken together, these data support a role for the NRG-1 receptor in human fetal lung development in vitro with HER2/HER3 heterodimers as the functional NRG-1 receptor and HER activation important in regulating epithelial cell proliferation. NRG-1 Decreases Fetal Lung SP-A Production In Vitro NRG-1 has also been shown to affect cellular differentiation (41). Therefore, we examined the effect of NRG-1(177–244) on the production of SP-A, as a marker of type II cell differentiation in human fetal lung. Using the 35-kD monomeric form of the protein, Western blot results were quantified by digital analysis and graphed in Figure 7. Control tissue maintained in serum-free Waymouth medium had significant levels of SP-A. The addition of NRG-1(177–244) to fetal lung explants at 10⫺8 M for 24 h reduced SP-A levels 53% (P ⬍ 0.05). The NRG-1 suppression of SP-A was dose dependent, and no longer significant at 10⫺12 M. This concentration is well below the Kd of the high affinity (10⫺10 M) receptor, and loss of SP-A suppression is expected if the effect is modulated through the high affinity receptor. The positive control, explants cultured with dBt-cAMP, demonstrated increased amounts of SP-A. To ensure that the change in SP-A was an effect of NRG-1 on type II pneumocytes, immunohistochemistry was performed on the tissue to identify SP-A–expressing cells. Only cells with type II pneumocyte morphology (epithelial layer in location, lamellar bodies) had SP-A immunoreactivity at any time in the culture system (D0, D4, and D6).

Discussion In this study, we found that HER2 and HER3 were both present in midgestation human fetal lung samples and their expression levels increased during explant differentiation in vitro. In addition, the HER2 and HER3 receptors were colocalized in lung tissue epithelial cells. We did not detect HER4 by either immunoblot analysis or RT-PCR. These data support HER2/HER3 heterodimers as the most likely NRG-1 receptor present in the developing human lung epithelium. The NRG-1 receptor formed is functional and capable of signal transduction in the fetal lung epithelium as evidenced by changes in receptor phosphotyrosine content after NRG-1(177–244) exposure. Ultimately, this signal results in increased epithelial cell proliferation, increased volume density of epithelial cells in lung explants, and decreased SP-A production. NRG-1, the ligand for high affinity HER2/HER3, is also present in the fetal lung. Interestingly, NRG-1 is expressed in the pulmonary epithelial cell compartment together with HER2 and HER3. Integrating the presence of the functional receptor and ligand, and the effect on explant epithelial cell proliferation and phenotype after receptor activation in vitro led us to postulate that an autocrine HER activation process regulates pulmonary epithelial cell proliferation and differentiation in the developing lung. As the tissue is undifferentiated at the time of explant culture initiation, NRG-1 exposure appears to affect the tissue’s normal autodifferentiation process, not cause dedifferentiation. Our analyses were confined to the pseudoglandular stage of lung development and type II pneumocyte differentiation in the canalicular stage (50). However, the receptors are present in the adult lung (34–37) and may play a general role in the regulation of pulmonary epithelial cell growth throughout life. The decreased SP-A production induced by NRG-1 identifies a specific effect of NRG-1 on type II pneumocytes. Activation of the receptor may either directly suppress SP-A production or affect the type II pneumocyte differentiation process, resulting in decreased SP-A production. Autocrine regulation of cell proliferation and differentiation by the HER/NRG-1 system has been postulated in other organ development. Schwann cells produce and secrete NRG-1, and express HER2 and HER3 (51–53). Their expression is coordinately regulated in response to axon damage, suggesting an autocrine interaction of Schwann

Figure 7. The effect of NRG-1(177–244) on SP-A production by human fetal lung explants. Human fetal lung explants were maintained for 4 d in culture with media alone, with NRG-1(177–244), or 1 ␮M dBtcAMP. Equal amounts of protein from the explants were suspended in Laemmli buffer, electrophoresed through a 4–15% SDS-PAGE gel, blotted onto nitrocellulose, and membranes probed for SP-A. Bound antibody was detected with a secondary antibody conjugated to alkaline phosphatase followed by substrate. (A) Blots from four separate experiments were quantified by digital imaging and plotted. Data were compared using an unpaired, two-tailed Student’s t test; *P ⬍ 0.05 compared with unstimulated control. (B) A representative Western blot.


cell NRG and its HER complex. Developing muscle synthesizes NRG and also expresses HER2 and HER3 (54). Stimulation of HER2/HER3 heterodimers induces synaptic expression of acetylcholine receptor genes, suggesting autocrine regulation of synapse development. The developing murine mammary gland also expresses all components of the HER signaling system (HER2, HER3, HER4, and NRG-1) predominantly in the epithelial cell layer (55). Much like our analysis of the lung, receptor and NRG-1 expression patterns were dynamic, implying a developmentally specialized function for each receptor. Taken together, these findings suggest that autocrine activation of the HER2/HER3 heterodimer may play a broad role in epithelial tissues during organ and gland development. In contrast, the NRG-1/HER interaction in brain and heart organogenesis appears to occur through a paracrine process. In the developing central nervous system, NRG-1 is expressed in the hindbrain (rhombomeres 2, 4, and 6) and migrating cranial cells (41), whereas HER4, a low affinity NRG-1 receptor, is expressed in rhombomeres 3 and 5 by the migrating neural crest cells (42). NRG-1 is postulated to induce proliferation of neural crest cells from rhombomeres 3 and 5 toward rhombomeres 2, 4, and 6. NRG-1 is also expressed in the developing endocardium, whereas HER2 and HER4 are expressed in underlying myocardium. The interaction of NRG-1 with HER2 and HER4 appears to control myocardial cell outgrowth and subsequent cardiac trabeculae development. The current study does not address all issues concerning the HER/NRG-1 receptor-ligand axis in the lung. It is possible that some NRG-1 is made in an extrapulmonary location and transported to the lung. However, NRG-1 mRNA and protein expression increased in the pulmonary explants during time in culture, indicating de novo lungspecific synthesis of NRG-1 must occur. The role of soluble versus membrane-bound NRG-1 also remains unclear. We did not attempt to determine if a lung-specific NRG-1 isoform exists. Our antibodies and PCR primers were designed to identify all members of the NRG-1 family, and NRG-1(177–244) has the biologic activity of all isoforms. Finally, the role of two other members of the NRG family, NRG-2 and NRG-3 (22, 23), in lung development was not addressed. NRG-2 may have a role, as this is another putative ligand for HER3 (22). Our data predict that NRG-3 will not have a role in lung development, as preliminary studies indicate that NRG-3 is a ligand for HER4, a receptor that we did not find expressed in the developing lung. Though we do not know the biologic significance of HER2 and HER3 homodimers, they may have a role in lung development or differentiation. HER2 is present in excess of the other members of the HER family, as determined by our immunoblot and immunohistochemistry studies. Therefore, HER2 homodimers may form along with HER2/HER3 heterodimers. Currently there is no known ligand for HER2 homodimers, so the role of this receptor combination must await identification of a specific ligand. HER3 homodimers can bind NRG-1; however, its tyrosine kinase domain is inactive and as a homodimer complex would be unable to lead to signal transduction. Thus, the biologic relevance of HER2 and HER3 homodimers in the developing lung remains unclear.

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In summary, our studies support the formation of a HER2/HER3 heterodimer creating a high affinity NRG-1 receptor in the developing human lung. The receptor is modulated over time in our in vitro explant system, suggesting a specialized receptor function, and the same epithelial cell layer that expresses the receptor produces the ligand. Activation of the receptor results in signal transduction, increased epithelial cell proliferation, increased epithelial cell volume density, and decreased expression of a type II pneumocyte differentiation marker, SP-A. A precise phenotypic role has not been completely elucidated in these studies. However, we speculate that this receptorligand system plays a role in regulation of epithelial cell proliferation in the pseudoglandular stage of lung development and type II epithelial cell differentiation in the canalicular stage. Acknowledgments: The authors would like to acknowledge Susan Wiechert, Kelli Goss, and Katherine Lower for their expert technical assistance.

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