Introduction

During fetal development, lung epithelial cells actively secrete fluid, thereby filling the developing lung. Vectorial Cl− transport-driven pulmonary fluid accumulation supports lung growth by establishing an intra-pulmonary pressure that promotes cellular proliferation. Animal studies with intrauterine tracheal drainage as well as tracheal occlusion demonstrated the pivotal relationship between fluid accumulation and fetal lung development1. A contribution of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) to this process has been suggested2,3,4. More precisely, during fetal lung development CFTR expression exhibits a time- and tissue-dependent expression pattern. The highest CFTR expression level was observed in the 1st and 2nd gestational trimester, while its expression gradually declines during the 3rd trimester5,6. Epithelial cell proliferation was accelerated by Cftr over-expression in the pseudoglandular stage resulting in an enhanced lung growth3. Prior to birth, the fetal lung fluid has to be removed to enable air breathing. Alveolar fluid clearance (AFC) is driven by epithelial Na+ transport accomplished by epithelial Na+ channels (ENaC) in the apical membrane compartment and the Na,K-ATPase in the basolateral membrane compartment of alveolar type II (ATII) cells. ENaC consist of three homologous subunits, α-, β-, and γ-ENaC7, and the Na–K-ATPase is composed of α1- and β1-subunits in ATII cells8. Vectorial Na+ transport establishes an osmotic driving force causing fluid absorption from the air spaces into the interstitium. In premature newborns a decreased AFC has been shown9, possibly due to a lower expression of epithelial Na+ channels10. In addition to surfactant deficiency, AFC insufficiency contributes to the development of the respiratory distress syndrome (RDS)10. Importantly, a sex ratio of 1:1.711,12 was observed for the RDS incidence, with males developing RDS significantly more frequently compared with female infants of the same gestational age, raising male mortality13. Up until now, male sex remains an independent risk factor for RDS development11,12. We have previously shown sex-specific differences in alveolar Na+ transport. Male sex was associated with lower Na+ transport and reduced levels of the ENaC and Na–K–ATPase subunits in fetal distal lung epithelial (FDLE) cells14. Na+ transport in female cells was more responsive to female sex steroids possibly contributing to the observed female advantage15, while androgens and glucocorticoids were either lacking any effect or equally stimulated Na+ transport in both sexes15,16. Another important growth factor during fetal lung development is the epidermal growth factor (EGF). EGF, via its receptor (EGFR), enhances terminal branch formation and stimulates proliferation and differentiation of epithelial and mesenchymal cells in culture17. EGFR deficient mice exhibit a neonatal lethal pulmonary phenotype and present a reduced branching as well as condensed lungs with collapsed alveoli, resembling a human neonatal RDS phenotype18,19. EGFR is the prototypic member of the ErbB (erythroblastic oncogene B) family of receptor tyrosine kinases (reviewed in20). To date, three other members of this family have been identified: ErbB2, ErbB3, and ErbB4, which are capable of forming homodimers, heterodimers, and possibly higher-order oligomers. Despite their large degree of structural homology, ErbB family members differ from each other in their patterns of expression, ligand specificity, and intracellular substrates. Complementing these receptors is a family of ligands: e.g. EGF and transforming growth factor-α (TGF-α) bind only to ErbB1 (EGFR), heparin-binding EGF (HB-EGF) binds to both ErbB1 and ErbB4, and neuregulin 1 (NRG1) which binds to ErbB3 and ErbB4. ErbB2 has no known ligand and acts through dimerization with one of the other three receptors, while ErbB3 is kinase impaired. Downstream ErbB signaling includes phosphoinositide 3-kinases (PI3-K)/ protein kinase B (AKT), mitogen-activated protein kinase (MAPK) signaling, and the phospholipase C (PLC) pathway21, all of which are known to affect ENaC functions.

Sex-specific responses to EGF have been previously noted regarding surfactant synthesis22,23,24 and an impact on epithelial Na+ transport has been described by others25,26. However, a potential sex-specific effect of EGF on Na+ transport in fetal alveolar cells is currently unknown. We therefore aimed to determine the impact of EGF and its related ligands on Na+ transport in sex-specific FDLE cells and reveal contributing pathways in this context.

Methods

Cell isolation and culture

Sprague–Dawley rats were obtained from the Medical Experimental Center (MEZ) of Leipzig University. Animals were kept in rooms with a 12 h light–dark cycle, constant temperature (22 °C) and humidity (55%). Food and water were supplied ad libitum. At gestational day E20-21 (term E = 22) pregnant rats were anesthetized by CO2 inhalation and euthanized by Pentobarbital injection. Separation of male and female fetuses was done through visual determination, as described before14. All experimental procedures were approved by the institutional review board (IRB: Landesdirektion Leipzig, permit number: T23/15) and complied with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.

FDLE cells, a model of fetal ATII cells, were isolated from fetal lungs as described previously27,28. Male and female fetuses were derived from the same litter in equal numbers and prepared separately. Fetal lungs were mechanically dissociated with razor blades. The resulting cell suspension was enzymatically digested by incubation with 0.125% trypsin (Fisher Scientific, Schwerte, Germany) and 0.4 mg/ml DNAse (CellSystems, Troisdorf, Germany) in HBSS (Fisher Scientific) for 10 min at 37 °C, followed by MEM containing 0.1% collagenase (CellSystems) and DNAse for 15 min at 37 °C. To separate adjacent lung fibroblasts from FDLE cells, the cell mix was plated twice for one hour at 37 °C in cell culture flasks. FDLE cells were then seeded on permeable Snapwell inserts (Costar, # 3407, surface area 1.1 cm2, Corning, NY) at a density of 106 cells per insert. For RNA and protein isolation, cells were seeded on larger inserts (ThinCert, #657641, surface area 4.6 cm2, Greiner Bio-One, Frickenhausen, Germany) at a density of 2 × 106 cells per insert. To generate sex-specific epithelial-fibroblast co-cultures, FDLE cells were seeded on inserts as described above, while the adjacent lung fibroblasts obtained by differential adhesion were seeded in the compartment below the insert at the bottom of the well plate. FDLE cells were cultured in serum-supplemented medium consisting of MEM with 10% FBS (Biochrom, Berlin, Germany), glutamine (2 mM, Fisher Scientific) and antibiotic–antimycotic (Fisher Scientific). Cell culture medium was changed to serum-free medium (SF-Med, Cellgro, Mediatech, Herndon, VA, USA) supplemented with either EGF (10 ng/ml, # 354,052, BD-Bioscience, San Jose, CA, USA), HB-EGF (50 ng/ml, # 97560, Biomol, Hamburg, Germany), TGF-α (10 ng/ml, # T8250-05, Biomol) or NRG1 (50 µg/ml, # SRP3055, Sigma-Aldrich, Taufkirchen, Germany) 24 h prior to analyses. The concentrations of estradiol (E2, 0.0037 µM) and progesterone (P, 2.8 µM) were chosen based on previous studies15,29. The antagonist Erlotinib (10 µM, # Cay10483-250, Biomol) was used to block EGFR and thereby the EGF-evoked response. Growth factors and the receptor antagonist were dissolved in dimethyl sulfoxide (DMSO). To exclude solvent influences on the evoked responses, control cells were treated with the same amount of the respective solvent.

Using chamber measurements

Ussing chamber measurements were performed five days after cell isolation, as previously reported14. Only monolayers with a transepithelial resistance (Rte) exceeding 300 Ω cm2 were included in the analyses. Electrophysiological solutions consisted of: 145 mM Na+, 5 mM K+, 1.2 mM Ca2+, 1.2 mM Mg2+, 125 mM Cl−, 25 mM HCO3−, 3.3 mM H2PO4− and 0.8 mM HPO42− (pH 7.4). For the basolateral solution, 10 mM glucose was used, while 10 mM mannitol was used in the apical solution. During measurements, the solutions were continuously bubbled with carbogen (5% CO2 and 95% O2). Short-circuit currents (ISC) were measured every 20 s with a transepithelial voltage clamp (Physiologic instruments, San Diego, CA, USA). After the ISC reached a stable plateau (Ibase), amiloride (10 µM, # A7410, Sigma-Aldrich) was applied to the apical chamber to assess the amiloride-insensitive ISC (Iamil-insens). The current reduction induced by amiloride (ΔIamil) was used as a measure of ENaC activity. Forskolin (10 µM, # F-6886, Sigma-Aldrich) was added to the apical compartment after amiloride application to increase the intracellular cyclic adenosine monophosphate (cAMP) concentration and thereby activate cAMP-sensitive ion channels like CFTR, contributing to the forskolin-induced ISC (ΔIforsk). Finally, CFTRinh172 (10 µM, # 3430, Bio-Techne, Wiesbaden-Nordenstadt, Germany) was applied apically to determine the CFTRinh172-sensitive ISC (ΔICFTR), a measure of CFTR activity. Amiloride was dissolved in water; forskolin and CFTRinh172 were prepared in DMSO.

mRNA expression analyses

RNA isolation, reverse transcription and real-time quantitative PCR (RT-qPCR) were done as described before16. Gene-specific primers are listed in Table 1.

Table 1 Primer sequences.
Full size table

Western blot analyses

FDLE cell inserts were placed on ice, and protein lysates prepared and analyzed as described elsewhere30. Phosphorylation of AKT was analyzed using antibodies against phospho-AKT at Ser473 (# 9271, Cell Signaling Technology, Frankfurt am Main, Germany), and AKT (# 9272, Cell Signaling Technology, both kindly provided by A. Garten). Phosphorylation of ERK1/2 (extracellular-signal regulated kinases) was analyzed using antibodies against phospho-ERK1/2 at Tyr202/Tyr204 (# 9101, Cell Signaling Technology), and ERK1/2 (# 9102, Cell Signaling Technology, both kindly provided by A. Garten). EGFR (C74B9, # 2646, Cell Signaling Technology) expression was measured and α-Tubulin (11H10, # 2125, Cell Signaling Technology) expression was used as a reference.

MTT

Metabolic activity in male and female FDLE cells stimulated with EGF was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as described before31.

Cell permeability assay

Epithelial permeability was assessed with FDLE cells cultured on Snapwell inserts. After two days in culture, the medium was changed to EGF-containing or control medium. After 24 h the medium was replaced by phenol red-free MEM (Fisher Scientific) in the lower compartment and phenol red-free MEM containing FITC-dextran (0.25 mg/ml, 3–5 kDa, # 46944, Sigma-Aldrich) in the upper insert compartment. Cells were incubated for another 24 h, followed by analysis of FITC-dextran fluorescence intensity in the lower compartment.

Statistical analysis

Differences between two groups were analyzed with the unpaired t-test. Otherwise, significant differences were determined by ANOVA with Dunnett’s post hoc test. A probability of p < 0.05 was considered significant for all statistical analyses. Statistical analysis was performed with GraphPad Prism software (GraphPad Prism 9.1.1, https://www.graphpad.com, GraphPad Software, La Jolla, CA, USA).

Results

Effect of EGF on Na+ and Cl− transporters in sex-specific FDLE cells

We addressed the effect of EGF on epithelial Na+ channels in male and female FDLE cells separately. Cells were cultured in SF-Med to exclude potential serum effects. In male and female FDLE cells, application of EGF significantly decreased α-ENaC, β-ENaC and γ-ENaC mRNA expression after 24 h compared with control cells (Fig. 1a, b). In detail, EGF reduced α-ENaC mRNA expression by approximately 30%, and β-ENaC and γ-ENaC mRNA expression by approximately 60% (p < 0.01; p < 0.001). EGF further reduced Na,K-ATPase-α1 in male FDLE cells by 10%, and Na,K-ATPase-β1 mRNA expression by 25% in both, male and female cells (p < 0.05; p < 0.001, Fig. 1c). In contrast to the reduction of Na+ transporter mRNA levels, EGF significantly elevated the mRNA expression of surfactant protein A (Sftpa), B (Sftpb) and C (Sftpc) in both sexes (Fig. 1d). Moreover, EGF increased Sftpa mRNA levels by 50%, Sftpb by more than 400%, and Sftpc by approximately 20–30% (p < 0.05; p < 0.01; p < 0.001), without any pronounced sex difference.

Figure 1
figure 1

EGF reduced mRNA expression of Na+ transporters in both sexes. Sex-specific FDLE cells were isolated from fetal rats, cultured in SF-Med and stimulated with EGF for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line) of relative fold change (RFC). In male (a) and female (b) FDLE cells, EGF significantly decreased α-ENaC, β-ENaC and γ-ENaC mRNA expression (n = 11–12; **p < 0.01; ***p < 0.001, t-test). (c) EGF also reduced Na,K-ATPase-α1 in male FDLE cells, and Na,K-ATPase-β1 mRNA expression in both, male and female cells (n = 8; *p < 0.05; ***p < 0.001, t-test). (d) Surfactant protein (Sftpa, Sftpb and Sftpc) mRNA expression was elevated by EGF in male and female FDLE cells (n = 8; *p < 0.05; **p < 0.01; ***p < 0.001, t-test). (Grey boxes) male; (yellow boxes) female.

Full size image

Ussing chamber analyses demonstrated that baseline ISC (Ibase) of male cells was significantly decreased by EGF after 24 h compared with untreated male controls (p < 0.01; Fig. 2a). The amiloride-sensitive ISC (ΔIamil) was also significantly decreased by EGF in male FDLE cells (p < 0.001). Similarly, EGF significantly decreased Ibase and ΔIamil in female FDLE cells (p < 0.01; p < 0.001; Fig. 2a). The results suggest that epithelial Na+ transport of male and female FDLE cells is equally reduced by EGF after 24 h. In contrast, epithelial barrier function with regard to Rte was highly increased by EGF in both sexes (p < 0.001; Fig. 2b). Furthermore, the amiloride-insensitive ISC (Iamil-insens), most likely Cl− transport, was strongly increased by EGF in male and female cells (p < 0.001; Fig. 2c). This is further supported by the significantly elevated forskolin-induced ISC (ΔIforsk) and the increased CFTRinh172-sensitive ISC (ΔICFTR) induced by EGF in female FDLE cells (p < 0.01; p < 0.001; Fig. 2d). Surprisingly, male FDLE cells did not show a significant increase of CFTR activity, since ΔIforsk and ΔICFTR were not affected by EGF addition. Finally, CFTR mRNA expression was strongly increased by EGF in both, male and female FDLE cells (p < 0.001; Fig. 2e). Taken together, EGF negatively regulated mRNA level and activity of Na+ transporters in both sexes after 24 h. On the other hand, EGF strongly elevated Cl− transport in females and CFTR mRNA expression in both, male and female FDLE cells.

Figure 2
figure 2

EGF decreased Na+ transport, but increased Cl− transport and CFTR mRNA expression. FDLE cells were cultured in SF-Med and stimulated with EGF for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) In male and female FDLE cells, EGF significantly decreased Ibase and ΔIamil (n = 52–59; **p < 0.01; ***p < 0.001 by t-test). EGF further significantly increased Rte (b) and Iamil-insens (c) in both sexes (***p < 0.001). (d) EGF strongly increased ΔIforsk and ΔICFTR, but only in female FDLE cells (n = 71–76; **p < 0.01; ***p < 0.001 by t-test). (e) EGF significantly increased CFTR mRNA expression in both sexes (n = 8; ***p < 0.001; t-test). (Grey boxes) male; (yellow boxes) female.

Full size image

Persistence of the EGF response was tested by stimulating FDLE cells with EGF for 24 h, followed by another 24 h culture in SF-Med without EGF. These experiments were done in mixed FDLE cells of both sexes. Culture with SF-Med restored Ibase and ΔIamil of initially EGF-treated cells to control levels (Fig. 3a). In contrast, EGF’s effect on Iamil-insens persisted even after 24 h culture in SF-Med, shown by the significantly higher Iamil-insens in formerly EGF-treated cells (p < 0.001). The elevated barrier function shown above was abolished by 24 h culture in SF-Med (Fig. 3b), suggesting that the effect of EGF on Na+ transport and Rte requires its continuous presence.

Figure 3
figure 3

Persistence of EGFs’ effect. FDLE cells (mixed sexes) were cultured in SF-Med and stimulated with or without EGF for 24 h, followed by a medium exchange to SF-Med lacking EGF in both groups and culture for another 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) The reducing effect of EGF on Ibase and ΔIamil did not persist after 24 h culture in SF-Med, while the increasing effect on Iamil-insens was retained in mixed FDLE cells (n = 26/27; ***p < 0.001 by t-test). (b) Furthermore, the stimulating effect of EGF on Rte was lost after 24 h in SF-Med.

Full size image

Sex-specific expression of EGFR

In the presence of fetal serum, female FDLE cells displayed a higher EGFR protein expression (p < 0.001; Fig. 4a). In FDLE cells cultured in SF-Med no sex-specific protein expression of EGFR was observed, and EGF application significantly downregulated EGFR protein expression after 24 h in both sexes (p < 0.001; Fig. 4b). In accordance, EGF also reduced EGFR mRNA expression in male and female cells by approximately 80% (p < 0.001; Fig. 4c), and EGFR mRNA expression was not sex-specific. The results thus suggest that EGFR expression is sex-specific only in the presence of fetal serum, while EGF equally reduces the expression of its own receptor in both sexes.

Figure 4
figure 4

Sex-specific and EGF-dependent expression of EGFR. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) EGFR protein expression was significantly higher in female FDLE cells compared to male cells in the presence of serum (n = 7; ***p < 0.001 by t-test). EGF application to FDLE cells cultured in SF-Med reduced EGFR protein (b) and mRNA expression (c) after 24 h in FDLE cells of both sexes (n = 6/8; ***p < 0.001 by t-test). (Grey boxes) male; (yellow boxes) female.

Full size image

Acute effects of EGF in sex-specific FDLE cells

Previous studies suggested a biphasic response with acute EGF effects differing from chronic effects. Sex-specific FDLE cells were thus cultured in SF-Med for 24 h prior to the addition of EGF for 45 min. In accordance to the chronic EGF treatment, Ibase and ΔIamil were significantly decreased by EGF after 45 min (p < 0.01; p < 0.001; Fig. 5a). However, this effect was only observed in male FDLE cells, while female FDLE cells were not affected by acute EGF addition. Moreover, Iamil-insens was significantly increased by EGF only in male cells (p < 0.05; Fig. 5b). Barrier function was not affected by short-term EGF treatment since the Rte was not different compared to controls or between sexes (Fig. 5c). Western blot demonstrated a significantly higher AKT phosphorylation in female FDLE cells after acute EGF treatment that was not observed in males (p < 0.05; Fig. 5d). ERK phosphorylation was significantly elevated by acute EGF application in both male and female cells, although the response to EGF with regard to ERK1/2 phosphorylation was stronger in male cells (p < 0.001; Fig. 5e). Since AKT phosphorylation is suggested to increase ENaC activity, and ERK1/2 phosphorylation downregulates ENaC, elevated pAKT in combination to a lower increase of pERK1/2 in females possibly prevents the decrease in ENaC activity that is observed in males after acute EGF treatment. These results show that at least for the acute effects of EGF sex-specific differences were observed with regard to Na+ and Cl− transport. EGFR protein expression was also decreased by short-term EGF exposure by approximately 30% in both male and female FDLE cells (p < 0.05; p < 0.001; Fig. 5f).

Figure 5
figure 5

Acute sex-specific effects of EGF via AKT-ERK1/2 signaling. FDLE cells were cultured in SF-Med and stimulated with EGF for 45 min. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) In male FDLE cells, EGF significantly decreased Ibase and ΔIamil after 45 min (n = 32–37; **p < 0.01; ***p < 0.001 by t-test), while female FDLE cells were not significantly affected by acute EGF application. (b) EGF acutely increased Iamil-insens in male FDLE cells (*p < 0.05), while Rte (c) was not affected within the experimental time frame. (d) EGF significantly increased AKT phosphorylation within 15 min only in female FDLE cells (n = 3; *p < 0.05 by t-test). (e) In contrast, EGF significantly increased ERK1/2 phosphorylation within 15 min in both, male and female FDLE cells (n = 3; ***p < 0.001 by t-test). (f) Short-term EGF exposure decreased EGFR protein expression (n = 10/9; *p < 0.05; ***p < 0.001 by t-test). (Grey boxes) male; (yellow boxes) female.

Full size image

In contrast to the acute effects of EGF, AKT phosphorylation was reduced in male FDLE cells after chronic (24 h) EGF treatment (p < 0.05; Fig. 6a), while AKT phosphorylation in females was not different between EGF-treated and untreated cells. Furthermore, ERK phosphorylation was not different in chronically EGF-treated and controls in both male and female FDLE cells (Fig. 6b). Thus, the acute effects of EGF on AKT/ERK phosphorylation differ from its chronic effects.

Figure 6
figure 6

Effect of chronic EGF exposure on AKT-ERK1/2 phosphorylation. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). FDLE cells were cultured in SF-Med and stimulated with EGF for 24 h. (a) EGF reduced AKT phosphorylation in male FDLE cells (n = 8; *p < 0.05 by t-test). (b) In contrast, chronic EGF exposure had no significant effect on ERK1/2 phosphorylation (n = 8).

Full size image

EGF affects barrier permeability and metabolic activity in sex-specific FDLE cells

As shown above, chronic EGF treatment strongly elevated Rte suggesting an improved barrier permeability. To confirm this observation a FITC-dextran assay was conducted showing a significantly lower epithelial permeability induced by EGF in both male and female FDLE cells (p < 0.001; Fig. 7a). The elevated Rte and the reduced permeability support an improved barrier function induced by EGF. On the other hand, metabolic activity determined by an MTT assay was elevated only in male cells, while female cells were not affected by EGF (p < 0.01; Fig. 7b). FDLE cell numbers after 24 and 48 h of EGF treatment were unchanged in both male and female cells, suggesting that EGF did not stimulate proliferation of epithelial monolayers (Fig. 7c).

Figure 7
figure 7

EGF decreased permeability in both sexes, but increased metabolic activity only in males. FDLE cells were cultured in SF-Med and stimulated with EGF for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) FITC-dextran assay demonstrated a significantly reduced permeability induced by EGF in male and female FDLE cells (n = 8; **p < 0.01; ***p < 0.001 by t-test). (b) EGF further increased the metabolic activity in male FDLE cells, in contrast to female cells, as shown with a MTT assay (n = 6; **p < 0.01 by t-test). (c) The cell number was not altered by EGF addition in male and female FDLE cells after 24 and 48 h (n = 6–8). (Grey boxes) male; (yellow boxes) female.

Full size image

Response to EGF in epithelial-mesenchymal co-cultures

EGF is known to stimulate the epithelial-mesenchymal interaction during fetal lung development. We therefore treated co-cultures of sex-specific FDLE cells and sex-specific fetal lung fibroblasts with EGF for 24 h. EGF significantly reduced Ibase and ΔIamil in both sexes (p < 0.001; Fig. 8a, b). Moreover, Iamil-insens was increased by EGF in male and female FDLE cells as shown above, lacking sex-specific effects (p < 0.01; p < 0.001; Fig. 8a, b). Epithelial barrier function with regard to Rte was highly increased by EGF only in males (p < 0.001; Fig. 8c), although the same trend was observed in females. EGFR protein expression in sex-specific fetal lung fibroblasts cultured in the presence (Fig. 8d) or absence of fetal serum (Fig. 8e) was not different between male and female fibroblasts. EGF application again significantly reduced EGFR protein levels in both male and female fibroblasts (p < 0.01; Fig. 8e).

Figure 8
figure 8

EGF decreased Na+ transport in epithelial-fibroblast co-cultures of both sexes. FDLE cells were co-cultured with fetal lung fibroblasts in SF-Med and stimulated with EGF for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). In male (a) and female (b) FDLE cells, EGF significantly decreased Ibase and ΔIamil and increased Iamil-insens (n = 26–32; **p < 0.01; ***p < 0.001 by t-test). (c) EGF significantly increased Rte only in male cells (***p < 0.001). (d) EGFR protein expression was not different between male and female fetal lung fibroblasts cultured in serum-supplemented medium (n = 6). (e) EGF application to fetal lung fibroblasts cultured in SF-Med strongly decreased EGFR protein levels after 24 h in both, male and female fibroblasts (n = 4; ***p < 0.001 by t-test). (Grey boxes) male; (yellow boxes) female.

Full size image

Effects of EGF after inhibition of EGFR with Erlotinib

Inhibition of EGFR with Erlotinib did not prevent the decrease of Na+ transport induced by EGF, but even further decreased Ibase and ΔIamil in both sexes (p < 0.05; p < 0.01; p < 0.001; Fig. 9a, b). On the other hand, Erlotinib prevented the increase of Iamil-insens induced by EGF (p < 0.01; p < 0.001; Fig. 9a, b). Furthermore, the significant increase of Rte induced by EGF was completely abolished by inhibition with Erlotinib (p < 0.001; Fig. 9c). Thus, Erlotinib prevents only some of the EGF-induced effects on epithelial ion transport, whereas other effects were even amplified.

Figure 9
figure 9

EGFR inhibitor prevented the effect of EGF on Iamil-insens and Rte, but not on Na+ transport. FDLE cells were cultured in SF-Med and stimulated with EGF with or without erlotinib for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). EGF significantly decreased Ibase and ΔIamil in both, male (a) and female (b) FDLE cells, which was further decreased by erlotinib (n = 26–31; *p < 0.05; **p < 0.01; ***p < 0.001 by t-test). In contrast, erlotinib prevented the significantly increased Iamil-insens induced by EGF in both sexes. (c) Erlotinib further prevented the significant increase of Rte induced by EGF in both sexes (***p < 0.001). (Grey boxes) male; (yellow boxes) female.

Full size image

Effects of EGF in combination with female sex steroids

Sex steroid, especially female sex steroids, have been previously shown by us to stimulate Na+ transport in FDLE cells15,29. Whether this stimulation is affected by the presence of EGF was analyzed in male and female FDLE cells treated with E2/P and EGF, alone or in combination. Female sex steroids prevented the reducing effect of EGF on Na+ transport. EGF significantly decreased ΔIamil in male FDLE cells, which was reversed by the addition of E2/P (p < 0.05; p < 0.01; Fig. 10a). Moreover E2/P stimulated ΔIamil in females and prevented the reduction induced by EGF (p < 0.05; p < 0.01). EGF significantly increased Iamil-insens in both male and female FDLE cells, which persisted in the presence of E2/P (p < 0.05; p < 0.001; Fig. 10b). EGF significantly increased Rte as shown above, while E2/P reduced Rte in male and female FDLE cells. EGF prevented this decrease of Rte induced by E2/P in both male and female FDLE cells (p < 0.01; p < 0.001; Fig. 10c). In conclusion, the presence of female sex steroids alters the response to EGF, independent of the cells’ sex. Notably, the concentration of female sex steroids in utero is equal for both sexes, as they are produced by the maternal placenta. Therefore, a sex specific effect of EGF in combination with female sex steroids is unlikely, at least for the fetal situation.

Figure 10
figure 10

Female sex steroids prevented the reducing effect of EGF on Na+ transport. FDLE cells were cultured in SF-Med and stimulated with EGF with or without female sex steroids (E2/P). Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). (a) EGF significantly decreased ΔIamil in male FDLE cells, which was reversed by the addition of E2/P (n = 16–18; *p < 0.05; **p < 0.01 by t-test). Moreover E2/P stimulated ΔIamil in females and prevented the reduction induced by EGF (n = 15–18; *p < 0.05; **p < 0.01). (b) EGF significantly increased Iamil-insens in both male and female FDLE cells, which persisted in the presence of E2/P (*p < 0.05; ***p < 0.001). (c) EGF significantly increased Rte, while E2/P reduced Rte in male and female FDLE cells. In addition, EGF prevented the decrease of Rte induced by E2/P (**p < 0.01; ***p < 0.001). (Grey boxes) male; (yellow boxes) female.

Full size image

Response of sex-specific FDLE cells to other ErbB ligands

Finally, other ErbB receptor ligands were analyzed in sex-specific FDLE cells. TGF-α exerted no effect on Ibase in male and female cells (Fig. 11a, b), which is in contrast to EGF as detailed above. However, ΔIamil was significantly decreased and Iamil-insens strongly increased by TGF-α in both sexes (p < 0.05; p < 0.001; Fig. 11a, b). We assume that the increased Iamil-insens together with the small decrease of ΔIamil counterbalanced total ISC and thus resulted in no net difference of Ibase. In accordance to EGF, TGF-α significantly increased Rte (p < 0.05; Fig. 11c). Thus, both EGFR (ErbB1) ligands, EGF and TGF-α, equally affect Na+ and Cl− transport, and barrier function in a sex-independent manner, even though not to the same degree. HB-EGF increased Iamil-insens and Rte in males, with a similar trend in females (p < 0.05; Fig. 11a, c), while Ibase and ΔIamil were not affected, neither in male nor in female FDLE cells (Fig. 11a–c). ErbB2 ligand NRG1 further increased Iamil-insens in male and female cells, while ΔIamil was reduced only in females (p < 0.05; p < 0.001; Fig. 11d). Barrier function with regard to Rte was not affected by NRG1 (Fig. 11e). In mixed FDLE cells, NRG1 further significantly increased ΔIforsk and ΔICFTR (p < 0.01; Fig. 11f). NRG1 therefore shares the Cl− transport stimulating properties of EGF, which were sex-independent.

Figure 11
figure 11

ErbB ligands equally increased amiloride-insensitive ISC in both sexes. FDLE cells were cultured in SF-Med and stimulated with TGF-α, HB-EGF or NRG1 for 24 h. Data are displayed as box and whiskers with the 10–90 percentile, mean (+) and median (horizontal line). TGF-α increased Iamil-insens and decreased ΔIamil in male (a) and female (b) FDLE cells (n = 23–38; ***p < 0.001 by t-test). HB-EGF increased Iamil-insens in male cells (n = 15–23; *p < 0.05). (c) TGF-α increased Rte in male and female cells, and HB-EGF increased Rte in male cells, with the same trend observed in females (*p < 0.05). (d) NRG1 increased Iamil-insens in male and female cells, while NRG1 decreased ΔIamil in female FDLE cells (n = 25–27; *p < 0.05; ***p < 0.001). (e) Rte was not affected by NRG1 in male and female cells. (f) NRG1 increased Iamil-insens, ΔIforsk and ΔICFTR in mixed FDLE cells (n = 39–43; **p < 0.01; ***p < 0.001). (Grey boxes) male; (yellow boxes) female; (white square) mix.

Full size image

Discussion

We aimed to determine whether EGF, a prominent mitogen during fetal lung development, exerts sex-specific effects on epithelial Na+ transport in fetal alveolar cells. EGF strongly reduced the ENaC subunit mRNA levels in both male and female FDLE cells. This was corroborated by a markedly reduced Na+ transport and ENaC activity, while amiloride-insensitive pathways as well as the barrier function (Rte) were raised by EGF. Notably, CFTR-mediated Cl− transport was elevated only in female cells, while CFTR mRNA levels were increased in male and female cells. The effects of EGF were reproduced with other ligands of the EGFR (TGF-α), ErbB3 (NRG1) and ErbB4 (HB-EGF, NRG1), diminishing Na+ transport in FDLE cells. In contrast to chronic effects of EGF after 24 h, acute effects of EGF were sex-specific, because Na+ transport was reduced only in males. Furthermore, pAKT was elevated only in female cells in response to acute EGF addition, while pERK1/2 was increased in both male and female cells. The study thus showed certain sex- and time-dependent effects on epithelial ion transport induced by EGF, while other analyzed aspects were similar between male and female FDLE cells.

Prior studies reported contradictive results about the effect of EGF on epithelial Na+ transport. In alveolar epithelial cells derived from adult male rats EGF was shown to increase net Na+ flux and the activity of the Na,K-ATPase via basolaterally located EGFR32. The elevated Na+ transport was attributed to an increased Na,K-ATPase-α1 and -β1 mRNA and protein expression33. Furthermore, in adult rat ATII cells EGF treatment increased the whole-cell conductance and density of ENaC channels, whereas mRNA expression of all three ENaC subunits was decreased34. In contrast, the majority of studies observed a negative effect of EGF on Na+ transport. EGF markedly and quickly decreased ENaC activity by lowering its open probability, paralleled by a reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) levels in ENaC-reconstituted CHO cells35. Anionic phospholipids such as PIP2 and phosphatidylinositol 3,4,5-trisphosphate (PIP3) were shown to regulate ENaC and the stimulation of PLC by EGFR activation decreased PIP2 and consequently ENaC activity35,36,37. EGF rapidly diminished amiloride-sensitive Na+ currents in perfused rabbit and mouse collecting tubules38,39, which was attributed to a rapid phosphorylation of ERK1/2. EGF further raised basal intracellular Ca2+ levels after 24 h and reduced ENaC function by a Ca2+-mediated process that affected trafficking and surface expression of ENaC in a human nasal airway epithelial cell line derived from a CF patient25. These contrasting results may suggest cell and/or tissue specific EGF-dependent regulation of ENaC activity. It is proposed that EGF increases Na+ absorption in the airways and the intestine, whereas it supposedly reduces Na+ transport in CCD cells, CF cells, CHO cells overexpressing ENaC and EGFR, and other kidney-derived cells40. In contrast to this, we observed an antagonizing effect of EGF on Na+ transport in alveolar epithelial cells of fetal origin, which suggests that EGF effects also depend on the developmental stage of the cells. Furthermore, chronic incubation with EGF reduced Na+ transport in both male and female cells, while short-term incubation reduced ENaC activity only in males. To our knowledge, this is the first study demonstrating a sex-specific effect of EGF on Na+ transport. Different modes of EGF-dependent modulation of ENaC-mediated Na+ absorption have been proposed. EGF caused a transient increase in Na+ transport in A6 cells, which was PI3-K dependent, followed by a chronic reduction of ENaC membrane abundance mediated through MAPK1/240,41. Inhibition of MEK1/2, thereby preventing ERK1/2 phosphorylation, stimulated the acute increase and abolished the chronic decrease of Na+ transport in response to EGF40. An increased pERK1/2 was demonstrated in male and female cells after short-term EGF exposure, whereas pAKT was significantly increased only in females. While activation of PI3-K/AKT signaling has been associated with enhanced ENaC activity, ERK1/2 signaling usually results in a decreased ENaC function. We suggest that the acutely increased pAKT counterbalances the diminishing effect of induced ERK1/2 signaling, resulting in no net changes of Na+ transport in females, whereas this compensatory effect is lacking in males leading to a decreased Na+ transport. Unlike the acute effects, chronic EGF application resulted in a reduction of pAKT in males, while no difference was observed for pERK1/2 in male and female FDLE cells after 24 h.

Importantly, EGF strongly stimulated the amiloride-insensitive fraction of epithelial ion transport. It has been previously shown that FDLE cells exhibit different amiloride-insensitive pathways, including secretion of Cl− and HCO3−28,42. Therein the presence of cAMP-activated Cl− and HCO3− secretion across rat FDLE cells was demonstrated, which was mediated via CFTR42. Raising intracellular cAMP levels by forskolin after inhibition of ENaC activity with amiloride increased ISC in female FDLE cells. Furthermore, CFTR activity was strongly increased by EGF, but only in females. Therefore, EGF increased amiloride-insensitive pathways in both sexes, but this increase was attributable to an enhanced CFTR activity only in female FDLE cells. In contrast to this, CFTR mRNA expression was elevated by EGF in both male and female FDLE cells. Short-term exposure to EGF elevated amiloride-insensitive currents only in males. The effects of EGF on Na+ transport and barrier function were normalized by removing EGF from the culture medium for 24 h, while the effect on amiloride-insensitive pathways persisted. In conclusion, EGF affects Na+ transport and amiloride-insensitive pathways in both sexes, but with varying magnitude and time specifics. Only few studies dealt with a relationship between EGF and epithelial Cl− secretion. In T84 cells acute EGF chronically increased ISC responses to a broad range of secretagogues, and bumetanide, an inhibitor of the Na,K,2Cl-cotransporter, abolished the effect of EGF, suggesting an enhanced Cl− secretion43. In addition, Cl− secretion was enhanced by EGF through upregulation of the Ca2+-dependent Cl− channel, transmembrane protein 16A (TMEM16A), in the apical membrane of colonic cells44. Another group demonstrated that EGF inhibited Ca2+-dependent Cl− secretion via stimulation of the PI3-K through recruitment of the regulatory PI3-K subunit p85 to ErbB2 in colonic epithelial cells45. Thus, regulation of epithelial Na+ transport and Cl− secretion by EGF is not consistent between different studies and cell-, sex- and developmental-specific effects have to be considered.

Notably, prolonged EGF application strongly stimulated Rte in both male and female cells, suggesting an improved barrier integrity that was confirmed by a permeability assay. Neither metabolic activity nor cell number was affected by EGF, suggesting that the enhanced barrier function is not due to an increased cellular proliferation. Other studies demonstrated that EGF increased Rte across adult alveolar epithelial cell monolayers and reduced the tight junction permeability32, possibly by regulating the expression of certain claudins46. In addition, EGF has been shown to be an effective intestinal regulator helping to protect intestinal barrier integrity via binding to EGFR and subsequent activation of MAPK/ERK, PI3-K/AKT, PLC-γ/PKC (protein kinase C), and STATS (signal transducers and activators of transcription substrates) signal pathways47.

In general, EGFR signaling is associated with lung growth and maturation, and a reduced EGFR expression was observed in hypoplastic fetal lungs48,49. In addition, lower EGF levels in the bronchoalveolar lavage fluid at birth were detected in infants with RDS or bronchopulmonary dysplasia compared with control infants50. On the other side, EGFR overactivation has been associated with acute respiratory distress syndrome (ARDS) and fibrosis development51,52. Protein expression of EGFR was higher in female FDLE cells. It has been shown previously that estrogen increases the uterine levels of EGFR53 and that EGFR density is higher in the female fetal rabbit lung, possibly due to a negative impact of androgens54. Chronic androgen treatment has been shown to reduce EGFR activity inhibiting surfactant protein gene expression in ATII cells and fetal lungs55,56,57,57. Serum-free cultivation of FDLE cells abolished the sex difference in EGFR protein expression. Thus, sex differences in EGFR protein expression were affected by the presence of fetal serum in the culture and possibly the fetal circulation. Both, the mRNA and protein expression of EGFR was strongly decreased by EGF in both sexes. This was observed after acute (45 min) and chronic (24 h) EGF treatment, although the extent was more pronounced after prolonged application. Similarly, EGFR protein expression was reduced by EGF in fetal lung fibroblasts, while no sex-specific expression was observed in the latter cell type. It can be assumed that this negative feedback regulation prevents hyperproliferation as shown in preterm baboon lungs with hyaline membrane disease possibly induced by elevated levels of the EGFR ligand TGF-α58.

Studies showed that EGF increased thymidine incorporation and DNA accumulation in fetal lung fibroblasts and ATII cells, and elevated choline incorporation into surfactant phosphatidylcholine23. EGF positively influenced the epithelial-mesenchymal interaction, and EGFR binding in fetal rat lung fibroblasts peaks on embryonic day 18–19, prior to surfactant surge56. EGFR inactivation or EGFR deficiency resulted in ATII cell immaturity apparent from an increased glycogen content and a reduced number of lamellar bodies59. EGF further stimulated Sftpc expression in normal but not in EGFR-deficient lungs60. It is suggested that EGF may elaborate a fibroblast-derived factor that stimulates surfactant synthesis in ATII cells61. We also demonstrated an enhanced mRNA expression of the surfactant proteins in response to EGF that was observed in male and female FDLE cells, lacking any pronounced sex difference. Previous studies observed sex differences in surfactant synthesis with males lagging behind, although both sexes achieved comparable responses in late gestation24. Our results could thus be explicable by the fact that FDLE cells are isolated from late gestation lungs during the saccular stage, while the surge in surfactant synthesis occurs during the canalicular stage. Furthermore, the prior studies addressed surfactant synthesis and not surfactant protein mRNA expression possibly explaining the differential outcomes.

As noted above, EGF supposedly exerts its effect on lung epithelial cells by affecting the epithelial-mesenchymal relationship. EGF has been shown to increase the proliferation of epithelial and mesenchymal cells in monoculture, and within the embryonic lung, the mesenchyme represents the major source of EGF as a paracrine factor acting on epithelial cells expressing EGFR17. Thereby epithelial proliferation and differentiation is stimulated. Due to this, we co-cultured FDLE cells with fetal lung fibroblasts in a sex-specific manner and repeated the preceding experiments. Despite the presence of fibroblasts, the effects of EGF on Na+ transport were essentially the same as we observed before. The presence of fibroblasts did not affect the response of epithelial Na+ transport to EGF, ruling out any indirect stimulating effects in this context.

Inhibition of EGFR with erlotinib prevented only some effects of EGF. Erlotinib inhibited the increase of amiloride-insensitive pathways and barrier function, while the reduction of Na+ transport was not affected. It is unclear why erlotinib is unable to prevent the reduced Na+ transport. However, the effects of erlotinib were not sex-specific. Erlotinib is a reversible inhibitor of EGFR that also exerts inhibitory activity on purified ErbB262. The inhibitory effect of erlotinib in ErbB2-expressing cells is suggested to arise at least in part through direct interaction with ErbB2 rather than indirectly through a process, that requires the presence of EGFR. Since ligandless ErbB2 also dimerizes with ErbB3 or ErbB4 and thereby exerts diverging biological actions, it might be conceivable that some EGF effects were affected by erlotinib while others differed.

Finally, female sex steroids prevented the reducing effect of EGF on Na+ transport in both male and female FDLE cells, while the effects of EGF on barrier function and Cl− transport persisted in the presence of female sex steroids. Since the fetal concentration of female sex steroids is equal for both sexes, as they are provided by the maternal placenta, no sex-specific effect of EGF in combination with female sex steroids is assumed. Concerning male sex steroids, no androgen receptor-specific mRNA expression was detected in male or female FDLE cells15. Thus, FDLE do not express androgen receptors during the saccular stage at which cell isolation was performed. Furthermore, flutamide, an androgen receptor antagonist showed no effect on Na+ transport in FDLE cells15. Together, the inability of flutamide to affect Na+ transport and the undetectable androgen receptor expression suggest a negligible impact of androgens on Na+ transport in FDLE cells.

Conclusions

EGF strongly reduced the ENaC mRNA expression and channel activity in both male and female FDLE cells. In contrast, amiloride-insensitive pathways as well as the barrier function were raised by EGF. While chronic effects were not sex-specific, short-term effects were limited to males. In conclusion, we showed certain sex- and time-dependent differences in epithelial ion transport induced by EGF, while other analyzed aspects were similar between male and female FDLE cells. From the results obtained in this study it cannot be assumed that EGF is responsible for the sex specific differences in Na+ transport observed in FDLE cells.