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ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.

Cigarette smoke exposure causes chronic oxidative lung damage. During pregnancy, fetal microchimeric cells traffic to the mother. Their numbers are increased at the site of acute injury. We hypothesized that milder chronic diffuse smoke injury would attract fetal cells to maternal lungs. We used a green-fluorescent-protein (GFP) mouse model to study the effects of cigarette smoke exposure on fetomaternal cell trafficking. Wild-type female mice were exposed to cigarette smoke for about 4 weeks and bred with homozygote GFP males. Cigarette smoke exposure continued until lungs were harvested and analyzed. Exposure to cigarette smoke led to macrophage accumulation in the maternal lung and significantly lower fetal weights. Cigarette smoke exposure influenced fetomaternal cell trafficking. It was associated with retention of GFP-positive fetal cells in the maternal lung and a significant reduction of fetal cells in maternal livers at gestational day 18, when fetomaternal cell trafficking peaks in the mouse model. Cells quickly clear postpartum, leaving only a few, difficult to detect, persisting microchimeric cells behind. In our study, we confirmed the postpartum clearance of cells in the maternal lungs, with no significant difference in both groups. We conclude that in the mouse model, cigarette smoke exposure during pregnancy leads to a retention of fetal microchimeric cells in the maternal lung, the site of injury. Further studies will be needed to elucidate the effect of cigarette smoke exposure on the phenotypic characteristics and function of these fetal microchimeric cells, and confirm its course in cigarette smoke exposure in humans.

Objective: Starting in 2013, all pediatric residents entering fellowship must be provided six educational units whose structure is to be determined by their individual career plans. We sought to determine whether (1) neonatology fellowship program directors (PDs) consistently identify certain weaknesses among incoming fellows and (2) neonatology fellowship PDs agree on the most beneficial activities in which pediatric residents should participate to improve preparation for entry into neonatology fellowships., Study Design: We sent a 21-question survey focused on the structure and implementation of the 6-unit curriculum to all members of the Organization of Neonatology Training Program Directors., Results: Sixty-seven percent of PDs responded. Seventy-five percent cited insufficient procedural skills as the primary weakness of incoming fellows. More than 80% rated additional training in clinical neonatology, including procedural and resuscitation training, as 'beneficial' or 'highly beneficial'. In contrast, fewer than 40% of PDs gave the same positive ratings to activities broadly focused on scholarship., Conclusions: The results of the survey may help guide pediatric residency programs as they undertake development of these new curricular initiatives for individual residents entering neonatology.

Stretch-induced differentiation of lung fetal type II epithelial cells is mediated through EGFR (ErbB1) via release of HB-EGF and TGF-α ligands. Employing an EGFR knock-out mice model, we further investigated the role of the ErbB family of receptors in mechanotranduction during lung development. Deletion of EGFR prevented endogenous and mechanical stretch-induced type II cell differentiation via the ERK pathway, which was rescued by overexpression of a constitutively active MEK. Interestingly, the expression of ErbB4, the only ErbB receptor that EGFR co-precipitates in wild-type cells, was decreased in EGFR-deficient type II cells. Similar to EGFR, ErbB4 was activated by stretch and participated in ERK phosphorylation and type II cell differentiation. However, neuregulin (NRG) or stretch-induced ErbB4 activation were blunted in EGFR-deficient cells and not rescued after ErbB4 overexpression, suggesting that induction of ErbB4 phosphorylation is EGFR-dependent. Finally, we addressed how shedding of ligands is regulated by EGFR. In knock-out cells, TGF-α, a ligand for EGFR, was not released by stretch, while HB-EGF, a ligand for EGFR and ErbB4, was shed by stretch although to a lower magnitude than in normal cells. Release of these ligands was inhibited by blocking EGFR and ERK pathway. In conclusion, our studies show that EGFR and ErbB4 regulate stretch-induced type II cell differentiation via ERK pathway. Interactions between these two receptors are important for mechanical signals in lung fetal type II cells. These studies provide novel insights into the cell signaling mechanisms regulating ErbB family receptors in lung cell differentiation.

Sufficient pulmonary surfactant production is required for the fetal-neonatal transition, especially in preterm infants. Neuregulin (NRG) and its transmembrane receptor ErbB4 positively regulate the onset of fetal surfactant synthesis. Details of this signaling process remain to be elucidated. ErbB4 is known to regulate gene expression in the mammary gland, where the receptor associates with the signal transducer and activator of transcription Stat5a to transactivate the β-casein gene promoter. We hypothesized that in the fetal lung, ErbB4 functions as a transcriptional regulator for surfactant protein B (Sftpb), the most critical surfactant protein gene. Re-expressing full-length ErbB4 in primary fetal ErbB4-depleted Type II epithelial cells led to an increased expression of Sftpb mRNA. This stimulatory effect required the nuclear translocation of ErbB4 and association with Stat5a, with the resultant binding to and activation of the Sftpb promoter. We conclude that ErbB4 directly regulates important aspects of fetal lung maturation that help prepare for the fetal-neonatal transition.

Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 μg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development.

Neuregulin is an important growth factor in fetal surfactant synthesis, and downregulation of its receptor, ErbB4, impairs fetal surfactant synthesis. We hypothesized that pulmonary ErbB4 deletion will affect the developing lung leading to an abnormal postnatal lung function. ErbB4-deleted lungs of 11- to 14-wk-old adult HER4heart mice, rescued from their lethal cardiac defects, were studied for the effect on lung function, alveolarization, and the surfactant system. ErbB4 deletion impairs lung function and structure in HER4heart mice resulting in a hyperreactive airway system and alveolar simplification, as seen in preterm infants with bronchopulmonary dysplasia. It also leads to a downregulation of surfactant protein D expression and an underlying chronic inflammation in these lungs. Our findings suggest that this animal model could be used to further study the pathogenesis of bronchopulmonary dysplasia and might help design protective interventions.

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.

Objective: ErbB receptors and their ligands play crucial roles in development. During late gestation, they might also be involved in the pathogenesis of prematurity-associated disorders. ErbB receptor dimerization leads to a diversity of biologic signals. We studied the expression and localization patterns of erbB receptors in the developing human umbilical endothelial cell system. It is still unclear, whether expression patterns might be developmentally regulated and depend on the cell type studied., Methods: Primary human umbilical venous endothelial cells (HUVEC) and arterial endothelial cells (HUAEC) were isolated between 24 and 42 weeks of gestation and used for immunoprecipitation, Western blotting, and confocal microscopy., Results: All four erbB receptors were present in HUVEC and HUAEC. Expression patterns were similar for cell types at gestational ages examined. ErbB4 always co-precipitated with erbB1 in both cell types independent of the gestational age. Confocal microscopy revealed that all erbB receptors were localized in the nucleus, erbB1 and erbB3 in the nucleoli, while erbB2 and erbB4 spared the nucleolar region. All receptors showed a tendency to co-localize. Growth factor stimulation altered localization patterns. Cellular subfractionation experiments for erbB4 largely confirmed microscopy results. Pretreatment with lipopolysaccharide enhanced this nuclear localization of erbB4, particularly of its intracellular domain., Conclusions: All erbB receptors are present in both HUVEC and HUAEC at all gestational ages tested. ErbB receptor expression patterns were independent of the developmental stage of the endothelial cell, at least in the third trimester. We speculate that endothelial erbB receptors might play a role in normal development in mid and late gestation. We also speculate that these findings, together with the known involvement of erbB receptors in development, inflammation, and angiogenesis, will open new avenues for erbB receptor-related research in the pathogenesis of fetal and neonatal inflammation-associated disorders.

Glucocorticoids stimulate foetal surfactant synthesis. Therefore, they are used in impending pre-term birth. One mechanism of action on surfactant synthesis is through the induction of neuregulin (NRG) secretion by foetal lung fibroblasts. The direct effects on signalling pathways, and specifically on erbB receptors in foetal type II cell surfactant synthesis, are less well understood. The present authors studied the effect of known promoters of foetal surfactant synthesis (namely dexamethasone and mature (i.e. NRG-containing) fibroblast-conditioned medium (FCM)) on erbB receptor activation, protein content and dimerisation patterns in foetal mouse lung type II cells. Dexamethasone inhibited surfactant synthesis in immature type II cells at day (d)16 of gestation, while the mature FCM had stimulatory effects. Both treatments directly stimulated surfactant synthesis in more mature (d17) cells. At this gestational day, dexamethasone had only a small effect on phosphorylation, but it stimulated the protein levels of all four erbB receptors. Dexamethasone effects were distinct from those of mature FCM, which stimulated both protein content and phosphorylation of all erbB receptors and of the signalling intermediate phospholipase Cgamma. Dexamethasone modulated erbB receptor dimerisation patterns, such that erbB2 became the main dimerisation partner for erbB4. In conclusion, dexamethasone signalling involves erbB receptors in foetal type II cells, in a manner similar to, but distinct from, neuregulin-containing fibroblast-conditioned medium signalling.

Bronchopulmonary dysplasia (BPD) and cerebral white matter damage (WMD) are neonatal disorders that occur most commonly in those who are born much before term. In a large multicenter database, we sought to determine whether the two disorders occur together more frequently than expected and whether BPD and other neonatal respiratory characteristics are more common among infants who develop ultrasound-defined WMD than among those who do not. In a sample of 904 infants who were born before the 30th week of gestation and survived until 36 wk postmenstrual age, we did not find a co-occurrence of BPD and WMD above what would be expected by chance. Confounding does not seem to account for this lack of association between WMD and BPD. In conclusion, our findings do not support the hypothesis that BPD contributes to the occurrence of sonographically defined WMD.

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

Epidermal growth factor (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-gamma (PLC-gamma) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-gamma phosphorylation 2.6-fold (day 17), 10.8-fold (day 19), and 4.2-fold (day 21). EGF stimulated (3)H-labeled diacylglycerol production in fibroblasts (beginning on day 18 in female and on day 19 in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-gamma phosphorylation by EGF. In conclusion, PLC-gamma expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-gamma activation by EGFR in fetal lung fibroblasts may be involved in EGF control of lung development.

During lung development there is tension between positive and negative regulators of fibroblast-epithelial communication controlling type II cell differentiation. A clinical consequence of imbalance of this tension is the increased risk for respiratory distress syndrome in male infants. We hypothesized that chronic intrauterine androgen exposure alters fetal lung fibroblast maturation by down-regulating epidermal growth factor receptor (EGF-R) activity and by up-regulating transforming growth factor-beta receptor (TGFbeta-R) activity, leading to an inhibition of surfactant protein B (SP-B) and -C (SP-C) gene expression in type II cells. We treated pregnant mice with dihydrotestosterone (DHT; 2 mg/day) or vehicle for 7 days, starting on gestational day 11. On day 18, EGF binding, EGF-R phosphorylation, TGFbeta-R binding, and TGFbeta1-induced cell proliferation were studied in sex-specific fibroblast cultures. SP-B and -C messenger RNA levels were measured in whole lungs. Chronic DHT treatment reduced both EGF binding (females to 78+/-8% and males to 65+/-9% of controls) and EGF-induced EGF-R phosphorylation. TGFbeta-R binding was increased (females to 173+/-39% and males to 280+/-64% of controls), and TGFbeta-induced cell proliferation was increased in female cells (231+/-57% of controls). SP-B and -C messenger RNA expression was reduced to 55+/-10% and 75+/-4%, respectively. We conclude that chronic DHT exposure beginning early in lung development alters the balance of growth factor signaling that regulates lung maturation.

Lung epithelial cell differentiation is predominantly regulated by mesenchymal-epithelial cell communication. We have previously shown that epidermal growth factor (EGF) positively influences this process, and that EGF receptor (EGF-R) binding in fetal rat lung fibroblasts peaks on d18-19 of gestation, just before the onset of augmented surfactant synthesis. This regulation of EGF-R in late gestation fetal lung fibroblasts may control the timing of mesenchymal-epithelial cell communication leading to surfactant synthesis. Hormones and growth factors exert positive and negative influences on lung development, but whether they regulate the EGF-R is unknown. We hypothesized that positive [EGF, cortisol, retinoic acid (RA)] and negative [transforming growth-factor-beta1 (TGF-beta1), dihydrotestosterone (DHT)] regulators of lung cell development regulate the EGF-R in the fetal lung. We studied EGF-R binding and protein abundance in sex-specific fetal rat lung fibroblasts cultured at d17, d19, and d21. EGF-R binding was significantly elevated after RA (both sexes d17 and d19, females d21) and after DHT (females d19) treatment. EGF and cortisol had minimal or inhibitory effects on EGF-R binding. Western blot analysis showed that the observed changes in EGF-R binding were associated with similar changes in EGF-R protein. We conclude that factors that affect lung maturation continue to regulate EGF-R in a developmental, sex-specific manner during late gestation.