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Androgens enhance airway branching but delay alveolar maturation contributing to increased respiratory morbidity in prematurely born male infants. Hoxb5 protein positively regulates airway branching in developing lung. In other organs, androgen regulation intersects with Hox proteins and TGFβ-SMAD signaling, but these interactions have not been studied in the lung. We hypothesized that androgen alteration of airway branching early in lung development requires Hoxb5 expression and that these androgen-Hoxb5 interactions occur partially through regional changes in TGFβsignaling. To evaluate acute effects of androgen and TGFβon Hoxb5, E11 whole fetal mouse lungs were cultured with dihydrotestosterone (DHT) with/without Hoxb5 siRNA or TGFβinhibitory antibody. Chronicin uteroDHT exposure was accomplished by exposing pregnant mice to DHT (subcutaneous pellet) from E11 to E18. DHT’s ability to enhance airway branching and alter phosphorylated SMAD2 cellular localization was partially dependent on Hoxb5. Hoxb5 inhibition also changed the cellular distribution of SMAD7 protein. Chronicin uteroDHT increased Hoxb5 and altered SMAD7 mesenchymal localization. TGFβinhibition enhanced airway branching, and Hoxb5 protein cellular localization was more diffuse. We conclude that DHT controls lung airway development partially through modulation of Hoxb5 protein expression and that this level of regulation involves interactions with TGFβsignaling.

Epidermal growth factor receptor signaling plays an important role in lung maturation. The authors hypothesized that specific protein kinase C (PKC) isoforms are expressed and activated by epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) (EGF receptor [EGFR] ligands) in fetal lung fibroblasts. The authors found 4 PKC isoforms expressed in gestational day 19 (d19) fetal rat lung fibroblasts, and focused on PKCalpha because of its developmental expression pattern. PKCalpha immunolocalization in d17, d19, and d21 fetal lung fibroblasts was similar to EGFR. PKCalpha expression decreased with lung maturation. EGF, but not TGFalpha, stimulated PKCalpha activation and membrane translocation. Further studies of PKCalpha functions in fetal lung development are clearly needed.

ErbB receptors are crucial for embryonic neuronal and cardiac development. ErbB receptor ligands neuregulin (NRG) and epidermal growth factor (EGF) play a major role in the developing lung, specifically in mesenchymal induced fetal surfactant synthesis by type II epithelial cells. Different erbB receptor ligands cause diverse biologic effects by stimulating specific erbB-dimers. It is not known how dimerization, cellular localization, and co-localization of erbB dimers are regulated in type II epithelial cells. We hypothesized that erbB receptors have a distinct dimerization, localization, and co-localization pattern in type II cells. In mouse type II epithelial cells, which express all four erbB receptors, erbB1 and erbB4 were the preferred dimerization partners. These dimerization patterns were ligand independent. Confocal microscopy showed these transmembrane receptors exhibited a strong nuclear localization. In non-stimulated cells, both erbB1 and erbB2 were predominantly localized to the nucleus and less intensely to the cytoplasm. However, erbB1 was mainly found in the nucleoli, whereas erbB2 spared the nucleolar region. ErbB3 was exclusively located in the nucleoli. ErbB4 was diffusely located in nucleus and cytoplasm, and like erbB2 spared the nucleolar region. Short stimulation with either EGF or NRG led to a more pronounced nuclear staining for erbB1, erbB2, and erbB4. All four receptors co-localized with each other after stimulation, but with varying intensity. The two known stimulators of fetal surfactant synthesis, NRG and NRG-containing fibroblast conditioned medium, changed cellular localization of the dimerization partners erbB4 and erbB2 in a distinct fashion. We conclude that erbB receptors have a receptor-specific localization and dimerization pattern in type II epithelial cells.

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-γ (PLC-γ) expression and activation by EGF is cell specific and developmentally regulated. PLC-γ immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-γ protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-γ phosphorylation 2.6-fold ( day 17), 10.8-fold ( day 19), and 4.2-fold ( day 21). EGF stimulated3H-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-γ phosphorylation by EGF. In conclusion, PLC-γ 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-γ 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-β receptor (TGFβ-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, TGFβ-R binding, and TGFβ1-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 ...

Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significantly increased TPL and DSPC content, and significantly decreased the expression of SP-A, SP-C, CuZnSOD, and catalase genes. Because of a crucial interplay of these factors for normal lung function at the time of birth, the molecular mechanisms by which these apparently opposing changes are accomplished warrant further investigation.

Organ-specific patterning is partly determined by Hox gene regulatory interactions with the extracellular matrix (ECM), cell adhesion and fibroblast growth factor (FGFs) signaling pathways but coordination of these mechanisms in lung development is unknown. We have previously shown that Hoxb-5 affects airway patterning during lung morphogenesis. Hoxb-5 regulation in fetal lung affects ECM expression of tenascin-C and alters FGF10 spatial and cellular expression. To test this hypothesis, gestational day 13.5 (Gd13.5) fetal mouse lung fibroblasts and whole lungs were cultured with Hoxb-5-specific small interfering RNA (siRNA). Western blots showed that siRNA-down regulation of Hoxb-5 led to decreased tenascin-C and FGF10 and was associated with increased Hoxb-4 and decreased Hoxb-6 protein levels. Hoxa-5 protein levels were not affected. Hoxb-5 siRNA-treated whole lung cultures had a significant decrease in total lung and peripheral branching region surface area. Immunostaining showed negligible levels of Hoxb-5 protein and tenascin-C, and loss of FGF10 spatial restriction. We conclude that Hoxb-5 helps regulate lung airway development through modulation of ECM expression of tenascin-C. ECM changes induced by Hoxb-5 may affect mesenchymal-epithelial cell signaling to alter spatial and cellular restriction of FGF10. Hoxb-5 may also affect lung airway branching indirectly by cross regulation of other Hoxb genes.

Androgen Controls Transforming Growth Factor-β Receptor and Epidermal Growth Factor Receptor Regulation and Surfactant Protein Gene Expression in the Developing Lung

In the fetal lung EGF-R activation stimulates the development of surfactant production via fibroblast-type II cell communication. EGF-R expression during fetal lung maturation is cell specific and developmentally regulated. Fetal rat lung fibroblasts but not type II cells exhibit a gestational increase in EGF-R that peaks at the time of maximum cell-cell communication stimulating surfactant synthesis. We hypothesize that cell specific EGF-R activation is important for the development of fibroblast-type II cell communication leading to surfactant production. We transducted immature rat lung fibroblasts (fetal days 17 and 19) which inherently express low (D17) and moderate (D19) levels of EGF-R activity, using retroviral constructs expressing wild type EGF-R (WT) or mutant EGF-R (MUT, lacking the intracellular signaling domain). Transduction efficiency was measured by specific EGF-R binding, which increased between 4- (D19) and 47-fold (D17) in transducted cells compared to controls. Fibroblast conditioned medium (FCM) was collected from transducted cells with or without EGF pretreatment, and its ability to stimulate choline incorporation into disaturated phosphatidylcholine (DSPC) studied in MLE12 cells (mouse lung epithelial cell line expressing surfactant). Choline incorporation into DSPC was stimulated by FCM obtained from D19 control cells treated with EGF (138±12%) and from both D17 and D19 fibroblasts transducted with WT EGF-R, but not by FCM from cells transducted with MUT EGF-R (Table shows% stimulation from control, mean±SEM). However, EGF-R activation with EGF downregulated this stimulatory effect (Table). These data show that increased EGF-R expression in fetal lung fibroblasts activates fibroblast-epithelial cell communication stimulating surfactant synthesis. Immature fibroblasts with low EGF-R levels may possess intact intracellular signal transduction pathways which can be utilized by the ectopically expressed WT EGF-R. Lack of stimulation with MUT EGF-R indicates a receptor mediated mechanism. EGF treatment of transducted cells may cause EGF-R downregulation and loss of signal transduction stimulating cell-cell communication. We conclude that EGF-R regulates development of cell-cell communication in fetal lung maturation.

Effect of Epidermal Growth Factor (EGF) on the Development of SP-A, SP-B, and SP-C mRNA expression in Fetal Rabbit Lung 244