The p66Shc adapter protein regulates the morphogenesis and epithelial maturation of fetal mouse lungs.

Many signaling pathways are mediated by Shc adapter proteins that, in turn, are expressed as three isoforms with distinct functions. The p66(Shc) isoform antagonizes proliferation, regulates oxidative stress, and mediates apoptosis. It is highly expressed in the canalicular but not the later stages of mouse lung development, and its expression persists in bronchopulmonary dysplasia, a chronic disease associated with premature birth. These observations suggest that p66(Shc) has a developmental function. However, constitutive p66(Shc) deletion yields no morphological phenotype, and the structure of the Shc gene precludes its inducible deletion. To elucidate its function in lung development, we transfected p66(Shc) or nonsilencing small-interfering RNA (siRNA) into the epithelia of embryonic day 11 mouse lungs that were then cultured for 3 days and analyzed morphometrically. To assess cellular proliferation and epithelial differentiation, lung explants were immunostained and immunoblotted for p66(Shc), proliferating cell nuclear antigen (PCNA), the proximal airway differentiation antigens Clara cell 10-kDa protein (CC10) and thyroid transcription factor (TTF)-1, and the alveolar surfactant proteins (SP)-A, -B, and -C. Explants transfected with nonsilencing siRNA demonstrated specific epithelial uptake and normal morphological development relative to uninjected controls. In contrast, transfection with p66(Shc) siRNA significantly increased lumenal cross-sectional areas, decreased branching, and increased epithelial proliferation (P < 0.05 for all). Relative to controls, the expression of SP-B, SP-C, CC10, and TTF-1 was decreased by p66(Shc) knockdown. SP-A was not expressed in either control or treated lungs. These data suggest that p66(Shc) attenuates epithelial proliferation while promoting both distal and proximal epithelial maturation.

Association of bronchopulmonary sequestration with expression of the homeobox protein Hoxb-5.

Bronchopulmonary sequestration (BPS) is caused by the abnormal development of an accessory lung diverticulum from the foregut very early in embryogenesis. The developmental abnormalities seen with BPS suggest that this anomaly is caused by abnormal expression of homeobox genes, which control axial identity and organ-specific patterning during embryogenesis. The authors previously have shown that the homeobox gene Hoxb-5 is necessary for normal airway branching during lung development. The authors now report that BPS is associated with aberrant developmental expression of Hoxb-5 protein, suggesting that this Hox gene is involved in the development of BPS.

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung.

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10(-6) M) and Hoxb-5 antisense oligonucleotide (20 microM) treatment each significantly decreased branching morphogenesis (P<0. 001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189+/-20% (mean+/-S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.

Growth factors and dexamethasone regulate Hoxb5 protein in cultured murine fetal lungs.

Studies on lung morphogenesis have indicated a role of homeobox (Hox) genes in the regulation of lung development. In the present study, we attempted to modulate the synthesis of Hoxb5 protein in cultured murine fetal lungs after mechanical or chemical stimuli. Murine fetuses at gestational day 14 (GD14) were removed from pregnant CD-1 mice, and lungs were excised and cultured for 7 days in BGJb media. The experimental groups were 1) untreated, unligated; 2) tracheal ligation; 3) supplemented media with either epidermal growth factor (EGF; 10 ng/ml), transforming growth factor (TGF)-beta 1 (2 ng/ml), dexamethasone (10 nM), EGF + TGF-beta 1, or EGF + TGF-beta 1 + dexamethasone. After 3 or 7 days, the cultured lungs were compared with in vivo lungs. Immunoblotting signals at 3 days in culture were stronger than those at 7 days. Western blot analyses showed that ligation, EGF, TGF-beta 1, and EGF + TGF-beta 1 downregulated Hoxb5 protein to approximately 20-70% of Hoxb5 protein levels in unligated, untreated cultured lungs. Furthermore, dexamethasone alone or in combination with EGF and TGF-beta 1 downregulated Hoxb5 protein by > 90% (P < 0.05) signal strength, similar to that seen in GD19 or in neonatal lungs. Immunostaining showed that Hoxb5 protein was expressed strongly in the lung mesenchyme at early stages in gestation. However, by GD19 and in neonates, it was present only in specific epithelial cells. A persistent level of Hoxb5 protein in the mesenchyme after EGF or TGF-beta 1 treatments or tracheal ligation was noted. Hoxb5 protein was significantly downregulated by EGF + TGF-beta 1, and it was least in lungs after dexamethasone or EGF + TGF-beta 1 + dexamethasone treatment. The decrease in Hoxb5 protein was significant only in the groups with dexamethasone added to the media. Thus immunostaining results parallel those of immunoblotting. The degree of Hoxb5 downregulation by dexamethasone or EGF + TGF-beta 1 + dexamethasone was similar to that seen in vivo in very late gestation, which correlated to the advancing structural development of the lung.

Expression and activity of epidermal growth factor receptor in late fetal rat lung is cell- and sex-specific.

Epidermal growth factor (EGF) augments late fetal lung maturation by advancing the ontogeny of fetal lung development and by stimulating surfactant synthesis. Previous studies have indicated that fibroblastalveolar epithelial cell communications mediate surfactant synthesis in the fetal lung and EGF acts through such a mechanism. We investigated the hypothesis that is differential activity and expression of the epidermal growth factor receptor (EGF-R) in fetal lung fibroblasts during the canalicular stage of lung development mediates EGF effects. To test this hypothesis, we examined fetal rat lung fibroblasts (FLFs) and type II cells of late gestation (canalicular and saccular stages; 17-22 days) by EGF-R binding techniques, SDS-PAGE, and Western blot analysis. Specific EGF binding increased 181% in day 18 female FLFs, with male FLFs exhibiting a similar increase on day 19. In contrast, specific EGF binding was low in type II cells, did not increase during late gestation, and there were no sex-specific differences. SDS-PAGE and Western blot analysis revealed a predominant 170-kDa EGF-R band in fibroblasts that increased with gestation (peak = 19 days), and was stronger in females. Immunoprecipitation of EGF-treated cells demonstrated the tyrosine kinase activity of the identified receptor. In contrast, type II cells showed minimal signal that did not increase until day 21 of gestation. We also examined whole fetal lung sections by immunohistochemistry to determine cell-specific expression of the EGF-R in vivo. Immunohistochemistry revealed specific EGF-R staining in columnar and cuboidal epithelia of small conducting airways and in mesenchyme of epithelial-mesenchymal borders (including subepithelial mesenchyme). In contrast, alveolar epithelia showed minimal staining, while subalveolar mesenchyme EGF-R staining peaked at day 19 of gestation. We conclude that cell-specific and sex-specific differences in EGF-R binding and EGF-R immunolocalization appears in the fetal lung at a developmental stage that is critical for alveolar epithelial cell differentiation. The results suggest a role for EGF-R activation in late fetal alveolar epithelial cell maturation, which is mediated through mesenchymal-epithelial cell communication.

Growth factor control of growth and epithelial differentiation in embryonic lungs.

embryonic lung cultures were exposed to either EGF (10 ng/ml) or TGF beta 1 (2 ng/ml) for 72 h, and branching morphogenesis, cell proliferation, and epithelial differentiation (the expression of DSPC synthesis and of surfactant protein C (SP-C) mRNA) were studied. EGF treatment stimulated branching morphogenesis (measured as the number of terminal left lung buds), epithelial differentiation, and cell proliferation. Branching morphogenesis was increased compared to controls after 48 h of culture by 47% and after 72 h by 34% (P < 0.0005). Choline incorporation into DSPC was stimulated by 343% (P = 0.05). SP-C expression was increased sixfold. Thymidine incorporation was stimulated by 49% (P < 0.05). The effects of EGF on thymidine labeling were distributed among epithelial cells of the airway walls and of the branching tips, and also the mesenchyme (P < 0.01 for each area compared to controls). In contrast, TGF beta 1 did not alter the number of terminal left lung buds, inhibited choline incorporation into DSPC by 35% (P < 0.05), and had no effect on thymidine incorporation (87% of control). There was increased thymidine labeling at the branching tips (P < 0.01), while other areas were not different from controls. We conclude that both EGF and TGF beta 1 affect the development of branching morphogenesis and of epithelial differentiation in the embryonic lung.

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate.

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.