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We compared proliferation (growth) and differentiation (development) related proteins in normal and hypoplastic fetal murine lungs. The hypoplastic lungs were created in CD-1 fetal mice by nitrofen exposure (25 mg per pregnant mouse given intragastrically on gestational day 8 [Gd8]), as published earlier. The lungs were harvested at Gd14, 16, 19 and from neonates. Immunoblot analyses were carried out for transcription factors (oncogenic proteins, nuclear receptor, and transmembrane receptor proteins) in severely hypoplastic murine fetal lungs with coexistent diaphragmatic hernia, and results were compared with those derived from normal lungs of equivalent age. These proteins have proposed roles in the regulation of proliferation and differentiation processes of fetal lungs. We have shown that the product of the oncogene c-myc was reduced in hypoplastic lungs at all stages of gestation, whereas c-Fos protein levels were variable. These proteins are known to regulate transcription of various developmental proteins, such as those responsible for proliferation and differentiation. Further, the nuclear transcription factors thyroid transcription factor-1 (TITF-1) and glucocorticoid receptor (GR) were reduced, and thyroid hormone receptor (TR) and retinoic acid receptors (RARs) were inhibited in severely hypoplastic lungs compared to normal lungs of equivalent gestational stage, except in neonatal lungs, where signals for RARs were seen. TITF-1 is known to localize in bronchial epithelial cells in developing lungs. It is restricted to type II pneumocytes with gestational development in the normal lungs and regulates surfactant proteins. Earlier, we have reported that surfactant proteins are reduced in hypoplastic lungs. In the current study, reduced GR and TITF-1 proteins may play a role in reducing surfactant proteins in the hypoplastic lungs. The significant inhibition in TR and RARalpha in the severely hypoplastic lungs reflects on affected epithelial cell maturation and alveolar formation, respectively. Altered RARbeta levels correlate with affected lung growth and branching morphogenesis of nitrofen-exposed lungs. A transmembrane receptor protein EGFR was reduced in hypoplastic lungs, suggesting the involvement of altered mesenchymal-epithelial signal transduction pathways. We conclude (1) Our data suggest altered levels of various nuclear transcription factors in the murine fetal hypoplastic lungs; (2) Reduced levels TITF-1 protein in hypoplastic lungs may have caused the functional immaturity of distal lung, immature airways and thus may affect overall differentiation of lungs. These results correlated with low levels of surfactant proteins in these lungs; (3) TR and RAR inhibition indicate their roles through reduced or retarded proliferation and differentiation processes in the severely hypoplastic lungs; (4) GR down-regulation in developing fetal murine hypoplastic lungs indicate delayed development, and GR up-regulation in affected neonates may be induced by stress/stretch caused at birth due to air-breathing; (5) Down- regulation of EGFR indicate altered mesenchymal-epithelial interactions and possible influence on lung proliferation and differentiation., (Copyright 2001 Wiley-Liss, Inc.)

The purpose of this study was to identify differentially expressed genes in normal and nitrofen-induced hypoplastic lungs in fetal mice. Such genes may play a role in the regulation of lung development. CD-1 pregnant dams were gavaged with 25 mg of nitrofen on gestational day (Gd) 8 to induce pulmonary hypoplasia and diaphragmatic hernia (DH). Normal and nitrofen-treated fetuses were removed on Gd 14 and Gd 16. Lungs were examined in all nitrofen-exposed fetuses and only those that had developed severely hypoplastic lungs with coexistent diaphragmatic hernia were taken for molecular analyses. RNA was extracted from normal and nitrofen-treated lungs, reverse transcribed, and PCR-amplified using 48 combinations of anchor and arbitrary primers for each condition. The resulting cDNAs from normal and hypoplastic lungs were run on 6% polyacrylamide differential display gels. In Gd 14 lungs, we observed 10 differentially expressed cDNA bands, of which 6 were identified to be inhibited and 4 were reduced in the hypoplastic lungs compared to normal fetal lungs. From the Gd 16 lungs, a total of 29 differentially expressed cDNA bands were found, of which 11 were reduced, 4 were inhibited, 11 were enhanced, and 3 were induced in the hypoplastic compared to the normal lungs. All 39 differentially expressed cDNAs were cloned, sequenced, and identified through BLAST searches. Among the sequences that were identified, results were as follows: 1) Hypoplastic Gd 14 lungs had two unknown cDNA sequences with reduced/inhibited expressions, whereas one was a known sequence having 77% similarity with a promoter region regulating various cytokines such as IL-1, IL-2, and IL-11. The expression of this sequence was inhibited in the hypoplastic lungs. This sequence also had similarity to lipid-binding proteins. 2) On Gd 16, hypoplastic lungs had one cDNA sequence with reduced expression which had 82% similarity with thyroid hormone receptor gene exon 1 and two other cDNA sequences with enhanced expressions. One of these enhanced cDNA sequences in hypoplastic lungs had 98% similarity with the fibroblast growth factor receptor-3 gene, and the other was an unknown sequence. Northern blot hybridizations were performed to confirm the differential expression of the two sequences of interest, which were identified as thyroid hormone receptor and fibroblast growth factor (FGF) receptor-3. Overall, out of a total of 39 RT-PCR products (i.e., cDNAs), the abundance of which was altered by nitrofen, 6 were found to be homologous to sequences in Gen Bank through BLAST searches. These 6 sequences became the products of interest, and 3 of these 6 products were similar to previously identified genes. Our results may shed some light on regulatory aspects of lung development and open avenues for treatment of hypoplastic lungs and other respiratory problems in human neonates.

Transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation, and epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, are important regulators of cell differentiation; however, their distinct role(s) in lung development and their mechanisms of action are not well understood. We evaluated the effects of these factors on lung morphogenesis in murine fetal lungs at gestational day 14 (time:zero) and again after 7 days in culture. Baseline controls were cultured after tracheal transection in supplemented BGJb medium, and other tracheally transected lungs were cultured following addition of EGF (10 ng/ml BGJb), TGF-beta1 (2 ng/ml BFJb), or with both in combination added to the medium. The control lungs in culture had poorly developed airways and an absence of defined acinar structures. The addition of EGF resulted in hyperplasia of primary airways with stunted outgrowths, monopodial branching, and absence of distinct acinar structures. Addition of TGF-beta1 alone, led to significant elongation of primary airways, without normal airway branching; however, terminal dipodial branching was seen and the prospective pulmonary acini were well defined. Combination of these growth factors (GF) resulted in a more normal branching pattern and differentiation, suggesting their epigenetic role in lung morphogenesis and mutual interactive mechanisms that regulate lung development. These lungs had more abundant and larger lamellar bodies than those after other treatments. Control lungs remained immature with prominent glycogen aggregates with occasional dense lamellar bodies. The total protein and DNA contents were highest with EGF treatment, followed by combination treatment; these observations were supported by immunohistochemical localization of proliferating cell nuclear antigen, an indication of the proliferative state of tissues. All the surfactant proteins were relatively unaltered and their messages were up-regulated for SP-A, but down-regulated for SP-B and SP-C in the lungs treated with growth factors. In conclusion, we have demonstrated enhanced biochemical and structural development of lungs treated in vitro with GF, and propose that further research in this area may lead to therapeutic uses of GF alone or in combination with other agents for the treatment of newborn respiratory distress due to lung immaturity or hypoplastic lung development.