Down-regulation of regulatory proteins for differentiation and proliferation in murine fetal hypoplastic lungs: altered mesenchymal-epithelial interactions.

Abstract

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.