Identification of glucocorticoid‐regulated genes that control cell proliferation during murine respiratory development

Development of the mammalian fetal lung is a complex process requiring cell proliferation and differentiation leading to the formation of a complex morphological structure that is populated with specific respiratory cell lineages. These processes are facilitated through interactions between mesenchymal cells, epithelial cells and the extracellular matrix (ECM) and are regulated, particularly during the latter stages of development, by mechanical stimuli. The influence of circulating systemic hormones such as retinoids, thyroid hormone and glucocorticoids are also critical, and together these signalling pathways help to induce the migration, proliferation and differentiation of pulmonary cells as well as to determine the architecture of the respiratory tree (Minoo & King, 1994; Mendelson, 2000). The actions of glucocorticoids on lung development have been extensively studied, particularly their role in enhancing surfactant production and promoting distal alveolar development (Grummer & Zachman, 1998; Nakamura et al. 2000; Flecknoe et al. 2004). Synthetic glucocorticoids, such as betamethasone and dexamethasone are used antenatally to reduce the incidence of preterm infants suffering respiratory distress (Liggins & Howie, 1972; Lyons & Garite, 2002; Jobe & Soll, 2004), yet their benefits are countered by reported side-effects in non-human primates. These include reduced body growth, disrupted immune cell proliferation and perturbed central nervous system development, particularly following the use of multiple doses (Coe & Lubach, 2005). Glucocorticoids exert the majority of their effects through binding to the intracellular glucocorticoid receptor (GR), which acts as a ligand-dependent transcription factor. The GR belongs to the steroid receptor family, a subgroup of the nuclear receptor superfamily (Robinson-Rechavi et al. 2003). Once bound by ligand, the GR dimerizes and translocates to the nucleus where it can directly or indirectly regulate expression of specific target genes (McKenna & O'Malley, 2002; Robinson-Rechavi et al. 2003). Treatment of fetal rat lung explants with dexamethasone retards growth, distorts branching, dilates proximal tubules, and reduces proliferation of epithelial cells within the distal tubules (Oshika et al. 1998). Furthermore, a number of biochemical and morphological features associated with accelerated maturation are observed following dexamethasone treatment. These features include flattened or cuboidal epithelial cells lining the distal tubules, rudimentary septa and large airspaces, compressed and attenuated mesenchymal tissue between adjacent epithelial tubules and increased transcription of genes associated with epithelial growth and differentiation including surfactant proteins A, B and C (Sftpa, Sftpb and Sftpc), secretory globulin 1a1 (Scgb1a1, CC10) and fibroblast growth factor 7 (Fgf 7). We and others have previously generated GR-null mice via gene targeting (Cole et al. 1995; Brewer et al. 2002). On a complete 129sv or C57BL/6 genetic background, all GR-null mice die at birth, due to respiratory dysfunction with severe atelectasis. We further demonstrated that the lungs of GR-null fetal mice have higher proportions of type-II and undifferentiated alveolar epithelial cells (AECs) as well as peri-saccular hypercellularity leading to increased DNA content (Cole et al. 2004). These findings suggest that GR activation is not essential for differentiation into the type-II AEC phenotype, but plays an important role in regulating lung cell proliferation and lung structural development and in enhancing the differentiation of primordial AECs. Although glucocorticoid signalling can induce both cell cycle arrest and apoptosis in different tissues (Sanchez et al. 1993; Baghdassarian et al. 1998; Zilberman et al. 2004), it is unclear how these mechanisms are involved in the glucocorticoid-induced maturation of the developing lung. To further investigate the role of glucocorticoid signalling in fetal lung development, we have now analysed GR-null mice to explore a possible role for glucocorticoid signalling in the regulation of cell proliferation or controlled cell death. We find that the lungs of day 18.5 p.c. GR-null fetal mice display significantly higher rates of cellular proliferation relative to wild-type littermates. To identify genes potentially responsible for the hyperproliferation, as well as other glucocorticoid-induced gene networks, we have used mouse whole genome microarrays on fetal lung tissue. We show that processes regulating cell proliferation, cell division and the cell cycle are among the most significantly altered ontologies in the lung of day 18.5 p.c. GR-null fetal mice. Quantitative Real Time PCR (qRT-PCR) of selected genes supported this conclusion and demonstrated that mRNA levels for p21CIP1, a well characterized cyclin-dependent kinase inhibitor (Corroyer et al. 1997), and Fgf 7 (Ulich et al. 1994), a known stimulator of pulmonary surfactant production during late fetal lung development, were both significantly reduced. On the other hand, mRNA levels for midkine (a retinoic acid induced lung growth factor; Kaplan et al. 2003) were significantly increased. We conclude that glucocorticoid signalling regulates hypercellularity of the fetal lung by affecting genes involved specifically in proliferation and not in apoptosis.