Interactions of glucocorticoid receptor signalling and retinoic acid receptor signalling during murine lung development

Abstract

Endogenous glucocorticoid (GC) signalling and retinoic acid (RA) signalling are essential to mammalian lung development, regulating crucial processes through the glucocorticoid receptor (GR) and retinoic acid receptors (RARs). However, exogenous administration of these molecules can severely affect the timing and magnitude of normal developmental events in the lung. The use of exogenous, synthetic GCs is widely accepted to accelerate lung maturation and improve lung function in very preterm infants, although adverse effects of exogenous GC administration have been observed in the lung as well as other developing organs in both human and rodent studies. More specifically, multiple doses of dexamethasone (a synthetic GC) disrupt alveologenesis in rodent models. This indicates that more research is warranted to elucidate the mechanisms behind these adverse effects of GC signalling. The ability of exogenous all-trans-RA (atRA) to antagonise and prevent this dexamethasone-induced disruption of alveolarisation provides an excellent opportunity to investigate the adverse effects of exogenous GCs on the developing lung. Using transgenic GR-null mice I have further examined interactions of GR and RAR signalling in the late gestation developing lung. The glucocorticoid receptor and the retinoic acid receptors regulate transcription of specific target genes by binding directly to the promoter/enhancer regions of these genes. In this study, atRA was administered ex vivo to E18.5 lung explants from GR-null fetal mice. A whole genome expression microarray analysis was performed to identify genes that are transcriptionally activated by atRA and antagonised by GR in the developing saccular lung. This analysis identified only two genes, Rarb and Kbtbd11, that displayed significant changes in transcript levels dependent on an interaction between atRA treatment and GR-genotype. While the function of Kbtbd11 during lung development is unknown, GR-induced negative regulation of Rarb levels has direct implications on RA6 induced signalling. Further analyses of the various Rarb transcripts identified the proximal Rarb2/4 P2 promoter as the site of this antagonism and chromatin immunoprecipitation analyses concluded that GR associates with the DNA at this promoter. To complement the ex vivo lung explants study, atRA was also administered in utero to pregnant GR-null mice during the canalicular and saccular stages of lung development, at E16.5, E17.5 and E18.5. While the genetic effects observed in the ex vivo study were present in this in utero study, morphometric analyses of these lungs did not detect any observable changes in lung morphology following atRA-administration. Further analysis of the transcript levels of various RAR-signalling regulators found that Crbp1 (cellular retinol binding protein 1) and Crabp1 (cellular retinoic acid binding protein 1) transcript levels are significantly increased following atRA-administration only in the absence of GR. These two proteins are involved in retinoid storage and synthesis, and affect levels of available RA ligand. Together these results show that GR interferes at several levels of retinoid signalling, obstructing both retinoid synthesis and RAR expression. This data provides evidence of an intimate, antagonistic relationship between these two members of the nuclear receptor superfamily.