SUN-015 Glucocorticoid Induction And Direct Genomic Regulation Of The Transcriptional Regulators Hif3a And Zbtb16 During Fetal Mouse Lung Development

Development of a functional human lung requires regulation of cell growth, cellular proliferation and differentiation in specific germ layer compartments. Important endocrine regulators of lung development include glucocorticoid (GC) steroids and the potent synthetic GC betamethasone is used to treat the deficits of human preterm birth. Previous studies with conditional mouse knockouts of the glucocorticoid receptor (GR) gene have established the mesenchymal compartment of the lung as the most important target for GC-signalling. To identify the direct GC gene targets and cell pathways responsible we have stimulated primary cell cultures of fetal rat lung mesenchymal fibroblasts for six hours with betamethasone and corticosterone, and analysed cellular responses using RNA-seq and GR-ChIP-Seq analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared to corticosterone. 487 genes were significantly stimulated by betamethasone or corticosterone, with 453 by both steroids, indicating strong overlap in regulation. Whole genome betamethasone/GR ChIP-seq analysis identified approximately 175 GR-binding sites across the genome, with most containing a conserved canonical 15bp GRE sequence. GREs were located near previously characterised GR-targets genes such as Per1, Sgk1, Fkbp5 and Dusp1, and also near many GC-induced genes identified by RNA-seq. Among these genes were two important transcriptional regulators, Hif3a and Zbtb16. Both were strongly induced at the RNA level and contained prominent GR binding sites. Hif3a mRNA levels were induced 5 fold in fetal lung fibroblasts by both corticosterone and betamethasone, and a strong GRE (AGGACATTCCGTCCT) was identified 1.5kb upstream of the TSS. Analysis in conditional GR-deficient mice showed markedly reduced expression of Hif3a in the fetal lung of complete GR-null mice. Zbtb16 mRNA levels were induced 6 fold in fetal fibroblasts by both corticosterone and betamethasone, and two GREs (AGAACACACTGTACC/ GGTACACTCTGTACT) were identified in intron B, 80-90kb downstream of the TSS of the Zbtb16 gene. Analysis in the lung of conditional GR-deficient mice showed reduced expression of Zbtb16 in both complete GR-null and lung mesenchymal-GR-deficient mice. Both transcription factors have been previously demonstrated to control cell proliferation pathways. Overall, cell pathway analysis has identified cell proliferation and cytoskeletal/cell matrix remodelling pathways as key processes stimulated in response to betamethasone. These results demonstrate that glucocorticoids induce higher levels and activity of specific cell regulatory pathways in the fetal lung via controlling transcription factor networks to ultimately contribute to the normal program of lung development.