Endocrine Abstracts

Development of a functional human lung requires regulation of cell growth, proliferation and differentiation in specific germ layer compartments. Important endocrine regulators of respiratory development include glucocorticoid (GC) steroids and the potent synthetic GC betamethasone is commonly used antenatally to treat the deficits of very preterm human birth. Previous studies with conditional mouse knockouts of the glucocorticoid receptor (GR) gene have established the mesenchymal compartment of the lung as the critical target for GC/GR signalling. To identify the direct betamethasone induced GR binding sites in the genome and associated potential gene targets we have stimulated primary cell cultures of fetal rat lung mesenchymal fibroblasts for six hours with betamethasone and analysed cellular responses using GR-ChIP-Seq and RNA-Seq analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared to corticosterone. Whole genome betamethasone/GR ChIP-seq analysis identified approximately 165 GR-binding sites across the genome, with nearly all containing a highly conserved canonical 15 bp glucocorticoid response element (GRE) sequence. GREs were located near previously characterised GR-targets genes such as Per1, Sgk1, Fkbp5 and Dusp1, near many GC-induced genes identified with RNA-Seq, and also near many novel genes, that also included non-protein coding miRNA and lincRNA genes. One of the strongest induced genes at the mRNA level was a transcription factor called Zbtb16 whose mRNA levels were induced 60 fold in fetal lung fibroblasts by 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 markedly reduced expression of Zbtb16 in both complete GR-null and lung mesenchymal-GR-deficient mice. These results demonstrate that glucocorticoids induce higher levels and activity of specific cell regulatory pathways in the fetal lung by controlling cell signalling networks to ultimately contribute to the normal program of lung development.