Glucocorticoid signalling via the glucocorticoid receptor (GR) is essential for normal lung development. Previous work using conditional mouse knockouts of the GR gene established that GR activity in the mesenchymal compartment of the lung is required for normal respiratory development. Screens for differentially expressed genes in the mesenchymal GR-deficient lung (GRmesKO) have identified the extracellular matrix (ECM) proteoglycan Versican (Vcan) as a potential GR-regulated repressed gene target. As well as an important structural ECM protein, Vcan has an important role as a cell surface receptor for the epithelial-derived growth factor midkine to regulate cell proliferation during organogenesis. Alternative exon splicing of the Vcan gene generate 5 isoforms V0, V1, V2, V3 and V4 that vary in structure and function.
We hypothesised that the severe mesenchymal cell hyperplasia observed in the saccular-stage GRmesKO fetal mouse lung is in part due to the lack of GR-mediated repression of Vcan levels. We performed isoform specific qPCR and immunohistochemistry on the GRmesKO fetal lung. We observed that all Vcan isoform mRNA levels in the fetal mouse lung decline from E14.5 to P0.5. We also show that the V1 isoform containing the beta domain of Vcan is far more abundant in E16.5 lung than E18.5.
All four isoform mRNA levels showed a 2-3 fold increase in E18.5 GRmesKO lungs relative to controls. We detect a strong increase in Vcan protein accumulation in GRmesKO and GRnull lung compared to WT controls. To further demonstrate the role of Vcan in lung cell proliferation we performed siRNA mediated knockdown of Vcan expression in primary rat lung fibroblasts. At 60 hours post-treatment, we observed a significant decrease in cell proliferation rate. In summary, glucocorticoid steroids regulate repression of the ECM protein Vcan to contribute to coordinated normal respiratory development in mammals.