Glucocorticoids mediate anti-inflammatory effects through the ubiquitously expressed glucocorticoid receptor (GR). The tissue-specific inhibition of proinflammatory pathways by glucocorticoids may be conferred by the expression of different GR isoforms regulated by GR gene exon 1 promoter variants. In the human placenta we have shown sexually dimorphic differences in expression and localization of several GR isoforms with increased expression of GRα-A, GRα-C3 and GRα-D3 in female placentae which was associated with anti-inflammatory glucocorticoid effects. Conversely male placentae had high GRβ, GR-A and GR-P expression with increased pro-inflammatory gene expression in vivo. We hypothesised that selective regulation of GR isoforms was through differential upregulation of GR Exon 1 regions which induces a shift from anti-inflammatory actions of glucocorticoids to upregulation of proinflammatory pathways.
Methods: BeWo cells were treated with hydrocortisone, dexamethasone or budesonide (1µM) or LPS (1ug/ml) alone or in combination for 4 and 24 hours. Protein lysates were analysed using western blot while the GR exon 1 variants mRNA was measured using QRT-PCR.
Results: Glucocorticoid treatment resulted in an increased expression of GRα-A, GRα-C3, GRα-D1 and D3. A significant increase in GRαD1 protein was observed at 4 hours with LPS treatment. However, LPS and glucocorticoids combination resulted into increased GRαD3 expression. Changes in GR isoform expression were associated with upregulation of selective GR promoters. LPS stimulation resulted in a 15-fold induction of GR promoter 1E region compared to control treatment. Budesonide treatment selectively upregulated exon 1J and 1I promoter regions.
Conclusion: Selective switching between GR isoforms following an inflammatory challenge suggests a specific role for GRα-D1 in promoting a proinflammatory response. The observed change in GRα-D1 expression may be associated with increased Exon1 E expression. The increased expression of Exon 1E and GRα-D1 may be responsible for the shift from glucocorticoid induced anti-inflammatory effects to the activation of proinflammatory pathways.