The interaction of the mineralocorticoid and glucocorticoid receptor in the nucleus and at DNA: Transcriptional implications for the glucocorticoid response of stress-associated brain regions

JOHN R. POOLEY; DIEGO M PRESMAN; SUNDAR GANESAN; A. SHESHASAYEE; R. LOUIS SCHILTZ; OZLEM KESKIN; STAFFORD L LIGHTMAN; GORDON L. HAGER

Chicago, Illinois

Congreso; 96th Annual meeting of the Endocrine Society; 2014

The Endocrine Society

Limited 11beta-hydroxysteroid dehydrogenase in brain allows glucocorticoid actions to be mediated by two nuclear hormone receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). MR and GR bind endogenously circulating glucocorticoids, share a hormone response element, and are co-expressed in the same neurons in several brain areas involved in HPA axis and stress regulation (e.g. hippocampus, PVN). This arrangement opens the possibility for MR/GR cooperation in the regulation of gene expression, a prospect first reported two decades previously when MR-GR interaction was observed in vitro. We have expanded on this work utilizing microscopy in conjunction with a unique cell line (3617ChMR) to study MR-GR interactions in vivo. 3617ChMR expresses GFP-tagged GR and mCherry-MR under tetracycline regulation and includes a tandem array of the MMTV long terminal repeat driving a Ras cDNA. Accumulation of fluorescent MR/GR at the chromatinized, stably integrated array structure (800-1200 GREs) is observable microscopically. The fluorescence lifetime (FLIM) approach to Forster resonance energy transfer (FRET) and fluctuation analysis by cross-correlation number and brightness assay (ccN&B) were used to assess MR-GR interactions in living cells. Initially confirming an interaction of MR-GR biochemically by co-immunoprecipitation, we show MR-GR interactions occur in the nucleus of living cells (FLIM-FRET, ccN&B) but crucially also at a chromatinized DNA template (ccN&B). This result strong supports the expectation that the interacting complex plays a transcriptional role in co-expressing cells. We compared the patterns of array-associated Ras and endogenous gene expression between MR+GR (interacting) and GR only (non-interacting) conditions by RT-PCR. Results indicate that the MR-GR interaction confers a different pattern of gene induction compared to that mediated by GR alone under a physiologically relevant hormone presentation (ultradian pulse). Curiously, examination of the stoichiometry of the complex formed in the nucleoplasm (N&B method) indicated the MR-GR interaction in the living cell may not be the anticipated heterodimer. We conclude that an interaction of MR and GR within the living cell participates in transcriptional regulation in response to endogenous glucocorticoids. More work is required to determine how hormone signals carried through the MR-GR complex differ in output from those carried by MR or GR alone.