Abstract
11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11beta-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11beta-HSD2, we coexpressed epitope-tagged MR and 11beta-HSD2 in HEK-293 cells lacking 11beta-HSD2 activity and analyzed their subcellular localization by fluorescence microscopy. When expressed alone in the absence of hormones, the MR was both cytoplasmic and nuclear. However, when coexpressed with 11beta-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11beta-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11beta-HSD2, but not with 11beta-HSD1 or other steroid-metabolizing enzymes. Aldosterone induced rapid nuclear translocation of the MR, whereas moderate cortisol concentrations (10-200 nm) did not activate the receptor, due to 11beta-HSD2-dependent oxidation to cortisone. Compromised 11beta-HSD2 activity (due to genetic mutations, the presence of inhibitors, or saturating cortisol concentrations) led to cortisol-induced nuclear accumulation of the MR. Surprisingly, the 11beta-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11beta-HSD2-dependent mechanism. Our results provide evidence that 11beta-HSD2, besides inactivating 11beta-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation.
Highlights
The mineralocorticoid receptor (MR)1 and the glucocorticoid receptor (GR) both belong to the steroid/thyroid receptor superfamily
In patients exhibiting loss-of-function mutations in the gene encoding 11-HSD2, e.g. individuals suffering from the syndrome of apparent mineralocorticoid excess (AME) [18, 19], or in mice lacking 11-HSD2 [27], deficiency of 11-HSD2 allows 11-hydroxyglucocorticoids to bind to the MR, leading to sodium retention, hypokalemia, and severe hypertension
We transiently expressed the epitope-tagged MR in HEK-293 cells in the absence of hormone and 11-HSD2 and observed a heterogeneous MR distribution pattern (Fig. 1). This is in line with the findings of Fejes-Toth et al [10], who reported a heterogeneous localization pattern of a green fluorescent protein-MR chimeric construct in the absence of steroids in transfected CV1 or Chinese hamster ovary cells, both lacking endogenous 11-HSD2 activity
Summary
MR, 11-HSD2 MR, 11-HSD2, 10 nM aldosterone MR, 11-HSD2, 10 nM cortisol MR, 11-HSD2, 10 nM corticosterone MR, 11-HSD2, 2.5 M glycyrrhetinic acid MR, 11-HSD2, 100 M furosemide. There is evidence from several in vivo studies that the aldosterone-induced salt retention is blunted by the co-administration of glucocorticoids, suggesting an MR antagonist-like action of glucocorticoids (40 – 44). We evaluated the impact of 11-HSD enzyme expression on the intracellular distribution of the MR and analyzed the corticosteroid-induced nuclear translocation of the receptor under various conditions. 11HSD2 tightly regulated the access of aldosterone to the MR by inactivating 11-hydroxyglucocorticoids, whereby the formed 11-keto products blunted the aldosterone-induced nuclear translocation of the MR
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