Human 11β-hydroxysteroid dehydrogenase: Studies on the stably transfected isoforms and localization of the type 2 isozyme within renal tissue

Abstract

The type 1 and type 2 isoforms of human 11β-hydroxysteroid dehydrogenase (11β-HSD) play a crucial role, respectively, in modulating glucocorticoid and mineralocorticoid hormone action. Deficiency of the 11β-HSD2 isoform, as described in the syndrome of apparent mineralocorticoid excess and following liquorice (glycyrrhetinic acid) or carbenoxolone ingestion, results in hypertension in which cortisol acts as a potent mineralocorticoid. Several studies have addressed the effects of progesterone, glycyrrhetinic acid, and their derivatives on 11β-HSD activity, but these were largely undertaken before the characterization of the 11β-HSD isoforms. The aim of this study was to evaluate the localization of 11β-HSD2 in human kidney and to study the effects of progesterone, glycyrrhetinic acid, and their related compounds on stable transfectants of the human 11β-HSD isoforms. Using an in-house sheep antibody against human 11β-HSD2, immunoperoxidase studies localized 11β-HSD2 to renal cortical and medullary collecting ducts. Glomeruli, vascular structures, loops of Henle, and proximal tubules were all negative. Confocal laser microscopy studies indicated both a cytoplasmic and nuclear localization for the enzyme within renal collecting ducts. The nuclear staining, which was intranuclear and was not associated with the nuclear membrane, accounted for 40% of the total cellular 11β-HSD2 immunoreactivity. Kinetic analysis of 11β-HSD activity in fetal kidney 293 cells stably transfected with h11β-HSD1 pcDNA3 or 11β-HSD2 pCR3 , indicated, respectively, low-affinity dehydrogenase/oxoreductase activity (K m for F. 1.8 μM: K m for E, 270 nM) and high-affinity dehydrogenase activity (K m for F. 190 nM). The reductase activity of 11β-HSD1 was inhibited by 11α-hydroxyprogesterone > carbenoxolone = glycyrrhetinic acid = progesterone > 11β-hydroxyprogesterone. The dehydrogenase activity of 11β-HSD2 was inhibited 11α-hydroxyprogesterone = 11β-hydroxyprogesterone > glycyrrhetinic acid > carbenoxolone = progesterone. 11β-HSD2, expressed in the renal collecting duct, serves to protect the mineralocorticoid receptor (MR) in an autocrine fashion. The demonstration of a nuclear localization for what was thought to be principally a microsomal enzyme suggests that interaction between the MR and its ligand (either aldosterone or cortisol) may be a nuclear rather than a cytoplasmic event. The inhibitory effects of progesterone, glycyrrhetinic acid, and related compounds on 11β-HSD1 and 2 were similar, and it remains to be seen what implication these findings have for 11β-HSD1 action in tissues such as the liver and gonad and renal 11β-HSD2 activity in relation to sodium homeostasis and blood pressure control.