Immunodetection of 11β-Hydroxysteroid Dehydrogenase Type 2 in Human Mineralocorticoid Target Tissues: Evidence for Nuclear Localization*

Abstract

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Expression of 11beta-hydroxysteroid dehydrogenase (11betaHSD) proteins in luteinizing human granulosa-lutein cells

“…1). However, the same antibody detected a protein of approximately 48 kDa in lysates prepared from type 2 11 HSD-transfected HEK2 cells, consistent with previous studies using human kidney tissue lysates (Shimojo et al 1997).…”

Glucocorticoid Measurements in Health and Disease - Metabolic Implications and the Potential of 24-h Urine Analyses

“…The enzyme 11b-hydroxysteroid dehydrogenase (11b-HSD) catalyses the reversible interconversion of cortisol to its hormonally inactive 11-oxo metabolite cortisone [16][17][18][19]. Two isoforms of the enzyme exist 11b-HSD1 and 11b-HSD2 [20,21].…”

Interactions between dehydroepiandrosterone and glucocorticoid metabolism in pig kidney: Nuclear and microsomal 11β-hydroxysteroid dehydrogenases

“…11βHSD activity has been observed in nuclei, mitochondrial and microsomal organelles from normal human and rat placenta [5] and choriocarcinoma cells [6]. In human kidney nuclei, immuno-reactive human 11βHSD2 accounted for 40% of total cellular 11βHSD2 protein content [7,8]. In kidney from rat and sheep, a third dehydrogenase (11βHSD3) was shown to be present in membranes of Golgi apparatus and mitochondria, while 11βHSD2 is preferentially located in the nuclear membrane [9,10].…”