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

Abstract 11β-Hydroxysteroid dehydrogenase (11βHSD) 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 11βHSD2 gene, suggest that it is the 11βHSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11βHSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11βHSD2 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 11βHSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11βHSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 μm indicated significant 11βHSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11βHSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11βHSD2, 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.