Extranuclear localization of endogenous 11beta-hydroxysteroid dehydrogenase-2 in aldosterone target cells.

ABSTRACT

Type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2) plays a key role in conferring aldosterone selectivity on the mineralocorticoid receptor (MR) by inactivating intracellular glucocorticoids before they can occupy the MR. 11betaHSD2 is a microsomal enzyme expressed in aldosterone target cells, although its subcellular distribution is still unclear. The goal of this study was to determine the subcellular localization of the endogenous 11betaHSD2 in renal aldosterone target cells. We generated an antibody against rabbit 11betaHSD2 and used it in combination with a nuclear marker and confocal laser scanning microscopy. On Western blots the antibody recognized a single band of approximately 41 kDa in the renal cortical collecting duct, outer medullary collecting duct, submandibular gland and adrenal cortex, whereas the colon, liver, renal medulla, and heart were negative. Immunohistochemistry showed specific reaction in the known aldosterone target cells of the kidney (connecting tubule, cortical collecting duct, and outer medullary collecting duct) with no signals over glomeruli, proximal nephron segments, and blood vessels. Staining for 11betaHSD2 was very weak in rabbit colon, and no immunoreactivity could be detected in the heart and brain. Confocal microscopy of kidney sections costained with the 11betaHSD2 antibody and the nuclear marker propidium iodide demonstrated that 11betaHSD2 is in the cytoplasmic compartment with no evidence for nuclear localization. Subcellular localization of 11betaHSD2 to a cytoplasmic compartment seems ideal for fulfilling its biological function, i.e. the efficient inactivation of intracellular glucocorticoids before they occupy MRs, which are predominantly cytoplasmic in the absence of hormone.