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• W2038665356 abstract "11β-Hydroxysteroid dehydrogenase (11β-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11β-HSD2, we coexpressed epitope-tagged MR and 11β-HSD2 in HEK-293 cells lacking 11β-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 11β-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11β-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11β-HSD2, but not with 11β-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 11β-HSD2-dependent oxidation to cortisone. Compromised 11β-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 11β-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11β-HSD2-dependent mechanism. Our results provide evidence that 11β-HSD2, besides inactivating 11β-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation. 11β-Hydroxysteroid dehydrogenase (11β-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11β-HSD2, we coexpressed epitope-tagged MR and 11β-HSD2 in HEK-293 cells lacking 11β-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 11β-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11β-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11β-HSD2, but not with 11β-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 11β-HSD2-dependent oxidation to cortisone. Compromised 11β-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 11β-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11β-HSD2-dependent mechanism. Our results provide evidence that 11β-HSD2, besides inactivating 11β-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation. mineralocorticoid receptor glucocorticoid receptor androgen receptor 11β-hydroxysteroid dehydrogenase endoplasmic reticulum apparent mineralocorticoid excess hemagglutinin The mineralocorticoid receptor (MR)1 and the glucocorticoid receptor (GR) both belong to the steroid/thyroid receptor superfamily. These ligand-dependent transcription factors are generally located in the nucleus even in the absence of hormone, with the exception of the GR, the androgen receptor (AR), and the MR. The localization of the MR in the absence of hormone is controversial (1Guiochon-Mantel A. Delabre K. Lescop P. Milgrom E. J. Steroid Biochem. Mol. Biol. 1996; 56: 3-9Crossref PubMed Scopus (80) Google Scholar). Several investigators reported mainly cytoplasmic localization of the MR in different tissues or transfected cells using immunohistochemistry and immunofluorescence analysis (2Binart N. Lombes M. Rafestin-Oblin M.E. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10681-10685Crossref PubMed Scopus (55) Google Scholar, 3Alnemri E.S. Maksymowych A.B. Robertson N.M. Litwack G. J. Biol. Chem. 1991; 266: 18072-18081Abstract Full Text PDF PubMed Google Scholar, 4Robertson N.M. Schulman G. Karnik S. Alnemri E. Litwack G. Mol. Endocrinol. 1993; 7: 1226-1239Crossref PubMed Scopus (61) Google Scholar, 5Lombes M. Binart N. Delahaye F. Baulieu E.E. Rafestin-Oblin M.E. Biochem. J. 1994; 302: 191-197Crossref PubMed Scopus (72) Google Scholar). In contrast, others observed both nuclear and cytoplasmic distribution of the native MR in cells from normal or adrenalectomized animals (6Krozowski Z.S. Rundle S.E. Wallace C. Castell M.J. Shen J.H. Dowling J. Funder J.W. Smith A.I. Endocrinology. 1989; 125: 192-198Crossref PubMed Scopus (90) Google Scholar, 7Lombes M. Farman N. Oblin M.E. Baulieu E.E. Bonvalet J.P. Erlanger B.F. Gasc J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1086-1088Crossref PubMed Scopus (105) Google Scholar, 8Farman N. Oblin M.E. Lombes M. Delahaye F. Westphal H.M. Bonvalet J.P. Gasc J.M. Am. J. Physiol. 1991; 260: C226-C233Crossref PubMed Google Scholar, 9Sasano H. Fukushima K. Sasaki I. Matsuno S. Nagura H. Krozowski Z.S. J. Endocrinol. 1992; 132: 305-310Crossref PubMed Scopus (57) Google Scholar) or of the recombinant MR in transfected cell lines (10Fejes-Toth G. Pearce D. Naray-Fejes-Toth A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2973-2978Crossref PubMed Scopus (214) Google Scholar). The unliganded MR is part of a soluble hetero-oligomeric complex that includes hsp90, hsp70, and other associated proteins (11Trapp T. Holsboer F. Trends Pharmacol. Sci. 1996; 17: 145-149Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 12Couette B. Fagart J. Jalaguier S. Lombes M. Souque A. Rafestin-Oblin M.E. Biochem. J. 1996; 315: 421-427Crossref PubMed Scopus (67) Google Scholar). The presence of hsp90 in the receptor complex seems to prevent activation of the unliganded MR. Binding of aldosterone initiates a conformational activation within the ligand-binding domain of the receptor that leads to the dissociation of several associated proteins from the receptor, followed by dimerization and nuclear translocation of the activated receptor. Variations in the expression level of associated proteins necessary for nuclear translocation of the MR or differences in the expression levels of proteins regulating intracellular free corticosteroid concentrations might be responsible for the observed controversial findings obtained from different tissues or cell systems. Furthermore, cross-reactivity of the applied anti-MR antibodies with other members of the steroid/thyroid receptor family cannot be excluded in some of the previous investigations. The mineralocorticoid aldosterone and the glucocorticoids cortisol and corticosterone have similar affinities for binding to the MR (13Krozowski Z.S. Funder J.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6056-6060Crossref PubMed Scopus (513) Google Scholar, 14Beaumont K. Fanestil D.D. Endocrinology. 1983; 113: 2043-2051Crossref PubMed Scopus (171) Google Scholar, 15Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1651) Google Scholar, 16Myles K. Funder J.W. Am. J. Physiol. 1994; 267: E268-E272PubMed Google Scholar). Although the circulating concentrations of cortisol (in humans) and corticosterone (in rodents) are 100–1000 times higher than those of aldosterone (17Holbrook M.M. Dale S.L. Melby J.C. J. Steroid Biochem. 1980; 13: 1355-1358Crossref PubMed Scopus (14) Google Scholar), cortisol and corticosterone do not lead to MR activation under normal conditions. This is due to the 11β-hydroxysteroid dehydrogenase (11β-HSD) activity present in most MR-expressing cells. Two 11β-HSD enzymes, both localizing to the ER membrane, have been described so far (for review, see Refs. 18White P.C. Mune T. Agarwal A.K. Endocr. Rev. 1997; 18: 135-156Crossref PubMed Scopus (548) Google Scholar and 19Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (450) Google Scholar). 11β-HSD1 is ubiquitously expressed, with the highest activity in the liver (20Lakshmi V. Monder C. Endocrinology. 1988; 123: 2390-2398Crossref PubMed Scopus (319) Google Scholar, 21Agarwal A.K. Monder C. Eckstein B. White P.C. J. Biol. Chem. 1989; 264: 18939-18943Abstract Full Text PDF PubMed Google Scholar). In vivo, it acts predominantly as a reductase, but it efficiently oxidates 11β-hydroxyglucocorticoids when measured in intact cells or in cell lysates. The catalytic domain of 11β-HSD1 is directed toward the ER lumen (22Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). 11β-HSD1 knockout mice were resistant to hyperglycemia provoked by obesity or stress, but these animals did not display severe defects in glucocorticoid metabolism (24Kotelevtsev Y. Holmes M.C. Burchell A. Houston P.M. Schmoll D. Jamieson P. Best R. Brown R. Edwards C.R. Seckl J.R. Mullins J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14924-14929Crossref PubMed Scopus (818) Google Scholar). In contrast, 11β-HSD2 is mainly expressed in the kidney and in other mineralocorticoid-sensitive tissues and catalyzes exclusively the oxidation of 11β-hydroxyglucocorticoids (25Albiston A.L. Obeyesekere V.R. Smith R.E. Krozowski Z.S. Mol. Cell. Endocrinol. 1994; 105: R11-R17Crossref PubMed Scopus (743) Google Scholar). Its catalytic moiety protrudes into the cytoplasm (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 26Naray-Fejes-Toth A. Fejes-Toth G. Endocrinology. 1998; 139: 2955-2959Crossref PubMed Google Scholar). In patients exhibiting loss-of-function mutations in the gene encoding 11β-HSD2, e.g. individuals suffering from the syndrome of apparent mineralocorticoid excess (AME) (18White P.C. Mune T. Agarwal A.K. Endocr. Rev. 1997; 18: 135-156Crossref PubMed Scopus (548) Google Scholar, 19Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (450) Google Scholar), or in mice lacking 11β-HSD2 (27Kotelevtsev Y. Brown R.W. Fleming S. Kenyon C. Edwards C.R. Seckl J.R. Mullins J.J. J. Clin. Invest. 1999; 103: 683-689Crossref PubMed Scopus (252) Google Scholar), deficiency of 11β-HSD2 allows 11β-hydroxyglucocorticoids to bind to the MR, leading to sodium retention, hypokalemia, and severe hypertension. A similar type of hypertension is induced by ingestion of licorice, which contains glycyrrhetinic acid, a potent inhibitor of 11β-HSD2 (28Monder C. Stewart P.M. Lakshmi V. Valentino R. Burt D. Edwards C.R. Endocrinology. 1989; 125: 1046-1053Crossref PubMed Scopus (335) Google Scholar, 29Souness G.W. Morris D.J. Endocrinology. 1989; 124: 1588-1590Crossref PubMed Scopus (75) Google Scholar, 30Gomez-Sanchez E.P. Gomez-Sanchez C.E. Am. J. Physiol. 1992; 263: E1125-E1130PubMed Google Scholar, 31Lindsay R.S. Lindsay R.M. Edwards C.R. Seckl J.R. Hypertension. 1996; 27: 1200-1204Crossref PubMed Scopus (277) Google Scholar). Elevated concentrations of endogenous 11β-HSD2 inhibitors, such as certain bile acids (32Buhler H. Perschel F.H. Fitzner R. Hierholzer K. Steroids. 1994; 59: 131-135Crossref PubMed Scopus (32) Google Scholar, 33Escher G. Nawrocki A. Staub T. Vishwanath B.S. Frey B.M. Reichen J. Frey F.J. Gastroenterology. 1998; 114: 175-184Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 34Ackermann D. Vogt B. Escher G. Dick B. Reichen J. Frey B.M. Frey F.J. Hepatology. 1999; 30: 623-629Crossref PubMed Scopus (45) Google Scholar) and progesterone metabolites (35Souness G.W. Latif S.A. Laurenzo J.L. Morris D.J. Endocrinology. 1995; 136: 1809-1812Crossref PubMed Google Scholar, 36Quinkler M. Johanssen S. Grossmann C. Bahr V. Muller M. Oelkers W. Diederich S. J. Clin. Endocrinol. Metab. 1999; 84: 4165-4171Crossref PubMed Google Scholar), and the uptake of exogenous inhibitors, such as glycyrrhetinic acid, carbenoxolone (28Monder C. Stewart P.M. Lakshmi V. Valentino R. Burt D. Edwards C.R. Endocrinology. 1989; 125: 1046-1053Crossref PubMed Scopus (335) Google Scholar, 29Souness G.W. Morris D.J. Endocrinology. 1989; 124: 1588-1590Crossref PubMed Scopus (75) Google Scholar, 30Gomez-Sanchez E.P. Gomez-Sanchez C.E. Am. J. Physiol. 1992; 263: E1125-E1130PubMed Google Scholar, 31Lindsay R.S. Lindsay R.M. Edwards C.R. Seckl J.R. Hypertension. 1996; 27: 1200-1204Crossref PubMed Scopus (277) Google Scholar), grapefruit flavonoids (37Zhang Y.D. Lorenzo B. Reidenberg M.M. J. Steroid Biochem. Mol. Biol. 1994; 49: 81-85Crossref PubMed Scopus (39) Google Scholar), and furosemide (38Escher G. Meyer K.V. Vishwanath B.S. Frey B.M. Frey F.J. Endocrinology. 1995; 136: 1759-1765Crossref PubMed Scopus (31) Google Scholar,39Fuster D. Escher G. Vogt B. Ackermann D. Dick B. Frey B.M. Frey F.J. Endocrinology. 1998; 139: 3849-3854Crossref PubMed Scopus (32) Google Scholar), have been causally linked with glucocorticoid-induced activation of the MR. By binding to the MR, aldosterone and, under certain circumstances, glucocorticoids play a major role in the regulation of sodium and potassium homeostasis. Whereas aldosterone normally elicits sodium retention and kaliuresis, 11β-hydroxyglucocorticoids cause kaliuresis without sodium retention. 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 (40Kenyon C.J. Saccoccio N.A. Morris D.J. Clin. Sci. 1984; 67: 329-335Crossref PubMed Scopus (24) Google Scholar, 41Brem A.S. Matheson K.L. Barnes J.L. Morris D.J. Am. J. Physiol. 1991; 261: F873-F879PubMed Google Scholar, 42Young M. Fullerton M. Dilley R. Funder J. J. Clin. Invest. 1994; 93: 2578-2583Crossref PubMed Scopus (431) Google Scholar, 43Funder J.W. Kidney Int. 2000; 57: 1358-1363Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 44Morris D.J. Souness G.W. Brem A.S. Oblin M.E. Kidney Int. 2000; 57: 1370-1373Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). More recently, evidence for distinct physiological effects of aldosterone in aldosterone target tissues and non-epithelial tissues was presented (43Funder J.W. Kidney Int. 2000; 57: 1358-1363Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). However, the mechanisms underlying these observations remain unclear. In this study, we evaluated the impact of 11β-HSD enzyme expression on the intracellular distribution of the MR and analyzed the corticosteroid-induced nuclear translocation of the receptor under various conditions. The results suggest that the MR is specifically associated with 11β-HSD2 at the ER membrane in the absence of corticosteroids. Moreover, 11β-HSD2 tightly regulated the access of aldosterone to the MR by inactivating 11β-hydroxyglucocorticoids, whereby the formed 11-keto products blunted the aldosterone-induced nuclear translocation of the MR. Wild-type and FLAG epitope-tagged human 11β-HSD1 and 11β-HSD2 expression plasmids were constructed as described (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Their activities were indistinguishable from those of their wild-type enzymes. Constructs expressing the mutant 11β-HSD2 proteins ΔL114,E115 (45Odermatt A. Dick B. Arnold P. Zaehner T. Plueschke V. Deregibus M.N. Repetto H. Frey B.M. Frey F.J. Ferrari P. J. Clin. Endocrinol. Metab. 2001; 86: 1247-1252PubMed Google Scholar) and R337C (46Wilson R.C. Krozowski Z.S. Li K. Obeyesekere V.R. Razzaghy-Azar M. Harbison M.D. Wei J.Q. Shackleton C.H. Funder J.W. New M.I. J. Clin. Endocrinol. Metab. 1995; 80: 2263-2266Crossref PubMed Google Scholar) were generated by polymerase chain reaction-based mutagenesis. The mutant 11β-HSD1 protein K5S,K6S was described previously (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The plasmid RShMR, expressing the full-length human MR, was a generous gift from Dr. R. M. Evans (Salk Institute, La Jolla, CA) (15Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1651) Google Scholar). The tagged human MR was obtained by insertion of the nonapeptide hemagglutinin (HA) epitope with the sequence YPYDVPDYA at the carboxyl-terminal end just upstream of the stop codon. The human sarcolipin construct containing a FLAG tag at the C terminus was described previously (47Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The N-terminally FLAG epitope-tagged human AR construct was generously provided by Dr. J. J. Palvimo (Institute of Biomedicine, Helsinki, Finland) (48Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (371) Google Scholar). Expression constructs encoding rat 3α-hydroxysteroid dehydrogenase (49Pawlowski J.E. Huizinga M. Penning T.M. J. Biol. Chem. 1991; 266: 8820-8825Abstract Full Text PDF PubMed Google Scholar), human steroid 5α-reductase type I (50Andersson S. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3640-3644Crossref PubMed Scopus (502) Google Scholar), mouse CYP7A1 cholesterol 7α-hydroxylase (51Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 271: 18017-18023Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and mouse CYP7B1 oxysterol 7α-hydroxylase (52Schwarz M. Lund E.G. Setchell K.D. Kayden H.J. Zerwekh J.E. Bjorkhem I. Herz J. Russell D.W. J. Biol. Chem. 1996; 271: 18024-18031Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) were a generous gift from Dr. D. W. Russell (Texas Southwestern Medical Center, Dallas). All constructs were verified by sequencing. HEK-293 cells were grown on glass coverslips in six-well plates containing 2 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transient transfections were performed using the calcium phosphate precipitation method with 0.5 µg of MR cDNA and 0.5 µg of 11β-HSD2 cDNA per well. When expressing the MR alone, 0.5 µg of empty pcDNA3 vector DNA was added as a carrier. Transient transfection resulted in 30–35% of 11β-HSD2- and MR-positive cells. Six hours post-transfection, cells were washed twice with Hanks' solution, followed by incubation in medium that was charcoal-stripped twice. Analysis of the medium by gas chromatography/mass spectrometry revealed the absence of corticosteroids. 2B. Dick, unpublished data. HEK-293 cells were transiently cotransfected with 50 ng of plasmid MMTV-lacZ (53Satoh K. Galli I. Ariga H. Nucleic Acids Res. 1993; 21: 4429-4430Crossref PubMed Scopus (7) Google Scholar), carrying the bacterial β-galactosidase gene (lacZ) under the control of the mouse mammary tumor virus promoter, and either 450 ng of plasmid RShMR, encoding the human MR (15Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1651) Google Scholar), or the HA-tagged MR cDNA construct. Dulbecco's modified Eagle's medium was replaced by steroid-free medium at 24 h post-transfection. Another 24 h later, cortisol dissolved in methanol or the corresponding amount of methanol was added, and cells were incubated for another 16 h prior to analysis by the Dual-Light assay (Tropix, Inc., Foster City, CA). 11β-HSD enzyme activity was measured in intact cells as described (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Briefly, transfected HEK-293 cells incubated in charcoal-treated Dulbecco's modified Eagle's medium for 48 h were washed once with Hanks' solution and resuspended in prewarmed (37 °C) steroid-free medium. Dehydrogenase activity was measured in a final volume of 20 µl containing 30 nCi of [3H]corticosterone and unlabeled corticosterone at different concentrations ranging from 25 nm to 2 µm. The reaction was started by mixing the cell suspension with the reaction mixture. Incubation was for 10–30 min, and the conversion of corticosterone to 11-dehydrocorticosterone was determined by thin-layer chromatography. Enzyme kinetics were analyzed by the Eadie-Hofstee linear transformation of the Michaelis-Menten equation. The protein expression of the different 11β-HSD constructs was compared by semiquantitative Western blot analysis. Transfected cells grown in charcoal-treated medium for 14 h were incubated in the presence or absence of the appropriate concentrations of 11β-HSD2 inhibitors for 5 min as indicated, followed by the addition of corticosteroids. Cells were incubated for 45 min at 37 °C and washed once with 150 mm sodium phosphate (pH 7.4) and 120 mmsucrose, followed by fixation with 4% paraformaldehyde for 10 min. Immunostaining was performed as described (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). FLAG epitope-tagged proteins and HA-tagged proteins were detected using mouse monoclonal anti-FLAG antibody M2 (Sigma, Buchs, Switzerland) and a rat monoclonal anti-HA antibody (Roche Molecular Biochemicals, Rotkreuz, Switzerland) as first antibodies, respectively, and anti-mouse antibody Alexa-488 and anti-rat antibody Alexa-594 (Molecular Probes, Inc., Leiden, The Netherlands) as secondary antibodies, respectively. In coexpression experiments with the AR, human 11β-HSD2 was detected with a rabbit polyclonal antibody generously provided by Dr. Z. N. Kyossev (Division of Nephrology, University of Arkansas, Little Rock, AK) (54Kyossev Z. Walker P.D. Reeves W.B. Kidney Int. 1996; 49: 271-281Abstract Full Text PDF PubMed Scopus (73) Google Scholar) and anti-rabbit secondary antibody Alexa-594. Immunofluorescence was detected using a Model LSM410 confocal microscope (Carl Zeiss, Göttingen, Germany). Quantitation of intracellular localization of the MR or AR was performed by counting the number of cells within an area selected under the transmitted light and determination of positively staining cells that were classified into three different categories: predominantly cytoplasmic staining, comparable intensity of nuclear and cytoplasmic staining, and exclusively nuclear staining. Results were obtained from at least three independent transfection experiments, in which between 400 and 500 stained cells were determined for each sample. To investigate the intracellular distribution of the MR in the absence of hormone, we transiently expressed the C-terminally HA epitope-tagged MR in HEK-293 cells and analyzed receptor localization by immunostaining and confocal microscopy. We confirmed the lack of endogenous 11β-HSD activity in HEK-293 cells (23Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The transcriptional activities of wild-type and HA-tagged MRs, determined by a chemiluminescent reporter gene assay, were indistinguishable. No MR transcriptional activity was detected in untransfected cells (data not shown). The anti-HA antibody, specifically recognizing the nonapeptide epitope YPYDVPDYA, was used for fluorescence microscopy detection. Incubation of untransfected HEK-293 cells with the anti-HA antibody did not produce any signal; therefore, the problem of cross-reactivity with other receptors inherent with antibodies raised directly against the MR could be avoided. Cells transiently expressing the MR and grown in steroid-free medium exhibited both cytoplasmic and nuclear presence of the receptor (TableI). A heterogeneous distribution pattern with significant cell-to-cell differences was observed (Fig.1). Low concentrations of aldosterone, cortisol, or corticosterone mediated complete nuclear translocation of the receptor (Table I).Table ICorticosteroid- and 11β-HSD2-dependent intracellular distribution of the MRPositive cellsNN/CC%MR303337MR, 10 nmaldosterone10000MR, 10 nmcortisol10000MR, 10 nmcorticosterone10000MR, 11β-HSD20199MR, 11β-HSD2, 10 nm aldosterone731710MR, 11β-HSD2, 10 nm cortisol0298MR, 11β-HSD2, 10 nm corticosterone0298MR, 11β-HSD2, 2.5 µm glycyrrhetinic acid0595MR, 11β-HSD2, 100 µm furosemide0496MR, ΔL114,E1152593MR, ΔL114,E115, 10 nm cortisol73189MR, ΔL114,E115, 50 nm cortisol9631MR, R337C1495MR, R337C, 10 nm cortisol83134MR, R337C, 50 nm cortisol9721MR, 11β-HSD1253342MR, 11β-HSD1, 10 nmcortisol9064MR, 11β-HSD1 mutant K5S,K6S303634MR, 3α-hydroxysteroid dehydrogenase294427MR, steroid 5α-reductase type I332938MR, CYP7B1 oxysterol 7α-hydroxylase67258MR, CYP7A1 cholesterol 7α-hydroxylase553411AR195724AR, 11β-HSD2245323AR, 100 nmtestosterone10000AR, 11β-HSD2, 100 nmtestosterone10000HEK-293 cells were transfected with the HA-tagged MR or cotransfected with the HA-tagged MR and FLAG-tagged wild-type or mutant 11β-HSD2, FLAG-tagged wild-type or mutant 11β-HSD1, or unrelated steroid-metabolizing enzymes. Transfected cells were grown for 14 h in steroid-free medium prior to the addition of corticosteroid hormones or inhibitors of 11β-HSD2 and incubation for another 45 min. Immunostaining using antibodies against the HA and FLAG epitopes was performed as described under “Experimental Procedures.” Alternatively, the FLAG-tagged AR was expressed alone or with wild-type 11β-HSD2 both in the absence and presence of testosterone. Cells staining positively for the MR were divided into three categories: N, predominantly nuclear; N/C, nuclear and cytoplasmic; C, predominantly cytoplasmic. Results represent the percentage of fluorescent cells relative to total cells, whereby 400–500 fluorescent cells were determined. Open table in a new tab HEK-293 cells were transfected with the HA-tagged MR or cotransfected with the HA-tagged MR and FLAG-tagged wild-type or mutant 11β-HSD2, FLAG-tagged wild-type or mutant 11β-HSD1, or unrelated steroid-metabolizing enzymes. Transfected cells were grown for 14 h in steroid-free medium prior to the addition of corticosteroid hormones or inhibitors of 11β-HSD2 and incubation for another 45 min. Immunostaining using antibodies against the HA and FLAG epitopes was performed as described under “Experimental Procedures.” Alternatively, the FLAG-tagged AR was expressed alone or with wild-type 11β-HSD2 both in the absence and presence of testosterone. Cells staining positively for the MR were divided into three categories: N, predominantly nuclear; N/C, nuclear and cytoplasmic; C, predominantly cytoplasmic. Results represent the percentage of fluorescent cells relative to total cells, whereby 400–500 fluorescent cells were determined. In most mineralocorticoid-sensitive cells, the MR is coexpressed with 11β-HSD2. Therefore, we analyzed the impact of 11β-HSD2 expression on the intracellular distribution of the MR. Dual staining of cells incubated in the absence of hormone and expressing both the HA-tagged MR and FLAG-tagged 11β-HSD2 and subsequent analysis by confocal microscopy revealed a localization pattern that was dramatically different from that observed when the MR was expressed in the absence of 11β-HSD2. The MR displayed a typically reticular distribution pattern, with no MR present in the nucleus, suggesting association of the receptor with the ER membrane in the absence of aldosterone (TableI and Fig. 2, A–C). An overlay image of the expression of the MR and 11β-HSD2 at high resolution indicated the colocalization of both proteins at the ER membrane (Fig. 2, D–F). The ER membrane association of the MR was analyzed further by coexpressing the HA-tagged MR with untagged wild-type 11β-HSD2 and FLAG-tagged sarcolipin, a small proteolipid known to be located exclusively in the ER membrane (47Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Both the MR and sarcolipin showed almost identical reticular expression patterns (Fig. 2, G–I), indicating ER membrane association of the MR. Sarcolipin alone did not affect the intracellular distribution of the MR since, in the absence of 11β-HSD2, a heterogeneous MR distribution comparable to that shown in Fig. 1 was observed. The MR also displayed exclusively reticular localization when coexpressed in the absence of corticosteroids with 11β-HSD2 mutant ΔL114,E115 or R337C, both of which were derived from patients suffering from AME and which retained only very low dehydrogenase activity (Table I) (45Odermatt A. Dick B. Arnold P. Zaehner T. Plueschke V. Deregibus M.N. Repetto H. Frey B.M. Frey F.J. Ferrari P. J. Clin. Endocrinol. Metab. 2001; 86: 1247-1252PubMed Google Scholar,46Wilson R.C. Krozowski Z.S. Li K. Obeyesekere V.R. Razzaghy-Azar M. Harbison M.D. Wei J.Q. Shackleton C.H. Funder J.W. New M.I. J. Clin. Endocrinol. Metab. 1995; 80: 2263-2266Crossref PubMed Google Scholar). Independent of the presence of 11β-HSD2, the MR translocated into the nucleus upon addition of" @default.
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• W2038665356 title "The Intracellular Localization of the Mineralocorticoid Receptor Is Regulated by 11β-Hydroxysteroid Dehydrogenase Type 2" @default.
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