ELSEVIER
MCE
Molecular and Cellular Endocrinology
Expression of 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD-2) in the developing human adrenal gland and human adrenal cortical carcinoma and adenoma*
Catherine L. Coulter a,*, Robin E. Smith ª, Michael Stowasser b, Hironobu Sasano ”, Zygmunt S. Krozowski ª, Richard D. Gordon b
a Laboratory of Molecular Hypertension, Baker Medical Research Institute, Prahran, Victoria 3181, Australia
b Hypertension Unit, University Department of Medicine, Greenslopes Hospital, Brisbane, Queensland 4120, Australia
c Department of Pathology, School of Medicine, Tohoku University, Sendai 98077, Japan
Received 19 January 1999; accepted 19 March 1999
Abstract
The aim of this study was to investigate the ontogeny of localization of 116HSD-2 protein in the human adrenal gland. In addition, we have investigated the effects of abnormal adrenal function on 11BHSD-2 by determining the pattern of localization of 11BHSD-2 protein, and the amount and level of expression of 116HSD-2 mRNA and protein in human adrenal cortical carcinoma and adenoma. In the human foetal adrenal gland 11BHSD-2 immunoreactivity (11ßHSD-2-ir) was detected in the foetal zone, whereas in normal adult adrenal glands 11ßHSD-2-ir was not detected by immunocytochemistry. In adrenal cortical carcinoma and adenoma, 11ßHSD-2-ir was detectable in specific regions, which have been identified as steroid synthesizing cells using 3ßHSD-ir as a marker. In adrenal cortical carcinoma and adenoma, 11HSD-2 mRNA and 11ßHSD-2 protein were detected by nuclease protection analysis and by western blot analysis, respectively. In summary, 11ßHSD-2-ir was detected in the foetal zone of the mid-gestation human foetal adrenal, whereas, 11ßHSD-2-ir was not detectable in the postnatal or normal adult adrenal gland. 11ßHSD-2 protein and mRNA was induced in adult human adrenal cortical carcinoma and adenoma. The induction of expression of 11ßHSD-2 in the adrenal cortex suggests a possible role in regulating abnormal adrenal steroidogenic function in these patients. @ 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cortisol; Steroids; Metabolism; Synthesis
1. Introduction
Two isoforms of 11 beta hydroxysteroid dehydroge- nase (11ßHSD; type 1 and type 2) have been described which catalyze the conversion of cortisol to its inactive metabolite, cortisone. 11ßHSD-2 is expressed in miner- alocorticoid-sensitive tissues, where it is thought to confer aldosterone specificity on the mineralocorticoid receptor (White et al., 1997). In the human and mouse, 11ßHSD-2 is expressed widely in the foetus and the
placenta, where it likely protects the foetus against the detrimental effects of early exposure to maternal gluco- corticoids (Stewart et al., 1994; Brown et al., 1996). In the sheep, whilst expression of 11}HSD-1 is absent, the expression of 11ßHSD-2 is high in both the foetal and adult adrenal cortex (Yang, 1995). In the rat, it has been demonstrated that both the 116HSD-1 and 11ßHSD-2 isoforms are expressed in the adrenal gland (Li et al., 1996; Shimojo et al., 1996a,b). Recent studies have provided evidence that in the rat 11ßHSD plays a role in the regulation of basal and stimulated steroid secretion from the adrenal gland (Musajo et al., 1996) and that ACTH induces adrenal 11ßHSD expression (Lecybyl et al., 1998). In the human and mouse foetus, previous studies have demonstrated abundant expres- sion of 116HSD-2 mRNA and 11ßHSD activity in the
# Presented in part at the 8th Adrenal Cortex Conference, Orford, Quebec, Canada, June, 1998.
* Corresponding author. Present address: Department of Physiol- ogy, University of Adelaide GPO Box 498, Adelaide, South Australia 5005, Australia; tel .: + 61-3-8303-5342; fax: + 61-8-8303-3356.
E-mail address: ccoulter@physiol.adelaide.edu.au (C.L. Coulter)
adrenal cortex during development; whereas, near term 11ßHSD-2 expression and activity decrease to unde- tectable levels (Murphy, 1981; Stewart et al., 1994; Brown et al., 1996). Whilst a recent study demonstrated that 11ßHSD-1 immunoreactivity is present in adult human adrenal cortex (Ricketts et al., 1998), previous studies have failed to demonstrate the presence of 11ßHSD activity (Murphy, 1981; Shimojo et al., 1996c), 11HSD-1 mRNA, 116HSD-2 mRNA or 11@HSD-2 protein (Shimojo et al., 1996a,c, 1997). Only after am- plification of the cDNA for the 11(HSD-2 gene by using the technique of RT-PCR, has the expression of 11ßHSD-2 mRNA in the human adult adrenal been detected (Mazzocchi et al., 1998). It is clear that there are marked species differences in the expression of the 11ßHSD isoforms in the adrenal and the role of these enzymes in the adrenal gland remain to be determined. In the present study, we have determined the develop- mental pattern of 11ßHSD-2 protein localization. In addition, we have measured 11ßHSD-2 mRNA and protein and determined the localization of 11ßHSD-2 protein in human adrenal cortical carcinoma and adenoma.
2. Materials and methods
2.1. Tissue collection
Human foetal adrenal glands between 10 and 24 weeks gestation (n = 10), postnatal adrenal glands (n = 2), normal adult adrenal glands (n=4) and adrenal cortical carcinoma (n= 16) were retrieved from the surgical pathology files at Tokohu University Hospital, Sendai, Japan and the University of California, San Francisco, CA, USA. Human liver and kidney biopsy samples were collected and frozen in liquid nitrogen for use as negative and positive controls for the analysis of 11ßHSD-2.
Specimens of adult human aldosterone-producing adrenal cortical carcinoma (n = 3) and adrenal ade- noma (n = 6) were retrieved at surgery at the University Department of Medicine, Greenslopes Hospital, Bris- bane, Australia. Where sufficient tissue was available, from each specimen, a portion was fixed in 10% forma- lin and embedded in paraffin and a portion was frozen in liquid nitrogen for analysis of 11ßHSD-2 protein by western immunoblot and 11ßHSD-2 mRNA by nucle- ase protection analysis. At the Hypertension Unit, Uni- versity of Queensland, all patients with primary aldosteronism presented with hypertension and showed elevated aldosterone/renin ratios and failure of plasma aldosterone to be suppressed with fludrocortisone test- ing. All patients who had surgery underwent adrenal venous sampling with lateralization of aldosterone
overproduction to the side of the affected adrenal and contralateral suppression. One patient with an aldos- terone-secreting adrenal carcinoma also showed ele- vated plasma cortisol concentrations. Another patient with an aldosterone-secreting adrenal carcinoma had both elevated plasma cortisol and dehydroepiandros- terone-sulfate concentrations.
2.2. Immunocytochemistry
To localize 11ßHSD-2 protein in human adrenal gland, we used a modification of the peroxidase anti- peroxidase method as described previously (Coulter et al., 1996). Sections (5 um) were cut from fixed adrenals embedded in paraffin and mounted. Immediately prior to immunocytochemical staining, the sections were de- paraffinized in xylene and hydrated through graded alcohols to Tris-buffered saline (TBS; 0.1 M, pH 7.4). To reduce endogenous peroxidase activity as well as permeablize the cell membranes and allow access of the antibody, the sections were pre-treated with TBS con- taining H2O2 (3%), saponin (0.1%) and lysine (0.02 M) for 20 min at RT and then washed in three changes of TBS. The sections were then pre-treated with TBS containing normal goat serum (3%, NGS) and bovine serum albumin (1%, BSA) for 15 min. The antiserum was diluted in 3% NGS/1% BSA in TBS. The sections were incubated with polyclonal antibodies raised against human type-2 11ßHSD (10 µg/ml) overnight at 4℃ in a humidified chamber. The polyclonal antibody to 11ßHSD-2 was raised in rabbits against a synthetic peptide corresponding to the last 16 amino acid residues of the human 11ßHSD-2 and has been charac- terized previously (Krozowski et al., 1995; Smith et al., 1996). To determine non-specific staining, non-immune rabbit serum and buffer controls were included. The next day, sections were washed in TBS, incubated with 3% NGS/1% BSA in TBS for 15 min and then incu- bated with goat-anti-rabbit immunoglobulin (GAR; 1: 100) for 30 min at RT. Sections were then washed in TBS and incubated with rabbit peroxidase anti-peroxi- dase (PAP; 1:100) for 30 min. To increase the intensity of the immunocytochemical signal, the GAR (1:100) and PAP (1:100) steps were repeated before the sections were washed with TBS and Tris-buffer alone. The sections were then treated with metal enhanced 3,5-di- aminobenzide tetrahydrochloride (Pierce, Rockford, IL, USA) and the appearance of the light brown product was observed under a light microscope. The same reac- tion time with DAB was used for sections of normal adrenal, adrenal carcinoma and adrenal adenoma. The sections were counterstained with Mayer’s Hematoxylin (10 s), rinsed and then dehydrated in graded alcohols, cleared in xylene and mounted.
2.3. Western blot
Sample preparation and western blot analysis was performed essentially as described previously (Smith et al., 1997). Total tissue homogenates were prepared from frozen human adrenal cortical carcinoma (n = 3) and adenoma specimens (n = 6). Homogenates of modified Chinese hamster ovary cells (Heffernan and Dennis, 1991) transfected with an expression plasmid containing recombinant human 11ßHSD-2 and ho- mogenates of adult human kidney were also prepared to be used as positive controls. Tissues were homoge- nized in five volumes of phosphate-buffered saline (PBS; 0.01 M, pH 7.4) containing sucrose (0.25 M), KCI (140 mM) and phenylmethylsulfonyl fluoride (1 mM) using a Ika-Ultraturrex T25 homogenizer (Janke and Kunkle, Stauten, Germany).
The tissue homogenate was centrifuged at 1500 x g for 1 min at 4℃ and the supernatant was then cen- trifuged at 100,000 x g for 1 h at 4℃ to isolate the microsomal pellets. Protein concentration of the micro- somal pellets was detemmined using the Bradford method using Bio-Rad protein dye (Bio-Rad Laborato- ries, Richmond, CA) and calibration against gamma- globulin (0-20 µg) as the protein standard.
Adrenal microsome pellets were dissolved by boiling in 10% sodium dodecyl sulfate (SDS) and 75 µg protein per lane was loaded and subjected to 5-15% gradient SDS-polyacrylamide gel electrophoresis under reduc- ing conditions. Proteins were transferred to a nitrocellu- lose membrane (0.2 um, Schleicher & Schuell Inc., Darmstadt, Germany) at 4℃ for 2 h using a Transfor system (Hoeffer Scientific Instruments, San Francisco, CA). To reduce non-specific binding, the membrane was incubated for 2 h at RT in PBS (0.1 M, pH 7.4) containing skim-milk powder (0.5%) and Tween-20 (0.1%). Then incubated overnight at 4℃ with a human 11ßHSD-2 antibody (1 µg/ml) diluted in PBS (0.1 M, pH 7.4) containing skim-milk powder (0.5%) and Tween 20 (0.1%). The membrane was then incubated at RT for 60 min with a 1:5000 dilution of goat anti-rab- bit IgG antibody conjugated to horseradish peroxidase. The membrane was then washed in PBS containing Tween 20 (0.1%) for 60 min before detection using a chemiluminescent kit (DuPont New England Nuclear, Boston, MA, USA) according to the manufacturers instructions.
2.4. Nuclease protection analysis
The plasmid construct used for the generation of an antisense riboprobe consisted of 500 bp of PtsI frag- ment of human 11HSD-2 ligated into the PstI site of pGEM-3Z and linearization with HindIII. The human 11ßHSD-2 antisense riboprobe was labelled with 32P- UTP synthesized using T7 polymerase and a Gemini
Riboprobe Synthesis kit (Promega, Madison, WI). A human pTRI-GAPDH template (Albion, Inc., TX) was used to generate an antisense RNA probe to be used as an internal control. The GAPDH antisense riboprobe was labelled with 32P-UTP using T7 poly- merase according to the manufacturers instructions.
Solution hybridization and nuclease protection anal- ysis was performed essentially as described previously (Albiston et al., 1994). Briefly, 5 × 104 cpm of ribo- probe was added to 20 µg of total tissue RNA or transfer RNA (tRNA), denatured for 5 min at 85°℃ and hybridized at 60℃ overnight. Each reaction mix- ture was then digested with 400 units of S1 nuclease for 50 min at 37℃. Reaction products were analyzed on a 4% polyacrylamide, 8 M Urea sequencing gel. Protected fragment sizes were calculated from a sequencing ladder run in parallel.
3. Results
3.1. Localization of 11ßHSD-2-ir
11ßHSD-2-ir was detected in the foetal zone of the human foetal adrenal between 10 and 24 weeks gesta- tion (Fig. 1A-D). No staining for 11HSD-2 was present in the outer definitive zone or in the clusters of adrenal medullary cells which invade the adrenal cortex during this period of development (Fig. 1B and D). In postnatal and normal adult human adrenal glands, 11HSD-2-ir was not detected in either the cortex or medulla (Fig. 1E and F).
In adrenal cortical carcinoma and adenoma, cyto- plasmic staining for 11ßHSD-2 was detectable in spe- cific regions, which we have identified as steroid synthesizing cells using an antibody raised against the steroidogenic synthesizing enzyme 3ß hydroxysteroid dehydrogenase (3ßHSD) as a marker (Fig. 1G-R). The immunostaining for 11ßHSD-2 was more widely dis- tributed in adrenal cortical carcinoma specimens (Fig. 1J-K) when compared to adrenal adenoma specimens (Fig. 1P-R). However, we did not observe any correla- tion between the intensity or distribution of 11ßHSD-2- ir in the adrenal cortical carcinoma and adenoma specimens compared to the patient’s plasma concentra- tions of adrenal steroid hormones i.e. aldosterone, cor- tisol and dehydroepiandrosterone-sulphate.
3.2. Western blot
By Western blot analysis, 11ßHSD-2 was detected at the expected size of 41 kDa in all three adrenal cortical carcinoma specimens and five of six adrenal adenoma specimens. The intensity of the 11ßHSD-2-immunore- active 41 kDa band was considerably lower in the adrenal specimens (Fig. 2, lanes 1-9) when compared
11ßHSD-2
FZ
CDZ+4F-
A
10 wks
B
10 wks
C
16 wks
CDZ- 4 FZ -
C
ZG
D
24 wks
E
Postnatal
F
Adult
3ßHSD
G
H
I
11ßHSD-2
J
K
L
3ßHSD
M
N
O
11ßHSD-2
P
Q
R
kDa
50 -
36 -
+ 11 BHSD-2
KC 987654321
to the human kidney (Fig. 2, lane K) or the modified Chinese hamster ovary cell line transfected with 11ßHSD-2 (Fig. 2, lane C).
An additional 11ßHSD-2-ir band at approximately 38 kDa was also detected in the human kidney, in one of three adrenal cortical carcinoma samples and five of six adrenal adenoma samples. The 11ßHSD-2-ir band at 38 kDa was not detected in the modified Chinese hamster ovary cell line transfected with 11ßHSD-2 and therefore may reflect proteolytic breakdown of 11ßHSD-2 in the human tissue samples.
3.3. Nuclease protection analysis
In human kidney, adrenal cortical carcinoma and adenoma, 11ßHSD-2 mRNA was detectable by nucle- ase protection analysis. The level of expression of 11ßHSD-2 mRNA was considerably lower in the speci- mens of adrenal cortical carcinoma and adenoma than in the human kidney. For one patient with an adrenal adenoma, 11ßHSD-2 mRNA was not detectable by nuclease protection analysis (Fig. 3, lane 6) nor was the 11ßHSD-2-ir 41 kDa protein band detectable by west- ern blot (Fig. 2, lane 7). The lack of 116HSD-2 mRNA may reflect reduced RNA loading as shown by the level of the control GAP mRNA. In the human liver, 11ßHSD-2 mRNA was not detectable by nuclease pro- tection analysis consistent with our previous data (Al- biston et al., 1994).
4. Discussion
In the human foetus and the placenta, high levels of 11ßHSD-2 mRNA and 11ßHSD-2 activity are present, where it has been suggested they protect the foetus against the detrimental effects of early exposure to maternal glucocorticoids (Stewart et al., 1994). Near term, 11ßHSD-2 activity is greatly restricted, primarily to the kidney and the placenta (Murphy, 1981). In the present study, we found that 11ßHSD-2-ir was localized to the foetal zone of the human foetal adrenal. Our data are consistent with previous studies which have demonstrated high expression of 11ßHSD-2 mRNA and 11ßHSD-2 activity in the human foetal adrenal at mid-gestation (Murphy, 1981; Stewart et al., 1994). In contrast to the foetus, we found that 11ßHSD-2-ir was not detected in the postnatal or adult human adrenal. Our data parallel previous findings which demonstrated a lack of 11ßHSD-2 activity and 11ßHSD-2-ir in the postnatal human adrenal gland (Murphy, 1981; Shi- mojo et al., 1996c, 1997). It is interesting that in the mouse, there is a developmental regulation of adrenal 11HSD-2 expression similar to that which has been observed in the human. Brown and coworkers (Brown et al., 1996) found a similar developmental pattern of adrenal 11ßHSD-2 expression in the mouse using in situ hybridization, where 11ßHSD-2 mRNA was ex- pressed up to embryonic day 14.5, then was unde- tectable for the remainder of gestation. In species such
Fig. 1. (A-F) are photomicrographs of sections of human adrenal glands at 10 weeks gestation (A and B), 16 weeks gestation (C), 24 weeks gestation (D), 8 weeks postnatally (E), and adult (F) immunostained with an antibody to 11ßHSD-2, where positive staining is dark brown. All sections have been counterstained with Mayer’s hematoxylin to stain the cell nuclei blue. In (A-C) arrow heads identify examples of cytoplasmic 11HSD-2-ir. In (B and D) long arrows identify the whorls of adrenomedullary cells which do not stain positively for 11BHSD-2-ir. (G-R) Photomicrographs of sections of human adrenal cortical carcinoma (G-L) and adenoma (M-R). For each of the adrenal carcinoma and adenoma specimens, there are two serial sections where the upper panel has been immunostained with an antibody to 3BHSD and the panel immediately below stained with an antibody to 11BHSD-2. The serial sections are (G and J), (H and K), (I and L), (M and P), (N and Q) and (O and R), and arrow heads identify an example of a cell which stained positively for both 3BHSD and 11(HSD-2 in the upper compared to the lower panel, respectively. G, H, J and K are from the same patient as lane # 1 Figs. 2 and 3. (I and L), are from the same patient as lane # 3 in Figs. 2 and 3. (M and P) are from the same patient as lane # 4 in Fig. 2. (N and Q) are from the same patient as lane # 6 in Fig. 2 and lane # 5 in Fig. 3. (O and R) are from the same patient as lane # 7 in Fig. 2 and lane #6 in Fig. 3. The scale bar represents 100 um. C, adrenal capsule; DZ, definitive zone; FZ, foetal zone; ZG, zona glomerulosa.
Liver
Kidney
1
2
3
4
5
6
11B-HSD2 mRNA
GAP mRNA
as the sheep, there does not appear to be a developmen- tal pattern of 11ßHSD-2 expression, as 11HSD-2 mRNA is detected in the foetal adrenal cortex through- out gestation and in the adult adrenal cortex (Yang, 1995). The factors which may be responsible for the inhibition of 11ßHSD-2 in the postnatal human adrenal gland remain to be elucidated.
In the human foetal adrenal gland, 11ßHSD-2-ir is expressed in the foetal zone, in cells which we have shown do not express 3HSD-ir or 3ßHSD mRNA (Mesiano et al., 1993). The foetal zone of the human foetal adrenal gland is responsible for the secretion of large amounts of the androgen dehydroepiandros- terone-sulphate and there is little evidence that the foetal adrenal gland can synthesize cortisol until after 24 weeks gestation (Mesiano et al., 1993). These data would suggest that 11HSD-2 expression in the human foetal adrenal gland plays a role in modulating the effects of maternal adrenal glucocorticoids.
In the present study, in the normal adult human adrenal gland we did not observe 11ßHSD-2-ir, whereas in human adrenal cortical carcinoma and ade- noma we found that 11HSD-2 mRNA, protein and immunoreactivity were present. In adrenal cortical car- cinoma and adenoma, 11ßHSD-2 staining was cyto- plasmic and in specific regions which have been identified as steroid synthesizing cells using 3ßHSD-ir as a marker. The presence of 3HSD-ir would suggest that these 11(HSD-2-ir adrenal cells are capable of steroidogenesis, although a full analysis of all the en- zymes in the adrenal steroidogenic pathway has not yet been determined. It is interesting that in the human foetal adrenal gland the induction of expression of 3ßHSD parallels the down-regulation of 11BHSD-2 activity. These data suggest that in human adrenal carcinoma and adenoma, the induction of expression or removal of inhibition of expression of 11ßHSD-2 does not involve the same factors which are responsible for
the down-regulation of 116HSD-2 in the human foetal adrenal. It may be that the expression of 11ßHSD-2 in adrenocortical tumours is caused by the lack of the mechanisms which normally regulate adrenal steroid synthesis and secretion. Recent studies in the rat adrenocortical cells in vitro, have shown that ACTH can stimulate the expression of 11ßHSD mRNA (Lecy- byl et al., 1998). However, the factors which induce 11ßHSD-2 in human adrenal cortical carcinoma and adenoma remain to be determined.
In the human kidney and in some of the specimens of adrenal cortical carcinoma and adenoma, we observed the presence of an additional 11ßHSD-2-ir band at approximately 38 kDa by Western blot analysis. Previ- ously, we have shown that an N-terminal deletion of 11ßHSD-2 yields a product of 11ßHSD-2-ir at approxi- mately 38 kDa (Obeyesekere et al., 1997). This 38 kDa form of 11ßHSD-2 has reduced enzymatic activity in tissue homogenates (Obeyesekere et al., 1997). Taken together, these data suggest that there is a smaller molecular form of the 11HSD-2 in vivo, which is less stable. Whether the presence of a 38 kDa form of 11ßHSD-2 reflects a normal mechanism for the inacti- vation of 11ßHSD-2, requires further investigation.
In conclusion, the developmental pattern of localiza- tion of 11ßHSD-2-ir in the human adrenal gland paral- lels the developmental expression of 11ßHSD-2 mRNA (Stewart et al., 1994; Mazzocchi et al., 1998) and 11ßHSD-2 activity (Murphy, 1981). The induction of expression of 11ßHSD-2 in the adrenal cortical car- cinomas and adenomas may suggest a possible role in regulating abnormal adrenal steroidogenic function in these patients. Alternatively, it may suggest that the de-regulation of 11ßHSD-2 expression is caused by the underlying pathological state of the adrenal gland.
Acknowledgements
This work was supported by the National health and Medical Research Council of Australia by a C.J. Mar- tin Fellowship to Dr Catherine L. Coulter and a Block grant to the Baker Medical Research Institute and by the National Heart Foundation of Australia by a Clin- ical Research Fellowship to Dr Michael Stowasser. The authors acknowledge the assistance of Dr David Cohn (Queensland Medical Laboratory) for preparation of pathological adrenal tissue specimens and Dr Robert Jaffe (University of California, San Francisco) for assis- tance in obtaining the human foetal and postnatal adrenal specimens. We thank Drs Kevin Li and Rod Dilley (Baker Medical Research Institute) for assistance with the western blot analysis and photomicrography, respectively.
References
Albiston, A.L., Obeyesekere, V.R., Smith, R.E., Krozowski, Z.S., 1994. Cloning and tissue distribution of the human 11 beta-hy- droxysteroid dehydrogenase type 2 enzyme. Mol. Cell Endocrinol. 105, R11-R17.
Brown, R.W., Diaz, R., Robson, A.C., Kotelevtsev, Y.V., Mullins, J.J., Kaufman, M.H., Seckl, J.R., 1996. The ontogeny of 11 beta-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor gene expression reveal intricate control of glucocorticoid action in development. Endocrinology 137, 794-797.
Coulter, C.L., Read, L.C., Carr, B.R., Tarantal, A.F., Barry, S., Styne, D.M., 1996. A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo. J. Clin. Endocrinol. Metab. 81, 1254-1260.
Heffernan, M., Dennis, J.W., 1991. Polyoma and hamster papo- vavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. Nucleic Acids Res. 19, 85-92.
Krozowski, Z., Maguire, J.A., Stein-Oakley, A.N., Dowling, J., Smith, R.E., Andrews, R.K., 1995. Immunohistochemical local- ization of the 11 beta-hydroxysteroid dehydrogenase type 11 enzyme in human kidney and placenta. J. Clin. Endocrinol. Metab. 80, 2203-2209.
Lecybyl, R., Jagodzinski, P., Krozowski, Z., Trzeciak, W., 1998. Proceedings of the 8th Adrenal Cortex Conference Orford, Que- bec, Canada.
Li, K.X.Z., Smith, R.E., Ferrari, P., Funder, J.W., Krozowski, Z.S., 1996. Rat 11 beta-hydroxysteroid dehydrogenase type 2 enzyme is expressed at low levels in the placenta and is modulated by adrenal steroids in the kidney. Mol. Cell Endocrinol. 120, 67-75. Mazzocchi, G., Rossi, G.P., Neri, G., Malendowicz, L.K., Albertin, G., Nussdorfer, G.G., 1998. 11 beta-hydroxysteroid dehydroge- nase expression and activity in the human adrenal cortex. FASEB J. 12, 1533-1539.
Mesiano, S., Coulter, C.L., Jaffe, R.B., 1993. Localization of cy- tochrome P450 cholesterol side chain cleavage, cytochrome P450 17a-hydroxylase /17,20-lyase, and 3ß-hydroxysteroid dehydroge- nase/isomerase steroidogenic enzymes in the human and rhesus monkey fetal adrenal gland: reappraisal of functional zonation. J. Clin. Endocrinol. Metab. 77, 1184-1189.
Murphy, B.E., 1981. Ontogeny of cortisol-cortisone interconversion in human tissues: a role for cortisone in human fetal development.
J. Steroid Biochem. 14, 811-817.
Musajo, F., Neri, G., Tortorella, C., Mazzocchi, G., Nussdorfer, G.G., 1996. Intra-adrenal 11 beta-hydroxysteroid dehydrogenase plays a role in the regulation of corticosteroid secretion: an in vitro study in the rat. Life Sci. 59, 1401-1406.
Obeyesekere, V.R., Li, K.X., Ferrari, P., Krozowski, Z., 1997. Trun- cation of the N- and C-terminal regions of the human 11 beta-hy- droxysteroid dehydrogenase type 2 enzyme and effects on solubility and bidirectional enzyme activity. Mol. Cell Endocrinol. 131, 173-182.
Ricketts, M.L., Verhaeg, J.M., Bujalska, I., Howie, A.J., Rainey, W.E., Stewart, P.M., 1998. Immunohistochemical localization of type 1 11 beta-hydroxysteroid dehydrogenase in human tissues. J. Clin. Endocrinol. Metab. 83, 1325-1335.
Shimojo, M., Condon, J., Whorwood, C.B., Stewart, P.M., 1996a. Proceedings of the 7th Adrenal Cortex Conference Crieff, Scot- land, UK.
Shimojo, M., Whorwood, C.B., Stewart, P.M., 1996b. 11 beta-Hy- droxysteroid dehydrogenase in the rat adrenal. J. Mol. En- docrinol. 17, 121-130.
Shimojo, M., Condon, J., Whorwood, C.B., Stewart, P.M., 1996c. Adrenal 11 beta-hydroxysteroid dehydrogenase. Endocr. Res. 22, 771-780.
Shimojo, M., Ricketts, M.L., Petrelli, M.D., Moradi, P., Johnson, G.D., Bradwell, A.R., Hewison, M., Howie, A.J., Stewart, P.M., 1997. Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization. Endocrinology 138, 1305-1311.
Smith, R.E., Maguire, J.A., SteinOakley, A.N., Sasano, H., Taka- hashi, K.I., Fukushima, K., Krozowski, Z.S., 1996. Localization of 11 beta-hydroxysteroid dehydrogenase type 11 in human ep- ithelial tissues. J. Clin. Endocrinol. Metab. 81, 3244-3248.
Smith, R.E., Li, K.X.Z., Andrews, R.K., Krozowski, Z., 1997. Im- munohistochemical and molecular characterization of the rat 11 beta-hydroxysteroid dehydrogenase type 11 enzyme. Endocrinol- ogy 138, 540-547.
Stewart, P.M., Murry, B.A., Mason, J.I., 1994. Type 2 11ßhydroxysteroid dehydrogenase in human fetal tissues. J. Clin. Endocrol. Metab. 78, 1529-1532.
White, P.C., Mune, T., Agarwal, A.K., 1997. 11BHydroxysteroid dehyrogenase and the syndrome of apparent mineralocorticoid excess. Endocr. Rev. 18, 135-156.
Yang, K., 1995. Ovine 11 beta-hydroxysteroid dehydrogenase: from gene to function. Endocr. Res. 21, 367-377.
.