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Type 2 11β-hydroxysteroid dehydrogenase in foetal and adult life
ft. Steroid Biochem. Molec. Biol. Vol. 55, No. 5/6, pp. 465-471, 1995
Pergamon
0960-0760(95)00195-6
Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00
Type 2 l l f l - H y d r o x y s t e r o i d D e h y d r o g e n a s e in Foetal and Adult Life P a u l M S t e w a r t , 1. C h r i s t o p h e r
B . W h o r w o o d 2 a n d J. I a n M a s o n 2
tDepartment of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, U.K. and 2Green Center for Reproductive Bioi'ogy Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, T X 75235-9051, U.S.A. Two i s o f o r m s of l : t p - h y d r o x y s t e r o i d d e h y d r o g e n a s e ( l l p - H S D ) catalyse the i n t e r c o n v e r s i o n of active cortisol to inactive cortisone; l l f l - H S D 1 is a low affinity, N A D P ( H ) - d e p e n d e n t d e h y d r o g e n a s e / o x o - r e d u c t a s e , a n d l l f l - H S D 2 a high affinity, N A D - d e p e n d e n t d e h y d r o g e n a s e . Because o f the i m p o r t a n c e of 11fl-HSD in r e g u l a t i n g c o r t i c o s t e r o i d h o r m o n e action, we have a n a l y s e d the distrib u t i o n of the l l f l - H S D i s o f o r m s in h u m a n a d u l t a n d foetal tissues (including placenta), and, in a d d i t i o n have p e r f o r m e d a series of s u b s t r a t e specificity studies on the novel, k i d n e y l l f l - H S D 2 i s o f o r m . Using an R T - P C R a p p r o a c h , we failed to detect l l ~ - H S D 1 m R N A in a n y h u m a n m i d - g e s t a t i o n a l foetal tissues. In c o n t r a s t 11fl-HSD2 m R N A was p r e s e n t in foetal lung, a d r e n a l , colon a n d kidney. In a d u l t tissues l l f l - H S D 2 gene expression was confined to the m i n e r a l o c o r t i c o i d t a r g e t tissues, k i d n e y a n d colon, whilst ll/~-HSD1 was expressed p r e d o m i n a n t l y in glucocorticoid t a r g e t tissues, liver, lung, p i t u i t a r y a n d c e r e b e l l u m . In h u m a n k i d n e y h o m o g e n a t e s , l l - h y d r o x y l a t e d p r o g e s t e r o n e derivatives, g l y c y r r h e t i n i c acid, c o r t i c o s t e r o n e a n d the " e n d p r o d u c t s " cortisone a n d l l - d e h y d r o c o r t i c o s t e r o n e were p o t e n t i n h i b i t o r s o f the N A D - d e p e n d e n t conversion of cortisol to cortisone. F i n a l l y high levels of l lfl-HSD2 m R N A a n d activity were observed in t e r m placentae, which c o r r e l a t e d positively with foetal weight. The tissue-specific d i s t r i b u t i o n of the ll/$-HSD i s o f o r m s is in keeping with t h e i r differential roles, l l f l - H S D 1 r e g u l a t i n g glucocorticoid h o r m o n e action a n d ll/~-HSD2 m i n e r a l o c o r t i c o i d h o r m o n e action. The c o r r e l a t i o n of l l f l - H S D 2 activity in the p l a c e n t a with foetal weight suggests, in addition, a crucial role for this e n z y m e in foetal d e v e l o p m e n t , possibly in m e d i a t i n g o n t o g e n y of the foetal h y p o t h a l a m o - p i t u i t a r y - a d r e n a l axis.
J. Steroid Biochem. Molec. Biol., Vol. 55, No. 5/6, pp. 465-471, 1995
INTRODUCTION l lfl-Hydroxysteroid dehydrogenase (llfl-HSD) is a member of the short-chain alcohol dehydrogenase family responsible for the interconversion of cortisol (F) to hormonally inactive cortisone (E). The biological role of the enzyme in man has come from clinical observations of the enzyme-deficient state (syndrome of Apparent Mineralocorticoid Excess, liquorice ingestion) [1], from which it is clear that l l f l - H S D plays a crucial role in conferring specificity upon the mineralocorticoid receptor (MR) [2, 3]. In addition, the enzyme is able to regulate the access of cortisol to the glucocorticoid receptor at several tissue sites [4--6]. 1 lfl-HSD was first characterized and cloned from rat liver [7];
Proceedings of the Workshop on the Molecular and Cell Biology of Hydroxysteroid Dehydrogenases, Hannover, Germany, 19--22 April 1995. *Correspondence to P. M. Stewart. 465
understandably therefore many of the initial functional, distribution and regulation studies were carried out in rodents. Recent studies, however, have increasingly focussed on 1lfl-HSD activity in human tissues, where two l lfl-HSD isoforms have been described and cloned. Type 1 l l f l - H S D (llfl-HSD1) was cloned from a human testis cDNA library using the rat liver 1 lfl-HSD cDNA as a probe [8]. This enzyme encodes for a low affinity, NADP/NADPH-dependent dehydrogenase/oxoreductase (apparent K m for F, 2 #M; K m for E, 0.3 ~tM) [9, 10]. Direct evidence for the existence of a second l l f l - H S D isoform (llfl-HSD2) has come from the analysis of 1 lfl-HSD activity in rabbit [11, 12] and rat kidney [13], and from further characterization of 1 lfl-HSD expression in human placenta [14], and in the mineralocorticoid target tissues, human kidney [10] and rectal colon [15]. These studies revealed that l lfl-HSD2, in contrast to l lfl-HSD1, is a high affinity (apparent K m for F, 50 nM), NAD-dependent
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unidirectional dehydrogenase. H u m a n 11fl-HSD2 c D N A was subsequently obtained by expression cloning from human kidney tissue [16]. In this manuscript we present data on the substrate specificity of 1 l f l - H S D 2 and summarize our results on the distribution of human 1 l f l - H S D 1 and 1 l f l - H S D 2 in adult and foetal tissues. In addition, we present data to suggest that placental 1 l f l - H S D 2 plays an important role in foetal development, specifically with respect to the regulation of foetal adrenal steroidogenesis. METHODS
H u m a n foetal tissues and decidua (16-19 weeks gestation) were obtained at elective abortion, in accordance with the Donors Anatomical Gift Act of the State of Texas. Fresh placentae were also obtained from 27 healthy deliveries (gestational age 38.5-41 weeks) and the birth weight noted for each infant. Surface blood clots were removed from the placenta which was then weighed intact. A specimen of each placenta was dissected from foetal membranes and stored at - 70°C for a maximum period of 8 weeks. In each case tissue was obtained from a central part of the placenta; preliminary experiments measuring l l f l - H S D activity at seven separate sites in a single placenta indicated that activity varied by < 8% at various sites within the placenta. Placentae were thawed simultaneously and homogenized in ice-cold 0.154 M KC1 buffer using an Ultraturrax homogenizer (Janke and Kunchel, Germany). Fresh foetal kidneys (n = 4-6) were similarly homogenized. Written consent from the woman about to be delivered or aborted was obtained using a consent form and protocol approved by the H u m a n Research Review Committee of the University of Texas Southwestern Medical Center. Adult human tissues were obtained from operative specimens in accordance with guidelines agreed by the University of Birmingham Pathology Department and local Ethics Committee and were snap frozen at - 70°C for m R N A analysis. 1 l f l - H S D activity studies Placentae and kidney homogenates were centrifuged for 10 min at 1000 g to sediment large tissue fragments and a protein assay performed on the supernatant (Coomassie blue kit, Biorad Laboratories, Richmond, CA, U.S.A.). Homogenates (0.25 mg protein/ml) were incubated in 0.5ml phosphate buffer (0.1M, p H 7.6), containing 50,000cpm [3H]cortisol (sp. act. 3 . 2 T B q / m m o l , Amersham International, Arlington Heights, IL, U.S.A.), 0.1 M F and 400 # M N A D for 30 min at 37°C in a shaking water bath. Based on our earlier studies detailing the co-factor preference and a p p a r e n t K m for the type 1 and 2 l l f l - H S D isoforms [10, 15], assays with 0.1/xM F and N A D were designed to detect type 2 activity, which is the predominant, if not exclusive, isoform in placenta and foetal kidney [10, 14]. Aliquots were extracted into 10 volumes of
dichloromethane and steroids separated by T L C using ethanol: chloroform (8:92) as a mobile phase. T h e T L C plates were analyzed on a Bioscan radioimaging detector, the fractional conversion of F to E calculated and enzyme activity expressed as pmol E (or F) formed/mg, protein for each homogenate/h. T o assess the substrate specificity of l l f l - H S D 2 , a series of possible substrates/competitive inhibitors were incubated at varying concentrations (10-5-10-1°M) with human foetal kidney homogenates together with 0.1 # M F in triplicate as described above and each experiment repeated three times. Stock solutions of all substrates/inhibitors were prepared in absolute ethanol, diluted in phosphate buffer, and added to the homogenates such that the final ethanol concentration in the incubate was always < 0.1 To. Reverse transcriptase-PCR of human tissue R N A
R N A was isolated from foetal and adult human tissues using a commercially available single step extraction method (RNAzol B, Biotecx Laboratories, Houston, T X , U.S.A.). First strand D N A was synthesized from 10 # g total R N A using avian myeloblastoma viral reverse transcriptase (RT) (Promega, Madison, WI, U.S.A.), driven primer extension from 3' antisense oligos against the human l l f l - H S D I (5' A C T T G C T T G C A G A A T A G G - 3 ' ) and h l l f l - H S D 2 cDNA's ( 5 ' - T C A C T G A C T C T G T C T T G A A G C - 3 ' ) [8, 16]. 10% of this reaction served as a template for the PCR amplification of either a 571 bp fragment of h l l f l - H S D 1 using the 3' oligo with the 5' sense oligo (5'-CTCGAGTCGGATGGCTTTTTATG-3') or a 477 bp fragment of h l l f l - H S D 2 using the 3' oligo with the 5' oligo ( 5 ' - A C C G T A T T G G A G T T G A A C A G C 3'), and involved 35 cycles of denaturation (94°C for 1 rain), annealing (47°C for 1 min) and extension (72°C for 2 min). Negative control experiments included the omission of the R T and template RNA. As a positive control, 8 ng of either the 1 l f l - H S D 1 / p c D N A I vector or 8 ng of the h l l f l - H S D 2 / p G E M 4 Z vector were used Table 1. Substrate specificity o f human kidney type 2
Ilfl-HSD. The ICsoof a variety of competing steroids is shown
IC5o Potent inhibitor /substrate 1l f l - O H - p r o g e s t e r o n e G l yc yrrhe t i ni c acid 1l ~ - O H - p r o g e s t e r o n e Corticosterone (B) Carbenoxolone 11-dehydrocorticosterone (A)
1.0 x 10
1.6 1.8 1.8 6.3 7.8
x × x x x
8M
10 8 M 10 SM 10 -s M 10-SM 10-SM
Moderate inhibitor /substrate Deoxycorticosterone Cortisone (E)
2.1 × 1 0 - 7 M 2.4 x 1 0 - 7 M
Weak inhibitor /subs tr ate Progesterone Dexamethasone 11 fl - O H - a n d r o s t e n e d i o n e
5.7 x I 0 - T M 8.7 × 1 0 - T M 8.3 x 10 6 M
Type 2 11B-HSD in Foetal and Adult Life as templates in separate PCR reactions. To confirm the presence of RNA of adequate integrity for RT, parallel R T - P C R experiments were performed to amplify a 652bp fragment of the ubiquitously expressed g,, subunit of human Na-K-ATPase [17] using the 3' antisense oligo ( 5 ' - G G C A A T T C T T C C C A T CACAGT-Y) and the 5' sense oligo (5'-ATATGGAACAGACTTGAGCCG-3').
467
HSD2 and presumably act as substrates. The liquorice derivatives, glycyrrhetinic acid and carbenoxolone were also potent inhibitors of activity. Corticosterone (B) is also a substrate for l l3-HSD2, but, interestingly, this enzyme appears to be inhibited by its own product, i.e. by E and ll-dehydrocorticosterone (A). Finally DOC, progesterone, dexamethasone and 113hydroxyandrostenedione inhibit l l3-HSD2 activity with ICs0s of approx. 10 -6 M.
]RESULTS
Analysis of II~-HSD1 and l l 3 - H S D 2 expression in foetal and adult tissues
Inhibition of renal 1lfl-HSD2 activity Table 1 shows the IC50 for a variety of steroids on renal l l/%HSD2 activity. As shown, 11~- and l l~/hydroxyprogesterone were potent inhibitors of 11/%
Figure l(a and b) details the distribution of l l3HSD2 gene expression in human foetal and adult Foetal
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5!~,i Fig. 1. P a r a l l e l R ' r - P C R a n a l y s e s o f m R N A e x p r e s s i o n i n h u m a n f o e t a l t i s s u e s (a) a n d h u m a n a d u l t t i s s u e s (b). U s i n g s p e c i f i c oligos a g a i n s t 11/~-HSD1, 11/~-HSD2 a n d t h e ~tl s u b u n i t o f N a K - A T P a s e , P C R a m p l i f i c a t i o n o f D N A f r a g m e n t s of sizes 571, 477 a n d 652 bp, r e s p e c t i v e l y , w a s p e r f o r m e d . T h e d e t e c t i o n of t h e u b i q u i t o u s l y e x p r e s s e d =1 m R N A s e r v e d as a c o n t r o l f o r t h e p r e s e n c e o f r e v e r s e - t r a n s c r i b a b l e RNA.
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tissues. While previous studies have failed to demonstrate 1 l f l - H S D 1 m R N A or activity in mid-gestational foetal tissues [18], l l[3-HSD2 activity appeared to be present in many foetal tissues and the R T - P C R studies would support this notion, l l[3-HSD2 m R N A was detected in foetal adrenal, lung, colon and kidney, but was absent in foetal heart and liver. In adult tissues, 1 l f l - H S D 2 gene expression was confined exclusively to the placenta and to the classical mineralocorticoid target tissues, kidney cortex and medulla, colon and salivary gland. In contrast, 1 I[3-HSD1 was localized to the predominantly glucocorticoid target tissues, lung, liver, spleen, pituitary and cerebellum. Some 1113HSD1 m R N A was present in kidney medulla and placenta, but parallel enzyme activity and N o r t h e r n blot studies (data not shown) revealed that this isoform was present in low amounts. No ll[3-HSD1 m R N A was detected in kidney cortex, colon or salivary gland. Placental 1 l [ 3 - H S D 2 activity and foetal weight
Details of the term birth weights and placental weights from the 27 term placentas are given in Table 2. As shown there was a wide broad distribution of term foetal weight (2580-4990g) and placental weight (434-1120 g). Indeed 7 foetuses were above the 90th percentile for " n o r m a l " gestational-matched controis, and 4 were below the 10th percentile. T h e capacity of the placenta to metabolize 11-hydroxylated C21 steroids varied between 1.73-7.95 #mol F/min. T h e r e was a significant positive correlation between foetal weight and l l[3-HSD activity (r =0.408, P = 0.034), but no significant correlation between placental weight and l l[3-HSD activity (r =0.043, P >0.05). (Parallel studies indicated no correlation between placental 3[3-hydroxysteroid dehydrogenase (3[3-HSD) activity and foetal or placental weight.) When placental l l [ 3 - H S D 2 activity was expressed in terms of total activity/placenta, the correlation between foetal weight and l l[3-HSD activity became statistically stronger (r = 0.784, P < 0.001), but this could be explained on the basis of a positive correlation between foetal and placental weight (r = 0.672, P < 0.001).
DISCUSSION 1113-HSD2 has been cloned and characterized from human kidney [10, 16]. T h e enzyme shares little sequence homology with 1 I[3-HSD1, and is a kinetically distinct isoform, l l[3-HSD1 possesses relatively low affinity, N A D P - d e p e n d e n t dehydrogenase activity. Furthermore, E has a higher affinity for 1113HSD1 than F, suggesting that in vivo this enzyme may principally act as a reductase. In contrast, 1113H S D 2 is a high affinity, N A D - d e p e n d e n t unidirectional dehydrogenase. As documented here, l l[3-HSD2 is inhibited by liquorice derivatives, and appears also to be inhibited by end-product (i.e. E and A). Furthermore, dexamethasone and hydroxylderivatives of progesterone are also substrates for l l[3-HSD2. This may be of relevance in some tissues not evaluated in this study, such as the ovary, where high affinity l l[3-HSD activity has been reported. T h e ontogeny of human ll[3-HSD1 and 2 is likely to be of interest. In adult tissues, l l[3-HSD2 expression is confined to the classical mineralocorticoid target tissues, kidney, colon and salivary gland. In contrast l l[3-HSD1 was expressed in predominantly glucocorticoid target tissues, liver, lung, pituitary and cerebellum. 11[3-HSD1 m R N A was detected in adult kidney medulla using the sensitive technique of R T - P C R , but parallel activity studies have indicated that the contribution of this isoform to total 1 I[3-HSD activity within the renal medulla is very low. Furthermore, we have consistently failed to demonstrate 1 I[3HSD1 m R N A in kidney cortex, rectal colon, and salivary gland. Our earlier studies had failed to demonstrate 1 I[3-HSD1 m R N A or activity in mid-gestational foetal tissues [18], and only low levels of ll[3-HSD1 activity (if any) in term placenta. T h e R T - P C R data presented here are consistent with the expression of l l [ 3 - H S D 2 in human foetal lung, adrenal, kidney, colon and placenta. F r o m the examination of 1 I[3-HSD gene expression in these two distinct developmental periods, the ontogeny of 11[3-HSD1 would seem to be a late gestational or post-natal event. In contrast, l l[3-HSD2 may "switch off" in some tissues
Table 2. Term birth weight, placental weight and placental 1113-HSD activity in 2 7 placentas. Results are expressed as mean +_SE and ranges. 1113-HSD rate was expressed as pmol E formed/h/mg.protein, and for "total" 1113-HSD activity, the 113-HSD rate was multiplied by the placental weight
Parameter
Mean + SE
Placental weight (g) Term foetal weight (g) 11/~-HSD rate (pmol E formed/h/ mg.homogenate protein) Total ll/3-HSD (/~mol/min/placenta)
585 + 29 3441 +__105 312 _ 14
434-1120 2580--4990 194--448
Range
3.04 + 0 . 2 3
1.73-7.95
Type 2 llfl-HSD in Foetal and Adult Life postnatally (e.g. lung) to be expressed in the adult only in MR-target tissues. These tissue localization studies have enabled us to clarify the role of the 1 l f l - H SD isoforms. 1 lfl-HSD2, in adult life is predominantly found in the classical mineralocorticoid-target tissues; its cellular localization within kidney and colon to collecting ducts [19] and surface epithelial cells [15] respectively, indicates that it maintains aldosterone-selectivity upon the MR in an autocrine fashion. In contrast, l l f l - H S D 1 is found principally in glucocorticoid target tissues, where its role may be to generate active F from E or to inactivate particularly high circulating F levels occurring for example at times of stress. Thus, factors which regulate the tissue-specific expression and regulation of these distinct isoforms will be of major importance in the analysis of corticosteroid hormone action. It is now clear from this and other studies that l lfl-HSD2 plays a key role in foetal development. 1 l f l- HS D activity has been analysed in the placenta and foetal membranes since the 1960's [20-24], but it is only very recently that this activity (with the exception of maternally-derived decidua) has been found to reflect the expression of the l lfl-HSD2 isoform [14,24]. It has been suggested that l l f l - H S D may serve to protect the developing foetus from the deleterious effects of maternal cortisol excess. T he unusual combination of a low birth weight and large placenta predicting adult hypertension in man [25], and a direct correlation between placental l l f l - H SD activity and
I O .ER I Estriol
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foetal weight and inverse correlation with placental weight in the rodent [26], suggests that defective placental 1 l fl -H SD activity may play a role in the subsequent development of hypertension in the offspring [27]. Our data suggest that this may not be the case. Firstly, l lfl-HSD2 activity and gene expression is present in many foetal tissues in addition to the placenta, suggesting that they can protect themselves from any cortisol excess in an autocrine or paracrine fashion [18]. Secondly, we have failed to identify any relationship between human placental l lfl-HSD activity and placental weight in man. Finally, if foetal glucocorticoid excess, mediated through defective placental 1lflH SD activity, was to be a factor in the development of hypertension, then one would expect evidence of suppression of the hypothalamo-pituitary-adrenal (HPA) axis in the low birth weight offspring, yet animal and human studies have, if anything, shown the opposite, ie. activation of the new-born HPA axis [28, 29]. The capacity for the placenta to metabolize cortisol is immense, with an average term placenta capable of converting 3/~mol of cortisol to cortisone per minute. Allowing for a placental whole blood flow of 500-700ml/min from both foetus and mother, we compute that a maximum of 1 pmol cortisol/min is delivered to the placenta. Thus, rather than considering placental l lfl-HSD as protecting the developing foetus from maternally-derived F, we suggest that it co-ordinates a far more crucial role in sustaining foetal adrenal steroidogenesis (Fig. 2). Such a scenario has
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Fig. 2. Steroidogenesis f r o m the foeto-placental unit illustrating the crucial role played by placental ll~$-HSD. The capacity of the placenta to m e t a b o l i s e F to E is i m m e n s e and this increased m e t a b o l i c clearance of cortisol stimulates foetal A C T H secretion and hence adrenal growth and steroidogenesis through the negative feedback m e c h a n i s m . Post.-partum, following r e m o v a l of the placenta, the foetal adrenal involutes, and circulating ACTH, DHEA and cortisone levels fall in the new-born.
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b e e n p r e s e n t e d for th e f o e t o - p l a c e n t a l u n i t o f the b a b o o n by P e p e a n d A l b r e c h t [30]. H e r e , as g e s t a t i o n a dvan ces , o e s t r o g e n i n d u c t i o n o f p l a c e n t a l 11 ~ - H S D activity i n d u c e s a c t i v a t i o n o f th e H P A - a x i s [31], w i t h i n c r e a s e d t r a n s c r i p t i o n o f t h e foetal P O M C gene, en h a n c e d A C T H s e c r e t i o n [32] a n d i n c r e a s e d foetal a d r e n a l s t e r o i d o g e n e s i s . B e c a u s e m a t e r n a l o e s t r o g e n is d e r i v e d p r e d o m i n a n t l y f r o m foetal a d r e n a l D H E A S , this m a y serve as a " f a s t f o r w a r d " r e g u l a t o r y process. D a t a f r o m earlier studies w o u l d s u p p o r t such a c o n c e p t in m a n [33-36]. T h e relative size o f t h e foetal a d r e n a l g l a n d is v e r y large c o m p a r e d to t h e a d u lt g l an d a n d foetal A C T H levels are also e l e v a t e d c o m p a r e d to a dul t values. S ev e r a l studies, p r i n c i p a l l y t h o s e f r o m a n e n c e p h a l i c foetuses, i n d i c a t e that foetal ad r en al g r o w t h an d s t e r o i d o g e n e s i s is u n d e r the c o n t r o l o f A C T H , b u t t h e cause o f the e l e v a t e d levels has n e v e r b e e n established. W h i l s t th e foetal a d r e n a l e x h i b i t s relative a t t e n u a t i o n o f 3 / 3 - H S D activity, a c c o u n t i n g for the large s e c r e t i o n o f D H E A a n d D H E A S , cortisol is s y n t h e s i z e d b y the foetal adrenal, w i t h c i r c u l a t i n g levels rising f r o m a p p r o x . 5 0 n m o l / 1 at 20 weeks ge st at i o n to 150o200 nmol/1 at t e r m . T h e close c o r r e lation b e t w e e n the h i g h foetal c o r t i s o n e levels ( 1 5 0 - 2 0 0 n m o l / 1 ) a n d D H E A levels t h r o u g h o u t gest a t i o n suggests t h a t the t r o p h i c s t i m u l u s to th e foetal H P A axis is actually e n h a n c e d cortisol m e t a b o l i s m r a t h e r t h a n i m p a i r e d s e c r e t i o n [37]. Finally, p o s t p a r turn, after r e m o v a l o f the p l a c e n t a and t h e r e f o r e the m a j o r s o u r c e o f cortisol clearance, th e foetal a d r en al r a p i d l y i n v o l u t e s , a n d c o r t i s o n e a n d A C T H levels fall i m m e d i a t e l y to n o r m a l a d u lt levels (50 nmol/1 a nd < 5 pmol/1, r e s p e c t i v e l y ) [37, 38]. O u r o b s e r v e d c o r r e l a t i o n b e t w e e n p la c e n t a l l l ~ - H S D activity an d b i r t h w e i g h t m a y s i m p l y be a reflection that larger foetuses h a v e larger adrenals. T h e l l / ~ - H S D story has c o m e a l o n g way since Carl M o n d e r first p u r i f i e d w h a t we n o w k n o w to be l l f i H S D 1 f r o m rat l i v e r m i c r o s o m e s . I n m a n , 1 l f i - H S D 1 seems likely to r e g u l a t e t h e access o f cortisol to the G R a nd m a y i n d e e d p r i m a r i l y act as an o x o - r e d u c t a s e . I n c o n t r a s t l l / 3 - H S D 2 confers specficity u p o n th e M R , a nd defects in its activity m a y e x p la in some f o r m s o f h y p e r t e n s i v e such as t h e s y n d r o m e o f A p p a r e n t M i n e r a l o c o r t i c o i d Excess. F i n a l l y t h e h i g h e x p r e s s i o n o f this i s o f o r m in h u m a n p l a c e n t a m a y be a p r i n c i p a l factor d r i v i n g foetal ad r en a l s t e r o i d o g e n e s i s a n d h e n c e o n g o ing gestation.
REFERENCES 1. Stewart P. M.: ll/3-hydroxysteroid dehydrogenase. In Hormones, Enzymes and Receptors, (Edited by M. C. Sheppard and P. M. Stewart) Balliere's Clin. Endocr. Metab., London (1994) Vol. 8, pp. 357-378. 2. Edwards C. R. W.~ Stewart P. M., Burr D., Brett L., McIntyre M. A., Sutanto W. S., DeKloet E. R. and Monder C.: Localisation of 11/3-hydroxysteroid dehydrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet ii (1988) 986-989.
3. Funder J. W., Pearce P. T., Smith R. and Smith A. I.: Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242 (1988) 583-585. 4. Fuller P. J. and Verity K.: Colonic sodium-potassium adenosine triphosphate subunit gene expression: ontogeny and regulation by adrenocortical steroids. Endocrinology 127 (1990) 32-38. 5. Whorwood C. B., Sheppard M. C. and Stewart P. M.: Licorice inhibits 11/3-hydroxysteroid dehydrogenase messenger ribonucleic acid levels and potentiates glucocorticoid hormone action. Endocrinology 132 (1993) 2287-2292. 6. Monder C., Miroff Y., Marandici A. and Hardy M.: 11/3Hydroxysteroid dehydrogenase alleviates glucocorticoidmediated inhibition of steroidogenesis in rat Leydig cells. Endocrinology 134 (1994) 1199-1204. 7. Agarwal A. K., Monder C., Eckstein B. and White P. C.: Cloning and expression of rat cDNA encoding corticosteroid l l/3-dehydrogenase. J. Biol. Chem. 264 (1989) 18,939-18,943. 8. Tannin G. M., Agarwal A. K., Monder C., New M. I. and White P. C.: The human gene for 1l~-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J. Biol. Chem. 266 (1991) 16,653-16,658. 9. Moore C. D. C., Mellon S. H., Murai J., Siiteri P. K. and Miller W. L.: Structure and function of the hepatic form of 11/3hydroxysteroid dehydrogenase in the squirrel monkey, an animal model of glucocorticoid resistance. Endocrinology 133 (1993) 368-375. 10. Stewart P. M., Murry B. A. and Mason J. I.: Human kidney ll/3-hydroxysteroid dehydrogenase is a high affinity NADdependent enzyme and differs from the cloned "type I" isoform. J. Clin. Endocr. Metab. 79 (1994) 480-484. 11. Bonvalet J. P., Doignon I., Blot-Chabaud M., Pradelles P. and Farman N.: Distribution of 11/3-hydroxysteroid dehydrogenase along the rabbit nephron. J. Clin. Invest. 86 (1990) 832-837. 12. Rusvai E. and Fejes-Toth A. N.: A new isoform of 11/3-hydroxysteroid dehydrogenase in aldosterone target cells. J. Biol. Chem. 268 (1993) 10,717-10,720. 13. Mercer W. and Krozowski Z. S.: Localization of an 11/3hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology 130 (1992) 540-543. 14. Brown R. W., Chapman K. E., Edwards C. R. W. and Seckl J. R.: Human placental l l/3-hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform. Endocrinology 132 (1993) 2614-2621. 15. Whorwood C. B., Ricketts M. L. and Stewart P. M. Epithelial cell localization of type 2 11/3-hydroxysteroid dehydrogenase in rat and human colon. Endocrinology 135 (1994) 2533-2541. 16. Albiston A. L., Obeyesekere V. R., Smith R. E. and Krozowski Z. S.: Cloning and tissue distribution of the human 11/3hydroxysteroid dehydrogenase type 2 enzyme. Molec. Cell Endocr. 105 (1994) Rll-R17. 17. Kawakami K., Ohta T., Nojima H. and Nagano K.: Primary structure of the ~-subunit of human Na,K-ATPase deduced from a cDNA sequence. J. Biochem 100 (1986) 389-397. 18. Stewart P. M., Murry B. A. and Mason J. I.: Type 2 11/3hydroxysteroid dehydrogenase in human foetal tissues. J. Clin. Endocr. Metab. 78 (1994) 1529-1532. 19. Whorwood C. B., Mason J. I., Ricketts M. L., Howie A. L. and Stewart P. M.: Detection of human ll/3-hydroxysteroid dehydrogenase isoforms using RT-]PCR and localization of the type 2 isoform to renal collecting ducts. Molec. Cell. Endocr. 110 (1995) R7-R12. 20. Osinski P. A.: Steroid 11/3-ol dehydrogenase in human placenta. Nature 187 (1960) 777. 21. Bernal A. L. and Craft I. L.: Corticosteroid metabolism in vitro by human placenta, foetal membranes and decidua in early and late gestation. Placenta 2 (1981) 279-285. 22. Giannopoulus G., Jackson K. and Tulchinsky D.: Glucocorticoid metabolism in human placenta, decidua, myometrium and foetal membranes. J. Steroid Biochem. 17 (1982) 371-374. 23. Murphy B. E. P.: Chorionic membrane as an extra-adrenal source of foetal cortisol in human amniotic fluid. Nature 266 (1977) 179-181. 24. Stewart P. M., Rogerson F. M. and Mason J. I.: Type 2 11/3-hydroxysteroid dehydrogenase mRNA and activity in human placenta and foetal membranes: Its relationship to birth
T y p e 2 11/%HSD in Foetal and A d u l t Life
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26. 27.
28.
29.
30. 31.
weight and putative role in foetal adrenal steroidogenesis.J. Clin. Endocr. Metab. 80 (199:5) 885-890. Barker D. J. P., Bull A. R., Osmond C. O. and Simmonds S. J.: Foetal and placental size and risk of hypertension in adult life. Br. Med. J. 301 (1990) 259-262. Benediktsson R., Lind~ay R. S., Noble J., Seckl J. R. and Edwards C. R. W.: Glucocorticoid exposure in utero: new model for adult hypertension. Lancet 341 (1993) 339-341. Edwards C. R. W., Benediktsson R., Lindsay R. S. and Seckl J. R.: Dysfunction of placental glucocorticoid barrier: link between foetal environment and adult hypertension? Lancet 341 (1993) 355-357. Klemcke H. G., Lunstra D. D., Brown-Borg H. M., Borg K. E. and Christenson R. K.: Association between low birth weight and increased adrenocortical function in neonatal pigs. J. Anim. Sci. 71 (1993) 1010-1018. Goland R. S., Jozak S., Warren W. B., Conwell I. M., Stark R. I. and Tropper P. J.: Elevated levels of umbilical cord plasma corticotrophin releasing hormone in growth-retarded foetuses. J. Clin. Endocr. Metab. 77 (1993) 1174-1179. Pepe G. J. and Albrecht E. D.: Regulation of the primate foetal adrenal cortex. Endocrine Rev. 11 (1994) 151-176. Baggia S., Albrecht E. D. and Pepe G. J.: Regulation of ll/~-hydroxysteroid dehydrogenase activity in the baboon placenta by estrogen. Endocrinology 126 (1990) 2742-2748.
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32. Pepe G. J., Davies W. A. and Albrecht E. D.: Activation of the baboon foetal pituitary-adrenocortical axis at midgestation by estrogen: enhancement of foetal pituitary proopiomelanocortin mRNA expression. Endocrinology 135 (1994) 2581-2587. 33. Beitins I. Z., Bayard F., Ances I. G., Kowarski A. and Migeon C. J.: The metabolic clearance rate, blood production, interconversion and transplacental passage of cortisol and cortisone in pregnancy near term. Pediat. Res. 7 (1973) 509-519. 34. Cart B. R. and Casey M. L.: Growth of the adrenal gland of the normal human foetus during early gestation. Early Hum Dev. 6 (1982) 121-124. 35. Winter J. S. D.: Foetal and neonatal adrenocortical development. In The Adrenal Gland 2nd Edition (Edited by V. H. T. James). Raven Press Ltd, New York: (1992) pp. 87-104. 36. Winters A. J., Oliver C., Colston C. et al.: Plasma ACTH levels in the human foetus and neonate as related to age and parturition. J. Clin. Endocr. Metab 39 (1974) 269-273. 37. Fisher D. A.: Endocrinology of foetal development. In, Williams Textbook of Endocrinology. 8th edition (Edited by J. D. Wilson and D. W. Foster). WB Saunders Company, PA: (1992) pp. 1049-1077. 38. Bro-Rasmussen F., Buus O. and Trolle D.: Ratio of cortisone/cortisol in mother and infant at birth. Acta Endocr. 40 (1962) 579-583.
Pergamon
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Type 2 l l f l - H y d r o x y s t e r o i d D e h y d r o g e n a s e in Foetal and Adult Life P a u l M S t e w a r t , 1. C h r i s t o p h e r
B . W h o r w o o d 2 a n d J. I a n M a s o n 2
tDepartment of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, U.K. and 2Green Center for Reproductive Bioi'ogy Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, T X 75235-9051, U.S.A. Two i s o f o r m s of l : t p - h y d r o x y s t e r o i d d e h y d r o g e n a s e ( l l p - H S D ) catalyse the i n t e r c o n v e r s i o n of active cortisol to inactive cortisone; l l f l - H S D 1 is a low affinity, N A D P ( H ) - d e p e n d e n t d e h y d r o g e n a s e / o x o - r e d u c t a s e , a n d l l f l - H S D 2 a high affinity, N A D - d e p e n d e n t d e h y d r o g e n a s e . Because o f the i m p o r t a n c e of 11fl-HSD in r e g u l a t i n g c o r t i c o s t e r o i d h o r m o n e action, we have a n a l y s e d the distrib u t i o n of the l l f l - H S D i s o f o r m s in h u m a n a d u l t a n d foetal tissues (including placenta), and, in a d d i t i o n have p e r f o r m e d a series of s u b s t r a t e specificity studies on the novel, k i d n e y l l f l - H S D 2 i s o f o r m . Using an R T - P C R a p p r o a c h , we failed to detect l l ~ - H S D 1 m R N A in a n y h u m a n m i d - g e s t a t i o n a l foetal tissues. In c o n t r a s t 11fl-HSD2 m R N A was p r e s e n t in foetal lung, a d r e n a l , colon a n d kidney. In a d u l t tissues l l f l - H S D 2 gene expression was confined to the m i n e r a l o c o r t i c o i d t a r g e t tissues, k i d n e y a n d colon, whilst ll/~-HSD1 was expressed p r e d o m i n a n t l y in glucocorticoid t a r g e t tissues, liver, lung, p i t u i t a r y a n d c e r e b e l l u m . In h u m a n k i d n e y h o m o g e n a t e s , l l - h y d r o x y l a t e d p r o g e s t e r o n e derivatives, g l y c y r r h e t i n i c acid, c o r t i c o s t e r o n e a n d the " e n d p r o d u c t s " cortisone a n d l l - d e h y d r o c o r t i c o s t e r o n e were p o t e n t i n h i b i t o r s o f the N A D - d e p e n d e n t conversion of cortisol to cortisone. F i n a l l y high levels of l lfl-HSD2 m R N A a n d activity were observed in t e r m placentae, which c o r r e l a t e d positively with foetal weight. The tissue-specific d i s t r i b u t i o n of the ll/$-HSD i s o f o r m s is in keeping with t h e i r differential roles, l l f l - H S D 1 r e g u l a t i n g glucocorticoid h o r m o n e action a n d ll/~-HSD2 m i n e r a l o c o r t i c o i d h o r m o n e action. The c o r r e l a t i o n of l l f l - H S D 2 activity in the p l a c e n t a with foetal weight suggests, in addition, a crucial role for this e n z y m e in foetal d e v e l o p m e n t , possibly in m e d i a t i n g o n t o g e n y of the foetal h y p o t h a l a m o - p i t u i t a r y - a d r e n a l axis.
J. Steroid Biochem. Molec. Biol., Vol. 55, No. 5/6, pp. 465-471, 1995
INTRODUCTION l lfl-Hydroxysteroid dehydrogenase (llfl-HSD) is a member of the short-chain alcohol dehydrogenase family responsible for the interconversion of cortisol (F) to hormonally inactive cortisone (E). The biological role of the enzyme in man has come from clinical observations of the enzyme-deficient state (syndrome of Apparent Mineralocorticoid Excess, liquorice ingestion) [1], from which it is clear that l l f l - H S D plays a crucial role in conferring specificity upon the mineralocorticoid receptor (MR) [2, 3]. In addition, the enzyme is able to regulate the access of cortisol to the glucocorticoid receptor at several tissue sites [4--6]. 1 lfl-HSD was first characterized and cloned from rat liver [7];
Proceedings of the Workshop on the Molecular and Cell Biology of Hydroxysteroid Dehydrogenases, Hannover, Germany, 19--22 April 1995. *Correspondence to P. M. Stewart. 465
understandably therefore many of the initial functional, distribution and regulation studies were carried out in rodents. Recent studies, however, have increasingly focussed on 1lfl-HSD activity in human tissues, where two l lfl-HSD isoforms have been described and cloned. Type 1 l l f l - H S D (llfl-HSD1) was cloned from a human testis cDNA library using the rat liver 1 lfl-HSD cDNA as a probe [8]. This enzyme encodes for a low affinity, NADP/NADPH-dependent dehydrogenase/oxoreductase (apparent K m for F, 2 #M; K m for E, 0.3 ~tM) [9, 10]. Direct evidence for the existence of a second l l f l - H S D isoform (llfl-HSD2) has come from the analysis of 1 lfl-HSD activity in rabbit [11, 12] and rat kidney [13], and from further characterization of 1 lfl-HSD expression in human placenta [14], and in the mineralocorticoid target tissues, human kidney [10] and rectal colon [15]. These studies revealed that l lfl-HSD2, in contrast to l lfl-HSD1, is a high affinity (apparent K m for F, 50 nM), NAD-dependent
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unidirectional dehydrogenase. H u m a n 11fl-HSD2 c D N A was subsequently obtained by expression cloning from human kidney tissue [16]. In this manuscript we present data on the substrate specificity of 1 l f l - H S D 2 and summarize our results on the distribution of human 1 l f l - H S D 1 and 1 l f l - H S D 2 in adult and foetal tissues. In addition, we present data to suggest that placental 1 l f l - H S D 2 plays an important role in foetal development, specifically with respect to the regulation of foetal adrenal steroidogenesis. METHODS
H u m a n foetal tissues and decidua (16-19 weeks gestation) were obtained at elective abortion, in accordance with the Donors Anatomical Gift Act of the State of Texas. Fresh placentae were also obtained from 27 healthy deliveries (gestational age 38.5-41 weeks) and the birth weight noted for each infant. Surface blood clots were removed from the placenta which was then weighed intact. A specimen of each placenta was dissected from foetal membranes and stored at - 70°C for a maximum period of 8 weeks. In each case tissue was obtained from a central part of the placenta; preliminary experiments measuring l l f l - H S D activity at seven separate sites in a single placenta indicated that activity varied by < 8% at various sites within the placenta. Placentae were thawed simultaneously and homogenized in ice-cold 0.154 M KC1 buffer using an Ultraturrax homogenizer (Janke and Kunchel, Germany). Fresh foetal kidneys (n = 4-6) were similarly homogenized. Written consent from the woman about to be delivered or aborted was obtained using a consent form and protocol approved by the H u m a n Research Review Committee of the University of Texas Southwestern Medical Center. Adult human tissues were obtained from operative specimens in accordance with guidelines agreed by the University of Birmingham Pathology Department and local Ethics Committee and were snap frozen at - 70°C for m R N A analysis. 1 l f l - H S D activity studies Placentae and kidney homogenates were centrifuged for 10 min at 1000 g to sediment large tissue fragments and a protein assay performed on the supernatant (Coomassie blue kit, Biorad Laboratories, Richmond, CA, U.S.A.). Homogenates (0.25 mg protein/ml) were incubated in 0.5ml phosphate buffer (0.1M, p H 7.6), containing 50,000cpm [3H]cortisol (sp. act. 3 . 2 T B q / m m o l , Amersham International, Arlington Heights, IL, U.S.A.), 0.1 M F and 400 # M N A D for 30 min at 37°C in a shaking water bath. Based on our earlier studies detailing the co-factor preference and a p p a r e n t K m for the type 1 and 2 l l f l - H S D isoforms [10, 15], assays with 0.1/xM F and N A D were designed to detect type 2 activity, which is the predominant, if not exclusive, isoform in placenta and foetal kidney [10, 14]. Aliquots were extracted into 10 volumes of
dichloromethane and steroids separated by T L C using ethanol: chloroform (8:92) as a mobile phase. T h e T L C plates were analyzed on a Bioscan radioimaging detector, the fractional conversion of F to E calculated and enzyme activity expressed as pmol E (or F) formed/mg, protein for each homogenate/h. T o assess the substrate specificity of l l f l - H S D 2 , a series of possible substrates/competitive inhibitors were incubated at varying concentrations (10-5-10-1°M) with human foetal kidney homogenates together with 0.1 # M F in triplicate as described above and each experiment repeated three times. Stock solutions of all substrates/inhibitors were prepared in absolute ethanol, diluted in phosphate buffer, and added to the homogenates such that the final ethanol concentration in the incubate was always < 0.1 To. Reverse transcriptase-PCR of human tissue R N A
R N A was isolated from foetal and adult human tissues using a commercially available single step extraction method (RNAzol B, Biotecx Laboratories, Houston, T X , U.S.A.). First strand D N A was synthesized from 10 # g total R N A using avian myeloblastoma viral reverse transcriptase (RT) (Promega, Madison, WI, U.S.A.), driven primer extension from 3' antisense oligos against the human l l f l - H S D I (5' A C T T G C T T G C A G A A T A G G - 3 ' ) and h l l f l - H S D 2 cDNA's ( 5 ' - T C A C T G A C T C T G T C T T G A A G C - 3 ' ) [8, 16]. 10% of this reaction served as a template for the PCR amplification of either a 571 bp fragment of h l l f l - H S D 1 using the 3' oligo with the 5' sense oligo (5'-CTCGAGTCGGATGGCTTTTTATG-3') or a 477 bp fragment of h l l f l - H S D 2 using the 3' oligo with the 5' oligo ( 5 ' - A C C G T A T T G G A G T T G A A C A G C 3'), and involved 35 cycles of denaturation (94°C for 1 rain), annealing (47°C for 1 min) and extension (72°C for 2 min). Negative control experiments included the omission of the R T and template RNA. As a positive control, 8 ng of either the 1 l f l - H S D 1 / p c D N A I vector or 8 ng of the h l l f l - H S D 2 / p G E M 4 Z vector were used Table 1. Substrate specificity o f human kidney type 2
Ilfl-HSD. The ICsoof a variety of competing steroids is shown
IC5o Potent inhibitor /substrate 1l f l - O H - p r o g e s t e r o n e G l yc yrrhe t i ni c acid 1l ~ - O H - p r o g e s t e r o n e Corticosterone (B) Carbenoxolone 11-dehydrocorticosterone (A)
1.0 x 10
1.6 1.8 1.8 6.3 7.8
x × x x x
8M
10 8 M 10 SM 10 -s M 10-SM 10-SM
Moderate inhibitor /substrate Deoxycorticosterone Cortisone (E)
2.1 × 1 0 - 7 M 2.4 x 1 0 - 7 M
Weak inhibitor /subs tr ate Progesterone Dexamethasone 11 fl - O H - a n d r o s t e n e d i o n e
5.7 x I 0 - T M 8.7 × 1 0 - T M 8.3 x 10 6 M
Type 2 11B-HSD in Foetal and Adult Life as templates in separate PCR reactions. To confirm the presence of RNA of adequate integrity for RT, parallel R T - P C R experiments were performed to amplify a 652bp fragment of the ubiquitously expressed g,, subunit of human Na-K-ATPase [17] using the 3' antisense oligo ( 5 ' - G G C A A T T C T T C C C A T CACAGT-Y) and the 5' sense oligo (5'-ATATGGAACAGACTTGAGCCG-3').
467
HSD2 and presumably act as substrates. The liquorice derivatives, glycyrrhetinic acid and carbenoxolone were also potent inhibitors of activity. Corticosterone (B) is also a substrate for l l3-HSD2, but, interestingly, this enzyme appears to be inhibited by its own product, i.e. by E and ll-dehydrocorticosterone (A). Finally DOC, progesterone, dexamethasone and 113hydroxyandrostenedione inhibit l l3-HSD2 activity with ICs0s of approx. 10 -6 M.
]RESULTS
Analysis of II~-HSD1 and l l 3 - H S D 2 expression in foetal and adult tissues
Inhibition of renal 1lfl-HSD2 activity Table 1 shows the IC50 for a variety of steroids on renal l l/%HSD2 activity. As shown, 11~- and l l~/hydroxyprogesterone were potent inhibitors of 11/%
Figure l(a and b) details the distribution of l l3HSD2 gene expression in human foetal and adult Foetal
(a)
,
Type2
,
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~
477bp - -
Na+/K + ATPase (x1
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--
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=
>,
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5!~,i Fig. 1. P a r a l l e l R ' r - P C R a n a l y s e s o f m R N A e x p r e s s i o n i n h u m a n f o e t a l t i s s u e s (a) a n d h u m a n a d u l t t i s s u e s (b). U s i n g s p e c i f i c oligos a g a i n s t 11/~-HSD1, 11/~-HSD2 a n d t h e ~tl s u b u n i t o f N a K - A T P a s e , P C R a m p l i f i c a t i o n o f D N A f r a g m e n t s of sizes 571, 477 a n d 652 bp, r e s p e c t i v e l y , w a s p e r f o r m e d . T h e d e t e c t i o n of t h e u b i q u i t o u s l y e x p r e s s e d =1 m R N A s e r v e d as a c o n t r o l f o r t h e p r e s e n c e o f r e v e r s e - t r a n s c r i b a b l e RNA.
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tissues. While previous studies have failed to demonstrate 1 l f l - H S D 1 m R N A or activity in mid-gestational foetal tissues [18], l l[3-HSD2 activity appeared to be present in many foetal tissues and the R T - P C R studies would support this notion, l l[3-HSD2 m R N A was detected in foetal adrenal, lung, colon and kidney, but was absent in foetal heart and liver. In adult tissues, 1 l f l - H S D 2 gene expression was confined exclusively to the placenta and to the classical mineralocorticoid target tissues, kidney cortex and medulla, colon and salivary gland. In contrast, 1 I[3-HSD1 was localized to the predominantly glucocorticoid target tissues, lung, liver, spleen, pituitary and cerebellum. Some 1113HSD1 m R N A was present in kidney medulla and placenta, but parallel enzyme activity and N o r t h e r n blot studies (data not shown) revealed that this isoform was present in low amounts. No ll[3-HSD1 m R N A was detected in kidney cortex, colon or salivary gland. Placental 1 l [ 3 - H S D 2 activity and foetal weight
Details of the term birth weights and placental weights from the 27 term placentas are given in Table 2. As shown there was a wide broad distribution of term foetal weight (2580-4990g) and placental weight (434-1120 g). Indeed 7 foetuses were above the 90th percentile for " n o r m a l " gestational-matched controis, and 4 were below the 10th percentile. T h e capacity of the placenta to metabolize 11-hydroxylated C21 steroids varied between 1.73-7.95 #mol F/min. T h e r e was a significant positive correlation between foetal weight and l l[3-HSD activity (r =0.408, P = 0.034), but no significant correlation between placental weight and l l[3-HSD activity (r =0.043, P >0.05). (Parallel studies indicated no correlation between placental 3[3-hydroxysteroid dehydrogenase (3[3-HSD) activity and foetal or placental weight.) When placental l l [ 3 - H S D 2 activity was expressed in terms of total activity/placenta, the correlation between foetal weight and l l[3-HSD activity became statistically stronger (r = 0.784, P < 0.001), but this could be explained on the basis of a positive correlation between foetal and placental weight (r = 0.672, P < 0.001).
DISCUSSION 1113-HSD2 has been cloned and characterized from human kidney [10, 16]. T h e enzyme shares little sequence homology with 1 I[3-HSD1, and is a kinetically distinct isoform, l l[3-HSD1 possesses relatively low affinity, N A D P - d e p e n d e n t dehydrogenase activity. Furthermore, E has a higher affinity for 1113HSD1 than F, suggesting that in vivo this enzyme may principally act as a reductase. In contrast, 1113H S D 2 is a high affinity, N A D - d e p e n d e n t unidirectional dehydrogenase. As documented here, l l[3-HSD2 is inhibited by liquorice derivatives, and appears also to be inhibited by end-product (i.e. E and A). Furthermore, dexamethasone and hydroxylderivatives of progesterone are also substrates for l l[3-HSD2. This may be of relevance in some tissues not evaluated in this study, such as the ovary, where high affinity l l[3-HSD activity has been reported. T h e ontogeny of human ll[3-HSD1 and 2 is likely to be of interest. In adult tissues, l l[3-HSD2 expression is confined to the classical mineralocorticoid target tissues, kidney, colon and salivary gland. In contrast l l[3-HSD1 was expressed in predominantly glucocorticoid target tissues, liver, lung, pituitary and cerebellum. 11[3-HSD1 m R N A was detected in adult kidney medulla using the sensitive technique of R T - P C R , but parallel activity studies have indicated that the contribution of this isoform to total 1 I[3-HSD activity within the renal medulla is very low. Furthermore, we have consistently failed to demonstrate 1 I[3HSD1 m R N A in kidney cortex, rectal colon, and salivary gland. Our earlier studies had failed to demonstrate 1 I[3-HSD1 m R N A or activity in mid-gestational foetal tissues [18], and only low levels of ll[3-HSD1 activity (if any) in term placenta. T h e R T - P C R data presented here are consistent with the expression of l l [ 3 - H S D 2 in human foetal lung, adrenal, kidney, colon and placenta. F r o m the examination of 1 I[3-HSD gene expression in these two distinct developmental periods, the ontogeny of 11[3-HSD1 would seem to be a late gestational or post-natal event. In contrast, l l[3-HSD2 may "switch off" in some tissues
Table 2. Term birth weight, placental weight and placental 1113-HSD activity in 2 7 placentas. Results are expressed as mean +_SE and ranges. 1113-HSD rate was expressed as pmol E formed/h/mg.protein, and for "total" 1113-HSD activity, the 113-HSD rate was multiplied by the placental weight
Parameter
Mean + SE
Placental weight (g) Term foetal weight (g) 11/~-HSD rate (pmol E formed/h/ mg.homogenate protein) Total ll/3-HSD (/~mol/min/placenta)
585 + 29 3441 +__105 312 _ 14
434-1120 2580--4990 194--448
Range
3.04 + 0 . 2 3
1.73-7.95
Type 2 llfl-HSD in Foetal and Adult Life postnatally (e.g. lung) to be expressed in the adult only in MR-target tissues. These tissue localization studies have enabled us to clarify the role of the 1 l f l - H SD isoforms. 1 lfl-HSD2, in adult life is predominantly found in the classical mineralocorticoid-target tissues; its cellular localization within kidney and colon to collecting ducts [19] and surface epithelial cells [15] respectively, indicates that it maintains aldosterone-selectivity upon the MR in an autocrine fashion. In contrast, l l f l - H S D 1 is found principally in glucocorticoid target tissues, where its role may be to generate active F from E or to inactivate particularly high circulating F levels occurring for example at times of stress. Thus, factors which regulate the tissue-specific expression and regulation of these distinct isoforms will be of major importance in the analysis of corticosteroid hormone action. It is now clear from this and other studies that l lfl-HSD2 plays a key role in foetal development. 1 l f l- HS D activity has been analysed in the placenta and foetal membranes since the 1960's [20-24], but it is only very recently that this activity (with the exception of maternally-derived decidua) has been found to reflect the expression of the l lfl-HSD2 isoform [14,24]. It has been suggested that l l f l - H S D may serve to protect the developing foetus from the deleterious effects of maternal cortisol excess. T he unusual combination of a low birth weight and large placenta predicting adult hypertension in man [25], and a direct correlation between placental l l f l - H SD activity and
I O .ER I Estriol
Estrone
[ CENTA I ~
~
Estriol
~-
DHEA
-91--
foetal weight and inverse correlation with placental weight in the rodent [26], suggests that defective placental 1 l fl -H SD activity may play a role in the subsequent development of hypertension in the offspring [27]. Our data suggest that this may not be the case. Firstly, l lfl-HSD2 activity and gene expression is present in many foetal tissues in addition to the placenta, suggesting that they can protect themselves from any cortisol excess in an autocrine or paracrine fashion [18]. Secondly, we have failed to identify any relationship between human placental l lfl-HSD activity and placental weight in man. Finally, if foetal glucocorticoid excess, mediated through defective placental 1lflH SD activity, was to be a factor in the development of hypertension, then one would expect evidence of suppression of the hypothalamo-pituitary-adrenal (HPA) axis in the low birth weight offspring, yet animal and human studies have, if anything, shown the opposite, ie. activation of the new-born HPA axis [28, 29]. The capacity for the placenta to metabolize cortisol is immense, with an average term placenta capable of converting 3/~mol of cortisol to cortisone per minute. Allowing for a placental whole blood flow of 500-700ml/min from both foetus and mother, we compute that a maximum of 1 pmol cortisol/min is delivered to the placenta. Thus, rather than considering placental l lfl-HSD as protecting the developing foetus from maternally-derived F, we suggest that it co-ordinates a far more crucial role in sustaining foetal adrenal steroidogenesis (Fig. 2). Such a scenario has
I 16-OH DHEAS
Estrone
469
DHEAS
DHEA ~
FOETUS Cholesterol
1 ACTH .~" \
\
Pregnenolone
17-OH pregnenolone ID IlS
Estradiol
~
Estradiol
17-0H progesterone
1 1-deoxycortisol
CORTISOL ~
CORTISOL ~
CORTISONE
CORTISOL
CORTISONE
Fig. 2. Steroidogenesis f r o m the foeto-placental unit illustrating the crucial role played by placental ll~$-HSD. The capacity of the placenta to m e t a b o l i s e F to E is i m m e n s e and this increased m e t a b o l i c clearance of cortisol stimulates foetal A C T H secretion and hence adrenal growth and steroidogenesis through the negative feedback m e c h a n i s m . Post.-partum, following r e m o v a l of the placenta, the foetal adrenal involutes, and circulating ACTH, DHEA and cortisone levels fall in the new-born.
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b e e n p r e s e n t e d for th e f o e t o - p l a c e n t a l u n i t o f the b a b o o n by P e p e a n d A l b r e c h t [30]. H e r e , as g e s t a t i o n a dvan ces , o e s t r o g e n i n d u c t i o n o f p l a c e n t a l 11 ~ - H S D activity i n d u c e s a c t i v a t i o n o f th e H P A - a x i s [31], w i t h i n c r e a s e d t r a n s c r i p t i o n o f t h e foetal P O M C gene, en h a n c e d A C T H s e c r e t i o n [32] a n d i n c r e a s e d foetal a d r e n a l s t e r o i d o g e n e s i s . B e c a u s e m a t e r n a l o e s t r o g e n is d e r i v e d p r e d o m i n a n t l y f r o m foetal a d r e n a l D H E A S , this m a y serve as a " f a s t f o r w a r d " r e g u l a t o r y process. D a t a f r o m earlier studies w o u l d s u p p o r t such a c o n c e p t in m a n [33-36]. T h e relative size o f t h e foetal a d r e n a l g l a n d is v e r y large c o m p a r e d to t h e a d u lt g l an d a n d foetal A C T H levels are also e l e v a t e d c o m p a r e d to a dul t values. S ev e r a l studies, p r i n c i p a l l y t h o s e f r o m a n e n c e p h a l i c foetuses, i n d i c a t e that foetal ad r en al g r o w t h an d s t e r o i d o g e n e s i s is u n d e r the c o n t r o l o f A C T H , b u t t h e cause o f the e l e v a t e d levels has n e v e r b e e n established. W h i l s t th e foetal a d r e n a l e x h i b i t s relative a t t e n u a t i o n o f 3 / 3 - H S D activity, a c c o u n t i n g for the large s e c r e t i o n o f D H E A a n d D H E A S , cortisol is s y n t h e s i z e d b y the foetal adrenal, w i t h c i r c u l a t i n g levels rising f r o m a p p r o x . 5 0 n m o l / 1 at 20 weeks ge st at i o n to 150o200 nmol/1 at t e r m . T h e close c o r r e lation b e t w e e n the h i g h foetal c o r t i s o n e levels ( 1 5 0 - 2 0 0 n m o l / 1 ) a n d D H E A levels t h r o u g h o u t gest a t i o n suggests t h a t the t r o p h i c s t i m u l u s to th e foetal H P A axis is actually e n h a n c e d cortisol m e t a b o l i s m r a t h e r t h a n i m p a i r e d s e c r e t i o n [37]. Finally, p o s t p a r turn, after r e m o v a l o f the p l a c e n t a and t h e r e f o r e the m a j o r s o u r c e o f cortisol clearance, th e foetal a d r en al r a p i d l y i n v o l u t e s , a n d c o r t i s o n e a n d A C T H levels fall i m m e d i a t e l y to n o r m a l a d u lt levels (50 nmol/1 a nd < 5 pmol/1, r e s p e c t i v e l y ) [37, 38]. O u r o b s e r v e d c o r r e l a t i o n b e t w e e n p la c e n t a l l l ~ - H S D activity an d b i r t h w e i g h t m a y s i m p l y be a reflection that larger foetuses h a v e larger adrenals. T h e l l / ~ - H S D story has c o m e a l o n g way since Carl M o n d e r first p u r i f i e d w h a t we n o w k n o w to be l l f i H S D 1 f r o m rat l i v e r m i c r o s o m e s . I n m a n , 1 l f i - H S D 1 seems likely to r e g u l a t e t h e access o f cortisol to the G R a nd m a y i n d e e d p r i m a r i l y act as an o x o - r e d u c t a s e . I n c o n t r a s t l l / 3 - H S D 2 confers specficity u p o n th e M R , a nd defects in its activity m a y e x p la in some f o r m s o f h y p e r t e n s i v e such as t h e s y n d r o m e o f A p p a r e n t M i n e r a l o c o r t i c o i d Excess. F i n a l l y t h e h i g h e x p r e s s i o n o f this i s o f o r m in h u m a n p l a c e n t a m a y be a p r i n c i p a l factor d r i v i n g foetal ad r en a l s t e r o i d o g e n e s i s a n d h e n c e o n g o ing gestation.
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3. Funder J. W., Pearce P. T., Smith R. and Smith A. I.: Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242 (1988) 583-585. 4. Fuller P. J. and Verity K.: Colonic sodium-potassium adenosine triphosphate subunit gene expression: ontogeny and regulation by adrenocortical steroids. Endocrinology 127 (1990) 32-38. 5. Whorwood C. B., Sheppard M. C. and Stewart P. M.: Licorice inhibits 11/3-hydroxysteroid dehydrogenase messenger ribonucleic acid levels and potentiates glucocorticoid hormone action. Endocrinology 132 (1993) 2287-2292. 6. Monder C., Miroff Y., Marandici A. and Hardy M.: 11/3Hydroxysteroid dehydrogenase alleviates glucocorticoidmediated inhibition of steroidogenesis in rat Leydig cells. Endocrinology 134 (1994) 1199-1204. 7. Agarwal A. K., Monder C., Eckstein B. and White P. C.: Cloning and expression of rat cDNA encoding corticosteroid l l/3-dehydrogenase. J. Biol. Chem. 264 (1989) 18,939-18,943. 8. Tannin G. M., Agarwal A. K., Monder C., New M. I. and White P. C.: The human gene for 1l~-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J. Biol. Chem. 266 (1991) 16,653-16,658. 9. Moore C. D. C., Mellon S. H., Murai J., Siiteri P. K. and Miller W. L.: Structure and function of the hepatic form of 11/3hydroxysteroid dehydrogenase in the squirrel monkey, an animal model of glucocorticoid resistance. Endocrinology 133 (1993) 368-375. 10. Stewart P. M., Murry B. A. and Mason J. I.: Human kidney ll/3-hydroxysteroid dehydrogenase is a high affinity NADdependent enzyme and differs from the cloned "type I" isoform. J. Clin. Endocr. Metab. 79 (1994) 480-484. 11. Bonvalet J. P., Doignon I., Blot-Chabaud M., Pradelles P. and Farman N.: Distribution of 11/3-hydroxysteroid dehydrogenase along the rabbit nephron. J. Clin. Invest. 86 (1990) 832-837. 12. Rusvai E. and Fejes-Toth A. N.: A new isoform of 11/3-hydroxysteroid dehydrogenase in aldosterone target cells. J. Biol. Chem. 268 (1993) 10,717-10,720. 13. Mercer W. and Krozowski Z. S.: Localization of an 11/3hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology 130 (1992) 540-543. 14. Brown R. W., Chapman K. E., Edwards C. R. W. and Seckl J. R.: Human placental l l/3-hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform. Endocrinology 132 (1993) 2614-2621. 15. Whorwood C. B., Ricketts M. L. and Stewart P. M. Epithelial cell localization of type 2 11/3-hydroxysteroid dehydrogenase in rat and human colon. Endocrinology 135 (1994) 2533-2541. 16. Albiston A. L., Obeyesekere V. R., Smith R. E. and Krozowski Z. S.: Cloning and tissue distribution of the human 11/3hydroxysteroid dehydrogenase type 2 enzyme. Molec. Cell Endocr. 105 (1994) Rll-R17. 17. Kawakami K., Ohta T., Nojima H. and Nagano K.: Primary structure of the ~-subunit of human Na,K-ATPase deduced from a cDNA sequence. J. Biochem 100 (1986) 389-397. 18. Stewart P. M., Murry B. A. and Mason J. I.: Type 2 11/3hydroxysteroid dehydrogenase in human foetal tissues. J. Clin. Endocr. Metab. 78 (1994) 1529-1532. 19. Whorwood C. B., Mason J. I., Ricketts M. L., Howie A. L. and Stewart P. M.: Detection of human ll/3-hydroxysteroid dehydrogenase isoforms using RT-]PCR and localization of the type 2 isoform to renal collecting ducts. Molec. Cell. Endocr. 110 (1995) R7-R12. 20. Osinski P. A.: Steroid 11/3-ol dehydrogenase in human placenta. Nature 187 (1960) 777. 21. Bernal A. L. and Craft I. L.: Corticosteroid metabolism in vitro by human placenta, foetal membranes and decidua in early and late gestation. Placenta 2 (1981) 279-285. 22. Giannopoulus G., Jackson K. and Tulchinsky D.: Glucocorticoid metabolism in human placenta, decidua, myometrium and foetal membranes. J. Steroid Biochem. 17 (1982) 371-374. 23. Murphy B. E. P.: Chorionic membrane as an extra-adrenal source of foetal cortisol in human amniotic fluid. Nature 266 (1977) 179-181. 24. Stewart P. M., Rogerson F. M. and Mason J. I.: Type 2 11/3-hydroxysteroid dehydrogenase mRNA and activity in human placenta and foetal membranes: Its relationship to birth
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