The human 11β-hydroxysteroid dehydrogenase type II enzyme: Comparisons with other species and localization to the distal nephron

~

Pergamon

ft. Steroid Biochem. Molec. Biol. Vol. 55, No. 5/6, pp. 457-464, 1995 0960-0760(95)00194-8

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00

The H u m a n l lfl-Hydroxysteroid Dehydrogenase Type II Enzyme: Comparisons with Other Species and Localization to the Distal Nephron Z. K r o z o w s k i , 1. A. L. A l b i s t o n f l V. R. O b e y e s e k e r e , 1 R. K. A n d r e w s 2 a n d R. E. S m i t h 1 1Laboratory of Molecular Hypertension and 2 Vascular Biology Laboratory, Baker Institute of Medical Research, Prahran 3181, Australia Effective g l u c o c o r t i c o i d i n a c t i v a t i o n is c u r r e n t l y t h o u g h t to be an i n d i s p e n s a b l e f e a t u r e o f m i n e r a l o c o r t i c o i d t a r g e t cells. T h e e n z y m e 11/ ~ - hydroxyst eroi d d e h y d r o g e n a s e (11fl-HSD) i n a c t i v a t e s glucoc o r t i c o i d s a n d p r e v e n t s t h e m f r o m b i n d i n g to t he n o n - s e l e c t i v e m i n e r a l o c o r t i c o i d r e c e p t o r . In t h e k i d n e y it is th e NAD d e p e n d e n t high affinity i s o f o r m (11fl-HSD2) w h i c h is t h o u g h t to e n d o w specificity on th e r e c e p t o r . T h e r e c e n t cl oni ng o f t he h u m a n , sheep a n d r a b b i t 11fl-HSD2 e n z y m e s p e r m i t s a c o m p a r i s o n o f t h e e n z y m e f r o m t he t h r e e species. H u m a n a n d r a b b i t e n z y m e s a r e 87% i d e n t i c a l a n d o f s i m i l a r length, while t he h u m a n a n d sheep e n z y m e s have only 75% i d e n t i t y . T h e last 12 r e s i d u e s in all t h r e e species w e r e f o u n d to be hi ghl y d i v e r g e n t , b u t m o s t o f t he ovine d i s h o m o l o g y c a n be a c c o u n t e d f o r by t he d e l e t i o n o f a single n u c l e o t i d e t o w a r d t he C - t e r m i n u s o f t he p r o t e i n r e s u l t i n g in a shift in r e a d i n g f r a m e g e n e r a t i n g a p r o t e i n 27 r e s i d u e s l o n g e r t h a n t he h u m a n i s o f o r m . N u m e r o u s o t h e r de l e t i ons w e r e also o b s e r v e d in this r e g i o n o f t he sheep cDNA sequence. F u r t h e r m o r e , t h e r a b b i t cDNA also d i s p l a y e d a l a r g e d e g r e e o f d i s h o m o l o g y w i t h t he h u m a n s e q u e n c e a s h o r t d i s t a n c e d o w n s t r e a m f r o m t he t e r m i n a t i o n codon. C o n s e r v e d o v e r l a p p i n g c y t o p l a s m i c t r a n s l o c a t i o n signals; w e r e o b s e r v e d in all t h r e e species, suggesting a t o p o l o g y w h e r e b y t he e n z y m e is a n c h o r e d into t h e e n d o p l a s m i c r e t i c u l u m by m u l t i p l e h y d r o p h o b i c r e g i o n s in t he N - t e r m i n u s a n d t h e b u lk o f t h e 11fl-HSD2 p e p t i d e is sited in t he c y t o p l a s m . A p o l y c l o n a l a n t i b o d y g e n e r a t e d a g a i n s t t h e C - t e r m i n u s o f h u m a n 11fl-HSD2 was used to localize t he e n z y m e w i t h i n t h e kidney. A high level o f i m m u n o r e a c t i v i t y was o b s e r v e d in distal t u b u l e s a n d collecting ducts, localizing t he e n z y m e to t h e s a m e p a r t o f t he n e p h r o n as t he m i n e r a l o c o r t i c o i d r e c e p t o r . M o d e r a t e levels o f st ai ni ng w e r e also seen in v a s c u l a r s m o o t h m u s c l e cells. T h e s e r e s u l t s s u p p o r t t h e n o t i o n t h a t 11fl-HSD2 is an a u t o c r i n e p r o t e c t o r o f the m i n e r a l o c o r t i c o i d r e c e p t o r a n d t h a t it plays an i m p o r t a n t ro le in cardiovascular homeostatic mechanisms.

J. Steroid Biochem. Molec. Biol., Vol. 55, No. 5/6, pp. 457-464, 1995

INTRODUCTION T h e study of the reguh,tion of active hormone concentrations at the level of the target cell has recently become an important subject. In the kidney glucocorticoids are inactivated [1,2] by the enzyme l l ft hydroxysteroid dehydrogenase ( l l f l - H S D ) allowing aldosterone to occupy the non-selective mineralocortiProceedings of the Workshop on the Molecular and Cell Biology of Hydroxysteroid Dehydrogenases, Hannover, Germany, 19-22 April 1995. *Correspondence to Z. Krozowski. 457

coid receptor [3]. A low affinity isoform of the enzyme ( l l f l - H S D 1 ) exists in the liver where it performs the reverse reaction, producing active glucocorticoid by converting cortisone to cortisol. In the distal tubule of the kidney, however, a high affinity unidirectional l l f - H S D isoform ( l l f - H S D 2 ) converts glucocorticolds to their inactive ll-keto metabolites [4]. Inhibition of 1l f l - H S D activity by licorice leads to sodium retention, hypokalemia and elevated blood pressure [5], symptoms similar to those observed in the congenital syndrome of apparent mineralocorticoid excess (AME) where patients also display low cortisone to cortisol

458

Z. Krozowski et al.

ratios and reduced 5fl-reductase metabolites in the urine [6]. T h e parallel modulation of these two enzymes has led some workers to suggest that a common circulating factor may be responsible [7]. However, recent clinical data has failed to show any correlation between inhibitors extracted from urine, and mineralocorticoid receptor activation and l l f l - H S D activity [8]. Numerous studies have provided evidence for a high affinity N A D - d e p e n d e n t l l f l - H S D enzyme distinct from the N A D P dependent l l f l - H S D 1 [9, 10-13]. An early study showed that the rat kidney contained an N A D - d e p e n d e n t activity localized to the distal nephron [9], clearly distinct from the medullay localization of l l f l - H S D 1 . In retrospect, the rat kidney proved to be a complex tissue to study with both isoforms of the enzyme present in high amounts, while human kidney appears not to contain appreciable amounts of the 1 l f l - H S D 1 isoform [14]. While the role of l l f l - H S D 1 in the rat medulla remains unknown it is possible that, consistent with transfection studies with the cloned c D N A [15], this enzyme serves as an oxidoreductase in the proximal tubule. T h e human 1 l f l - H S D 2 enzyme was recently cloned from a kidney expression library [16]. Characterization of the cloned species showed the enzyme to be N A D dependent, have a high affinity for glucocorticoids, to be exclusively dehydrogenase in directionality and inhibitable by glycyrrhetinic acid, the active component of licorice. N o r t h e r n blot analysis showed the l lflH S D 2 gene to be highly expressed in kidney and placenta, with undetectable levels in the liver. These results are in excellent agreement with previous in vitro studies, confirming the authenticity of the cloned enzyme [9, 11-13]. T h e l l f l - H S D 1 and l l f l - H S D 2 enzymes belong to the short-chain alcohol dehydrogenase superfamily [17, 18] but there is only 14% identity between the two isoforms. Instead, 1 l f l - H S D 2 is almost closely related to 17fl-HSD2 [19] with which it is 34% identical. Ovine and rabbit l l f l - H S D 2 enzymes [20, 21] have also been cloned recently and this study will compare all three species. Further work from our laboratory has focused on the localization of the enzyme within various tissues. We have recently raised a polyclonal antibody to human l l f l - H S D 2 and performed immunohistochemicial studies in the kidney and placenta [22]. T h e present report also includes additional localization studies in the kidney. MATERIALS AND METHODS

Cloning strategy Full details of the cloning strategy have been presented elsewhere [16]. Briefly, a human kidney c D N A library in pcDNA1 was transfected into C H O P [23] cells. T h r e e days later radioactive corticosterone was added to the culture medium, incubated overnight and the medium assayed for the presence of steroid metab-

olites by thin layer chromatography. Positive pools were identified and a single clone isolated by sibling selection. Immunohistochemistry A peptide corresponding to the last 15 residues of human 1 I ~ - H S D 2 was synthesized. Generation of the antiserum, immunopurification of the antibody and immunohistochemistry were performed as previously described [22]. RESULTS

T h e human l l f l - H S D 2 c D N A clone isolated by expression screening is 1872 bases in length and contains an open reading frame of 405 amino acids (Fig. 1). T w o features of the c D N A sequence are immediately apparent. First, the 5' untranslated region is highly G C rich, suggesting the possibility of significant m R N A secondary structure. However, computer analysis for secondary structure revealed only one pair of seven nucleotide elements which may form a significant stem in the classical stern and loop structure. Secondly, nucleotides surrounding the initiator codon ( G G C C G C C A T G G ) conform well with the Kozak [24] consensus sequence ( G C C A G C C A U G G ) . A comparison of the human, rabbit and sheep 11~H S D 2 enzymes is shown in Fig. 2. T h e respective lengths of these proteins are 405, 406 and 427 residues. T h e extra length of the ovine protein is due to frame shifts resulting from the deletions of nucleotides starting at a position corresponding to 1181 nucleotides in the human cDNA. T h e r e is an 87% identity between human and rabbit sequences and a 75% identity between human and sheep. T h e highest degree of sequence dissimilarity is seen in the C-terminal region, in particular the last 12 residues in each sequence are highly divergent. However, despite frame shifts in the ovine sequence comparison of the three proteins by the Clustal4 algorithm [25] shows an interesting alignment of Proline residues in this region. Five positions contain conserved prolines in all three species over a stretch of 25 amino acids. Potential N-linked glycosylation sites are not conserved and occur at residues 394-396 in the human, at 272-274 in the rabbit, and at 96-98 and 245-247 in the sheep. In the human enzyme there are cytoplasmic translocation signals, Arg-Arg, at positions 335, 336 and 359, 360. T h e motif at position 335 is conserved in the rabbit while the one at position 359 is conserved in the sheep sequence. We further explored the divergence of the ovine and human C-terminal regions by aligning the corresponding c D N A sequences (Fig. 3). T h e ovine sequence was found to contain numerous deletions and one insertion of three nucleotides when compared to the human cDNA. Overall the region 1164--1330 nucleotides in the sheep contains the highest degree of divergence with the human c D N A while sequences immediately 5' and

11~-HSD2

3' remain highly conserved. T h e overall degree of conservation suggests that the differences are not due to splicing events. Comparison of the rabbit and h u m a n sequences over the same region showed a high degree of dishomology beginning at nucleotide 1380 in the h u m a n sequence, well beyond the termination codons in both species (results not shown). T h e high degree of dissimilarity here may be due to either a gene insertion event or to R N A splicing, As part of o n - g o i n g studies to characterize the 11/~H S D 2 e n z y m e we have raised a polyclonal antibody

in K i d n e y

459

( H U H 2 3 ) to a synthetic peptide deduced from the h u m a n c D N A . Figure 4 shows a low power view of a section of h u m a n kidney cortex stained with the immunopurified antiserum. Intense staining was observed in distal convoluted tubules and collecting ducts. Occasionally populations of distal tubules showed only moderate staining. In Fig. 5 is s h o w n a high power magnification of a section of h u m a n kidney cortex. Again intense staining was observed in distal convoluted tubules while smooth muscle cells of an artery showed moderate reactivity.

m ~ G CGCGCCCCAGGCCGGTGTACCCCCGCACTCCGCGCCCCGGCCTAGAAGCTCTCTCTCCCCGCTCCCCGGCCCGGCCCCC i0

20

30

40

50

60

70

80

MET E R W P W P S G G A W L L V CCCCGCCCCGCCcCAGCCCGCTGGGCCGCCATGGAGCGCTGGCCTTGGCCGTCGGGCGGCGCCTGGCTGCTCGTGGCTGC 90 100 110 120 130 140 150

A

A 160

R A L L Q L L R S D L R L G R P L L A A L A L L A CCGCGCGCTGCTGCAGCTGCTGCGCTCAGACCTGCGTCTGGGCCGCCCGCTGCTGGCGGCGCTGGCGCTGCTGGCCGCGC 170 180 190 200 210 220 230 L D W L C Q R L L P P P A A L A V L A A A G W I A TCGACTGGCTGTGCCAGCGCCTGCTGCCCCCGCCGGCcGCACTCGCCGTGCTGGCCGCCGCCGGCTGGATCGCGTTGTCC 250 260 270 280 290 300 310

A 240 L

S 320

R L A R P Q R L P V A T R A V L I T G C D S G F G K E CG C C T G G C G C G C C C G C A G C G C C T G C C G G T G G C C A C T CGCGCGGTGCTCATCACCGGCTGTGACTCTGGTTTTGGCAAGGA 330 340 350 360 370 380 390 400 T A K K L 0 SMET G F T V L A T V L E L N S P G A I E GACGG CCAAG AAACTGGACTC CATGGGCTTCACGGTG CTGGC CACCGTATTGGAG TTGAACAGCC CCGGTG CCATCGAGC 410 420 430 440 450 460 470 480 L R T C C S P R L R L L QMET D L T K P G D I TGCGTACCTGCTGCTCC CCTCGCCTAAGGCTGCTGCAGATGGACCTGACCAAACCAGGAGACATTAG 490 500 510 520 530 540

S

R L L E CCGCTTGCTAGAG 550 560

F T K A H T T S T G L W G L V N M A G H N S V V A D A TTCACCAAGGCCCACAC CACCAGCACCGGCCTGTGGGGCCTCGTCAACAACGCAGGC CACAATGAAGTAGTTGCTGATGC 570 580 590 600 610 620 630 640 E L S P V A T F R S CXETE V N F F GGAGCTGTCTC CAGTGGC CACTTTCCGTAGCTGCATGGAGGTGAATTTCT~GCG 650 660 670 680 690

G

A

L E L T K G L CTCGAGCTGACCAAGGGCCTCC 700 710 720

L P L L R S S R G R I V T V G S P A G DMET P ¥ P C TGCC CCTGCTGCGCAGCTCAAGGGGCCGCATCGTGACTGTGGGGAGCCCAGCGGGGGACATGCCATATCCGTGCTTG 730 740 750 760 770 780 790 A Y G T S K A A V A L LMET D T F S C E L L P W G G C CTATGGAACCTCCAAAGCGGCCGTGGCGCTACTCATGGACACATTCAGCTGTGAACTCCTTCCCTGG~AAGGT 810 820 830 840 850 860 870

L

V

G GGG 800 K

V 880

S I I Q P G C ~ K T E S V R N V G Q W E K R K Q L CAGCATCATCCAG CCTGGCTGCTTCAAGACAGAGTCAGTGAGAAACGTGGGTCAGTGGGAAAAGCGCAAGCAATTGCTGC 890 900 910 920 930 940 950

L 960

L A H L P Q E L L Q A Y G K D Y I E H L H G Q F L H S TGG CCAAC CTGCC TCAAGAGCTGCTGCAGGCCTACGGCAAGGACTACATCGAGCACTTGCATGGGCAGTTCCTGCACTCG 970 980 990 1000 1010 1020 1030 1040 L R L A MET S D L T P V V D A I T D A L L A A R P CTACGCCTGGCCATGTCCGACCTCACCCCAGTTGTAGATGCCATCACAGATGCGCTGCTGGCAGCTCGGCCCCGCCGC 1050 1060 1070 1080 1090 1100 1110 Y Y P G Q G L G LMETY F I H Y ¥ L P E G L R R R CTATTACCCCGGCCAGGGCCTGGGGCTCATGTACTTCATCCACTACTACCTGCCTGAAGGCCTGCGGCGCCC~CTGC 1130 1140 1150 1160 1170 1180 1190 Q A F F I S S C L P R A L Q P G Q P G T T AGG CCTTCTTCATCAGTCACTGTCTGCCTCGAGCACTGCAGCCTGGCCAGCCTGGCACTACCC 1210 1220 1230 1240 1250 1260 O D P N L S P G P S P CAGGACCCAAACCTGAGCCCCGGCCCTTCCCCAG 1290 1300 1310

R

R

R CG 1120

F

L 1200

P P Q D A A CACCACAGGACGCAGCC 1270 1280

A V A R CAGTGGCTCGGTGAGC CATGTGCACCTATGGCCCAGCCACTGCAGC 1320 1330 1340 1350 1360

ACAGGAGG CTCCG TGAGCCTTGGTTCCTCCCCGAAAACCCCCAGCATTACGATC 1370 1380 1390 1400 1410

CCCCAAGTGTCCTGGACCCTGGCCTA 1420 1430 1440

AAGAATCC CACCCCCACTTCATGC 1450 1460

CCACTGCCGATGCCCAATCCAGGCCCGGTGAGGCCAAGGTTTCCC AGTGAGCCTCT 1470 1480 1490 1500 1510 1520

GCG CCTCTCCACTGTTTCATGAGC 1530 1540

CCAAACACCCT CCTGGCACAACGCTCTACCCTGCAG CTTGGAGAACTCCGCTGGAT 1550 1560 1570 1580 1590 1600

GGGAGTCTCATG CAAGACTTCAC TGCAGC CTTTCACAGGACTCTGCAGATAGTGC 1610 1620 1630 1640 1650

CTCTGCAAACTAAGGAGTGACTAGG 1660 1670 1680

TGGGTTGGGGACCCCCTCAGGATTGTTTCTCGGCACCAGTGCCTCAGTGCTGCAATTGAGGGCTAAATCCCAAGTGTCTC 1690 1700 1710 1720 1730 1740 1750 1760 TTGACTGGCTCAAGAATTAGGGCCCCAACTACACACCCCCAAGCCACAGGGAAGCATGTACTGTACTTCCCAATTG 1770 1780 1790 1800 1810 1820 1830 A'FI"I"I'#~T ~ A G A C AAA'FI-I-Fr A~I~T C T r C 1850 1860 1870

T

CCAC 1840

~ 1880

Fig. 1. C o m p l e t e ,:DNA and deduced protein sequences o f the h u m a n l l / ~ - H S D 2 e n z y m e . A r r o w s indicate the position and extent of s e c o n d a r y structure f o r m a t i o n in the 5'-untranslated region.

460

Z. Krozowski et al. HUM-HSD2 RAB-HSD2 SHE-HSD2

MERWPWPSGGAWLLVAARALLQLLRSDLRLGRPLLAALALLAALDWLCQRLLPPPAALAV MERWPWPSGGAWLLVAARALIQLLRADLRLGRPLLAALALLAALDWLCQSLLPPSAALAV MESWPWPSGGAWLLVAARALLQLLRADLRLGRPLLAALALLAALDWLCQRLLPPLAALAV ** * * * * * * * * * * * * * * * * * **** * * * * * * * * * * * * * * * * * * * * * * * **** * * * * * •

°

•

.

HUM-HSD2 RAB-HSD2 SHE-HSD2

LAAAGWIALSRLARPQRLPVATRAVLITGCDSGFGKETAKKLDSMGFTVLATVLELNSPG LAAAGWIALSRLARPQRLPVATRAVLITGCDSGFGKETAKKLDAMGFTVLATVLEMNGPG LAATGWIVLSRLARPQPLPVATRAVLITGCDSGFGNATAKKLDAMGFTVLATVLDLNSPG ************************************************************

HUM-HSD2 RAB-HSD2 SHE-HSD2

AIELRTCCSPRLRLLQMDLTKPGDISRLLEFTKAHTTSTGLWGLVNNAGHNEVVADAELS ALELRACCSPRLKLLQMDLTKPADISRALEFTKAHTTSTGLWGLVNNAGHNDWADVELS ALELRACCSSRLQLLQMDLTKPADISRVLEFTKVHTASTGLWGLVNNAGQNIFVADAELC *************************** *********************** ***o**o

HUM-HSD2 RAB-HSD2 SHE-HSD2

PVATFRSCMEVNFFGALELTKGLLPLLRSSRGRIVTVGSPAGDMPYPCLGAYGTSKAAVA PVATFRNCMEVNFFGALELTKGLLPLLHHSRGRIVTLGSPAGEMPYPCLAAYGTSKAAMA PVATFRTCMEVNFFGALEMTKGLLPLLRRSSGRIVTVSSPAGDMPFPCLAAYGTSKAALA **************************** *******************************

HUM-HSD2 RAB-HSD2 SHE-HSD2

LLMDTFSCELLPWGVKVSIIQPGCFKTESVRNVGQWEKRKQLLLANLPQELLQAYGKDYI LLMDAFSCELLPWGVKVSVIQPGCFKTESVSNVSHWEQRKQLLLANLPGELRQAYGEDYI LLMGNFSCELLPWGVKVSIILPACFKTESVKDVHQWEERKQQLLATLPQELLQAYGEDYI

HUM-HSD2 RAB-HSD2 SHE-HSD2

EHLHGQFLHSLRLAMSDLTPVVDAITDALLAARPRRRYYPGQGLGLMYFIHYYLPEG-LR EHLHREFLHSLRLALPDLSPWDAITDALLAARPRPRYYPGRGLGLMYFIHYYLPEG-LR EHLNGQFLHSLSQALPDLSPWDAITDALLAAQPRRRYYPGHGLGLIYFIHYYLPEGCGR ***• ***** *. ** * * * * * * * * * * * * * ** ***** **** * * * * * * * * * * * •

HUM-HSD2 RAB-HSD2 SHE-HSD2

o

.

•

,

,

o

RRFLQAFFISHC ..... L P - - R A L Q P G Q - P G T T P P Q D A A Q D - P N L S P G P S P A V A R..... R R F L Q S F F I I P C . . . . . L P - - R A L R P G Q - P G A T P A P D T A Q D N P N P N P D P S L V G A R. . . . . VSCSPSSSVPMCQEHYRLPAWPYLCPGHSPGPRPQTGPLSHCPVSRAHVEQLQQRRFLVP **. **. *. ,. * .° .* .

HUM-HSD2 RAB-HSD2 SHE-HSD2

.

..

•

*

* *

*

LLFFQVF

Fig. 2. Multiple a l i g n m e n t of the h u m a n (HUM-HSD2), rabbit (RAB-HSD2) and sheep (SHE-HSD2) 11fl-HSD2 protein sequences [16, 21, 20]. Asterisks denote positions of identity in all three enzymes and dots indicate conservative changes. A l i g n m e n t was p e r f o r m e d by the CLUSTAL4 a l g o r i t h m [25].

Furthermore, HUH23 also showed some staining of visceral epithelial cells of the outer capillary loop of a glomerulus. Figure 6 shows a high powered view of a renal artery stained with HUH23 more clearly illustrating staining of all smooth muscle cells surrounding the blood vessel. We have previously validated the specificity of HUH23 immunoreactivity in the kidney by

preincubation of the antibody with the immunizing peptide [22]. DISCUSSION

The recent expression cloning of the l lfl-HSD2 enzyme from three species has permitted a comparison

HUM-HSD2 SHE-HSD2

1209 1097

TTCATCAGTCACTGTCTGCCTCGAGCACTGCAGCCTGGCCAGCCTGGCACTACCCCACCA TTCATCAGTCCCTATGTGCCAAGAGCACTACAGGCTG-CCAGCCTGGCCTTACCTCTGCC

HUM-HSD2 SHE-HSD2

1269 1156

CAGGACGCAGCCCAGGACCCAAACCTGAGCCCCGGCCCTTCCCCAGCAGTGGCTCGG~GA~ CGGGACATA-CCCAGGACCAAGGCCCCAGACTGGACCCCTCTCC--CACTG-CCCAGTGA

HUM-HSD2 SHE-HSD2

1329 1212

GCCATGTGCACCTATGGCCCAGCCACTGCAGCACAGGAGGCTCCGTGAGCCTTGGTTCCT GC-AGG-GCACATGTAG---AGCAGCTCCAGCAGAGGAGGTTCCTTGTGCCCTTGCTCTT

HUM-HSD2 SHE-HSD2

1389 1267

CCCCGAAAACCCCCAGCATTACGATCCCCCAAGT--GTCCTGGACCCTGGCCTAAAGAAT CTTCCA GGTATTI~--~CCCC~GGTCTGCCCTAGA~C'I~GCCC~i~AC

HUM-HSD2 SHE-HSD2

1447 1319

CCCACCCCCACTTCATGCCCACTGCCGATGCC-CAATCCAGGCCCGGTGAGGCCAAGGTT CCCAACCC ...... ATGC--ACTGCCGATGCCACAGGCCAGGCCTGGTGAGGTGAAGGCT

Fig.3.A•ignment•fhuman(H•M-HSD2)andsheep(SHE-HSD2)cDNAsequ•nc•ssurr•undingtcrminati•n

codons. S t o p c o d o n s a r e b o x e d .

Alignmentwasper~rmedbythe

CLUSTAL4algorithm[25].

l lfl-HSD2 in Kidney of protein sequences. H u m a n and rabbit sequences are of similar length and most highly conserved save for 12 residues at the C-terminus. T h e sheep sequence differs from both these proteins in that it is significantly longer due to single nucleotide deletions which result in frame shifts in the coding sequence. These deletions may have arisen as part of the same evolutionary process which gave rise to the more extensive divergence further downstream. T h e frame shifts in the sheep c D N A sequence produce a non-conserved peptide of 69 amino acids starting from residue 358. However, despite the changes in reading frmnes comparison of l l f l - H S D 2 from all three species showed an interesting alignment of prolines in the C-terminal region. Proline residues are often found at fl-turns, suggesting that 1 l f l - H S D 2 is extensively folded in this region. Little is currently known about the topology of the microsomal l l f l - H S D 2 enzyme. Recent studies on the l l f l - H S D 1 isoform ihave revealed a single transmembrane segment at the N-terminus, with the bulk of the polypeptide chain projecting into the lumen of endoplasmic reticulum [26]. H y d r o p a t h y analysis of the l l f l - H S D 2 protein sequence suggests that the enzyme is anchored by three transmembrane segments at the N-terminus [27]. T h e identification of conserved

461

cytoplasmic translocation signals suggests that l lflH S D 2 projects into the cytoplasmic side of the endoplasmic reticulum. T h e two isoforms of 1 l f l - H S D thus appear to be localized within different intracellular compartments. Previous studies on the 1 l f l - H S D 1 enzyme in the rat showed that it was localized in proximal tubules [28] while the mineralocorticoid receptor was sited in distal tubular elements [29], leading workers to suggest a paracrine mode of protection. However, other studies have shown that 11fl-HSD1 is unlikely to protect the receptor on the grounds of its proximal tubular localization, low affinity for glucocorticoids and the observation that it acts as an oxidoreductase in mineralocorticoid responsive toad bladder cells [15]. In the present study we used a highly specific antibody [22] to demonstrate that 11fl-HSD2 colocalizes with the mineralocorticoid receptor in distal tubules and collecting ducts; these results suggest a mechanism consisting of autocrine protection of the receptor: Occasionally distal tubules showed only moderate staining. This suggests that the concentration of the enzyme varies between populations of tubules. This variation may be a reflection of a normally low expression in some distal tubules or it may be due to the

Fig. 4. I m m u n o h i ~ ; t o c h e m i c a l localization of 11fl-HSD2 in a section o f h u m a n kidney cortex using the HUH23 antibody. D, distal convoluted tubule; C, collecting duct; G, g l o m e r u l u s ; T, distal tubules ( m a g n i f i c a t i o n x 124).

462

Z. Krozowski et al.

modulation of enzyme expression by the surrounding milieu. T h e role of the low amounts of l l f l - H S D 2 staining in the visceral epithelial cells of the outer capillary loop of the glomerulus m u s t remain speculative, but it is interesting to note that the mesangial cells do not appear to be immunoreactive with the H U H 2 3 antiserum. Studies on the 1 l f l - H S D activity in isolated tubules and glomeruli from the h u m a n kidney have also shown considerable variability [30]. Smooth muscle cells of the vasculature also showed moderate staining with the H U H 2 3 antibody. T h e rat mesenteric vascular arcade has previously been shown to be highly aldosterone-selective in vivo, and in vitro shows considerable levels of l l f l - H S D activity [31]. T h e s e observations are consistent with a more recent study suggesting that glucocorticoids and mineralocorticoids modulate vascular smooth muscle cell contractility by affecting the N a ÷ and/or Ca 2÷ transport systems in these cells [32]. In vivo studies in h u m a n subjects also support an important role for 1 l f l - H S D 2 activity in the vasculature [33]. By including A M E patients in their study, and the use of enzyme inhibitors, these authors concluded that 1 l f l - H S D modulates the access of cortisol to vascular receptors and thereby influences vascular sensitivity to noradrenaline, confir-

ming previous results obtained in a study on cortisol induced hypertension in m a n [34]. In another study, 11fl-HSD activity was found to be defective in a proportion of patients with essential hypertension. T h e defect was not associated with mineralocorticoid excess and it was suggested that it m a y cause hypertension by increasing exposure of vascular steroid receptors to cortisol [35]. Given the present identification of l l f l H S D 2 in both the distal tubule and vascular smooth muscle cells the above observation in hypertensive patients would suggest that the enzyme may be differentially regulated at the two sites. T h e recent expression cloning of 11fl-HSD2 from the kidneys of three species and the localization of the enzyme to the distal nephron and vasculature has implications for the type I form of A M E . T h e fact that the same protein was cloned in all three instances suggests that it is the most abundant renal 11fl-HSD in these species. T h e identification of l l f l - H S D 2 in mineralocorticoid target cells and vascular smooth muscle cells is consistent with a defect in this enzyme leading to sodium retention and the development of hypertension in these patients. W h a t remains to be explained is why 5fl-reductase activities are also compromised in this syndrome and in some hypertensives

Fig. 5. HUH23 localization o f 11fl-HSD2 in h u m a n kidney cortex. A, artery; D, distal c o n v o l u t e d tubule; E, visceral epithelial cells; G, glomerulus; V, vascular s m o o t h m u s c l e cells ( m a g n i f i c a t i o n x 310).

11/%HSD2 in K i d n e y

463

Fig. 6. Staining of an interlobular renal artery with the HUH23 antibody. A, artery; V, vascular s m o o t h m u s c l e cells (magnification × 310).

[36]. O n e p l a u s i b l e e x p l a n a t i o n m a y b e t h a t e l e v a t e d l e v e l s o f c o r t i s o l d o w n - r e g u l a t e e x p r e s s i o n o f t h e 5/3reductase gene. In the type II form ofAME, 11/3-HSD activity appears normal, but decreased 5/3-reductase a c t i v i t y r e s u l t s in a l o w e r c l e a r a n c e o f c o r t i s o l p r o d u c i n g s y m p t o m s i d e n t i c a l to t h o s e s e e n in t y p e I p a t i e n t s [6]. T h i s i m p l i e s that: m u t a t i o n s in t h e 5 / 3 - r e d u c t a s e g e n e m a y r e s u l t in t h e A M E t y p e I I s y n d r o m e a n d s u g g e s t s t h a t 5 / 3 - r e d u c t i o n is also p a r t o f t h e m e c h a n ism protecting the mineralocorticoid receptor from o c c u p a t i o n b y g l u c o c o r t i c o i d s . S t u d i e s are c u r r e n t l y u n d e r w a y in o u r l a b o r a t o r y to i d e n t i f y m u t a t i o n s in t h e 1 1 / 3 - H S D 2 g e n e in A M E p a t i e n t s a n d to d e t e r m i n e whether glucocorticoids modulate 5/3-reductase gene expression. Acknowledgements--The authors are in debt to Professor John Funder for ideas and stimulating discussion, and to Alicia SteinOakley, Julie Maguire and Dr John Dowling for assistance with immunostaining studies.

REFERENCES 1. Funder J. W., Pearce P. T., Smith R. and Smith A. I.: Mineralocorticoid actioJa: target tissue specificity is enzyme, not receptor, mediated. Science 242 (1988) 583-585.

2. Edwards C. R., Stewart P. M., Burr D., Brett L., McIntyre M. A., Sutanto W. S., deKloet E. R. and Monder C.: Localisation of 11 beta-hydroxysteroid dehydrogenase--tissue specific protector of the mineralocorticoid receptor. Lancet 2 (1988) 3. 986-989. Krozowski Z. S. and Funder J. W.: Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc. Nam. Acad. Sci. U.S.A. 80 (1983) 6056--6060. 4. Naray-Fejes-Toth A., Watlington C. O. and Fejes-Toth G.: 11 beta-Hydroxysteroid dehydrogenase activity in the renal target cells of aldosterone. Endocrinology 129 (1991) 17-21. 5. Stewart P. M., Wallace A. M., Valentino R., Burt D., Shackleton C. H. and Edwards C. R. W.: Mineralocorticoid activity of liquorice: llbeta-hydroxysteroid dehydrogenase comes of age. Lancet ii (1987) 821-824. 6. Ulick S., Tedde R. and Wang J. Z.: Defective ring-A reduction of cortisol as the major metabolic error in the syndrome of apparent mineralocorticoid excess. J. Clin. Endocr. Metab. 74 (1992) 593-599. 7. Morris D. J., Semafuko W. E., Latif S. A., Vogel B., Grimes C. A. and Sheff M. F.: Detection of glycyrrhetinic acid-like factors (GALFs) in human urine. Hypertension 20 (1992) 356-360. 8. Walker B. R., Aggarwal I., Stewart P. M., Padfield P. L. and Edwards C.: Endogenous inhibitors of 11 beta-hydroxysteroid dehydrogenase in hypertension. J. Clin. Endocr. Metab. 80 (1995) 529-533. 9. Mercer W. R. and Krozowski Z. S.: Localization of an 11 beta hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology 130 (1992) 540-543. 10. Walker B. R., Campbell J. C., Williams B. C. and Edwards C. R.: Tissue-specific distribution of the NAD(+)-dependent

464

11.

12.

13.

14.

15.

16.

17. 18. 19.

20.

21.

22.

Z. K r o z o w s k i et al. isoform of 11 beta-hydroxysteroid dehydrogenase. Endocrinology 131 (1992) 970-972. Rusvai E. and Naray-Fejes-Toth A.: A new isoform of ll-betahydroxysteroid dehydrogenase in aldosterone target cells, ft. Biol. Chem. 268 (1993) 10,717-10,720. Brown R. W., Chapman K. E., Edwards C. and Seckl J. R.: Human placental 11-beta-hydroxysteroid dehydrogenase-evidence for and partial purification of a distinct NAD-dependent isoform. Endocrinology 132 (1993) 2614-2621. Stewart P. M., Murry B. A. and Mason J. I.: Human kidney 11 beta-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform, ft. Clin. Endocr. Metab. 79 (1994) 480-484. Tannin G. M., Agarwal A. K., Monder C., New M. I. and White P. C.: The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. ft. Biol. Chem. 266 (1991) 16,653-16,658. Duperrex H., Kenouch S., Gaeggeler H. P., Seckl J. R., Edwards C.,, Farman N. and Rossier B. C.: Rat Liver llbetahydroxysteroid dehydrogenase complementary deoxyribonucleic acid encodes oxoreductase activity in a mineralocorticoidresponsive toad bladder cell line. Endocrinology 132 (1993) 612-619. Albiston A. L., Obeyesekere V. R., Smith R. E. and Krozowski Z. S.: Cloning and tissue distribution of the human 11 betahydroxysteroid dehydrogenase type 2 enzyme. Molec. Cell Endocr. 105 (1994) RI1-R17. Krozowski Z.: l lbeta-Hydroxysteroid dehydrogenase and the short-chain alcohol dehydrogenase (SCAD) superfamily. Molec. Cell Endocr. 84 (1992) C25-C31. Krozowski Z.: The short-chain alcohol dehydrogenase superfamily: variations on a common theme. J. Steroid Biochem. Molec. Biol. 51 (1994) 125-130. Wu L., Einstein M., Geissler W. M., Chan H. K., Elliston K. O. and Andersson S.: Expression cloning and characterization of 17beta-hydroxysteroid dehydrogenase Type 2, a microsomal enzyme possessing 20alpha-hydroxysteroid dehydrogenase activity. J. Biol. Chem. 268 (1993) 12,964-12,969. Agarwal A. K., Mune T., Monder C. and White P. C.: NAD( + )-dependent isoform of 11 beta-hydroxysteroid dehydrogenase---cloning and characterization of cDNA from sheep kidney. J. Biol. Chem. 269 (1994) 25,959-25,962. Naray-Fejes-Toth A. and Fejes-Toth G.: Expression cloning of the aldosterone target cell-specific 1lbeta-hydroxysteroid dehydrogenase from rabbit collecting duct cells. Endocrinology 136 (1995) 2579-2586. Krozowski Z., Maguire J. A., Stein-Oakley A. N., Dowling J., Smith R. E. and Andrews R. K.: Immunohistochemical localization of the llbeta-hydroxysteroid dehydrogenase type II enzyme in human kidney and placenta. J C E M 80 (1995) 2203-2209.

23. Heffernan M. and Dermis J. W.: Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. Nucl. Acids Res. 19 (1991) 85-92. 24. Kozak M.: At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Molec. Biol. 196 (1987) 947-950. 25. Higgins D. G. and Sharp P. M.: Fast and sensitive multiple sequence alignments on a microcomputer. C A B I O S 5 (1989) 151-153. 26. Ozols J.: Lumenal orientation and post-translational modifications of the liver microsomal 11 beta-hydroxysteroid dehydrogenase, ft. Biol. Chem. 270 (1995) 2305-2312. 27. Albiston A. L., Obeyesekere V. R., Smith R. E. and Krozowski Z. S.: Cloning of the llbetaHSD type H enzyme from human kidney. Endocr. Res. 21 (1995) 399-409. 28. Rundle S. F., Funder J. W., Lakshmi V. and Monder C.: The intrarenal localization of mineralocorticoid receptors and 11-beta dehydrogenase: immunocytochemical studies. Endocrinology 125 (1989) 1700-1704. 29. Krozowski Z. S., Rundle S. E., Wallace C., Castell M. J., Shen J. H., Dowling J., Funder J. W. and Smith A. I.: Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence. Endocrinology 125 (1989) 192-198. 30. Kenouch S., Alfaidy N., Bonvalet J. P. and Farman N.: Expression of 1lbeta-Ohsd along the nephron of mammals and humans. Steroids 59 (1994) 100-104. 31. Funder J. W., Pearce P. T., Smith R. and Campbell J.: Vascular type I aldosterone binding sites are physiological mineralocorticoid receptors. Endocrinology 125 (1989) 2224-2226. 32. Kornel L.: The role of vascular steroid receptors in the control of vascular contractility and peripheral vascular resistance. J. Steroid Biochem. Molec. Biol. 45 (1993) 195-203. 33. Walker B. R., Connacher A. A., Webb D. J. and Edwards C.: Glucocorticoids and blood pressure~a role for the cortisol/cortisone shuttle in the control of vascular tone in man. Clin. Sci. 83 (1992) 171-178. 34. Sudhir K., Jennings G. L., Esler M. D., Korner P. I., Blombery P. A., Lambert G. W., Scoggins B. and Whitworth J. A.: Hydrocortisone-induced hypertension in humans: pressor responsiveness and sympathetic function. Hypertension 13 (1989) 416--421. 35. Walker B. R., Stewart P. M., Shackleton C., Padfield P. L. and Edwards C.: Deficient inactivation of cortisol by ll-betahydroxysteroid dehydrogenase in essential hypertension. Clin. Endocr. 39 (1993) 221-227. 36. Soro A., Ingrain M. C., Tonolo G., Glorioso N. and Fraser R.: Evidence of coexisting changes in 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activity in subjects with untreated essential hypertension. Hypertension 25 (1995) 67-70.