Eur. J. Biochem. 249, 355-360 (1997) 0 FEBS 1997

Minireview series The 11P-hydroxysteroid dehydrogenase system, a determinant

of glucocorticoid and mineralocorticoid action

Function, gene organization and protein structures of llp-hydroxysteroid dehydrogenase isoforms Udo C. T. OPPERMANN, Bengt PERSSON and Hans JORNVALL Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

(Received 26 May125 July 1997) - EJB 97 075310

Enzymatic interconversion of active and inactive glucocorticoid hormone is important, and is carried out physiologically by 1 1P-hydroxysteroid dehydrogenase (1 1P-HSD) isoforms, explaining their role in cellular and toxicological processes. Two forms of the enzyme, 11P-HSD-1 and 11P-HSD-2, belonging to the protein superfamily of short-chain dehydrogenases/reductases, have been structurally and functionally characterised. Although displaying dehydrogenase and reductase activities in v i m , the dominant in vivo function of the type-I enzyme might be to work as a reductase, thus generating active cortisol from inactive cortisone precursors. On the other hand, for adrenal glucocorticoids the type-2 enzyme seems to be exclusively a dehydrogenase and, by inactivating glucocorticoids, confers specificity to peripheral mineralocorticoid receptors.

Keywords: glucocorticoid metabolism; 1 IP-hydroxysteroid dehydrogenase; hydroxysteroid dehydroge- nase; short-chain dehydrogenaselreductase; glucocorticoid therapy.

Recent discoveries emphasise the importance of steroid me- tabolism in the cellular and molecular control of hormone action. Hydsoxysteroid dehydrogenases (HSD) play a key role in these events because hydroxyl and keto groups often are the activity- determining chemical functions of steroid hormones. This estab- lishes these enzymes as important prereceptor regulators of sig- nalling pathways, in particular HSD acting on steroid hydroxyl groups at positions 3a, 3P, 118 and 17P, as ‘chemical switches’ for ‘receptor-active’ and ‘receptor-inactive’ hormone forms [ 1 - 51. These developments in the fields of endocrinology and me- tabolism offer new insights into physiological and pathophysio- logical mechanisms and give hope for an improvement in the pharmacotherapy of steroid-hormone-related diseases, including hormone-dependent cancer forms, cardiovascular diseases and inflammatory processes. This review focusses on the enzyme 1 1P-hydroxysteroid dehydrogenase (1 1P-HSD) and summarises our knowledge of the different forms of 11P-HSD and their in- volvement in glucocorticoid and xenobiotic phase-I metabolism.

Forms and functions of lla-HSD

The NAD(P)H-dependent interconversion of ‘receptor- active’ glucocorticoids secreted by the adrenals (cortisol in hu-

Correspondence to U. C . T. Oppermann, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stock- holm, Sweden

Fax: f 4 6 8 33 74 62.

man, corticosterone in rodents) to the ‘inert’ 11-0x0 congeners (cortisone in human, dehydrocorticosterone in rodents) is achieved by the enzyme 11P-HSD (Fig. 1) and has been de- scribed several decades ago [6]. Pioneering work by Monder and White and their groups [ l , 7-11], followed by others [12-171 gave a detailed characterisation of 11P-HSD enzymes. Thus far, two isoforms (1 1P-HSD-I and 11P-HSD-2) have been isolated and analysed at a molecular level from different mammalian spe- cies. For both isoforms, the primary structures have been deter- mined by a combined approach involving molecular biology and protein chemistry [ l , 7, 13, 171. In primary-culture systems, with close to in vivo situations, the two 11P-HSD forms seem to act in an antagonistic manner, i.e. type 1 acts mainly as an ll-oxo- reductase, producing active hormone [12], and type 2 works as an NAD’-dependent 1 1P-dehydrogenase, inactivating the active hormone cortisol (corticosterone). The precise tissue-specific physiological roles of 11P-HSD-1 are however still obscure and have to be defined [ 181. Another physiological function of 11p- HSD-1 might be its involvement in xenobiotic carbonyl reduc- tion [19].

The role of 11P-HSD-2 as a ‘protector’ of mineralocorticoid receptors against occupancy of high levels of circulating corti- sol, which if not inactivated would act as a mineralocorticoid- receptor ligand instead of the mineralocorticoid aldosterone, is established by genetic analysis of patients with the rare type-I1 ‘apparent mineralocortiocid excess syndrome’, having defects in the 11P-HSD-2 gene [8].

Gene and protein structures of llp-HSD E-mail: udo.oppermann@mbb.ki.se Abbreviations. HSD, hydroxysteroid dehydrogenase ; ER, endoplas-

mic reticulum: SDR. short-chain dehvdrogenase/reductase. Human and rodent genes for 11P-HSD-1 and 11P-HSD-2 Enzyme. 1 l,&Hy&oxysteroid dehydrogenase (EC 1 .I. 1.146). have been cloned and their chromosomal localisations deter-

356 Oppermann et al. (Eus J. Biochem. 249)

Cortisone Cortisol

4

I 1 P-HSD

0

Dehydrocorticosterone Corticos terone

Fig. 1. lip-Hydroxysteroid dehydrogenase catalyses the NAD(P)H dependent interconversion of cortisol (active hormone) and cortisone (inactive hormone) in humans, and of corticosterone (active hormone) and dehydrocorticosterone (inactive hormone) in rodents, thus constituting a prereceptor mechanism of hormonal control.

1lP-HSD-1

transmembrane SDR domain domain

1 1 P-HSD-2

untrunszated

transcription products

gene

transcription products

protein

transmembrane SDR domain domain

Fig. 2. Genomic organisation of the human type-1 and type-2 ll,&HSD genes. Differential splicing and promoter usage give rise to three forms of 11P-HSD-I in vertebrates (IA, 1B and 1C). Translation products of 11P-HSD-lB and 11p-HSD-IC have not been described. No splicing variant of the type 2 enzyme has been detected.

Oppermann et al. ( E m J . Biuchern. 249) 357

Fig. 3. Primary- and tertiary-structure elements important in ll/?-HSD and SDR enzymes, demonstrated by comparison of the human ll/?- HSD isozyme sequences (derived from their cDNAs) with that of 3d20p-HSD from Streptomyces hydrogenans. Parts of the nucleotide-binding site (N), the active site (A) with a S-Y-K triad (asterisks), and of the seven /3 strands (arrows), corresponding to their positions in 3d20P-HSD are indicated in the sequence alignment (A) and the three-dimensional structure (B) of 3d20P-HSD (PDB code 2HSD; N and A in red, complexed NAD' in magenta). Asn-linked glycosylation sites in the 11P-HSD-1 and 11P-HSD-2 sequences are boxed. The transmembrane segments in 118 HSD most proximal (M) to the SDR-core domain were identified by the TMAP program [34]. The 11P-HSD-2 sequence contains two further possible transmembrane segments (residues 4-47), an N-terminal segment (dotted) and a middle segment (dotted box), the latter being similar (9 identities of 15 residues; residues 33-47 compared with 53-67) to the third trammembrane domain. (A) Created with the program PRALIN; (B) made with the program ICM (version 2.6, Molsoft, Metuchen, NJ).

mined [7, 9, 171. The human 11P-HSD-1 gene (1lBHSD) con- sists of six exons, spanning approximately 9 kb, and maps to chromosome 1. On the other hand, the human 11P-HSD-2 gene (HSDIIK) consists of five exons, has an approximate size of 6.2 kb (Fig. 2), and is located on the long arm of chromosome 16 (16q22). In the case of 11P-HSD-1, differential splicing and promoter usage give rise to the transcription of three tissue-spe- cific products, designated 11P-HSD-1A, B and C [15, 201. Only the 11P-HSD-1A product seems to be translated into an enzy- matically active form [15], and this can be partially explained by the route of synthesis of the type-I enzyme and the general structure of short-chain dehydrogenasesheductases (SDR). The 1B and 1C mRNA species might be involved in the cell-specific regulation of expression of the 1A form or, if translated at all,

act as steroid-binding proteins, involved in intracellular hormone trafficking ; however, experimental proof for these alternatives is missing. Enzymatic activity for the 1B and 1C forms was not detectable when expressed in eukaryotic cells [15, 201. No such transcription multiplicity has been detected for the I IHSDK gene, where only two tissue-specific transcription start sites in the 5' untranslated region have been determined, further high- lighting the distant relationship between the two 11P-HSD genes. Fig. 2 illustrates schematically the gene structures and the resulting transcription and translation products of 11P-HSD-1

Structural analyses reveal that both enzyme forms belong to the protein superfamily of SDR, of which over 100 mechanisti- cally related enzymes have been characterised from prokaryotic

and 11P-HSD-2.

358 Oppermann et al. (Eul: J. Biochem. 249)

Table 1. Characteristics of mammalian 11P-HSD forms.

Form Reaction direction with Coenzyme Molecular Kinetic constants Locali- Modification Tissue distribution mdSS On with gluco- zation

adrenocorti- dexametha- SDWPAGE corticoids coids sone

kDa

Type 1 dehydrogenase reductase NAD(H), 34 low affinity ER glycosylation ; and reductase in vitro NAD(P)H (= 1 PM) further modifi-

cations pos- sible

Type 2 dehydrogenase dehydrogenase NAD(H) 40 high affinity ER gl ycosylation ? and reductase (= 10 nM) in vitro

widespread peripheral and central distribu- tion, mainly liver, kid- ney, lung, brain mainly MR-depen- dent epithelial tissues: distal tubules, parotis, salivary glands, colon

and eukaryotic sources [5, 7, 13, 211. All known three-dimen- sional structures reveal a similar one-domain folding pattern [21] (Fig. 3 B). Well-conserved structural elements form the ba- sis for SDR membership and are restricted to particular sequence segments, indicating a common fold, active site and reaction mechanism [21]. X-ray analysis, chemical modifications, site- directed mutagenesis and sequence comparisons established most of the conserved segments to form essential parts of the ‘Rossmann fold’ coenzyme-binding site and the catalytic center, often with a seven ,&stranded motif and a catalytically active ‘triad’ of Ser, Tyr and Lys residues [21, 251. These common structural SDR motifs, present in human lip-HSD-1 and l l p - HSD-2, are shown in Fig. 3. Monomeric and oligomeric SDR enzymes are described, but no experimental analyses are avail- able concerning the oligomerisation of 11P-HSD-1 and 1 lp-

Table 1 summarizes structural and functional characteristics of the type-I and type-2 11p-HSD forms. 11P-HSD-1 and I lp- HSD-2 share about 25 % amino acid identities in the about 260- residue SDR-core region. Both forms contain the common struc- tural SDR motifs centered around the nucleotide-binding site and the catalytic triad (Fig. 3) [21]. N-terminal extension of this SDR core by hydrophobic sequence segments (1 1p-HSD-1 A, =30 amino acid residues; 11P-HSD-2, =80 amino acid resi- dues) and Asn-linked glycosylation of 11P-HSD-lA result in the observed mass difference (“6 kDa) between the mature type- 1 and type-2 products. Importantly, this also determines their subcellular localisation in the endoplasmic reticulum (ER). The hydrophobic N-terminal segment or the C-terminal region of the type-2 enzyme extending the SDR-core domain (Fig. 3A), could be shorter than expected, since analysis of the corresponding cDNA predicts a molecular mass of 44 kDa rather than the ap- parent 40 kDa observed upon SDS/PAGE. The 11p-HSD-1A N- terminal segment contains a signal-anchor motif, which is re- sponsible for translocation and glycosylation of the polypeptide chain, since the 11p-HSD-1B form, lacking the N-terminal do- main, does not get translocated and cotranslationally modified. Thus, the role of the N-terminal hydrophobic domain is to con- tribute to a transmembrane domain, probably also responsible for ER retention, and to allow the lumenal N-linked oligosaccha- ride modification. Glycosylation, in turn, might be responsible for correct protein folding, achieved by ER chaperones such as binding protein, calnexin or calreticulin, and protein stability. Using rat 11p-HSD-1 cDNA it could be shown that mutation of glycosylation sites [I I] or inhibition of the glycosyl transferase step [lo] in a heterologous expression system severely affected enzymatic activity. The occurrence of Asn-linked carbohydrates in ll&HSD-l was the reason why a lumenal orientation was

HSD-2.

suggested, even in the absence of experimental evidence [22]. Existence of multiple enzyme species because of protein modifi- cation is possible [22] and a feature common to several SDR enzymes [21]. In contrast to the situation with the type-1 en- zyme, no modification has been detected for 11P-HSD-2, al- though motifs for Asn-linked oligosaccharide transfer are pre- sent in the primary structure. Inspection of the sequence [16] and analysis of fluorescent protein constructs [ 141 has given strong evidence that the bulk of the protein is facing the cytosolic com- partment and that it is anchored in the ER by the N-terminal hydrophobic domain. Further factors probably contribute to the cellular topology, since both isoforms have also been localised to the nuclear compartment [ 12, 231. Conflicting experimental evidence, including apparent lack of immunoreactivity against antisera, lack of detectable type-1 or type-2 mRNA transcripts, differential coenzyme dependence and differential substrate af- finities, suggest that further tissue-specific 1 ID-HSD forms exist [12], the latest examples being llP-HSD activity in lymphoid organs and choriocarcinoma cells [24, 261. Considering the multiplicity within the SDR family, this possibility of further 11p-HSD forms does not seem inconsistent.

Reaction direction of 11P-HSD

The fundamental importance of the preferential reaction di- rection catalysed by 11P-HSD forms is indicated by that this reaction can cause ‘switches’ between the active and the inactive forms of the glucocorticoid hormones. Since the reaction in prin- ciple is freely reversible, the preferential reaction direction of each isozyme form is crucial. There is general consensus that the type-2 form acts as a hormone-inactivating enzyme, since in vitro and in vivo data suggest it to function exclusively as an 11P-dehydrogenase of adrenocorticoids [8, 12, 181. However, with the synthetic glucocorticoid dexamethasone, dehydroge- nase and reductase activities of 1 ID-HSD-2 could be detected [27-291. The preferential reaction direction of the type-2 form, which, at least in vitro, can act as a reductase and dehydrogenase of cortisone and cortisol, respectively, seems to be dependent on cellular context, translational modifications, subcellular environ- ment, and substrate. Since 11p-HSD-I catalyses only reduction of 1 I-dehydrodexamethasone contrary to the type-2 form [27, 291, differential clinical effects between synthetic and endoge- nous glucocorticoids might in part be explained by the different metabolic properties of 11P-HSD isoforms. In most primary cul- ture systems, l@-HSD-1 acts as a reductase, thus suggesting the in vivo role to be an activator of cortisone precursors. However, upon cell disruption and homogenisation 1 ID-dehydrogenase ac- tivity is detectable in addition to the reductase activity, indicat-

Oppermann et al. (Eur: J. Biochem. 249) 359

ing the importance of cellular and local enzyme environments, such as redox state, coenzyme availability or microsomal topol- ogy [ 121. Differential recovery of dehydrogenase and reductase activities upon protein purification of the type-I form initially suggested the existence of two proteins. However, the successful cloning and heterologous expression of rat type 1 1 1P-HSD, and the analysis of the purified mouse type-I enzyme revealed that both activities reside within one protein [lo, 301. Distinct cata- lytic sites were then suggested to answer the unresolved question of differential loss of activity upon experimental manipulations. Distinct sites, however, seem unlikely, given the general molecu- lar architecture of SDR HSD enzymes. Instead, the explanation of the different dehydrogenaseheductase observations may be the occurrence of heterogeneities in the form of protein modifi- cations between enzyme forms catalysing the two reactions. In this context, site-directed mutagenetic replacement of single res- idues in the related SDR enzyme, 3P/17P-HSD from Comumo- nus testosteroni, results in the preferential loss of reaction direc- tion and suggests that critical sequence elements are responsible for the reaction direction in SDR proteins [25]. Furthermore, N- linked glycosylation might play a role, since mutagenetic re- placements of glycosylation sites reveal differential effects on the reaction direction in the rat type-1 enzyme [ I l l . Thus, glyco- sylation seems to be necessary for correct folding, stability and hence regulation of enzyme activity.

Differential hormonal regulation of dehydrogenase and re- ductase activities of the type-I enzyme was demonstrated re- cently in a primary-culture system of rat Leydig cells, where 11P-HSD-I seems to work primarily as a dehydrogenase inacti- vating cortisol [31]. Although the absence of simultaneous ex- pression of the type-2 enzyme was not completely ruled out in that study, but later work [32] and these results suggest a com- plex role of 11P-HSD forms in the cellular regulation of gluco- corticoid action. Furthermore, clinical studies reveal an impair- ment of hepatic 1 1P-hydroxysteroid dehydrogenase activity in alcoholic and chronic liver disease, which may partly explain the pseudo-Cushing symptoms observed in these patients and indicates the in vivo role of 1 1P-HSD in hepatic oxidative corti- sol metabolism [33].

Perspectives

Construction and characteiization of transgenic or knock-out animals, detection of further possible enzyme forms, analysis of factors influencing the reaction direction, and tertiary-structure determinations are the challenging tasks in the immediate future. They will improve our understanding of the specific roles that 11P-HSD isoforms play in the cellular physiology, and should aid in the design of specific agonists and antagonists of value in pharmacotherapy of glucocorticoid-related diseases.

Support by the Swedish Medical Research Council (project 13X- 3532 and 03P-11312), Deutsche Forschung.s~eme~nschu~t and Swedish Foundation for International Cooperation in Research and Higher Educa- tion is gratefully acknowledged.

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