Aldosterone biosynthesis in bovine, rat and human adrenal: commonalities and challenges

Molecular and Cellular Endocrinology, 78 (1991) C119-Cl24 Q 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50

MOLCEL

Cl19

02569

At the Cutting Edge

Aldosterone

biosynthesis in bovine, rat and human adrenal: commonalities and challenges Jiirg Miiller

Steroid Laboratol?: Department

ofMedicine, (Accepted

Key words: Cytochrome

Multifunctional

P-4.50,,,;

steroidogenic

Corticosterone

methyl

enzymes

J, Miiller, University

8 May 1991)

oxidation;

A generation ago, many endocrinologists (myself included) thought that there were many different steroidogenic enzymes, with a specific enzyme responsible for each step in the biosynthetic pathways leading to various steroid hormones. Pregnenolone, for example, was considered to be produced from cholesterol by the sequential actions of ‘22K_hydroxylase’, ‘2Oa-hydroxylase’ and ‘20,22-lyase’. Similarly, the three-step conversion of deoxycorticosterone to aldosterone was assumed to be regulated by three different enzymes, i.e. ‘11/3-hydroxylase’, ‘l%hydroxylase’ and ‘18-hydroxydehydrogenase’. Moreover, the pattern of urinary and plasma steroids observed in patients with congenital adrenal hyperplasia indicated the existence of two different substratespecific or zone-specific ‘1 ~~-hydro~lases’ (Zachmann et al., 1971; Levine et al, 1980) and ‘21-hydroxylases’ (Kuhnle et al., 1981). During the last decade, the major steroidogenie enzymes have been successfully isolated and structurally identified by molecular biological techniques (Miller, 1988). The results of these investigations allow the foIlowing generalizations: (11 The number of steroidogenic enzymes is relatively small.

Address for correspondence: Department of Medicine, Zurich, Switzerland.

Unkersity Hospital, CH-8091 Zurich, Switzerland

Steroid Laboratory, Hospital, CH-8091

Zona

glomerulosa;

Zona

fasciculata

(2) Most of them catalyze more than one biosynthetic step. (3) Within each animal species, they are identical in different steroid hormone-producing glands and cells. Thus, cytochrome P-450,,, the cholesterol side-chain cleaving enzyme, catalyzes all three steps involved in the conversion of choIestero1 to pregnenolone (Shikita and Hall, 1973a, b; Hume et al., 1984). Adrenocortical cytochrome P-450,,,, which controls 17n-hydroxylation in the cortisol biosynthetic pathway, is identical to that regulating 17~-hydro~lation and the 17,20-lyase reaction in the androgen biosynthetic pathway in the testis (Zuber et al., 1986a, b; Chung et al., 1987). Both reactions required for the conversion of pregnenolone to progesterone, i.e. 3/Lhydroxysteroid dehydrogenation and steroid-S-ene-4-ene isomerization, are also catalyzed by a single enzyme (Ford and Engel, 1974; Ishii-Ohba et al., 1986). The most versatile steroidogenic enzyme, however, appears to be bovine cytochrome PPreparations of this enzyme, isolated from 4%,* adrenocortical mitochondria, purified to electrophoretic homogeneity and reconstituted with adrenodoxin and adrenodoxin reductase, have been found to catalyze all of the following reactions: (1) Il/3-h&~~~&~tiun of deoxycorticosterone (DOO, DOC-21-sulfate (Ingelman-Sundberg et al., 19781, ll-deoxycortisol or androstenedione (Sat0 et al., 1978), (2) 28-hydroxylution of DOC (Sate et al., 1978;

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Watanuki et al., 1978) or corticosterone (Wada et al., 1984; Yanagibashi et al., 1986). (3) ~9-~y~~~~y~~~~u~ of androstenedi~ne (Sate et al., 1978) or lo-hydro~-DOC (Okamoto et al.. 1982) (4) 6&hy&oxytntion of androstenedione (Ingelman-Sundberg et al., 1978) (5) conl’ersion to aldosterone of corticosterone (Wada et al., 1984) or l%hydroxycorticosterone (Wada et al., 1985; Yanagibashi et al., 1986) and (6) aromatization of 19-oxoandrostenedione (Suhara et al., 1986). Enzymolo~ biosynthesis

of the

final

steps

of aldosterone

The observation that bovine and porcine cy tochromes P-450, rs were capable of catalyzing a two-step conversion of corticosterone to aldosterone (Wada et al., 1984; Yanagibashi et al., 1986) appeared to invalidate previous theories about a specific aldosterone-producing enzyme. The urinary steroid patterns observed in patients with inherited disorders of the terminal portion of the aldosterone biosynthetic pathway, as well as experimental evidence (obtained largely from studies in rats) had suggested the existence of a ‘corticosterone methyl oxidase’ (Ulick, 1976) or a ‘cytochrome P-450,,,,’ (Hornsby and Crivello, 19X3), i.e. a mitochondrial mixed-function oxidase occurring only in the zona glomerulosa and catalyzing exclusively the conversion of corticosteronc to 18-hydroxcorticosterone (l&OH-B) and to aldosterone. During the purification of and bovine cytochrome P-450, Iti from porcine adrenocortical tissue, Yanagibashi et al. (1986) found no aldosterone-producing activity in extramitochondr~al fractions or in mito~hondrial fractions not containing Il.&/ lo-hydroxylase activity; they thus concluded that cytochrome Pwas the only enzyme capable of synthesiz450,,, ing aldosterone in these glands. In addition, an antibody raised against cytochrome P-4.50, ,p inhibited the conversion of corticosterone to 18OH-B and to aldosterone by reconstituted zona glomerulosa mitochondrial extracts. Intact mitochondria from bovine or porcine zona fasciculata were reported to convert corticosterone to aldosterone either at an immeasur-

ably low rate, or at least at a substantially lower rate than zona glomerulosa mitochondria, in spite of similar 1 I,& and lo-hydroxylase activities (Yanagibashi et al., 1986; Ohnishi et al., 1988). In contrast, solubilized and reconstituted mitochondrial extracts from the two zones were equally active in catalyzing all three steps in the conversion of deoxycorticosterone to aldosterone. This finding was interpreted as indicating that cytochrome P-450,, p from both zones had the same intrinsic aldosterone-producing activity, but that within zona fasciculata mitochondria this activity was selectively suppressed by unknown local inhibitors. The situation was completely different in the rat adrenal. Neither solubilized and reconstituted zona fasciculata extracts, nor a purified zona fasciculata cytochrome P-450, ,B preparation, converted deoxycorticosterone or corticosterone to aldosterone (Ohnishi et al., 1988; Lauber and Miiller, 1989; Ogishima et al., 1989b). Aldosterone-producing activity was restricted to zona glomerulosa mitochondria and was clearly due to the presence of a second form of cytochrome P-450,,,. This enzyme differed from the major form of rat cytochrome P-450,,, (molecular weight 51,000) by a lower molecular weight (49,O~) and by a broader range of catalytic activities, which included corticosterone methyl oxidations 1 and 2 (Lauber and Mullet-, 1989; Ogishima et al., 1989b). The enzyme was first discovered as a mitochondrial protein by Meuli and Miller (1983) and was later found to crossreact with a monoclonal antibody raised against bovine adrenocortical cytochrome P-450, ,p (Lauber et al., 1987). The extensive structural similarity between the 51 kDa and the 49 kDa forms of the enzyme on peptide mapping after partial proteolysis suggested that the 49 kDa protein might be formed from the 51 kDa protein by a posttranslational processing mechanism (Lauber and Miiller, 1989). This hypothesis was consistent with the observation that during potassium repletion of potassium-deficient rats. the gradual appearance of the 49 kDa protein coincided with a progressive loss of immunorcactivity and enzymatic activity of the zona glomerulosa 51 kDa protein. The possibility of the two forms deriving from a single precursor was laid to rest, however, when two

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separate groups of investigators succeeded in cloning and sequencing cDNAs encoding the two forms of the enzyme (Nonaka et al., 1989; Imai et al., 1990; Matsukawa et al., 1990). The deduced amino acid sequences show extensive homology (83%) but also distinct differences, which clearly indicate that the 51 kDa and the 49 kDa proteins are encoded by two different genes. Long-term in the rat

regulation

of aldosterone

biosynthesis

From a variety of experimental studies, it has long been accepted that the two final steps of aldosterone biosynthesis in the rat zona glomeruRosa are rate-limiting, variable and subject to complex and multifactorial physiological control (Miilier, 1988). The first indication of a molecular basis for these mechanisms was the gradual appearance of a zona glomerulosa mitochondrial protein accompanying the gradual increase in corticosterone methyl oxidase 1 and 2 activity during potassium repletion of potassium-deficient rats (Meuli and Miiller, 1983). Later, the same mitochondrial 49 kDa protein was found to be increased during sodium restriction and to appear in cultured rat zona glomerulosa cells simultaneously with the induction of aldosterone biosynthesis by high extracellular potassium (Meuli and Miller, 1984; Miiller et al., 1989). In view of the recently established identity of the 49 kDa protein with the aldosterone-producing species of rat cytochrome P-450,,,, the factors controlling its expression are potentially of great importance in terms of the adaptation of aldosterone secretion to changes in sodium and potassium intake. The marked increase in the zona glomeruIosa level of cytochrome P-450, rp mRNA with potassium repletion of potassium-deficient rats suggests that potassium controls the biosynthesis of the 49 kDa enzyme probably at the transcriptional level (Lauber et al., 1990). This mechanism is consistent with the time course of the reappearance of aldosterone-biosynthetic activity and the mitochondrial 49 kDa protein, during both potassium repletion in vivo and potassium-induced aldosterone biosynthesis in cultured zona glomerulosa cells in vitro. There is indirect evidence that two separate

control systems independently regulate the expression of the two genes encoding the two forms of cytochrome P-450, I8 in rat zona glomerulosa cells. Thus, potassium repletion has no effect on the mitochondrial content of 51 kDa protein, but induces the 49 kDa protein (Meuli and Miiller, 1983). Given alone, ACTH induces only the biosynthetic activities characteristic of the 51 kDa enzyme in cultured zona glomerulosa cells (Miiller et al., 1991). Prolonged treatment of rats with a high dose of ACTH, however, results in alterations of zona glomerulosa steroid biosynthesis suggestive of induction of the 51 kDa enzyme and repression of the gene encoding the 49 kDa enzyme (Miiller, 1978; Lauber et al., 1990). The rat model

As shown in Fig. 1, the existence of two cytochrome P-450, ,p enzymes, with different catalytic activities, fully explains the zona glomeruloss specificity of aldosterone secretion in the rat adrenal cortex. Expression of the gene encoding the 49 kDa enzyme appears to be a characteristic of zona glomerulosa cells not shared by the cells of the inner zones. The variabili~ of the expression of this gene, and its control by specific stimulators - and perhaps also inhibitors - probably both play an important role in the weli-known rat rona glomerulosa

18-OH-DQC

-

zona fasciculata

I

18-OH-3 I

18-W

-DOC

I

Aldo cytochromes Fig. 1. Biosynthetic steps (hatched arrows) and the

P-45O,,p

catalyzed by the 51 kDa form 49 kDa form (black arrows) of

cytochrome P-450,,, in the glomerulosa and fasciculata ZOIKS of the rat adrenal cortex. DOC, deoxycorticosterone; B, corticosterone; I8-OH-DOC, lo-hydro~-11-deo~corticosterone; l&OH-B, 18~hydro~corticosterone; Aido, aldosterone.

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variability of the response in aldosterone secretion to a constant dose of a given stimulator, and the dependence of this response on sodium and potassium intake.

The bovine model

Two different ~yto&hrome P-450,,, enzymes encoded by two different genes are also found in the bovine adrenal cortex. However, they have similar catalytic activities and are evenly distributed among the different zones of the gland (Fig. 2). Hashimoto et al. (1989) detected at least four different potential genes for bovine cytochrome P-450,,,. Two of them turned out to be pseudogenes, encoding non-functional truncated forms of the enzyme. Morohashi et al. (1987) and Kirita et al. (1988) succeeded in cloning and sequencing two different cDNAs encoding two distinct forms of complete bovine cytochrome P-450,,,. Later, the corresponding proteins, with molecular weights of 49,500 and 48,500, were isolated from adrenocorticai mitochondria (Ogishima et al., 1989aI. Both forms of the enzyme were found in a single gtand at almost equal amounts in all zones of the adrenal cortex. In reconstituted enzyme systems, both proteins converted DOC to

zona glomerulosa

1a-uH-ouc

ggg

la-OH-8

18-OH-OOC

I cyioctiromes

P -450,,p

Fig. 2. Biosynthetic steps catalyzed by the 49.5 kDa form (hatched arrows) and the 48.5 kDa form (black arrows) of cytochrome P-450, ,p in the glomerulosa and fasciculata zones of the bovine adrenal cortex. See legend to Fig. 1 for an explanation of abbreviations.

corticosterone, 1%OH-DOC, 18-0)3-B and aldosterone, The 49.5 kDa form was three times as active as the 48.5 kDa form in the production of aldosterone and l&OH-B but somewhat less active in the production of corticosterone and 1% OH-DOC. Mitochondria of transfected COS-7 cells expressing either cDNA converted DOC to corticosterone, 1X-OH-DOC, 19-OH-DOC and l&OH-B; corticosterone to aldosterone; and 1Ideoxycortisol to cortisol (Morohashi et al., 1990). In the intact bovine adrenal gland, the zona glomerulosa specificity of aldosterone production cannot be explained by the zonal distribution of the two forms of cytochrome P-450,,,. The selective suppression of the aldosterone-synthetic activity inherent to both forms of the enzyme in zona fasciculata mitochondria must be due to yet unknown local factors. In this regard, it is possible that phospholipids, calmodulin and ascorbic acid may play important roles in the mitochondrial regulation of the final step in aldosterone biosynthesis. The addition of phospholipids to a reconstituted system of purified bovine cytochrome P-450 1Ip markedly stimulates the conversion of corticosterone to aldosterone and to a lesser extent - the conversion of corticosterone to 18-OH-B (Ohnishi et al., 1984). According to Ogishima et al. (1989a), the presence of a phospholipid (phosphatidylcholine) is essential for the aldosterone-producing activity, but not for the lip- and Whydroxylase activities of reconstituted bovine cytochrome P-450 preparations. Under similar experimental conditions, calmodulin decreases the conversion of corticosterone to aldosterone, while increasing the conversion of corticosterone to l&OH-B (Ohnishi et al., 1986). In the presence of ascorbate and NADH, bovine adrenocortica1 mitochondria CORvert DOC to aldosterone at an increased rate, whereas the conversion of DOC to corticosterone, l&OH-DOC and 1%OH-B remains unaltered (Yanagibashi et al., 1990). In addition, these authors showed that the concentration of semidehydroascorbate reductase is considerably higher in zona glomerulosa mitochondria than in zona fasciculata mitochondria, so that ascorbate may facilitate the last step of aldosterone biosynthesis in zona glomerulosa mitochondcia by providing a source of reducing equivalents.

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A human

model?

The gene encoding human cytochrome Pwas localized on the long arm of chromo450,,, some 8 by the use of a partial cDNA clone from a human adrenal library (Chua et al., 1987). Later, this gene was isolated together with a linked second gene (Mornet et al., 1989); the nucleotide sequences of these two genes are 95% homologous in coding regions. Screening of total human adrenal RNA with specific oligonucleotide probes indicated that only one of these two genes was expressed. However, Kawamoto et al. (1990) have succeeded in isolating a cDNA clone corresponding to the second gene from a library derived from a human aldosterone-producing adrenocortical adenoma. When cDNAs encoding the two forms of human cytochrome P-450,,, were expressed in COS-7 cells, both enzymes showed a similar activity in catalyzing the conversions of DOC to corticosterone and 18-OH-DOC. However, only the ‘tumor form’ of the enzyme converted DOC to aldosterone to a measurable extent, and was considerably more active in converting DOC to 18-OH-B. In the light of these observations, the human adrenal cortex appears to correspond much more closely with the ‘rat model’ (Fig. 11 than with the ‘bovine model’ (Fig. 2). A final cautionary note, however, should be sounded, in that the validity of the rat model in man has yet to be directly tested. For example, at present, there is no evidence that the expression of the gene encoding the second, aldosteroneproducing form of cytochrome P-450,,, is restricted to the zona glomerulosa - nor even that the gene is transcribed at all in non-neoplastic adrenocortical cells. Acknowledgements

The author’s recent work cited in this article was performed in collaboration with Markus Lauber, Christoph Schmid and Marianne BiiniSchnetzler and has been supported by Research Grants No. 3.882-0.85 and 32.25326.88 from the Swiss National Foundation for Scientific Research. The skillful technical assistance of Heidi Seiler, Gaby Haesler-Vetsch, Dora Schmid and

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