J. Steroid Biochem. Molec. Biol. Vol. 47, No. 1-6, pp. 213-221, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain. Pergamon Press Ltd

S E P A R A T E I N D U C T I O N O F T H E T W O I S O Z Y M E S

O F C Y T O C H R O M E P-4501~ I N R A T A D R E N A L

Z O N A G L O M E R U L O S A C E L L S

JURG MOLLER* and MAJA OERTLE

Steroid Laboratory, Department of Internal Medicine, University Hospital, 8091 Zfirich, Switzerland

Snmmary- - In the rat adrenal cortex, two isozymes of cytochrome P-45011 # (CYP11B1 and CYPI IB2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their Y-flanking regions. CYPI 1B 1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 1 lfl- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and/n vitro, the expression of the genes encoding CYP11BI and CYPI 1 B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYPl lB1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYPI1B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion, In vitro, only CYP11B 1 activity was detectable and responsive to ACTH treatment in zona giomerulosa cells cultured at a potassium concentration of 6.4 mmol/l. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration (~> 12 mmol/l).

"11~-HYDROXYLASE", "ALDOSTERONE

SYNTHASE" AND "CORTICOSTERONE METHYL

OXIDASE"

When Conradin Meuli[1] observed a novel mitochondrial protein with an apparent Mw of 49,000 (49K protein), which was restricted to the zona glomerulosa of the rat adrenal cortex and which appeared and disappeared in parallel with aldosterone biosynthesis during exper- imental alterations of potassium intake, he was convinced that he had discovered "cortico- sterone methyl oxidase". Up to that time (1983), this was a purely hypothetical enzyme, assumed to catalyze the final two steps of aldosterone biosynthesis, i.e. the 18-hydroxylation of corticosterone and the 18-hydroxydehydrogena- tion of 18-hydroxyeorticosterone. The existence of such an enzyme---a third steroidogenic mito- chondrial cytochrome P-450--had been postu- lated on the basis of indirect experimental evidence obtained mainly in rats (for a review

Proceedings o f the l l th International Symposium of the Journal o f Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Seefeld, Austria, 30 May-2 June 1993.

*To whom correspondence should be addressed.

see [2]) and of clinical observations made in patients with inborn errors of aldosterone bio- synthesis [3, 4]. However, by 1990 it had been clearly established by immunological crossreac- tivity [5], by assaying catalytic activities of re- constituted purified enzyme protein [6, 7] and finally by the expression of eDNA on COS-7 cells [8], that the rat zona glomerulosa mito- chondrial 49K protein was not simply a "eorticosterone methyl oxidase" but rather an isozyme of "11 fl-hydroxylase" with an extended range of catalytic activities, which included corticosterone methyl oxidation 1 and 2. In contrast, the major form of rat "1 lfl-hydroxyl- ase" isolated from zona fasciculata mitoehon- dria converted deoxycorticosterone (DOC) only to eorticosterone and 18-hydroxy-DOC (18- OH-DOC).

Whereas Lauber and coworkers[5, 6] had considered the possibility that the aldosterone- producing form (49K) might be a derivative of the non-aldosterone producing form (51K) of the enzyme, generated by a posttranslational proteolytic process, the cloning and sequencing of complete complementary and genomic DNAs encoding rat "11 ]~-hydroxylase" (CYP 11B 1) and "aldosterone synthase"

213

214 JORG MOLLER and MAJ^ OEgTLE

(CYPllB2) clearly established that these are two different proteins encoded by two related but separate genes [9, 12]. The homology be- tween these two genes is much higher in their coding (88%) than in their 5'-flanking (52%) nucleotide sequences, indicating that their tran- scription is separately regulated by different mechanisms (Table 1). An evident manifestation of this separate regulation is the fact that C YPI IB! is expressed in all the zones of the rat adrenal cortex, but CYPIIB2 only in the zona glomerulosa [13]. A third rat CYPI lB gene, i.e. rat CYPIIB3, has been cloned from genomic DNA libraries [11, 14]. It is highly homologous to CYPIIB2 in its coding regions, but has almost the same sequence as the CYPIlB1 gene in its 5' upstream region. According to available evidence, the CYPIIB3 gene is not spon- taneously expressed and encodes a protein with- out steroidogenic activity.

Two isozymes of cytochrome P-450H~ corresponding in their catalytic activities to rat CYPllB1 and CYPIlB2 have also been found in human and mouse adrenal glands and in aldosterone-producing adrenocortical adenomas [15,19]. In cultured human zona glomerulosa cells, both CYPIlB1 and CYPIlB2 genes were expressed[17]. In the mouse adrenal zona glomerulosa, however, only transcripts encoding the aldosterone- producing isozyme were found by in situ hybrid- ization [19].

The situation is completely different in a number of other animal species, such as cattle and pigs. Thus, the major forms of bovine or porcine "1 lfl-hydroxylase", irrespective of zonal origin, display all the catalytic activities of rat CYPllB2 (including aldosterone pro- duction) upon extraction from mitochondria and reconstitution or upon expression in COS-7 cells[20-22]. Out of five different C Y P l l B genes isolated from bovine genome, 3 turned

out to be pseudogenes[23]. The 2 CYPllB isozymes expressed in the bovine adrenal cortex have similar catalytic activities and are equally distributed among the different zones [22, 24]. Apparently, their inherent aldosterone biosyn- thetic activity is suppressed in the inner zones of the adrenal cortex by a yet unknown intramito- chondrial mechanism.

ZONE SPECIFICITY OF ALDOSTERONE BIOSYNTHESIS AND ITS REGULATION

The concept of a functional zonation of the mammalian adrenal cortex, i.e. considering the zona glomerulosa the "mineralocorticoid- secreting gland" and the zona fasciculata the major "glucocorticoid-secreting gland" was proposed long before the discovery of aldoster- one on the basis of purely morphological observations[25-27]. This concept was later supported by direct evidence showing that the zona glomerulosa of the rat or beef adrenal cortex was the only source of aldosterone whereas the glucocorticoids corticosterone and cortisol were mainly produced by the zona fasciculata [28-30]. In species such as man, rat and mouse, this specificity is obviously due to the fact that the zona glomerulosa cell is the only cell in which the gene encoding the aldos- terone-producing type of cytochrome P-450.~, i.e. CYPIlB2, is expressed. In other animal species such as cattle and pig, the endogenous aldosterone-producing activity of the cyto- chrome(s) P-4501~ present in all the zones of the adrenal cortex is continuously blocked in the zona fasciculata and zona reticularis mitochon- dria [20, 21] by a yet unknown regulatory device.

In the rat adrenal cortex, there is a clear-cut functional zonation not only in secretory prod- ucts but also in the regulation of their bio- synthesis. Whereas the control of aldosterone

Table 1. Properties of rat cytochromes P-450Hp'

CYP11BI CYP11B2 ("1 lfl-Hydroxylase') ("Aldosterone synthase")

Genes

cDNA Mature Protein

Amino acids Molecular weight

Occurence in adrenal cortex Conversion of DOC to

88% Homology in coding regions 81% Homology in mature proteins 52% Homology in 5" flanking regions 2765 bp 2824 bp

475 476 54,608 54,273 All zones Zona giomerulosa Corticosterone Corticosterone 18-OH-DOC 18-OH-DOC 19-OH-DOC 18-OH-B

Aldosterone

"According to Okamoto and Nonaka [51] and Mukai et al. [12].

Induction of the 2 isozymes of P-450 ttB

production is unquestionably multifactorial and • • potentially involves a large number of different 3so stimulators and inhibitors, the biosynthetic ac- tivity of the zona fasciculata is regulated almost e8o exclusively by the stimulatory action of a single hormone, i.e. the pituitary peptide adreno- corticotropin (ACTH). In contrast to ACTH, 21o which enhances corticosteroid production equally in zona glomerulosa and zona fascicu- 14o lata cells, angiotensin II, potassium ions and serotonin stimulate steroidogenesis selectively in rat zona glomerulosa cells [31]. Similarly, atrial 7o natriuretic peptides blocks (stimulated) steroid production in zona glomerulosa cells, only [32]. The zone specificity of some of these regulators is less extensive in the bovine adrenal cortex than in the rat adrenal cortex. Thus, the pro- duction of cortisol by bovine zona fasciculata cells was found to be stimulated by angiotensin II [33] and to be inhibited by atrial natriuretic peptide [34].

THE ROLE OF ACTH

Beside its short-term stimulatory action on steroidogenesis in zona glomerulosa and zona fasciculata cells, ACTH also has a long-term effect on steroid hormone production by influ- encing the expression of genes encoding steroidogenic enzymes. In primary cultures of bovine adrenocortical cells, the expression of the genes encoding cytochromes P-450scc, P- 45011p, P450c21 and P-45017~, (i.e. CYPll,4, CYP l lB I, CYP21A and CYP17), adrenodoxin and 38-hydroxysteroid dehydrogenase/ A 5-4 isomerase is markedly increased--mainly at the transcriptional level--by ACTH [35-40].

Treatment of intact rats with a high dose of ACTH for 4 days led to a progressive decrease in the conversion of DOC to 18-hydroxycorti- costerone and aldosterone by zona glomerulosa tissue (Fig. 1, [41]), which was preceded by a complete disappearance of CYPllB2 mRNA and was accompanied by a biphasic increase in the zona glomerulosa CYPllB1 mRNA level (Fig. 2,[42]). Zona fasciculata CYPl lA and CYP11B1 mRNA levels were not altered. Treat- ment of sodium-deficient rats with dexametha- sone for 2 days led to decreases in zona glomerulosa and zona fasciculata CYPllB1, CYPllA1 and CYP21A mRNA levels (which were prevented by the simultaneous adminis- tration of ACTH), but did not affect the zona glomerulosa CYPI 1B2 mRNA level [42].

215

I T

O A

lO00

800

600

400

200

I 1 I 2 I 3 I 4 I ~ags o f ACT~.~ t reatment

Fig. 1. Effect of 4 days of ACTH treatment [ACTH(1-24)Zn, 0.2 mg/kg per day s.c.] of normally fed rats on the capsular conversions of tritiated DOC ([3H]DOC; 50nmol/ml) to aldosterone (Aldo, O), 18-hy- droxycorticosterone (18-OH-B, A), corticosterone (B, O) and 18-OH-DOC (A). Mean values (n =3) +SD are shown. Left scale Aldo and 18-OH-B production. Right

scale: B and 18-OH-DOC production. From Ref. [41].

POTASSIUM STATUS

In the zona glomerulosa of sodium-replete, potassium-deficient rats, the conversion of DOC to 18-hydroxycorticosterone and aldosterone was completely suppressed (Fig. 3), the mito- chondrial CYP11B2 protein was absent, and the level of CYPllB2 mRNA was unmeasurably low (Fig. 4, [1, 41,42]). Within 4 days of potass- ium repletion, aldosterone biosynthesis returned to normal (Fig. 3) and the CYP11B2 mitochon- drial 49K protein gradually reappeared [1, 6]. On the first day of potassium repletion zona glomerulosa CYPI 1B2 mRNA rose to 20 times the level seen in normally fed rats (Fig. 4, [42]). This high level was maintained during the fol- lowing days. At the same time, the zona glomerulosa CYP11B1 mRNA concentration gradually decreased to reach about 10% of the original level on day 4 of potassium repletion. Potassium depletion or repletion did not affect the zona fasciculata CYPllB1 mRNA or the zona glomerulosa or fasciculata CYPllA1 mRNA levels [41, 42].

SODIUM STATUS AND THE RENIN-ANGIOTENSIN SYSTEM

Dietary sodium restriction of rats led to an increased activity of the final steps of aldoster- one biosynthesis[43], an increased concen- tration of mitochondrial CYPllB2 protein

216

3.6 kb

JCrRG MOLLER and MAJA OERTLE

I z . g . z . f . r | ,

0 1 2 4 0 1 2 4

P 450aldo mRNA

f d a y s of

A C T H - t reatment

3.6 kb D , -

P 45011P, mRNA

I z , g . I z . f .

Fig. 2. Effect of 1, 2 and 4 days of ACTH treatment of normally fed rats on zona glomerulosa (z.g.) and zona fasciculata (z.f.) CYPI1B2 (P450mo) and CYPllB1 (P450ttp) mRNA levels. Northern blot

autoradiography. Data from Ref. [42]. 1, liver.

[44, 45] and increased total adrenal and zona glomerulosa levels of CYPllB2 mRNA[42, 46-48]. Total adrenal or zona fasciculata levels ofCYP11BI mRNA were not altered by sodium deficiency. However, we observed a marked decrease in this parameter in the zona glomeru- losa after 2 weeks of sodium restriction [42]. In reverse, substantially diminished CYPIlB2 ac- companied by markedly increased CYPllB1

?SO

600 !

150

T T I e I 3 I 4 I dags of KCI Orinking

Fig. 3. Effect of 4 days of potassium repletion of potassium- deficient rats on capsular adrenal conversions of tritiated DOC ([3H]DOC; 50nmol/ml) to aldosterone (Aldo, 0) , 18-hydroxycorticosterone (18-OH-B, &), eortieosterone (B, O) and 18-OH-DOC (A). Mean values (n = 3) +SD are

shown. From Ref. [41].

mRNA levels were found in the zona glomeru- losa of rats treated for 2 weeks with a mineralo- corticoid (deoxycorticosterone trimethyl acetate). Sodium deficiency or mineralocorti- coid treatment did not, however, affect the zona glomerulosa levels of CYPllAI or CYP21A1 mRNA. These observations indicate that the expression of CYP 11B 1 in the zona glomeru- losa is regulated not only by ACTH but also by unknown additional factors.

Treatment with a converting-enzyme inhibi- tor, completely prevented the increases in aldos- terone biosynthesis, mitochondrial CYPI1B2 protein and zona glomerulosa CYP11B2 mRNA levels induced in rats by sodium restric- tion [45, 48]. Converting enzyme inhibitors also blocked (totally or partially) increases in these parameters elicited by a high potassium intake. According to these observations, the increased expression of the CYPI1B2 gene elicited by these dietary measures is either mediated by angiotensin II or, at least, requires the presence of angiotensin II.

INFORMATION FROM A CELL CULTURE MODEL

Cellular mechanisms involved in the regu- lation of CYPI1B1 and C Y P I l B 2 gene ex- pression are studied more appropriately under in vitro than under in vivo experimental con- ditions. In primary cultures of rat adrenal zona glomerulosa cells CYPI 1B1 and CYP11B2 can be independently induced by ACTH and by

3.6 kb

Induction of the 2 isozymes of P-450.p

I z . g . z . f . ,.- ~ !

0 1 2 4 0 1 2 4

P 450aldo mRNA

f d a y s of

K repletion

3 .6 kb D,- P450,18 m R N A

I I I I z . g . I z,f .

Fig. 4. Effect of 1, 2 and 4 days of KC1 drinking of potassium depleted rats on zona glomerulosa (z.g.) and zona fasciculata (z.f.) CYPI 1 B2 (P 450alao ) and CYPI 1BI (P 4501]B) mRNA levels. Northern blot

autoradiography. Data from Ref. [42]. 1, liver.

217

a high extracellular potassium concentration, respectively.

Under baseline conditions (i.e. in a medium with a potassium concentration of 6.4 mmol/1), the cells lose their capacity of aldosterone biosynthesis completely within a few days of monolayer culture but continue to produce corticosterone and 18-OH-DOC from endogen- ous precursors or from added DOC [49, 50]. Upon increasing the potassium concentration in the medium to 12-32 mmol/1, the conversion of DOC to aldosterone and 18-hydroxycorticos- terone becomes measurable, but only after a delay of 24 h (Fig. 5). These two conversions increase more extensively during the next few days and reach a plateau after 4 to 7 days. The induction of aldosterone biosynthetic activity is associated with the appearance of CYPllB2 immunoreactive protein [50] and CYP11B2 mRNA (Fig. 6).

Addition of ACTH to the incubation medium ([K + ] = 6.4 retool/l) for 4 h led to an increase in CYPllBl- type activity, which continued for 2 days and returned to its original level within a week. This effect of ACTH could be imitated by added monobutyryl cyclic AMP and could be blocked by actinomycin D and cycloheximide (but not by nifedipine or atrial natriuretic pep- tide). The induction of CYP11B2-type activity by a high extracellular potassium concentration was inhibited by actinomycin D, cycloheximide, nifedipine and atrial natriuretic peptide. These observations indicate that within the rat zona

glomerulosa cell the induction of CYP 11B 1 by ACTH and the induction of CYP11 B2 by extra- cellular potassium are mediated by cyclic AMP and by intracellular calcium ions, respectively (Fig. 8).

Prolonged exposure to a high concentration of ACTH of zona glomerulosa cultured at a high potassium concentration did not suppress

0 A

3.0

T

0.6 7.8

0.4 1.2

o.2 l .rK:l I

..______,/,/ 7 - I 8 I 9 I m I 11 I

dags

Fig. 5. Effect of increasing the medium potassium concen- tration on the conversions of tritiated DOC (13H]DOC; 140~mol/ml) to aldosterone (hJdo, O), 18-hydroxycorti. costerone (18-OH-B, A), corticosterone (13, O) and 18-OH- DOC (A), Rat zona glomerulosa cells were kept in primary culture for 11 days. Potassium concentration was 6.4 mmol/l on days 1-6 and was raised to 32mmol/! on day 7. Radioactive precursor was added for I h. Mean values of 3

culture dishes + SEM. For methodology, see Ref. [50].

218 JORG M~LLER and MAJA OERTLE

Rat P-450aldo mRNA

.4.. 4 .8 kb

~ . 1.9 kb

1 2 3 4 5 6 7

K - - q, , i, F G L

A C T H I * I

Fig. 6. Effects of ACTH exposure (10-Smmol/1, for 4h, on day 4; lane 2), of a high potassium concentration in the culture medium (18 retool/1 on days 7 to 10; lane 3) or both (lane 4) on CYP11B2 (P 450~do) mRNA level in rat zona glomerulosa cells kept in primary culture. F: zona fasciculata, G: zona glomerulosa (fresh tissue from normally fed rats), L: liver. Northern blot autoradiography. Sec Ref. [42]

and [50] for methodology.

A Ido

1.40

1.05

O. 70

0.35

.~ ,,/'~ •

I ACTH, I O ' ' M I

/ __-o'" 'l w ~

7 9 I I

B 28

21

14

?

I

13 " 15 " " 1 7 19 21 23 25 da..qs

Fig. 7. Effect o£ a high potassium concentration and ACTH in the medium on the conversion of tritiated DOC ([3H]DOC; ]40 nmol/m]) to aldosteron¢ (Aldo, O) and co~icosterone (B, O) by cultured rat zona

glomerulosa cells. Mean values of 3 culture dishes + SEM. See Ref. [50] for methodology.

Induction of the 2 isozymes of P-450HB 219

I ACTH

c A M P

r a f a d r e n o l z o n a g lomer 'u los=9 c e l l I

n / f e d / p i n e - ~-

A N P - - = . - c y c / o h e x i m i d e , - ,= - - a c f i n o m l / c i n D - =

mRNA • = - - c, gclohexirnide

51K enzyme

DOC coPHcosfer'one

#":Tet 1 1÷

5c '9it L 1

m R N A

1 4 9 K e n z y m e

DOC a / d o s f e r o n e

Fig. 8. Hypothetical mechanisms of induction of CYPI 1BI (51K enzyme) by ACTH and of CYPI 1B2 (49K enzyme) by extracellular potassium concentration ([K + ],) in cultured rat zona glomerulosa cells. ANP: atrial natriuretic peptide, [Ca2+]~: intracellular calcium concentration, Based on unpublished

observations.

C Y P l l B 2 act iv i ty , a n d even h a d a t e m p o r a r y

s t i m u l a t o r y effect o n a l d o s t e r o n e b iosyn thes i s (Fig. 7). Acco rd ing ly , the nega t ive effect o f p r o l o n g e d A C T H t r e a t m e n t o n C Y P I I B 2

express ion in v ivo does n o t seem to be due to a di rect a c t i on o f the pep t ide o n the z o n a g l o m e r u l o s a cell b u t r a the r invo lves a n e x t r a a d r e n a l m e c h a n i s m .

Acknowledgements--The authors' work described and cited in this article has been supported by Research Grants 32.25326.88 and 32-32315.91 from the Swiss National Foundation for Scientific Research. The skilful technical assistance of Heidi Seiler, Dora Schmid and Iris Dannenmann-Glesti is gratefully acknowledged.

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