Final Steps Of Aldosterone Biosynthesis Research Articles

Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Regulation of aldosterone biosynthesis: the end of the road?

1. Because aldosterone is the minority hormone of the adrenal cortex, its proper function depends on protective physiological mechanisms. These include a particular site of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex as well as a complex multifactorial control system, which adapts aldosterone production to acute and chronic changes in body sodium and potassium content, irrespective of pituitary adrenocorticotropic hormone (ACTH) secretion. 2. Over the past decade, an important element of these mechanisms has been identified in the form of the enzyme involved in the final steps of aldosterone biosynthesis. In species such as the human, rat and mouse, the conversion of deoxycorticosterone to aldosterone is catalysed by an isozyme (CYP11B2) of cytochrome P450(11) beta (CYP11B1). The gene encoding this enzyme is expressed only in the zona glomerulosa. Its transcription is enhanced by sodium deficiency and potassium intake, but is suppressed by long-term administration of high doses of ACTH. 3. In contrast, the gene encoding CYP11B1 (i.e. the major (non-aldosterone-producing) type of the enzyme) is expressed mainly in the zona fasciculata and its expression depends on physiological concentrations of ACTH. 4. In other animal species (cattle, pig, sheep), the major forms of cytochrome P450(11) beta have an inherent aldosterone-synthesizing activity, which is, however, selectively suppressed in mitochondria of zona fasciculata cells. 5. The elucidation of the mechanisms involved in this suppression or of those mediating alterations in CYP11B2 expression in response to physiological stimuli may be important areas of future research on the regulation of aldosterone biosynthesis.

Aldosterone: The minority hormone of the adrenal cortex

In comparison with the glucocorticoids cortisol and corticosterone, the mineralocorticoid aldosterone is a minority hormone of the mammalian adrenal cortex, and its proper function is dependent upon protective physiological mechanisms. These include a particular site of aldosterone synthesis in zona glomerulosa cells as well as a complex multifactorial control system, which adapts aldosterone production to acute and chronic changes in body sodium and potassium contents, irrespective of pituitary ACTH secretion. In the course of the last few years, an important element of these mechanisms has been identified in the form of the enzyme involved in the final steps of aldosterone biosynthesis. In species such as the human, rat, and mouse, the conversion of deoxycorticosterone to aldosterone is catalyzed by an isozyme (CYP11B2) of cytochrome P450 11β (CYP11B1). The gene encoding this enzyme is expressed only in the zona glomerulosa. Its transcription is enhanced by sodium deficiency and potassium intake, but is suppressed by long-term administration of high doses of ACTH. In contrast, the gene encoding CYP11B1, i.e., the major (non-aldosterone—producing) type of the enzyme, is expressed mainly in the zona fasciculata, and its expression depends on physiological concentrations of ACTH. In other animal species (cattle, pig), the major forms of cytochrome P450 11β have an inherent aldosterone-synthesizing activity, which is, however, selectively suppressed in mitochondria of zona fasciculata cells by yet unknown mechanisms.

Final steps of aldosterone biosynthesis: Molecular solution of a physiological problem

The final two steps of aldosterone biosynthesis play a key role in the complex physiological adaptation of aldosterone secretion to changes in sodium and potassium content of the mammalian organism. The nature and identity of the enzyme catalyzing these steps have only recently been established. In the rat as well as in the human adrenal, two types of cytochrome P-450 11 ß are encoded by two different genes. The major type of the enzyme catalyzes only the conversion of deoxycorticosterone to corticosterone or 18-hydroxy-11-deoxycorticosterone; it is present in all the zones of the adrenal cortex. The second type of the enzyme catalyzes the three steps involved in the conversion of deoxycorticosterone to aldosterone and occurs only in the zona glomerulosa. In rat zona glomerulosa cells, separate control systems independently regulate the expression of the two genes, according to long-term in vivo experiments or to experiments with primary cultures of zona glomerulosa cells. Expression of the non-aldosterone-producing enzyme is induced by ACTH, whereas the expression of the aldosterone-producing enzyme is dependent on the extracellular potassium concentration.

Aldosterone biosynthesis and cytochrome P-450 11β: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-450 11β has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11β-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K. protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-450 11β. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-450 11β in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.

A potassium-induced mitochondrial protein related to aldosterone biosynthesis.

Late steps of aldosterone biosynthesis, i.e., the conversions of corticosterone to 18-hydroxycorticosterone and aldosterone, are catalyzed by a mitochondrial cytochrome P-450. Resumption of potassium intake by potassium-depleted rats resulted within 2 days in a marked stimulation of these conversions, as reflected by increased production of aldosterone and 18-hydroxycorticosterone and decreased outputs of deoxycorticosterone, corticosterone, and 18-hydroxy-11-deoxycorticosterone by incubated capsular portions of the adrenal glands. The stimulation of aldosterone biosynthesis was accompanied by the appearance of a protein with a molecular weight of about 49,000 in the mitochondria of the zona glomerulosa but not of the inner zones of the adrenal cortex. Over 48 h of potassium repletion, the amount of this protein increased in parallel with the activity of the final steps of aldosterone biosynthesis. According to its molecular weight, its zone specificity, and the time course of its appearance, this protein might represent the steroid 18-methyl oxidase (cytochrome P-450CMO for corticosterone methyl oxidase) that catalyzes the conversion of corticosterone to 18-hydroxycorticosterone and aldosterone.

Potassium intake and aldosterone biosynthesis: The role of cytochrome P-450

K + Repletion for 48 h of rats previously kept on a low K + diet for 2 weeks specifically increased the conversion of corticosterone into aldosterone and 18-hydroxycorticosterone by incubated capsular fractions of rat adrenal tissue. This increase in the activity of the final steps of aldosterone biosynthesis was not accompanied by an increase in capsular adrenal mitochondrial cytochrome P-450 concentration. By contrast, an increased corticosterone-induced absorbance change (BI) was consistently found in capsular adrenal mitochondria upon K + repletion. In addition, a type I-like absorbance change was induced with 18-hydroxy-11-deoxycorticosterone but not with 18-hydroxycorticosterone. Therefore, K + repletion of K + depleted rats specifically increased the binding of corticosterone and possibly 18-hydroxy-11-deoxy-corticosterone to the 18-methyl oxydase enzyme complex. Whether this increased binding was due to an increase in enzyme protein concentration or due to a better availability of the substrate to the enzyme, could not be decided from these experiments.

EFFECTS OF PROLONGED INFUSIONS OF POTASSIUM CHLORIDE, ADRENOCORTICOTROPHIN OR ANGIOTENSIN II UPON SERUM ALDOSTERONE CONCENTRATION AND THE CONVERSION OF CORTICOSTERONE TO ALDOSTERONE IN RATS

The temporal relation between alterations in serum aldosterone and in the conversion of labelled corticosterone to aldosterone by incubated adrenal tissue was studied in conscious rats receiving long-term infusions of KCl, ACTH or angiotensin II. When potassium-deficient rats were given KCl, a marked increase in serum aldosterone was observed only after 12 h, i.e. at a time when the conversion of corticosterone to aldosterone had become normal. After 24 h of ACTH infusion into sodium- and potassium-replete rats the serum aldosterone was markedly elevated, whereas the conversion of corticosterone to aldosterone was significantly decreased. After 48 h of continued ACTH infusion the serum aldosterone returned to normal and there was a further decrease in the conversion rate. A 24-h angiotensin II infusion into sodium- and potassium-replete rats induced significant increases in both the serum aldosterone and the conversion. After 48 h of continued angiotensin infusion the serum aldosterone returned to normal while the conversion and the blood pressure remained elevated. These results indicate that the activity of the enzymes involved in the final steps of aldosterone biosynthesis may become rate-limiting for the secretion of aldosterone during potassium deficiency and during prolonged ACTH treatment. On the other hand, the observed transiency of aldosterone stimulation by exogenous angiotensin II was not due to a suppression of the final steps of aldosterone biosynthesis and remains unexplained.

Multifactorial regulation of the final steps of aldosterone biosynthesis in the rat

Several known stimulators, including angiotensin II, potassium ions, serotonin and ACTH, act directly on the zona glomerulosa of the rat adrenal cortex during short-term incubation experiments. They rapidly enhance an early biosynthetic step involved in the conversion of cholesterol to pregnenolone. The steroidogenic response elicited by these agents depends on the activity of the enzymes necessary for the final stages of aldosterone biosynthesis, in particular the 18-hydroxylation of corticosterone. The activity of these enzymes is in turn regulated by unknown mediators, which act in response to alterations in sodium and potassium balance. According to indirect evidence, the serum potassium appears to be an important but not the only regulator. Angiotensin II and serotonin may also have a long-term stimulating effect on the final steps of aldosterone biosynthesis. By contrast, treatment of rats with ACTH for a few days causes a marked decrease in the conversion of tritiated corticosterone to 18-hydroxycorticosterone and aldosterone.

Effects of hypophysectomy with or without ACTH maintenance therapy on the final steps of aldosterone biosynthesis in the rat.

ABSTRACT The effects of hypophysectomy, low-dose ACTH administration or both on aldosterone biosynthesis by incubated adrenal tissue were studied in rats during different dietary regimens. Six days after hypophysectomy without any replacement therapy, the capsular adrenals of rats receiving a complete diet converted tritiated corticosterone to aldosterone and 18-hydroxycorticosterone at a normal and at a moderately increased rate, respectively, and produced normal amounts of aldosterone from endogenous precursors. Hypophysectomy, with ACTH maintenance therapy, resulted in significant decreases in these parameters. In rats kept on a sodium-deficient diet, hypophysectomy alone did not alter the conversion rates, but led to a decreased output of endogenous aldosterone. ACTH treatment of intact as well as of hypophysectomized sodium-restricted animals was followed by a decreased incorporation of tritiated corticosterone into aldosterone and 18-hydroxycorticosterone. In rats kept on a potassium-deficient diet, hypophysectomy induced an 800 % increase in the capsular adrenal conversion of added corticosterone to aldosterone and 18-hydroxycorticosterone; this was partially prevented by ACTH treatment. Hypophysectomy with or without ACTH treatment did not impair restoration to the normal of the apparent capsular adrenal 18-hydroxylase activity in response to resumed potassium intake by potassium-restricted rats. These results indicate that neither the maintenance nor the induction of an increased activity of the enzymes involved in the final steps of aldosterone biosynthesis due to sodium restriction or to an increased potassium intake, depend on a functioning pituitary gland. On the other hand, even small doses of exogenous ACTH, which are just sufficient for the maintenance of a normal adrenal weight in hypophysectomized animals, can cause significant decreases in the activity of these enzymes.

Effect of potassium intake on the final steps of aldosterone biosynthesis in the rat. 3. Combined sodium and potassium restriction.

ABSTRACT Capsular adrenals ("zona glomerulosa") of rats which had been kept on a sodium- and potassium-deficient diet and which were markedly hypokalaemic, converted tritiated corticosterone to 18-hydroxycorticosterone and aldosterone, and tritiated cortexolone to cortisol at the same respective rates as the capsular adrenals of sodium- and potassium-replete animals. Aldosterone production from endogenous precursors was elevated under basal conditions of incubation, but not under stimulation by added serotonin. Corticosterone and deoxycorticosterone outputs were normal during incubation with or without serotonin. Capsular adrenals of rats which had been kept first on a potassium-deficient diet for two weeks and then on a sodium- and potassium-deficient diet for two weeks converted 18 times more tritiated corticosterone to 18-hydroxycorticosterone and aldosterone and produced 5 times more aldosterone from endogenous precursors than the tissue of rats which had been kept on the potassium-deficient diet for the whole period, although the serum potassium was similarly low in both groups. These results indicate that under simple potassium restriction as well as under combined sodium and potassium restriction, neither the plasma potassium concentration nor the total body potassium is the only regulator of the activity of the enzymes involved in the final steps of aldosterone biosynthesis.