Cyp11b1 Null Mouse, a Model of Congenital Adrenal Hyperplasia

Abstract

Patients with congenital adrenal hyperplasia arising from mutations of 11␤-hydroxylase, the final enzyme in the glucocorticoid biosynthetic pathway, exhibit glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo-pituitary-adrenal activity, and excess mineralocorticoid activity caused by the accumulation of deoxycorticosterone. A mouse model, in which exons 3-7 of Cyp11b1 (the gene encoding 11␤-hydroxylase) were replaced with cDNA encoding enhanced cyan fluorescent protein, was generated to investigate the underlying disease mechanisms. Enhanced cyan fluorescent protein was expressed appropriately in the zona fasciculata of the adrenal gland, and targeted knock-out was confirmed by urinary steroid profiles and, immunocytochemically, by the absence of 11␤-hydroxylase. The null mice exhibited glucocorticoid deficiency, mineralocorticoid excess, adrenal hyperplasia, mild hypertension, and hypokalemia. They also displayed glucose intolerance. Because rodents do not synthesize adrenal androgens, changes in reproductive function such as genital virilization of females were not anticipated. However, adult homozygote females were infertile, their ovaries showing an absence of corpora lutea and a central proliferation of disorganized steroidogenic tissue. Null females responded normally to superovulation, suggesting that raised systemic progesterone levels also contribute to infertility problems. The model reveals previously unrecognized phenotypic subtleties of congenital adrenal hyperplasia.The final steps leading to the production of glucocorticoids and mineralocorticoids are undertaken by 11␤-hydroxylase and aldosterone synthase, encoded by two closely linked genes, Cyp11b1 and Cyp11b2, respectively, that share 95% sequence homology. In rodents, the common substrate, deoxycorticosterone, is converted into the mineralocorticoid, aldosterone, in the adrenal zona glomerulosa by aldosterone synthase and into corticosterone, the main glucocorticoid, by 11␤-hydroxylase in the zona fasiculata. Cortisol rather than corticosterone is the main glucocorticoid in humans because Cyp17 is expressed in the zona fasciculata. Unlike rodents, human adrenals produce significant amounts of the androgen dehydroepiandrosterone (DHEA). 2 Patients with congenital adrenal hyperplasia (CAH) have a markedly reduced capacity to produce glucocorticoids. In ϳ90% of cases CAH is caused by deficiency of 21-hydroxylase, the penultimate enzyme in the pathways of both aldosterone and cortisol synthesis (1). In 8% of CAH cases, point mutations in or deletion of Cyp11b1 drastically reduce or completely destroy 11␤-hydroxylase activity (2-4).Adrenal hyperplasia is a consequence of increased ACTH secretion in the absence of normal negative feedback control by glucocorticoids of the hypothalamo-pituitary-adrenal axis. In 21-hydroxylase-deficient patients, ACTH drives the production of progesterone and 17-hydroxyprogesterone and channels earlier 17-hydroxylated intermediates in the glucocorticoid pathway toward the synthesis of adr...

Nonclassic 21-hydroxylase deficiency presenting as endometrial hyperplasia with uterine bleeding in a 67-year-old woman

“…We also postulate that there was no atypia or neoplasia because the progesterone effect was antimitotic at the cellular level. Of note, there is a knockout mouse model of nonvirilizing non-salt-wasting congenital adrenal hyperplasia that demonstrates chronic elevations of 17-OHP associated with both endometrial hyperplasia and adenomyosis (6). That model bears a number of similarities to the present case, including hyerpertension based on mild elevation of mineralocorticoid, absence of virilization, absence of glucose intolerance, regular menses, and subfertility.…”

Osilodrostat (LCI699), a potent 11β-hydroxylase inhibitor, administered in combination with the multireceptor-targeted somatostatin analog pasireotide: A 13-week study in rats

“…Increases in mean adrenal weight in female rats receiving osilodrostat monotherapy were associated with the development of adrenocortical hypertrophy (zona fasciculata); this histological phenotype has been previously noted in 11β-hydroxylase null mice (Mullins et al, 2009). The development of adrenocortical hypertrophy may result from accumulation of cholesterol and steroid precursors due to inhibition of adrenocortical steroid biosynthesis by osilodrostat or, alternatively, represent a trophic effect of increased pituitary production of ACTH in the absence of normal negative feedback by glucocorticoids (Harvey and Sutcliffe, 2010).…”

“…may be attributable to the inhibitory effect of osilodrostat on aromatase activity (IC 50 for human enzyme, 1.7 μM; Novartis Pharma AG, unpublished data), which is responsible for catalyzing the conversion of testosterone into estradiol (Nunez et al, 1996). It is also possible that the effects were contributed by Cyp11b1 inhibition, as Cyp11b1 null mice also had abnormal ovaries, which contained pre-ovulatory follicles but no clearly defined corpora lutea, and the ovary was filled with lobular amorphous cells, likely nonsecreting luteinized granulosa cells (Mullins et al, 2009). Although no histological or mean weight changes for the heart were reported in this study, it is important to note that pasireotide has been shown to cause QT c prolongation in clinical settings (Breitschaft et al, 2014), whereas preclinical studies of osilodrostat have identified doserelated QT c prolongation in monkeys (Novartis Pharma AG, unpublished data). It is, therefore, necessary to establish the cardiac safety profile of pasireotide and osilodrostat in combination before the combination therapy can be applied to the broader patient population.…”

Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney

“…Mice were killed by CO 2 inhalation after restraint stress, maximizing plasma corticosterone levels. Heparinized blood was collected by cardiac puncture, and plasma was stored for measurement of electrolytes, osmolality (freezing point depression), and hormones (32). …”