Fumiko Mitani

Adrenocortical Zonation in Humans under Normal and Pathological Conditions

CONTEXT Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. OBJECTIVE The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. RESULTS Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushings syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. CONCLUSION Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus–pituitary–adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R–MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

The undifferentiated cell zone is a stem cell zone in adult rat adrenal cortex

The adrenal cortex of mammals has been known to consist of three morphologically and functionally distinct zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF) and the zona reticularis (zR), each of which secretes a specific corticosteroid different from those produced by the other two zones. We found previously, however, that an additional zone existed between zG and zF of adult rat adrenal cortex and that the cells in that zone were in a functionally undifferentiated state as an adrenocortical cell [Endocrinology 135, (1994) 431]: they were incapable of synthesizing highly active forms of corticosteroids, such as aldosterone and corticosterone, although they could produce their precursors. Hence, we named the zone as the undifferentiated cell zone (zU) of the adrenal cortex. Here we show that zU and its surroundings, i.e. the innermost portion of zG and the outermost portion of zF are the sites for cell replication in adult rat adrenal cortex and that the cells raised there migrate to other regions. Such cell replications in this region occur regardless of physiological conditions, such as the rise and fall of hormonal stimuli and circadian fluctuation of adrenocortical activities. On the bases of these and other findings previously described, we propose that zU is the stem cell zone of the adult rat adrenal cortex. Our recent success in isolating novel cell lines, which display an undifferentiated phenotype similar to that of zU cells, could facilitate the exploration of molecular mechanisms for the differentiation and development of the adrenocortical cells.

Cytochrome P450 in adrenocortical mitochondria

SummaryCytochrome P450 in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11β-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P450 each catalyzing a specified reaction have been purified to a homogeneous state, that is, P450scc having cholesterol side chain cleavage activity and P45011β having 11β-hydroxylation activity. The properties of these purified P450s as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P450 in the mitochondria) preparation in situ.Among many findings, both purified cytochromes P450 were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P450. These effects are important from the point of view that the primary role of the heme of cytochrome P450 is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P450 in the reconstituted system have also been described Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.

Localization of p450aldo and p45011β in normal and regenerating rat adrenal cortex

A novel layer of cells that do not contain both P450aldo and P45011 beta has been discovered between the zonae glomerulosa and fasciculata of the rat adrenal cortex. Since P450aldo and P45011 beta are the enzymes responsible for the formation of aldosterone and corticosterone, respectively, the cells in that zone are presumably inert in synthesizing both aldosterone and corticosterone, in other words, the layer is composed of cells that have no zone-specific endocrine function as an adrenocortical component. Cytologically, the layer consists of tightly packed cells, which contain a lesser amount of lipid droplet than the cells in the other zones, and appears as a white ring or a white zone in the double immunostaining with anti P450aldo and anti P45011 beta. Upon angiotensin II-stimulation evoked by Na-deficiency, the number of the zona glomerulosa cells expressing P450aldo increases for the initial 2 or 3 days and then the P450aldo-containing zona glomerulosa cells begin to proliferate. Thus angiotensin II serves as a proliferator of the zona glomerulosa cells of the rat adrenal cortex. During the period, the thickness of the white zone decreases for initial 3 days and becomes constant after 5 or 6 days, being about 5% of the total cell number of the adrenal cortex. When localization of replicating cells was examined in the adrenal cortex, they were found to be concentrated in and around the white zone. Then the pulse-chase experiments with BrdU showed that the labeled cells migrated out of the white zone and into the zonae fasciculata and reticularis. The localization of the replicating cells in the regenerating adrenal cortex was also around the region between the zonae glomerulosa and fasciculata. On the basis of these findings, we suggest that the newly discovered cell layer (the white zone) is the stem cell zone of the rat adrenal cortex.

Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex.

The CYP11B1 gene, which encodes steroid 11 beta-monooxygenase, which is responsible for the synthesis of cortisol and corticosterone, the major glucocorticoids in mammals, is expressed specifically in the zona fasciculata of the adrenal cortex. We have analyzed the promoter region of the rat CYP11B1 gene by using a transient-expression system with adrenocortical Y1 cells and have identified a positive regulatory region. The region contained two adjacent sites for the binding of Y1-cell nuclear proteins: the binding site for an AP-1 transcription factor composed of JunD and a Fos-related protein, and the site for Ad4-binding protein (Ad4BP). The binding of the AP-1 factor to the regulatory region had a suppressive effect on that of Ad4BP in the nuclear extracts. Mutational analyses revealed that the transcriptional activation of the CYP11B1 gene promoter in Y1 cells was attributable to the AP-1 site but not to the Ad4 site. Subsequently, nuclear extracts of the zona fasciculata cells from the rat adrenal cortex were found to contain both AP-1 factor and Ad4BP, whose binding properties to the regulatory region were almost identical to those of the two factors in the Y1-cell nuclear extracts. Moreover, immunohistochemical analyses of rat adrenal cortices showed that the AP-1 factor was present in the nuclei of CYP11B1-expressing cells in the zona fasciculata but not in the nuclei of cells in the other zones. From these results, we propose that the AP-1 transcription factor found in this study plays an important role in the zone-specific expression of the CYP11B1 gene in rat adrenal cortex.

Effects of long term stimulation of ACTH- and angiotensin II-secretions on the rat adrenal cortex

In the rat adrenal cortex, aldosterone synthase cytochrome P450 (P450aldo), a mineralocorticoid synthesizing enzyme, localizes in the zona glomerulosa (zG), while cytochrome P45011 beta (P45011 beta), a glucocorticoid synthesizing enzyme, localizes in the zonae fasciculata-reticularis (zFR). In between zG and zF, a cell-layer which contains neither P450aldo nor P45011 beta is present, where replicating cells were abundant as judged by the incorporation of bromodeoxyuridine (BrdU) and/or by detecting PCNA in their nuclei. When plasma ACTH level of the rat was raised 3-fold for 2-3 weeks by the administration of metyrapone, a potent inhibitor of glucocorticoid formation, most of zG cells containing P450aldo disappeared, while zF cells with P45011 beta increased. Under the conditions, the cell-layer without P450aldo and P45011 beta became very thin, and replicating cells were mainly in the outermost portion of zF. When angiotensin II secretion was also stimulated for 2-3 weeks by feeding the rats on Na-deficient diet, the P450aldo-containing cells proliferated to form a thicker zG (7-8 cells-thick from 1-2), while the width of zF containing P45011 beta decreased slightly. Coincidently the cell-layer devoid of P450aldo and P45011 beta became thin, though slightly, and numbers of replicating cells significantly increased in and around the inner edge of the proliferated zG. When both ACTH and angiotensin II secretions were stimulated simultaneously, the cell-layer without P450aldo and P45011 beta almost disappeared and replicating cells were around the boundary of zG and zF. Based on these results we propose that the cell-layer between zG and zF devoid of P450aldo and P45011 beta is the stem cell layer of rat adrenal cortex.

Expression of cytochromes P450aldo and P45011β in rat adrenal gland during late gestational and neonatal stages

The development of the rat adrenal gland during late gestational and neonatal stages was studied by following the expression of aldosterone synthase cytochrome P450 (P450aldo) and glucocorticoid-synthesizing cytochrome P450 (P45011 beta). Cells expressing P450aldo, a functional marker for the mineralocorticoid-synthesizing zona glomerulosa, were not detected until day 20 of fetal age, i.e., 2 days before birth, although the zona glomerulosa cells were histologically recognizable at the 18th day of gestation. The intensity of P450aldo staining thereafter became stronger with age in the outer portion of the cortex. Cells expressing P45011 beta, a marker for the glucocorticoid-producing zona fasciculata, were present in the fetal adrenals on the 18th day. P45011 beta-positive cells were distributed over the whole adrenal gland and intermingled with the cells containing tyrosine hydroxylase, a marker enzyme for medullary cells. The P45011 beta-positive and tyrosine hydroxylase-positive cells began to separate on the 20th day, and were completely resolved from each other around the third day after birth. Expression of P450aldo and P45011 beta, together with that of tyrosine hydroxylase, thus serves as a suitable marker for studying the development of the adrenal gland.

Adrenocortical zonation factor 1 is a novel matricellular protein promoting integrin-mediated adhesion of adrenocortical and vascular smooth muscle cells

Expression of a previously cloned secretory protein named adrenocortical zonation factor 1 (AZ‐1, also called Tin‐ag‐RP or lipocalin 7) is tightly linked with the zonal differentiation of adrenocortical cells. It is also present in vascular smooth muscle (VSM), although its function has remained unknown. In this study, the location of AZ‐1 was specified to the basal laminae along adrenocortical sinusoidal capillaries and surrounding VSM cells in the arterial system, consistent with the fact that AZ‐1 was extractable under denaturing conditions as a 52 kDa polypeptide. Purified recombinant AZ‐1 exhibited abilities to bind to fibronectins via the first type III repeat (anastellin) and to collagens with affinities in submicromolar ranges. AZ‐1 immobilized on substratum or bound to collagens or anastellin promoted adhesion and spreading of adrenocortical cells. Although VSM cells spread on AZ‐1 slowly, AZ‐1 bound to anastellin facilitated the spreading. The adhesion activity of AZ‐1 was mediated by a subset of integrins, including α1β1, α2β1, and α5β1, in a cell type‐specific manner. Collectively with the putative role of AZ‐1 in the adrenocortical zonation, we propose that AZ‐1 potentially regulates functions of adrenocortical and VSM cells by modulating cell–matrix interactions.

Magnetic and natural circular dichroism spectra of cytochromes P-45011β and P-450scc purified from bovine adrenal cortex

Magnetic (MCD) and natural circular dichroism (CD) spectra various complexes of cytochrome P-450(11) beta (P-450(11) beta) and cytochrome P-450scc (P-450scc) from bovine adrenal cortex were measured from 250 nm to 700 nm. MCD and CD spectral contours of cytochromes P-450(11) beta and P-450scc in the Soret and visible regions were, as a whole, analogous to those of cytochromes P-450 from rabbit liver microsomes and also from Pseudomonas putida in their high-spin ferric, high-spin ferrous and ferrous-CO complexes. MCD spectrum of the low-spin ferric P-450scc free from the substrate, cholesterol, was very similar to that caused by addition of 20 alpha-hydroxycholesterol, a reaction intermediate. However, it was distinct from those of the low-spin ferric P-450(11) beta and P-450scc complexes caused by addition of external nitrogenous ligands. The electronic states of the heme in the low-spin ferric P-450 free from substrates seemed to be subtly different from those of low-spin complexes coordinated with external nitrogenous ligands. Soret CD spectra of ferric low-spin complexes were not so different from each other. Upon reduction of high-spin ferric P-450(11) beta or P-450scc, the Soret CD magnitudes increased significantly in contrast with those of other P-450s, the Soret CD magnitudes of which decrease upon reduction. This may reflect an increased proximity of the neighbouring aromatic groups upon reduction of high-spin P-450(11) beta or P-450scc. High substrate specificity of adrenal P-450s compared with liver P-450s can be explained in view of the above findings. THe CD spectra in the near ultraviolet region (250-350 nm) were found to be quite sensitive to the spin change for ferric P-450scc, while the MCD spectra in this region did not reflect substantially the spin state of the enzyme. MCD parameters of cytochrome P-450s were compared to those of other hemoproteins in diagrams describing selected MCD spectral values of hemoproteins so far available and were discussed in connection with the structures of the heme environment of P-450.