3ßHSD and CYB5A double positive adrenocortical cells during adrenal development/aging.

Androstenedione is a common precursor of sex steroids produced and secreted in the human adrenal gland and produced by 3ß-hydroxysteroid dehydrogenase (3ßHSD), 17ß-hydroxylase/17,20-lyase (CYP17) and cytochrome b5 (CYB5A). 3ßHSD is expressed in the zona glomerulosa (ZG) and fasciculata (ZF), CYP17 in the ZF and zona reticularis (ZR) and CYB5A in the ZR, respectively. We previously demonstrated the presence of cortical parenchymal cells co-expressing 3ßHSD and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex and hypothesized that these cells may play an important role in androstenedione production in human adrenal gland. Age-related morphologic development of these hybrid cells has, however, not been studied. Therefore, in this study, 48 human adrenal specimens from various age groups were retrieved. Double-immunohistochemical analyses were used in order to study the correlation between this hybrid cell type and age. In both male and female adrenal cortex, the means of total adrenocortical area, the area positive for CYB5A and its ratio reached highest peak in the 21-40-year-old (y.o.) group. The greatest overlap between 3ßHSD and CYB5A in both total and relative area was present in the 13-20 y.o. group. For all the markers mentioned above, statistically significant differences were detected among the different age groups examined (p < 0.05). These findings indicated that both area and ratio of 3ßHSD and CYB5A double positive cells, which could represent the hybrid cells of ZF and ZR, are correlated with human adrenal development and could subsequently influence age-related serum androstenedione levels.

Estrogen-related receptor α in normal adrenal cortex and adrenocortical tumors: involvement in development and oncogenesis.

AIMS: The nuclear hormone receptor estrogen-related receptor α (ERRα) regulates the activation of mitochondrial genes in various human tissues, but its role in the adrenal gland and its disorders has not been defined. Therefore, we examined ERRα expression in both normal adrenal cortex (NAC) and adrenocortical tumor (ACT) in order to study the possible correlation of ERRα with adrenal development and tumor development. METHODS: Human adrenal specimens (non-pathological fetal n=7; non-pathological post-birth n=40; aldosterone producing adenoma (APA) n=11; cortisol producing adenoma (CPA) n=11; adrenocortical carcinoma (ACC) n=8) were immunohistochemically examined in this study. NAC (n=13) and ACT (n=28) frozen tissue specimens were also available for studying ERRα mRNA levels. KEY FINDINGS: In fetal NAC tissues, ERRα labeling index (LI) in fetal zone (FZ) was significantly higher that that in neocortex (NC), and the differences among age groups for overall mean LI was statistically significant when analyzed according to individual cortical layers. ERRα LI was also significantly higher in ACC than in other types of ACT. ERRα mRNA was detected in NAC and all types of ACT. SIGNIFICANCE: Results of our present study suggest a possible role of ERRα in adrenal development and ACC.

Human adrenal cells that express both 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) contribute to adrenal androstenedione production.

Androstenedione is one of several weak androgens produced in the human adrenal gland. 3ß-Hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) are both required for androstenedione production. However, previous studies demonstrated the expression of HSD3B2 within the zona glomerulosa (ZG) and fasciculata (ZF) but low levels in the zona reticularis. In contrast, CYB5A expression increases in the zona reticularis (ZR) in human adrenal glands. Although their colocalization has been reported in gonadal theca and Leydig cells this has not been studied in the human adrenal. Therefore, we immonolocalized HSD3B2 and CYB5A in normal human adrenal glands and first demonstrated their co-expression in the cortical cells located at the border between the ZF and ZR in normal human adrenal. Results of in vitro studies using the human adrenal H295R cells treated with the HSD3B2 inhibitor, trilostane, also demonstrated a markedly decreased androstenedione production. Decreasing CYB5A mRNA using its corresponding siRNA also resulted in significant inhibition of androstenedione production in the H295R cells. These findings together indicate that there are a group of cells co-expressing HSD3B2 and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex, and these hybrid cortical cells may play an important role in androstenedione production in human adrenal gland.

Peroxisome proliferator-activated receptor-{gamma} suppresses CYP11B2 expression and aldosterone production.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor for the antidiabetic agent thiazolidinedione, which exerts various physiological activities, independent of lowering blood glucose. However, the role of PPARγ in aldosterone production has not been clarified. The objective of this study was to investigate the effect of PPARγ on aldosterone synthase gene (CYP11B2) expression and aldosterone production. Localization of PPARγ expression in normal adrenal cortex was determined by immunohistochemistry. Aldosterone production and CYP11B2 expression levels were determined using human adrenocortical carcinoma H295R cells. Pioglitazone suppressed angiotensin II-induced aldosterone secretion and CYP11B2 expression. PPARγ was expressed in zona glomerulosa in human normal adrenal gland. PPARγ overexpression enhanced pioglitazone-mediated CYP11B2 transrepression. The pioglitazone-mediated suppression of aldosterone secretion and CYP11B2 expression were canceled by PPARγ L466A/E469A mutant. Pioglitazone also suppressed potassium-mediated CYP11B2 induction, but not N6-2'-O-dibutyladenosine-3',5'-cyclic monophosphate stimulation. Rosiglitazone and GW1929 also suppressed CYP11B2 transactivation. Mutation analysis revealed that the Ad1/CRE element in CYP11B2 5'-flanking region was responsible for the pioglitazone-mediated transrepression. Pioglitazone suppressed ionomycin and a truncated constitutively active form Ca(2+)/calmodulin-dependent kinase I (CaMKI)-mediated CYP11B2 transcriptional activation. A CaMK inhibitor KN-93 attenuated pioglitazone-mediated CYP11B2 transrepression. PPARγ suppresses CYP11B2 expression and aldosterone secretion.

Development of the human adrenal zona reticularis: morphometric and immunohistochemical studies from birth to adolescence.

Age-related morphologic development of human adrenal zona reticularis (ZR) has not been well examined. Therefore, in this study, 44 human young adrenal autopsy specimens retrieved from large archival files (n=252) were examined for immunohistochemical and morphometric analyses. Results demonstrated that ZR became discernible around 4 years of age, and both thickness and ratio per total cortex of ZR increased in an age-dependent fashion thereafter, although there was no significant increment in total thickness of developing adrenal cortex. We further evaluated immunoreactivity of both KI67 and BCL2 in order to clarify the equilibrium between cell proliferation and apoptosis in the homeostasis of developing human adrenals. Results demonstrated that proliferative adrenocortical cells were predominantly detected in the zona glomerulosa and partly in outer zona fasciculata (ZF) before 4 years of age and in ZR after 4 years of age, but the number of these cells markedly decreased around 20 years of age. The number of BCL2-positive cells increased in ZR and decreased in ZF during development. Adrenal androgen synthesizing type 5 17beta-hydroxysteroid dehydrogenase (HSD17B5 or AKR1C3 as listed in the Hugo Database) was almost confined to ZR of human adrenals throughout development. HSD17B5 immunoreactivity in ZR became discernible and increased from around 9 years of age. Results of our present study support the theory of age-dependent adrenocortical cell migration and also indicated that ZR development is not only associated with adrenarche, but may play important roles in an initiation of puberty.