Development of monoclonal antibodies against the human 3β-hydroxysteroid dehydrogenase/isomerase isozymes

Abstract

The human 3β-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzymes catalyze the conversion of 3β-hydroxy Δ 5-6 steroids into 3-keto Δ 4-5 steroids, which is required for the synthesis of the mature steroid hormones secreted by the adrenal and gonads. The human has 2 isozymes, the HSD3B1 that is traditionally located in placenta and extra-adrenal tissues and the HSD3B2 that is expressed in the adrenal and gonads. Mice with both cryptochrome 1 and 2 genes deletion were recently found to have salt-sensitive hypertension and hyperaldosteronism. These deletions were also associated with overexpression of the Hsd3b6 enzyme, the homolog of the human HSD3B1, in the zona glomerulosa which was believed to explain the hyperaldosteronism. A report using antibodies against human HSD3B1 suggested that it was expressed in the zona glomerulosa of normal human adrenals and in patients with idiopathic hyperaldosteronism and the HSD3B2 expressed in both the zona fasciculata and glomerulosa. We have developed specific monoclonal antibodies against the human HSD3B1 and HSD3B2 isozymes and found that the main enzyme expressed in the zona glomerulosa was the HSD3B2. Faint staining of the adrenal was also obtained using the anti-HSD3B1antibody only at high concentrations of antibody. This study fails to confirm that HSD3B1 expression in the human zona glomerulosa and double immunofluorescence clearly shows that the HSD3B2 is expressed in the zona glomerulosa and fasciculata and in the zona glomerulosa HSD3B2 is co-expressed with aldosterone synthase (CYP11B2).

In situ metabolomics of aldosterone-producing adenomas

“…Furthermore, our result showed a positive correlation between staining intensities of HSD3B2 and CYP11B2 in APAs, which is consistent with the features of ZG, in which coexpression of HSD3B2 and CYP11B2 has been reported (22). Previous studies have aimed to classify APAs with regard to genotypes using various approaches, including clinical (23), histopathological (20,21), steroidogenic (14), and molecular parameters (8,24).…”

Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor

“…Indeed, CAC significantly increased the conversion of pregnenolone to progesterone, leading to the increase of testosterone levels, at 50 μmol/L. The homolog of rat 3β‐HSD1 in ALCs in 3β‐HSD6 (encoded by Hsd3b6 ) in mouse MLTC‐1 Leydig cells 41 . Apparently, Hsd3b6 was not affected in MLTC‐1 cells after CAC treatment.…”

Role of Cryptochrome-1 and Cryptochrome-2 in Aldosterone-Producing Adenomas and Adrenocortical Cells

“…Immunohistochemistry studies in normal human adrenals showed that HSD3B2 was the predominant isoform, while HSD3B1 localized mainly in the outermost layer zona glomerulosa [ 8 , 13 , 14 ]. In adrenal pathology, HSD3B1 appeared to be strongly expressed in the hyperplastic zona glomerulosa cells of BAH samples, while its expression was low in a series of eight APAs, composed predominantly of zona fasciculata-like cells [ 13 ].…”

“…HSD3B catalyzes the conversion of pregnenolone to progesterone, an enzymatic reaction required for aldosterone biosynthesis [ 11 ]; two different HSD3B isoforms are expressed in man— HSD3B1 is mainly expressed in the placenta, while HSD3B2 localizes primarily in adrenals and gonads [ 12 ]. Immunohistochemistry studies in normal human adrenals showed that HSD3B2 is the predominant isoform, expressed through the zona glomerulosa and the zona fasciculata (ZF), while HSD3B1 displays faint immunoreactivity, predominantly in the outermost layer zona glomerulosa (ZG) [ 8 , 13 , 14 ]. Moreover, in APA samples, HSD3B1 expression was significantly correlated with the expression of the rate-limiting enzyme for aldosterone production—aldosterone synthase (CYP11B2) [ 15 ].…”