Analysis of Testosterone Binding with Cytochrome P450 11β-Hydroxylase and Its Variants Through Computational Approach

Effects of 4- tert-pentylphenol on the gene expression of P450 11β-hydroxylase in the gonad of medaka ( Oryzias latipes)

The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7 cells are used for transfection experiments to analyze the actions of functional molecules including steroids. However, a preliminary study suggested that COS-7 cells metabolize [3H]testosterone to [3H]androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the present study investigated the expression of steroidogenic enzymes and the metabolism of steroids in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase, 17β-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome P450 11β-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β, and 17β-HSD actively metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17β-HSD activity toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.

Gene expression profiling during spermatogenesis in early maturing male Atlantic salmon parr testes

Cloning and Expression of a Rat Cytochrome P-450 11β-Hydroxylase/Aldosterone Synthase (CYP11B2) cDNA Variant

In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase, cytochrome P450 17alpha-hydroxylase/17,20-lyase, cytochrome P450 11beta-hydroxylase, 11beta-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17alpha-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17alpha-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout.

Compensatory adrenal growth after unilateral adrenalectomy (ULA) leads to adrenocortical hyperplasia. Because zonal growth contributions are not clear, we characterized the phenotype of cortical cells that proliferate using immunofluorescence histochemistry and zone-specific cell counting. Rats underwent ULA, sham adrenalectomy (sham), or no surgery and were killed at 2 or 5 days. Adrenals were weighed and sections immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Adrenal weight increased after ULA compared with sham and no surgery at both time points, and there was no difference between sham and no surgery. However, either ULA or sham increased Ki67-positive cells in the outer fasciculata at both time points compared with no surgery. Outer fasciculata-restricted proliferation is thus associated with adrenal weight gain in ULA but not sham. Experiment repetition using proliferating cell nuclear antigen and bromodeoxyuridine showed similar results. After ULA, adrenal DNA, RNA, and protein increased at both time points, whereas after sham, only adrenal DNA increased at 2 days. Compensatory growth thus results from hyperplasia and hypertrophy, whereas sham induces only a transient adrenal hyperplasia. Dexamethasone pretreatment prevented the increase in adrenal weight after ULA and blocked Ki67 labeling in the outer fasciculata but not zona glomerulosa in all groups. These results clearly show that the outer fasciculata is the primary adrenal zone responsible for compensatory growth, responding to steroid-suppressible stress signals that alone are ineffective in increasing adrenal mass.

Effects of chronic manipulation of adrenocorticotropic hormone levels in Chinook salmon on expression of interrenal steroidogenic acute regulatory protein and steroidogenic enzymes

In situ hybridization was used to examine cellular differentiation during rat adrenal regeneration, defining zona glomerulosa [cytochrome P-450 aldosterone synthase (P-450aldo) mRNA positive], zona fasciculata [cytochrome P-450 11beta-hydroxylase (P-45011beta) mRNA positive], or zona intermedia [negative for both but 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA positive]. After unilateral adrenal enucleation with contralateral adrenalectomy (ULE/ULA), the expression of all mRNA was reduced at 2 days. From 5 to 10 days, P-45011beta and 3beta-HSD mRNA increased while P-450aldo remained low; at 20 days, all mRNA were increased. From 2 to 10 days, cells adjacent to the capsule showed intermedia cell differentiation; by 20 days, the subcapsular glomerulosa cells reappeared. This suggests that after enucleation the glomerulosa dedifferentiates to zona intermedia. The experiment was repeated in rats where the postenucleation ACTH rise was prevented. Rats underwent ULE with sham ULA (ULE/SULA) or ULE/SULA with ACTH treatment. Adrenals from ULE/SULA rats expressed increased P-450aldo mRNA at 10 days and reduced P-45011beta mRNA and adrenal weight at 30 days. ACTH treatment reversed the pattern toward that seen in ULE/ULA. These findings show that the enucleation-induced dedifferentitation of the glomerulosa cell may result in part from elevated plasma ACTH and that prevention of dedifferentiation may result in impaired regeneration.

Pre-clinical Cushing's syndrome (pre-CS) is sometimes seen with adrenal cortical tumors. An 80-year-old woman had severe hypertension and hypokalemia, the typical clinical features of primary aldosteronism, but detailed hormonal examinations revealed the accompanying pre-CS. After adrenalectomy by laparoscopy, her blood pressure was remarkably reduced and the hypokalemia also recovered. The tumor consisted of mostly light clear cells and scattered dark compact cells resembling islands. Abundant expression of cytochrome P450 aldosterone synthase in the clear cells and cytochrome P450 11beta-hydroxylase in the dark cells was detected by immunohistochemical studies, which confirmed that clear cells can produce aldosterone and compact cells can produce cortisol.

A quantitative analysis of zone-specific proliferation was done to determine the recovery of adrenal cortical zonation during regeneration after enucleation. Adult male rats underwent adrenal enucleation [unilateral enucleation (ULE)] or sham surgery, both accompanied by contralateral adrenalectomy. At 2, 5, 10, and 28 days, blood and adrenals were collected to assess functional recovery. Adrenal sections were immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P-450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P-45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Recovery of fasciculata secretory function occurred by 28 days as reflected by plasma ACTH and corticosterone, whereas glomerulosa function reflected by plasma aldosterone remained low at 28 days. At 5 days, ULE adrenals showed increased Ki67+ cells in the glomerulosa and inner fasciculata, whereas at 10 and 28 days increased proliferation was restricted to the outer fasciculata. These data show that enucleation results in transient elevations in glomerulosa and inner fasciculata cell proliferation followed by a delayed increase in the outer fasciculata. To assess adrenal growth in enucleated adrenals previously suppressed by the presence of an intact adrenal, rats underwent ULE and sham surgery; after 4 wk, the intact adrenal was removed and enucleated adrenals were collected at 2, 5, and 10 days. Overall, proliferation was delayed in this model, but at 5 days, Ki67+ cells increased in the outer fasciculata, whereas by 10 days, increased proliferation occurred in the outer and inner fasciculata. The key novel finding of increased proliferation in the inner fasciculata suggests that the delayed growth of the enucleated adrenal results in part from a regenerative response.

The in vitro metabolism of 11β-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway

Dynamic expression of 11β-hydroxylase during testicular development, recrudescence and after hCG induction, in vivo and in vitro in catfish, Clarias batrachus

We previously reported elevated adipose leptin expression, plasma leptin concentrations, and adrenocortical leptin receptor expression in the long-term hypoxic (LTH) ovine fetus. This study addressed whether leptin antagonist (LA) administration to LTH fetal sheep altered expression of key genes governing cortisol synthesis. Ewes were maintained at high altitude (3,820 meters) from 40 to 130 days gestation (dG), returned to Loma Linda University, and implanted with a maternal tracheal catheter. Reduced Po2 was maintained by nitrogen infusion. On 132 dG, LTH (n = 11) and age-matched, normoxic control (n = 11) fetuses underwent vascular catheter implantation. At 138 dG, fetuses were continuously infused with either saline or the LA (1.5 mg·kg(-1)·day(-1)) for 4 days and samples collected for blood gases, ACTH, and cortisol. Fetal adrenal cortex was collected for determination of steriodogenic acute regulatory protein (StAR), ACTH, and leptin receptor, cholesterol side-chain cleavage (CYP11A1), cytochrome P-450 11β-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17β-hydroxysteroid dehydrogenase (HSD3B) expression. In the saline-infused LTH fetuses, StAR, ACTH receptor, CYP11A1, and CYP17 expression was significantly lower compared with control (P < 0.05), whereas levels of CYP11B1, CYP21, and HSD3B mRNA were similar between groups. LA infusion restored expression of StAR, pSTAT3, CYP11A1, and CYP17, but not ACTH receptor, to normal ontogenic levels in the LTH group while having no effect on control fetuses. Neither fetal plasma ACTH nor cortisol concentrations were altered by LA infusion. We speculate that while leptin plays a role in governing expression of key enzymes and StAR in response to LTH, other factors play a role in modulating cortisol synthesis in these fetuses.

Adrenal cytochrome P450 11β-hydroxylase (CYP11B1) is a mitochondrial enzyme that catalyzes the final step of glucocorticoid synthesis, converting 11-deoxycortisol (S) and deoxycorticosterone (DOC) to cortisol (F) and corticosterone (CORT), respectively. CYP11B1 is predominantly located in the zona fasciculata of the adrenal gland with studies also showing that CYP11B1 catalyzes the conversion of androstenedione (A4) and testosterone (T) to 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT), respectively. Adrenal vein sampling in women has shown that 11OHA4 turnover is high, with a marked increase after treatment with ACTH which is known to upregulate CYP11B1 expression. We hypothesized that CYP11B1’s affinity for A4 as a substrate may be favored over glucocorticoids since 11OHA4 is one of the major androgens produced in the adrenalThis study aimed to elucidate the kinetic parameters (Km and Vmax) for A4 and T with respect to CYP11B1. A4 and T (0.2 µM to 5 µM) were assayed in HEK-293 cells transiently transfected with CYP11B1 and the adrenodoxin redox partner. Data was used to generate progress curves and fitted to the Michaelis-Menten equation. A4 had the lowest Km (0.21 μM) with a significantly higher Vmax (315.77 pmol/min/mg protein) in comparison to T, S and DOC. Results suggest that A4 binds more readily to CYP11B1 resulting in the high turnover of substrate. The androgenic activity of CYP11B1 catalyzed steroid products was determined using a luciferase assay conducted in CV1 cells. Both A4 and 11OHA4 showed no androgenic activity, however, the 11βHSD2 product of 11OHA4, 11-ketoandrostenedione (11KA4), elicited a response at 100 nM. 11OHT was an androgen receptor (AR) agonist, however, exhibiting a lower response when compared to T and 11-ketotestosterone over all concentrations tested (1, 10 and 100 nM). As confirmation of CYP11B1 activity in the adrenal, circulatory steroid levels were determined in healthy female subjects (n=88). CORT, F and 11OHA4 were measured at a frequency of 70–100%, compared to 11OHT (40–70%), while 11βHSD2 activity produced 11KA4 (<10%). In conclusion, the catalytic efficiency of CYP11B1 towards A4 is higher compared to T and the classical substrates. The high substrate affinity and turnover provides evidence for 11OHA4 in adrenal vein samples. Concurrently, our analysis suggests that agonism is diminished by the presence of the C11- hydroxyl group, while AR agonistic activity is gained upon its conversion to a keto group. Furthermore, these androgens could perhaps modulate cortisol production in the adrenal due to potential competition between precursor substrates.

Exercise prevents the increased anxiety-like behavior in lactational di-(2-ethylhexyl) phthalate-exposed female rats in late adolescence by improving the regulation of hypothalamus-pituitary-adrenal axis