C19 Steroid Production Research Articles

SAT-742 Characterising the Metabolism, Glucuronidation and Sulfation of C11-oxy C19 Steroids

The metabolism of 11β-hydroxyandrostenedione (11OHA4), a major adrenal C19 steroid, was first characterised in our in vitro prostate models showing that 11OHA4, catalysed by 11βHSDs, 17βHSDs and 5α-reductases, yields potent androgens, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) in the 11OHA4-pathway [1]. Findings have since led to the analysis of C11-oxy steroids in PCOS, CAH and 21OHD. However, the only circulating C11-oxy steroids included to date have been 11OHA4, 11keto-androstenedione (11KA4), 11β-hydroxytestosterone (11OHT) and 11KT, with 11KT reported as the only potent androgen produced from 11OHA4. We have identified higher levels of 11KDHT compared to 11KT in prostate cancer tissue and benign prostatic hyperplasia tissue and serum, with data suggesting impeded glucuronidation of the C11-oxy androgens [2,3]. The assessment of 11KDHT and the inactivation/conjugation of the C11-oxy steroids in clinical conditions is therefore crucial.We investigated the metabolism of testosterone, 11KT, 11OHT, dihydrotestosterone, 11KDHT and 11OHDHT in JEG-3 placenta choriocarcinoma, MCF-7 BUS and T-47D breast cancer cells, focusing on glucuronidation and sulfation. Steroids were assayed at 1 µM and metabolites were quantified using UPC2-MS/MS. Conjugated steroids were not detected in JEG-3 cells with DHT (0.6 µM remaining) metabolised to 5α-androstane-3α,17β-diol and androsterone (AST), and 11KDHT (0.9 µM remaining) to 11OHAST and 11KAST. 11OHA4 was converted to 11KA4 (12%) and 11KT (2.5%); and 11KT to 11KDHT (14%). In MCF-7 BUS cells, DHT was significantly glucuronidated, whereas 11KDHT was not. 11KAST was the only steroid in the MCF-7 BUS and T-47D cells that was significantly sulfated (p<0.05). In parallel we investigated sulfation in the LNCaP prostate model. Comparing sulfated to glucuronidated levels, only DHT was sulfated, 26%. Analysis showed that C19 steroids were significantly conjugated (glucuronidated + sulfated) compared to the C11-oxy C19 steroids.As there exists an intricate interplay between steroid production and inactivation, impacting pre- and post-receptor activation, efficient conjugation would limit adverse downstream effects. Our data demonstrates the production and impeded conjugation of active C11-oxy C19 steroids, allowing the prolonged presence of androgenic steroids in the cellular microenvironment. Identified for the first time is the 11OHA4-pathway in placenta and breast cancer cells, and the sulfation of 11KAST. Characterising steroidogenic pathways in in vitro models paves the direction for in vivo studies associated with characterising clinical disorders and disease, which the C11-oxy C19 steroids and their intermediates, including inactivated and conjugated end-products, have highlighted. [1] Bloem, et al. JSBMB 2015, 153; [2] Du Toit & Swart. MCE 2018, 461; [3] Du Toit & Swart, JSBMB 2020, 105497.

SAT-019 Clinical Disorders and Diseases Marked by Elevated Adrenal C11-oxy C21 and C11-oxy C19 Steroid Levels

Adrenal steroid hormones have been implicated in numerous clinical conditions with adrenal androgens marked as major contributors in the progression of androgen-dependent diseases and disorders such as congenital adrenal hyperplasia, 21-hydroxylase deficiency, polycystic ovary syndrome (PCOS) and castration-resistant prostate cancer. Although adrenal C19 androgens, androstenedione and testosterone, have been routinely monitored in patients under clinical care, it is only recently that the contribution of the C11-oxy androgens has come to the attention of clinicians. Adrenal 11β-hydroxyandrostenedione (11OHA4) is systemically converted by steroidogenic enzymes to produce potent C11-oxy C19 steroids, notably 11keto-testosterone and 11keto-dihydrotestosterone (11KDHT) in the 11OHA4-pathway. Furthermore, we recently identified adrenal C11-oxy C21 steroids as potential contributors to androgen excess via the C11-oxy C21 backdoor pathway −11β-hydroxyprogesterone and 11keto-progesterone (11KPROG), together with 21-deoxycortisol and 21-deoxycortisone are converted to 11KDHT, thus potentially adding to the androgen pool in disease states. Using a high-throughput ultra performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the separation and quantification of C11-oxy steroids and their downstream metabolites, we were able to simultaneously analyze 51 steroids: 31 C21 and 20 C19, which include the C11-oxy C21 and C11-oxy C19 steroids, in a single 8 min chromatographic step. Our in vivo data show, in neonatal plasma, at birth, detected C11-oxy C21 steroids were 2-fold higher than C11-oxy C19 steroids, ~755.6 and ~393.1 ng/dL, respectively. Conversely, in PCOS patients, the C11-oxy C19 steroids were present at higher levels compared to the C11-oxy C21 steroids and also compared to controls, showing a greater shunt towards the production of C19 and C11-oxy C19 steroids. In benign prostatic hyperplasia patients, suffering from a relatively undefined condition in terms of adrenal steroids, the C11-oxy C19 steroid levels were 8-fold higher in circulation compared to the C11-oxy C21 steroids, with prominent C11-oxy steroids quantified as 11KPROG (190.5 ng/dL), 11keto-dihydroprogesterone (278.9 ng/dL), 11OHA4 (2598.8 ng/dL), 11keto-androstenedione (415.4 ng/dL) and 11KDHT (183.7 ng/dL). We propose that the 11OHA4- and C11-oxy C21 backdoor pathways may identify novel steroid biomarkers in clinically defined and undefined conditions. Indeed, accurate quantification of these steroid hormones has been facilitated by UPC2-MS/MS with comprehensive steroid profile analysis potentially aiding neonatal screening, identifying rare enzymatic deficiencies, determining hyperandrogenism in males and females −promising routinely applicable superiority over other analytical methods, with this study underscoring this promise.

Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.

Adrenal C11-oxy C21 steroids contribute to the C11-oxy C19 steroid pool via the backdoor pathway in the biosynthesis and metabolism of 21-deoxycortisol and 21-deoxycortisone

21-Hydroxylase deficiency presents with increased levels of cytochrome P450 21‐hydroxylase substrates, progesterone and 17α-hydroxyprogesterone, which have been implicated in the production of androgens via the backdoor pathway. This study shows the biosynthesis of C11-oxy C21 steroids, 21-deoxycortisol and 21-deoxycortisone, and their metabolism by steroidogenic enzymes in the backdoor pathway yielding novel steroid metabolites: 5α‐pregnan-11β,17α-diol-3,20-dione; 5α-pregnan-17α-ol-3,11,20-trione; 5α‐pregnan-3α,11β,17α-triol-20-one and 5α-pregnan-3α,17α-diol-11,20-dione. The metabolism of 21-deoxycortisol was validated in LNCaP cells expressing the relevant steroidogenic enzymes showing for the first time that the steroid, produced at high levels in 21OHD, is metabolised via the C11-oxy derivatives of 5α‐pregnan-17α-ol-3,20-dione and 5α‐pregnan-3α,17α-diol-20-one to substrates for the lyase activity of CYP17A1, leading to the production of C11-oxy C19 steroids. 21-Deoxycortisol thus contributes to the pool of potent androgens in 21OHD, with novel steroid metabolites also presenting possible biomarkers in disease identification.

11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown.We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids – 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

Chemical and biochemical approaches to the production of 7-hydroxylated C19-steroids.

Abstract Interest has developed into 7-hydroxylated derivatives of 3β-hydroxylated C19-steroids, such as dehydroepiandrosterone (DHEA) and epiandrosterone because of their effects on inflammation, immune response, and cell repair. These steroids are not currently available from commercial sources, and it is necessary to produce them for relevant studies. We report here the chemical and biochemical approaches that were used for their production. Simplified chemical approaches lead to the production of 7α-/7β-hydroxy-DHEA and 7β-hydroxy-epiandrosterone in gram quantities, which are made available to researchers. Biochemical approaches were used to produce isotope-labeled compounds. Thus, 2H-, 3H-, and 14C-labeled 7α-/7β-hydroxy-DHEA and 7β-hydroxy-epiandrosterone could be produced in quantities sufficient for use in investigations into their mode of action.

Effects of Anticonvulsants on Human P450c17 (17α‐Hydroxylase/17,20 Lyase) and 3β‐Hydroxysteroid Dehydrogenase Type 2

Women with epilepsy apparently have a higher incidence of polycystic ovary syndrome (PCOS) than do women without epilepsy. Whether the underlying disease or the antiepileptic drug (AED) treatment is responsible for this increased risk is unknown, although clinical reports implicate valproic acid (VPA) as a potential cause. The steroidogenic enzymes 3beta HSDII (3beta-hydroxysteroid dehydrogenase) and P450c17 (17alpha-hydroxylase/17,20 lyase) are essential for C19 steroid biosynthesis, which is enhanced during adrenarche and in PCOS. To determine whether the AEDs VPA, carbamazepine (CBZ), topiramate (TPM), or lamotrigine (LYG) directly affect the activities of human 3beta HSDII and P450c17, we added them to yeast expressing human P450c17 or 3beta HSDII and assayed enzymatic activities in the microsomal fraction. Concentrations of VPA < or = 10 mM had no effect on activities of P450c17; however, VPA inhibited 3beta HSDII activity starting at 0.3 mM (reference serum unbound concentration, 0.035-0.1 mM) with an IC50 of 10.1 mM. CBZ, TPM, and LTG did not influence 3beta HSDII or P450c17 activities at typical reference serum unbound concentrations, but did inhibit 3beta HSDII and P450c17 at concentrations >10-fold higher. None of the tested AEDs influenced 3beta HSDII or P450c17 activities at concentrations normally used in AED therapy. However, VPA started to inhibit 3beta HSDII activity at concentrations 3 times above the typical reference serum unbound concentration. Because inhibition of 3beta HSDII activity will shift steroidogenesis toward C19 steroid production when P450c17 activities are unchanged, very high doses of VPA may promote C19 steroid biosynthesis, thus resembling PCOS. CBZ, TPM, and LTG influenced 3beta HSDII and P450c17 only at toxic concentrations.

Molecular cloning and expression of guinea pig cytochrome P450c21 cDNA (steroid 21-hydroxylase) isolated from the adrenals

In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid and glucocorticoid syntheses, P450c21 plays an important role, because it is localized at the key branch between glucocorticoids and C19 steroid production. Its implication in congenital adrenal hyperplasia is also of great clinical interest. In this study, in addition to describing the isolation of the P450c21 cDNA from guinea pig (GP) adrenal and comparing it to those from other species, we report on its tissue-distribution and on the activity of the recombinant protein towards progesterone and 17-hydroxyprogesterone. The guinea pig P450c21 includes the full-length coding region (1464 nucleotide) that is translated to a protein of 488 amino acids. The clone shares highly conserved regions with other species. The guinea pig P450c21 cDNA hybridized with a major transcript of 2.1 kb and with two minor related transcripts of 1.8 and 1.5 kb and was found to be adrenal-specific among the various tissues analyzed. Characterization of the enzymatic activity by transient transfection of the guinea pig P450c21 cDNA in human embryonic kidney 293 cells indicated a net preference for the 21-hydroxylation of 17-hydroxyprogesterone in comparison to the progesterone substrate. Assays showed a maximum conversion rate of 12.5% for the conversion of progesterone into deoxycorticosterone (mineralocorticoid pathway), whereas the guinea pig P450c21 demonstrated a higher activity with 17α-hydroxyprogesterone, with 55% of 11-deoxycortisol formation (glucocorticoid pathway) after 48 h. Adrenocorticotropin and an analogue of the second messenger cyclic adenosine monophosphate specifically increased the abundance of P450c21 mRNA levels in guinea pig adrenal cells.

MARMOSETS EXPRESS A FETAL ZONE AT BIRTH BUT NO ZR IN ADULTHOOD

The newborn marmoset produces large amounts of DHEAS that fall to low= undetectable levels in adults. Is there a fetal zone in newborns and ZR in adults, distinct from the ZF by low=absent 3b-HSD and higher cyt b5? We used IHC to stain adrenals from one day (n ¼ 2) vs. one year-old (n ¼ 4) marmosets for b5, reductase, P450c17, and 3b-HSD. Expression of b5 in 1 d-olds was undetectable in the ZG=ZF and strong in the inner half of the cortex, suggesting a fetal zone (FTZ). Reductase stained slightly in the ZG=ZF and strongly in the FTZ. P450c17 expression was undetectable in the ZG, faint in the ZF, and strong in the FTZ. 3bHSD stained in the outer cortex but not in the FTZ. In 1 y-old adrenals, b5 staining was undetectable in any region of the cortex. Reductase stained slightly in the ZG and stronger in the ZF. P450c17 was undetectable in the ZG and positive in the ZF. 3b-HSD staining was highest in the ZG and uniform through the ZF to the medullary interface. These results suggest the newborn marmoset adrenal, like the human, contains a significant FTZ geared to C19 steroid production while the adult shows a ZG and ZF, but no ZR.

Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum.

Testis fragments from Bufo arenarum were incubated with [7(n)-(3)H]pregnenolone (P5), [1,2-(3)H]dehydroepiandrosterone (DHEA) and [1,2,6.7-(3)H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3beta-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17chi-hydroxylase, C17-20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17beta-hydroxysteroid dehydrogenase (17betaHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17betaHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17betaHSD could be due to the reduction of C19-substrates. The activity of 5chi-reductase was inhibited by CNK only if results from P5 and DHEA were considered.

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies.

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.

Identification and developmental expression of the estrogen receptor alpha and beta in the baboon fetal adrenal gland.

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones ofthe baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor alpha mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8+/-40.3 attomoles/microg RNA) compared with mid (27.4+/-5.4 attomoles/microg RNA) gestation. Moreover, estrogen receptor alpha mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6+/-0.5 silver grains/0.025 mm2) than in the fetal zone (8.3+/-1.5 grains/0.025 mm2) late in gestation. The mRNA for the beta-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor alpha and beta proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor alpha and beta were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.

Adrenarche results from development of a 3beta-hydroxysteroid dehydrogenase-deficient adrenal reticularis.

Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

Expression of 11?-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal

To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. The RNase protection assay was designed to distinguish specific bands that corresponded to CYP11B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C19 steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.

17 alpha-Hydroxylase and chromogranin A in 6th week human fetal adrenals.

The development of the human fetal adrenals starts in the 6th week gestational age and adrenal C19 steroid production becomes of major importance for the maintenance of the pregnancy. Therefore, in the present study, human fetal adrenals at 6 weeks of gestational age were immunostained for 17 alpha-hydroxylase, the key enzyme for the production of C19-steroids. In parallel, chromaffin cells were characterized by immunohistochemical staining for chromogranin A, the major soluble protein in adrenal chromaffin granules. Large 17 alpha-hydroxylase-immunoreactive cells were found in the center of the adrenal anlagen during the 6th week of gestation. At the same developmental stage, chromaffin cells with a neuronal-like appearance occurred in the paraortic area and started to invade the adrenal primordium. Our results show that, even at week 6 gestational age, when chromaffin cells start to enter the adrenal anlagen, human adrenals already contain differentiated, 17 alpha-hydroxylase immunoreactive cortical cells which were located to the center of the primordium.

Immunohistochemical analysis of AT1 receptor versus P450c17 and 3 beta HSD expression in ovine adrenals.

Previous studies in ovine adrenocortical cells in vitro have shown angiotensin II (AII) receptors are expressed on the zona fasciculata (ZF) cells and are functionally coupled to phosphoinositidase C and increased [Ca2+]i, but AII stimulation does not cause an acute change in cortisol biosynthesis. AII can, however, chronically regulate differential expression of P450c17 and 3 beta HSD in ovine adrenocortical cells in vitro. We have stained ovine adrenal sections with specific antisera to the angiotensin II Type-1 receptor (AT1-R), as well as P450c17 and 3 beta HSD in order to further test the hypothesis that changes in AT1-R expression underlie changes in zonal expression of P450c17 and 3 beta HSD in vivo. AT1-R expression was found to be highest in the outermost layer of cells (zona glomerulosa, ZG) which stained negatively for P450c17 and only faintly positive for 3 beta HSD, as expected. The adjacent layer of cells (ZF) stained much less strongly for AT1-R but stronger for P450c17 and 3 beta HSD. These findings are consistent with our previously reported in vitro expression data, and suggest that the transition from ZG to ZF phenotype, i.e. increased P450c17 and 3 beta HSD expression, may require reduced expression of AT1-R, but maintenance of reduced levels of AT1-R expression in the ovine ZF still allows for differential control of the P450c17: 3 beta HSD ratio. Thus, even though there is no acute cortisol response to AII alone in these cells, AII stimulation can oppose C19 steroid production in the face of cortisol biosynthesis by the ZF in response to agonists such as ACTH.

Inhibin and activin differentially regulate androgen production and 17α-hydroxylase expression in human ovarian thecal-like tumor cells

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-beta superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 microM) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3 beta HSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3 beta HSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1.5-fold) and activity (twofold) but with minimal effects on the mRNA for 3 beta HSD and P450scc. HOTT cell responses to activin (0.05-50 ng/ml) and inhibin (0.05-50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

Adrenal androgen biosynthesis with special attention to P450c17.

In vitro studies of human adrenal androgen synthesis are limited because of the difficulties in obtaining adrenals. We describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19-steroid production. The cells were characterized with regard to responsiveness to a variety of agents as measured by steroid secretion and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17) expression, a key enzyme in C19-steroid production. Forskolin and dibutyryl cAMP, which were more effective than ACTH, enhanced the production of DHEA and androstenedione over a 48-hour treatment period. Agents that act by increasing intracellular calcium (angiotensin II and K+ ions) as well as protein kinase A pathway activators (ACTH, forskolin, and dibutyryl cAMP) individually increased the mRNA levels and activity of P450c17. In addition, angiotensin II but not K+ ions attenuated the increased expression promoted by the kinase A agonists. Thus, the complexity of human adrenal P450c17 expression through multiple signaling pathways may contribute importantly to the diverse patterns of human adrenocortical steroidogenesis.