Comparison of aldosterone production among human adrenocortical cell lines.

Several human adrenocortical cell lines have been used as model systems for aldosterone production. However, these cell lines have not been directly compared with each other. Human adrenal cell lines SW13, CAR47, the NCI-H295 and its sub-strains and sub-clones were compared with regard to aldosterone production and aldosterone synthase (CYP11B2) expression. Culture media was collected 48 h after incubation, aldosterone secretion was measured and the data were normalized to the amount of cell protein. RNA was isolated for microarray analysis and quantitative RT-PCR (qPCR). The cell lines with the highest aldosterone production were further tested with regard to angiotensin II (Ang II) stimulation. Neither aldosterone nor CYP11B2 transcript were detected in SW13 or CAR47 cells. The aldosterone production by the NCI-H295, H295A, H295R-S1, H295R-S2, H295R-S3, HAC13, HAC15 and HAC50 were 119, 1, 6, 826, 18, 139, 412, and 1 334 (pmol/mg protein/48 h), respectively. H295A and H295R-S1 expressed less CYP11B2 than the commonly used H295R-S3 cells; while NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 expressed 24-, 14-, 3-, 10-, and 35-fold higher CYP11B2 compared with the H295R-S3 cells. When treated with Ang II, NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 showed significantly higher aldosterone production than the basal level (p<0.05). A comparison of the available human adrenal cell lines indicates that the H295R-S2 and the clonal cell lines, HAC13, HAC15 and HAC50 produced the highest levels of aldosterone and responded well to Ang II.

Contrasting effects of eplerenone and spironolactone on adrenal cell steroidogenesis.

Spironolactone and eplerenone are widely used as mineralocorticoid antagonists. Spironolactone has several nonspecific actions including inhibition of androgen receptor and steroid hormone biosynthesis. While studies have shown that eplerenone does not exhibit nonspecific actions on androgen receptor, its effects on steroid hormone production have not been reported. Herein, the effects of eplerenone (0.1-30 microM) and spironolactone (0.1-30 microM) on steroid production were examined in human adrenocortical H295R cells. Spironolactone inhibited basal production of cortisol (91%) and aldosterone (53%). Treatment of H295R cells with angiotensin II (Ang II) for 24 h increased aldosterone production by 11-fold. Spironolactone inhibited Ang II stimulation of aldosterone production by 80%. Addition of pregnenolone increased aldosterone (9-fold) and cortisol (3-fold) production. Spironolactone inhibited pregnenolone metabolism to aldosterone (67%) and cortisol (74%). The inhibitory effects of spironolactone occurred at concentrations far higher than those needed to block mineralocorticoid receptor, suggesting an action directly on the enzymes involved in steroid production. In contrast, eplerenone did not inhibit basal, Ang II, forskolin, pregnenolone-stimulated cortisol, or aldosterone production. Together, these data demonstrate that opposed to spironolactone, pharmacologic concentrations of eplerenone do not inhibit adrenal cell aldosterone or cortisol production.

DAX-1A (NR0B1A) expression levels are extremely low compared to DAX-1 (NR0B1) in human steroidogenic tissues.

The orphan nuclear receptor DAX-1 (dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1; NR0B1) is known for its role in human development, specifically sex determination and steroidogenesis. Several recent publications have described an alternatively spliced form of DAX-1 called DAX-1A ( NR0B1A). DAX-1A is encoded by exons 1 and 2A of DAX-1, with exon 2A located within the DAX-1 intron 1. DAX-1A expression has been observed in several tissues, including adrenal gland, ovary, and testis. Transfection studies have further shown that DAX-1A has an inhibitory effect on DAX-1, suggesting a role for DAX-1A in the regulation of adrenal and gonadal differentiation/function. However, the relative level of DAX-1 versus DAX-1A transcripts still remains unclear. Herein, we developed and performed quantitative real-time RT-PCR to measure DAX-1 and DAX-1A mRNA expression levels in H295R human adrenal carcinoma cell lines, human adult and fetal adrenal glands, corpus luteum, testis, whole pre- and postmenopausal ovaries, ovarian follicles, placenta, liver, and kidney. These mRNA expression levels were quantified using DAX-1 and DAX-1A standard curves. In addition, Western blotting analysis was performed to examine both DAX-1 and DAX-1A protein levels in H295R cells, adrenal glands, corpus luteum, and liver. Both DAX-1 and DAX-1A mRNA were detected in all samples of H295R cells, human fetal and adult adrenals, testis, ovary, ovarian follicles, and corpus luteum. However, DAX-1 mRNA levels were significantly higher (> 37-fold) than that seen for DAX-1A (p<0.01). DAX-1A mRNA expression levels were undetectable in human liver, placenta, and kidney. Western blotting analysis results demonstrated that DAX-1 protein was predominantly expressed in H295R cells, human adult adrenal, and corpus luteum. These results suggest that in comparison to DAX-1A, DAX-1 is, by far, the predominant mRNA isoform found in human adrenal glands and gonads.

Pituitary homeobox factor 1, a novel transcription factor in the adrenal regulating steroid 11beta-hydroxylase.

Pituitary homeobox 1 (Ptx1/Pitx1) is a homeodomain-containing transcription factor present throughout pituitary development. Ptx1/Pitx1 interacts with steroidogenic factor 1 (SF-1) in the regulation of pituitary gene expression. SF-1 also plays a critical role in the transcription of enzymes involved in adrenal steroidogenesis. Therefore, we analyzed the presence and role of Ptx1/Pitx1 in human adrenal cortex. Both Ptx1/Pitx1 and SF-1 mRNA were expressed in the human adrenal gland, and immuno-electron microscopy demonstrated the presence of Ptx1/Pitx1 protein in the nucleus of adrenocortical cells. Computer analysis revealed the presence of Ptx1/Pitx1 signal sequences within the promoter region of human 11beta hydroxylase ( hCYP11B1). To examine the role of Ptx1/Pitx1 in the regulation of the genes, we prepared reporter constructs using the 5'-flanking DNA of the hCYP11B1 gene and transfected them into Y-1 mouse adrenocortical cells, HeLa and CV-1 cells. Ptx1/Pitx1 stimulation of hCYP11B1 reporter activity (3-fold over basal) in Y-1 cells was equal to that observed with SF-1. The hCYP11B1 promoter activity in Y-1 cells was not synergistically increased by co-transfection with both Ptx1/Pitx1 and SF-1. Both basal and ACTH-stimulated hCYP11B1 reporter activities in Y-1 cells were increased by co-transfection with either Ptx1/Pitx1 or SF-1 expression vectors. In contrast, co-transfection with both Ptx1/Pitx1 and SF-1 synergistically increased hCYP11B1 promoter activity in HeLa and CV-1 cells (5-fold and 20-fold over basal, respectively). In conclusion, this study represents the first demonstration for a role of Ptx1/Pitx1 in the regulation of transcription of enzymes involved in adrenal steroidogenesis.

Inhibition of Src tyrosine kinase stimulates adrenal androgen production.

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.

Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes.

In the current study we test the hypothesis that liver receptor homologue-1 (LRH; designated NR5A2) is involved in the regulation of steroid hormone production. The potential role of LRH was assessed by first examining expression in human steroidogenic tissues and second by examining effects on transcription of genes encoding enzymes involved in steroidogenesis. LRH is closely related to steroidogenic factor 1 (SF1; designated NR5A1), which is expressed in most steroidogenic tissues and regulates expression of several steroid-metabolizing enzymes. LRH transcripts were expressed at high levels in the human ovary and testis. Adrenal and placenta expressed much lower levels of LRH than either ovary or liver. To examine the effects of LRH on steroidogenic capacity we used reporter constructs prepared with the 5'-flanking region of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A1), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), 11beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Co-transfection of these reporter constructs with LRH expression vector demonstrated that like SF1, LRH enhanced reporter activity driven by flanking DNA from StAR, CYP11A1, CYP17, HSD3B2, and CYP11B1. Reporter constructs driven by CYP11A1 and CYP17 were increased the most by co-transfection with LRH and SF1. Of the promoters examined only HSD3B2 was more sensitive to LRH than SF1. The high level of ovarian and testicular LRH expression make it likely that LRH plays an important role in the regulation of gonadal function.

Differential regulation of aldosterone synthase and 11beta-hydroxylase transcription by steroidogenic factor-1.

11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.

Gene profiling of human fetal and adult adrenals.

The mechanisms that lead to the steroidogenic differences in the human fetal adrenal (HFA) and adult adrenal gland are not known. However, gene expression clearly plays a critical role in defining their distinct steroidogenic and structural phenotypes. We used DNA microarrays to compare expression levels of several thousand transcripts between the HFA and adult adrenal gland. Total RNA was isolated from 18 HFA and 12 adult adrenal glands. Samples of total RNA were used to make five pools of poly A+ RNA (mRNA). Gene profiling was done using five independent microarrays that contained between 7075 and 9182 cDNA elements. Sixty-nine transcripts were found to have a greater than 2.5-fold difference in expression between HFA and adult adrenals. The largest differences were observed for transcripts that encode IGF-II (25-fold higher in HFA) and 3beta-hydroxysteroid dehydrogenase (24-fold higher in adult). Among the other genes, transcripts related to sterol biosynthesis or to growth and development were higher in the HFA than adult adrenals. Transcripts concerned with cellular immunity and signal transduction were preferentially expressed in the adult adrenal. The vast majority of the 69 transcripts have not been studied with regard to adrenal function. Thus, these gene profiles provide valuable information that could help define the mechanisms that control adrenal function.

Liver receptor homologue-1 is expressed in the adrenal and can regulate transcription of 11 beta-hydroxylase.

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.

Metformin directly inhibits androgen production in human thecal cells.

OBJECTIVE: To examine the direct effect of metformin on thecal cell androgen production. SETTING: Basic science research laboratory, University of Texas Southwestern, Dallas, Texas. INTERVENTION(S): Human ovarian theca-like tumor cells were treated with various concentrations of metformin in the presence and absence of forskolin for 48 hours. MAIN OUTCOME MEASURE(S): Media were collected, and radioimmunoassay (RIA) for progesterone, 17 alpha-hydroxyprogesterone (17OHP), androstenedione, and testosterone was performed. The effect of metformin on the expression of various enzymes involved in theca cell steroidogenesis was examined. RESULT(S): Metformin (50 microM and 200 microM) significantly inhibited androstenedione production from both forskolin-stimulated and unstimulated theca cells. Testosterone production was also significantly inhibited in forskolin-treated cells in the presence of 200 microM of metformin-treated compared with forskolin-only-treated cells. Western blot analysis revealed that metformin significantly inhibited the expression of steroidogenic acute regulatory (StAR) protein and 17 alpha-hydroxylase (CYP17) expression in cells stimulated with forskolin compared with forskolin treatment alone. There was no significant change in either 3beta-hydroxysteroid dehydrogenase (3 beta HSD) or cholesterol side-chain cleavage (CYP11A1) protein expression. Northern analysis revealed a significant decrease in the expression of CYP17 mRNA in forskolin-stimulated cells treated with metformin (200 microM) compared with forskolin-only-treated cells, however, there was no significant change in steroidogenic acute regulatory protein mRNA expression. CONCLUSION(S): Our results suggest that metformin may have a direct effect on thecal cells' androgen production.

A role for src tyrosine kinase in regulating adrenal aldosterone production.

Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regulators of aldosterone production are angiotensin II (Ang IotaIota) and potassium (K(+)). Ang IotaIota stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang IotaIota activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 microM-10 microM) inhibited basal, Ang IotaIota, K(+) and dibutyryladenosine cyclic monophosphate (dbcAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 microM) for 6, 12, 24, and 48 h revealed a time-dependent inhibition of aldosterone production. Inhibition by PP2 (0.3-10 microM) was also observed for the metabolism of 22R-hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non-specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3beta-hydroxysteroid dehydrogenase (3betaHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 3betaHSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [(14)C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17alpha-hydroxyprogesterone. To determine if this apparent increase in 17alpha-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3betaHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechanism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.

Expression profiles of SF-1, DAX1, and CYP17 in the human fetal adrenal gland: potential interactions in gene regulation.

Cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(C17)) is a critical branchpoint enzyme for steroid hormone biosynthesis. During human gestation, P450(C17) is required for the production of dehydroepiandrostenedione sulfate by the fetal adrenal cortex and for testicular production of androgens that mediate male sexual differentiation. In this study, we investigate the regulation of the human CYP17 gene by two orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX1. In human embryos, SF-1 and DAX1 are expressed throughout the developing adrenal cortex from its inception at 33 days post conception (dpc). In contrast, P450(C17) expression, which commences between 41 and 44 dpc, is limited to the fetal zone. The 5'-flanking region of the human CYP17 gene contains three functional SF-1 elements that collectively mediate a > or =25-fold induction of promoter activity by SF-1. In constructs containing all three functional SF-1 elements, DAX1 inhibited this activation by > or =55%. In the presence of only one or two SF-1 elements, DAX1 inhibition was lost even though SF-1 transactivation persisted. These data suggest that efficient repression of SF-1-mediated activation of the human CYP17 gene by DAX1 requires multiple SF-1 elements. Opposing effects of SF-1 and DAX1 may fine tune the differential responses of various SF-1 target genes in different endocrine tissues.

Transcriptional regulation of human 11beta-hydroxylase (hCYP11B1).

Steroid 11beta-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11beta-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5'-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing -1102 bp of hCYP11B1 5'-flanking DNA (pB1-1102). An element at position -71/-64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. In addition, Western analysis of H295R and adrenal lysates demonstrated expression of high levels of ATF-2 and ATF-1. CRE-binding protein levels varied among the strains of H295R cells tested. Transcription of CYP11B1 also appeared to be regulated by steroidogenic factor-1 (SF-1). Luciferase reporter gene activity was increased after cotransfection with expression vectors containing SF-1. An element in hCYP11B1 at positions 242/-234 (CCAAGGCTC), previously termed Ad4, was required for maximal induction by SF-1 and was found to bind SF-1 in electrophoretic mobility shift assays. The key role for SF-1 in hCYP11B1 transcription is in contrast to its lack of an effect on expression of the hCYP11B2 (aldosterone synthase) isozyme. The differential effects of SF-1 on transcription of hCYP11B1 and hCYP11B2 may be one of the mechanisms controlling differential expression of these isozymes within the zonae fasciculata and glomerulosa of the human adrenal cortex.

Bone morphogenetic protein inhibits ovarian androgen production.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-4 on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of androstenedione and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-4. The accumulation of androstenedione, progesterone, and 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in androstenedione secretion. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively. In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3betaHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-4 inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor beta superfamily, including activin and inhibin, had minimal effect on androstenedione production in the absence of forskolin. In the presence of forskolin, activin inhibited androstenedione production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-4 inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced androstenedione accumulation and increased progesterone production. These effects of BMP-4 seem similar to those caused by activin, another member of the transforming growth factor-beta superfamily of proteins.

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.

Transforming growth factor beta inhibits steroidogenic acute regulatory (StAR) protein expression in human ovarian thecal cells.

In this study, we investigated the effects of TGFbeta1 on steroidogensis and expression of the steroidogenic acute regulatory (StAR) protein which regulates an important early step in the steroidogenic pathway. We utilized a human ovarian thecal like tumor (HOTT) cell model and investigated the effects of activin-A, inhibin-A, or TGFbeta1 in the presence of forskolin and the effect of dibutyryl cyclic AMP (dbcAMP) on steroid accumulation in the culture medium. Cells were also treated with different concentration of TGFbeta1 in the presence of forskolin, combined steroid production was measured at the end of 48 h and after 3 h incubation with 22R-hydroxycholesterol. In the presence of TGFbeta1 there was a dose-dependent inhibition of androstenedione production. Inhibition in combined steroid production was apparent at the highest concentration of TGFbeta1 tested. In the presence of 22R-hydroxycholesterol, combined steroid production was significantly inhibited at lower concentrations. TGFbeta1 inhibited StAR protein expression in a concentration dependent manner. There was also a similar inhibition in StAR mRNA. These results suggest that the effect of TGFbeta1 on steroid production and possibly follicular development may be in part due to its effects on StAR expression.

The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal.

Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.

Regulation of human CYP11B2 and CYP11B1: comparing the role of the common CRE/Ad1 element.

In humans, the final steps in corticosteroid production results from the activity of aldosterone synthase in the glomerulosa and 11beta-hydroxylase in the fasciculata. The regional expression of these isozymes is believed to result from transcriptional regulation of the aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) genes. Previous studies suggest that the primary cis-element needed for agonist enhanced transcription of the CYP11B genes shares high sequence similarity to a consensus cAMP Response Element (CRE). Here the role of the CRE/Adl was studied. Reporter constructs prepared with the 5'flanking DNA of hCYP11B2 and hCYP11B1 were transfected into NCI-H295R (H295R) adrenocortical tumor cells. Both hCYP11B2 and hCYP11B1 driven reporter constructs responded in a similar manner to treatment with angiotensin II, potassium, dbcAMP, or forskolin. Mutation of the hCYP11B1 CRE/Adl element decreased basal reporter expression and decreased response to agonist. Mutation of the hCYP11B2 CRE/Adl element caused a loss of basal expression but retained response to agonist suggesting a role for other cis-elements in hormonal regulation of hCYP11B2. In addition, both cis-elements were able to form complexes with in vitro prepared CRE binding (CREB) protein, activating transcription factor (ATF)-1 and ATF-2 in mobility shift assays. However, only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. Taken together these data suggest that the CRE/Adl element plays an important role in the transcriptional regulation of both hCYP11B genes but does not play an important role in the regional distribution of the two isozymes within the adrenal.

Adrenal zonation: clues from 11beta-hydroxylase and aldosterone synthase.

Aldosterone and cortisol are the major mineralocorticoid and glucocorticoid produced by the human adrenal. Circulating levels of angiotensin II and potassium control the adrenal production of aldosterone, while the production of cortisol is controlled mainly by adrenocorticotropin. The capacity of the adrenal cortex to differentially produce aldosterone and cortisol relies to a large degree on the expression of aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1). CYP11B2 catalyzes the final steps in the biosynthesis of aldosterone and is expressed solely in the glomerulosa of the adrenal cortex, while CYP11B1 catalyzes the final steps in the biosynthesis of cortisol and is expressed in the fasciculata/reticularis. The zonal expression of these two isozymes appears to result from transcriptional regulation of the two genes. Herein, the recent progress in defining the cellular mechanisms that regulate transcription of these two isozymes and thus the capacity of the adrenal gland to differentially produce aldosterone and cortisol is discussed.

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.