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Endocrinology Vol. 148, No. 8 3722-3729
Copyright © 2007 by The Endocrine Society

B-Type Natriuretic Peptide Inhibited Angiotensin II-Stimulated Cholesterol Biosynthesis, Cholesterol Transfer, and Steroidogenesis in Primary Human Adrenocortical Cells

Faquan Liang1, Ann M. Kapoun1, Andrew Lam, Debby L. Damm, Diana Quan, Maile O’Connell and Andrew A. Protter

Scios, Inc., Fremont, California 94555

Address all correspondence and requests for reprints to: Andrew A. Protter, Ph.D., Scios Inc., 6500 Paseo Padre Parkway, Fremont, California 94555. E-mail: andyprotter{at}mac.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we demonstrate that B-type natriuretic peptide (BNP) opposed angiotensin II (Ang II)-stimulated de novo cholesterol biosynthesis, cellular cholesterol uptake, cholesterol transfer to the inner mitochondrial membrane, and steroidogenesis, which are required for biosynthesis of steroid hormones such as aldosterone and cortisol in primary human adrenocortical cells. BNP dose-dependently stimulated intracellular cGMP production with an EC50 of 11 nM, implying that human adrenocortical cells express the guanylyl cyclase A receptor. cDNA microarray and real-time RT-PCR analyses revealed that BNP inhibited Ang II-stimulated genes related to cholesterol biosynthesis (acetoacetyl coenzyme A thiolase, HMG coenzyme A synthase 1, HMG coenzyme A reductase, isopentenyl-diphosphate {Delta}-isomerase, lanosterol synthase, sterol-4C-methyl oxidase, and emopamil binding protein/sterol isomerase), cholesterol uptake from circulating lipoproteins (scavenger receptor class B type I and low-density lipoprotein receptor), cholesterol transfer to the inner mitochondrial membrane (steroidogenic acute regulatory protein), and steroidogenesis (ferredoxin 1,3ß-hydroxysteroid dehydrogenase, glutathione transferase A3, CYP19A1, CYP11B1, and CYP11B2). Consistent with the microarray and real-time PCR results, BNP also blocked Ang II-induced binding of 125I-labeled low-density lipoprotein and 125I-labeled high-density lipoprotein to human adrenocortical cells. Furthermore, BNP markedly inhibited Ang II-stimulated release of estradiol, aldosterone, and cortisol from cultured primary human adrenocortical cells. These findings demonstrate that BNP opposes Ang II-induced steroidogenesis via multiple steps from cholesterol supply and transfer to the final formation of steroid hormones. This study provides new insights into the cellular mechanisms by which BNP modulates Ang II-induced steroidogenesis in the adrenal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN-ALDOSTERONE system has been implicated in a series of cardiovascular diseases. Angiotensin II (Ang II) is a vasoactive peptide hormone that potently stimulates aldosterone synthesis in the adrenal cortical cells, which results in sodium and water retention, cardiac fibrosis, and inflammation, leading to hypertension, heart failure, and renal failure (1, 2, 3, 4, 5). It also stimulates cortisol release from human adrenal cells, which may also contribute to the pathophysiology of this neurohormone (6, 7).

Adrenal steroidogenesis starts from cholesterol, which is a common precursor of all steroid hormones. Cholesterol can be provided either by de novo biosynthesis from acetyl coenzyme A or circulating low- and high-density lipoproteins. The uptake of low-density lipoprotein (LDL) involves binding of the LDL particles to specific cell surface receptors termed LDL receptors (LDLR) followed by internalization of the lipoprotein-receptor complex and lysosomal hydrolysis leading to the release of cholesterol esters (8). In contrast, the delivery of cholesterol from high-density lipoprotein (HDL) takes place through scavenger receptor class B type I (SR-BI) on the cell surface without the internalization and degradation of lipoprotein particles (9, 10, 11). The interaction of HDL with SR-BI leads to the formation of a nonaqueous channel through which cholesterol esters move down their concentration gradient to the plasma membrane (10). The lipid-poor HDL then dissociate from the cells and reenter the circulation. SR-BI also binds native LDL and modified LDL and mediates the cholesterol supply from LDL to steroidogenic cells and other types of cells (12, 13). Cholesterol is converted to pregnenolone, which is used for the synthesis of three main families of steroid hormones including aldosterone, cortisol, and sex hormones.

Atrial natriuretic peptide and B-type natriuretic peptide (BNP) are cardiac-derived peptide hormones that counteract the renin-angiotensin-aldosterone system, endothelin, and hemodynamic forces that promote the progression of heart failure. Both atrial natriuretic peptideand BNP have been previously shown to inhibit Ang II-induced aldosterone synthesis in adrenal glomerulosa cells (14, 15, 16). However, it remains unclear whether Ang II and natriuretic peptides regulate the cholesterol biosynthesis, cholesterol transfer, and steroid synthetic pathways that are required for adrenal steroidogenesis. Of note, most human adrenal cell studies were performed using the H295R cell line, a transformed human adrenocorticocarcinoma cell. This transformed cell line may not appropriately reflect the features of primary human adrenal cells. In this study, we isolated primary human adrenocortical cells from human adrenal gland and demonstrated that BNP exerted a broad functional opposition to Ang II, including cholesterol biosynthesis, cholesterol uptake, cholesterol transfer, and steroidogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ang II was purchased from Sigma Chemical Co. (St. Louis, MO). Human BNP was obtained from American Peptide Co. (Sunnyvale, CA). [125I]HDL and [125I]LDL were obtained from Biomedical Technologies (Stoughton, MA). All other reagents were provided by commercial sources.

Isolation and culture of human adrenocortical cells
Three human adrenal glands donated by a 50-yr-old white man (donor 1), a 20-yr-old white man (donor 2), and a 63-yr-old white man (donor 3) were purchased from Tissue Transformation Technology (Edison, NJ), kept in oxygenized buffer, and shipped to Scios Inc. within 24 h. Adrenal cortex was dissected and digested with 0.25% collagenase I. The freshly isolated cells were cultured in DMEM containing 10% fetal bovine serum for 3 d. Cells were changed to serum-free medium and treated with 200 nM human BNP in the presence or absence of 100 nM Ang II for 24 h. Pooled biological duplicate cells isolated from donor 1 were used for microarray analysis. Cultured human adrenocortical cells were mixed populations including glomerulosa cells, fasciculata cells, and reticularis cells that synthesize all types of steroid hormones.

Intracellular cGMP assay
Human adrenocortical cells were preincubated with 0.1 mM 3-isobutyl-1-methylxanthine for 1 h before treatment of increasing concentrations of BNP for 10 min. Cells were lysed with 0.1 M HCl at room temperature for 20 min. The lysates were centrifuged, and the levels of cGMP in the supernatant were measured using a cGMP enzyme immunoassay kit from Assay Designs (Ann Arbor, MI).

cDNA microarray
Gene expression profiles were determined as previously described (17) from cDNA microarrays containing 8600 elements derived from clones isolated from normalized cDNA libraries or purchased from ResGen (Invitrogen Life Technologies, Carlsbad, CA). Fluorescently labeled cDNA probes were prepared from pooled mRNAs generated from duplicate wells of cells from four groups: control (unstimulated), 100 nM Ang II, 200 nM BNP, and the combination of Ang II and BNP. Quadruplicate hybridizations were performed. Differential expression values were expressed as the ratio of the median of background-subtracted fluorescent intensity of the experimental RNA to the median of background-subtracted fluorescent intensity of the control RNA. For ratios of 1.0 or more, the ratio was expressed as a positive value. For ratios less than 1.0, the ratio was expressed as the negative reciprocal (i.e. a ratio of 0.5 = –2.0).

Real-time RT-PCR
Real-time RT-PCR was performed in a two-step manner. cDNA synthesis and real-time detection were carried out in a PTC-100 Thermal Cycler (MJ Research, Waltham, MA) and an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA), respectively. Random hexamers (QIAGEN, Valencia, CA) were used to generate cDNA from 200 ng RNA, as described in Applied Biosystems User Bulletin No. 2. TaqMan PCR Core Reagent Kit or TaqMan Universal PCR Master Mix (Applied Biosystems) was used in subsequent PCR according to the manufacturer’s protocols. Relative quantitation of gene expression was performed using the relative standard curve method. All real-time RT-PCR were performed in triplicate.

Sequence-specific primers and probes were designed using Primer Express version 2 software (Applied Biosystems). Sequences of primers and probes are shown in Table 1Go. Expression levels were normalized to 18S rRNA. The selection of 18S rRNA as an endogenous control was based on an evaluation of the {Delta}CT levels (Applied Biosystems document no. 4308134C) of the following six housekeeping genes: cyclophilin A, 18S, GAPDH, ß-actin, ß-glucuronidase, and hypoxanthine guanine phosphoribosyl transferase. The {Delta}CT levels of 18S did not differ significantly between treatment conditions; thus, they were expressed at constant levels between samples (data not shown).


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  		TABLE 1. Real-time RT-PCR primers and probes

 
Binding of 125I-labeled lipoproteins
Human adrenocortical cells were pretreated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h and then incubated with fresh serum-free medium containing different concentrations of [125I]LDL or [125I]HDL at 37 C for 1 h. For nonspecific binding, 2.5 mg/ml of unlabeled LDL or HDL was included in the medium. Cells were washed and lysed with 1 M NaOH. The radioactivity in the lysates was counted using a Beckman {gamma}-counter.

Steroid hormone measurement
Human adrenocortical cells were treated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h. The conditioned medium was saved for steroid measurements. The levels of cortisol and estradiol were measured using enzyme immunoassay kits provided by Assay Designs, Inc. The aldosterone content in the conditioned medium was measured using the aldosterone RIA kit from Diagnostic Products Inc. (Los Angeles, CA).

Statistical analysis
Data were analyzed by ANOVA with the Newman-Keuls test to assess statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BNP induced cGMP synthesis in human adrenocortical cells
BNP binds to type-A natriuretic peptide receptor, leading to the generation of cGMP, which mediates biological effects of the peptide. Cultured human adrenocortical cells were treated with increasing concentrations of BNP for 10 min and then subjected to cGMP assay. BNP dose-dependently stimulated intracellular cGMP production in human adrenocortical cells with an EC50 of 11 nM (Fig. 1Go), suggesting human adrenocortical cells express type-A natriuretic peptide receptor.


Figure 1
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  		FIG. 1. Effect of BNP on intracellular cGMP synthesis in primary human adrenocortical cells. Cells were preincubated with 0.1 mM 3-isobutyl-1-methylxanthine for 1 h and then treated with increasing concentrations of BNP for 10 min. Cells were lysed with 0.1 M HCl and subjected to intracellular cGMP assay.

 
Effect of BNP on Ang II-regulated gene expression
To determine the effect of BNP on Ang II-induced gene expression profiles in human adrenocortical cells, we performed a cDNA microarray analysis. The cells were treated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h. Arrays were probed in quadruplicate for a total of 12 hybridizations as follows: control vs. Ang II, Ang II vs. the combination of Ang II and BNP, and control vs. BNP. The cDNA microarray analysis revealed that Ang II treatment resulted in 80 gene expression changes with up-regulation of 49 genes and down-regulation of 31 genes in human adrenocortical cells. These differentially expressed genes represent about 1% of total genes on the array. BNP opposed 49% of Ang II-regulated genes in primary human adrenocortical cells. In addition, a small number of genes were down-regulated in the cells treated with BNP alone. A hierarchical cluster analysis was performed to identify gene expression patterns affected by Ang II and BNP. A visualization of this analysis is shown in Fig. 2Go. A complete listing of differentially expressed genes is provided in the table published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.


Figure 2
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  		FIG. 2. cDNA microarray analysis demonstrates a broad opposition of BNP to effects of Ang II in primary human adrenocortical cells. Cells were treated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h and collected for a cDNA microarray analysis described in Materials and Methods. A total of 80 differentially expressed genes were identified in the array. Gene expression patterns were generated using the hierarchical clustering algorithm contained in Spotfire software. Each row represents one of 80 genes, and each column represents the results from duplicate hybridizations. A, Control vs. Ang II (A II); B, Ang II vs. Ang II plus BNP; C, control vs. BNP. Normalized data values depicted in shades of red and green represent elevated and repressed expression, respectively.

 
BNP opposed Ang II-stimulated genes related to cholesterol biosynthesis, cholesterol uptake, cholesterol transfer, and steroidogenesis
The adrenal steroidogenesis requires de novo cholesterol biosynthesis, uptake of cholesterol into the cells, cholesterol transfer to mitochondria, and steroid synthesis. A number of genes responsible for cholesterol biosynthesis were up-regulated in the cells stimulated with Ang II. This regulation was inhibited by cotreatment with BNP (Table 2Go). These genes include acetoacetyl coenzyme A thiolase (ACAT2), HMG-coenzyme A synthase 1 (HMGCS1), isopentenyl-diphosphate {Delta}-isomerase (IPP isomerase: IDI1), lanosterol synthase (LSS), sterol–4 C-methyl oxidase (SC4MOL), and emopamil binding protein (EBP) or sterol-{Delta}8-{Delta}7-isomerase. In addition, farnesyl-diphosphate farnesyltransferase 1 (FDFT1) catalyzes the farnesylation of a series of products including H-Ras, N-Ras, K-Ras, Rho B, Rho D, and Rho E, which are involved in cell signaling processes. This gene was also induced by Ang II and blocked by BNP.


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  		TABLE 2. Gene expression clusters induced by Ang II and opposed by BNP in primary human adrenocortical cells

 
SB-RI and LDLR are two major membrane receptors responsible for uptake of cholesterol from circulating lipoproteins into steroidogenic cells. The cDNA microarray analysis demonstrated that Ang II induced the expression of both SR-BI (3-fold increase) and LDLR (1.8-fold increase), and BNP markedly inhibited the induction of SR-BI and LDLR in primary human adrenocortical cells stimulated with Ang II (Table 2Go).

The steroidogenic acute regulatory protein (StAR) modulates the acute stimulation of steroid synthesis in steroidogenic cells (18, 19, 20). StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where it is converted to pregnenolone by the cholesterol side-chain cleavage cytochrome P450scc system. Here, we showed that StAR expression was also up-regulated by Ang II, and BNP inhibited this induction in primary human adrenocortical cells (Table 2Go).

Several genes involved in the steroid synthetic pathway were induced by Ang II and opposed by BNP (Table 2Go). Another rate-limiting step for steroid synthesis is the conversion of cholesterol to pregnenolone, catalyzed by the cholesterol side-chain cleavage cytochrome P450scc system. This system consists of three components: ferredoxin (FDX), ferredox reductase (FDXR), and the terminal cytochrome P450. Ang II stimulated the expressions of both FDX and its reductase FDXR, which were opposed by BNP. 3ß-Hydroxysteroid dehydrogenase (HSD3B1) isoenzymes are responsible for the oxidation and isomerization of {Delta}5–3ß-hydroxysteroid precursors into {Delta}4-ketosteroids, thus catalyzing an essential step required for the synthesis of all active steroid hormones. Recently, a novel human glutathione transferase (GST) of the {alpha}-class denoted GST A3-3 was identified to have the same function as HSD3B1, but with about 230 times the efficiency to catalyze the isomerization reaction (21, 22). The expression of these two enzymes was stimulated by Ang II, and the stimulation was inhibited by BNP. Moreover, the genes encoding the enzymes involved in the final step of aldosterone, cortisol, and estradiol synthesis (CYP11B1, CYP11B2, and CYP19A1) were also up-regulated in the cells treated with Ang II, and this induction was blocked when cotreated with BNP. CYP11B2 was analyzed by real-time RT-PCR because this gene was not printed in this array.

Genes involved in cell growth and differentiation were also regulated by Ang II and affected by BNP. These include insulin-induced gene 1 (INSIG1), integrin {alpha}7 (ITGA7), inhibin {alpha} (INHA), CDC2-related protein kinase 7 (CRK7), CDC 42 binding protein kinase {alpha} (CDC42BPA), ring finger protein 1 (RING1), and matrix Gla protein (MGP). These genes may associate with Ang II-stimulated adrenocortical cell growth or hyperplasia, which may be also involved in the excess of steroid hormone synthesis.

Validation of microarray by real-time RT-PCR
To verify the microarray data, real-time RT-PCR was performed to analyze the expression of eight representative genes involved in cholesterol supply, cholesterol transfer, and steroidogenesis. Consistent with the microarray results, real-time RT-PCR analyses confirmed that the genes encoding HMGCS1, SR-BI, LDLR, StAR, CYP11B1, CYP11B2, and CYP19A1 were up-regulated by Ang II, and this induction was inhibited by BNP in cultured primary human adrenocortical cells isolated from three individual donors (Fig. 3Go). HMG coenzyme A reductase (HMGCR) is a rate-limiting enzyme that converts HMG-CoA to mevalonate during cholesterol biosynthesis. It has been previously shown that the expression and activity of this enzyme were highly induced by ACTH in human adrenal cortex (23, 24). The expression of HMGCR did not meet expression fold criteria used for our microarray analysis, but real-time PCR demonstrated that Ang II stimulated a 1.5- to 3-fold increase in HMGCR mRNA. BNP completely opposed this stimulation in the cultured cells from all three donors. The differential results for HMGCR may reflect the lower sensitivity of the microarray compared with real-time RT-PCR, suggesting also that other gene expression differences may exist that are not detected by the microarray analysis.


Figure 3
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  		FIG. 3. Validation of cDNA microarray data by real-time RT-PCR. Human adrenocortical cells from three individual donors were treated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h and collected for real-time RT-PCR analysis as described in Materials and Methods. Eight representative genes including HMGCS1, HMGCR, SR-BI, LDLR, StAR, CYP11B1, CYP11B2, and CYP19A1 were analyzed.

 
Effect of BNP on lipoprotein binding stimulated by Ang II
Our microarray and real-time RT-PCR data demonstrated that BNP opposed Ang II-stimulated expression of SR-BI and LDLR in human adrenocortical cells. Because cholesterol uptake requires binding of lipoproteins to these two receptors, we performed a lipoprotein binding assay. Cultured primary human adrenocortical cells were pretreated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h and then incubated with increasing concentrations of [125I]LDL or [125I]HDL at 37 C for 1 h as described in Materials and Methods. Consistent with microarray and real-time RT-PCR results, the binding of [125I]LDL or [125I]HDL was significantly increased in the cells pretreated with Ang II and decreased when the cells were pretreated with Ang II in the presence of BNP (Fig. 4Go). There was no significant change of lipoprotein binding in the cells pretreated with BNP alone.


Figure 4
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  		FIG. 4. BNP inhibited Ang II-induced binding of LDL and HDL to human adrenocortical cells. Cells were treated with 100 nM Ang II (AII) in the presence or absence of 200 nM BNP for 24 h before incubation with different concentrations of [125I]LDL or [125I]HDL for 1 h at 37 C. After washing, cells were dissolved with 1 M NaOH, and the radioactivity in the cells was counted. The data are presented as mean ± SD from three separate experiments; *, P < 0.01 vs. control; **, P < 0.01 vs. Ang II.

 
BNP inhibited Ang II-induced steroidogenesis
To further examine the effect of BNP on Ang II-dependent steroidogenesis, we measured steroid production in the conditioned medium of cultured primary human adrenocortical cells. Treatment of the cells with 100 nM Ang II for 24 h resulted in a significant increase in the release of aldosterone, cortisol, and estradiol. BNP significantly lowered Ang II-induced release of these steroid hormones from human adrenocortical cells (Fig. 5Go). Noteworthily, we observed that the level of cortisol was 1000-fold higher than the level of aldosterone in the conditioned medium collected from cultured primary human adrenocortical cells, suggesting cortisol may be also critical for contributing to the pathophysiology of Ang II.


Figure 5
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  		FIG. 5. Effect of BNP on Ang II-stimulated steroidogenesis in primary human adrenocortical cells from three individual donors. Cells were treated with 100 nM Ang II in the presence or absence of 200 nM BNP for 24 h. For aldosterone production, cells were treated with increasing concentrations of BNP in the presence or absence of 100 nM Ang II.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate for the first time that BNP broadly opposes Ang II-stimulated steroidogenesis, including de novo cholesterol biosynthesis, uptake of cholesterol into adrenal cells, transfer of cholesterol into mitochondria, and steroid synthesis in primary human adrenocortical cells.

De novo cholesterol biosynthesis
One source of cholesterol supply is provided by de novo synthesis within the adrenocortical cells starting from acetyl coenzyme A. This synthetic pathway requires many enzymes and their cooperative regulation in the cell. Previously, it was not known whether Ang II and natriuretic peptides affect the expression of these enzymes and modulate cholesterol biosynthesis in adrenal cells. Here, we provided the first demonstration that Ang II increased cholesterol synthesis through up-regulation of several enzyme genes including ACAT2, HMGCS1, HMGCR1, IDI1, LSS, SC4MOL, and EBP, and BNP opposed Ang II-induced cholesterol synthesis by inhibiting the expression of these up-regulated enzyme genes in human adrenocortical cells. These findings suggest that BNP may inhibit Ang II-induced adrenal steroidogenesis through modulation of de novo cholesterol biosynthesis.

Cholesterol uptake
Another major source of cholesterol supply for adrenal steroidogenesis is from circulating lipoproteins such as LDL and HDL. Uptake of cholesterol from LDL is mainly mediated through LDLR in the surface of adrenocortical cells, whereas the membrane receptor SR-BI is responsible for delivery of cholesterol from HDL into the cells (8, 9, 10, 11). Regulation of SR-BI and LDLR by Ang II has been previously studied in the human NCI-H295R adrenocortical carcinoma cell line. Cherradi et al. (25) reported that Ang II increased SR-BI expression with minimal effect on LDLR in the NCI-H295R cell line. Similarly, Pilon et al. (26) showed that Ang II stimulated SR-BI, but not LDLR, expression in the NCI-H295 cell line. Interestingly, they found that Ang II increased not only HDL binding and HDL-derived cholesterol uptake but also LDL binding and LDL-derived cholesterol uptake when the LDLR pathway was inhibited. This suggests that SR-BI may also mediate cholesterol uptake from LDL for steroidogenesis. Consistent with their findings, our data demonstrated that Ang II induced SR-BI expression and increased the binding and uptake of both HDL and LDL in primary human adrenocortical cells. The difference from their studies is that our microarray data showed Ang II significantly stimulated LDLR expression, and the result was confirmed by real-time RT-PCR from the cells isolated from three individual donors. The discrepant results may be derived from different cell types used in each study. Although multiple pathways for steroidogenesis were confirmed in NCI-295 cells, this transformed cell line may not completely reflect the features of human adrenocortical cells.

The study reported here demonstrated that BNP inhibited Ang II-induced expression of SR-BI and LDLR as well as uptake of cholesterol from HDL and LDL into human adrenocortical cells, providing another cellular mechanism by which BNP suppresses Ang II-stimulated steroidogenesis.

Cholesterol transfer into mitochondria
The rate-limiting step in the activation of steroidogenesis is the delivery of cholesterol from the mitochondrial outer membrane to the inner membrane where cholesterol undergoes an enzymatic cascade leading eventually to the formation of steroids. Identification of mutations in humans and mouse knockout studies demonstrated that StAR is critical for facilitation of cholesterol movement to mitochondria for steroidogenesis. A number of studies showed that the expression of StAR was up-regulated by Ang II in human NCI-H295R adrenocortical carcinoma cells (27, 28, 29). In this study, we demonstrated that BNP completely blocked Ang II-stimulated StAR expression in human adrenocortical cells, indicating BNP may also control Ang II-induced adrenal steroidogenesis by limitation of cholesterol transfer to the mitochondrial inner membrane for initiation of steroid synthesis.

Steroidogenesis
Three novel findings related to the steroid synthetic pathway were reported in this study: 1) Ang II up-regulated a number of enzymes involved in steroid synthesis, including FDX1, FDXR, 3ßHSD1, GSTA3, CYP19A1, CYP11B1, and CYP11B2; 2) BNP inhibited the stimulation of these enzymes by Ang II in primary human adrenocortical cells; and 3) BNP inhibited the release of aldosterone, cortisol, and estradiol in human adrenocortical cells. The excess in aldosterone and cortisol synthesis has been linked to a series of cardiovascular diseases such as hypertension, atherosclerosis, heart failure, and renal failure. The inhibitory effect of BNP on Ang II-stimulated aldosterone and cortisol production suggests a beneficial role for BNP in treatment of cardiovascular diseases. Interestingly, we found that CYP19A1 or aromatase responsible for the conversion of adrenal androgen to estrogen was strongly up-regulated in human adrenocortical cells treated with Ang II and inhibited when cotreated with BNP. These results were further confirmed by the measurement of estrogen release in the conditioned media. Consistent with our findings, Kalenga et al. (30) reported that Ang II induced the secretion of estradiol from human placenta, and this effect seems to be linked to the stimulation of local androgen aromatization. The biological significance of Ang II-induced estrogen release is still unclear. Studies from Koh et al. (31) showed that women with the high-activity genotype of the ACE gene had an increased risk of developing breast cancer when compared with those possessing low-activity ACE genotype, suggesting that the renin-angiotensin system may link to breast cancer incidence. The aromatase inhibitors have been used to treat advanced breast cancer, especially in postmenopausal women (32).

In conclusion, this study demonstrates that BNP counterbalances Ang II-induced steroidogenesis via multiple steps from the cholesterol supply and transfer to the final formation of steroid hormones (Fig. 6Go). These findings provide new insights into the cellular mechanisms by which BNP modulates Ang II-induced steroidogenesis in the adrenal gland.


Figure 6
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  		FIG. 6. Scheme depicting the inhibitory effects of BNP on Ang II-stimulated cholesterol biosynthesis, cholesterol uptake, cholesterol transfer, and steroidogenesis in human adrenocortical cells.

 


   Footnotes
 
Disclosure statement: All the authors have nothing to disclose.

First Published Online May 3, 2007

1 F.L. and A.M.K. contributed equally to this work. Back

Abbreviations: Ang II, Angiotensin II; BNP, B-type natriuretic peptide; FDX, ferredoxin; FDXR, ferredox reductase; GST, glutathione transferase; HDL, high-density lipoprotein; HMGCR, HMG coenzyme A reductase; LDL, low-density lipoprotein; LDLR, LDL receptor; SR-BI, scavenger receptor class B type I; StAR, steroidogenic acute regulatory protein.

Received November 29, 2006.

Accepted for publication April 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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