Adrenal Androgen Biosynthesis with Special Attention to P450c17ª
J. IAN MASON,b,d IAN M. BIRD,c,d AND WILLIAM E. RAINEYd
b Department of Clinical Biochemistry University of Edinburgh Edinburgh Royal Infirmary NHS Trust Edinburgh EH3 9YW, Scotland
“Department of Obstetrics & Gynecology University of Wisconsin-Madison Meriter/Park Hospital Madison, Wisconsin 53715
d Cecil H. & Ida Green Center for Reproductive Biology Sciences and Departments of Obstetrics & Gynecology and Biochemistry University of Texas Southwestern Medical Center Dallas, Texas 75235
The human adrenal gland is unique in its high secretion rate of dehydroepiandros- terone (DHEA) and its sulfoconjugate, dehydroepiandrosterone sulfate (DHEAS).1.2 Extremely high production rates of these C19-steroids occur in the fetal adrenal, whereas minimal secretion occurs from the adrenal cortex of the child until adrenar- chy. The synthesis of adrenal C19-steroids then increases, attaining a maximum in the young adult. Thereafter, a slow decline in adrenal C19-steroid secretion continues through the remainder of adult life, while C21-steroid secretion is little affected.
The last decade has seen considerable progress in the understanding of the molecular mechanisms controlling the expression of adrenocortical steroidogenic enzymes.3.4 Nonetheless, the nature of mechanisms specifically controlling DHEA and DHEAS synthesis by human adrenocortical cells remains largely unresolved. Primary cultures of both fetal and adult human adrenocortical cells have acted as models for evaluating human adrenal DHEA production.5-8 These cells, however, have certain limitations because of the availability and age of tissue and the difficulty in obtaining sufficient cells for molecular studies. Notwithstanding potential differ- ences in the nature of tumor or immortalized cells and primary parenchymal cells, a human adrenocortical cell line would facilitate investigations into the molecular parameters that regulate DHEA synthesis. The human adrenocortical cell line NCI-H295 appears to have retained the capacity to express all adrenal steroidogenic enzymes. Gazdar and colleagues9 established the NCI-H295 cell line from an invasive primary adrenocortical carcinoma. The patient from whom the cell line was established showed signs of mineralocorticoid, glucocorticoid, and androgen excess. The tumor (a 14 x 13 x 11-cm right adrenal mass) was surgically removed in
“These studies were supported in part by the National Institutes of Health (AG08175 and DK43140), American Heart Association (Texas Affiliate 93R-082), and Merck Sharp and Dohme. I. M. B. was also supported by National Institute of Health training grant T32-HD- 07190.
b Address for correspondence.
December 1980. A portion of the adrenal tumor was finely minced and placed in culture in microwells using a variety of growth media. Because of fibroblast growth, a population of tumor cells that grew as a suspension was used to establish the H295 cell line. The initial description of the steroidogenic properties of the H295 cell line was performed after the cells had been in culture for 7-10 years. More than 30 steroids were detected in the culture medium from H295 cells, of which about 20 were identified. Importantly, depending on the culture conditions, the major steroids produced by these cells were glucocorticoids and C19-steroids. Thus, these cells represented the first report of a human adrenal cell line which maintained the ability to secrete both C19- and C21-adrenocortical steroids. The ability of H295 cells to secrete C19-steroids in response to activators of the protein kinase A signaling pathway as well as to express transcripts for the range of adrenal steroid enzymes was later demonstrated. 10,11
Herein, we demonstrate that these H295 cells also retain a range of hormonal responsiveness and secrete steroids similar to those seen in normal human adrenocor- tical cells. The ability of these cells to produce cortisol, androstenedione, DHEA, and DHEAS suggests that these cells may well act as a model system to determine the factors controlling the production of C19-steroids, the so-called adrenal andro- gens, and glucocorticosteroids.
MATERIALS AND METHODS
Cell Culture and Experimental Treatment
Human NCI-H295 adrenal tumor cells were obtained from the American Type Culture Collection (Rockville, Maryland). Cells were maintained in an equal mixture (v/v) of Dulbecco’s modified Eagle’s and Ham’s F12 media (DME-F12) containing insulin (1 µg/ml), transferrin (1 ug/ml), selenium (1 ng/ml), linoleic acid (1 µg/ml), BSA (1.25 mg/m) added in the form of 1% ITS plus (Collaborative Research, Bedford, Massachusetts), and 2% Ultroser SF (Sepracor, Inc., Marlborough, Massa- chusetts) and antibiotics. Stock cultures were grown at 37℃ on 75-cm2 tissue culture plates (Costar, Cambridge, Massachusetts) in a humidified atmosphere of air (5 l/min) supplemented with carbon dioxide (0.2 l/min). Selection of an H295 cell population which remained attached during culture has been described. For experi- ments, cells were subcultured and after 48 hours rinsed and placed in fresh serum-free medium containing 0.01% BSA (defined medium) and treated with ACTH (Cortrosyn), forskolin, or dibutyryl cyclic AMP (dbcAMP). Cortrosyn (ACTH 1-24) was obtained from Organon (West Orange, New Jersey). Forskolin, angioten- sin II (AII), 12-O-tetradecanoylphorbol 13-acetate (TPA), and dbcAMP were obtained from Sigma Chemical Co. (St. Louis, Missouri).
Analysis of Steroidogenic Enzyme Activity
Cells were washed in DME-F12 and incubated for 6 hours with 0.5 ml of medium consisting of DME-F12 and antibiotics supplemented with 2.5 uM pregnenolone, 100,000 dpm/ml [3H]pregnenolone (New England Nuclear-DuPont), and a potent 3ß-hydroxysteroid dehydrogenase inhibitor, 4MA (17B-N,N-diethylcarbamoyl-4- diethyl-4-aza-5x-androstane-3-one, 1 µM; Merck Research Laboratories, Rahway, New Jersey). At the end of the incubation, medium was recovered and extracted with
methylene chloride (2 x 3 ml), and the combined extracts were dried under air, reconstituted in methylene chloride (0.5 ml), and dried before final reconstitution (0.05 ml). Samples were then applied to Silica Gel 60 F254 plastic-backed TLC plates (EM Science, Gibbstown, New Jersey) and developed twice in chloroform-ethyl acetate (90:10, vol/vol). 17a-Hydroxylase activity was computed from the fractional conversion of pregnenolone to 17a-hydroxypregnenolone and DHEA, as identified against authentic standards. Results were expressed as nanomoles per milligram of protein per 2 hours.
Stimulation of Steroid Secretion and Analysis of Steroids
To assess the effects of treatment on steroid secretion, experiments were performed in media containing 0.5% LPSR-1 (Sigma Chemical Co.) containing 1% antibiotics. Cells subcultured onto 24-well plates were maintained for 24 hours in DMEM/F12 containing 0.5% LPSR-1. Medium was then renewed (1 ml/well) and treatment begun with the agents shown for a 48-hour period. The DHEA content of media recovered from each well was determined using an assay kit from Diagnostic Products Corp, Los Angeles, California. The cortisol or aldosterone contents of media were determined against cortisol or aldosterone standards, respectively, prepared in defined medium, using coated tube immunoassays from Diagnostic System Laboratories, Webster, Texas. Results of steroid assays were normalized to cellular protein per well and expressed as nanomoles per milligram of cell protein.
Protein Determination
Cells were solubilized in Tris-HCI (50 mM, pH 7.4), containing NaCl (150 mM), SDS (1%), EGTA (5 mM), MgCl2 (0.5 mM), MnCl2 (0.5 mM), and phenylmethylsul- fonylfluoride (PMSF, 0.2 mM) and stored frozen at -20℃. Protein content of samples was then determined by bicinchoninic acid protein assay, using the BCA assay kit (Pierce, Rockford, Illinois).
Protein Separation and Immunoblotting Analysis
Protein samples were separated by polyacrylamide (12%) SDS gel electrophore- sis at constant voltage. Proteins were then transferred to Immobilon P membrane (Millipore, Milford, Massachusetts) using a Transblot apparatus (Hoefer, San Francisco, California) at 100 V for 1.5 hours, in a transfer buffer of 20 mM Tris, 150 mM glycine, and 20% methanol. The Immobilon membrane was preblocked using a blocking buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40, 0.5% dried skimmed milk), before incubating with primary antibody (10 µg/ml in 10 mM Tris pH 7.4, 150 mM NaCl) for 2 hours. The membrane was then washed twice more in blocking buffer and incubated for 1 hour with [125]]-protein A (ICN Biomedicals, Irvine, CA) before final repeated washing (for 1 hour). Specific binding to the membrane was quantified by direct imaging (AMBIS) before final exposure to Kodak X-Omat AR film at -70℃. Primary antisera were those raised against human P450c17 (kindly provided by Dr. M. Waterman, Vanderbilt University) and human placental 3ß-HSD.
Northern Analysis
Cells on 100-mm culture dishes were treated as described and then lysed at 4℃ into 1 ml RNAzol B solution (Cinna Biotecx, Houston, Texas) before transfer to a microfuge tube. Phase separation was achieved by mixing with 0.15 ml CHCI3, incubation at 4℃ for 5 minutes, and centrifugation (12,000 x g; 20 minutes; 4℃). The upper phase (0.7 ml) was transferred to a second microfuge tube, and RNA was then precipitated by the addition of 0.8 ml isopropanol and standing for 1 hour at -20°C. RNA was recovered by centrifugation (30 minutes; 12,000 x g ; 4℃) and the recovered pellet was washed once in 75% ethanol (1.0 ml) before drying under air and dissolving in 1 mM EDTA, pH 7.0 (0.1 ml). After determination of recovery and purity by measuring absorbance at 260 and 280 nm, samples were precipitated by the addition of 1 ml absolute ethanol and 0.01 ml sodium acetate (3 M; pH 5.2) and stored at -70℃ before analysis.
Samples were separated by electrophoresis on gels containing 1.1% agarose in the presence of formaldehyde. The presence and integrity of the major RNA species were examined under ultraviolet light to ensure consistency between lanes. RNA was transferred to a Magna NT membrane (MSI) by pressure blotting (75 psi, 1 hour; PossiBlot Pressure Blotter, Stratagene, La Jolla, California) and cross-linked under UV light. Prehybridization was carried out at 42℃ overnight in a final buffer composition of 50% formamide, 5 x SSC, 1 x PE, and 50 mg/ml transfer RNA (20 × SSC contains 3.0 M NaCl and 0.3 M trisodium citrate, pH 7.0; 5 x PE contains 250 mM Tris-HCI [pH 7.5], 0.5% sodium pyrophosphate, 5% sodium dodecyl sulfate [SDS], 1% polyvinylpyrrolidone, 1% Ficoll, 25 mM EDTA, and 1% BSA). Hybridiza- tions were performed sequentially in the same buffer at 42℃ for 16-24 hours using antisense probes to 3B-HSD, then P450c17 and finally P450scc. Each antisense probe was labeled with [32P] by asymmetric PCR in the presence of [32P]dCTP; Amersham, Arlington Heights, Illinois. The blots were then washed in 2 x SSC containing 0.1% SDS at room temperature for 15 minutes and in 0.1 x SSC containing 0.1% SDS at room temperature for 2 × 30 minutes before direct radioimaging quantification of bound probe (Quantprobe V3.02, Ambis Systems Inc, San Diego, California) and exposure to film (Hyperfilm, Amersham). Blots were subsequently stripped by repeated washing in 0.1 x SSC/0.5% SDS at 65℃ and checked for lack of radioactivity before reprobing. Finally, all blots were probed for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA using an antisense probe generated by asymmetric polymerase chain reactions against bases 39-900 of the human cDNA and bound probe quantified as just described. Binding of G3PDH probe per lane was then used to normalize data for P450c17, P450scc, and 30-HSD mRNA against minor variations in the RNA loading of the lanes.
Probe Preparation
Antisense probes were prepared by polymerase chain reaction in a 50-ul volume under standard conditions, but with the following modifications: forward to reverse primers were added at a 1:100 ratio (0.3 and 30 pmol), the free dCTP concentration reduced to 5 u.M, and the addition of 50 uCi [32P]dCTP (3000 Ci/mmol, Amersham). Template was added at 10 ng per kilobase. Labeling was performed through 40 cycles. Incorporation of label was routinely 60-75% by this procedure. Templates and oligonucleotides were as follows: Human P450c17 probe template was pCD- 17&H26 and the forward and reverse oligonucleotides were 5’-GCACCAAGACTA- CAGTG-3’ and 5’-ACTGACGGTGAGATGAG-3’. Human 3ß-hydroxysteroid de-
hydrogenase (3B-HSD) probe template was the BglII/BglII sequence of human Type II cDNA (in PCR1000), and the forward and reverse oligonucleotides were 5’-CTCTCCAGCATCTTCTG-3’ and 5’-TCACTACTTCCAGCAGG-3’. Human cytochrome P450-side chain cleavage (P450scc) probe template was a complete cDNA in Bluescript, kindly provided by Dr. Michael Waterman (Vanderbilt Univer- sity). Forward and reverse oligonucleotides were 5’-TCTCCTGGTGACAATGG-3’ and 5’-CTTGCACCAGTGTCTTG-3’, respectively.
RESULTS
We have previously shown that treatment of H295 cells with elevated K+ or AII results in an increase in [Ca2+]i and increased aldosterone secretion.12,13 The data shown in FIGURE 1 extend these findings to show the effects of K+ (14 mM) on secretion of aldosterone, DHEA, and cortisol. Elevation of medium K+ to 14 mM resulted in a 3.7-fold increase in DHEA secretion, but also increased secretion of aldosterone (3.5-fold) and, to a lesser extent, cortisol (2.9-fold) over a 48-hour period. These findings differ from those for AII (10 nM), which gave a greater increase in aldosterone secretion (5.7-fold) but lesser increases in DHEA (2.2-fold) and cortisol (2.7-fold) secretions. The effects of K+ also contrast with those of dbcAMP which had the greatest effect on DHEA (23.0-fold) and cortisol (7.9-fold) secretions but a similar effect (3.3-fold) on aldosterone secretion. In contrast, treatment with TPA, a potent activator of protein kinase C, had little or no effect on DHEA, cortisol, or aldosterone secretion.
In addition to the stimulatory action of AII on aldosterone secretion in H295 cells, AII can also attenuate the positive effects of dbcAMP on DHEA and cortisol secretion. The data shown in FIGURE 1 confirm that in combination with dbcAMP, AII further increased aldosterone secretion, but attenuated the secretion of DHEA and cortisol over a 48-hour period. Combined treatment with dbcAMP and potas- sium also resulted in increased aldosterone, but it had a lesser attenuative effect on DHEA secretion and no effect on cortisol secretion compared to that of dbcAMP alone. However, combined treatment with TPA and dbcAMP resulted in almost no change in aldosterone secretion, but a marked attenuation in both DHEA and cortisol secretion.
Treatment of H295 cells with AII and dbcAMP or forskolin was previously shown to alter the expression of key steroid-metabolizing enzymes such as P450scc, P450c17, and 38-HSD25,34 within 20 hours. The results of northern blot analysis of changes in the levels of P450scc, P450c17, and 30-HSD mRNA in response to the various effectors are shown in FIGURE 2. Time-dependent increases in the levels of P450c17 mRNA were seen with as little as 3 hours of treatment with K+ and continued to increase thereafter (data not shown). Although more modest increases in P450scc were also seen, there was no effect on 30-HSD mRNA at any time. Treatment with forskolin or dbcAMP most potently increased the levels of mRNA for P450c17 and to a lesser extent P450scc and 3B-HSD. AII treatment also increased mRNA levels for 3B-HSD and had a lesser effect on mRNA for both P450c17 and P450scc. While increased K+ had no effect on 3B-HSD, the effect on P450c17 mRNA was greater than that of AII, but the effect on P450scc was similar in each case. On the other hand, phorbol ester treatment markedly increased 30-HSD mRNA levels while suppressing the basal level of P450c17 mRNA.
These changes in the level of P450c17 mRNA were paralleled by changes in 17a-hydroxylase activity. Increased K+ increased this activity to a level comparable to that in response to AII, while treatment with TPA resulted in a decrease in activity
3.0
*
*
Aldosterone
T
2.5
*
T
2.0
*
*
*
T
1.5
1.0
*
0.5
0
2.0
*
£
DHEA
*
1.0
£
*
*
*
+
+
0
-
80
70
*
*
Cortisol
60
50
*
40
30
*
·
20
*
10
0
Con
All
K+
TPA
dbcAMP
dbcAMP/All
dbcAMP/K+
dbcAMP/TPA
(FIG. 3). Although AII and especially TPA attenuated the marked dbcAMP- promoted increase in 17a-hydroxylase activity, increased K+ levels did not modulate the action of dbcAMP treatment on 17a-hydroxylase activity. It is noteworthy that all these changes in 17a-hydroxylase activity in response to 48 hours of treatment with AII, K+, and TPA alone or in combination with dbcAMP correlated closely with changes in DHEA and cortisol secretion (FIG. 1), both of which depend on 17a-hydroxylase for their synthesis.
The results of western immunoblot analysis of levels of P450c17 and 30-HSD proteins from H295 cells after various treatments are shown in FIGURE 4. Changes in P450c17 immunodetectable protein were consistent with those for P450c17 mRNA and the activity just described. Quantitatively, although each treatment increased the
level of immunodetectable 3B-HSD protein twofold above basal, combined treat- ment with forskolin and TPA was not additive as was observed with mRNA, suggesting that posttranscriptional regulation of enzyme expression may also be occurring. Although the probe employed for northern analysis cannot distinguish between type I and type II isoforms of human 3B-HSD, western blot analysis indicates that the principal isoform expressed in H295 cells after various treatments remains the type II isoform as judged by its increased mobility on gel compared to the type I isoform stably expressed in human embryonic kidney 293 cells.
DISCUSSION
The H295 cell has the apparent properties of a pluripotent adrenocortical cell which, on activation of the protein kinase A pathway with agents such as forskolin or dbcAMP, induces fasciculata/reticularis-like function, that is, increased cortisol/ DHEA secretion, due primarily to a marked increase in 17a-hydroxylase expres- sion.10,12 Alternatively, treatment with AII and subsequent activation of phospholi- pase C develop a glomerulosa-like functionality characterized by elevated aldosterone secretion, markedly increased aldosterone synthase expression, but low 17x-
8
P450c17
*
*
6
4
*
*
±
mRNA (Fold over control)
2
£
+
0
6
*
38-HSD
*
4
*
*
2
0
5
P450scc
4
3
*
2
*
1
0
Con
All
K+
TPA
dbcAMP
Forsk
4
*
17a-hydroxylase activity (nmol/mg.h)
3
2
1
0
Con
All
K+
TPA
dbcAMP
dbcAMP/All
dbcAMP/K+
dbcAMP/TPA
hydroxylase expression and little secretion of 17a-hydroxylated steroids. 10,14 Treatment with K+, which acts to increase [Ca2+]i through the opening of dihydropyridine- sensitive Ca2+ channels in H295 cells,13 also results in increased dehydro- epiandrosterone secretion in addition to increased aldosterone production.12 The results of this study show further that this increase in DHEA secretion in response to K+ was accompanied by an increase in P450c17 mRNA, as observed previously in response to AII.14,15 Surprisingly, however, treatment of H295 cells with K+ also increased expression of 17a-hydroxylase, as measured at the level of mRNA and activity, together with increased secretion of DHEA as well as cortisol. These effects of K+ on 17a-hydroxylase expression exceed those of AII, so enhancing overall steroidogenic capacity rather than just aldosterone secretion.
Increased expression of 17a-hydroxylase is regulated by agents such as ACTH in vivo and forskolin or dbcAMP in vitro, which increase cellular cAMP and/or activate
protein kinase A.3,4,16 Agents such as AII, however, activate phosphoinositidase C in adrenocortical cells, resulting in increased [Ca2+ ]i (through both release of intracel- lular stores and influx through plasma membrane Ca2+ channels) and activation of protein kinase C (through release of diacylglycerol).17,18 Studies using H295 cells loaded with Fura 2 previously showed that K+ increases [Ca2+]; through the opening of nifedipine-sensitive voltage-dependent Ca2+ channels in the plasma membrane. 13 Thus, as seen in other mammalian adrenocortical cells, 19 treatment with K+ activates a Ca2+ signaling pathway alone, whereas treatment with AII additionally stimulates protein kinase C and neither is classically regarded as signaling through direct coupling to adenylyl cyclase. However, in many cells “cross-talking” occurs between signaling pathways, and we previously hypothesized that AII may increase 17a- hydroxylase expression through indirectly increasing cAMP levels.20 Our findings in this study suggest that such an explanation for the action of K+ on 17a-hydroxylase expression is not valid. In addition to monitoring the effects of these and other agents on 17a-hydroxylase expression, we also studied their effects on P450scc and 3ß-HSD at the level of mRNA. Both P450scc and 3B-HSD expression are increased in response to activation of protein kinase A, and 3B-HSD expression is also increased by protein kinase C activation in these cells12 (also, Bird, Rainey, and Mason, unpublished data). While K+ increased 17a-hydroxylase and, to a lesser extent, P450scc mRNA, it was without effect on levels of 3B-HSD mRNA. Thus, K+ must have induced 17a-hydroxylase expression through a non-cAMP signaling mechanism which was not protein kinase C-mediated because TPA suppressed 17a-hydroxylase expression and increased 3B-HSD expression. Other evidence suggests that K+ can regulate 17a-hydroxylase through a Ca2+ second messenger system (data not shown). First, BAYK8644 treatment reproduced the effects of K+ on P450c17 mRNA, 17a-hydroxylase activity, and steroid secretion. Secondly, calcium channel blocker nifedipine blocked the action of K+ on P450c17 mRNA. Thus, it seems likely that the intracellular signal controlling K+-induced P450c17 expression is Ca2+. (Previous studies in H295 cells using the calcium ionophore A23187 also reported a modest
Basal
Forsk
Forsk/TPA
.293/Type 1
TPA
293
44 KD →
# 3BHSD
55 KD -
+ P450c17
increase in P450c17 and P450scc mRNA in 6-12 hours, but a decline thereafter, an effect that may be attributed to long-term cellular toxicity by A23187,21 as reported in other steroidogenic cells.22) Because AII also increases [Ca2+]; and TPA does not reproduce this effect in H295 cells, it also seems likely that the small increase in P450c17 expression observed in response to AII treatment is also a direct conse- quence of increased [Ca2+ ]i.
The expression of P450c17 is principally transcriptionally regulated23,24 and much is known about the regulatory elements that control transcription.16,24 Two cAMP- responsive elements (CRS1 and CRS2) have been demonstrated in the bovine CYP17 gene, and studies of bovine CYP17 constructs suggest that attenuation of expression by protein kinase C is mediated through a sequence identical to or overlapping CRS1.25 Our studies in H295 cells, as in bovine adrenocortical cells, show that combinations of dbcAMP or forskolin with AII result in attenuated expression of P450c17 and a corresponding increase in aldosterone secretion to- gether with a marked decrease in 17«-hydroxylated steroids such as DHEA or cortisol. This action of AII is apparently mediated through the protein kinase C pathway because TPA is also a potent attenuator of dbcAMP or forskolin-induced P450c17 expression. In contrast to the action of AII and TPA, however, combined treatment of H295 cells with dbcAMP and K+ failed to attenuate and in fact marginally increased dbcAMP-induced P450c17 expression and cortisol secretion. Although some decrease in DHEA secretion was observed, this was much less than that seen in response to dbcAMP with AII or TPA. Our data suggest that there may be an additional Ca2+ responsive element yet to be identified in the CYP17 gene in the human and possibly other mammalian species. Furthermore, activation of this element by increases in [Ca2+]; does not appear to impair increased transcription in response to cAMP via CRS1 or CRS2 sequences. Thus, these findings suggest that the control of transcription of genes encoding key steroidogenic enzymes may occur in response to changes in [Ca2+]i as well as the more well characterized protein kinase A and protein kinase C signaling pathways, and so reveals a further level of complexity in the control of zonal function in the adrenal cortex.
SUMMARY
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 17a-hydroxylase cyto- chrome P450 (P450c17) expression, a key enzyme in C19-steroid production. Forsko- lin 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) individu- ally 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.
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