Regulation of Human Adrenal Carcinoma Cell (NCI-H295) Production of C19 Steroids*
WILLIAM E. RAINEY, IAN M. BIRD, C. SAWETAWAN, NEIL A. HANLEY, JOHN L. MCCARTHY, ELIZABETH A. McGEE, REBECCA WESTER, AND J. IAN MASON
Departments of Obstetrics and Gynecology and Biochemistry and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235
ABSTRACT
The regulation of biosynthesis of the adrenal C19 steroids (the so- called adrenal androgens) remains unclear. Understanding adrenal production of C19 steroids is important when the benefits of these steroids are considered on processes and diseases associated with aging. In vitro studies defining the mechanisms that regulate the production of human adrenal C19 steroids have been limited because of the diffi- culties in obtaining adrenal tissue. A cell line that retains differentiated adrenal functions would greatly facilitate research in this area. Herein, we describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19 and C21 steroid production. The cells were characterized with regard to ACTH, forsko- lin, and dibutyryl cAMP (dbcAMP) responsiveness, as measured by increased cAMP production, synthesis of steroids, and induction of 17a-hydroxylase cytochrome P450 (P450c17). Forskolin and dbcAMP, which were more effective than ACTH, enhanced the production of cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione over a 48-h treatment period. Comparison of the
relative amounts of measured steroid secreted under forskolin treat- ment indicated that the primary product was cortisol (70%), followed by androstenedione (14%), DHEA (9%), and DHEAS (7%). Cortisol was also demonstrated to be the major steroid product by examination of UV-detectable steroids after high performance liquid chromato- graphic separation. The increases in steroid production caused by ACTH, forskolin, and dbcAMP occurred in a concentration- and time- dependent manner. A key enzyme in the production of C19 steroids is P450c17. ACTH, forskolin, and dbcAMP increased the activity of 17a- hydroxylase by approximately 2.5-, 10-, and 10-fold, respectively. These effects on enzyme activity occurred in a concentration-dependent man- ner and coincided with increased levels of P450c17 mRNA. In summary, H295 cells should provide a much-needed model to study mechanisms controlling the secretion of glucocorticoids and C19 steroids, because steroid production in these cells is hormonally controlled and associated with the induction of P450c17. (J Clin Endocrinol Metab 77: 731-737, 1993)
T HE HUMAN adrenal gland is unique in its high secretion rate of dehydroepiandrosterone (DHEA) and its sulfo- conjugate DHEA sulfate (DHEAS) (reviewed in Refs. 1 and 2). The last decade has seen considerable progress in the understanding of the molecular mechanisms controlling the expression of adrenocortical steroidogenic enzymes. None- theless, problems concerning the mechanisms that control DHEA and DHEAS synthesis by human adrenocortical cells remain to be solved. Primary cultures of both fetal and adult human adrenocortical cells have acted as models for evalu- ating human adrenal DHEA production (3-6). However, these cells have certain limitations due to availability and age of tissue and the difficulty obtaining sufficient numbers of cells for biomolecular studies. A human adrenocortical cell line would facilitate investigations into the molecular param- eters that regulate DHEA synthesis. Recently, a report de- scribed a human adrenocortical carcinoma cell line (NCI- H295) that retains adrenal steroidogenic enzyme expression (7). Herein, we demonstrate that H295 cells also retain hor- monal responsiveness and secrete steroids similar to those
Received December 31, 1992. Accepted May 11, 1993.
Address requests for reprints to: Dr. William E. Rainey, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
* This work was supported in part by NIH Grant AG-08175 and American Heart Association Texas Affiliate Grant 91G-082.
secreted by normal human adrenocortical 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 relative pro- duction of glucocorticoids and C19 steroids, the so-called adrenal androgens.
Materials and Methods
Cell culture and experimental treatment
Human NCI-H295 adrenal tumor cells were obtained from the Amer- ican Type Culture Collection (Rockville, MD). Cells were maintained in an equal mixture (vol/vol) of Dulbecco’s Modified Eagle’s and Ham’s F- 12 Media (DME-F12) containing insulin (1 ug/mL), transferrin (1 ug/ mL), selenium (1 ng/ml), linoleic acid (1 ug/mL), BSA (1.25 mg/mL) added in the form of 1% ITS plus (Collaborative Research, Bedford, MA), 2% Ultroser SF (Sepracor, Inc., Marlborough, MA), and antibiotics. Stock cultures were grown at 37 C on 75-cm2 tissue culture plates (Costar, Cambridge, MA) in a humidified atmosphere of air (5 L/min) supple- mented with carbon dioxide (0.2 L/min). Selection of an H295 cell strain that retained attachment during culture was accomplished by changing medium routinely over a 3-month period and maintaining only the cells that were attached to the culture dishes. For experiments, cells were subcultured and, after 48 h, rinsed, placed in fresh serum-free medium containing 0.01% BSA (defined medium), and treated with ACTH (Cortrosyn), forskolin, or dibutyryl cAMP (dbcAMP). Cortrosyn [ACTH- (1-24)] was obtained from Organon (West Orange, NJ). Forskolin, an- giotensin-II (AII), and dbcAMP were obtained from Sigma Chemical Co. (St. Louis, MO).
Analysis of steroids, cAMP, and protein
The cortisol content of the medium was determined in duplicate against cortisol standards prepared in defined (serum-free) medium using a coated tube [125]]cortisol-linked immunoassay (ICN Biomedicals, Costa Mesa, CA). DHEA and DHEAS were determined using assay kits from Diagnostic Products Corp. (Los Angeles, CA). Androstenedione was determined using an assay kit from Diagnostic Systems Corp. (Houston, TX). The results of the steroid assays were expressed as nanomoles per mg cell protein. The medium content of cAMP was determined using a specific RIA from Advanced Magnetics (Cam- bridge, MA). The acetylated procedure, for increased sensitivity, was used in this assay. Results were expressed as picomoles per mg protein. For protein determination, cells were solubilized in Tris-HCI (50 mmol/ L; pH 7.4) containing NaCI (150 mmol/L), sodium dodecyl sulfate (1%), EGTA (5 mmol/L), MgCl2 (0.5 mmol/L), MnCl2 (0.5 mmol/L), and phenylmethylsulfonylfluoride (0.2 mmol/L) and stored frozen at -70 C. The protein content of the samples was then determined by bicin- choninic acid protein assay, using the BCA assay kit (Pierce, Rock- ford, IL).
HPLC analysis of steroids
Aliquots (9 mL) of the incubation media were extracted with 5 vol dichloromethane. After removing the aqueous phase, the organic phase was back-extracted with 1 ml distilled water. The dried dichloromethane extract was dissolved in 0.15 mL absolute methanol, and a 70-pL aliquot of methanol was placed in a microfuge tube to which 35 uL of water was added. The tube was vortexed and microfuged. Steroids in the aqueous methanol medium were separated by high performance liquid chromatography (HPLC). The HPLC was carried out using Waters Associates (Milford, MA) apparatus (6000A solvent pump, C-18 uBond- apak reverse phase column, and model 440 absorbance detector at 254 nm to identify 44-3-ketosteroids). Isocratic elution with 60% aqueous methanol (vol/vol) at 0.9 mL/min was used to effect separation of the steroids. Analysis of steroids was made by comparing peak elution times to those of authentic steroid standards.
Thin layer chromatographic (TLC) identification of [H] pregnenolone metabolites
Cells maintained and treated as described above were then incubated for 4 h with serum-free medium containing [3H]pregnenolone (150,000 dpm/mL) with 0.25 umol/L unlabeled pregnenolone added. No enzyme inhibitors were present. At the end of incubation, medium was extracted with dichloromethane (twice, 3 mL each time), dried, and redissolved in a small volume of dichloromethane. The organic extracts were divided for separation by two TLC procedures. Samples were applied to silica gel plates (Keiselgel 60, F254, EM Separations, Gibbstown, NJ) and developed twice in solvent 1 (chloroform-ethyl acetate, 90:10, vol/vol) or developed once in solvent system 2 (chloroform-ethyl acetate-meth- anol, 90:10:2, vol/vol). Lanes containing radiolabeled samples were then scanned using a BioScan detector. Data were collected in 256 channels/ lane to a total of 10,000 counts (~1 h/lane). Peaks were identified by comparison to authenticated standards run on the same plate.
Analysis of steroidogenic enzyme activity
Cells were rinsed in DME-F12 and incubated for 6 h with 0.5 mL medium consisting of DME-F12 supplemented with 2.5 umol/L preg- nenolone, 100,000 dpm/mL [7-3H]pregnenolone (New England Nuclear- DuPont, Boston, MA), and a potent 5a-reductase/36-hydroxysteroid dehydrogenase inhibitor, 178-N,N-diethylcarbamoyl-4-diethyl-4-aza- 5a-androstane-3-one (1 µmol/L; Merck, Sharp, and Dohme, West Point, PA). At the end of the incubation, the medium was recovered and extracted as described above. Samples were then applied to TLC plates and developed twice in solvent system 1 (above). 17a-Hydroxyl- ase activity was computed from the fractional conversion of pregneno- lone to 17a-hydroxypregnenolone and DHEA, as identified against authentic standards. Results were expressed as nanomoles per mg pro- tein/2 h.
Analysis of mRNA for 17a-hydroxylase cytochrome P450 (P450c17)
RNA extraction, gel electrophoresis, and Northern analysis for P450c17 were performed exactly as described previously (8, 9). Blots were washed in 2 × sodium citrate buffer-0.1% sodium dodecyl sulfate and twice in 0.1 × sodium citrate buffer-0.1% sodium dodecyl sulfate (30 min each). Blots were then exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) at -70 C, and where required, radio- activity was quantified using an Ambis Radioanalytic Imaging System (Quantprobe V3.02, Ambis Systems, Inc., San Diego, CA).
Statistics
Statistical analyses of the data were performed using analysis of variance, followed by Newman-Keuls multiple comparison analysis.
Results
cAMP and steroid production
Initially, we examined the effects of hormone treatments on H295 cell morphology. Previously, we and others had shown that treatment with agonists of the protein kinase-A pathway causes human adrenal cells in culture to undergo retraction from the culture dish (termed cell rounding) (3, 10). H295 cells underwent a characteristic rounding response after treatment for 24 h with dbcAMP (1 mmol/L) or forsko- lin (10 umol/L; Fig. 1). Treatment with ACTH (0.1 µmol/L) had no discernible effect (data not shown).
Next, the ability of H295 cells to produce cAMP after hormonal treatment was examined. Both forskolin (20 umol/ L) and ACTH (0.1 umol/L) caused significant increases in cAMP production by H295 cells (Table 1). Forskolin caused a 14-fold increase, but ACTH caused only a 2-fold change in cAMP production. The addition of a phosphodiesterase
A) Control
B) Forskolin
C) dbcAMP
| Treatment | cAMP production (pmol/mg protein · 2 h) | |
|---|---|---|
| Without IBMX | With IBMX | |
| Basal | 0.84 ± 0.03 | 3.04 ± 0.14 |
| ACTH | 1.73 ± 0.07 | 6.96 ± 0.42 |
| Forskolin | 12.02 ± 0.50 | 32.72 ± 4.87 |
H295 cells were treated for 2 h in medium containing ACTH (0.1 umol/L) or forskolin (20 umol/L). Values represent the mean ± SE for six different dishes of cells and are representative of three independent experiments. Where indicated, the phosphodiesterase inhibitor isobu- tylmethylxanthine (IBMX) was included in the incubation medium at a concentration of 0.5 mmol/L.
inhibitor (isobutylmethylxanthine; 0.5 mmol/L) caused a doubling of the amount of cAMP measured under each of the conditions tested, although the fold increase was similar to that seen in the absence of inhibitor.
The ability of H295 cells to produce adrenal-specific ste- roids was evaluated. Cells were initially treated for 48 h with angiotensin-II (0.1 µmol/L), ACTH (0.1 umol/L), dbcAMP (1 mmol/L), and forskolin (20 umol/L). Medium contents of cortisol, androstenedione, DHEA, and DHEAS were deter- mined by RIA. The agonists of the protein kinase-A pathway (ACTH, dbcAMP, and forskolin) increased the production, with respect to basal values, of all steroids examined; how- ever, the increase in ACTH-stimulated DHEAS was not significant (Fig. 2). Forskolin and dbcAMP stimulated steroi- dogenesis to a greater degree than ACTH. During forskolin treatment, cortisol was the major steroid product, represent- ing 70% of those quantified, whereas the C19 steroids (an- drostenedione, DHEA, and DHEAS) totaled 30%. Because H295 cells have been reported to produce multiple steroids,
the UV-detectable steroids secreted into the experimental medium were examined. H295 cells were treated for 48 h alone or with forskolin. Figure 3 illustrates the UV-detectable steroids produced in the last 24 h of treatment. Cortisol was found to be the major steroid product, followed by 118- hydroxyandrostenedione.
Further analysis showed that the effects of ACTH, dbcAMP, and forskolin on steroid secretion were concentra- tion dependent (Fig. 4). The average maximally effective concentration was 50 nmol/L for ACTH, between 7-20 umol/L for forskolin, and 0.3 mmol/L for dbcAMP. The stimulation of steroidogenesis was also shown to be time dependent (Fig. 5). Significant increases in all steroids meas- ured were found after 6 h of treatment with ACTH, forskolin, or dbcAMP (Fig. 5). The accumulation of new steroid prod- ucts in the incubation medium continued through 72 h of treatment.
Regulation of 17a-hydroxylase activity
A pivotal enzyme in the production of C19 steroids as well as cortisol in mammalian adrenal cells is P450c17 (11, 12). To establish further whether P450c17 levels were central to determining the steroids formed by H295 cells, we analyzed the products from cells incubated with [3H]pregnenolone (Fig. 6). Control cells produced mainly 11-deoxycorticoster- one, corticosterone, and progesterone. However, cells pre- treated with the kinase-A agonist dbcAMP produced mainly cortisol, 11-deoxycortisol, androstenedione, and 118-hydrox- yandrostenedione, with lesser quantities of 17a-hydroxy- pregnenolone and DHEA.
Further experiments were designed to determine whether H295 cell regulation of P450c17 was similar to that previ-
BASAL
AH
ACTH
Forskolin
dbcAMP
0
2
4
6
8
10
12
14
CORTISOL (nmol/mg protein)
BASAL
All
ACTH
Forskolin
dbcAMP
0.0
0.4
0.8
1.2
1.6
2.0
ANDROSTENEDIONE (nmol/mg protein)
BASAL
All
ACTH
Forskolin
dbcAMP
0.0
0.2
0.4
0.6
0.8
1.0
DHEA (nmol/mg protein)
BASAL
All
ACTH
Forskolin
dbcAMP
0.0
0.2
0.4
0.6
0.8
1.0
1.2
DHEAS (nmol/mg protein)
20
1 Cortisol
Absorbance 254 nm (Arbitrary Units)
18
2 11BOH-Androstenedione
3 Unknown
1
4 Corticosterone
16
5 11-Deoxycortisol
6 Androstenedione
14
12
10
8
2
6
4
3
5
6
2
4
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Retention Time (min)
ously seen in ovine, bovine, and fetal human adrenocortical cells (13-16). H295 cells were treated for 48 h in serum-free medium containing increasing concentrations of ACTH, for- skolin, and dbcAMP. P450c17 activity was determined by examining the cellular conversion of radiolabeled pregnen- olone to 17a-hydroxypregnenolone and DHEA in the pres- ence of a 36-hydroxysteroid dehydrogenase inhibitor (Fig. 7). ACTH, forskolin, and dbcAMP caused significant in- creases in the activity of P450c17. Maximal stimulation of activity was seen using ACTH (10 nmol/L), forskolin (1 umol/L), and dbcAMP (0.3 mmol/L). Forskolin and dbcAMP caused the largest increase in activity (~10-fold), whereas ACTH was able to increase activity only 2.5-fold.
Regulation of mRNA expression for P450c17
Increases in P450c17 activity in ovine, bovine, and fetal human adrenal cells have been associated with changes in P450c17 mRNA levels (13-16). Therefore, we examined the regulation of H295 cell P450c17 mRNA levels. H295 cells were treated with ACTH (0.1 umol/L), forskolin (20 umol/ L), and dbcAMP (1 mmol/L) for 24 h. Examination of P450c17 mRNA by Northern analysis demonstrated that each factor increased the expression of P450c17 mRNA (Fig. 8). Forskolin and dbcAMP were most effective, increasing levels of P450c17 mRNA by 11- and 14-fold, respectively,
11.0
10.0
9.0
0 --- ACTH
CORTISOL (fold stimulation)
8.0
Forskolin
7.0
dbcAMP
6.0
5.0
4.0
3.0
2.0
1.0
0.0
BASAL
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
28.0
24.0
20.0
DHEA
(fold stimulation)
16.0
12.0
8.0
4.0
0.0
BASAL
11
10
-9
-8
-7
-5
-5
-4
-3
-2
18.0
16.0
ANDROSTENEDIONE (fold stimulation)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
BASAL
-11
10
-9
-8
-7
-6
-5
-4
-3
-2
Log[Agonist](M)
above basal values. ACTH also increased mRNA, but only by 3.5-fold above basal levels.
Discussion
Currently, the only steroidogenically active cell line avail- able for the study of adrenocortical cell function is the Y-1 mouse adrenal tumor cell. These cells have been extremely useful for examining molecular events controlling adrenal cell function; however, they are deficient in P450c17, and so
24.0
Basal
CORTISOL (nmol/mg protein)
₹
20.0
ACTH
₹
Forskolin
I
16.0
dbcAMP
12.0
8.0
8
₹
.
4.0
0.0
01 3 6
12
24
48
72
TIME (hours)
5.0
DHEA (nmol/mg protein)
T
4.0
₹
3.0
?
2.0
1.0
O
0.0
01 3 6
12
24
48
72
ANDROSTENEDIONE (nmol/mg protein)
16.0
*
12.0
8.0
a
4.0
8
0.0
01 3
6
12
24
48
72
TIME (hours)
do not produce glucocorticoids or C19 steroids (17). The H295 human adrenocortical cell line, which has been in cell culture for over 10 yr, was recently described to produce a variety of steroid hormones (7). Herein, we demonstrate that the H295 cell line should provide an in vitro model to help ascertain factors controlling adrenal androgen production in addition to glucocorticoids. Such studies should be instru- mental in defining the mechanisms involved in normal and pathological adrenal function.
1000
Solvent
1
1 Cortisol
2 Corticosterone
800
6
3 11-Deoxycortisol
1
4 11BOH-Androstenedione
5 17aOH-Pregnenolone
600
3
6 11-Deoxycorticosterone
8
9
7 Dehydroepiandrosterone
Counts per channel
400
2
8 Pregnenolone
4
9 Androstenedione
10 Progesterone
200
Origin
5
7
10
1
0
1000
Solvent 2
6,8
800
600
3
1
400
2
9
4
6
200
Origin
10
5
7
8
0
0
2
4
6
8
10
12
14
16
18
20
Distance (cm)
P450c17 ACTIVITY (nmol/mg/2h)
7.0
6.0
ACTH
Forskolin
5.0
dbcAMP
5
4.0
3.0
2.0
1.0
2
0.0
BASAL
-11 -10
9
8
·
7
-6
-5
-4
-3
-2
Log[Agonist](M)
Initial experiments determined the ability of H295 cells to respond to treatment with ACTH, forskolin, or dbcAMP. Previously, fetal and adult human adrenal cells were shown
~Control
~ ACTH
~dbcAMP
~ Forskolin
28S
18S -
P450c17
Counts Per Band
224
779
3263
2537
28S
18S →
to exhibit a retraction from the substrate, known as “round- ing,” after treatment with agents that increase intracellular CAMP (3, 10). Greater than 80% of the H295 cells retracted from the culture substratum in response to 24 h of treatment with either dbcAMP or forskolin. Interestingly, ACTH caused no detectable change in H295 cell morphology. The lack of response to ACTH treatment probably relates to the minor effect of this hormone on cAMP production in H295 cells. The reason for the insensitivity of these cells to ACTH is unknown, but probably involves decreased expression of ACTH receptor. The maintenance of ACTH receptor expres- sion appears to be controlled by agonists of cAMP production in other cell culture systems (18-20). Thus, under normal culture conditions, ACTH receptor expression may be low.
H295 cells also retained the ability to produce steroid hormones after treatment with ACTH, forskolin, or dbcAMP. ACTH was less effective than forskolin or dbcAMP in stim- ulating steroidogenesis. Forskolin and dbcAMP caused in- creased production of all steroids examined within 3 h of treatment, indicating that the H295 cell could act as a model for acute as well as chronic steroid production. In the current study, experiments were carried out in the absence of low
density lipoprotein, thus causing the cells to rely on intracel- lular stores or endogenous production of cholesterol for steroid synthesis. The ability to use endogenously produced cholesterol for substrate is similar to that of primary cultures of fetal human adrenocortical cells (21).
Data from both HPLC and RIA analyses indicate that the major steroid produced after treatment with dbcAMP or forskolin was cortisol. The ability of the H295 cells to produce cortisol indicates the presence of 17a-hydroxylase, 21-hy- droxylase, and 118-hydroxylase enzymes. The mRNAs for these enzymes have been detected in the H295 cell line (22), although these enzymes were lost after long term culture of fetal human adrenal cells (4). In addition, over 30% of the steroid products examined here were C19 steroids. The adult human adrenal in vivo can produce almost equal amounts of glucocorticoids and C19 steroids; however, the relative ratio between these steroids changes greatly during adrenarche and puberty and throughout the course of human aging. The H295 cell should provide an excellent in vitro model to study regulation of the relative amounts of glucocorticoids vs. C19 steroids secreted by adrenal cells.
We investigated the regulation of P450c17 expression, an enzyme that appears to be pivotal in determining the relative production of C19 steroids (11-12). ACTH, dbcAMP, and forskolin increased the activity of P450c17 in a concentra- tion-dependent manner. The increase in activity was paral- leled by an increase in the mRNA level for P450c17. This ability of H295 cells to respond to agonists of the protein kinase-A pathway by induction of P450c17 is similar to that seen in primary cultures of bovine, ovine, and fetal human adrenocortical cells (13-16) and agrees with the findings of Staeles et al. (22).
In conclusion, this study demonstrates that the H295 adrenocortical cell line can serve as an in vitro model for human adrenal cell function. After treatment with agonists of the protein kinase-A pathway, these cells increase the production of glucocorticoids and adrenal androgens as well as the expression of P450c17. The accessibility of a function- ing human adrenal cell line should expedite examination of the pharmacological, biochemical, and molecular biological mechanisms that control adrenal steroid biosynthesis.
Acknowledgment
The authors gratefully acknowledge the expert editorial assistance of E. Ann Whisenand.
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