Human NCI-H295 Adrenocortical Carcinoma Cells: A Model for Angiotensin-II-Responsive Aldosterone Secretion*
IAN M. BIRD, NEIL A. HANLEY, R. ANN WORD, J. MICHAEL MATHIS, JOHN L. MCCARTHY, J. IAN MASON, AND WILLIAM E. RAINEY
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
Excessive secretion of aldosterone from the adrenal results in the most common form of endocrine hypertension. An understanding of the regulatory processes involved in aldosterone synthesis and release is needed to define the biomolecular mechanisms controlling excessive production of aldosterone. However, in vitro studies regarding the regulatory mechanisms of human aldosterone production have been limited because of difficulties in obtaining tissue and the subsequent isolation of aldosterone-secreting glomerulosa cells. Herein we describe an adrenocortical carcinoma cell line, NCI-H295, which provides a suitable angiotensin-II (AII)-responsive model system to investigate the acute and chronic regulation of aldosterone synthesis. The cells were characterized with regard to the effects of AII on second messenger systems, aldosterone release, and levels of aldosterone synthase (P450c18) mRNA. In the presence of lithium, AII caused a rapid, but
transient, increase in the production of inositol tris- and bisphosphates, whereas a prolonged gradual accumulation of inositol monophosphate occurred. Treatment with AII resulted in a 4.5-fold increase in total inositol phosphates in a concentration-dependent manner and an in- crease in intracellular cytoplasmic free Ca2+. Significant increases in aldosterone (3.5-fold) were detected within 1 h of AII addition. Aldo- sterone release occurred in a concentration- and time-dependent man- ner. The type 1 AII (AT1) receptor was shown to be responsible for activation of phosphoinositidase-C, increased intracellular free Ca2+, and aldosterone production, as determined by use of the AT1 receptor antagonist DuP753. In addition, AII treatment resulted in a time- dependent increase in levels of P450c18 mRNA, as detected by RNAse protection assay. In summary, NCI-H295 cells provide a valuable model system to define mechanisms regulating human aldosterone production. (Endocrinology 133: 1555-1561, 1993)
A LDOSTERONE, produced by cells within the glomeru- losa zone of the adrenal gland, is a major regulator of sodium homeostasis. Of clinical relevance, abnormal produc- tion can result in certain disease states. Excessive secretion of aldosterone is manifested by salt retention and hyperten- sion; deficient production leads to the salt wasting character- istic of Addison’s disease. In understanding the relevant etiology for these disorders, knowledge of the regulatory mechanisms of aldosterone secretion is of paramount impor- tance.
Present knowledge regarding the regulation of aldosterone production is derived from in vitro studies in rat and bovine glomerulosa cell preparations. Those studies have shown that the primary regulator of aldosterone production is an- giotensin-II (AII). Binding of AII to the AII receptor in these cells results in rapid activation of phosphoinositidase-C and increased intracellular free Ca2+ ([Ca2+];), which, in turn, promotes the conversion of cholesterol into pregnenolone by the cholesterol side-chain cleavage enzyme (1, 2). Thereafter, pregnenolone is rapidly metabolized to aldosterone by the sequential action of 30-hydroxysteroid dehydrogenase, 21-
Address all correspondence and requests for reprints to: Dr. William Rainey, Departments of Obstetrics and Gynecology and Biochemistry, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
* This work was supported in part by American Heart Association Texas Affiliate Grant 91G082 and NIH Grant AG-08175.
hydroxylase, 118-hydroxylase, and an enzyme uniquely re- quired for aldosterone production, aldosterone synthase. Thus, aldosterone secretion is a complex process and poten- tially subject to many levels of regulation.
Investigations into the regulation of aldosterone produc- tion have been hampered by the progressive inability of adrenal zona glomerulosa cell cultures to respond to All and produce aldosterone (3-5). Hence, the constant requirement for freshly acquired tissue along with the lack of readily available human tissue and the difficulties associated with the isolation of glomerulosa cells have been a major obstacle to defining the pharmacological, biochemical, and molecular biological mechanisms that regulate aldosterone production in human glomerulosa cells. Thus, although studies have confirmed that AII stimulates aldosterone production from freshly isolated human glomerulosa cells (5-8), a complete understanding of the regulation of aldosterone synthesis in vivo has not been possible. Notably, there is a need for a human cell model that retains its ability to secrete aldosterone in response to AII. Recently, a human adrenocortical carci- noma-derived cell line (NCI-H295) has become available that secretes multiple steroids (9). Herein we report that this human cell model system, which is commercially available, secretes aldosterone in response to AII and other agents. The stimulation of aldosterone secretion is mediated via the type 1 AII (AT1) receptor coupled to polyphosphoinositidase-C, and this response is accompanied by an increase in [Ca2+]i. Furthermore, AII induces a time-dependent increase in levels of P450c18 mRNA.
Materials and Methods
Cell culture
NCI-H295 cells, obtained from the American Type Culture Collection (Rockville, MD), were maintained in Dulbecco’s Modified Eagle’s-Ham’s F-12 medium (DME/F12) supplemented with insulin (1 µg/ml), trans- ferrin (1 µg/ml), selenium (1 ng/ml), linoleic acid (1 µg/ml; 1% ITS plus, Collaborative Research, Bedford, MA), 2% UltroSer SF (Sepracor, Inc., Marlborough, MA), and antibiotics. Cells were maintained and grown on 75-cm2 flasks at 37 C under an atmosphere of 5% CO2-95% air. During the initial 3 months of culture, only attached cells were retained when medium was changed. For subsequent experiments from the resultant stocks, cells were subcultured, and after 48 h, medium was renewed as required. Where aldosterone secretion was studied in re- sponse to agonists, cell medium was removed and replaced with serum- free medium (DME/F12 containing antibiotics and 0.01% BSA), and cells were cultured for a further 24 h. Cells were then rinsed and treated in the same medium. At the end of the incubation, medium was removed and stored frozen for subsequent assay.
Protein and steroid determination
Cells were solubilized in Tris-HCI (50 mM; pH 7.4) containing NaCl (150 mm), sodium dodecyl sulfate (1%), EGTA (5 mm), MgCl2 (0.5 mm), MnCl2 (0.5 mm), and phenylmethylsulfonylfluoride (0.2 mm) and stored frozen at -70 C. The protein content of the samples was determined by the bicinchoninic acid protein assay, using the BCA assay kit (Pierce, Rockford, IL). The aldosterone content of recovered medium was deter- mined by RIA (Diagnostic Products Corp., Los Angeles, CA). The aldo- sterone antisera used in this kit exhibited the following relevant cross- reactivities: 18-hydroxycorticosterone, 0.033%; corticosterone, 0.002%; 11-deoxycorticosterone, 0.006%; 11-deoxycortisol, 0.0004%; and corti- sol, no detectable cross-reactivity.
HPLC separation of steroid products
Incubation medium was extracted with 5 vol dichloromethane, and solvent was removed under nitrogen before redissolving the extracts in absolute methanol (0.15 ml). Steroids were separated by isocratic elution from a C18 Bondapak reverse phase column using 60% methanol and monitoring absorbance of eluant at 254 nm, exactly as described previ- ously (10). Fractions were collected at 15-sec intervals, and solvent was removed before redissolving the dried material in medium for RIA. Authenticated standards were also separated on the same column by the same procedure to assist in identification of unknown products.
Detection of aldosterone synthase mRNA by RNAse protection assay
A unique DNA fragment corresponding to the 3’-untranslated region of the human P450c18 cDNA (1545-1807) (11, 12) was isolated by polymerase chain reaction from human adult adrenal cDNA (Clonetech, Palo Alto, CA) and subcloned into the transcription vector pBSK (Stra- tagene, La Jolla, CA). This fragment contains approximately 86% simi- larity to the 3’-untranslated region of the human P450c11 cDNA (13). Template DNA containing the P450c18 fragment was linearized with BamHI, and antisense RNA was synthesized using T7 RNA polymerase in the presence of [a-32P]UTP. RNase protection assays were performed, as described previously (14), by hybridizing 20 ug total RNA with 1 x 106 cpm labeled antisense RNA probes at 45 C for 16 h. After RNase digestion (20 µg/ml at 30 ℃ for 30 min), RNA samples were analyzed on 5% polyacrylamide-7 M urea gels.
Determination of [Ca2+]; using fura-2-loaded H295 cells
Cells were plated onto glass coverslips and cultured in growth me- dium, as described above, for 3 days. Cells were then loaded with fura- 2/AM ester (5 uM; Molecular Probes, Eugene, OR) in buffer (130 mm NaCl, 4.8 mm KCI, 1 mm MgCl2, 1.5 mm CaCl2, 1 mM Na2HPO4, 15 mm glucose, 1 mg/ml BSA, and 10 mm HEPES, pH 7.4). Loading was
achieved over 45 min at 37 C under a 5% CO2-95% O2 atmosphere. Cells were then rinsed and incubated for a further 20 min in the same buffer. Coverslips were mounted on a Teflon frame in a cuvette con- taining buffer without fura-2 or albumin. The frame ensured a 30° angle of the coverslip to the incident/excitation fluorimeter beam. All fluores- cence measurements were made using a Perkin-Elmer 650-10S fluores- cence spectrophotometer (Norwalk, CT) while the buffer was stirred with an electronically controlled paddle to ensure rapid mixing of added reagents. Excitation and emission wavelengths were 340 nm (slit width, 5 nm) and 510 nm (slit width, 5 nm), respectively. The autofluorescence of cells and buffers was also measured and subtracted where necessary. [Ca2+], was computed based on the method of Grynkiewicz et al. (15) and Tsien et al. (16) using the formula: [Ca2+]; = Ka(F - Fmin)/(Fmax - F), where Ka = 224 nM; F is the observed 340 nm fluorescence; Fmax is the 340 nm fluorescence at a Ca2+ concentration (millimolar) sufficient to saturate fura-2, and Fmin is the 340 nm fluorescence at a Ca2+ concentra- tion (nanomolar) sufficient to give no significant binding to fura-2. A single excitation wavelength was used because the fluorimeter was not equipped with an automated filter wheel. Concentrations calculated by this method did not differ by more than 5% from those calculated by the ratio method when excitation wavelength was changed manually (17).
Studies of acute phosphoinositidase-C activation
Cells cultured on 24-well plates were prelabeled during the last 48 h of culture by supplementing the growth medium with 10 uCi/ml [3H] inositol (Amersham International, Arlington Heights, IL). Before treat- ment, cells were rinsed once in DME/F12 medium and incubated for 30 min in DME/F12 containing 10 mm LiCl (to inhibit phosphoinositol phosphatases). Cells were then treated in a final volume of 0.5 ml with the treatments shown, and incubated for 30 min. At the end of this time, 0.25 ml ice-cold perchloric acid (15%) was added to each well to denature proteins and lyse the cells. The base of each well was scraped, and the well contents were recovered, with a 0.5-ml water wash, into a microfuge tube (1.5 ml). Precipitated material was pelleted by centrifu- gation (12,000 × g; 2 min), and the supernatant was transferred to a glass tube. The aqueous phase (containing inositol and phosphoinositols) was neutralized by mixing with freon-octylamine, as described previ- ously (18).
Separation of [3H]inositol from combined [3H]inositol phosphates was carried out on columns of AG1X8 anion exchange resin. Samples were loaded onto individual columns (0.25 ml resin/column), and all unbound [3H]inositol was eluted with water (twice; 4 ml). The combined eluates from the column loading and washing (10 ml, containing all of the [3H] inositol) were collected into a single 20-ml vial. The collecting vial was then removed and replaced, and each column was eluted with 1 M ammonium formate-0.1 M formic acid (twice; 2 ml) to recover the bound [3H]inositol phosphates (up to inositol tetrakisphosphate). Alternatively, where separation of headgroups into different classes [inositol mono (InsP)-, bis (InsP2)-, tris (InsP3)-, and tetrakis (InsP4)-phosphates] was required, the column volume was increased to 0.6 ml, and separation was achieved by elution five times with 2 ml of each of the following: water only (free inositol), 60 mm ammonium formate-5 mm disodium tetraborate (GroPIns), 200 mm ammonium formate-0.1 M formic acid (InsP), 400 mm ammonium formate-0.1 M formic acid (InsP2), 800 mM ammonium formate-0.1 M formic acid (InsP3) and 1.2 M ammonium formate-0.1 M formic acid (InsP4). The radioactivity of all samples was determined by liquid scintillation counting for 5 min each or less if a counting error of 1% outside 2 sD (to 40,000 counts) could be achieved in that time. All values shown are corrected for volume.
[225 I] AII binding displacement
A modified method of Viard et al. (19) was followed on cells cultured in 12-well plates. Radiolabeled AII (125]; 2000 Ci/mmol) was obtained from Amersham International, and 0.05 uCi was added to each well. Binding was carried out in 0.5 ml binding medium (DME/F12 containing 0.5% BSA and 0.1% bacitracin, pH 7.4) for 1 h at 37 C. At the end of this period, wells were washed in DME/F12 medium (22 C; three times) before cell lysis in 0.5 M NaOH containing deoxycholate (0.4%). Radio-
activity associated with the cell lysates was then determined in a y- counter.
Statistical analysis
Statistical analysis of the data was accomplished using analysis of variance, followed by Newman-Keuls multiple comparison analysis.
Results
Treatment of H295 cells with AII (10-7 M) resulted in a time-dependent increase in aldosterone secretion over 72 h, as detected by RIA (Fig. 1). This increase was detectable within 1 h of treatment and continued to rise linearly for up to 3 h. After 3 h, the medium aldosterone content increased more rapidly, and this increase continued for up to 72 h. Although ACTH (10-7 M) promoted a similar acute rise in aldosterone during the initial 3 h of treatment, the long term production of aldosterone was much less marked than that in response to AII. The AII stimulation of aldosterone pro- duction was concentration dependent (Fig. 2), reaching a maximal 22-fold above basal at 10-8 M AII. A marked stim- ulation was still observed at concentrations as low as 10-11 M AII.
To confirm that the cells were indeed producing aldoste- rone and the RIA used for quantification of aldosterone was specific, medium from cells stimulated for 24 h with AII (10-7 M) was extracted with dichloromethane, and the ex-
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tracts were analyzed by reverse phase HPLC. Several UV- absorbing products were detected (Fig. 3), but only one was detectable by RIA. This UV-absorbing and RIA-detectable peak had an identical retention time to the aldosterone standard run on the same system.
The production of aldosterone uniquely requires the expression of aldosterone synthase, and a previous report described the presence of mRNA for P450c18 in these cells. Using RNAse protection assay methodology, we studied the time-dependent effect of AII on levels of P450c18 mRNA. Results showed that All treatment increased message levels in as little as 3 h, and levels remained elevated for at least 12 h after stimulation (Fig. 4).
To investigate the possibility that activation of phosphoi- nositidase-C occurred in response to AII, [3H]inositol-prela- beled H295 cells were stimulated for 30 min in the presence of Li+. A concentration-dependent increase in combined [3H] inositol phosphates was observed (Fig. 2), reaching a maxi- mum of 4-fold above the basal level at 10-7 M and with a threshold at 10-10 M. The time-dependent formation of the individual classes of [3H]phosphoinositols was also studied (Fig. 5). Within the first 2 min of stimulation, there was a rapid rise in [3H]InsP3 and [3H]InsP2, which fell to basal levels by 5 min. Whereas [3H]InsP4 changed little during stimula- tion, a rise in [3H]InsP was observed by 2 min, and this was sustained throughout stimulation. These findings are con- sistent with the activation of a polyphosphoinositide-specific phosphoinositidase-C (1, 2). In contrast, there was no change in [3H]GroPIns, confirming that the response was not indi- rectly due to activation of phospholipase-A2. An interesting finding was that there was no detectable change in labeling of the phosphoinositides during treatment (data not shown),
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but this was accounted for by the corresponding time-de- pendent drop in free [3H]inositol (Fig. 5). Thus, phosphoi- nositide resynthesis kept pace with degradation in these cells.
In addition to activation of phosphoinositidase-C, AII treatment caused an acute increase in the concentration of [Ca2+]. Treatment with AII (10-8 M) resulted in a rapid increase in [Ca2+]; from 78 ± 3 to 409 ± 28 nm (n = 5; mean ± SE; Fig. 6). Thereafter, [Ca2+]; declined to near-basal levels within 60 sec.
To characterize further the AII receptor subtype mediating these responses, we studied the effects of the selective AT1 and AT2 antagonists DuP753 and PD123319 on AII binding, activation of phosphoinositidase-C, acute aldosterone secre- tion, and increases in [Ca2+]. DuP753 (10-5 M) effectively displaced all specifically bound [125I]AII and blocked the effects of AII (10-8 M) on activation of phosphoinositidase- C, increased [Ca2+]i, and aldosterone secretion (Figs. 6 and 7). In contrast, PD123319 (10-5 M) reduced the responses by no more than 10%.
Discussion
The major finding of this study is that the NCI-H295 adrenocortical cell line can act as an in vitro human model for AII-regulated aldosterone production. We show that un-
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der these culture conditions, the cells exhibit multiple differ- entiated functions associated with adrenal glomerulosa cells. The accessibility of a human cell line to study aldosterone production should allow additional studies of the pharma- cological, biochemical and molecular biological mechanisms controlling normal and physiological aldosterone production.
The recent description of the H295 cell line demonstrated that these cells retained the ability to produce steroid hor- mones from exogenously added pregnenolone (9). Herein we demonstrate that these cells secrete aldosterone, and that the production is regulated by AII in a time- and dose- dependent manner. Increased aldosterone secretion is de- tectable within 1 h, indicating that no induction of steroid- metabolizing enzymes was required for this response. After 3 h, however, the rate of aldosterone secretion increased further. ACTH treatment reproduced the initial increase in aldosterone secretion over the first 3 h, but not the increased rate of secretion thereafter. In bovine and rat adrenocortical cell models, ACTH is known to act through the cAMP- signaling pathway, whereas AII acts through the phosphoi- nositidase-C pathway. Both pathways can promote an acute increase in aldosterone secretion (20), but the two agents have markedly different chronic effects on expression of steroid-metabolizing enzymes. In cultured fetal bovine adre- nocortical cells, which secrete aldosterone, AII preferentially induces aldosterone synthase activity (21), whereas ACTH preferentially induces 17a-hydroxylase activity (22). The re- cent findings by ourselves and others (10, 23) have confirmed that NCI-H295 cells respond to stimulation with kinase-A activators, such as ACTH, forskolin, and (Bu)2CAMP, with a strong induction of 17a-hydroxylase activity and that this
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response is accompanied by a marked increase in the pro- duction of 17-hydroxylated steroids. In contrast AII stimu- lation has little or no effect on induction of 17a-hydroxylase or production of 17-hydroxysteroids by these cells. We show here that AII does, however, stimulate an increase in message for P450c18, and this increase occurs within a few hours of stimulation.
By examining the production of [3H]phosphoinositols in H295 cells we have shown that AII stimulates the rapid, but transient, production of [3H]InsP3 and [3H]InsP2. Although there is little change in [3H]InsP4, sustained production of [3H]InsP occurs in the presence of Li+. Such a response is characteristic of a phosphoinositidase-C acting on the poly- phosphoinositides, as previously described in rat and bovine glomerulosa cell preparations (1, 2). The rapid phosphoinos- itidase-C response was also accompanied by a rapid increase in [Ca2+]i, again paralleling findings in rat and bovine cell
model systems (20). The dose dependency of the phosphoi- nositidase-C response was similar to that in rat and bovine models, but required AII at concentrations 10 times higher than required for aldosterone secretion. The reason for this dissociation between aldosterone secretion and phosphoi- nositide response remains unclear, but this increased sensi- tivity of human glomerulosa cells to All has also been noted previously (5).
In the rat and bovine model systems, AII is known to activate phosphoinositidase-C, increase [Ca2+]i, and stimulate aldosterone secretion via an AT1 receptor (21, 22, 24-26). Using the selective AT1 antagonist DuP753 (Losartin) and the AT2 receptor antagonist PD123319, we have shown that only DuP753 effectively inhibits the binding of AII to its receptor, abolishes the stimulation of both phosphoinositi- dase-C and [Ca2+]i, and markedly inhibits AII stimulation of aldosterone secretion in H295 cells. Thus, the AII receptor coupled to phosphoinositidase-C is of the AT1 subtype, and this receptor-phosphoinositidase-C complex mediates the stimulatory action of All on aldosterone secretion. Consistent with these findings, a cDNA probe to the AT1 receptor protein-coding region hybridized to a 2.4-kilobase message in the total RNA fraction isolated from H295 cells (data not shown). This message is the same size as that reported for AT1 mRNA from rat (25) and human (27) tissues. The ability to detect messages in total RNA by Northern analysis also suggests that AT1 mRNA is abundant in H295 cells.
In conclusion, our study demonstrates for the first time that the NCI-H295 human adrenocortical carcinoma cell line retains functional control of aldosterone production via the AII receptor. AII stimulates a concentration- and time-de- pendent secretion of aldosterone that is mediated via an AT1 receptor functionally coupled to a polyphosphoinositide- specific phosphoinositidase-C. Activation of this phosphoi- nositidase-C is accompanied by a rapid increase in cellular [Ca2+]i. AII treatment of these cells also results in an increase in message for aldosterone synthase. These previously un-
a) Binding
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reported features for human adrenocortical carcinoma cells are similar to those described in the rat and bovine glomer- ulosa cell systems. We believe that the H295 cell line will provide an invaluable model system in which to define the control mechanisms regulating the production of aldoste- rone. The availability of these cells will also greatly expedite the dissection of molecular mechanisms controlling expres- sion of trophic hormone receptors and steroidogenic en- zymes, which, in turn, determine the long term production of aldosterone.
Acknowledgments
The authors gratefully acknowledge the expert editorial assistance of E. Ann Whisenand. We would also like to thank DuPont for the DuP753 (Losartin) antagonist, and Parke-Davis for the PD123319 antagonist used in this study.
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