Autocrine-Paracrine Role of Endothelin-1 in the Regulation of Aldosterone Synthase Expression and Intracellular Ca2+ in Human Adrenocortical Carcinoma NCI-H295 Cells*
GIAN PAOLO ROSSI, GIOVANNA ALBERTIN, SERGIO BOVA, ANNA S. BELLONI, FRANCESCO FALLO, UBERTO PAGOTTO, LUCIA TREVISI, GIORGIO PALÙ, ACHILLE C. PESSINA, AND GASTONE G. NUSSDORFER
Departments of Clinical and Experimental Medicine, Pharmacology (S.B., L.T.), Division of Endocrinology (F.F.), Institutes of Microbiology (G.P.) and Anatomy (A.S.B., G.G.N.), University of Padua Medical School, Padova, Italy; and Max Planck Institute of Psychiatry, Clinical Institute (U.P.), Munich, Germany
ABSTRACT
The role played by endothelin (ET-1) and its receptor subtypes A and B (ETA and ETB) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse tran- scription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ETA, and ETB complementary DNAs consis- tently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respec- tively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ETB agonist IRL-1620; this effect was not in-
hibited by either the ETA antagonist BQ-123 or the ETB antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ETB, but not ETA, activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active ET-1 biosynthetic path- way and are provided with ETA and ETB receptors; 2) ET-1 regulates in an autocrine/paracrine fashion the secretion of aldosterone by NCI-H295 cells by enhancing both AS transcription and raising the intracellular Ca2+ concentration; and 3) the former effect of ET-1 probably involves the activation of both receptor subtypes, whereas calcium response is exclusively mediated by the ETB receptor. (En- docrinology 138: 4421-4426, 1997)
E NDOTHELIN-1 (ET-1), the prototype of a family of 21- amino acid residue peptide hormones (1), exerts mul- tiple biological effects, including very potent vasoconstric- tion, mitogenesis, stimulation of protooncogene expression, inhibition of renin secretion, and stimulation of cat- echolamines, vasopressin, and aldosterone secretion (for re- view, see Refs. 2 and 3). The latter effect was observed both in vivo in animals and in vitro, and is potentially important in conditions where enhanced ET-1 and aldosterone secre- tions coexist, such as severe and/ or malignant hypertension, congestive heart failure, and hypoxia (for review, see Ref. 4). We recently demonstrated the expression of the genes for prepro-ET-1 and of its receptors A and B (ETA and ETB) in homogenates of the normal adrenal cortex of rats and hu- mans as well as in aldosterone-producing tumors (5-7) and showed that the mineralocorticoid secretagogue effect of ET-1 is mediated in the rat by the ETB receptor (6; for review,
see Ref. 8). However, whether this also applies to human adrenocortical cells is not known at present.
A human adrenocortical tumor cell line (NCI-H295) is now available (9) that expresses angiotensin II AT1 receptor func- tionally coupled to phosphoinositidase C, secretes aldoste- rone in response to angiotensin II, and has been widely used for investigating the regulation of adrenal steroidogenesis in vitro (10-13). Hence, we hypothesized that NCI-H295 cells can be useful for investigating the role of ET-1 in the regu- lation of human adrenocortical function.
This study was, therefore, designed to investigate whether NCI-H295 cells 1) express the genes for prepro-ET-1, human ET-1 converting enzyme-1 (hECE-1), and ETA and ETB re- ceptors; 2) are endowed with a functional ET-1 biosynthetic pathway; 3) possess functional ETA and ETB receptor sub- types; and 4) regulate the expression of the aldosterone syn- thase (AS) gene in response to ET-1.
Materials and Methods
Reagents
Unless otherwise stated, laboratory reagents were obtained from Sigma Chemical Co. (Milan, Italy). ET-1 was obtained from either Pen- insula Laboratories (Merseyside, UK) or Neosystem Laboratories (Stras- bourg, France). The ET agonists and antagonists used were BQ-123, a selective ETA antagonist (14); BQ-788, a potent and selective ETB an- tagonist (15); sarafotoxin 6C, a weak ETB agonist (16); and IRL-1620, a potent ETB agonist (17). They were purchased from Neosystem Labo-
Address all correspondence and requests for reprints to: Gian Paolo Rossi, M.D., F.A.C.C., Clinica Medica 1, Dipartimento di Medicina Clinica e Sperimentale, University Hospital via Giustiniani, 2, 35126 Padova, Italy. E-mail: gprossi@ipdunidx.unipd.it.
* Presented in part at the 77th Scientific Meeting of The Endocrine Society, Washington, D.C., June 14-17, 1995. This work was supported by Regione Veneto, Giunta Regionale, Ricerca Finalizzata (Venice, Italy) (Contract no. 91.00.218 PF41 115.06.654, to G.P.R.).
ratories. [125I]ET-1 (SA, 2000 Ci/mmol) was purchased from Amersham Laboratories (Aylesbury, UK).
Cell culture
Human NCI-H295 adrenal carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD; catalogue no. CRL 10296). Cells were initially maintained in RPMI 1640 medium (Life Technologies, Eggenstein, Germany; catalogue no. 041-02404M) sup- plemented with 2% FCS, insulin (0.005 mg/ml; Monotard, Novo Nor- disk Farmaceutici, Rome, Italy), transferrin (0.01 mg/ml), sodium se- lenite (30 nm), and antibiotics (penicillin-streptomycin). Cells were maintained and grown on 25-cm2 flasks at 37 C under an atmosphere of 5% CO2-95% air. During the initial 2 months of culture, only attached cells were retained when medium was changed. Cells used for the experiments described were routinely maintained as monolayer cul- tures. When the responses of cells to agonists were studied, the medium was removed and replaced with serum-free medium and 0.02% BSA.
NCI-H295 cells were used for RNA extraction by the guanidium isothiocyanate method. RNA was precipitated by the addition of 500 pl ethanol and 20 pl 3 M sodium acetate and standing for 1 h at -80 C. It was recovered by centrifugation (15 min, 12,000 × g, 4 C), and the pellet was washed once in 75% ethanol (1.0 ml) before being dried under vacuum and dissolved in diethylpyrocarbonate-water. After isolation, total RNA was checked for integrity by 1.5% agarose gel electrophoresis and quantified by measurement of UV absorbance at 260 nm.
Reverse transcription-PCR (RT-PCR)
For use in the PCR, total RNA was reversely transcribed to comple- mentary DNA (cDNA) with random hexamers (2.5 µM), as previously detailed (5). After incubation at 42 C for 15 min, temperature was raised to 95 C for 5 min and then quickly decreased to 5 C for 5 min. For amplification of the resulting cDNA, 20 ul of the reverse transcription mixture were used. The sample volume was increased to 100 pl with a solution containing 50 mm KC1, 10 mm Tris (pH 8.3), 2 mm MgCl2, and 0.2 µM of up- and downstream primers as well as 2.5 U Taq polymerase (AmpliTaq DNA Polymerase, Perkin-Elmer/Cetus, Norwalk, CT). Am- plification of prepro-ET-1, ETA, ETB, and hECE-1 cDNA was carried out with the primers and the thermal profiles previously reported (5, 18), using a Delphi 1000 Thermal Cycler (MJ Research, Waterston, MA). The specificity of the amplification products for the gene of interest was confirmed by hybridization with the following cDNA-specific probes: hECE-1, 5’-TTG GAC TTT GAG ACG GCA CTG GC-3’; ETA receptor, 5’-CCT CAA CCT CTG CGC TCT TAG TGT-3’; and ETB receptor, 5’-TCC TGC CTT GTG TTC GTG CTG GGG-3’. As a positive control, amplification of a 838-bp fragment of the human ß-actin gene was carried out in parallel, as described previously (5). As false positive results of RT-PCR for the ß-actin gene, due to amplification of retro- pseudogenes, have been reported (19), in parallel experiments ETA and ETB receptor PCR was carried out with no prior RT to further rule out the possibility of amplifying genomic DNA.
Immunocytochemistry
NCI-H295 cells were cultured in double slide flasks at a concentration of 20,000 cells/well in RPMI 1640 with all ingredients and 2% FCS for 4 days, then washed in PBS and cultured for 24 h in RPMI supplemented only with antibiotics, glutamine, selenium, and transferrin. Immuno- cytochemistry was performed with minor modifications as previously described (20). Briefly, two antibodies for ET-1 were used: a rabbit polyclonal (Peninsula, Heidelberg, Germany) and a mouse monoclonal (Dianova, Hamburg, Germany; cross-reactivity for ET-3, 7% and 2%, respectively). Rabbit and swine serum and biotinylated swine antirabbit and antimouse IgGs were obtained from Dako (Hamburg, Germany). After medium removal and several washes in cold PBS, the cell mono- layer was rapidly dried and fixed in 4% paraformaldehyde, and en- dogenous peroxidase was blocked with 1% H2O2. The sections were then preincubated in rabbit or swine serum (1:10), followed by application of polyclonal or monoclonal ET-1 antibody (1:1500 and 1:125) overnight at 4 C. After incubation with the respective biotinylated IgGs (1:300) and the streptavidin-biotin-horseradish peroxidase complex (1:100) for 1 h at room temperature each, the enzymatic reaction was performed for 5-10
min at room temperature using 0.1% 3,3-diaminobenzidine tetrahydro- chloride (diluted in PBS with 0.05% hydrogen peroxide) as chromogen. After immunostaining, the sections were inserted for dehydration in a series with increasing concentrations of ethanol-xylol, and the coverslips were attached with Eukitt (Riedel de Haen, Seelze, Germany). For im- munocytochemical detection, negative controls with omission of the primary antibody were run in parallel.
Autoradiography
Cell monolayers were immediately frozen at -30 C by immersion in isopentane and stored at -80 C. They were processed according to the method of Kuhar (21) with minor modifications (6). ET-1-binding sites were labeled in vitro by incubation for 120 min with 100 PM [125]]ET-1; nonspecific binding was determined by adding 1 µM unlabeled ET-1. Selective [125I]ET-1 binding to ETA and ETB was studied by adding 100 nM BQ-123 and sarafotoxin 6C, respectively. The reaction was stopped by washing the samples three times in 50 nm Tris-HCl buffer. After rinsing, the sections were rapidly dried, fixed in paraformaldehyde vapors at 80 C for 120 min, and coated with NTB2 Kodak nuclear emulsion (Eastman Kodak, Rochester, NY). The autoradiograms were exposed for 2 weeks at 4 C and then developed with undiluted D19 Kodak developer. They were stained with hematoxylin-eosin, and ob- served and photographed with a Leitz Laborlux microscope (Leitz, Rockleigh, NJ). For the purpose of quantitation, autoradiograms were analyzed by computer-assisted densitometry using a camera-connected microscope and a personal computer equipped with software specifi- cally written for this purpose (Studio Casti Imaging, Venice, Italy). Given the uneven distribution of cells on monolayers, only areas correspond- ing to identifiable cells were outlined and measured. For each autora- diogram (n = 4), 10-18 areas of about 55,000 um2 (79,500 pixels) were analyzed. The density of the ETA subtype was assessed in the presence of the ETB weak agonist sarafotoxin 6C (500 nM), and that of ETB was determined in the presence of BQ-123 (500 nM).
Gene expression
NCI-H295 cells (5 × 105 cells/well) were incubated for 24 h at 37 C with ET-1 (10-8 M) or IRL-1620 (10-7 M) and with ET-1 (10-8 M) plus BQ-123 or BQ-788 (10-6 M). At the end of the incubation period, cells were harvested, and RNA was extracted and reversed transcribed (see above). PCR amplification was performed (see above) with 21-mer 5’- ACATTGGTACAGGTTTTCCTC-3’ (sense) and 5’-CAGATGCAAGAC- TAGTTAATC-3’ (antisense) (22). To evaluate the kinetics of the ampli- fication reaction, 5-ul aliquots of the amplification mixture were collected after every two cycles starting from the 20th up to the 38th cycle. Aliquots of the PCR products were transferred to a nylon mem- brane (Immobilon-S, Millipore, Milan, Italy) by a slot blot apparatus (Milliblot, Millipore) and UV cross-linked (Stratagene UV-Crosslinker 1800, Stratagene-Duotech, Milan, Italy). Detection of the digoxigenin- labeled amplification products on the nylon membrane was carried out by high affinity antidigoxigenin antibody Fab fragments conjugated to alkaline phosphatase using a chemiluminescent detection kit (DIG Lu- minescent Detection Kit, Boehringer Mannheim, Mannheim, Germany). Light generated via dephosphorylation of the chemiluminescent sub- strate (CSPD, Boehringer Mannheim) was used to impress x-ray films (BioMax MR-1 film, Sigma) with a 20-min exposure, as in an autora- diographic procedure (5). Quantification of the PCR products was car- ried out by measuring the integrated optical density of the autoradiog- raphies with an image analyzer IBAS 2000 (Zeiss, Unterkochen, Germany). Plots of the integrated optical density vs. the number of cycles were then elaborated; the cycle number (N50) that corresponded to the half-maximal PCR products was also calculated as an estimate of the amount of initial amplifiable template of the gene (5, 23). Comparison of the N50 of cells exposed to the different agonists and antagonists was carried out by one-way ANOVA followed by Bonferroni post-hoc test; the significance level was set at 0.05.
Measurement of intracellular Ca2+ concentration ([Ca2+];)
[Ca2+]i was measured by fluorometry in a double wavelength mode in a Perkin-Elmer LS-50B spectrofluorometer equipped with a magnetic
stirrer and a temperature regulator (Perkin-Elmer, Norwalk, CT). Cell monolayers were loaded with fura-2/acetoxymethyl ester (fura-2/AM; Molecular Probes, Eugene, OR) as described by Bird et al. (24). Briefly, NCI-H295 cells were plated on 8 × 30-mm glass coverslips and cultured in growth medium, as described above, for 7 days. For cell loading, coverslips were incubated in a buffer (130 mm NaCl, 4.8 mm KCI, 1 mM MgCl2, 1.5 mM CaCl2, 1 mM NaH2PO4, 15 mM glucose, 1 mg/ml BSA, and 10 mm HEPES, pH 7.4) containing 5 µM fura-2/ AM for 45 min at 37 C in a 5% CO2-95% O2 atmosphere. Coverslips were then transferred to a customized holder, inserted into a quartz cuvette, and placed into the spectrofluorometer, where they were superfused at a rate of 5 ml/min with the same buffer without BSA (25). The cells were left to recover for 20 min before starting the experiment. [Ca2+]i was calculated by the formula of Grynkiewicz et al. (26): [Ca2+]; = {[(R - Rmin)/(Rmax - R)] X (Sf/Sp) × Kal, where Ka is the dissociation constant of fura-2 and was assumed to be 225 nM, R is the 340/380 nm ratio of fura-2 fluorescence measured in the cells, Rmax is the 340/380 nm ratio in the presence of a saturating calcium concentra- tion, Rmin is the 340/380 nm ratio of fura-2 fluorescence in a Ca2+-free solution containing EGTA, and Sf/Sp is the ratio of Ca2+-free to Ca2+-bound fura-2 fluorescence at 380 nm. Calibration was per- formed by using fura free acid. Rmin was determined in a solution containing 115 mm KCI, 10 mM NaCl, 2 mm MgSO4, 10 mm K2H2- EGTA, and 10 mm K2-3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.2. For the determination of Rmax, 2 mM CaCl2 was added to the medium, and CaK2-EGTA was substituted for K2H2-EGTA. Rmax/ Rmin, and Sf/Sp values were 17.36, 1, and 7.81, respectively.
The effect of ET-1 (10 nm) and the ETB agonist IRL 1620 (100 nm) on [Ca2+]; was assessed with and without 10-min pretreatment with BQ-788 (0.8 mm) and/or BQ-123 (1 mm). As exposure to ET-1 was reported to induce homologous down-regulation (27), each experiment was carried out on a different preparation of NCI-H295 cells. Each set of experiments was carried out four times on different cell preparations with consistent results.
Results
Analysis of prepro-ET-1, ETA, ETB, and hECE-1 messenger RNA (mRNA) levels
RT-PCR analysis of RNA from NCI-H295 cells showed prepro-ET-1, hECE-1, ETA, ETB, and AS mRNA in all samples examined (Fig. 1, A and B). The specificity of the amplifica- tion products obtained was confirmed by 1) hybridization with cDNA-specific probes, 2) size identity, and 3) lack of amplification of each cDNA when diethylpyrocarbonate- water was used instead of mRNA as the template for RT-PCR.
Immunocytochemistry
Cell monolayers showed a strong ET-1 immunoreactivity when incubated with either the polyclonal (not shown) or the monoclonal anti-ET-1 primary antibody (Fig. 2A), whereas no staining was seen when the primary antibody was omit- ted (Fig. 2B).
Autoradiographic studies
Specific binding of [125]]ET-1 was demonstrated by com- parison of total binding (Fig. 3A) and binding in the presence of excess cold ET-1 (Fig. 3B). The binding was partially dis- placed by both the ETA antagonist BQ-123 (Fig. 3C) and the ETB antagonists BQ-788 (Fig. 3D). Quantitative densitometric analysis of the autoradiograms revealed that the ETB subtype accounted for the majority (mean ± SEM, 72.6 ± 9%) and the ETA accounted for the minority (37.4 + 5%) of the total [125I]ET-1-specific binding.
A
DNA Size Marker
prepro-ET-1
ETA receptor
ETB receptor
DNA Size Marker
ECE-1
H2O
ßActin
883 442
760
B
DNA Size Marker
CYP11B1
CYP11B1
Aldo Synthase
Aldo Synthase
H2O
1114 -
501 +
+320
Effect of ET-1 on expression of the AS gene
The kinetics of amplification of the AS cDNA are reported in Fig. 4, where the amount of amplified DNA (log integrated optical density units) is plotted vs. the number of PCR cycles. The curve corresponding to the ET-1-treated cells cDNA was shifted to the left compared with that in the controls (Fig. 4), thereby indicating a higher initial abun- dance of AS mRNA (i.e. an enhanced expression of the AS gene) (23); however, the slopes of all curves were similar, suggesting a similar efficiency of amplification. The spe- cific ETB agonist IRL-1620 displayed a similar shift to the left compared with the control curve. The concomitant
A
B
A
B
C
D
exposure to ET-1 and either BQ-123 or BQ-788 did not abolish the stimulatory effect of ET-1. The N50, i.e. the number of cycles at which half-maximal amplification was attained, was significantly lower for ET-1 (25.00 ± 0.24 SEM; P = 0.006) and IRL-1620 (26.18 ± 0.59; P < 0.05) than
for the controls (28.90 ± 0.20). Similarly, the N50 of cells treated with both ET-1 plus BQ-788 (25.61 ± 0.84) and ET-1 plus BQ-123 (26.04 ± 0.70) was lower than the control value, although not significantly so, due to the larger dispersion of the data.
1600
Amount of amplified DNA (Log integrated O.D. units)
40
Control
ET-1
IRL-1620
ET1 + BQ788
ET-1+BQ-123
1
20
22
24
26
28
30
32
34
36
38
PCR Cycle
Effect of ET-1 on [Ca2+];
The exposure of NCI-H295 cells to ET-1 induced a 95% increase in [Ca2+]; (from 64.3 ± 6 to 127.3 ± 19 SEM; n = 4; Fig. 5A). This effect rapidly disappeared with repeated ex- posure to ET-1 and was abolished by 10-min pretreatment with the BQ-788 (Fig. 5B), whereas it was not affected by pretreatment with BQ-123 (Fig. 5C). Either BQ alone did not modify resting [Ca2+] ¡. The exposure of the cells to IRL 1620 induced a 91% increase in [Ca2+]; (from 65 ± 7 to 124 ± 22; n = 3; Fig. 5D). This effect was prevented by 10-min pre- treatment with BQ-788 (data not shown).
Discussion
Identification of the functional role of ETs and their re- ceptor subtypes in the regulation of aldosterone biosynthesis and secretion in humans has been prevented to date by the lack of suitable models of investigation. From this stand- point, the availability of a cell line capable of synthesizing aldosterone in a regulated fashion and expressing both ET receptor subtypes would be very desirable.
Our coupled cell biology, immunocytochemical, and au- toradiographic study shows that NCI-H295 cells not only express the mRNAs of prepro-ET-1 and hECE-1 and can synthesize immunoreactive ET-1, a finding in partial agree- ment with the results of Li et al. (28) in adrenocortical car- cinomas, but also that they can express and translate into functional proteins both ETA and ETB receptor subtypes. Thus, these results strongly suggest that ET-1 may act as an autocrine-paracrine factor in the regulation of NCI-H295 cell function.
This contention is supported by the demonstration that
125
A
100
ET-1
75
50
125
B
100
[Ca2+ ], (nM)
ET-1
75
50
125
C
100
ET-1
75
50
D
125
100
IRL-1620
75
50
0
50
100
150
200
250
300
Time (sec)
ET-1 is able to enhance the expression of the AS gene through a mechanism that is likely to mainly involve the ETB receptor subtype. In fact, our findings indicate that ET-1 exposure induces a higher initial abundance of AS mRNA in the NCI- H295 cells, and the ETB agonist IRL-1620 is equipotent to ET-1 as far as this effect is concerned. It must be pointed out, however, that exposure to either the specific ETA antagonist
BQ-123 or the specific ETB antagonist BQ-788 alone could not abolish the stimulatory effect of ET-1, thereby suggesting that both receptor subtypes are somehow involved in the medi- ation of this effect of ET-1.
As concerns the intracellular event brought about by ET receptor activation, we found that activation of the ETB re- ceptor subtype by either ET-1 or IRL 1620 leads to a clear-cut increase in [Ca2+]], which vanished with repeated ET-1 stim- ulation, probably due to homologous desensitization (27). This effect was abolished by pretreating the cells with the specific ETB antagonist BQ-788 alone or in combination with BQ-123, but not by treatment with the specific ETA antagonist BQ-123 alone, thereby confirming that the ET-1-induced in- crease in [Ca2+]¡ is an ETB receptor-mediated effect. Calcium mobilization is deemed to play an important role in the control of steroidogenesis in the adrenal cortex (29); accord- ingly, the bulk of the evidence indicates that activation of the ETB receptor subtype mediates the aldosterone secretagogue effect of ET-1 (for review, see Ref. 8).
Hence, the question arises of what are the cellular events associated with ETA receptor subtype activation and ensuing stimulation of AS gene expression. The fact that no evident increase in [Ca2+]; and aldosterone secretion was elicited by ET-1 activation of the ETA subtype is intriguing and obvi- ously worthy of further investigation.
In conclusion, our results provide evidence for the con- comitant expression AS and the prepro-ET-1, hECE-1, ETA, and ETB receptor genes in NCI-H295 adrenocortical carci- noma-derived cells. Furthermore, they demonstrate that NCI-H295 cells synthesize mature ET-1 and express both the ETA and ETB receptor subtypes at the protein level. Both ET receptors are able to bind [125I]ET-1 and can mediate the secretagogue effect of ET-1 on aldosterone by acting in an autocrine-paracrine fashion at the transcriptional level of AS; however, only ETB activation was found to be associated with a sizable increase in [Ca2+] ;. Thus, these findings indi- cate that NCI-H295 cells may be a useful model for inves- tigating the role of ET-1 and its receptor subtypes in the regulation of adrenocortical function in humans as well as the potential autocrine-paracrine role of ETs in the patho- genesis of adrenal tumors.
Acknowledgments
We gratefully acknowledge the expertise and help with the molecular and cellular techniques of Susanna Hofman of the Department of Clin- ical and Experimental Medicine and Katia Pillon of the Division of Endocrinology of our university.
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