ELSEVIER
Human Adrenocortical NCI-H295 Cells Express VIP Receptors. Steroidogenic Effect of Vasoactive Intestinal Peptide (VIP)
A. HAIDAN,*1 U. HILBERS,* S.R. BORNSTEIN,*+ M. EHRHART-BORNSTEIN **
*Department of Internal Medicine III, University of Leipzig, 04103 Leipzig, Germany ¡NIH, NICHD and ¿NIH, NIMH, Bethesda, MD 20892
Received 9 March 1998; Accepted 9 July 1998
HAIDAN, A., U. HILBERS, S. R. BORNSTEIN AND M. EHRHART-BORNSTEIN. Human adrenocortical NCI-H295 cells express VIP receptors. Steroidogenic effect of vasoactive intestinal peptide (VIP). PEPTIDES 19(9) 1511-1517, 1998 .- VIP receptors are frequently overexpressed by various endocrine tumors. In this study the expression of VIP receptors in the human adrenocortical carcinoma cell line NCI-H295 and their involvement in the regulation of steroidogenesis was investigated. NCI-H295 cells express VIP1 and VIP2 receptors as demonstrated by RT-PCR, whereas they do not express VIP itself. The receptors are functionally coupled to steroidogenesis since VIP (10-9 M to 10-6 M) exerted a dose-dependent stimulatory effect on the release of aldosterone, cortisol, and DHEA. VIP increased ACTH-stimulated releases of aldosterone and cortisol. The proliferation rate of NCI-H295 cells was not affected by VIP. These data show that NCI-H295 cells express both forms of the VIP receptor and that VIP is involved in an ACTH-independent regulation of steroidogenesis in the adrenal tumor cells. @ 1998 Elsevier Science Inc.
NCI-H295 Steroidogenesis VIP VIP receptors
VIP is a 28-amino acid peptide of the glucagon-secretin family with a broad range of biologic activities (32,33) including regulation of hormone secretion (for review, see Reference 10) and both stimulation and inhibition of neo- plastic growth in different organs (11,17,21-23,39). Besides its physiological functions, peptides of this family could be involved in tumorigenesis, since their receptors are fre- quently overexpressed in endocrine tumors (29,37). There- fore, radiolabeled VIP became a promising imaging agent for tumor scanning in the last few years (30,35,36). How- ever, little is known about the effect of VIP on the hormone production and the proliferation of tumors that over-express VIP receptors. It is even possible that VIP receptors on neoplastic cells are functionally uncoupled to an effect on hormone secretion as observed in rat insulinoma cells (1).
In the human adrenal gland, primary Cushing’s syn- dromes have been described that were the result of an
adrenocortical over-responsiveness or aberrant expression of neuropeptide receptors such as the vasopressin receptor (19,25) and the receptor for gastric inhibitory peptide (GIP), a peptide that belongs to the glucagon-secretin family to- gether with VIP (18,31). Considering the fact that the vari- ety and number of human primary tumors that express VIP receptors are higher than for any other neuropeptide recep- tor known (29), VIP may be involved in ACTH-indepen- dent primary Cushing’s syndrome.
In the normal adrenal gland, VIP is produced in and released from nerve fibers and chromaffin cells in various species including humans (10). The neuropeptide stimulates steroidogenesis in the normal human and rat adrenal via the release of catecholamines from the medulla, suggesting the presence of VIP receptors mainly on chromaffin cells (2,7,16).
This study was designed to investigate the possible role
1 Requests for reprints should be addressed to A. Haidan, Medizinische Klinik und Poliklinik III der Universität Leipzig, Philipp-Rosenthal-Str. 27, 04103 Leipzig, Germany.
of VIP in the regulation of steroidogenesis in adrenocortical tumor cells. NCI-H295 cells, which are derived from an invasive primary human adrenocortical carcinoma, are ca- pable of producing all corticosteroids, i.e., androgens, glu- cocorticoids, and mineralocorticoids (12). NCI-H295 cells express the full complement of human adrenocortical en- zymes (26) as well as mRNA transcripts for the ACTH receptor (24) and the angiotensin II type 1 receptor (4). Steroidogenic activity is increased by the adrenocortical agonists angiotensin II (3), forskolin, dibutyryl-cyclic AMP and, to a lesser extent, by ACTH (27).
Three questions should be answered: first, are VIP re- ceptors and VIP itself expressed in NCI-H295 cells; second, does VIP affect the hormone secretion of this carcinoma cell line; and third, is VIP involved in the regulation of prolif- eration in these cells.
METHOD
Reagents
Unless otherwise indicated, all reagents were purchased from Sigma Chemical Company (München, Germany).
PCR Experiments
RNA isolation and cDNA synthesis were carried out as described previously (13). To remove traces of DNA, the RNA was treated with DNase before reverse transcription. For PCR amplification of the first strand cDNA, the follow- ing primers were used. 1) VIP1 receptor: forward primer 5’-CGC GGA TCC GCG GAG TGT GẠC TAT GTG CAG AT-3’, and the reverse primer 5’-CGG GGT ACC CCG CCT CAG GAT GAA GGA TAT GA-3’; 2) VIP2 receptor: forward primer 5’-GCG CGA AAT TAA CCC TCA CTA AAG TTC ACC CAG AAT GCC GAT TTC A-3’, and the reverse primer 5’-CTG TAA TAC GAC TCA CTA TAG GGA ATG ATG CAG TAC TGC AGG AAG A-3’; 3) VIP: forward primer 5’-TCA CTC AGA TGC AGT CTT CA-3’, and the reverse primer 5’-AGC CTT TGG TGA TTA GCA AT-3’.
The forward primer for the VIP1 receptor is located in exon 2 of the VIP1 receptor gene according to the sequence deposited under accession number U11080 (EMBL) and the reverse primer in exon 5 (EMBL: U11083). The length of the PCR product was calculated using the mRNA sequence (EMBL: L13288). The VIP2 receptor gene sequence is not available until now. Therefore only the mRNA sequence (EMBL: L36566) could be used for primer design. To verify that no genomic DNA was amplified, a control with mRNA before reverse transcription was performed. No PCR prod- uct could be seen in these controls. The forward primer for VIP is located in exon 5 of the VIP gene (M11552) and the reverse primer in exon 7 (M11554). The size of the PCR product was calculated by adding the lengths of the ampli- fied fragments of exon 5 and 7 and the length of exon 6 (M11553).
All fragments were amplified for 40 cycles in 25 ul containing 1× PCR buffer, 0.2 mmol/l deoxy-NTPs, 7.5 pmol each of forward and reverse primers, 0.026 U/pl Expand™M High Fidelity PCR System enzyme mix (Boehr- inger, Mannheim, Germany), 4 mM MgCl2 for VIP1 recep- tor, 2.75 mM MgCl2 for the VIP2 receptor, and 1.25 mM MgCl2 for VIP. The amplification conditions were as fol- lows: denaturation at 94℃ for 15 s, annealing of the primers for 30 s at 60℃ (VIP1 receptor), at 55°℃ (VIP2 receptor), or at 57℃ for VIP, and elongation at 72℃ for 45 s. Before starting the PCR, the samples were denatured at 94℃ for 3 min. The final elongation step was prolonged to 7 min.
The size of the PCR-fragment was determined after electrophoresis and ethidium bromide staining with a 100-bp ladder (Gibco, Eggenstein, Germany). The identity of every fragment was confirmed by digestion with two restriction enzymes (Serva, Heidelberg, Germany).
Cell Culture and Incubation Procedure
Human adrenal cells in primary culture that were used for RNA isolation were prepared and maintained as previously described (13).
NCI-H295 carcinoma cells (ATCC, Rockville, MD) were maintained in RPMI 1640 Medium (Gibco, Eggen- stein. Germany) containing hydrocortisone (3.6 µg/1), insu- lin (5 mg/l), transferrin (100 mg/l), B-estradiol (2.7 µg/1), selenite (5 µg/1), 2% fetal calf, and antibiotics. The cells were grown at 37°℃ in a 5% CO2 humidified atmosphere under routinely changed media.
The cells were incubated with VIP (human, porcine, rat; Peninsula, Merseyside, UK) and/or ACTH1-24 (Synacthen, Ciba-Geigy, Wehr, Germany) in serum-free medium con- taining ascorbic acid (10-7 M), transferrin (100 mg/l), BSA (0.01%), and bacitracin (0.01%).
Hormone Measurements
Hormone concentrations in the incubation media were mea- sured by radioimmunoassay (RIA), using the following kits. Cortisol-RIA (Biermann, Bad Nauheim, Germany); sensi- tivity: 5.5 nmol/l; cross-reactivity: cortisol 100%, pred- nisolone 76%, 11-deoxycortisol 11.4%, prednisone 2.3%, other steroids < 1%; intra- and interassay variations: 5.1 and 6.4%, respectively. Active DHEA (Diagnostic Systems Laboratories, Webster, TX, USA); sensitivity: 0.07 ng/ml; cross-reactivity: dehydroepiandrosterone (DHEA) 100%, other steroids < 0.88%; intra- and interassay variations: 10.6 and 10.2%, respectively. Aldosteron-RIA (Biermann, Bad Nauheim, Germany); sensitivity: 44.4 pmol/l; cross- reactivity: aldosterone 100%, other steroids < 0.033%; intra- and interassay variations: 5 and 10.4%, respectively.
VIP concentrations at the beginning and at the end of the incubation period were measured using the VIP-RIA kit from Euro-Diagnostica (Malmö, Sweden); sensitivity: 3 pmol/l; cross-reactivity: VIP 1-28 100%, VIP 1-6, 1-18,
and 1-22 < 2.5%, VIP 11-28 83.3%, VIP 7-28 90.9%, VIP 18-28 71.4%, other peptides < 0.01%; intra- and interassay variations: 6.0 and 8.5%, respectively.
[3H]thymidine Incorporation Assay
Cells were plated on 96-well dishes at a density of 105 cells/well and incubated for 24 h with VIP in serum-free medium or in 2% FCS containing medium. To assess cell growth, a [3H]thymidine assay was performed as described previously (14). Cells were incubated with 2.5 mCi/ml [3H]thymidine (Peninsula, Belmont, CA) for 24 h. All equipment was purchased from Packard (Meriden, CT). Cells were removed from the culture plate by pipetting the suspension up and down for several times and harvested with the Filtermate 196. After drying the filter with the harvested cells at 60℃ for 1 h, a plastic scintillator sheet (FlexiScint) was placed over the filter, again placed in an oven at 70°℃ for 30 min and counted in a Microplate Scintillation & Luminescence Counter (TopCount).
WST-1 Assay
The colorimetric assay for the quantification of cell prolif- eration and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells was performed according to the manufacturer’s protocol of the cell proliferation reagent WST-1 (4-[3-(4- Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben- zene disulfonate; Boehringer, Mannheim, Germany). Cells were plated on 24 well dishes at a density of 2.5 × 105 cells/810 pl/well and incubated for 24 h with VIP in serum- free medium or in 2% FCS containing medium. 90 pl WST-1 reagent were added for the last 45 min of incuba- tion. At the end of the incubation, media were removed and floating cells were pelleted by centrifugation. Absorbances of the supernatants were measured at 450 nm against a background control using the UV/VIS Spectrometer Lambda Bio (Perkin-Elmer, Weiterstadt, Germany). For the blank, medium was incubated with WST-1 for 45 min in the absence of cells. The reference wave length was 690 nm.
Statistical Analysis
Results are expressed as means ± SEM and statistical significance was determined by analysis of variance (ANOVA) using the software package SPSS for Windows, version 6. Differences were considered significant when p < 0.05, very significant when p < 0.01, and highly significant when p < 0.001. All experiments were repeated for a minimum of 3 times (n) using four wells per experi- ment.
RESULTS
VIP1 and VIP2 receptor mRNA expression could be dem- onstrated by RT-PCR in NCI-H295 cells. The amplification products had the expected lengths of 482 bp for the VIP1
600 bp
1
2
3
4
5
6
7
8
receptor and 627 bp for the VIP2 receptor (Fig. 1). No fragments were detected in fibroblasts, which do not express VIP receptors. The identity of both fragments was proven by digestion with two restriction enzymes. Digestion of the VIP1 receptor PCR product with BstXI led to a 104-bp and a 378-bp fragment and with MspI to a 144-bp and a 338 bp-fragment. The VIP2 receptor PCR product was digested with BanI resulting in a 179-bp and a 448 bp-fragment and with Bsp1286I resulting in a 184-bp and 443-bp fragment (data not shown).
NCI-H295 cells did not express VIP mRNA as shown by RT-PCR (Fig. 2). In both positive controls, human adrenal cells in primary culture and normal human adrenal tissue,
600 bp
1 2 3 4 5
the expected cDNA fragment of 769 bp was amplified. To confirm the identity of the amplified fragment, the PCR product was digested with EcoRI resulting in a 185-bp and a 584-bp fragment and with SspI resulting in four fragments (65 bp, 87 bp, 217 bp, and 400 bp).
Under basal conditions the cells released 5.38 ± 0.39 nmol/l aldosterone, 456 + 40 nmol/l cortisol, and 0.53 ± 0.03 ng/l DHEA in 24 h. VIP caused a dose-dependent increase in hormone release at concentrations from 10-9 M to 10-6 M. VIP at 10-6 M stimulated aldosterone secretion to 149 ± 8% (Fig. 3 A), cortisol secretion to 147 ± 7% (Fig. 3 B), and DHEA secretion to 124 ± 5% of basal secretion (Fig. 3 C). ACTH stimulated the release of aldosterone, cortisol, and DHEA in a dose-dependent manner. Maximal stimulation of the NCI-H295 cells was achieved with 10-7 M ACTH (184 ± 9%, 179 ± 8%, and 182 ± 8%, respec- tively; Fig. 3). VIP (10-6 M) significantly increased the secretion of aldosterone (p < 0.001) and cortisol (p<0.01) from ACTH-stimulated cells and it led to a slight, but not significant, increase of DHEA secretion from ACTH-stim- ulated cells (Fig. 3).
To monitor a possible degradation of VIP during the incubation, its concentrations in the serum-free medium were measured at the beginning (1.07 ± 0.05 × 10-6 M) and after 24 h of stimulation (0.92 ± 0.02 × 10-6 M). No significant loss of VIP was detected.
Incubation of the cells with 10 6 M VIP in serum-free medium as well as in the presence of 2% FCS for 24 h did not change the proliferation rate of the NCI-H295 cells as determined by [3H]thymidine incorporation (Fig. 4 A) and by the WST-1 cell proliferation assay (Fig. 4 B).
DISCUSSION
VIP has gained increasing importance as a neuropeptide hormone in the regulation of endocrine tissues. It exerts its tissue-specific effects via two different receptors, the VIP1 and VIP2 receptor which both bind pituitary adenylate- cyclase activating peptide (PACAP) and VIP and are des- ignated type II PACAP receptors. The type II PACAP receptors have the same high affinity to VIP and both PACAP forms, while the type I PACAP receptor recognizes PACAP-27 and PACAP-38 with a high affinity and VIP with a low affinity (34). Our data show that the adrenocor- tical cell line NCI-H295 expresses both type II PACAP receptors, the VIP1 and VIP2 receptor. This confirms re- cently published data by Bodart et al. (5), who suggested that VIP, PACAP-27, and PACAP-38 stimulated aldoste- rone production is mediated by type II PACAP/VIP recep- tors in this cell line due to the fact VIP and PACAP stimulated with roughly the same ED50 and via the accu- mulation of cAMP.
In the normal adrenal, VIP-induced stimulation of corti- costeroid secretion seems to depend on the intact structure of the adrenal and a VIP-stimulated catecholamine release
aldosterone secretion (% of basal secretion)
275
…
250
A
without ACTH
ZZZ
ACTH 10-8 M
225
ACTH 10-7 M
200
…
175
150
T
125
*
* T
T
100
T
cortisol secretion (% of basal secretion)
220
B
200
180
..
I
160
T
T
140
T
**
120
T
100
80
DHEA secretion (% of basal secretion)
200
C
**
180
160
**
T
140
**
**
T
T
120
T
T
100
T
80
Co
-9
-8
-7
-6
-6
-6
VIP concentration (log mol/l)
80
A
control
2.0
control
70
VIP 10-6 M
absorbance (A450 nm - A690 nm)
B
VIP 10-6 M
60
1.8
CPM/103
50
1.6
40
30
1.4
20
10
1.2
0
serum-free
2 % FCS
0
serum-free
2 % FCS
from the adrenal medulla. VIP did not affect steroid secre- tion by isolated rat adrenocortical cells, but stimulated al- dosterone release from intact adrenals in a catecholamine- dependent manner (16). Also in normal human adrenal cells in primary culture, VIP stimulated corticosteroid secretion mainly indirectly via catecholamines released from adreno- medullary chromaffin cells (7). These data suggest that, in contrast to the observed expression of VIP receptors in the NCI-H295 cell line, in the normal human adrenal VIP receptors are predominantly located on chromaffin cells. Besides the expression of VIP receptors, adrenal tumor NCI-H295 cells and adrenomedullary chromaffin cells have other proteins in common that normally are not expressed by adrenocortical cells such as synaptophysin and neuronal cell adhesion molecule (9), suggesting a neuroendocrine differentiation of adrenal tumors.
These VIP receptors had no influence on the proliferation rate of the NCI-H295 cells, since stimulation with VIP did neither influence the incorporation of [3H]thymidine nor the cleavage of WST-1. However, the VIP receptors proved to be functionally active. Incubation with VIP had a pro- nounced stimulatory effect on steroid secretion. It stimu- lated the release of mineralocorticoids (aldosterone), glu- cocorticoids (cortisol), and androgens (DHEA). The ability of VIP to increase ACTH-stimulated aldosterone and cor- tisol release suggests that VIP binds to a receptor distinct from the ACTH-receptor, which is in accordance with the RT-PCR results. VIP only slightly increased ACTH-stimu- lated DHEA secretion which could be due to the low stim- ulatory effect of VIP on the secretion of that hormone.
NCI-H295 cells did not express VIP excluding an auto- crine stimulation. In the human adrenal VIP is produced in
the adrenal medulla, by a subpopulation of the catechol- amine-containing chromaffin cells (15). In our model of the perfused porcine pancreas, it reached high local concentra- tions within the adrenal following splanchnic nerve stimu- lation (8). Since adrenal cortex and adrenal medulla are interwoven to an astonishing degree in the human adrenal (6), VIP secreted from chromaffin cells could stimulate VIP receptors on cortical cells in a paracrine manner. Therefore, a stimulatory influence on adrenocortical cells with an ab- errant expression of VIP receptors is very likely.
Mutations in adrenocortical trophic factors and their re- ceptors have been suggested to be involved in adrenocorti- cal tumorigenesis (20). The ACTH receptor gene has been extensively studied but no structural mutations were de- tected in the great majority of these tumors (28). However, evidence is accumulating that ectopic expression of receptor for neuropeptides or cytokines is associated with adreno- cortical tumorigenesis (18,19,25,31,38). The present study shows that the human adrenocortical carcinoma cell line expresses active VIP receptors, namely VIP1 and VIP2 receptors. These receptors are involved in the regulation of steroidogenesis in these cells. Therefore, in cases of corti- cotropin-independent Cushing’s syndrome, the involvement of VIP receptors should be considered.
ACKNOWLEDGEMENTS
We wish to thank Sandy Laue for her expert technical assistance. This work was supported by the Wilhelm-Sander Stiftung (grant 95.033.1 to MEB), the DFG (Eh 161/2-4), and by the Bundesministerium für Bildung, Forschung und Technologie (BMB+F), Interdisciplinary Center for Clin- ical Research at the University of Leipzig (01KS9504, B1). SRB is recip- ient of a Heisenberg grant from the Deutsche Forschungsgemeinschaft.
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