ELSEVIER
Production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1, by cultured human adrenocortical carcinoma cells
Kazuhiro Takahashia,*, Ayako Yoshinoyaª, Osamu Murakamib, Kazuhito Totsuneb, Shigeki Shibaharaª
aDepartment of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
bSecond Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan Received 14 September 1999; accepted 28 October 1999
Abstract
The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmuno- assay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 ± 1.6 fmol/10 5 cells/24 h and 11.0 ± 0.8 fmol/10 5 cells/24 h, respectively, mean ± SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10-9-10-7 mol/l), angiotensin II (10-9-10-7 mol/l), or dexamethasone (10-8-10-6 mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-« (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immu- noreactive-endothelin levels in the culture medium. Interferon-y (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)ß (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines. @ 2000 Elsevier Science Inc. All rights reserved.
Keywords: Adrenocortical tumors; Tumor necrosis factor; Calcitonin gene-related peptide; Corticotropin-releasing hormone; Neuropeptide Y; Urocortin
1. Introduction
Adrenal medulla and pheochromocytomas are known to produce and secrete various kinds of neuropeptides and vasoactive peptides, such as neuropeptide Y (NPY) [1,16], vasoactive intestinal polypeptide (VIP) [23], pituitary ade- nylate-cyclase activating polypeptide (PACAP), [30] and natriuretic peptides [35,36]. On the other hand, information on the production of peptides by adrenal cortex and adre- nocortical tumors is limited. Accumulating evidence shows that normal adrenal cortex and adrenocortical tumors pro- duce and secrete some peptides.
* Corresponding author. Tel .: +81-22-717-8116; fax: +82-22-717- 8118. E-mail address: ktaka-md@mail.cc.tohoku.ac.jp (K. Takahashi).
Adrenomedullin (ADM) is a potent vasodilator peptide, that was originally isolated from human pheochromocytoma [11,12]. We and other investigators have recently reported that ADM mRNA was expressed in adrenocortical tumors, such as aldosterone-producing tumors and adrenocortical carcinomas [15,34]. ADM mRNA was detected in cultured human adrenal carcinoma H295 cells by RT-PCR method [18]. We have shown the production and secretion of ADM by SW-13 adrenocortical carcinoma cells [34]. The expres- sion of ADM mRNA and/or ADM peptide was shown in the adrenal cortex of rat and mouse [4,10]. Endothelin-1 (ET-1), a potent vasoconstrictor peptide discovered from vascular endothelial cells [8,38], is also produced by adrenal cortex and adrenocortical tumors [6,14,22]. On the other hand, the regulation of secretion of ADM and ET-1 by adrenocortical tumors has not been studied in detail. We have therefore
studied the regulation of the secretion of two vasoactive peptides, ADM and ET-1, in SW-13 human adrenocortical carcinoma cells.
2. Materials and methods
2.1. Materials
Recombinant human interferon-y (IFN-y) (IFN-y-la, ImunomaxR-y) was a gift from Shionogi Co. (Osaka, Ja- pan). Human IL-1B (IL-1B) was a gift from Otsuka Phar- maceutical Co. (Tokushima, Japan). Human adrenocortical adenocarcinoma cell line SW-13 [13], and human chorio- carcinoma cell line, BeWo were obtained from Health Sci- ence Research Resources Bank of Japan Health Sciences Foundation (Osaka, Japan). Human glioblastoma cell line T98G was obtained from the American Type Culture Col- lection (Rockville, MD, USA). Human neuroblastoma cell line IMR-32 was obtained from the Cancer Cell Repository, Institute of Development, Aging, and Cancer, Tohoku Uni- versity. Angiotensin II was from Peptide Institute, Inc. (Osaka Japan); ACTH was from Peninsula Laboratories, Inc. (Belmont, CA, USA); ADM, ET-1, and other peptides were from either Peptide Institute, Inc. or Peninsula Labo- ratories, Inc. Human TNF-a (TNF-a) was obtained from Boehringer Mannheim Biochemica (Mannheim, Germany); dexamethasone from Wako Pure Chemical Industries, Ltd (Osaka, Japan); [a-32P]dCTP from Amersham Pharmacia Biotech (Tokyo, Japan); [125]]-Na from Daiichi Kagaku Co. (Tokyo, Japan); restriction endonucleases from Takara Shuzo (Otsu, Japan), Boehringer Mannheim Biochemica (Mannheim, Germany) and New England BioLabs (Bev- erly, MA, USA).
2.2. Cell culture
SW-13 cells were cultivated at 37℃ under 5% CO2 in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum, as previously reported [34]. After the me- dium was replaced, cells were treated with ACTH (10-9- 10-7 mol/l), angiotensin II (10-9-10-7 mol/l) or dexameth- asone (10-8-10-6 mol/l) for 24 h. In another experiment, cells were cultured with IFN-y (10 and 100 U/ml), TNF-a (2 and 20 ng/ml) or IL-1ß (1 and 10 ng/ml) for 24 h. The cells were harvested for RNA extraction and the culture media were collected for the measurement of peptides.
2.3. Radioimmunoassay
The peptides in the culture media were extracted using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) as previously reported [31]. The extract was reconstituted with assay buffer [0.1 mol/l phosphate buffer, pH 7.5 containing 0.1% (v/v) bovine serum albumin (BSA), 0.2% (v/v) Triton X-100, and 0.1% (w/v) sodium azide] and assayed. The
radioimmunoassay of ADM was performed as previously reported [24,31] using the antiserum (No 102) against hu- man ADM-(1-52) [31]. Cross reaction with CGRP, ET-1 and other peptides was less than 0.001%. IR-ET was mea- sured by radioimmunoassay using antibody against ET-1 (BP6), as previously reported [28,29]. Cross-reaction with big ET-1 (1-38), ET-2, and ET-3 was 0.1%, 60% and 70%, respectively. Radioimmunoassays of CGRP [19,27], corti- cotropin-releasing hormone (CRH) [21], NPY [20], and urocortin [33] were performed as previously reported.
Chromatographic characterization of IR-ET in the cul- ture medium was performed by reverse phase high-perfor- mance liquid chromatography (HPLC) using a uBondapak C18 column (3.9 mm x 300 mm, Waters). The culture medium extract was reconstituted with water containing 0.1% (v/v) trifluoroacetic acid and loaded onto the column. Reverse phase HPLC was performed with a linear gradient of acetonitrile containing 0.1% (v/v) trifluoroacetic acid from 10% to 60% at a flow rate of 1 ml/min/fraction over 50 min. Each fraction (1 ml) was collected, dried by air, and assayed.
2.4. Northern blot analysis
Total RNA was extracted from cultured cells by the guanidine thiocyanate-cesium chloride method and sub- jected to Northern blot analysis, as previously reported [31]. The Northern probe for human ADM mRNA was the Hind III/EcoRI cDNA fragment of pBS-hAM2 [31]. Northern probe for ET-1 mRNA was the BamH1/EcoR1 cDNA frag- ment of pBT-hET-1 [28]. The expression of B-actin mRNA was examined as an internal control.
Radioactive signals were detected by exposing the filters to X-ray films (X-AR5; Kodak, Rochester, NY, USA). The intensity of hybridization signals was quantified with a Bioimage analyzer (BAS 1500; Fuji Film, Tokyo, Japan). The intensity representing ADM mRNA and ET-1 mRNA was normalized with the intensity of B-actin band, and the normalized values in various samples were compared.
2.5. Statistics
Data are shown as mean ± SEM, unless otherwise stated. Statistical analysis was performed by one-way anal- ysis of variance, followed by Fisher’s protected least sig- nificant difference.
3. Results
Northern blot analysis showed the expression of ADM mRNA and ET-1 mRNA in SW-13 cells (Fig. 1). The expression level of ADM mRNA in SW-13 cells was about 50% of T98G glioblastoma cells [32], but higher than BeWo human choriocarcinoma cells and IMR-32 human neuro- blastoma cells. On the other hand, ET-1 mRNA was clearly
SW-13
T98G
BeWo
IMR-32
ADM mRNA
ET-1 mRNA
B-actin mRNA
expressed only in SW-13 cells. Expression of ET-1 mRNA was very low in T98G cells and was not detectable in BeWo cells and IMR-32 cells.
Both IR-ADM and IR-ET were detected in the culture medium of SW-13 cells (27.7 ± 1.6 fmol/105 cells/24 h and 11.0 ± 0.8 fmol/10 5 cells/24 h, mean ± SEM, n = 6) (Table 1). Reverse phase HPLC of the IR-ET in the medium extract showed an immunoreactive peak eluting in the po- sition of ET-1, indicating that the IR-ET in the medium was ET-1 (Fig. 2). On the other hand, no significant amount of CGRP, CRH, NPY, or urocortin was secreted by SW-13 cells (Table 1).
Treatment with ACTH (10-9-10-7 mol/l) or angiotensin II (10-9-10-7 mol/l) for 24 h had no significant effects on IR-ADM levels or IR-ET levels in the culture medium of SW-13 (Fig. 3) (IR-ADM, F(6, 28) = 0.708, P> 0.6 and IR-ET, F(6, 28) = 0.652, P > 0.6). Treatment with dexa- methasone (10-8-10-6 mol/l) also had no significant effects on them (Fig. 4) (IR-ADM, F(3, 16) = 2.298, P > 0.1 and IR-ET, F(3, 16) = 0.238, P > 0.8). Northern blot analysis showed that ADM mRNA and ET-1 mRNA levels were not noticeably changed by these treatments (data not shown).
| Adrenomedullin | 27.7 ± 1.6 fmol/105 cells/24 h |
| Endothelin-1 | 11.0 ± 0.8 fmol/105 cells/24 h |
| Calcitonin gene-related peptide | N.D. |
| Corticotropin-releasing hormone | N.D. |
| Neuropeptide Y | N.D. |
| Urocortin | N.D. |
N.D., not detectable; No significant difference in the peptide concentra- tions was noted between the unconditioned medium and the medium cultured for 24 h by SW-13 cells (P > 0.1).
3 1 2
100
VW
80
80
60
IR-ET (fmol/ml)
60
ACN (%)
40
40
20
20
0
0
0
10 20 30 40 50
minutes
Treatment with one of three cytokines, IFN-y, TNF-a, and IL-1B, modulated IR-ADM levels and IR-ET levels in the culture medium differently (Fig. 5) (IR-ADM, F(6, 21) = 2.802, P < 0.05, and IR-ET, F(6, 21) = 5.666, P < 0.002). Treatment with TNF-« (20 ng/ml) increased signif-
A
30
IR-ADM (fmol/105 cells/24h)
20
10
0
control
10 -9
10 -8
10 -7
10 -9
10 -8
≤ 10 -7
ACTH
Ang II
B
12
(fmol/105 cells/24h)
10
8
IR-ET
6
4
2
0
control
10 -9
10 -8
10 -7
10 -9
10 -8
10 -7
ACTH
Ang II
(M)
A
40
IR-ADM (fmol/10 5 cells/24h)
30
20
10
0
control 10 -8 10 -7 10 -6 Dexamethasone (M)
B
12
(fmol/105 cells/24h)
10
8
IR-ET
6
4
2
0
control
10 -8 10 -7 10 -6 Dexamethasone (M)
icantly both IR-ADM levels and IR-ET levels in the culture medium (P < 0.05 and P < 0.002, respectively) (Fig. 5A and B). IFN-y (100 U/ml) increased IR-ET levels in the culture medium (P < 0.002), but not IR-ADM levels. On the other hand, IL-1B (10 ng/ml) increased IR-ADM levels in the culture medium (P < 0.05), but not IR-ET levels in SW-13 cells.
Northern blot analysis showed that treatment with TNF-a (20 ng/ml) caused about 20% increase in the expres- sion level of ADM mRNA (Fig. 6). Treatment with IFN-y (100 U/ml) and TNF-« (20 ng/ml) caused about 70% and 50% increases in the expression levels of ET-1 mRNA, respectively.
4. Discussion
The present study has shown the production and secre- tion of two vasoactive peptides, ADM and ET-1, by SW-13 human adrenocortical carcinoma cells. Neuroendocrine properties of adrenocortical cells, in particular the cells of the zona glomerulosa, have been proposed by Ehrhardt- Bornstein and Hilbers [3], who showed expression of neural cell adhesion molecule in normal adrenal cortex and the
A
*
40
*
IR-ADM (fmol/105 cells/24h)
30
20
10
0
control
IFN 10U/ml
IFN 100U/ml
TNF 1ng/ml
TNF 10ng/ml
IL 1ng/ml
IL 10ng/ml
B
20
(fmol/10 5 cells/24h)
*
* *
15
IR-ET
10
5
0
control
IFN 10U/ml
IFN 100U/ml
TNF 1ng/ml
TNF 10ng/ml
IL 1ng/ml
IL 10ng/ml
human adrenocortical cell line, NCI-H295. ADM and ET-1, and their specific receptors are expressed in the brain [24, 25,29], and therefore, our findings on ADM and ET-1 in SW-13 cells may suggest the neuroendocrine properties of SW-13 cells. On the other hand, no significant amount of other neuropeptides, including CGRP, NPY, CRH, and uro- cortin, was secreted by SW-13 cells. These neuropeptides were produced in adrenal medulla and pheochromocytoma [1,16,19]. In this regard, ADM and ET-1 are the two unique peptides that are produced both in adrenal cortex and me- dulla, and by tumors derived from them.
It was reported that dexamethasone increased expression levels of ADM mRNA in rat vascular endothelial cells and vascular smooth muscle cells [7,9]. Angiotensin II increased ADM production by rat vascular smooth muscle cells [26]. In contrast to these reports, dexamethasone or angiotensin II had no noticeable effects on the secretion of ADM and ET-1 by SW-13 cells in the present study. This may be due to the difference of cell types.
Control
IFN-Y
TNF-a. IL-1B
ADM mRNA
ET-1 mRNA
ß-actin mRNA
The secretion of ADM and ET-1 was differently stimu- lated by cytokines. TNF-a increased the secretion of both ADM and ET-1 by SW-13 cells. IL-16 increased the secre- tion of only ADM, while IFN-y increased the secretion of only ET-1. Almost parallel changes were observed between the mRNA expression levels and IR-peptide levels in the medium in SW-13 cells with cytokine treatment, except for the effect of IL-1B (10 ng/ml) on ADM mRNA. The in- crease in IR-ADM in the medium by IL-1ß (10 ng/ml) was not large, and therefore the change in ADM mRNA by IL-1ß might not be detected by Northern blot analysis. Our findings suggest that adrenocortical production of ADM and ET-1 is regulated by cytokines rather than ACTH and an- giotensin II. In some pathologic conditions, such as severe infectious diseases, elevated plasma levels of cytokines may affect adrenocortical production of ADM and ET-1, which may modulate the secretion of aldosterone or cortisol by the adrenal cortex. ET-1 is known to stimulate aldosterone secretion [17,22]. On the other hand, ADM inhibited aldo- sterone secretion by dispersed rat adrenal glomerulosa cells [37]. ADM stimulated corticosterone release from rat adre- nal glands by raising blood flow, and enhanced aldosterone secretion from rat and human adrenal glands, possibly via the release of catecholamines by adrenal chromaffin cells [2,17].
The present study has shown that SW-13 human adre- nocortical carcinoma cells produce and secrete two vasoac- tive peptides, ADM and ET-1, and that the secretion of these two peptides is modulated differently by cytokines.
Acknowledgments
We thank Prof. S.R. Bloom, Dr M.A. Ghatei, and Dr P.M. Jones (Hammersmith Hospital, London) for their gift
of the endothelin antiserum (BP6) and the plasmid contain- ing the endothelin-1 cDNA (pBT-hET-1). This study has been supported partly by a Grant-in-Aid for Scientific Re- search on Priority Areas (A) from the Ministry of Educa- tion, Science, Sports, and Culture of Japan, and by the Gonryou Medical Foundation.
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