ELSEVIER
Expression of Adrenomedullin mRNA in Adrenocortical Tumors and Secretion of Adrenomedullin by Cultured Adrenocortical Carcinoma Cells
KAZUHIRO TAKAHASHI,*1 FUMITOSHI SATOH,¡ MASAHIKO SONE,*į KAZUHITO TOTSUNE, ¡ ZENEI ARIHARA,¡ TAKAO NOSHIRO,¡ TORAICHI MOURI§ AND OSAMU MURAKAMI;
*Department of Molecular Biology and Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan
Department of Internal Medicine, National Iwate Hospital, Ichinoseki, Iwate 021-0015, Japan §Division of Neuroendocrinology, Mouri Clinic, Natori, Miyagi 981-1224, Japan
Received 29 May 1998; Accepted 24 July 1998
TAKAHASHI, K., F. SATOH, M. SONE, K. TOTSUNE, Z. ARIHARA, T. NOSHIRO, T. MOURI AND O. MURAKAMI. Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells. PEPTIDES 19(10) 1719-1724, 1998 .- Immunoreactive-ad- renomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non- functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochro- mocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 + 1.2 pmol/g ww (mean + SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/105 cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedul- lin was not detectable in the extract of SW-13 cells (<0.09 fmol/105 cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors. @ 1998 Elsevier Science Inc.
Adrenomedullin Aldosterone
Radioimmunoassay
Northern blot Adrenal Adrenocortical Carcinoma
ADRENOMEDULLIN (ADM) is a potent vasodilator pep- tide, that was originally isolated from human pheochromo- cytoma, based on its stimulating action on platelet cAMP production (6,7). The C-terminal portion-(16-52) of ADM
shares about 27% homology with calcitonin gene-related peptide (CGRP).
High levels of immunoreactive-adrenomedullin (IR- ADM) and ADM mRNA are found in the normal adrenal
Requests for reprints should be addressed to Kazuhiro Takahashi, MD, Department of Molecular Biology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 Japan.
glands, in particular adrenal medulla, and in tumor tissues of pheochromocytomas (3,7,18,20,30). Immunocytochemistry of ADM in human adrenal glands showed positive ADM- immunostaining in the medulla, but not in the cortex (19). Tumor tissue concentrations of IR-ADM were very low or undetectable in aldosterone-producing adrenocortical ade- nomas and cortisol-producing adrenocortical adenomas (20).
On the other hand, it has recently been reported that ADM mRNA was expressed in adrenocortical tumors, such as aldosterone-producing tumors and adrenocortical carci- nomas and that ADM was secreted from primary cultures of adrenocortical tumors (10). The expression of ADM mRNA and/or ADM peptide was shown in the rat adrenal zona glomerulosa (5) and in the adrenal cortex (zona reticularis and zona fasiculata) of rat and mouse (2). ADM mRNA was also detected in cultured human adrenal carcinoma H295 cells by RT-PCR method (14). ADM is produced and se- creted not only by pheochromocytomas but also by various kinds of tumors, including glioblastomas, pulmonary tu- mors, and colonic carcinomas (12,14-16,26,27).
In the present study, we have studied ADM mRNA expression and IR-ADM concentrations in the tumor tissues of adrenocortical tumors to clarify cause(s) for the discrep- ant results in ADM and ADM mRNA levels in the tumor tissues of adrenocortical tumors. In addition, we have stud- ied possible production and secretion of ADM by a human adrenocortical carcinoma cell line, SW-13.
METHOD
Tissues
Tumor tissues of adrenocortical tumors were obtained at surgery from 31 patients with adrenocortical tumors, con- sisting of 17 aldosterone-producing adenomas, 7 cortisol- producing adenomas, 3 non-functioning adenomas, and 4 adrenocortical carcinomas (two cortisol-producing and two non-functioning). Tumor tissues of 5 pheochromocytomas and normal parts of adrenal glands obtained from 4 patients with aldosterone-producing adenomas were included as positive controls. This study has been approved by the Ethics Committee on human study of Tohoku University School of Medicine. The tissues were stored at -80℃ until extraction of RNA and peptides.
Peptides were extracted from all tumor tissues and nor- mal parts of adrenal glands. Total RNA was extracted from four aldosterone-producing adenomas, two cortisol-produc- ing adenomas, one non-functioning adenoma, four adreno- cortical carcinomas, five pheochromocytomas, and four nor- mal parts of adrenal glands.
Cell culture
Human adrenocortical adenocarcinoma cell line, SW-13 (8) was obtained from Health Science Research Resources
Bank of Japan Health Sciences Foundation (Osaka, Japan). SW-13 cells were cultivated at 37℃ under 5% CO2 in Leibovitz’s L-15 medium supplemented with 10% fetal calf serum.
Radioimmunoassay
The peptides were extracted from tissues as previously reported (24). The peptides in the culture media and cul- tured cells were extracted as previously reported (26,27). 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, 0.1% (w/v) sodium azide and 3.3% (w/v) dextran T-40] and assayed. The radioimmunoassay was performed as previously re- ported (18,26) using the antiserum (No. 102) against human ADM-(1-52) (19). Cross reaction with CGRP and other peptides was less than 0.001%.
Chromatographic characterization of IR-ADM in the cul- ture medium was performed by Sephadex G-50 (superfine) column chromatography and reverse phase high-perfor- mance liquid chromatography (HPLC) using a uBondapak C18 column (3.9 mm × 300 mm, Waters, Milford, MA, USA). The culture medium extract was reconstituted in 1 mol/l acetic acid containing 0.5% (w/v) BSA, and loaded onto the Sephadex G-50 column (10 × 560 mm). The column was eluted with 1 mol/l acetic acid containing 0.5% (w/v) BSA at a flow rate of 6 ml/h. Fractions (0.82 ml/ fraction) were collected, dried by air, reconstituted in assay buffer and assayed.
For the reverse phase HPLC analysis, the extract was reconstituted with water containing 0.1% (v/v) trifluoroace- tic acid and loaded onto the column. The column was eluted 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.
Northern blot analysis
Total RNA was extracted from tissues and cultured cells by the guanidine thiocyanate-cesium chloride method and was subjected to Northern blot analysis, as previously reported (26). The Northern probe for human ADM mRNA was the Hind III/EcoRI cDNA fragment of pBS-hAM2 (26). The expression of ß-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). The inten- sity of hybridization signals was quantified with a Bioimage analyzer (BAS 1000; Fuji Film, Tokyo, Japan). The inten- sity representing ADM mRNA was normalized with the intensity of ß-actin band, and the normalized values in various samples were compared.
A
Aldo 1
Aldo 2
Aldo 3
Aldo 4
Normal 1
Normal 2
Normal 3
Normal 4
Non 1
Cortisol 1
Cortisol 2
Carcinoma 1
Carcinoma 2
Carcinoma 3
Carcinoma 4
ADM mRNA
₿ actin mRNA
B
Cortisol 2
Aldo 1
Aldo 3
Pheo 1
Pheo 2
Pheo 3
Pheo 4
Pheo 5
ADM mRNA
₿ actin mRNA
RESULTS
Northern blot analysis showed the expression of ADM mRNA in tumor tissues of 4 aldosterone-producing adeno- mas (Case 1-4), 2 cortisol-producing adenomas (Case 1 and 2), one non-functioning adenoma and three out of 4 adre- nocortical carcinomas (Case 1, 3, and 4) (Fig. 1A). The expression levels of ADM mRNA in aldosterone-producing adenomas were approximately 20%-60% of the levels in normal parts of adrenal glands (Case 1-4) (cortex and medulla). ADM mRNA was highly expressed in pheochro- mocytomas, in particular in Case 1, 3, 4 and 5 (Fig. 1B). The expression levels of ADM mRNA in adrenocortical adeno- mas (a corisol-producing adenoma, Case 2, and aldosterone producing adenomas, Case 1 and 3) were approximately 15%-30% of the levels of these pheochromocytomas (Cases 1, 3, 4 and 5).
On the other hand, IR-ADM was not detectable in the
| IR-ADM (pmol/g ww) | |
|---|---|
| Aldosterone-producing adenoma | |
| Case 1 | ND |
| Case 2 | ND |
| Case 3 | ND |
| Case 4 | ND |
| Normal parts of adrenal gland | |
| Case 1 | 8.46 |
| Case 2 | 8.65 |
| Case 3 | 11.3 |
| Case 4 | 8.52 |
| Non-functioning adenoma | |
| Case 1 | ND |
| Contisol-producing adenoma | |
| Case 1 | ND |
| Case 2 | ND |
| Adrenocortical carcinoma | |
| Case 1 | ND |
| Case 2 | ND |
| Case 3 | ND |
| Case 4 | ND |
| Pheochromocytoma | |
| Case 1 | 0.44 |
| Case 2 | 1.7 |
| Case 3 | 113.1 |
| Case 4 | 198.2 |
| Case 5 | 73.0 |
ND = not detectable (<0.12 pmol/g ww).
tumor tissues of adrenocortical tumors by radioimmunoas- say (<0.12 pmol/g wet weight (ww)) (Table 1). IR-ADM concentrations in the tumor tissues of pheochromocytomas ranged from 0.44 to 198.2 pmol/g ww and those in normal parts of adrenal glands were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) (Fig. 1). We therefore studied IR-ADM con- centrations of other 20 adrenocortical tumors. IR-ADM was detected only in 2 out of 13 aldosterone-producing adeno- mas (1.05 and 0.22 pmol/g ww) and in one out of 5 cortisol- producing adenomas (1.43 pmol/g ww), but not detected in two non-functioning adenomas.
We then studied possible production and secretion of ADM by an adrenocortical adenocarcinoma cell line, SW- 13, in order to clarify whether tumor cells of adrenocortical tumors themselves produce and secrete ADM. ADM mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and its expression level was approximately 50% of the level of a pheochromocytoma Case 3 (Fig. 2). IR-ADM was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/105 cells/24 h = 113.0 ± 4.2 pmol/l, mean ± SEM, n = 4), while IR-ADM concentrations in the unconditioned medium were very low (4.5 ± 0.6 pmol/l), indicating an active secretion of IR-ADM by SW-13 cells. IR-ADM accumulated in the culture medium of SW-13 cells time-dependently (Fig. 3). On the other hand, IR-ADM
SW 13
Pheo 3
ADM mRNA
ß actin mRNA
was not detected in the cell extract of SW-13 cells (<0.09 fmol/105 cells).
Sephadex G-50 column chromatography of the culture medium extract showed a single immunoreactive peak elut- ing in the position of synthetic human ADM-(1-52) (Fig. 4A). Reverse phase HPLC showed two peaks, one peak eluting earlier and one eluting in an identical position to synthetic human ADM-(1-52) (Fig. 4B). ADM with an oxidized methionine, which was generated by incubating ADM with 3% (v/v) H2O2 for 1 h at room temperature, was eluted in the same position as ADM.
DISCUSSION
The present study has shown the expression of ADM mRNA in tumor tissues of various adrenocortical tumors. These findings are compatible with the recent report by Liu
80
IR-ADM (fmol/10 5 cells)
60
40
20
0
4 H
8 H
24 H
32 H
A
2000
Vo
ADM
V
V
IR-ADM (fmol/fraction)
1500
1000
500
0
0
10
20
30
40
50
60
fraction
B
ADM
80
V
80
IR-ADM (fmol/ml)
60
60
ACN (%)
40
40
20
20
0
10
20
40
50
0
0
30
minutes
et al. (10). On the other hand, IR-ADM was not detectable in the tumor tissues of 90% cases of adrenocortical tumors, which confirmed the findings in our previous report (20). This discrepancy between ADM mRNA expression levels and IR-ADM levels in these tumors may be explained by the rapid secretion of ADM without long-term storage after the translation in adrenocortical tumors. To address the hypothesis that ADM is secreted rapidly after the translation from adrenocortical tumors, we have measured IR-ADM levels in the culture medium of SW-13 and in the cell extracts of SW-13 cells. IR-ADM levels in the culture medium of SW-13 were 48.9 + 1.8 fmol/105 cells/24 h, while cellular content of IR-ADM of SW-13 cells was less than 0.09 fmol/105 cells. This finding indicated that SW-13 cells secreted at least 20 times more ADM into the medium for one hour than the amount of their intracellular storage.
Sephadex G-50 column chromatography confirmed the identity of IR-ADM in the culture medium of SW-13.
However, reverse phase HPLC showed a peak eluting ear- lier than ADM, in addition to a peak in the position of human ADM-(1-52). Material eluting earlier on HPLC may represent ADM with some minor modification. Such peaks eluting earlier were also found in the culture medium of DLD-1 colorectal adenocarcinoma cells (16).
The findings on the production and secretion of ADM by SW-13 adrenocortical carcinoma cells suggested that ADM mRNA detected in the tumor tissues of adrenocortical tu- mors is mainly derived from tumor cells themselves. On the other hand, we could not exclude the possibility that ADM mRNA in the vascular endothelial cells and vascular smooth muscle cells in the tumor contributed partly to the expres- sion levels of ADM mRNA in the tumor tissues. It was reported that ADM was produced and secreted by cultured vascular endothelial cells and vascular smooth muscle cells (22,23).
The production and secretion of ADM by adrenocortical tumors support the hypothesis that adrenal cortical cells produce and secrete ADM and that ADM may act on the secretion of adrenal steroid hormones in a paracrine or autocrine fashion. It has recently been reported that ADM mRNA was expressed in the adrenal cortex of rat and mouse (2,5). ADM inhibited aldosterone secretion by dispersed adrenal glomerulosa cells in the rat (32). ADM stimulated corticosterone release from rat adrenal glands by raising blood flow rate, and enhanced aldosterone secretion from rat and human adrenal glands, possibly via the release of cat-
echolamines by adrenal chromaffin cells (1,13). In addition, it was reported that ADM is related to the proliferation of cells (4,31). Another possible pathophysiological role of ADM secreted from adrenocortical tumors may, therefore, be actions on the growth of tumor cells.
It is well-known that adrenal medulla and tumors derived from it produce and secrete various kinds of neuropeptides and vasoactive peptides, such as neuropeptide Y, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) (9,11,17,25). On the other hand, little is known on the production and secretion of regulatory peptides by adrenal cortex and adrenocortical tumors. In this regard, we have previously reported that cerebellin was highly expressed in the tumor tissues of cortisol-producing adrenocortical adenomas (21). In addi- tion, high concentrations of B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were detected in the tumor tissues of adrenocortical adenomas (28,29). Some peptides produced in the adrenal cortex may consti- tute hitherto unknown regulatory system on the production and secretion of adrenal steroid hormones.
ACKNOWLEDGEMENTS
We are grateful to Ms. Kikuchi for her secretarial assistance. This study has been supported partly by a Grant-in-aid for Scientific Research (C) (No. 09670117) from the Ministry of Education, Science, Sports and Culture of Japan (to K. T.), a Grant-in-aid for Brain Science Research from the Ministry of Health and Welfare of Japan (to K. T.), and by the Gonryou Medical Foundation (to K. T.).
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