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Expression of urotensin II and its receptor in adrenal tumors and stimulation of proliferation of cultured tumor cells by urotensin II
Kazuhiro Takahashi ª,*, Kazuhito Totsune b, Osamu Murakami b, Zenei Arihara b, Takao Noshirod, Yutaka Hayashi℃, Shigeki Shibahara ª
a Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
b Department of Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan
” Department of Pediatric Oncology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan d Department of Medicine, Sano Ishikai Hospial, Sano, Tochigi 327-0832, Japan
Received 5 August 2002; accepted 23 September 2002
Abstract
Urotensin II is a potent vasoactive peptide, which was originally isolated from fish urophysis. We studied expression of urotensin II and its receptor mRNAs in the tumor tissues of adrenocortical tumors, pheochromocytomas and neuroblastomas. Effects of exogenously added urotensin II on cell proliferation were studied in a human adrenocortical carcinoma cell line, SW-13 and a human renal cell car- cinoma cell line, VMRC-RCW. The reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of urotensin II and its receptor mRNAs in all the samples examined; seven pheochromocytomas, nine adrenocortical adenomas (four with primary aldostero- nism, four with Cushing syndrome and one with non-functioning adenoma), four adrenocortical carcinomas, one ganglioneuroblastoma and five neuroblastomas, as well as four normal portions of adrenal glands (cortex and medulla). Urotensin II-like immunoreactivity was detected in one of eight adrenocortical adenomas, two of four adrenocortical carcinomas, one of six pheochromocytomas, and one of five neuroblastomas by radioimmunoassay, but not in normal portions of adrenal glands (detection limit; 0.2 pmol/g wet weight). Treatment with urotensin II for 24 h significantly increased number of SW-13 cells (at 10-8 and 10-7 mol/l) and VMRC-RCW cells (at 10-8 mol/l). These findings raise the possibility that urotensin II may act as an autocrine/paracrine growth stimulating factor in adrenal tumors.
C) 2003 Elsevier Science Inc. All rights reserved.
Keywords: Urotensin II; Adrenal; Tumors; Pheochromocytoma; Neuroblastoma; Adrenocortical; Growth
1. Introduction
Urotensin II (U-II) is a “somatostatin-like” cyclic peptide which was originally isolated from fish urophysis [12]. The reverse pharmacological approach has shown that human U-II is an agonist for the orphan receptor GPR14 [2]. U-II is a potent vasoconstrictor peptide and its potency of vasocon- striction is one order of magnitude greater than endothelin-1 [2,7]. On the other hand, U-II elicited vasodilatation in some arteries, possibly through the release of endothelium-derived hyperpolarizing factor and nitric oxide [3,16]. In addition to vasoactive effects, this peptide has a positive inotropic action [13] and stimulates proliferation of vascular smooth muscle cells [14,27,28]. Furthermore, it inhibited insulin release from the perfused rat pancreas [15].
U-II is expressed in various human tissues including adrenal glands [2,8,25]. U-II-like immunoreactivity is present in human plasma and urine [8,25]. It is well known that neural crest-derived tumors, such as pheochromocy- tomas, neuroblastomas and ganglioneuroblastomas, express a variety of neuropeptides, vasoactive peptides and their receptors [1,9,21,22]. Moreover, adrenocortical tumors ex- press some peptides, for example, adrenomedullin and endothelin-1 [17,19,24]. We have shown that U-II and U-II receptor mRNAs are expressed in human tumor cell lines of various origins, including SW-13 human adrenocortical carcinoma cells [18,20]. There has been, however, no re- port on expression of U-II and U-II receptor in the tumor tissues of adrenal tumors, neuroblastomas and ganglioneu- roblastomas. We, therefore, studied expression of U-II and U-II receptor in the tumor tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and the presence of U-II-like immunoreactivity by radioimmunoassay. Because
* Corresponding author. Tel .: +81-22-717-8116; fax: +81-22-717-8118. E-mail address: ktaka-md@mail.cc.tohoku.ac.jp (K. Takahashi).
U-II stimulates proliferation of vascular smooth muscle cells [14,27,28], we also studied effects of U-II on prolif- eration of SW-13 adrenocortical carcinoma cells as well as VMRC-RCW renal cell carcinoma cells.
2. Methods and materials
2.1. Tissues
This study has been approved by the Ethics Committee of Tohoku University School of Medicine. Tumor tis- sues comprising seven benign pheochromocytomas, nine adrenocortical adenomas (four with primary aldosteronism, four with Cushing syndrome and one non-functioning adenoma), four adrenocortical carcinomas, one gan- glioneuroblastoma and five neuroblastomas were obtained at surgery. Morphologically normal portions of adrenal glands (cortex and medulla) were obtained at surgery from four patients with primary aldosteronism. Informed consent was obtained from each subject, or parents of the infant subjects in cases of neuroblastoma and gan- glioneuroblastoma. The tissues were immediately frozen and stored at -80℃ prior to extraction of RNA and peptides.
2.2. RT-PCR
Total RNA was extracted from tissues by the guanidine thiocyanate-cesium chloride method. RT-PCR analysis of U-II and U-II receptor mRNAs was performed as previously reported [20,25]. The sense primer for U-II was 5’-CTTT- CAACTCTCAGCACCTCAT-3’ (nucleotide numbers 122/ 143) and the anti-sense primer was 5’-CCTAGTTTTTCTC- CACACTGTT-3’ (complementary to 485/506) (Gene Bank accession no. AF104118). The sense primer for U-II receptor (human GPR14) was 5’-CCCCAACGCAACCCTCAACA-3’ (nucleotide numbers 81-100) and the antisense primer was 5’-GGTCGCGGTAGTTCCTGGTGA-3’. (complemen- tary to 953-973) (Gene Bank accession no. AF140631). Expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was examined as an internal control. Am- plification products were run on a 5% polyacrylamide gel electrophoresis, stained with ethidium bromide, visualized with an ultraviolet transluminator, and photographed.
2.3. Peptide extraction and radioimmunoassay
Four normal portions of adrenal glands (cortex and medu- lla) and tumor tissues of six pheochromocytomas, seven adrenocortical adenomas (four with primary aldosteronism,
(A)
adrenal 1
adrenal 2
adrenal 3
adrenal 4
pheo 1
pheo 2
pheo 3
pheo 4
pheo 5
pheo 6
pheo 7
NB 1
NB 2
NB 3
NB 4
NB 5
NB 6
M
400
U-II
900
U-Il receptor
1000
(bp)
G3PDH
(B)
adenoma 1 (PA)
adenoma 2 (PA)
adenoma 3 (PA)
adenoma 4 (PA)
adenoma 5 (Cushing)
adenoma 6 (Cushing)
adenoma 7 (Cushing)
adenoma 8 (Cushing)
adenoma 9 (non)
carcinoma 1
carcinoma 2
carcinoma 3
carcinoma 4
M
400
U-II
900
U-Il receptor
1000
(bp)
G3PDH
two with Cushing syndrome and one non-functioning ade- noma), four adrenocorical carcinomas, one ganglioneurob- lastoma and five neuroblastomas were available for the peptide extraction. Tissues were extracted, as reported pre- viously [23]. Briefly, the tissue (approximately 750 mg) was boiled in 2 ml of 1 mol/l acetic acid for 10 min. Eight milliliter of 50% methanol in 0.5 mol/l acetic acid was added to each sample and the tissue was homogenized. The homogenate was centrifuged by 15,000 x g for 30 min. The supernatant was separated, dried by air, reconstituted in assay buffer (0.1 mol/l phosphate buffer, pH 7.5 containing 0.1% (w/v) bovine serum albumin, 0.2% (v/v) Triton X-100 and 0.1% (w/v) sodium azide) and assayed, as previously reported [20].
2.4. Cell culture and cell proliferation
SW-13 human adrenocortical carcinoma cells [6] were obtained from the Health Science Research Resources Bank of Japan Health Sciences Foundation (Osaka, Japan), and cultivated at 37℃ under 5% CO2 in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum. VMRC-RCW renal cell carcinoma cells were obtained from the Cancer Cell Repository, Institute of Development, Ag- ing and Cancer Tohoku University, and cultivated at 37 ℃ under 5% CO2 in RPMI medium 1640 supplemented with 10% fetal bovine serum [18].
The cells were seeded in 48-well plates at a density of 1 × 104 cells/ml (0.3 ml/well), and were grown for 72 h. Medium was then replaced by phenol-red-free Leibovitz’s L-15 medium supplemented with 0.5% fetal bovine serum (SW-13 cells) or phenol-red-free RPMI medium 1640 sup- plemented with 0.5% fetal bovine serum (VMRC-RCW cells), and further cultivated for 48 h. Medium was then replaced by phenol-red-free Leibovitz’s L-15 medium or RPMI medium 1640 with 0.5% fetal bovine serum con- taining human U-II (Peptide Institute, Osaka, Japan), and cultivated for 24 h.
Human adrenomedullin (Peptide Institute) was used as a control, because this peptide has a modulatory action on growth in various types of cells. Adrenomedullin stimulates proliferation of certain types of cells such as Swiss 3T3 cells [29] and cultured retinal pigment epithelial cells [26], whereas it inhibited proliferation of other types cells, such as vascular smooth muscle cells [5] and renal mesangial cells [4]. The cell number was assessed by using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), as previously reported [26]. Statistical analysis was performed by one-way analysis of variance followed by Fisher’s protected least significant difference.
3. Results
The RT-PCR analysis showed expression of U-II and U-II receptor mRNAs in all the samples examined; seven
pheochromocytomas (Fig. 1A), nine adrenocortical adeno- mas (four with primary aldosteronism, four with Cushing syndrome and one non-functioning adenoma) (Fig. 1B), four adrenocortical carcinomas (Fig. 1B), one ganglioneu- roblastoma and five neuroblastomas (Fig. 1A), as well as four normal portions of adrenal glands. No apparent differ- ence was noted in the expression of U-II and U-II receptor mRNAs between normal portions of adrenal glands and tumors, between pheochromocytomas and adrenocortical tumors, or between adrenocortical adenomas and carcino- mas. Negative controls using water instead of total RNA showed no bands in the RT-PCR of U-II and U-II receptor mRNAs (data not shown).
U-II-like immunoreactivity was detected in one (one case of Cushing syndrome) of eight adrenocortical adenomas, two of four adrenocortical carcinomas, one of six pheochromo- cytomas, and one neuroblastoma of five neuroblastomas/one ganglioneuroblastoma by radioimmunoassay (detection limit; 0.2 pmol/g wet weight) (Fig. 2). On the other hand, U-II-like immunoreactivity was not detected in four normal portions of adrenal glands (cortex and medulla) (Fig. 2).
Treatment of SW-13 cells with U-II (10-8 and 10-7 mol/l) for 24h increased cell number significantly (125 and 220% of control, respectively), whereas treatment with adrenomedullin increased cell number significantly only at a higher concentration (10-7 mol/l) (F(5, 30) = 50.925,
1.5
Ull-like immunoreactivity (pmol/g wet weight)
1.0
0.5
0.0
Normal adrenals
Cushing
PA
Non
carcinomas
Pheochromocytomas
NB/GNB
Adrenocortical tumors
200
Cell number
(% of control)
**
*
100
0
(A)
control
10-9
10-8
10-7
10-8
10-7
mol/l
U-II
AM
150
*
100
Cell number (% of control)
*
50
0
control
10-9
10-8
10-7
10-8
10-7
mol/l
(B)
U-II
AM
P < 0.0001) (Fig. 3A). U-II also stimulated proliferation of VMRC-RCW human renal carcinoma cells at a con- centration of 10-8 mol/l but not at 10-7 mol/l (F(5, 30 = 10.618, P < 0.0001) (Fig. 3B). Contrary to SW-13 cells, adrenomedullin decreased number of VMRC-RCW cells at a concentration of 10-7 mol/l (Fig. 3B).
4. Discussion
The present study has shown, for the first time, the ex- pression of U-II and U-II receptor in the tumor tissues
of adrenocortical adenomas, adrenocortical carcinomas, pheochromocytomas, a ganglioneuroblastoma and neuro- blastomas. Exogenously added U-II stimulated cell prolifer- ation not only in SW-13 adrenocortical carcinoma cells but also in VMRC-RCW renal cell carcinoma cells. We have previously reported expression of U-II and U-II receptor mRNAs in various human tumor cell lines [18,20]. Taken together, these results raise the possibility that U-II may act as an autocrine or paracrine growth stimulator in tumors. U-II secreted by the tumor may also promote angiogenesis in the tumor tissue because U-II stimulated proliferation of vascular smooth muscle cells [14,27,28]. Previous studies
have shown that human U-II has potent vasoactive proper- ties [2,3,7,16]. U-II is expressed in various human organs and is also present in plasma and urine [2,8,25]. U-II has therefore been supposed to regulate the circulation as an autocrine/paracrine factor and/or a circulating hormone. The present study has suggested novel pathophysiological roles of U-II in tumors.
Another possible role of U-II secreted from normal adrenal and adrenal tumors is an autocrine or paracrine action on the secretion of steroid hormones and cate- cholamines. It is known that some vasoactive peptides and neuropeptides affect the secretion of steroid hormones and/or catecholamines from normal adrenal and adrenal tumors [10,11]. For example, endothelin-1 stimulates the aldosterone secretion from the zona glomerulosa cells of adrenal cortex [11,19]. It is therefore plausible that U-II may also modulate the secretion of steroid hormones and/or catecholamines as an autocrine/paracrine factor. On the other hand, there was no noticeable difference in the expres- sion of U-II and U-II receptor between cortisol-secreting adenomas (Cushing syndrome) and aldosterone-secreting adenomas (primary aldosteronism), or between pheochro- mocytomas and adrenocortical tumors. More quantitative methods to detect mRNA expression levels, however, may reveal some differences in the expression levels of U-II and U-II receptor mRNAs in these tumor tissues.
We have previously reported that U-II and U-II receptor mRNAs were expressed in all samples of normal human tissues examined, including brain, pituitary, heart, adrenal, kidney and colon [25]. Two exons are located between the primers for U-II [20,25]. Contamination of genomic DNA into the RNA samples therefore does not explain the ubiq- uitous expression of U-II mRNA, although we could not deny this possibility completely in the RT-PCR of U-II receptor. Our previous report and the present study indicate that the genes of U-II and U-II receptor are ubiquitously expressed in various normal and tumor tissues, suggesting that U-II may be involved in the fundamental physiology of cells, such as survival. It is noteworthy, however, that U-II-like immunoreactivity was only detected in the tumor tissues (5 of 25 tumor tissues), but not in normal portions of adrenal glands, suggesting an increased production of U-II by tumor cells. This discrepant results may be due to a higher sensitivity of RT-PCR method than radioimmunoas- say. Moreover, U-II-like immunoreactivity was not detected by radioimmunoassay in brain tissues or cardiac tissues obtained at autopsy, although U-II mRNA was expressed in these samples (our unpublished observations).
The highest concentration of U-II-like immunoreactivity was found in one adenoma with Cushing syndrome, fol- lowed by one adrenocortical carcinoma, and these levels were about 1 pmol/g wet weight. The concentration of ex- ogenously added U-II that stimulates the proliferation of SW-13 cells or VMRC-RCW cells appears to be higher than these tumor tissue concentrations. It may be possible, however, that local concentrations of U-II around tumor
cells may be high enough to stimulate cell proliferation in some cases of tumors, because the tumor tissues contain various types of cells in addition to tumor cells. On the other hand, we could not deny the possibility that U-II-like immunoreactivity or U-II mRNA detected in the tumor tis- sues was partly derived from normal cells, such as vascular endothelial cells, because cultured vascular endothelial cells expressed U-II mRNA (our unpublished observations).
A higher concentration of U-II (10-7 mol/l) had no sig- nificant effects on number of VMRC-RCW cells. Watanabe et al. [28] reported that U-II stimulated on [3H]thymidine in- corporation into vascular smooth muscle cells, with a maxi- mal effect at a concentration of 50 nmol/l. They also showed that higher concentrations of U-II decreased [3H]thymidine incorporation significantly, possibly due to cytotoxic effects. A higher concentration of U-II (10-7 mol/l) may also be cy- totoxic to VMRC-RCW cells in our study. Adrenomedullin stimulated proliferation of SW-13 cells, but inhibited pro- liferation of VMRC-RCW cells. This is consistent with previous reports showing that effects of adrenomedullin on cell proliferation were different depending on cell types or perhaps on experimental conditions [4,5,26,29].
It is well known that adrenal medulla, pheochromocy- tomas, neuroblastomas and ganglioneuroblastomas express many types of neuropeptides and vasoactive peptides [1,9]. There is accumulating evidence showing that adrenocortical tumors secrete some peptides such as adrenomedullin and endothelin-1, which affects the cell growth and/or the steroid hormone secretion [11,17,19,24]. In the present study, U-II and U-II receptor mRNAs are expressed in adrenocortical tumors, which is in accord with the hypothesis that U-II also belongs to such adrenocortical regulatory peptides.
Acknowledgments
This work was supported in part by Grants-in-aid for Sci- entific Research (B) (no. 13470030) and (C) (no. 13671094), and a Grant-in-aid for Scientific Research on Priority Areas (A) (no. 13035005) from the Ministry of Education, Science, Sports and Culture of Japan, by Grants for the Renal Ane- mia Research (2000 and 2001), by a Research Grant from the HIROMI Medical Research Foundation (2001) and by a Research Grant from the Intelligent Cosmos (2002).
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