Endothelin-1 and adrenomedullin enhance the growth of human adrenocortical carcinoma-derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis
GIOVANNA ALBERTIN, GIANNI CARRARO, LUCIA PETRELLI, DIEGO GUIDOLIN, GIULIANO NERI and GASTONE G. NUSSDORFER
Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, I-35121 Padua, Italy Received September 17, 2004; Accepted November 5, 2004
Abstract. The human adrenocortical carcinoma-derived SW-13 cell line is currently used to study the interrelationships occurring between cytokines and growth factors and endothelins (ET) and adrenomedullin (AM). SW-13 cells express either ET-1 and AM or growth factors, and several cytokines stimulate ET-1 and AM release from SW-13 cells. However, neither the morphology and steroid-hormone secretion of SW-13 cells nor the expression of ET and AM receptors and the effects of ET and AM on SW-13 cell growth have been investigated. Electron microscopy showed that SW-13 cells were deprived of the typical organelles involved in steroid-hormone synthesis (i.e. mitochondrial with tubular cristae, smooth endoplasmic reticulum and lipid droplets), their prominent ultrastructural features being rough endo- plasmic reticulum cisternae, free ribosomes and mitochondria with laminar cristae. Accordingly, steroid-hormone secretion was very low: no cortisol was produced and only very small amounts of aldosterone and its precursors were released. No appreciable secretory response to physiological concentrations of ACTH was observed. Reverse transcription-polymerase chain reaction showed the expression of pro ET-1 and proAM genes, as well as detected the mRNAs of only the ET- and AM-receptor subtypes, which are currently thought to mediate the growth-promoting action of these peptides: i.e. the ETA and AM2 receptors. In keeping with these observations, both ET-1 and AM markedly stimulated the growth of SW-13 cells, by enhancing the proliferation and lowering the apoptosis rate. Taken together, our findings allow us to conclude that SW-13 cannot be used for investigating the mechanisms involved in the regulation of steroid-hormone secretion, but are a suitable and useful model to study the role of endogenous
ET and AM systems in the autocrine-paracrine control of human adrenocortical-cell growth.
Introduction
SW-13 cell line has been derived from human adrenocortical carcinoma (1) and is currently used in the experiments dealing with cytokines and growth factors. It has been demonstrated that SW-13 cells secrete FGF-2, TGF-a and PDGF, and their growth is stimulated by FGF-1, FGF-2 and interleukin (IL)-1, thereby suggesting the possibility of an autocrine growth control (2-6).
More recent investigations provided evidence that SW-13 cells can be used as a model to study the interrelationships occurring between inflammatory cytokines and endothelin (ET) and adrenomedullin (AM) systems in the control of human adrenocortical-cell growth. SW-13 cells were found to express ET-1 and AM (7,8) and to secrete sizeable amount of ET-1 and AM in the culture medium. TNF-a increases the release of both peptides, while interferon-y and IL-18 specifically raise the secretion of ET-1 and AM, respectively (9,10).
In light of finding indicating that ET-1 and AM exert a notable stimulating action on the growth of adrenocortical cells (11,12), the possibility cannot be ruled out that the up- regulation of ET-1 and AM secretion may at least partly mediate the growth-promoting action of cytokines and growth factors on SW-13 cells. Obviously, this hypothesis requires that ET-1 and AM receptors are expressed in SW-13 cells, and the present study was undertaken to address this issue.
Materials and methods
Reagents. Human ET-1 was purchased from Neosystem Laboratoires (Strasbourg, France), and human AM1-52 from Phoenix Pharmaceuticals (Belmont, CA). [3H]thymidine was provided by Perkin-Elmer Life Sciences (Boston, MA), and osmium tetroxide, glutaraldehyde and paraformaldehyde were obtained from Serva (Heidelberg, Germany). L-15 Leibovitz culture medium (L-15), fetal calf serum (FCS), phosphate buffered saline (PBS) and all other chemicals and reagents were purchased from Sigma Chemical Company (St. Louis, MO).
Correspondence to: Professor G.G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy
E-mail: gastone.nusdorfer@unipd.it
Key words: SW-13 cells, steroid-hormone secretion, endothelin-1, adrenomedullin, cell proliferation, cell apoptosis
| Primers | Sequence | Annealing temperature | Product size | Accession number |
|---|---|---|---|---|
| pET-1-865-5' | 5'-AAG ACA AAC CAG GTC GGA GA-3' | |||
| pET-1-1009-3' | 5'-TGG AGG CTA TGG CTT CAG AC-3' | 62℃ | 154 bp | XM_043856 |
| ETA-R-1088-5' | 5'-TCT CCA TCT GGA TCC TGT CC-3' | |||
| ETA-R-1437-3' | 5'-ATG AAG AGG GAA CCA GCA AA-3' | 60°℃ | 350 bp | XM_003485 |
| ETB-R-994-5' | 5'-TCT GCT TGC TTC ACT CCG TTC A-3' | |||
| ETB-R-1198-3' | 5'-GCC ACT TCC CGT CTC TGC TTT A-3' | 60℃ | 205 bp | NM_003991 |
| pAM-77-5' | 5'-ACT TCG GAG TTT TGC CAT TGC-3' | |||
| pAM-434-3' | 5'-CAT CCG GAC TGC TGT CTT C-3' | 55°℃ | 358 bp | BC015961 |
| PAM-112-5' | 5'-CAG TGA ATG GGA AGC CTC AT-3' | |||
| PAM-512-3' | 5'-CGG AAT AAC CAG AAG GGT TG-3' | 60℃ | 401 bp | AF035320 |
| CRLC-726-5' | 5'-AGA AGG CGT TTA CTG CAA CAG-3' | |||
| CRLC-996-3' | 5'-TCC CTG TCC AAT TAT GGT CAG-3' | 62°℃ | 271 bp | XM_043055 |
| L1-R-256-5' | 5'-GGT CCT CTT TGC CCT CTA CC-3' | |||
| L1-R-449-3' | 5'-AGG TGT AGT CCA GCG TGA CC-3' | 62°℃ | 194 bp | XM_006764 |
| RAMP1-212-5' | 5'-CAG GAC CAT CAG GAG CTA CA-3' | |||
| RAMP1-480-3' | 5'-CCT ACA CAA TGC CCT CAG TG-3' | 65°℃ | 269 bp | NM_005855 |
| RAMP2-144-5' | 5'-CGC CTC CTC CTT CTG CT-3' | |||
| RAMP2-340-3' | 5'-CTG CTA ATC ATG GCC CAG TC-3' | 57°℃ | 197 bp | XM_008206 |
| RAMP3-153-5' | 5'-AAG GCT TTC GCA GAC ATG AT-3' | |||
| RAMP3-403-3' | 5'-ATA ACG ATC AGC GGG ATG A-3' | 63°℃ | 251 bp | XM_004893 |
| GAPDH-393-5' | 5'-GAA GGC TGG GGC TCA TTT-3' | |||
| GAPDH-530-3' | 5'-CAG GAG GCA TTG CTG ATG AT-3' | 55°℃ | 138 bp | XM_006959 |
| R, receptor. |
Cell culture. SW-13 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured at 37℃ without CO2 in L-15 supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells between the 5th and 10th passage were plated in 35 mm tissue culture dishes (Gibco, Paisley, UK).
Electron microscopy. SW-13 cells were plated at a concen- tration of 5x105 cells/dish, and cultured for 24 h. Monolayers were fixed in 3% phosphate-buffered glutaraldehyde, post- fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in Epon-812. Thin (60-80 nm) sections were cut with an LKB SuperNova ultramicrotome (Reichert- Jung, Vienna, Austria), counterstained with lead-hydroxide, and observed in a Hitachi H-300 transmission electron microscope.
Steroid secretion. SW-13 cells were plated at a concentration of 5x105 cells/dish, and grown for 24 h. Medium was collected, and replaced with a fresh one containing ACTH (10-8-10-6 M), and cells were cultured for other 24 h. Pre-ACTH and post- ACTH culture media were frozen until hormone assay, which
was carried out by either quantitative high pressure liquid chromatography (HPLC) (13,14) or radioimmune assay (RIA) (15), using commercial kits for cortisol and aldosterone purchased from Bouty Diagnostici (Milan, Italy).
Reverse transcription (RT)-polymerase chain reaction (PCR). SW-13 cells were plated at a concentration of 5x105 cells/ dish, grown for 24 h, and then harvested and frozen. Total RNA was extracted from SW-13 cells, as well as from two previously collected and frozen normal human adrenal cortexes, and reverse transcribed to cDNA (16,17). The amplification of the resulting cDNA was carried out as described earlier (16,18). Briefly, in a thermal cycler (PCR Sprint; Hybaid, Ashford, UK), we used an initial denaturation step at 95℃ for 180 sec, followed by 40 cycles at 95℃ for 30 sec, annealing for 180 sec and an extension step at 72℃ for 30 sec. An additional extension step at 72°℃ for 5 min was then performed. As negative control, one PCR was carried out without prior RT of the RNA. Amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydro- genase (GAPDH) was performed as positive control. Detection of the PCR amplification products was carried out by size
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fractionation on 2% agarose gel electrophoresis. The primer sequences, annealing temperature and the expected size of amplicons are shown in Table I.
Cell proliferation and apoptosis. SW-13 cells were plated at a concentration of 5x105 cells/dish, and grown for 24 h. Medium was replaced with a fresh one containing or not ET-1 or AM (10-8 or 10-7 M), and cultured for further 24 h. For proliferation assay, cultures were incubated with [3H]thymidine (1 [Ci/ml) for 20 h, then cells were harvested, washed in PBS, and solubilized in 0.25% trypsin. The radioactivity was counted in a Wallac 1450 Microbeta Trilux Counter (Perkin-Elmer). For apoptosis assay, cultures were fixed in 4% paraformaldehyde, and apoptotic cells were detected by TUNEL assay (In Situ Cell Death Detection kit; Roche Molecular Biochemicals, Mannheim, Germany), as previously described (19).
Statistics. Data were expressed as means ± SEM of 4 or 6 separate experiments, and their statistical comparison was done by ANOVA, followed by the unpaired Student’s t-test.
Results
Electron microscopy showed that SW-13 cells were round and contained a multilobated nucleus with a prominent nucleolus (Fig. 1A). Irregularly-shaped mitochondria displayed laminar cristae (Figs. 1B and 2). Numerous rough endoplasmic reticulum (RER) cisternae were present in the cytoplasm, and many of them were dilated and showed a moderately electron- opaque content (Fig. 1B). Free ribosomes were exceedingly abundant, and a well developed Golgi apparatus was always located near the nucleus (Fig. 2). Neither lipid droplets nor smooth endoplasmic reticulum (SER) profiles could be seen.
HPLC assay demonstrated that SW-13 cells secreted small quantities of 11-deoxycorticosterone, corticosterone, 18- hydroxycorticosterone and aldosterone. ACTH (10-8 M) did
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| pmol/105 cells | Baseline | ACTH |
|---|---|---|
| 11-Deoxycortisol | ND | ND |
| Cortisol | ND | ND |
| 11-Deoxycorticosterone | 5.1±4.0 | 3.7±3.2 |
| Corticosterone | 4.0±3.3 | 5.6±2.5 |
| 18-Hydroxycorticosterone | 5.2±1.5 | 2.6±2.2 |
| Aldosterone | 1.2±1.4 | 1.7±0.6 |
Data are means ± SD (n=4). ND, not detected.
| pmol/105 cells | Baseline | ACTH 10-8 M | ACTH 10-7 M | ACTH 10-6 M |
|---|---|---|---|---|
| Aldosterone | 1.6±0.9 | 1.8±1.2 | 3.3±1.8ª | 3.7±2.6ª |
| Cortisol | ND | ND | ND | ND |
ªp<0.05 from baseline. Data are means ± SD (n = 6). ND, not detected.
not significantly affect steroid-hormone production (Table II). RIA confirmed that SW-13 cells did not produce cortisol, and additionally showed that supramaximal concentrations of ACTH (10-7 or 10-6 M) were able to evoke a 2-fold increase in aldosterone production (Table III).
RT-PCR allowed the detection of pro(p)ET-1 and ETA- receptor mRNAs in SW-13 cells; ETB-receptor expression was not seen (Fig. 3A). SW-13 cells expressed pAM, peptidyl- glycine a-amidating monooxygenase (PAM), L1-receptor, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP)3 mRNAs. The expression of RAMPI and RAMP2 genes was not observed (Fig. 3B). As expected, normal human adrenal cortex expressed either pET-1, pAM and PAM or all ET-1 and AM receptor subtypes (Fig. 3).
Both ET-1 and AM, independently of their concentration, significantly increased the proliferation rate of SW-13 cells (30-40%) (Fig. 4A). Both peptides markedly decreased the apoptosis rate, in a concentration-dependent manner (10-8 M, 56-60%; and 10-7 M, 73-82%) (Fig. 4B).
Discussion
The ultrastructural features of SW-13 cells have been previously studied only occasionally, and especially as far as their cytoskeleton is concerned (20). It is commonly accepted that all steroid hormone-secreting cells (adrenocortical cells, testis Leydig cells, and ovarian granulosa, thecal and lutein
size marker
PET-1
water
ETA
size marker
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ETB
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2
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0
2
0
350-
154
+-205
size marker
GAPDH
water
pAM
water
CPAM
water
size marker
2
b
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0
a b
358→
+401
138→
size marker
CRLR
L1-R
RAMP1
RAMP2
|RAMP3
water
water
size marker
water
water
water
2
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00
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269
194
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251
cells) possess common morphological features, that reflect their ability to synthesize steroid hormones from cholesterol, stored in lipid droplets, through a series of enzymes located on mitochondrial cristae, which are always of tubular type, and SER (11,21). The lack of lipid droplets and SER, and the presence of mitochondria with conventional laminar cristae rule out the possibility that SW-13 cells possess the classic phenotype of steroidogenic cells. On the contrary, the presence of abundant RER and free ribosomes indicates that SW-13 cells are rapidly growing cells, endowed with a very elevated rate of protein synthesis.
In keeping with the morphologic observations, SW-13 cells possess a very low capability of synthesizing adreno- cortical hormones: only aldosterone and its precursors (11- deoxycorticosterone and 18-hydroxycorticosterone) are produced, while cortisol, the main glucocorticoid hormone in humans, is not secreted, indicating that SW-13 are deprived of P450c17 activity (21). Taken together, these findings, along with the unresponsiveness of SW-13 cells to physiological concentrations of the main adrenocortical secretagogue ACTH, allow us to conclude that this cell line is not a suitable model for studying the mechanisms involved in the regulation of steroid-secretion in human adrenocortical cells.
According to the investigations reviewed in the Introduction (7-10), RT-PCR indicates that SW-13 cells express both pET-1 and pAM genes. Moreover, we provide the first evidence that these cells, like human adrenocortical and aldosteronoma cells (22,23), express the PAM gene, a
1750
3H-thymidine incorporation [com/103 cells-min]
proliferation rate
10-8 M
10-7 M
1500-
*
*
*
*
1250
1000
750
C
ET-1
AM
40
apoptosis rate
TUNEL positive cells [%]
30
10ª M
10-7 M
20-
*
*
10-
*
*
0
C
ET-1
AM
finding consistent with their capacity to release sizeable amounts of AM. PAM is involved in the last step of post- translational cleavage of pAM, i.e. the conversion of the inactive glycine-extended AM1-53 to the mature and active AM1-52 (24,25).
It is current knowledge that ET-1 acts on its target cells through two main subtypes of receptors, the ETA and ETB receptors (26), and AM via either LI-receptors or receptors produced by the interaction of CRLR with RAMPs. The interactions of CRLR with RAMPI produces a selective calcitonin gene-related peptide receptor, and that with RAMP2 and RAMP3 two selective AM receptors, named AMI and AM2 receptors, respectively (27,28). ETA and ETB receptors, as well as AMI and AM2 receptors have been previously shown to be expressed in human adrenocortical cells (22,29), and our RT-PCR findings confirm this contention. Evidence has been provided that ETB receptors mainly mediate the secretagogue effect of ET-1 on adreno- cortical cells (18,30), while ETA receptors are involved in the proliferogenic action of ET-1 (31,32). Preliminary findings suggest that the AM2 receptor plays a main role in the growth- promoting action of AM, at least in the rat thymus and the prostate carcinoma cell line DU-145 (33,34). Hence, our present observation that SW-13 cells express only ETA and AM2 (CRLR/RAMP3) receptors strongly suggest that endogenous ET-1 and AM systems are mainly involved in the control of SW-13 cell growth.
In agreement with this contention, we found that both ET-1 and AM stimulate the growth of SW-13 cells, not only by enhancing proliferation, but also by decreasing apoptosis. The former observation is rather expected, inasmuch as ET-1 and AM are known to enhance DNA replication of adreno- cortical cells cultured in vitro (35-37), and the latter one is not surprising for AM, which has been previously found to exert a marked antiapoptotic action on numerous cell types cultured in vitro, including human and rat zona glomerulosa cells (23,38). In contrast, the marked anti-apoptotic effect of ET-1 is rather astonishing, since this peptide, acting via ETB receptors coupled to the nitric oxide (NO) synthase cascade, has been reported to exert a pro-apoptotic action (39). However, there is proof that ETA receptors blunt NO production (39). This consideration makes it likely that in the absence of ETB receptors, the ET-1-mediated activation of ETA receptors may lower NO level in SW-13 cells, thereby leading to a decrease in their basal apoptosis rate.
Taken together, our findings allow us to conclude that SW-13 cells are a suitable model for investigating the involvement of endogenous ET and AM systems in the autocrine-paracrine control of human adrenocortical-cell growth. Moreover, the lack of ETB and AMI receptors in SW-13, if confirmed by analyzing their expression as proteins, could improve our knowledge on the role of ETA and AM2 in the mediation of the biological effects of ET and AM on adrenocortical cells.
Acknowledgements
This study was supported by the Ministero dell’Università e Ricerca Scientifica e Tecnologica (MURST) Grant FIRB No. RBAU-01-PPBS to GGN. The authors are indebted to Miss Alberta Coi for her outstanding secretarial support.
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