Neuropeptides B and W enhance the growth of human adrenocortical carcinoma-derived NCI-H295 cells by exerting MAPK p42/p44-mediated proliferogenic and antiapoptotic effects
PAOLA G. ANDREIS1, MARCIN RUCINSKI3, GIULIANO NERI1, MARIA TERESA CONCONI2, LUCIA PETRELLI1, PIER PAOLO PARNIGOTTO2, LUDWIK K. MALENDOWICZ3 and GASTONE G. NUSSDORFER1
1Department of Human Anatomy and Physiology, Section of Anatomy, 2Department of Pharmaceutical Sciences, University of Padua, I-35121 Padua, Italy; 3Department of Histology and Embryology, Poznan School of Medicine, PL-60781 Poznan, Poland
Received September 9, 2005; Accepted September 29, 2005
Abstract. Neuropeptides B and W (NPB and NPW) are endo- genous ligands of two G protein-coupled receptors, named GPR7 and GPR8. GPR7 and GPR8 are expressed in the adrenal cortex, and there is evidence that NPB and NPW stimulate glucocorticoid secretion from human adrenocortical cells by activating protein kinase (PK) A and PKC signaling. To gain insight into the role of NPB and NPW in human adrenal functional regulation, we have investigated their effects on the secretion and growth of the human adrenocortical carcinoma-derived NCI-H295 cell line. NCI-H295 cells were found to express both GPR7 and GPR8 mRNAs, but neither NPB nor NPW (up to 10-6 M) affected their secretory activity. In contrast, both peptides (from 10-10 to 10-6 M) enhanced the growth of NCI-H295 cells, by raising their proliferative activity and lowering their apoptotic deletion rate. NPB and NPW (10-6 M) stimulated tyrosine kinase (TK) and mitogen-activated PK (MAPK) p42/p44 activities in NCI-H295 cells. Both these effects were blocked by the TK inhibitor tyrphostin-23, while the MAPK p42/p44 inhibitor PD-98059 annulled only MAPK p42/p44 activation. The growth-stimulating effect of 10-6 M NPB and NPW were not affected by either the PKA and PKC inhibitors H-89 and calphostin-C or the MAPK p38 antagonist SB-293580, but were abolished by both tyrphostin-23 and PD-98059. Taken together, our findings allow us to conclude that GPR7 and GPR8 expressed in NCI-H295 cells: i) are, at variance with those present in normal human adrenocortical cells, uncoupled to PKA- and PKC-dependent cascades, thereby explaining the absence of any secretory response to
Correspondence to: Professor Gastone G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Via Gabelli 65, I-35121 Padua, Italy E-mail: gastone.nusdorfer@unipd.it
Key words: neuropeptides B and W, GPR7 and GPR8, NCI-H295 cells, cell proliferation, cell apoptosis, tyrosine kinase, mitogen- activated protein kinases
NPB and NPW; and ii) are coupled to the TK-dependent MAPK p42/p44 signaling, whose activation mediates the proliferogenic and antiapoptotic effect of NPB and NPW.
Introduction
Neuropeptides B and W (NPB and NPW) are 29- and 30- amino acid regulatory petides, which are identified as endo- genous ligands of the G protein receptors (GPRs) 7 and 8 (1,2). NPB and NPW bind and activate both receptors, but with contrasting affinities: NPB, GPR7>GPR8; and NPW, GPR8>GPR7 (3,4). GPR7 and GPR8 are expressed in human brain, especially in the hypothalamic suprachiasmatic, supra- optic, dorsomedial, ventromedial, and paraventricular nuclei (5), and NPW immunoreactivity has been detected in the rat hypothalamus and pituitary gland (6).
Findings indicate that NPB and NPW are involved in the central regulation of feeding behavior and energy homeostasis (7-11). Evidence has been accumulated that other peptides playing a similar role are able to control glucocorticoid secretion, acting at both the central and the peripheral branch of the hypothalamic-pituitary-adrenal axis: neuropeptide-Y (for review see ref. 12), leptin (13-17), orexins (18-22), chole- cystokinin (23-27), and beacon (28-34). Accordingly, NPB and NPW, although being unable to affect ACTH release from dispersed rat anterior-pituitary cells, were found to raise the blood level of corticosterone when administered intra- cerebroventricularly, thereby suggesting a hypothalamic locus of action (CRH and/or AVP release) (7,10). Moreover, NPB and NPW were found to enhance corticosterone secretion from cultured rat (35) and cortisol release from dispersed human adrenocortical cells (36).
Of great interest, Mazzocchi and co-workers (36) detected GPR7 and GPR8 mRNA expression in both zona glomerulosa (ZG) and zona fasciculata-reticularis cells of the human adrenals, but were unable to evidence any effect of NPB and NPW on aldosterone secretion from dispersed ZG cells, thus raising question on the biological role of these receptors in ZG cells. According to the cell migration theory (for review see ref. 37), ZG in mammals is the cambium layer involved in
| Primer | Sequence | Annealing temperature (℃) | Product size (bp) |
|---|---|---|---|
| GPR7 | |||
| Sense | 5'-CTTGGAGAGCTGGAAACGAG-3' | ||
| Antisense | 5'-GGACACAGATGGTGGACACG-3' | 60 | 898 |
| GPR8 | |||
| Sense | 5'-GCCACTGCCGTTCCTCTAT-3' | ||
| Antisense | 5'-GATGATGGGGGTGATGATGG-3' | 60 | 747 |
| GAPDH | |||
| Sense | 5'-CCCTTCATTGACCTCAACTA-3' | ||
| Antisense | 5'-CCAGTGAGCTTCCCGTTCA-3' | 58 | 585 |
adrenocortical cell renewal. Previous investigations showed that NPB and NPW stimulate proliferation of cultured rat adrenocortical cells (35). Hence, the involvement of GPR7 and GPR8 located in the ZG in the regulation of human adrenal growth appears to be an appealing possibility.
To address this issue, we decided to explore the possible growth effects of NPB and NPW on human carcinoma-derived NCI-H295 cells, whose physiology is currently thought to reflect that of normal human adrenocortical cells (for review see ref. 38). Our decision to use this cell line was due to the following reasons: i) the current increasing difficulty to obtain normal human adrenal cortexes; ii) human adrenocortical cells in primary culture have a limited life-span and rapidly shift from a steroid-secreting differentiated phenotype to a dedifferentiate fibroblast-like one; and iii) primary cultures are always contaminated by the presence of stromal cells (fibroblasts and endothelial cells).
Materials and methods
Reagents. NPB and NPW were purchased from Phoenix Pharmaceuticals (Belmont, CA), and Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium from Gibco (Paisley, UK). Medium 199 was obtained from Difco (Detroit, MI), and anti-p42/44 antibody from Santa Cruz Biotechnology (Santa Cruz, CA). H-89, calphostin-C, tyrphostin-23, PD- 98059 and SB-203580 were provided by Biomol (Milan, Italy). Dibutyryl-cyclic adenosine 3’,5’ monophosphate (db-cAMP), fetal calf serum (FCS), bovine serum albumine (BSA), phosphate-buffered saline (PBS), 5’-bromo-2’-deoxy- uridine (BrdU), 4’,6-diamino-2-phenylindole dilactate (DAPI), myelinic basic protein (MBP), and all other chemicals and laboratory reagents were purchased from Sigma Aldrich Corp. (St. Louis, MO).
NCI-H295 cell cultures. NCI-H295 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 75 cm2 flasks in DMEM-F12 supple- mented 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 dish (Gibco), and cultured at 37°℃ without CO2 for 24 h.
Electron microscopy. NCI-H295 cells were plated at a concentration of 5x105 cells/dish, and cultured for 24 h. Monolayers were fixed in 3% phosphate-buffered glutar- aldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in Epon-812 (39). Thin (60-80 nm) sections were cut with an LKB SuperNova ultramicrotome (Reichert-Jung, Vienna, Austria), counter- stained with lead-hydroxide, and observed in a Hitachi H-300 transmission electron microscope.
Reverse transcription (RT)-polymerase chain reaction (PCR). NCI-H295 cells were plated at a concentration of 5x105 cells/ dish, grown until confluency, and then harvested and frozen. Total RNA was extracted, and reverse transcribed to cDNA (40,41), and the amplification of the resulting cDNA was carried out as described earlier (42,43), using human GPR7 and GPR8 specific primers (36). Briefly, in a thermal cycler 489 DNA TC (Perkin-Elmer Life Sciences, Milan, Italy), we used an initial denaturation step at 95℃ for 180 sec, followed by 34 cycles of 94℃ for 60 sec, 60℃ for 120 sec, and 72℃ for 50 sec. An additional extension step at 72℃ for 5 min was then carried out. As negative control, one PCR was per- formed without prior RT of the RNA. Amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was carried out as positive control (36). Detection of the PCR amplification products was first performed by size fractionation on 2% agarose gel electrophoresis. After purification (QIA-Quick PCR purification kit; Qiagen, Hilden, Germany), amplicons were identified by sequencing (Alf sequencer; Pharmacia Biotech, Freiburg, Germany). The primer sequences, annealing temperature and the expected size of amplicons are shown in Table I.
Steroid-hormone secretion. NCI-H295A cells were plated at a concentration of 5x105 cells/dish, and grown until con- fluency. Medium was collected, and replaced with a fresh one containing NPB or NPW (from 10-12 to 10-6 M) or db- CAMP (10-8 M), and cultured for other 3 h. Pre-incubation and post-incubation culture media were frozen until hormone assay, which was carried out by quantitative high pressure liquid chromatography (HPLC) (44,45). Cortisol was also measured by radioimmune assay (RIA), using a cortisol RIA
N
*
*
N
*
*
A
M
M
M
*
G
G
N
N
B
kit (IRE-Sorin, Vercelli, Italy): sensitivity, 90 pmol/l; intra- and interassay CVs, 5.8 and 7.9% respectively.
Cell proliferation and apoptosis. NCI-H295A cells were plated at a concentration of 105 cells/dish, and grown until subconfluency. For apoptosis assay, cultures were grown in DMEM-F12 supplemented with 2.5% FCS (instead of 10%). Medium was replaced with a fresh one, and cultures were incubated for 24 h as follows: i) NPB or NPW (from 10-12 to 10-6 M) or without any peptide; and ii) H-89 (10-5 M), calphostin-C (10-5 M), tyrphostin-23 (10-5 M), PD-98059 (10-4 M) or SD-203580 (10-4 M) alone and in the presence of 10-6 M NPB or NPW. In the cell-proliferation experiments, during the last 12 h of incubation, BrdU was added to the culture medium at a final concentration of 10 uM (21,22). Cultures were fixed in 4% paraformaldehyde for 30 min. BrdU-positive (S-phase) cells were detected using the Cell
Proliferation Kit of Amersham Pharmacia (Aylesbury, UK) (46,47), and TUNEL-positive (apoptotic) cells using the In Situ Cell Death Detection Kit of Roche Molecular Biochemicals (Mannheim, Germany), as previously detailed (48). The percentages of positive cells were evaluated by counting 3000 cells per culture dish.
Tyrosine kinase (TK) and MAPK p42/p44 activity. NCI-H295 cells were plated at a concentration of 105 cells/dish, and grown until confluency. Cells were harvested, and put in Medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose, containing 5 mg/ml BSA. They were incubated (5x105 cells/ml) with 10-6 M NPB and NPW or without any peptide (baseline value). Other preparations were pre-incubated for 30 min with 10-5 M tyrphostin-23 or 104 M PD-98059, and then exposed to NPB or NPW. The incubation was carried out in a shaking bath at 37℃ for 15 min, and was stopped by two quick washes with ice-cold PBS. NCI-H295 cells were lysed and homogenized, as previously detailed (47,49,50). Homo- genates were centrifuged at 4℃ at 800 x g for 10 min, and then at 12000 x g for 15 min. Supernatants were removed, the protein concentration was determined by the Lowry method, and then were stored at -80℃. TK activity was assayed, using poly(Glu4,Tyr1) as substrate (51). MAPK p42/p44 activity was measured using an anti-MAPK p42/p44 polyclonal antibody and MBP as substrate (52).
Statistics. Data were expressed as means ± SEM of six separate experiments. The statistical comparison of results was done by ANOVA, followed by Duncan’s multiple range test.
Results
Electron microscopy showed that NCI-H295 cells contained numerous elongated mitochondria provided with lamellar cristae, several arrays of rough endoplasmic reticulum (RER) cisternae, abundant free ribosomes, and a well-developed Golgi apparatus. Smooth endoplasmic reticulum (SER) profiles and lipid droplets were not seen (Fig. 1).
RT-PCR evidenced the expression of both GRP7 and GRP8 mRNAs in NCI-H295 cells. No amplification of PCR mixture with water instead of RNA or without prior RT of RNA was observed, thereby confirming the specificity of the reaction (Fig. 2).
RIA demonstrated that neither NPB nor NPW from 10-12 to 10-6 M affected cortisol secretion from NCI-H295 cells, which in contrast were responsive to db-cAMP (Fig. 3). Quantitative HPLC assay showed that NPB and NPW (10-6 M) did not elicit significant changes in the basal secretion of all post-pregnenolone steroid hormones from NCI-H295 cells (Table II).
NPB and NPW concentration-dependently raised the proliferation rate of NCI-H295 cells, minimal and maximal effective concentrations being 10-10 M (about 2-fold increase) and 10-8/10-6 M (about 3/3.6-fold increase) (Fig. 4). NPB and NPW (10-6 M) decreased the number of TUNEL-positive NCI-H295 cells (Fig. 5). Quantitative assay demonstrated a significant lowering in the apoptosis rate with NPB con- centrations of 10-8 and 10-6 M (about 30/45% decrease) and a NPW concentration of 10-6 M (about 49% decrease) (Fig. 6).
size marker
GPR7
water
GPR8
water
GAPDH
water
898
+747
585→
10
cortisol secretion [nmol/mg protein · h]
**
8-
A, 10-12M
B, 10*10M
6.
C, 10™ªM
D, 10*6M
2
T
T
T
T
T
T
T
T
T
1-
0
A
B
C
D
A
B
C
D
bsl
NPB
NPW
db-cAMP
| pmoles/mg protein h | Baseline | NPB | NPW |
|---|---|---|---|
| Progesterone | 503±162 | 601±190 | 484±151 |
| 11-Deoxycortisol | 106±38 | 118±35 | 135±36 |
| Cortisol | 1602±595 | 1409±512 | 1585±606 |
| 11-Deoxycorticosterone | 96±30 | 85±28 | 106±36 |
| Corticosterone | 358±115 | 447±138 | 401±119 |
| 18-Hydroxycorticosterone | 309±125 | 262±108 | 333±120 |
| Aldosterone | 86±31 | 95±36 | 78±30 |
| Values are means ± SD (n=6). |
NPB and NPW (10-6 M) enhanced basal TK activity in NCI-H295 cells (about 82% increase), and the effect was abrogated by tyrphostin-23, but not PD-98059 (Fig. 7).
50
proliferation rate [% of BrdU-positive cells]
40-
A, 10-12M B, 10*1ºM C, 10ªM D, 10M
**
**
**
**
30-
*
20-
*
T
T
10-
T
T
0
A
B
C
D
A
B
C
D
bsl
NPB
bsl
NPW
A
B
C
Likewise both peptides (10-6 M) raised MAPK p42/p44 activity (about 2.9-fold increase), and the effect was annulled by either tyrphostin-23 or PD-98059 (Fig. 8).
The NPB- and NPW (10-6 M)-induced rise in the pro- liferation rate and decrease in the apoptosis rate were abrogated by both tyrphostin-23 and PD-98059, and unaffected by H-89, calphostin-C (Figs. 9 and 10) and SB-203580 (Fig. 11). Neither tyrphostin-23 nor PD-98059 were per se able to alter basal parameters (Figs. 9 and 10).
apoptosis rate [% of TUNEL-positive cells]
25-
A, 10’12M B, 10*10M C, 10ªM D, 10 M
20-
15
*
10-
**
**
5-
A
B
C
D
A
B
C
D
bsi
NPB
bsl
NPW
350
TK activity [fmol Pi/mg protein · min]
**
**
300-
**
A, controls
**
B, tyrphostin-23
C, PD-98059
250-
200-
a
150-
T
T
T
a
100
A
B
C
A
B
C
A
B
C
baseline
NPB
NPW
Discussion
Our study shows that NCI-H295 cells, like human adreno- cortical cells (36), express both GPR7 and GPR8, indicating that they could be a suitable model for investigating the adrenocortical effect of NPB and NPW in humans. However, in contrast to human adrenocortical cells (36), NCI-H295 cells do not display any sizeable secretory response to NPB and NPW, although exhibiting a normal glucocorticoid (cortisol) response to db-cAMP, the cell membrane permeable form of cAMP, the main mediator of the ACTH secretagogue effect (37). This observation, along with the demonstration that
ERK1/2 activity [fmol Pi/mg protein · min]
700-
**
**
600-
A, controls
B, tyrphostin-23
C, PD-98059
500-
400-
300-
a
T
a
T
T
a
a
200
A
B
C
A
B
C
A
B
C
baseline
NPB
NPW
50
proliferation rate [% of BrdU-positive cells]
40-
A, controls
B, H-89
**
**
C, calphostin-C
**
**
**
30-
D, tyrphostin-23
E. PD-98059
20-
a
1
T
T
&
4
10-
T
T
T
0
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
baseline
NPB
NPW
neither NPB nor NPW affect not only late, but also early steps of steroid synthesis, allow us to conclude that NCI- H295 GPR7 and GPR8 behave differently than normal human adrenocortical cells. Evidence indicates that NPB and NPW enhance cortisol secretion from human adreno- cortical cells by activating adenylate cyclase (AC) and phospholipase-C (PLC)-dependent cascades (36), and we tentatively suggest that GPR7 and GPR8 in NCI-H295 cells are uncoupled with AC and PLC, thereby being unable to elicit any secretagogue effect.
apoptosis rate [% of TUNEL-positive cells]
25-
A, controls B, H-89 C, calphostin-C D, tyrphostin-23 E, PD-98059
20
4
a
3
15-
a
10
*
*
**
**
**
**
5-
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
baseline
NPB
NPW
proliferation rate [% of BrdU-positive cells]
apoptosis rate [% of TUNEL-positive cells]
50-
25
A, controls B, SB-203580
40-
**
20
**
**
30
**
-15
20
-10
*
**
*
**
10
-5
A
B
A
B
A
B
A
B
A
B
A
B
bsl
NPB
NPW
bsl
NPB
NPW
The observed differences in the secretory behavior between NCI-H295 and human adrenocortical cells are not unexpected in light of our ultrastructural findings. It is commonly agreed that all steroid hormone-secreting cells (adrenocortical cells, testis Leydig cells, and ovarian granulosa, thecal and lutein 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 (for review see refs. 37,53). According to the only study at present available (54), NCI-H295 cells lack lipid droplets and SER, and contain mitochondria with conventional laminar cristae, indicating that they do not possess the classic morphologic phenotype of steroid secreting cells. On the contrary, the presence of abundant RER profiles and free
ribosomes suggest that NCI-H295 cells are rapidly growing cells, endowed with a very elevated rate of protein synthesis.
Our study provides evidence that GPR7 and GPR8 in NCI-H295 cells mediate a potent growth effect of their endo- genous ligands. In fact, NPB and NPW stimulate the growth of NCI-H295 cells, by enhancing their proliferation rate and decreasing their apoptosis rate. In this connection, we recall that a similar 2-fold mechanism has been found to underlie the adrenocortical growth promoting effect of other regulatory peptides, including endothelin-1 (ET-1) (39), adrenomedullin (AM) (39,55-58), ghrelin (47,50), and orexins (22). Further- more, our findings also provide insight into the mechanism(s) of this effect of NPB and NPW.
Like ET-1 (46), AM (59,60) and ghrelin (47,50), NPB and NPW activate both TK and MAPK p42/p44. The contention that MAPK p42/p44 activation depends on TK activation is supported by the fact that it is blocked not only by the MAPK p42/p44 inhibitor PD-98059 (61), but also by the TK inhibitor tyrphostin-23 (62). The following pieces of evidence strongly suggest that TK-dependent MAPK p42/p44 activation mediates the growth effect of NPB and NPW on NCI-H295 cells: i) tyrphostin-23 and PD-98059, at the concentrations abrogating the stimulating action of NPB and NPW on TK and MAPK p42/p44, respectively, annul the peptide-induced rise and decrease in the proliferation and apoptosis rates of NCI-H295 cells; and ii) these inhibitors are per se ineffective, which makes unlikely the possibility that these effects may ensue from their non-specific toxic action on NCI-H295 cells. Consistent findings indicate that MAPK p38 cascade is involved in the control of cell growth, and especially in the activation of apoptosis (63-65). However, our study appears to rule out the possibility that this may occur in NCI-H295 cells, inasmuch as the MAPK p38 antagonist SB-203580 (66) does not alter either the proliferogenic or the antiapoptotic effect of NPB and NPW.
The TK receptor is able to activate the MAPK p42/p44 cascade directly (63,67,68). However, evidence indicates that TK, behaving as a true G protein-coupled receptor, can also activate protein kinase (PK)A and PKC signaling (63,69-71). Accordingly, ET-1 has been found to stimulate proliferation of ZG cells via two independent pathways involving either a TK-dependent or a PKC-dependent MAPK p42/p44 cascade (46). Our present results make unlikely the possibility that PKA and PKC cascades play a relevant role in the growth effects of NPB and NPW. In fact, neither the PKA inhibitor H-89 (72) nor the PKC inhibitor calphostin-C (73) is able to blunt the proliferogenic and antiapoptotic effects of these peptides on NCI-H295 cells. Since AC/PKA and PLC/PKC cascades are involved in the secretagogue effect of NPB and NPW on human adrenocortical cells (36), these observations add support to the contention that in NCI-H295 cells GPR7 and GPR8 are uncoupled with these signaling pathways (see above). Of great interest, these findings are reminiscent of those obtained with adrenomedullin, proadrenomedullin N- terminal 20 peptide and ghrelin, which are regulatory peptides able to stimulate ZG cell growth through a MAPK p42/p44 pathway exclusively dependent on TK activation and unable to affect basal secretion of ZG cells (47,50,59,60,74,75).
Although NCI-H295 cell line physiology may not reflect that of in vivo adrenocortical cells (see above), taken together
our observations raise the appealing possibility that NPB and NPW, via GPR7 and GPR8, may act as growth and tumor promoters in the adrenal gland. Further studies are underway to assess the expression and function of GPR7 and GPR8 in hyperplastic and tumorous human adrenal cortex.
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