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Programme des Sciences Biomédicales and Gynecology-Obstetrics Department (J.R., A.M., J.L.), Faculté de Médecine, Université de Montréal; Départements des Sciences Biologiques (J.R., L.S., J.L.)and Chimie (S.St-P.), Université du Québec à Montréal, Canada, H3C 3P8
Address all correspondence and requests for reprints to: Prof. Julie Lafond, Université du Québec à Montréal, Département des Sciences Biologiques, C.P. 8888, Succursale "Centre-Ville," Montréal, Québec, Canada H3C 3P8. E-mail: lafond.julie{at}uquam.ca
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Introduction |
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During the last few years, significant interest has been given to placental CRF. This great interest arises from the apparent link between the production of this peptide and the duration of pregnancy (3). In that regard, several studies indicated that maternal concentration of CRF is elevated during pregnancy, reaching its highest level at the onset of labor (4, 5), particularly during an abnormal pregnancy complicated by preterm labor (6).
The syncytiotrophoblast layer appears to be, at least in the third trimester, the principal cellular source of CRF (7) and therefore, most studies dealing with the control of this peptide’s synthesis and release were conducted on trophoblast cells isolated from human term placenta. Isolated trophoblast cells from human term placenta undergo spontaneous syncytiotrophoblastic-like morphological and biochemical differentiation in vitro (8) and would reflect in vivo syncytiotrophoblast. Several studies (for a review , see Ref. 9) pointed glucocorticoids and progesterone as the major regulators of placental CRF synthesis. However, many other putative CRF secretagogues have been identified, such as acetylcholine, angiotensin II, arginine vasopressin, catecholamines, interleukin-1, NPY, oxytocin, and prostaglandins (10, 11, 12). Nitric oxide has been reported as a negative regulator (13).
Among these secretagogues, NPY, a peptide produced by cytotrophoblastic cells, amnion, chorion, and decidua (11, 14) is abundant in both plasma and amniotic fluids of woman throughout pregnancy (15). NPY binds to a mixed population of Y1 and Y3 receptors on the brush border membranes of human syncytiotrophoblast (16). These receptors are linked to multiple signal transduction cascades. Until now, no study intended to elucidate the mechanisms implicated in the modulation of placental CRF release. For that reason, we decided to investigate the mechanisms implicated in the NPY effects. The purpose of this study was to investigate the role of calcium signaling on the NPY induced CRF synthesis and release in cultured human trophoblast cells.
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Materials and Methods |
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75 g) was grossly minced, rinsed in HBSS containing the additives
mentioned above, and cut again into smaller pieces, before being rinsed
in 100 ml HBSS without calcium and magnesium on a rotary shaking
incubator (50 rpm) for 10 min at 32 C. The rinsed tissue was after
subjected to two consecutive digestions of 10 min in 120 ml of a
similar medium containing 0.125% trypsin (ICN Biochemicals, Inc., Montréal, Québec, Canada) and 0.02%
deoxyribonuclease type I (Roche Molecular Biochemicals,
Laval, Canada). Both supernatants were filtered over a
succession of 200 µm and 60 µm sieves to remove aggregates. The
digestion was stopped by the addition of 4% (vol/vol) of FCS
(HyClone Laboratories, Inc. Logan, UT). The cell
suspension was centrifuged at 400 g for 10 min. The red
blood cells were lysed in 140-mM ammonium
chloride-Tris-HCl buffer at 100 rpm for 15 min. The cells were than
centrifuged at 350 g for 5 min, resuspended in HBSS, layered over
a 10% percoll (Amersham Pharmacia Biotech, Baie
d’Urfé, Canada) and centrifuged at 450 g for 10 min. The
resulting pellet was washed twice in HBSS (Life Technologies, Inc.) and once in culture medium by centrifugation at 350 g
for 5 min. The isolated trophoblast cells were suspended in DMEM
containing FCS (10%), fatty acid-free BSA (0.5%) (ICN Biochemicals, Inc.), penicillin (100 U/ml), streptomycin (100
µg/ml), Amphotericin (2.5 µg/ml) and gentamycin (50
µg/ml). After cells counting and evaluation of viability by trypan
blue exclusion (viability usually greater than 90%), the cells were
plated at a density of 1.7 x 106 per well in 24 well
plates (Sarstedt, Montréal, Québec, Canada). The cells were
incubated in a humidified atmosphere (95% air and 5%
CO2 at 37 C). The culture medium was changed
after the first 3 h and changed every day thereafter. All
experiments were done at the fourth day of culture. On that day, cells
count was around 106 per well. This variation is inherent
to the methods and the model, but intrapreparation variability as
judged by protein determination of cellular lysate was not more than
15%.
Evaluation of trophoblast phenotype
Using the above isolation procedure, we usually obtained
cultures consisting of more than 90% trophoblast cells as judged by
morphological and immunocytochemical determination of
pan-cytokeratin-positive (epithelial cell marker;
Sigma, St. Louis, MO) and vimentin-positive cells
(mesenchymal cell marker; Sigma) (20). Briefly, cells were
growth for 12 h on plastic coverslips and were fixed for 10 min in
acetone at -20 C. After evaporation, endogenous peroxidase activity
was quenched by a 10 min incubation at 4 C with 3%
H2O2 in methanol.
Nonspecific binding was blocked by a 3 h incubation at room
temperature with 5% skimmed milk, 3% FCS and 1% BSA. Primary
antibody was diluted (1:200 for vimentin, 1:400 for cytokeratin) in TBS
containing 0.1% Tween and incubated for 1 h. The biotinylated
second antibody included in histochemical ABC kit
(Oncogene Research, Cambridge, MA) was diluted in the same
buffer and incubated for 1 h. An irrelevant antibody [trp E
(Ab-1)], included in the kit, was used as first antibody for the
control. All the following steps were done in accordance with the
Oncogene’s technical protocol, except that we also used
the DAB enhancer from Sigma.
Measurements of CRF release by cultured trophoblasts
The levels of CRF release in the medium and in the cells were
determined by RIA kit (Phoenix Pharmaceuticals, Inc.,
Mountain View, CA), following the Phoenix’s technical protocol. The
sensitivity of the CRF RIA kit was 23.9 ± 2.5 pg/tube. The
interassay and within-assay variation coefficients at 16 pg/tube were
8.4% and 3.8%, respectively. On the fourth day of culture, the
content of CRF was determined in the absence (basal) and the presence
of NPY for 3 h and 8 h. For all experiments, the treated
cells were compared with cells exposed to the appropriate vehicle(s).
Before each experiment, cells were serum starved for 1 h in DMEM
containing 0.1% BSA. Afterward, the medium was replaced by 200 µl of
medium containing a 2x concentration of the agent used as treatment.
Ten minutes later, NPY (200 µl of a 2 x 107
M stock) was added and incubated for the appropriate time.
The medium present in each well was collected, centrifuged at 350
x g for 5 min, evaporated to dryness before being stored at
-20 C until assayed. For the intracellular CRF content, cells were
lysed by the addition of 200 µl of HBSS containing 0.1% Nonidet
P-40, followed by a 5 sec sonication. The resulting medium was treated
as described above.
Evaluation of the promotion of the autonomous form of CaMKII
The method used was essentially described by Abraham et
al. (21) with minor modifications. Briefly, cells were serum
starved for 1 h in DMEM containing 0.1% BSA, than the medium was
changed for 200 µl of fresh medium and preincubated for 10 min.
Afterward, NPY (200 µl of a 2 x 10-7 M
stock) was added and the cells were incubated for different interval of
time. At the end of the incubation, the medium was aspired and replaced
by 75 µl of an ice-cold lysis buffer constituted of an 1:1 mixture of
calcium/magnesium-free HBSS and 50 mM MOPS buffer
containing 1% Nonidet P-40, 20 mM sodium
pyrophosphate, 4 mM dithiothreitol, 2
mM NaF, 2 mM EGTA, 1
mM ammonium molybdate, 200
µM PMSF, 20 µM
leupeptine, and 1 µg/ml aprotinin. Following a 5-sec sonication, the
protein content of the lysates (75.9 ± 11.4 µg/well) was
quantified by the BCA protein assay (Pierce Chemical Co.,
Rockford, IL). The autonomous CaMKII activity of the lysate was assayed
by incubating 5 µg of the lysate in a total 25 µl mixture
containing 50 µM autocamtide-2 and 10
µM [
-32P]ATP in 10
mM MOPS (pH 7.4) containing 10 mM
magnesium chloride and 1 mM EGTA, at room
temperature for 5 min. The reaction was stopped by the addition of 150
µl of ice cold 10% trichloroacetic acid. Samples were incubated for
30 min at 4 C and centrifuged at 10,000 x g for 3 min.
An aliquot (150 µl) of the supernatant was added to an equal volume
of 30% trichloroacetic acid and the mixture was adsorbed on a
MultiScreen phosphocellulose plates (Millipore Corp.,
Nepean, Canada). Following three washes, the filters were punched into
vials and counted using a liquid scintillation ß counter after
addition of 5 ml of Ready protein+ liquid scintillation cocktail
(Beckman Coulter, Inc., Missisauga, Canada). The specific
incorporation into the synthetic peptide was determined as the
differentiation in its presence or absence.
Evaluation of the ERKs activation
The phosphorylation status of ERKs is a quantitative indicator
of ERKs activation. Therefore, the evaluation of ERKs activation was
done by Western blot using an anti-phosphoERKs antibody purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Briefly,
cells were serum-starved for 3 h, preincubated for 10 min in 200
µl fresh serum-free medium and incubated in the presence of NPY
10-7 M, for an additional 0, 2.5, 5, 7.5, 10,
15, 30, and 180 min. At the end of the incubation, the medium was
aspired and replaced by 75 µl of SDS loading buffer containing 80
µM sodium vanadate, 80 µM sodium
pyrophosphate, 10 mM EDTA, 0.8 mg/ml benzamidine, 80
µM PMSF, 0.8 µg/ml pepstatin, and 0.4 µg/ml
aprotinin. The protein content of the lysates (80.2 ± 12.0
µg/well) was quantified by a modification of the Bradford method
(22). Proteins (10 µg) were loaded on an alkaline tricine-SDS-PAGE
system consisting of a 4% stacking gel and a 12% acrylamide-glycerol
separating gel (23), using a Mini-Protean II system (Bio-Rad Laboratories, Inc. Hercules, CA). After migration, proteins were
transferred to PVDF membrane (Roche Molecular Biochemicals) using a semidry system (Millipore Corp.). The membrane was blocked overnight at 4 C in
Tris-buffered saline containing 0.05% Tween (TBS-T), 5% skimmed milk,
3% FCS, and 1% BSA. These membranes were blotted for 1 h at room
temperature with the anti-phosphoERKs antibody (1:1000 in TBS-T),
washed three times with TBS-T, and incubated for 1 h in TBS-T
containing 0.5% skimmed milk, 0.3% FBS, and 0.1% BSA and a
horseradish peroxidase-coupled antirabbit IgG antibody (1:1250)
(Roche Molecular Biochemicals). Finally, the
immunoreactive bands were developed by chemiluminescent (Roche Molecular Biochemicals). The specific bands were quantified by
densitometry scanning of the x-ray film using the Personal Densitometer
(Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed
with ImageQuant software (Sunnyvale, CA).
Data analysis
For each cells preparation (n = 3–5), experiments were
done in duplicate or triplicate, as described, at a given condition or
time point. The mean of such replicates was used as a single datum
point for analysis. When time-course or dose-response protocols were
analyzed, ANOVA on repeated measures followed by Dunnett’s
posthoc test were used. Otherwise, Newman-Keuls test was
used to compare multiple condition experiments.
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, a
and b). For the 3 h incubation (Fig. 1a), only the CRF release was
significantly increased, the NPY effect was perceived at concentration
ranging from 10-8 M (p <
0.05) to 10-6 M (p < 0.01).
After a 8 h incubation with NPY, long enough to observe CRF
synthesis, the peptide increased both CRF synthesis (total CRF content)
and release at concentrations ranging from 10-8 to
10-6 M (Fig. 1b; P < 0.01).
Therefore, from now on, when we will mention NPY-induced CRF synthesis
we will always refer to the 8 h experiment because no modification
of total CRF content was observed during any of the 3 h
experiments (Figs. 1a, 2b, 3b, 4b, and 7b). The NPY (10-7
M) increased CRF release by 2- and 3-fold at 3 h and
8 h, respectively. The NPY potency to induce CRF release was
slightly different between the 3 h and 8 h incubations (with
PEC50 of 9.59± 0.61 and
8.93 ± 0.38 respectively; P < 0.05). However,
the NPY potency to induce CRF release at the longer incubation time was
identical to its potency to induce CRF synthesis
(PEC50 8.93 ±
0.62).
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, a, c, and
d) because BIBP 3226 prevented both effects. In most cell types, the
Y1 receptors are linked to PTX-sensitive G
proteins. Because no studies have reported this kind of coupling in
trophoblastic cells, we preincubated the cells overnight with 500 ng/ml
of PTX. As expected, the toxin eliminated the effect of NPY on both CRF
synthesis and release (Fig. 2, a, c, and d).
Implication of calcium in NPY-induced CRF synthesis and
release
To examine the involvement of intracellular calcium in NPY-induced
CRF synthesis and release in trophoblastic cells, we loaded the cells
(30 min) with 20 µM BAPTA/AM, an intracellular calcium
chelator. As shown in Fig. 3, a and c,
BAPTA prevents 50% of the NPY-induced CRF release at 3 h
(P < 0.05) and 75% at 8 h (P <
0.01). Nonetheless, no effect of the calcium chelation could be
discerned on CRF synthesis (Fig. 3d). The calcium implicated in the
NPY-induced CRF-release could originate from either extracellular
calcium influx or intracellular store release. Therefore, we
investigated both possibilities. Since depolarization-induced CRF
release has been reported (13), we investigated the involvement of
L-type calcium channels in the NPY effects. The result showed that the
L-type calcium channels specific blocker, nitrendipine (2
µM) did not prevent the effects of NPY on
either CRF synthesis or release (Fig. 3, a, c, and d). In
syncytiotrophoblasts, NPY is known to activate PLC-ß (16). Two
strategies have been elaborated to examine the contribution of the NPY
to InsP3-R pathway: 1) cells incubation with
U-73122 (5 µM), a inhibitor of G protein-coupled
receptors activation of PLC-ß (25), and 2) cells incubation with
xestospongin C (1 µM), an InsP3-R blocker
(26). As shown in Fig. 4, a and c, both
agents reduced CRF release after 3 h and 8 h
(P < 0.01). Surprisingly, U-73122 and xestospongin C
treatments eliminated (P < 0.001) the NPY effects on
CRF synthesis (Fig. 4d). Because prolonged stimulation of calcium
release will empty the stores without refilling them, we incubated the
cells with SKF 96365 (50 µM), a SOC influx
blocker (27). Figure 4, a and c, showed that SKF 96365 reduces the
NPY-induced CRF release by 65% after 3 h (P <
0.05) and 50% after 8 h (P < 0.05). Such an
effect of the blocker on NPY-induced CRF synthesis (Fig. 4d) was less
obvious since NPY-stimulated CRF release was still important
(P < 0.01).
Activation of CaMKII and ERKs by NPY
Because of the importance of calcium in the NPY-induced CRF
release, we postulated that calcium dependent kinases might be involved
in this process. Because we have evidences of PKCs (16) and
calcium-dependent ERKs activation (data not shown) by NPY, we
investigated the activation of both CaMKII and ERK by NPY to ascertain
their involvement in the signal transduction pathways connecting NPY to
CRF synthesis and release.
The CaMKII is characterized by an activation-dependent
autophosphorylation leading to the promotion of a calcium/calmodulin
independent activity (28). Therefore, a time-course experiment was
conducted to evaluate the induction of the autonomous form of the
kinase in presence of NPY (10-7 M). As shown
in Fig. 5, NPY produced a rapid promotion
of the autonomous form of CaMKII. This activation was already apparent
after 15 sec (P < 0.05) and the activity was
maintained for an additional 30 sec, before gradually returning to the
basal level.
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, the incubation of cells with NPY leads
to a sustained ERKs activation.
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, a and c,
cell incubation with BIM (50 nM), a nonisotype specific
inhibitor of PKCs (30), inhibited NPY-induced CRF release
(P < 0.05) but had no effect on CRF synthesis (Fig. 7d). Interestingly, the use of Gö6976 (1
µM), a specific inhibitor of the conventional
(calcium-dependent) and µ PKCs (31, 32), had no effect on both
NPY-induced release and synthesis (Fig. 7, a and c, and d). On the
other hand, CaMKII, another calcium-dependent kinase, appears to be
linked to both synthesis and release because the presence of AIP (1
µM), a specific inhibitor of this kinase (33),
reduced NPY-induced CRF release by 45% after 3 h and totally
prevented NPY-induced CRF synthesis and release after 8 h (Fig. 7, c and d). Finally, the cells incubation with PD 098056 (40
µM), an ERK kinase inhibitor (34), had no
significant effect on NPY-induced CRF synthesis or release (Fig. 7, a and c and d). |
Discussion |
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All studies took place on the fourth day of culture because preliminary experiments have showed that this period coincides with the peak of secretory competence for both CRF and hCG (data not shown). This is in accordance with the syncytiotrophoblastic origin of these peptides. The syncytiotrophoblast model used, even though costly, time-consuming, and variable from preparation to preparation, has the advantage to rely on nontransformed cells containing the endogenous CRF gene. The present study shows that addition of NPY to trophoblastic cells, on their fourth day in culture, results in an increase in CRF synthesis and release. The stimulation of CRF release by NPY from these cells during the 3-h incubation period is similar to previous result reported by Petraglia et al. (11). Moreover, we showed for the first time that NPY modulates CRF synthesis in trophoblastic cells. An up-regulation of CRF synthesis has been reported in Y2 receptor bearing SK-N-BE2 cells transfected with the human CRF promoter (37). Taking into account the up-regulated synthesis, the NPY effect on CRF release between 3 h and 8 h appears mainly derived from this novel synthesis. After consideration, it could mean that during a short incubation period (3 h), the NPY induces the release of preformed vesicles containing CRF, while longer incubation period (8 h) allows the production and the processing of new CRF by the trophoblastic cells.
The syncytiotrophoblast displays on its maternal surface two types of NPY binding sites, the Y1 and Y3 subtypes (16). Because NPY could stimulate CRF synthesis and release by interacting with either one of them, we explored this possibility with the highly specific Y1 antagonist, BIBP3226 (24). This molecule completely abolished the NPY-induced CRF synthesis and release, thus strongly supporting the involvement of Y1 receptor subtype in the neuropeptide effects. Our results also showed that a pretreatment of trophoblastic cells with PTX, known to inactivate Gi and Go proteins by ADP ribosylation (38), caused a total inhibition of the NPY-induced CRF synthesis and release, suggesting that some PTX-sensitive G-proteins are involved in this syncytiotrophoblast response to NPY. This result is not surprising because, in most cell types studied, the Y1 receptor is PTX-sensitive (39).
Calcium-dependent CRF secretion has already been suggested in placental explants and cells (13, 36, 40). In these studies, KCl induced a marked increase in CRF release, which could be attributable to a depolarization-induced calcium influx in secreting cells. In that respect, a study done on microdialyzed median eminence has shown that basal and KCl-induced CRF releases are mediated by L-type calcium channel activation (41). Because syncytiotrophoblast has been shown to possess these channels (42), as well as L-type calcium channels dependent secretion process (43, 44), we used BAPTA/AM and nitrendipine to investigate the possible involvement of calcium and these channels in the NPY effects. The reduction of NPY-induced CRF release in presence of BAPTA/AM strongly suggest the involvement of calcium in CRF release, whereas the involvement of L-type calcium channels is unlikely according to the nitrendipine ineffectiveness. The fact that this blocker has no effect on NPY-induced CRF release is not surprising because Y1 receptors, at the exception of those found in vascular smooth muscles, are generally linked to L-type calcium channels inhibition (39) and therefore cannot account for the stimulation.
Our previous study (16) has demonstrated that Y1 receptors of human syncytiotrophoblasts are linked to PLC-ß activation. This activation leads to the cleavage of PtdInsP2, which generates 1,2 diacylglycerol (an activator of cPKCs and nPKCs) and Ins (1, 4, 5)P3 (which has a permissive effect on IP3-R) (45). The inhibition of NPY-induced CRF release and synthesis by U-73122 indicates that PLC-ß activation is partially responsible for the NPY effect, and almost totally accountable for the NPY-induced CRF synthesis. However, a report showing a decrease of PYY binding to Y1 receptor following U-73122 incubation (46) prompted us to further investigate the involvement of InsP3-releasable intracellular stores, by using the InsP3-R blocker, xestospongin C. The results obtained with xestospongin C reflect those obtained using the aminosteroid inhibitor of PLC-ß, indicating that a decrease of NPY binding, while not excluded, cannot explain the inhibition seen with the use U-73122. Therefore, we propose that in trophoblastic cells, NPY binds to its receptors which in turn activates PLC-ß. The generated InsP3 binds its receptor, induces calcium release from the intracellular store, which is responsible for the NPY-induced CRF synthesis and release.
The use of SKF 96365 [an inhibitor of the receptor-mediated calcium influx, the voltage-operated calcium channels (47) and SOC influx (27)] was initially planned to investigate the influence of SOC calcium influx on the NPY-induced CRF synthesis and release. The lack of specificity of this wide-range tool is not a problem because many aspects of the calcium entry in syncytiotrophoblasts remain to be elucidated. Also, it is likely to involve multiple calcium influx pathways. In the first time, our results showed that SOC influx is implicated only in NPY-induced CRF release. In the presence of NPY, either SKF 96365 or BAPTA/AM should induce the emptying of the calcium stores, while the presence of U-73122 or xestospongin C should not. These results suggest that the emptying of the store mediated by NPY could induced the activation of a pathway implicated in CRF synthesis.
Our results obtained in presence of BAPTA lead also to another interrogation: Why the calcium influx, which should be induced by the presence of this chelator alone (48), is not enough by itself to induce the release of CRF? A possible explanation is that calcium influx following the emptying of intracellular stores, even if SKF 96365 seems to be as efficient as U-73122 or xestospongin C in reducing NPY-induced CRF release, is not the sole factor to its release, but rather a cofactor.
In the placenta, where PKCs are activated by NPY (16), nothing has been published on either CaMKII or ERKs activities. To our knowledge, our results report a novel relationship between phosphoERKs immunoreactivity or activity and G protein-coupled receptor in trophoblastic cells. Our results clearly show that NPY promotes the autonomous form of CaMKII. The kinetic of the stimulation is rapid and transient, meaning that either the NPY effect on CaMKII could be rapidly desensitized or that the receptor could be rapidly internalized. In this regard, ERKs activation has been reported to follow G protein-coupled receptor endocytosis (49).
ERKs are known as downstream effectors of Y1 receptors (50, 51). We have measured the ERKs activation by their phosphorylation status (52), in presence of NPY. Our results showed a biphasic activation of the ERKs by NPY, a first activation that is rapid, followed by a sustained phase. Usually, the studies on ERKs activity induced by G protein-coupled receptors look at short period because a peak of ERKs activity is normally found between 2 and 10 min. The significance of the sustained ERK activation is intriguing, but not uncommon for receptor linked to PTX-sensitive G proteins (53). Phosphatidylinositol 3-kinase, Ras- and PKC-dependent processes have been proposed for the Y1 receptor-mediated ERKs activation (50, 51). Nevertheless, a considerable amount of work has to be done to ascertain which of these or other pathways are implicated in trophoblastic cells.
Many studies, mainly done on hypothalamic cells, have involved protein kinase A (PKA) and PKCs as inducers of CRF synthesis and release (54, 55). However, in syncytiotrophoblast, there is only evidence of the involvement of PKC in the inhibition of CRF release (36). Our results indicate that CaMKII, ERKs, and calcium-independent PKCs are regulators of CRF release and/or synthesis. The use of the nonisotype specific PKC inhibitor, BIM, inhibited NPY-induced CRF release but not its synthesis, while the conventional and µ PKCs inhibitor, Gö6976, did not. This means that novel or atypical subtypes of PKCs are implicated in the NPY-induced CRF secretion and could represent the cofactors associated with SOC influx responsible for CRF release. However, the PKC isotypes activated following NPY receptors activation are not known in syncytiotrophoblast. Previous studies have shown the presence of the three subfamilies of PKCs in syncytiotrophoblast (56, 57). Because Y1 receptors are linked to PLC and to phosphatidylinositol 3-kinase (16), both novel and atypical PKCs could be involved. The nonimplication of PKCs in the CRF synthesis induced by NPY is probably tissue specific because PKCs have been shown to be implicated in CRF gene expression in transiently transfected chicken macrophages (58).
CaMKII is a ubiquitous enzyme that has been implicated in peptide hormone secretion (59, 60). Our results with AIP clearly showed the involvement of this kinase in NPY-induced CRF synthesis and release. The exact nature of the substrates involved in the CaMKII action on CRF release is still unclear. In mouse pancreatic B-cells, this kinase is involved in the early steps of the exocytotic machinery leading to insulin release (60). The CaMKII has been implicated in the increasing of the size of the readily releasable vesicle pool and in the decreasing time for the refilling of the vesicles. Then, the implication of CaMKII in the NPY-stimulated CRF synthesis is in accordance with previous studies (37, 61). In these reports, NPY has been shown to induce CaMKII activity, which phosphorylates the cAMP response element binding protein (CREB). The phosphorylated CREB interacts with the cAMP response element (CRE) within several genes and leads to their transcription (37). Because CREs are found in the CRF promoter (58, 62), we could hypothesize that a similar mechanism could be involved in trophoblastic cells. Nevertheless, there is still a considerable amount of work that has to be done to ascertain the role of CREB in the NPY-induced CRF synthesis.
In conclusion, our study indicated that Y1 receptor links NPY to CRF synthesis and release in trophoblastic cells. Furthermore, our results indicate that these NPY effects are involved the PLC-InsP3-R axis and the CaMKII. They also showed that calcium independent PKCs are implicated in the stimulatory effects of NPY on CRF release. Our results suggest the existence of a diversity of biochemical pathways connecting the Y1 receptor to the CRF synthesis and release. Nonetheless, further studies are necessary to elucidate the sequence of their involvement. Our results also suggest that we can add PTX-sensitive G protein-coupled receptors, like the NPY-Y1 receptor, to the growing list of modulators of CRF synthesis and release.
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Acknowledgments |
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Footnotes |
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2 Recipient of a FRSQ-FCAR santé doctoral studentship.
Received October 6, 1999.
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in human placenta: localization,
secretion, and binding sites. Endocrinology 124:2016–2022
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