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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3356-3364
Copyright © 2000 by The Endocrine Society

Original Studies

Activation of L-Type Calcium Channels Induces Corticotropin-Releasing Factor Secretion from Human Placental Trophoblasts1

Jacques Robidoux2, Lucie Simoneau, André Masse and Julie Lafond

Programme des Sciences Biomédicales de la Faculté de Médecine, Université de Montréal (J.R., A.M., J.L.), et Laboratoire de Physiologie Materno-Foetale, Département des Sciences Biologiques, Université du Québec à Montréal (J.R., L.S., J.L.), Montréal, Québec, 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ultimate outcome of pregnancy, parturition, is a well orchestrated process in which placental corticotropin-releasing factor (CRF) seems to play an important role. The objective of the present study was to investigate the involvement of L-type calcium channels and calcium-dependent signaling in the depolarization-induced CRF release from human syncytiotrophoblast. The basal secretion of CRF by trophoblastic cells, isolated from human term placenta, was maximal after their functional differentiation, which was monitored by hCG measurements. On the fourth day of culture, the basal CRF secretion of the cells in serum-free medium was linear between 2 and 8 h. Incubation of the trophoblasts with KCl, a depolarizing stimulus, or with Bay K8644, an L-type calcium channel agonist, for 3 or 8 h led to an increase in CRF secretion, but was without effect on its synthesis. This stimulated CRF release was calcium dependent, as it could be prevented by loading cells with 1,2-bis(0-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl) ester. Furthermore, the KCl-induced CRF secretion involved L-type calcium channels activation, as 2 µmol/L nitrendipine, an L-type specific blocker, abolished the stimulation. In trophoblasts, where we have previously shown calcium-dependent protein kinase C (cPKCs) activity, incubation with Bay K8644 also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase activities. In the present study we observed that CaMKII and cPKCs were linked to the Bay K8644-induced secretion of CRF, as only the autocamtide-2 related inhibitory peptide, a CaMKII inhibitor, and Gö6976, an inhibitor of µ and cPKCs partially prevented (30–78%) the activation of CRF release by Bay K8644. The use of PD 098056, an inhibitor of the ERKs kinases, showed no effect on CRF release. Taken together, these results support a depolarization-induced and calcium-dependent exocytotic-like secretion of CRF from human placental trophoblasts. In addition, CaMKII and cPKCs seem to be potential modulators or mediators of these calcium effects on CRF secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FUNCTIONAL placental unit, which includes fetal membranes (amnion and chorion), plays an important role in the maintenance of pregnancy, the growth of the fetus, and the timing of birth. This pivotal role of the placenta is largely accountable to its ability to produce a large variety of hormone and to its intimate relationship with both fetal and maternal compartments (1). This gives the embryonic organ the opportunity to act in paracrine and endocrine manners on both fetal and maternal physiology throughout pregnancy (2).

The ultimate outcome of pregnancy, parturition, is only partially elucidated. Considering the plethora of hormones, growth factors, and cytokines produced by the placental unit during spontaneous labor and delivery, this process appears to be well orchestrated (3). In the last few years, corticotropin-releasing factor (CRF), a 41-amino acid peptide component of the stress response in humans, has emerged as a potent regulator of parturition, if not one of the main coordinators of the placental clock controlling the duration of pregnancy and labor (4). In that regard, several studies indicated that the maternal plasma concentration of CRF is elevated during pregnancy, reaching its highest level at the onset of labor (5, 6), in particular during abnormal pregnancy complicated by preterm labor (7). Thus, as the outer layer of the chorionic villus, the placental syncytiotrophoblast, appears to be, especially in the third trimester, the principal source of CRF during pregnancy (8, 9), it is of interest to characterize the mechanisms involved in the regulation of CRF release by this multicompetent endocrine structure.

In most cell types, protein kinase A and protein kinase C (PKC) are major positive regulators of prepro-CRF transcription and CRF secretion (10, 11, 12, 13, 14). In addition, intracellular calcium and calcium influx via L-type calcium channels have been implicated in CRF release from hypothalamic cells (15, 16).

Three studies have shown that the membrane depolarization of placental trophoblasts increases CRF release (17, 18, 19). However, no study until now has explored directly the implication of protein kinases in the modulation of CRF release by trophoblastic cells. Therefore, we investigated the role of L-type calcium channels in the depolarization-induced CRF synthesis and release in cultured trophoblastic cells. Additional aims of the present study were to investigate the contributions of calcium calmodulin kinase II (CaMKII),calcium-dependent PKCs (cPKC)s, and extracellular regulated kinase (ERK1/2) in L-type calcium channel-dependent CRF secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Placental trophoblast isolation and culture

Normal term placentas from either vaginal or caesarian delivery were obtained in accordance with the established guidelines of the ethical committee of the Centre Hospitalier de l’Université de Montréal. After delivery, the placentas were kept in 500 mL cold (4 C) DMEM (Life Technologies, Inc., Burlington, Canada) containing penicillin (200 U/mL), streptomycin (200 µg/mL), emphotericin B (5 µg/mL), and gentamicin (50 µg/mL) all from Sigma (St. Louis, MO). The trophoblast cells were isolated as previously described by Thiede (20), following the modifications reported by Stromberg et al. (21), and Winkel et al. (22). Briefly, the villous tissue (~75 g) was grossly minced, rinsed in Hanks’ Balanced Salt Solution (HBSS; Life Technologies, Inc.) containing the additives mentioned above, and cut into smaller pieces before being rinsed in 100 mL HBSS without calcium and magnesium (HBSS1) on a rotary shaking incubator (50 rpm) for 10 min at 32 C. The rinsed tissue was subjected to two consecutive digestions of 10 min in 120 mL HBSS1 containing 0.125% trypsin (ICN Pharmaceuticals, Inc., Montréal, Canada) and 0.02% deoxyribonuclease type I (Roche Molecular Biochemicals, Laval, Canada). Both supernatants were filtered over a succession of 200- and 60-µm pore size sieves to remove aggregates. The digestion was stopped by the addition of 4% (vol/vol) FCS (HyClone Laboratories, Inc., Logan, UT). The cell suspension was centrifuged at 400 x g for 10 min. The red blood cells were lysed in 140 mmol/L ammonium chloride-Tris-HCl buffer at 100 rpm for 15 min. The cells were than centrifuged at 350 x g for 5 min, resuspended in HBSS, layered over a 10% Percoll (Amersham Pharmacia Biotech, Baie d’Urfé, Canada), and centrifuged at 450 x g for 10 min. The resulting pellet was washed twice in HBSS and once in DMEM by centrifugation at 350 x g for 5 min. The isolated trophoblast cells were suspended in DMEM containing FCS (10%), fatty acid-free BSA (0.5%; ICN Pharmaceuticals), penicillin (100 U/mL), streptomycin (100 µg/mL), Amphotericin (2.5 µg/mL), and gentamicin (50 µg/mL). After cell counting and evaluation of viability by trypan blue exclusion (viability usually >90%), the cells were plated at a density of 1.7 x 106/well in 24-well plates (Sarstedt, Montréal, Canada). The cells were incubated in a humidified atmosphere (95% air-5% CO2 at 37 C). The culture medium was changed after the first 3 h and every day thereafter. All experiments were performed on the fourth day of culture. On that day, the cell count was about 106/well; however, interpreparation variability was important. 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 16%.

Evaluation of trophoblast phenotype

Using the above isolation procedure, we commonly obtained cultures consisting of more than 90% trophoblast cells, as judged by morphological and immunocytochemical determination of pan-cytokeratinpositive (epithelial cell marker; Sigma) and vimentin-positive (mesenchymal cell marker; Sigma) cells (23). Briefly, cells were grown 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 tris-(hydroxymethyl)aminomethane-buffered saline (TBS) containing 0.1% Tween and incubated for 1 h. The biotinylated second antibody included in the 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 of the following steps were performed in accordance with the Oncogene technical protocol, except that we also used the diaminobenzidene enhancer from Sigma.

Measurements of hCG and CRF release by cultured trophoblasts

The levels of CRF and hCG release in the medium and in cells were determined, respectively, by RIA (Phoenix Pharmaceuticals, Inc., Mountain View, CA) and enzyme-linked immunosorbent assay (ELISA; Medicorp, Montréal, Canada) kits, following their respective protocols. The sensitivity of the CRF RIA kits was 24.2 ± 2.7 pg/tube, and the sensitivity of the hCG ELISA kit was 75 mIU/mL. The inter- and within-assay variation coefficients were, respectively, 9.0% and 5.6% at 16 pg/tube for CRF and 8% and 3.5% at 50 mIU/mL for hCG. For the everyday follow-up of hormone release, the cells were cultured in serum-containing DMEM containing 0.5% (days 1 and 2) or 0.1% (days 3–7) BSA. On the fourth day of culture, the CRF content was determined in the absence (basal) and the presence of either KCl (40 mmol/L) or Bay K8644 (5 µmol/L) for 3 and 8 h. For all experiments, the treated cells were compared to cells exposed to the appropriate vehicle(s). Before each experiment, cells were serum starved for 1 h in DMEM containing 0.1% BSA. After that, the medium was replaced by 200 µL medium containing the agent used as treatment. Ten minutes later, KCl or Bay K8644 (200 µL) was added and incubated for the appropriate time. The medium present in each well was collected, centrifuged at 350 x g for 5 min, and 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 HBSS containing 0.1% Nonidet P-40, followed by a 5-s sonication. The resulting medium was treated as described above.

Evaluation of promotion of the autonomous form of CaMKII

The method used was essentially that described by Abraham et al. (24) with minor modifications. Briefly, cells were serum starved for 1 h in DMEM containing 0.1% BSA, then the medium was exchanged for fresh medium (200 µL) and preincubated for 10 min. After that, Bay K8644 (200 µL of a 10 µmol/L stock) was added, and the cells were incubated for different periods of time. At the end of the incubation, the medium was aspired and replaced by 75 µL ice-cold lysis buffer constituted of a 1:1 mixture of calcium/magnesium-free HBSS and 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer containing 1% Nonidet P-40, 20 mmol/L sodium pyrophosphate, 4 mmol/L dithiothreitol, 2 mmol/L sodium fluoride, 2 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mmol/L ammonium molybdate, 200 µmol/L phenylmethylsulfonylfluoride, 20 µmol/L leupeptin, and 1 µg/mL aprotinin. After a 5-s sonication, the protein content of the lysates (83.8 ± 10.0 µg/well) was quantified by the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). The autonomous CaMKII activity of the lysate was assayed by incubating 5 µg lysate in a total 25 µL mixture containing 50 µmol/L autocamtide-2 and 10 µmol/L [{gamma}-32P]ATP in 10 mmol/L MOPS (pH 7.4) containing 10 mmol//L magnesium chloride and 1 mmol/L ethyleneglycol-bis([beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at room temperature for 5 min. The reaction was stopped by the addition of 150 µL ice-cold 10% trichloroacetic acid. Samples were incubated for 30 min at 4 C and centrifuged at 10,000 x g for 3 min. After that, 150 µL supernatant were added to an equal volume of 30% trichloroacetic acid before being adsorbed onto MultiScreen phosphocellulose plates (Millipore Corp., Nepean, Canada). 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.). After three washes, the filters were punched into vials and counted using a liquid scintillation ß-counter after the addition of 5 mL Ready Protein+ liquid scintillation cocktail (Beckman Coulter, Inc., Missisauga, Canada). The specific incorporation into synthetic peptide was determined as the difference in its presence and in its absence.

Evaluation of the ERK1/2 activation

The evaluation of ERK1/2 activation was made by Western blot using an antiphospho-ERK1/2 antibody purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Briefly, cells were serum-starved for 3 h and preincubated for 10 min in 200 µL fresh serum-free medium and in the presence of 5 µM Bay K8644 for different periods of time. At the end of the incubation, the medium was aspirated and replaced by 75 µL SDS loading buffer containing 80 µmol/L sodium vanadate, 80 µmol/L sodium pyrophosphate, 10 mmol/L ethylenediamine tetraacetate, 0.8 mg/mL benzamidine, 80 µ mol/L phenylmethylsulfonylfluoride, 0.8 µg/mL pepstatin, and 0.4 µg/mL aprotinin. The protein content of the lysates (75.0 ± 11.9 µg/well) was quantified by a modification of the Bradford method (25). 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 (26) using a Mini-Protean II system (Bio-Rad Laboratories, Inc., Hercules, CA). After migration, proteins were transferred to a polyvinylidene difluoride 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 antiphospho-ERK1/2 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, 0.1% BSA, and 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 densitometric scanning of the x-ray film using a Personal Densitometer (Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed with ImageQuant software.

For each cell preparation (n = 3, unless otherwise stated), experiments were performed in duplicate or triplicate at a given condition or time point. The mean of such replicate was used as a single datum point for analysis. When time-course protocols were analyzed, ANOVA on repeated measures followed by Dunnett’s post-hoc test were used. Otherwise, the Newman-Keuls test was used to compare multiple condition experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of basal CRF release by human placental trophoblasts in culture

To determine whether the cell preparations displayed CRF secretion competence, the basal 24-h secretion of CRF was followed over a period of 7 days. As shown in Fig. 1Go, basal CRF secretion by freshly isolated trophoblasts was modest over the first 48 h of culture. We observed a sustained peak on days 3 and 4, followed by a progressive decline to reach the initial level of secretion by the sixth day. To determine whether this increase in CRF secretion could be correlated with trophoblast differentiation into functional syncytiotrophoblast, we measured the production of hCG, an accepted marker of this differentiated state (27). As shown in Fig. 1Go, CRF release correlated with hCG production. This increase in the secretion of both peptides could not be attributed to cellular proliferation, as the trophoblastic cells did not incorporate thymidine significantly during the 3-h incubation performed 24 h after plating, and their nuclei and protein contents both declined by about 10% daily (data not shown). As the cells were fully competent to produce CRF on days 3–4 of culture, the subsequent experiments were all performed on the fourth day.



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  		Figure 1. Time course of basal CRF and hCG secretion by human placental trophoblasts in culture over a 7-day period. CRF and hCG 24-h secretions were determined by RIA and ELISA, respectively, as described in Materials and Methods. Data represent the mean ± SE from three different placental cell preparations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. basal).

 
We further characterized basal CRF release over a 24-h incubation period of the cells in serum-free DMEM. Figure 2Go shows an initial rapid CRF accumulation in the medium upon the first 2 h of incubation, followed by a modest, but linear, accumulation for the subsequent 6 h of incubation. The CRF accumulation in the medium reached a plateau after 24 h. Therefore, the subsequent experiments were conducted at 3 h (when CRF is able to be detected by the RIA kit) and at 8 h (still in the linear portion of the basal CRF release and enough to detect synthesis). The intracellular CRF content was not significantly different over the 24-h incubation period, which means that the cells were not depleted over time, even without serum (data not shown), and that constitutive basal synthesis and release of CRF were present.



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  		Figure 2. Time course of basal CRF secretion by cultured syncytiotrophoblasts over a 24-h period. CRF secretion was determined by RIA as described in Materials and Methods. Data represent the mean ± SE from three different placental cell preparations.

 
Regulation of CRF secretion after cell depolarization or L-type calcium channel activation

Potassium chloride-induced CRF secretion has been previously described in placental explants and cells (17, 18, 19). In the present study we wanted to establish that calcium influx was responsible for the KCl-induced CRF release and investigate the nature of the channels responsible for this calcium entry into the trophoblastic cells. Potassium chloride (40 mmol/L) increases the amount of CRF released in the medium by 70% after 3 or 8 h (P < 0.05; Fig. 3Go, A and C). Meanwhile, the total cellular CRF content (determined by adding released CRF and the intracellular content of the peptide) was not modified after KCl treatments (Fig. 3Go, B and D). To ensure that the KCl-induced CRF release was a calcium-dependent effect, we loaded the cells for 30 min with 20 µmol/L 1,2-bis(0-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl) ester (BAPTA/AM), an intracellular calcium chelator. As shown in Fig. 3Go, a and c, BAPTA totally prevented KCl-induced CRF release, but did not have any effect on basal CRF release. As the KCl effect most likely involves voltage-gated calcium channels, and the presence of L-type calcium channels has been reported in syncytiotrophoblast (28), we investigated the involvement of L-type calcium channels in the KCl effect on the CRF release. Nitrendipine (2 µmol/L), an L-type specific blocker, prevented the KCl-induced CRF release, but had no effect on the basal release of the peptide (Fig. 3Go, A and C). Furthermore, incubation of the cells with Bay K8644 (5 µmol/L; an L-type calcium channels agonist) also elicited a stimulation of CRF secretion, increasing the CRF released by 80% after 3 h (P < 0.05; Fig. 4AGo) and by 70% after 8 h (P < 0.01; Fig. 4CGo). Incubations of the cells with Bay K8644 was also without any effect on CRF synthesis (Fig. 4Go, B and D). As expected, preincubation with either BAPTA/AM or nitrendipine completely prevented the rise of CRF release induced by Bay K8644 (Fig. 4Go, A and C).



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  		Figure 3. Effect of BAPTA/AM and nitrendipine on KCl-induced CRF release. When appropriate, cells were loaded with BAPTA/AM (20 µmol/L) for 30 min and serum starved for 1 h. When present, nitrendipine (2 µmol/L) was added 10 min before KCl (40 mmol/L) and incubated for 3 h (A and B) or 8 h (C and D). CRF release (A and C) and total CRF content (B and D) were determined by RIA. Data represent the mean ± SE from three different placental cell preparations. *, P < 0.05 vs. basal; +, P < 0.05 vs. control; ++, P < 0.01 vs. control.

 


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  		Figure 4. Effect of BAPTA/AM and nitrendipine on Bay K8644-induced CRF release. When appropriate, cells were loaded with BAPTA/AM (20 µmol/L) for 30 min and serum starved for 1 h. When present, nitrendipine (2 µmol/L) was added 10 min before Bay K8644 (5 µmol/L) and incubated for 3 h (A and B) or 8 h (C and D). CRF release (A and C) and total CRF content (B and D) were determined by RIA. Data represent the mean ± SE from three different placental cell preparations for 3 h and for three different placental preparations for 8 h. *, P < 0.05 vs. basal; **, P < 0.01 vs. basal; +, P < 0.05 vs. control; ++, P < 0.01 vs. control.

 
Activation of CaMKII and ERK1/2 in response to L-type calcium channels activation

We investigated the activation of both CaMKII and ERK1/2 by Bay K8644 to ascertain their involvement in the signal transduction pathways connecting L-type calcium channels to CRF release.

CaMKII is characterized by an activation-dependent autophosphorylation, leading to the promotion of calcium/calmodulin-independent activity (29). A time-course experiment was conducted to evaluate the induction of the autonomous form of the kinase in the presence of Bay K8644 (5 µM). As shown in Fig. 5Go, the presence of Bay K8644 induces a slow, but important, promotion of the autonomous form of CaMKII, which reached a maximum after 120 s (1.24 ± 0.20 pg/min·mg protein vs. 0.58 ± 0.25; P < 0.05) and thereafter gradually returned to the basal level.



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  		Figure 5. Time course of CaMKII activation by Bay K8644. Cells were serum starved for 1 h. The medium was replaced by 200 µL fresh medium 10 min before the addition of Bay K8644 (5 µmol/L). At different intervals of time, the medium was retrieved, and ice-cold lysis buffer (75 µL) was added. The autonomous CaMKII activity was than measured as described in Materials and Methods. Data represent the mean ± SE from three different placental cell preparations. *, P < 0.05 vs. basal.

 
ERK1/2 are catalytically activated by phosphorylation (30), which can be monitored by Western blot using a phospho-ERK1/2 antibody. A time-course experiment was conducted to evaluate the induction of the phosphorylated form of the kinases in presence of Bay K8644 (5 µmol/L). As shown in Fig. 6Go, incubation of the cells with Bay K8644 led to a biphasic ERK1/2 activation. A first peak of activation was obtained after 7.5 min (P < 0.01), followed by a sustained activation phase from 30 min to 3 h. In contrast, the medium alone had no effect on ERK1/2 activity (not shown).



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  		Figure 6. Time course of ERK1/2 activation by Bay K8644. Cells were serum starved for 1 h. The medium was replaced by 200 µL fresh medium 10 min before the addition of Bay K8644 (5 µmol/L). At different intervals, the medium was retrieved, and ice-cold lysis buffer (75 µL) was added. The phospho-ERK1/2 immunoreactivity was than measured as described in Materials and Methods. Data represent the mean ± SE from three different placental cell preparations. **, P < 0.01 vs. basal.

 
Effect of protein kinase inhibitors on Bay K8644-induced CRF release

We evaluated the involvement of CaMKII and ERK activation in Bay K8644-induced CRF release. Furthermore, as we recently reported the presence of cPKC activity in syncytiotrophoblast (31), we also investigated the involvement of cPKCs in Bay K8644-induced CRF release. Cells were incubated in the presence of well defined inhibitors of these kinase families. As shown in Fig. 7Go, A and C, incubation of the cells with Gö6976 (1 µmol/L), a specific inhibitor of the conventional (calcium-dependent) and µ PKCs (32, 33), prevented most (50% at 3 h and 80% at 8 h) of the Bay K8644 effect on CRF release. Our results also showed that CaMKII, another calcium-dependent kinase, appears to be linked to this CRF release, as the presence of autocamtide-2 related inhibitory peptide (AIP; 1 µmol/L), a specific inhibitor of this kinase (34), partially blocked (50%) Bay K8644-induced CRF release for both incubation times (Fig. 7Go, A and C). However, incubation of the cells with PD 098056 (40 µmol/L), an ERK kinases inhibitor (35), was without effect on Bay K8644-induced CRF release at both incubation times (Fig. 7Go, A and C). Finally, neither of these inhibitors has any effect on basal CRF synthesis or release (Fig. 7Go, A–D).



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  		Figure 7. Effects of Gö6976, AIP, and PD 098056 on Bay K8644-induced CRF release. The cells were preincubated in serum-free DMEM for 1 h. Cells were serum starved for 1 h and Gö6976 (1 µmol/L), AIP (1 µmol/L), or PD 098056 (40 µmol/L) was added 10 min before Bay K8644 (5 µmol/L) and incubated for 3 h (A and B) or 8 h (C and D). CRF release (A and C) and total CRF content (B and D) were determined by RIA. Data represent the mean ± SE from three different placental cell preparations. *, P < 0.05 vs. basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Peptide secretion by endocrine cells occurs by at least two distinct pathways, described as the constitutive and regulated pathways by Kelly (36). In the latter, peptides are packaged and stored in moderate and high density secretory granules, which are released in response to the appropriate external stimuli (37). Peptides conveyed by the constitutive secretory pathway are packaged into secretory vesicles of low density and released at a constant rate at the cell surface (37). Calcium is the central second messenger in regulated peptide release and has been suggested to play a role in exocytosis by binding directly or via its complexion to calmodulin to cytoskeletal elements, granule proteins, calcium-dependent kinases, or calcium/calmodulin-dependent kinases. In syncytiotrophoblast, electron microscopic studies showed the existence of light and dense granules that emerge from the Golgi apparatus and travel through the cytoplasm until they reach the brush border plasma membrane, where they fuse in an exocytotic fashion (38, 39). Little is known regarding the pathways of CRF secretion in syncytiotrophoblast, and there is no ultrastructural study addressing vesicular localization of CRF.

As studies performed on placental explants as well as placental cells have suggested the existence of calcium-dependent CRF secretion (17, 18, 19), the present study was designed to ascertain the role of L-type calcium channels and subsequent calcium-dependent signaling on CRF synthesis and release by human trophoblastic cells. The results presented show that L-type calcium channels appear to be the sole route for calcium entry after a depolarizing stimuli. The results also demonstrate for the first time that a calcium influx through L-type calcium channels promotes the activation of both CaMKII and ERKs in trophoblastic cells, and that the former effect is partly responsible for the associated increase in CRF release. In addition, CRF release after L-type calcium channel activation implicates cPKCs.

First, we established the competence of our cell preparations in secreting CRF. Our results clearly showed that cytotrophoblasts freshly isolated from human term placenta secreted a low level of CRF. A parallelism was observed in their acquisition of CRF- and hCG-secreting competencies, two markers of functional differentiation into syncytiotrophoblast, reaching maximal values on the fourth day of culture. These results are in agreement with a previous study that reported an undetectable level of CRF in freshly isolated cytotrophoblasts (19) and correlate with the absence of immunohistochemical staining in cytotrophoblasts for third trimester placentas (9). The observed peak after 3 days of culture is also in agreement with the CRF immunohistochemical staining in intermediate trophoblasts and in the syncytiotrophoblast layer from third trimester placentas (9) and correlates with the peak in placental cell CRF production on the third day reported by Ni et al. (19). The explanation for the following decrease in the secretion of both peptides on days 5 and 6 is less obvious, but has been observed previously for hCG after 4–5 days of culture (40, 41).

Our study agrees with one study performed on placental explants (17) and two studies performed in the Smith laboratory on placental cells (18, 19) that reported that KCl induces a marked increase in CRF release, which suggests depolarization-induced calcium-dependent CRF release by the syncytiotrophoblast. Furthermore, the present results showed that KCl stimulation of CRF release from syncytiotrophoblast during both 3- and 8-h incubations was indeed calcium dependent and mediated via an L-type calcium channel activation, as BAPTA/AM and nitrendipine abolished this stimulation. Moreover, direct activation of L-type calcium channels by Bay K8644 mimicked the KCl-induced CRF release, which further substantiates the role of these channels in the regulation of release of this peptide by trophoblastic cells. The current results are in agreement with the presence of L-type calcium channels in human placenta (28) as well as the L-type calcium channel-dependent secretion process (28, 42, 43, 44). On the other hand, the important depolarization that should accompany the use of a high KCl concentration makes it unlikely that the T-type calcium channels that are also expressed in the placenta (45) play any significant role in the KCl-induced increase in CRF release. Interestingly, the presence of BAPTA alone, which should induce calcium influx (46), is not enough by itself to induce the release of CRF, which suggests that the route of calcium influx is an important determinant of CRF release. In addition, the absence of effect of nitrendipine on basal CRF release either means that there is a low spontaneous activity of the channels or that the threshold level of calcium in the vicinity of the secretory apparatus is not reached.

We provide the first evidence for the promotion of the autonomous form of CaMKII after activation of L-type calcium channels by Bay K8644 in syncytiotrophoblast. This kinase activation was surprisingly slow and transient. Although the mechanism involved in this slow kinetic was not explored, it could be possible that the microvillous nature of the apical route of calcium entry being rich in filamentous actin could constitute an effective calcium buffer that will slow down diffusion of the divalent cation from the entrance to a CaMKII located deeper in the cytoplasm (47). Nevertheless, it was rapidly desensitized, because despite continuous channels stimulation, CaMKII activity returns to its basal level by 180 s. In that respect, it is possible that the lowering of the activity during sustained calcium elevation arises from autophosphorylation on serine 314, which has been correlated with the lost of autonomy in hippocampal pyramidal neurons (48). An alternative hypothesis is that the sustained rise in intracellular calcium has activated a phosphatase, which, in turn, has dephosphorylated the threonine 286 and terminated the autonomous activity (49, 50).

We also showed for the first time a modest, but sustained, activation of ERK1/2 after the activation of L-type calcium channels by Bay K8644 in trophoblastic cells. Although the exact mechanism of that activation was beyond the scope of the present study, it appears that stimuli that can lead to sustained calcium entry can also lead to sustained ERK1/2 activity. In the present context we can speculate that ERK1/2 activation could result from a calcium-, calcium/calmodulin-, CaMKII-, or cPKC-dependent activation. In that respect, calcium-dependent and cPKC activations of the cytosolic tyrosine kinase pp125FAK or PYK2, which, in turn, initiates the formation of a Src-Grb2-Sos complex, leading to the activation of the Ras/Raf/MEK1/2/ERK1/2 cascade, have been described (51, 52). Raf has also been shown to be phosphorylated and activated by cPKCs. The activated Raf, in turn, leads to the phosphorylation and activation of MEK1/2, which are the principal ERK1/2 kinases (53). CaMKII as also been proposed to be implicated in ERK1/2 activation, an effect that appears to be upstream from MEK1/2 (54). Finally, calcium-dependent trans-activation of the epidermal growth factor receptor has been proposed to mediate ERK1/2 via the Grb2-Sos/Ras/Raf/MEK1/2 cascade (55).

Previous reports have shown that PKC activation by phorbol ester stimulates CRF release in perfused hypothalami (10) or in dispersed hypothalamic cells (56). The present study shows that PKCs are implicated in CRF release from human placental cells, as our results showed that an inhibition of µ and cPKCs markedly decreased CRF release. It is unlikely that inhibition of the µ subtype of PKC has any effect after L-type calcium channel activation, as they do not have a calcium-binding domain (57). However, cPKCs are responsive to calcium and are present in syncytiotrophoblast (29, 58, 59); thus, it is more plausible that these kinases, repetitively implicated in the secretion process (60), are indeed part of the mechanisms leading to CRF release in syncytiotrophoblast. Clearly, there are many potential targets for cPKCs in the secretory apparatus, which include cytoskeletal proteins as well as proteins known to be implicated in the last steps of exocytosis. However, we cannot exclude the possibility that the effect of the PKC inhibitor could be the result of cross-talk between cPKCs and either L-type calcium channels or CaMKII. Concerning L-type calcium channels, Fomina and Levitan have described a positive control on L-type calcium channels responsiveness by PKC basal activity (61). With regard to CaMKII, PKCs are known to phosphorylate many cytoskeletal elements that release calmodulin, which, in turn, becomes available to activate CaMKII (62). Nevertheless, even if the exact mechanism(s) involved is not clearly defined, our results give evidence of an important effect of cPKC as a CRF release modulator.

CaMKII is ubiquitous, and considerable experimental evidence implicates the enzyme in peptide hormone secretion (63, 64). The effect of AIP on CRF secretion clearly showed the involvement of CaMKII in the induction of CRF release after L-type calcium channel activation. The exact nature of the substrates involved in the CaMKII action on CRF release is still unclear, but in mouse pancreatic B cells, the kinase is involved at early steps of the exocytotic machinery leading to insulin release (63). The ERK1/2 activation by Bay K8644 led us to investigate its possible role in CRF release, but as with pancreatic ß-cells, where glucose leads to ERK1/2 activation but not to ERK1/2-dependent insulin release (65), we could not substantiate ERK1/2 involvement in CRF release by syncytiotrophoblast.

In summary, our data showed that activation of voltage-operated calcium channels directly or by a depolarizing stimulus induces CRF release from the syncytiotrophoblast. The calcium influx via these channels can lead to the activation of both CaMKII and ERKs in trophoblastic cells, and the former effect is partly responsible for the associated increase in CRF release. CRF release after L-type calcium channel activation also implicates cPKCs. Actually, it is not clear whether these ex vivo observations are any reflection of the in vivo situations, but they conclusively showed that calcium is effective to induce CRF release, and that other stimuli leading to cPKC and CaMKII activation could also be potent CRF secretagogs.


   Acknowledgments
 
We express our gratitude to Mrs. Christiane Paré (Chief of Nursing) and the staff of the Department of Obstetrics and Gynecology of Pavillon St. Luc of the Centre Hospitalier Universitaire de Montréal for the donation of placentas. We are also grateful to Dr. Robert Moreau for valuable criticism of the paper, and to Mélanie Laramée for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from Université du Québec à Montréal and March of Dimes Birth Defects Foundation of USA (to J.L.). Back

2 Recipient of a FRSQ-FCAR santé doctoral studentship. Back

Received January 13, 2000.

Revised May 15, 2000.

Accepted May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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