Mol. Hum. Reprod. Advance Access originally published online on June 13, 2006
Molecular Human Reproduction 2006 12(7):443-450; doi:10.1093/molehr/gal053
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Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro
Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX, USA
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, MRB 11.138, Galveston, TX 77555-1062, USA. E-mail: ydong{at}utmb.edu
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Abstract |
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Calcitonin gene-related peptide (CGRP) is a multifunctional peptide present in both maternal and fetal circulations in pregnancy. Its receptors have been identified on human trophoblast cells, but the role of CGRP in trophoblast differentiation remains unknown. This study was designed to determine the effect of CGRP on the differentiation of villous trophoblasts isolated from normal human term placentae. The morphological and functional differentiation of the trophoblast cells were assessed by desmosomal protein immunofluorescent staining and the quantification of hCG, estrogen and progesterone secretion. Results showed that (i) exposure of villous trophoblast cells to CGRP led to a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation; (ii) immunofluorescent staining with antidesmosomal antibody was identified at the boundaries between aggregated cytotrophoblast cells, and these stainings disappeared when cells fused to form syncytiotrophoblast cells; (iii) the formation of multinucleated syncytiums in primary cultured cytotrophoblasts was stimulated by CGRP as evidenced by the changes in antidesmosomal staining; (iv) CGRP increases trophoblast hCG secretion in a time- and dose-dependent manner, and this secretion was blocked by CGRP antagonist, CGRP8–37, and (v) both 17ß-estradiol (E2) and progesterone concentrations in the culture medium were increased by CGRP, and these increases were dose dependent. These observations suggest that CGRP may be involved in the morphological and functional differentiation of trophoblast cells, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.
Key words: CGRP/placenta/pregnancy/trophoblasts
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Introduction |
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Fetal trophoblast cells play an important role in the process of implantation and placentation in the human. With the progression of placentation, cytotrophoblasts differentiate in two pathways; the extravillous trophoblast cells undergo proliferation and migration to invade the maternal endometrium and myometrium and remodel the spiral arteries (Aplin, 1991
). Meanwhile, the villous cytotrophoblast cells undergo cellular fusion and differentiation to form syncytiotrophoblast (Potgens et al., 2002). The syncytiotrophoblast is a multinucleated continuous epithelial sheet extending over the entire villous surface. It is terminally differentiated and must be kept healthy by the continuous fusion of cytotrophoblast and the shedding of old nuclei through syncytial knot (Benirshke, 2000). The syncytiotrophoblasts are constantly bathed by the maternal blood from 
11 weeks of pregnancy and contribute to the majority of human placental transport, endocrine and immunoregulatory functions. Disturbed trophoblast fusion or abnormal turnover of trophoblast at the villous surface has been linked to pregnancy complications such as pre-eclampsia (Johansen et al., 1999). Therefore, the investigation of syncytial fusion in the human placenta is important to our understanding of maternal–fetal communication.
In vitro studies have shown that the villous differentiation is regulated by factors from extracellular matrix (Aplin et al., 2000; Minas et al., 2005). Epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), dexamethasone (Malassine and Cronier, 2002), estradiol (E2) (Cronier et al., 1999), leukaemia inhibitory factor (LIF) (Sawai et al., 1995) and hCG induce trophoblast differentiation (Morrish et al., 1987; Shi et al., 1993; Garcia-Lloret et al., 1994), whereas hypoxia, transforming growth factor ß1 (TGFß1) and endothelins inhibit trophoblast differentiation (Morrish et al., 1991; Alsat et al., 1996; Niger et al., 2004). However, the mechanisms regulating trophoblast fusion in vivo are not fully understood.
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide generated from alternative splicing of the primary RNA transcript of the gene-encoding calcitonin (CT)-CGRP (Wimalawansa, 1996). CGRP is a powerful endogenous vasodilator as well as an autocrine growth factor (Segond et al., 1992), which stimulates human umbilical vein endothelial cell proliferation (Haegerstrand et al., 1990). CGRP is widely distributed within nervous system tissues and is present in the sensory and autonomic nerve fibres, of which the sensory neurons show close relationship to blood vessels (Wimalawansa, 1996). During pregnancy, maternal plasma CGRP levels increase with gestational age (Stevenson et al., 1986), and fetal plasma CGRP concentration increases with infant birthweight (Parida et al., 1998), implying that CGRP might be involved in the maintenance of placental function and fetal growth.
CGRP acts at the cellular level by binding to a seven-transmembrane domain G-protein-coupled receptor (Wimalawansa, 1996). This receptor requires the coexpression of calcitonin receptor-like receptor (CALCRL) and a single transmembrane domain receptor activity modifying protein 1 (RAMP1) (McLatchie et al., 1998). Recent immunofluorescent studies have shown the presence of CALCRL and RAMP1 in trophoblast cells of human villous tissues (Dong et al., 2004) and the expression of CGRP protein in human decidua (Tsatsaris et al., 2002), which lacks innervation. This novel evidence leads us to postulate that one role of CGRP in the human placenta might be to regulate the differentiation of villous cytotrophoblast cells. This study tests this hypothesis using morphological, endocrinological and biochemical approaches.
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Materials and methods |
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Purification of villous term trophoblast cells
Placental tissues of term pregnancy (38–40 weeks, n = 18) were obtained from Caesarean sections. Use of tissues was approved by the Institutional Review Board at the University of Texas Medical Branch and conducted according to the Declaration of Helsinki Ethical Principles of 1973 (revised in 1983). Villous tissues were dissected free of membranes and vessels, rinsed and minced in Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS; GIBCO, Invitrogen, Grand Island, NY, USA). Cytotrophoblast cells were isolated after trypsin–DNase digestion and the discontinuation of Percoll gradient fractionation, using the method previously described by Kliman et al. (1986)
with modifications made by Levy et al. (2000) and Elchalal et al. (2004). The key step in the modification is that non-adherent cells and syncytial fragments were removed after 3–4 h by washing three times with culture medium. Briefly, pieces of term placental villous tissues were subjected to treatment with trypsin (0.125%)–DNase I (10 U/ml) (Sigma, St Louis, MO, USA). Cells were then resuspended in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (GIBCO), and layered over a Percoll gradient made from 70 to 5% Percoll (Sigma). Following centrifugation, the intermediate layer (1.048–1.062 g/ml) was carefully collected and plated on 100-mm culture dishes (3 x 104 cells/cm2). Three hours after being cultured at 37°C in humidified 5% CO2–95% air, the cells were carefully washed with culture medium to remove non-adherent cells (Levy et al., 2000). The purity of the isolated cells was 96% as tested by anti-cytokeratin 7 immunostaining (monoclonal antibody; DAKO Cytomation, Denmark). It is reported that cytokeratin 7 is the most tropho-specific cytokeratin known and is recommended as the marker of trophoblast population (Frank et al., 2001). The viability of the cells was >99% as assessed by the Trypan Blue exclusion test. The cells were plated in triplicate on 60-mm culture dishes (3 x 104 cells/cm2) in 3 ml of DMEM supplemented with 25 mM HEPES (Sigma), 20 mM glutamine, 10% fetal bovine serum, 100 IU/ml of penicillin and 100 µg/ml of streptomycin (Sigma).
Immunofluorescent staining
Purified isolated cytotrophoblast cells were cultured in Lab-Tek 2-well chamber slides (Nunc, Naperville, IL, USA) at 3 x 104 cells/cm2 for 24, 48 and 72 h in the presence or absence of CGRP (1 x 10–8 M). The cells fixed by 70% ice-cold acetone on chamber slides were exposed to cytokeratin 7 (mouse monoclonal, 1:200, DAKO Cytomation), CALCRL/RAMP1 (polyclonal, 1:200, produced by this laboratory) or desmosomal protein (mouse monoclonal, 1:100, Sigma), and they were incubated overnight in the cold room (4°C). After being washed with phosphate-buffered saline (PBS), the cells were incubated with the fluorescence-conjugated secondary antibody Alexa Fluor 488 (for cytokeratin 7 and desmosomal protein) or Alexa Fluor 594 (for CALCRL/RAMP1) (Molecular Probes, Eugene, OR, USA) at room temperature for 2 h. The slides were then rinsed with PBS, mounted using 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), covered with cover slips and viewed under an Olympus Microscope with Image ProPlus Software (Olympus Optical, Tokyo, Japan). The specificity of the affinity-purified primary polyclonal CALCRL and RAMP1 antibodies has been characterized by our previous work (Chauhan et al., 2004; Dong et al., 2004), which showed the linearity of the antibodies by loading increasing concentrations of the corresponding antigens in western blot analysis and only detecting bands of expected molecular weight. The respective bands were totally blocked by higher doses of antigens.
Radioimmunoassay of intracellular cAMP
Cells were cultured in 35-mm-well plates (3 x 104 cells/cm2) for 24 h and treated with various doses of CGRP (10–10–10–7 M) in the presence of 100 µM phosphodiesterase inhibitor and 3-isobutyl-1-methyl-xanthine (IBMX; Sigma) for 5 min. Cells treated with IBMX alone served as control. Reactions were terminated by replacing the medium with ice-cold ethanol. After brief sonication and centrifugation, the supernatant was concentrated in a speed vacuum pump and reconstituted in a 500-µl assay buffer. Cyclic AMP was quantified using cAMP 125I radioimmunoassay kit (The BiotractTM cAMP Assay System; Amersham Biosciences, Piscataway, NJ, USA) as per the manufacturer’s instructions. The cAMP standards (2–128 fmol/tube) and samples were acetylated by adding triethylamine/acetic anhydride (2:1 v/v, 5 µl/tube) before the assay. The assay is a standard competitive assay, and the results were expressed as femtomoles per cAMP million cells.
Syncytium formation assay
Syncytium formation was observed by immunostaining cells with antibody against desmosomal proteins (Sigma) as described by Douglas and King (1990). The staining of desmosomal proteins at the intercellular boundaries in aggregated cells progressively disappears, as the syncytium is formed. The nuclei contained in 100 syncytia in a random area were counted, and six slides were examined for each experimental condition. The results are expressed as the number of nuclei per syncytium.
Hormone assay
After 24 h of starvation in serum-free culture medium, cells were exposed to either varying doses of CGRP (10–10–10–7 M) for 48 h or 1 x 10–8 M CGRP for 24, 48 and 72 h. The culture medium was centrifuged, separated into aliquots and frozen for further determination of hCG, 17ß-E2 and progesterone. Media hCG levels were determined by immunoenzymetric assay (Alpha Diagnostics International, San Antonio, TX, USA). 17ß-E2 and progesterone concentrations in the media were measured by radioimmunoassay kits (Diagnostic Products, Los Angeles, CA, USA). Hormone productions were expressed as medium concentration per million cells for a given time. The cell number was counted before plating. Because trophoblast estrogen synthesis requires an androgen precursor, the effect of CGRP on trophoblast 17ß-E2 production was conducted with exogenous androgen in the form of androstenedione (0.025 µg/ml of androstenedine, 4-androsten-3,17-dione; Sigma).
Statistical analysis
Values were expressed as mean ± SEM. Testing of group values of cell number and hormone between treated and untreated control groups was determined by paired Student’s t-test. For multiple comparisons, the significance of the difference was tested by one-way analysis of variance (ANOVA), followed by post hoc test. P < 0.05 was considered statistically significant.
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Demonstration of trophoblast cell purity and viability
As assessed by cytokeratin 7-positive staining (Figure 1, green), 96% of the cells isolated and purified were trophoblast cells. Cell nuclei were counterstained with DAPI containing mounting medium (blue). The viability of the cells was determined by Trypan Blue exclusion test, which indicated that >99% of the trophoblast cells were alive.
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Existence of CGRP receptor components CALCRL and RAMP1 in the isolated trophoblast cells
As determined by immunofluorescent staining, CGRP receptor components CALCRL (A) and RAMP1 (B) were abundantly expressed by the primary cultured (24 h) cytotrophoblasts cells (red, Figure 2), indicating the presence of CGRP receptors on the cells. Replacement of the primary antibodies with pre-immune serum resulted in a negative staining (C), implying the specificity of the polyclonal antibodies.
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Induction of intracellular cAMP by CGRP exposure
To determine whether the CGRP receptor was coupled to adenylate cyclase in these purified isolated cytotrophoblast cells, we treated the cells with increasing concentrations of CGRP for 5 min. As shown in Figure 3, in the presence of IBMX (a cAMP phosphodiesterase inhibitor), CGRP (1 x 10–10–10–7 M) caused a dose-related increase in cAMP production, suggesting that adenylate cyclase activation may be involved in CGRP actions.
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Stimulation of syncytium formation in vitro by CGRP
Immunofluorescent staining with antidesmosomal antibody was identified at the boundaries between aggregated cytotrophoblast cells, and this staining disappeared when cells fused to form syncytiotrophoblast cells. As indicated by the disappearance of desmosomal immunostainings at the intracellular boundaries (Figure 4), multicellular aggregates are formed by 24 h of culture and become the dominant form by 48 h. At 72 h, the fusion occurred not only between the single cytotrophoblast but also between existing syncytia, leading to giant syncytia containing 10–20 nuclei. Compared with control (A1-3), CGRP exposure (B1-3) increases the number of syncytia and the number of nuclei per syncytium, indicating that CGRP may stimulate cytotrophoblast fusion and differentiation favouring placental maintenance and development.
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Quantitative analysis of CGRP-induced trophoblast morphological differentiation
To quantify the syncytium formation in control and treated cells, we scored 100 syncytia and counted the number of the nuclei in each syncytium. As shown in Figure 5A, no significant differences were observed in the number of syncytia with 3–5 or 6–8 nuclei between the CGRP-treated and control groups after 24 h of culture. Interestingly, after 48 h of CGRP exposure, the number of syncytium with more than nine nuclei was substantially increased compared with control groups (Figure 5B), and syncytia with 12–14 nuclei were apparent at 48 h only in CGRP-treated cultures. Meanwhile, the number of syncytia with 3–5 nuclei was substantially lower in CGRP groups, indicating the possibility that CGRP-induced trophoblast fusion occurred not only between single cytotrophoblasts but also between existing syncytia. Furthermore, at 72 h of CGRP exposure (Figure 5C), the number of syncytium with >12 nuclei was significantly increased compared with control, suggesting that CGRP stimulates human trophoblast cell morphological differentiation as evidenced by an increase in trophoblastic fusions.
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CGRP stimulates functional differentiation of trophoblast
As determined by immunoenzymetric assay, CGRP treatment increases trophoblast hCG production in a dose-dependent manner (Figure 6A). Further experiments demonstrated that CGRP time dependently increased trophoblast cell hCG production (Figure 6B), and this increase in hCG levels in cell culture medium was temporally associated with increased syncytium formation (Figure 4). In addition, both progesterone and 17ß-E2 production by trophoblast cells were dose dependently increased by CGRP (Figure 7A and B), suggesting that CGRP stimulates both morphological and functional differentiation of trophoblast cells. Meanwhile, CGRP antagonist, CGRP8–37, completely inhibited CGRP-induced trophoblast cell hCG, progesterone and E2 production (Figures 6A and 7A and B), indicating that CGRP actions on trophoblast hormone production were mediated via CGRP receptors.
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Discussion |
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During human pregnancy, the differentiation of cytotrophoblast into syncytiotrophoblast is a major determinant for placental development and fetal growth (Benirshke, 1990
). Although it is generally accepted that the cytotrophoblast layer of the placenta is the germinal zone from which syncytium is derived, the physiological mechanisms that control this differentiation are not fully understood. This study demonstrates that the incubation of cytotrophoblasts with CGRP for 48 h significantly enhanced the number of syncytium when compared with control. After 72 h of CGRP exposure, the number of syncytia with >12 nuclei was substantially increased compared with controls. Furthermore, coincident with the stimulation of syncytium formation, CGRP significantly increased hCG, progesterone and estrogen productions in these cells; these increases were completely inhibited by CGRP antagonist, CGRP8–37. Taken together, this study demonstrated that multifunctional peptide CGRP stimulates human villous cytotrophoblasts to aggregate and fuse to form multinucleated syncytiotrophoblasts that synthesize and secrete specific hormones required for fetal growth and development. This finding indicated that, in addition to the classical endocrine hormones and growth factors, CGRP can modulate the differentiation of trophoblast cells and thus play a role in human placental development.
Neuropeptides expressed by trophoblasts may play a role in regulating trophoblastic differentiation. Vasoactive intestinal peptide (VIP) mRNA was expressed by both cytotrophoblasts and syncytiotrophoblasts in first- and third-trimester human placentas (Marzioni et al., 2005), and VIP modulates the in vitro trophoblastic secretion of hCG and progesterone. Insulin-like growth factor-I (IGF-I) and its receptors are localized on both cytotrophoblasts and syncytiotrophoblasts (Maruo et al., 1995), and IGF-I stimulates the secretion of hCG and human placental lactogen (hPL) following the enhancement of the proliferative activity of trophoblasts, implying that neuropeptides may play a role in regulating trophoblast functional differentiations.
Evidence indicates that CGRP and its receptors are present at the human feto-maternal interface. Immunohistological analysis of first-trimester placental chorionic villi showed CGRP in decidual cells (Tsatsaris et al., 2002), and this was confirmed in cultured decidual cells by Southern blot analysis and immunohistochemistry and by radioimmunoassay in culture medium. CGRP receptor components CRLR and RAMP1 were detected by RT–PCR amplicons and immunohistochemistry from decidua and trophoblast of rat and human placenta (Dong et al., 2002; Tsatsaris et al., 2002), indicating that CGRP at the implantation site may act in a paracrine or autocrine manner. CGRP exhibits multifunctional properties in the human. CGRP stimulates the adhesiveness of human umbilical vein endothelial cells to isolated human neutrophils as well as to human monocytoid U937 cells in a dose- and time-dependent manner (Sung et al., 1992). In the study on embryonal teratocarcinoma cells, CGRP displayed a potent mitogenic and chemotactic property (Gerbaud et al., 1991), suggesting the involvement of CGRP in embryonic growth and development. Furthermore, CGRP has been demonstrated to stimulate the proliferation of human endothelial cells (Haegerstrand et al., 1990), implying that CGRP may be important for the formation of new vessels during physiological events such as implantation and placentation. This study provides evidence showing that CGRP stimulates human villous cytotrophoblast fusion and hCG and sex steroid production, suggesting that CGRP may be involved in the morphological and functional differentiation of the villous trophoblasts.
CGRP exhibits different effects on various systems, indicating the presence of several intracellular signalling pathways. In guinea pig ileum, CGRP-induced smooth muscle cell relaxation involves cAMP production (Rekik et al., 1997). In human colon smooth muscle cell, CGRP induces relaxation via both the cyclic guanosine monophosphate (cGMP) and cAMP pathways (Boyer et al., 1998). Cyclic AMP has been demonstrated to be involved in trophoblast differentiation. In primary cultures of human placental trophoblast cells, cAMP stimulates both aromatase activity and hCG secretion (Lobo and Bellino, 1989). Forskolin incubation caused a dose-dependent increase in intracellular and secreted cAMP and a co-ordinate fusion of trophoblast cells which yield syncytia containing hundreds of nuclei per cytoplasm (Wice et al., 1990). Furthermore, treatment of BeWo cells with 8-bromo-cyclic AMP increased the levels of hCG secretion and induced cell fusion leading to the formation of large syncytia (MacCalman et al., 1996). Our present study demonstrated that CGRP-stimulated human trophoblast cell differentiation is accompanied by an intracellular cAMP accumulation, implying that the effect of CGRP on trophoblast cell fusion and differentiation might be attributed to CGRP-induced intracellular cAMP production.
CGRP is primarily synthesized in the sensory neurons of dorsal root ganglia, which extends axons centrally to the spinal cord and peripherally to various organs (including blood vessels) and releases the peptide into the bloodstream (Wimalawansa, 1996). CGRP is present in the maternal circulation, and plasma CGRP levels rise during gestation (Stevenson et al., 1986). Recent immunohistochemical studies have shown the presence of CGRP in human decidua (Tsatsaris et al., 2002), indicating that this peptide appears to be locally produced at the human feto-maternal interface. Therefore, in addition to the circulating CGRP, the CGRP synthesized and released by decidual cells may stimulate trophoblast differentiation. Recent data revealed that in pre-eclampsia, CGRP levels in maternal and fetal circulation were lower (Halhali et al., 2001), and CGRP receptor expression in trophoblast cell was reduced compared with gestational age-matched control (Dong et al., 2005). We, therefore, postulated that the decrease in CGRP expression at the human feto-maternal interface may lead to a lower rate of cytotrophoblast fusion and an aged syncytium without enough new fusion events which lead to necrotic shedding seen in pre-eclampsia (Huppertz and Kingdom, 2004). In addition, as a potent vasodilator in both maternal and feto-placental circulation (Dong et al., 2005), CGRP may contribute to the maternal cardiovascular adaptation and low feto-placental vascular resistance in normal pregnancy. A decrease in CGRP levels may contribute to the increasing maternal blood pressure in pre-eclamptic pregnancies (Redman and Sargent, 2005).
In summary, we have demonstrated that CGRP is capable of inducing the morphological and functional differentiation of human villous cytotrophoblast cells in vitro. Although the cellular mechanisms by which CGRP promotes the formation of multinucleated syncytium remain poorly understood, our findings support a role for CGRP in the regulation and maturation of the villous cytotrophoblast at the term of pregnancy. This study reinforces the hypothesis that CGRP plays a role in the developmental regulation of functional differentiation of the trophoblast.
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Acknowledgements |
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The authors thank Mrs Cheryl R. Welch for administrative support and Elizabeth Martin for the assistance in the placental sample collections. For editorial and graphic assistance, we thank Ob/Gyn Publication, Grant and Media Support director and staff: R.G. McConnell, Kristi Barrett, John Helms and Mindy Loya. This study was supported by The National Institutes of Health Grants HL-70883, HL-58144.
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Submitted on December 7, 2005; resubmitted on May 12, 2006; accepted on May 16, 2006.
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