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Immunology |
Laboratory of Reproductive Immunology, Institute of Obstetrics and Gynecology, Fudan University, Shanghai 200011, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and analyzed the cell viability and signaling pathway. The results showed that rhSDF-1 increased the viability of trophoblast cells and the activation of extracellular signal-regulated kinases signaling pathway in vitro. Our findings suggest that first-trimester trophoblast cells express functional CXCR4/CXCL12, which may play an important role in early pregnancy such as stimulating trophoblast cell proliferation or differentiation in an autocrine manner.
cytokines, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines constitute a family of closely related chemoattractant cytokines. Several groups of chemokines have been described and two major groups can be distinguished based on the presence (CXC) or absence (CC) of a single amino acid between two cysteine residues near the N-terminus. Chemokine activity is mediated through cell surface receptors that are related to the 7-transmembrane-domain (7-TMD) receptor family. Although chemokine receptors are predominantly expressed by hematopoietic cells, certain types are also expressed by endothelial cells, epithelial cells, and neural cells. Chemokines and chemokine receptors have been implicated as pivotal players in many physiological and pathological situations. Some of the notable examples are their involvement in processes such as trafficking of immune cells to multiple target organs, central nervous system development, and HIV pathogenesis [9– 12]. Also, chemokines can modulate enzyme secretion, cell adhesion, cytotoxicity, tumor cell growth, degranulation, and T-cell activation [13, 14].
Like tumor cells, first-trimester trophoblast cells are able to proliferate, migrate, and invade. Therefore, it is interesting to speculate that chemokines at the materno-fetal interface may interact with chemokine receptors expressed by trophoblast cells so as to regulate the growth, differentiation, and functioning of the placenta in paracrine or autocrine manners. The expression of chemokine receptors by placenta, and particularly trophoblast cells, may also provide some useful insights into the cellular mechanisms of trans-placental HIV-1 infection and the prevention of vertical intrauterine transmission [15].
In this study, we first analyzed the transcription of chemokine receptors in freshly isolated first-trimester trophoblast cells. Among 18 chemokine receptors, CXCR4 and CXCR6 were found highly transcribed. As a chemokine receptor extraordinaire, CXCR4 and its specific ligand CXCL12 (stromal cell-derived factor-1; SDF-1) play an important role in hematopoiesis and developmental processes. In cells expressing both CXCR4 and CXCL12, CXCR4/ CXCL12 activation may stimulate cell proliferation in autocrine or paracrine manners [16, 17]. Therefore, we furthermore detected the expression of CXCR4 and CXCL12 in trophoblast cells by in situ hybridization, immunocytochemistry, and immunohistochemistry, treated trophoblast cells with recombinant human (rh)SDF-1 and analyzed the cell viability/proliferation and signaling pathway to identify the functional role of CXCR4/CXCL12 in trophoblast cells. Our findings demonstrate that first-trimester human trophoblast cells express functional CXCR4/CXCL12, which increase trophoblast cell viability and the activation of ERK1/ 2 signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First-trimester human placentas (gestational age, 5–10 wk) were obtained from clinically normal pregnancies, which were terminated for nonmedical reasons, at the Obstetrics and Gynecology Hospital of Fudan University. Each patient completed a signed, written consent form. The Fudan University Human Investigation Committee approved the form and the use of the placental tissue.
Trophoblast Cell Culture
First-trimester human placentas were separated carefully from the deciduas under a stereomicroscope, minced into small fragments, and incubated in Hanks balanced salt solution (HBSS) containing 0.25% trypsin (Life Technologies, Merelbeke, Belgium), 4.2 mM MgSO4, 25 mM HEPES, and 50 Kunits/ml DNase type IV (Life Technologies) for 10 min at 37°C with gentle agitation. Then the suspension was filtered (100-µm pores). Trypsin digestion was stopped with 10% fetal calf serum (FCS; Hyclone, Logan, UT). The remaining placental tissues were subject to another three cycles of 10-min trypsinization. The four resultant cell suspensions were pooled, centrifuged at 300 x g for 10 min, and resuspended in 4 ml DMEM (Dulbecco modified Eagle medium high D-glucose; Life Technologies). This suspension was layered over a preformed Percoll gradient (Pharmacia, Uppsala, Sweden) made up in HBSS. The gradient was made from 70% to 5% Percoll (vol/vol) in 5% steps of 2 ml each by dilutions of 90% Percoll with HBSS. The gradient was centrifuged at 1000 x g for 20 min. The middle layer (density of 1.048–1.062 g/ml) was removed and washed with DMEM. Cells were diluted with DMEM supplemented with 2 mM glutamine, 10% heat inactivated FCS, 25 mM HEPES, 100 UI/ml penicillin, and 100 µg/ml streptomycin.
The isolated human trophoblast cells were plated in plastic Petri dishes and incubated at 37°C for 40 min to allow the contaminating macrophages to adhere to the plastic. The nonadherent trophoblast cells were transferred to fresh plates as well as eight-chambered LabTek slides at a concentration of 2–4 x 105 cells/ml. Cells were cultured in DMEM supplemented with 2 mM glutamine, 10% heat-inactivated FCS, 25 mM HEPES, 100 UI/ml penicillin, and 100 µg/ml streptomycin at 37°C in 95% air and 5% CO2.
Reverse Transcription-Polymerase Chain Reaction Analysis
Trophoblast cells were isolated from pools of first-trimester placentas. Total cellular RNA was extracted from freshly purified trophoblast cells using Trizol reagent (Life Technologies, Merelbeke, Belgium).
One microgram total RNA was denatured, and reverse transcription was performed for 1 h at 42°C with 0.5 µg oligo(dT)18, 1.0 mM 4dNTP, 20 U RNasin Ribonuclease inhibitor (Promega, Madison, WI), 200 U Moloney virus-reverse transcriptase (Superscript II; Life Technologies, Paisley, United Kingdom), and 5x reaction buffer in a total of 20 µl. Amplification was performed with 5 µl cDNA, 0.2 mM dNTP, 1 mM MgCl2, 2.5 U AmpliTaq DNA Polymerase (Perkin-Elmer, Norwalk, CT), 0.8 mM specific sense and antisense primers, and 10x reaction buffer in a 50-µl reaction volume. The primers used for the detection of chemokine receptors and housekeeping gene GAPDH were indicated in Table 1. After 5 min precycle at 95°C, the reaction was followed by 30 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. When the final cycle was over, samples were kept at 72°C for 15 min to complete the synthesis. Polymerase chain reaction (PCR) reaction products were analyzed on 1.5% agarose gels and ethidium bromide-stained bands were photographed. The experiments were repeated three times.
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Immunocytochemistry
After 24 h of culture, trophoblast cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS, and permeabilized for 4 min in 0.3% Triton X-100-PBS. Cells were incubated with 7% goat serum in PBS for 30 min to reduce nonspecific binding. Primary antibodies diluted in PBS containing 1% BSA were added. Anti-human cytokeratin and anti-human vimentin monoclonal antibody (Sino-America Co. Ltd., Shanghai, China) were used as markers for cells of trophoblast lineage and nontrophoblast lineage, respectively. Anti-human CXCR4 monoclonal antibody (R&D Systems, Abingdon, U.K.) was also administered to detect whether trophoblast cells express CXCR4. After incubation with primary antibody overnight at 4°C, cells were washed in PBS-0.1% Tween, then incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Sino-America Co. Ltd.) for 2 h at room temperature. Slides were stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. The experiments were repeated five times.
In Situ Hybridization
Human CXCL12 and CXCR4 in situ hybridization kits were obtained from Boshide Co. Ltd. (Wuhan, China). The oligonucleotide probes correspond to three sequences in CXCL12 mRNA (5'-AGGTCGTGGTCGTGCTGGTCCTCGTGCTGA-TCAAGCATCTCAAAATTCTCAACACTCCAA-CAGGAGTACCTGGAGAAAGCTTTAAACAAG-3') and in CXCR4 mRNA (5'-CACCATCTACTCCATCATCTTCTTAACTGG-GATATATCTGTGACCGCTTCTACCCCAATG-TCCTTGGAGC CAAATTTAAAACCTCTGCCC-3'), respectively. Five first-trimester placentas collected for in situ hybridization were immediately fixed with 4% paraformaldehyde for 1 h at 4°C. They were then permeated with 12% sucrose and passed through an increasing concentration gradient of sucrose up to 18%. The specimens for frozen section were embedded with embedding compound, frozen at –80°C, and stored until further analysis. Frozen sections (8 µm thick) were prepared by a cryostat and treated with 10 µg/ml of proteinase K. Slides were incubated for 2 h at 37°C in a prehybridization buffer. Hybridization was performed under coverslips overnight at 40°C. Following hybridization, the coverslips were removed and slides were rinsed three times for 10 min with 2x SSC, then treated with 20 µg/ml RNase in 0.5 M sodium chloride and 10 mM Tris (pH 8.0) for 30 min at 37°C to digest nonhybridization RNA probe. They were then washed three times for 20 min in 0.1x SSC at 42°C. Bound mRNA was detected with alkaline-phosphatase-labeled antidigoxigenin goat antibody and developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium color development substrate.
Immunohistochemical Staining
Five first-trimester placentas were collected for immunohistochemical staining. From the frozen specimens, serial sections of 8 µm thickness were prepared in a cryostat and fixed with cold acetone for 5 min. They were then blocked with methanol containing 3% H2O2, sequentially 7% goat normal serum, and incubated with anti-CXCR4, anti-CXCL12 monoclonal antibody (R&D Systems) overnight at 4°C. They were then sequentially treated with a biotinylated rabbit anti-mouse antibody and HRP- labeled streptoavidin. Development was performed by treating the sections with a Liquid DAB-Plus Substrate kit (Sino-America Co. Ltd.). After counterstaining with hematoxylin, immunostaining of CXCR4 and CXCL12 on the tissue sections was detected by light microscopy.
ELISA
Supernatants of trophoblast cell cultures were harvested after 12, 24, 36, 48, and 60 h of culture. Each supernatant was centrifuged at 2000 x g and stored at –80°C. Enzyme-linked immunosorbent assays were performed with human SDF-1 kit (R&D Systems) according to the instructions of the manufacturer. The SDF-1 assay demonstrated a sensitivity of 18 pg/ml and an intraassay coefficient of variation of between 3.4% and 3.9%. Five individual samples were tested.
Cell Viability/Proliferation Assays
Two different techniques, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma Chemicals, St. Louis, MO) assay and counting of viable cell numbers, were applied to measure the effect of CXCL12 on trophoblast cell viability/proliferation. Isolated trophoblast cells were resuspended in DMEM with 10% FBS and seeded at a density of 3 x 104 cells/well in 96-well, flat-bottom microplates or 3 x 105 cells/ ml in plates. After 24 h of culture, the medium was replaced with DMEM containing 1% FBS, and then the culture carried on for 12 h before treatment. The medium was removed once again, and the cells were stimulated with recombinant human SDF-1 (rhSDF-1, R&D Systems), 100 ng/ml anti-CXCL12 monoclonal antibody, 100 ng/ml anti-CXCR4 monoclonal antibody, respectively, or stimulated with the combinations of rhSDF-1 and anti-CXCL12 or anti-CXCR4 antibody, at 37°C for 48 h. To investigate whether the activation of ERK1/2 was responsible for the effect of rhSDF-1 on trophoblast cells, PD98059 (50 µM; New England Biolabs, Beverly, MA) was added to some wells during the chemokine stimulation.
Following the treatment, trypan blue exclusion test was carried out to determine the viable cells, and cell numbers were counted with a Neubauer chamber hemocytometer, with quadruplicate counts performed for each treatment condition. The result was expressed as the ratio of the number of viable cells with treatment to that without treatment. The experiments were repeated four times.
For the MTT assay, 20 µl of the MTT reagent was added to each well of 96-well microplates and incubated at 37°C for 4 h. The medium was decanted, and 100 µl of acid-isopropyl alcohol was added to solubilize the reactive crystals. Absorbency was measured at a wavelength of 540 nm on an automatic microplate reader. The values of the treated cells were compared with the values generated from the untreated control cells and reported as the percentage viability. The experiments were repeated four times.
Western Blot
Western blot analysis was performed using anti-phospho-ERK1/2 and anti-ERK1/2 (total) antibodies (Cell Signaling Technology, Beverly, MA). After having been cultured in DMEM containing 10% FBS for 24 h, trophoblast cells were starved in DMEM supplemented with 1% FBS for 12 h and then stimulated with 100 ng/ml rhSDF-1 for the indicated period of time at 37°C. The cells were lysed in 1% NP-40, 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml Aprotinin, and 0.1% SDS for 10 min at 4°C. Nuclei were removed by centrifugation at 12 000 x g at 4°C for 10 min, and cell lysates were assayed for protein contents using the Bradford protein assay. Proteins (50 µg) were resuspended in sample buffer (2% SDS, 62.5 mM Tris, pH 6.8, 0.1% bromophenol blue and 2.5% 2-mercaptoethanol, 10% glycerol), separated on 10% SDS-polyacrylamide gels. The proteins were transferred to a nitrocellulose membrane by electrotransfer for 1 h. After being soaked in blocking buffer (1x TBS with blocking reagent [5% milk]), the membrane was incubated with primary antibody overnight at 4°C. Blots were developed using the HRP-linked secondary antibody and a chemiluminescent detection system. The experiments were repeated three times.
Statistical Analysis
All values were expressed as the mean ± SD. Data from the counting of cell numbers and MTT assay were performed using analysis of variance (one-way ANOVA), with application of the Dunnett test. Differences were accepted as significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 24 h of culture, we characterized the expression of cytokeratin and vimentin in these cells. As shown in Figure 1A, the cells we isolated were almost all stained for cytokeratin, whereas in Figure 1B, no cells were found stained with antivimentin antibody. We observed that the purity of isolated trophoblast cells was above 95%.
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Transcription of Chemokine Receptors in First-Trimester Trophoblast Cells
Using PCR primers specific for human chemokine receptors, we have analyzed the transcription of chemokine receptors in freshly isolated first-trimester trophoblast cells. Three independent experiments were done (including 15 placental samples), and the results were similar. Trophoblast cells transcribed CCR1, CCR3
CCR5, CCR8CCR9, CXCR1CXCR4, CXCR6, XCR1, and CX3CR1, and the ratios of the band intensities of chemokine receptors to that of GAPDH were 0.397 ± 0.049, 0.437 ± 0.056, 0.284 ± 0.056, 0.593 ± 0.071, 0.801 ± 0.122, 0.198 ± 0.023, 0.702 ± 0.100, 0.807 ± 0.113, 0.921 ± 0.098, 1.083 ± 0.110, 1.120 ± 0.246, 0.849 ± 0.150, and 0.725 ± 0.151, respectively. Among these chemokine receptors, CXCR4 and CXCR6 were highly transcribed in trophoblast cells (the band intensities of these two chemokine receptors were higher than that of GAPDH in each experiment). The mRNA of CCR2, CCR6, CCR7, CCR10, and CXCR5 were never detected in trophoblast cells (Fig. 2).
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Expression of CXCR4 and CXCL12 in First-Trimester Trophoblast Cells
After identifying CXCR4 transcription in isolated first- trimester trophoblast cells by reverse transcription-PCR (RT-PCR), we detected CXCR4 mRNA in tissue sections by in situ hybridization. Figure 3A shows that CXCR4 mRNA was localized in the trophoblast cells. We also analyzed the CXCR4 expression in trophoblast cells cultured for 24 h by immunocytochemistry. The results showed that the cytoplasm and cytomembrane of trophoblast cells were stained for CXCR4 as seen in Figure 1C. In addition, immunohistochemical staining for CXCR4 in the first-trimester placental tissues showed specific brown-colored staining in the cytoplasm and cytomembrane of villous cytotrophoblasts, syncytiotrophoblasts, and extravillous cytotrophoblasts (Fig. 4).
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Because CXCR4 and its specific ligand CXCL12 play an important role in many physiological and pathological situations, it is interesting to determine whether trophoblast cells also express CXCL12. In situ hybridization using antisense probe for CXCL12 revealed that CXCL12 mRNA was localized in the villous cytotrophoblasts, syncytiotrophoblasts, and extravillous cytotrophoblasts, where intense hybridization signal was seen. In control experiments, sense-strand RNA probe for CXCL12 did not hybridize to the placental cells (Fig. 3). Immunohistochemistry also confirmed that the first-trimester trophoblast cells express CXCL12 (Fig. 4C). Furthermore, we measured soluble SDF-1 levels in the culture medium. The supernatant medium of trophoblast cell cultures, which derived from five irrelevant first-trimester placentas, produced similar levels of SDF-1. The SDF-1 in supernatant medium cumulated over time, and its concentration was 334.8 ± 56.9 pg/ml after trophoblast cells had been cultured for 60 h (Fig. 5).
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Effect of rhSDF-1 on Trophoblast Cell Viability/Proliferation
The capacity of rhSDF-1 to affect trophoblast cell viability/proliferation was investigated by MTT assay and counting the cell numbers. Primary cultured first-trimester trophoblast cells were serum-starved for 12 h and then treated with increasing concentrations of rhSDF-1 (1–200 ng/ml) for 48 h. Both MTT assay and counting of cell numbers demonstrated that rhSDF-1 increased the number of viable trophoblast cells in a dose-dependent manner, and the absorbance revealed in MTT assay strongly correlated to the viable cell number (Fig. 6). Strikingly, addition of antibody for CXCL12 or CXCR4 completely neutralized the stimulatory effect of exogenously administered CXCL12. If rhSDF-1 was not given and trophoblast cells were treated with CXCL12 antibody or CXCR4 antibody alone, the viability of these cells was significantly lower than that of the cells not treated (Fig. 6C).
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CXCR4/CXCL12 Affect Trophoblast Cell Viability/ Proliferation Through the Activation of ERK1/2 Pathway
To study the intracellular pathway mediating the trophoblast cell viability/proliferation by rhSDF-1, we focused our attention on the activation of mitogen-activated protein kinases (MAPKs), which converts extracellular stimuli to intracellular signals that control gene expression, cell proliferation, and differentiation. In leucocytes and CXCR4 transfected cells, CXCL12 signaling induced phosphorylation of several cellular proteins, including MAPKs. We first measured the viability of cells in the presence of PD98059, which is a specific mitogen-activated ERK inhibitor. MTT assay showed that PD98059 completely inhibited the increasing viability induced by rhSDF-1 in trophoblast cells, and the cell viability was even lower than that of untreated cells (Fig. 6C). Based on this finding, we next, using phospho-specific antibodies, directly analyzed ERK1/2 activation in trophoblast cells treated with rhSDF-1 by Western blot assay. As shown in Figure 7, after being serum-starved for 12 h, trophoblast cells still had phosphorylated ERK1/ 2 forms without CXCL12 treatment. Phosphorylation of ERK1/2 increased significantly after rhSDF-1 was administered 10 min later. The peak of ERK1/2 phosphorylation lasted up to 30 min and then slowly declined, although after 2 h of treatment, it was still above the basal level. The analysis of lysates for the total expression of ERK1/2 ensured the equal loading of proteins in different lanes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we analyzed the transcription of 18 chemokine receptors in first-trimester trophoblast cells and found CXCR4 and CXCR6 mRNA to be highly expressed. Using immunocytochemistry, we confirmed CXCR4 expression in primary cultured trophoblast cells. In situ hybridization and immunohistochemical examination of cryosections of first-trimester placentas also revealed CXCR4 expression by trophoblast cells.
CXCR4 is an extraordinary chemokine receptor. Apart from its important impact on HIV-1 infection, CXCR4 and its specific ligand CXCL12 play a key role in lymphocyte trafficking and recruitment at sites of inflammation and in hematopoiesis and developmental processes such as organogenesis, vascularization, and embryogenesis [20, 21]. Mice that lack the CXCL12 gene die in utero with severe heart defects and have fewer B-cell progenitors, suggesting defects in the formation of leukocytes. In addition to defects in hematopoiesis and cardiogenesis and fetal lethality in utero, mice lacking the CXCR4 gene have defective formation of large vessels supplying the gastrointestinal tract, which may be due to defective vascular branching [22]. Recently, Bajetto et al. [16] have demonstrated that cultured astrocytes, which express CXCR4, are able to release CXCL12 after lipopolysaccharides treatment, and the concomitant expression of CXCR4 and CXCL12 lead to autocrine and paracrine regulation of cell growth. Human thyroid carcinoma cell lines ARO also express both CXCR4 and CXCL12. Migration of ARO cells is CXCL12 dependent in vitro and requires mainly the Gi-coupled ERK1 and ERK2 MAPK pathway [23]. Because, in our experiments, CXCR4 was expressed in trophoblast cells, we think it is interesting to test whether or not these cells also produce CXCL12. In situ hybridization and immunohistochemical analysis revealed that first-trimester trophoblast cells constitutively express CXCL12. In addition, primary cultured trophoblast cells continued to secrete soluble SDF-1 into the culture medium. The SDF-1 in supernatant medium cumulated over time and its concentration was 334.8 ± 56.9 pg/ml after trophoblast cells had been cultured for 60 h.
CXCL12 functions as a chemotactic factor for B and T lymphocytes and endothelial cells, stimulating cell migration through inducing reorganization of the actin cytoskeleton [24]. It is also identified as a growth factor for murine pre-B cells in the presence of interleukin-7 and, recently, it has been reported that CXCL12 develops the proliferative action of thrombopoietin in megakaryocytic progenitor cells and costimulates the proliferation of anti-CD3-activated CD41 T-cells [25, 26]. In human arterial endothelial cells, CXCL12 induces gene expression of early growth response-1 (Egr-1) and vascular endothelial growth factor (VEGF), and enhances VEGF-induced cell proliferation [27]. CXCL12 also directly stimulates the proliferation of cultured astrocytes, which strongly suggests that this chemokine might behave as a growth factor. More interestingly, Hall and Korach have demonstrated that CXCL12 represents a common component of estrogen-estrogen receptor signaling in both ovarian and breast cancer cells and that CXCL12 mediates the mitogenic effects of estradiol in these cells [28]. In our study, we evaluated whether or not rhSDF-1 has an effect on trophoblast cell survival, and the results indicated that rhSDF-1 increases the viability of serum-starved trophoblast cells in a dose-dependent manner, and this effect is inhibited by antibody to CXCL12 or CXCR4, if added. When trophoblast cells were treated with anti-CXCL12 or anti-CXCR4 antibody alone, the cell viability was significantly lower than that of control, which suggests that anti-CXCL12 or anti-CXCR4 antibody inhibits the functioning of endogenic CXCL12 secreted by trophoblast cells.
It is well known that CXCR4 triggers multiple intracellular signals in response to CXCL12, especially calcium mobilization and phosphorylation of ERK1/2 [29, 30]. From invertebrates to humans, the ERK MAP kinase module plays a central role in signaling cell growth, differentiation, and survival [31, 32]. The fidelity of signaling and the spatiotemporal activation are key determinants in generating precise biological responses. For example, in PC12 cells, nerve growth factor stimulation causes sustained activation of ERK, then induces cell differentiation, whereas epithelial growth factor stimulation induces cell proliferation due to transient activation of ERK [33]. With the generation of knockout mice for each of the ERK isoforms, Pages et al. illustrate that, besides controlling cell proliferation, the ERK cascade also controls cell differentiation and cell behavior [34]. ERK1–/– mice are viable, fertile, and of normal size, but there is a defect in thymocyte terminal differentiation in these animals [34]. On the contrary, disruption of the ERK2 locus leads to embryonic lethality early in mouse development after the implantation stage. ERK2 mutant embryos fail to form the ectoplacental cone and extraembryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus [31]. In this study, we investigated whether CXCL12 increases trophoblast cell viability through the activation of the ERK1/2 pathway. The effect that rhSDF-1 increases trophoblast cell viability in vitro is strongly inhibited by PD98059, which indicates that CXCR4 expressed in these cells functions through the MAPK signaling pathway. Moreover, we directly demonstrated the regulation of the ERK1/2 activity by rhSDF-1. Western blot analysis showed that rhSDF-1 caused a rapid and long-lasting (up to 120 min) activation of ERK. Interestingly, we have observed that a spontaneous activation of ERK1/2 was also evident in trophoblast cells after 12 h of serum starvation, suggesting that ERK1/2 may also be an important component of the basal trophoblast cell proliferation or differentiation. This finding coincides with the result from a recent study by Kita et al. that phosphorylated ERK1/2 is expressed in first-trimester villous cytotrophoblasts but not in syncytiotrophoblasts [35]. Because CXCL12 contributes to the cell viability and activates the ERK1/2 signaling pathway, we are of the view that endogenic CXCL12 may be at least partially responsible for the spontaneous activation of MAP kinase in these cells, which deserves to be explored further. In addition, CXCL12 may exert some different effects on trophoblast cells depending on the degree of cellular differentiation or the presence of other active cytokines and growth factors, although CXCR4/CXCL12 are expressed both in villous cytotrophoblasts and in syncytiotrophoblasts.
The expresssion of CXCR4/CXCL12 in trophoblast cells also provides some useful insights into the cellular mechanisms of transplacental HIV-1 infection. Despite the continuous circulation of HIV-1 and HIV-1-infected cells in the mothers during pregnancy, only a few infants are infected in utero, and most of these mother-to-child transmissions of HIV-1 occur during labor. More interestingly, syncytiotrophoblasts, which are in direct contact with the maternal blood, apparently select the transmission of R5 HIV strains (use the CCR5 as a coreceptor), but not X4 HIV strains (use the CXCR4 as a coreceptor), though they express CXCR4 during the pregnancy [18, 36, 37]. The reasons for the paradoxically low frequency of HIV transmission during pregnancy and selective transplacental transmissions are not fully understood. Because CXCL12 is constitutively expressed by syncytiotrophoblasts and the primary cultured trophoblast cells can spontaneously secrete CXCL12, we postulate that CXCL12 inhibits X4 HIV from infecting syncytiotrophoblasts, as mucosally derived CXCL12 could reduce X4 HIV transmission across mucosal sites [38].
In summary, we have analyzed the transcription of all identified chemokine receptors in first-trimester trophoblast cells. Among these chemokine receptors, CXCR4 mRNA was highly transcribed. We also confirmed that both CXCR4 and its specific ligand CXCL12 are expressed in trophoblast cells. Our experiments further demonstrated that CXCL12 increases trophoblast cell viability through the activation of the ERK1/2 pathway. These data suggest that CXCR4/CXCL12 play an important role in early pregnancy, such as stimulating trophoblast cell proliferation or differentiation in an autocrine manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 0086 21 63770768; djli{at}shmu.edu.cn
Received: 29 October 2003.
First decision: 23 November 2003.
Accepted: 10 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Y. Huang, X.-Y. Zhu, M.-R. Du, X. Wu, M.-Y. Wang, and D.-J. Li Chemokine CXCL16, a scavenger receptor, induces proliferation and invasion of first-trimester human trophoblast cells in an autocrine manner Hum. Reprod., April 1, 2006; 21(4): 1083 - 1091. [Abstract] [Full Text] [PDF]
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V. M. Abrahams, I. Visintin, P. B. Aldo, S. Guller, R. Romero, and G. Mor A Role for TLRs in the Regulation of Immune Cell Migration by First Trimester Trophoblast Cells J. Immunol., December 15, 2005; 175(12): 8096 - 8104. [Abstract] [Full Text] [PDF]
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