Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein.

We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI™ and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.

Human trophoblast in trisomy 21: a model for cell-cell fusion dynamic investigation.

Trophoblastic cell fusion is one essential step of the human trophoblast differentiation leading to formation of the syncytiotrophoblast, site of the numerous placental functions. This process is multifactorial and finely regulated. Using the physiological model of primary culture of trophoblastic cells isolated from human placenta, we have identified different membrane proteins directly involved in trophoblastic cell fusion: connexin 43, ZO-1 and recently syncytins. These fusogenic membrane retroviral envelop glycoproteins: syncytin-1 (encoded by the HERV-W gene) and syncytin-2 (encoded by the FRD gene) and their receptors are major factors involved in human placental development. Disturbances of syncytiotrophoblast formation are observed in trisomy 21-affected placentas. Overexpression of the copper/zinc superoxide dismutase (SOD-1), encoded by chromosome 21 as well as an abnormal hCG signaling are implicated in the defect of syncytiotrophoblast formation. This abnormal trophoblast fusion and differentiation in trisomy 21-affected placenta is reversible in vitro by different ways.

ZO-1 is involved in trophoblastic cell differentiation in human placenta.

Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.

Trisomy 21- affected placentas highlight prerequisite factors for human trophoblast fusion and differentiation.

Trophoblastic cell fusion is one essential step of the human trophoblast differentiation pathway and is a multifactorial and dynamic process finely regulated and still poorly known. Disturbances of syncytiotrophoblast formation are observed in numerous pathological clinical conditions such as preeclampsia, intrauterine growth retardation and trisomy 21. In this review, we summarize current knowledge of the different membrane proteins directly involved in trophoblastic cell fusion, which we identified by using the physiological model of primary culture of villous trophoblastic cells. Connexin 43 and gap junctional intercellular communication point to the role of molecular exchanges through connexin channels preceding membrane fusion. Zona occludens-1, which can interact with connexin 43, is also directly involved in trophoblast fusion. The recently identified fusogenic membrane retroviral envelop glycoproteins syncytin 1 (encoded by the HERV-W gene) and syncytin 2 (encoded by the FRD gene) and their receptors are major factors involved in human placental development . We describe the increasing number of factors promoting or inhibiting trophoblast fusion and differentiation and emphasize the role of human chorionic gonadotropin (hCG) and its receptor. Indeed, in trisomy 21 the dynamic process leading to membrane fusion is impaired due to an abnormal hCG signaling. This abnormal trophoblast fusion and differentiation in trisomy 21-affected placenta is reversible in vitro. Trisomy 21 trophoblastic cell culture may therefore be useful to identify the possible large number of prerequisite factors involved in trophoblast fusion, the limiting step of trophoblast differentiation.

Development and hormonal functions of the human placenta.

The human placenta is characterized by the intensity of the trophoblast invasion into the uterus wall and the specificity of its hormonal functions. Placental hormones are required for the establishment and maintenance of pregnancy, adaptation of the maternal organism to pregnancy and fetal growth. In the early placenta at the maternofetal interface, the human trophoblast differentiates along two pathways: 1/ the villous trophoblast pathway including the cytotrophoblastic cells which differentiate by fusion to form the syncytiotrophoblast that covers the entire surface of the villi; 2/ the extravillous trophoblast pathway. The cytotrophoblastic cells of the anchoring villi in contact with the uterus wall proliferate and then migrate into the decidua and the myometrium but also participate to the remodeling of the spiral arteries. During the first trimester of pregnancy the spiral arteries are plugged by trophoblastic cells, allowing the development of the fetoplacental unit in low oxygen environment. At this stage of pregnancy the extravillous trophoblast secretes a large amount of hormones such as particular hyperglycosylated forms of hCG directly involved in the quality of the placentation. At 10-12 weeks of pregnancy, the trophoblastic plugs are progressively dislocated and the syncytiotrophoblast starts to bath in maternal blood. It secretes the major part of its polypeptide hormones in maternal circulation taking over the maternal metabolism in order to increase the energetic flux to the fetus. As example the placental GH (growth hormone) secreted continuously by the syncytiotrophoblast is directly involved in the insulino-resistance of pregnancy. Capturing the cholesterol from the maternal lipoproteins, the syncytiotrophoblast synthesizes also large amount of progesterone essential for the uterine quiescence. Deprived of cytochrome P450 17alpha-hydroxylase-17:20 lyase, it uses the maternal and fetal adrenal androgens to synthesize estrogens. The differentiation and hormonal functions of the human trophoblast are regulated by the environmental O2 and reflect mammalian evolution.

Human endogenous retrovirus-FRD envelope protein (syncytin 2) expression in normal and trisomy 21-affected placenta.

Human trophoblast expresses two fusogenic retroviral envelope proteins, the widely studied syncytin 1, encoded by HERV-W and the recently characterized syncytin 2 encoded by HERV-FRD. Here we studied syncytin 2 in normal and Trisomy 21-affected placenta associated with abnormal trophoblast differentiation. Syncytin 2 immunolocalization was restricted throughout normal pregnancy to some villous cytotrophoblastic cells (CT). During the second trimester of pregnancy, syncytin 2 was immunolocalized in some cuboidal CT in T21 placentas, whereas in normal placentas it was observed in flat CT, extending into their cytoplasmic processes. In vitro, CT isolated from normal placenta fuse and differentiate into syncytiotrophoblast. At the same time, syncytin 2 transcript levels decreased significantly with syncytiotrophoblast formation. In contrast, CT isolated from T21-affected placentas fused and differentiated poorly and no variation in syncytin 2 transcript levels was observed. Syncytin 2 expression illustrates the abnormal trophoblast differentiation observed in placenta of fetal T21-affected pregnancies.

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration.

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

Human placenta as an endocrine organ.

The placenta is a unique, autonomous and transient organ. It ensures maternal-fetal exchanges and is also involved in maternal tolerance of feto-paternal antigens. The human placenta is characterized by the major invasion of the trophoblast, which comes in contact with the maternal blood, and by the intensity and the specificity of its endocrine functions. Placental hormones are required for the establishment and maintenance of pregnancy, adaptation of the maternal organism to pregnancy, fetal growth and well being, and development of the mechanisms involved in parturition. The endocrine tissue of the placenta is the syncytiotrophoblast, which covers the chorionic villi, and arises from the fusion of the cytotrophoblasts. In this review we will summarize the particulars of human syncytiotrophoblast development and endocrine functions.

Involvement of connexin 43 in human trophoblast cell fusion and differentiation.

The syncytiotrophoblast is the principal component of the human placenta involved in feto-maternal exchanges and hormone secretion. The syncytiotrophoblast arises from the fusion of villous cytotrophoblasts. We recently showed that functional gap junctional intercellular communication (GJIC) is an important prerequisite for syncytiotrophoblast formation and that connexin 43 (Cx43) is present in cytotrophoblasts and in the syncytiotrophoblast. To determine whether Cx43 is directly involved in trophoblast fusion, we used an antisense strategy in primary cultures of human villous cytotrophoblasts that spontaneously differentiate into the syncytiotrophoblast by cell fusion. We assessed the morphological and functional differentiation of trophoblasts by desmoplakin immunostaining, by quantifying hCG (human chorionic gonadotropin) production and by measuring the expression of specific trophoblast genes (hCG and HERV-W). Furthermore, we used the gap-FRAP (fluorescence recovery after photobleaching) method to investigate functional GJIC. Cytotrophoblasts treated with Cx43 antisense aggregated and fused poorly. Furthermore, less HERV-W env mRNA, hCGbeta mRNA and hCG secretion were detected in Cx43 antisense-treated cytotrophoblasts than in cells treated with scrambled antisense. Treatment with Cx43 antisense dramatically reduced the percentage of coupled trophoblast cells. Taken together, these results suggest that Cx43 is directly involved in human trophoblast cell-cell communication, fusion and differentiation.

Requirement of gap junctional intercellular communication for human villous trophoblast differentiation.

During pregnancy, the villous trophoblast develops from the fusion of cytotrophoblastic cells (CT) into a syncytiotrophoblast (ST), supporting the main physiological functions of the human placenta. Connexin43 (Cx43) is demonstrated in situ and in vitro in the villous trophoblast between CT and between CT and ST. Moreover, the presence of a gap junctional intercellular communication (GJIC) during in vitro trophoblast differentiation was previously demonstrated. Because the exchange of molecules through gap junctions is considered to play a major role in the control of cell and tissue differentiation, we studied the effects of a gap junctional uncoupler, heptanol, on morphological and functional trophoblast differentiation and on GJIC measured by the fluorescence recovery after photobleaching method. We found that when the GJIC was interrupted, CT still aggregated but fused poorly. This morphological effect was associated with a significant decrease of trophoblastic-specific gene expression (beta human chorionic gonadotropin and human chorionic somatomammotropin). This blocking action was reversible as demonstrated by recovery of GJIC and trophoblast differentiation process after heptanol removal. Moreover, the inhibition of the trophoblast differentiation did not affect Cx43 transcript expression and Cx43 protein expression. These data suggest that the molecular exchanges through gap junctions preceding cellular fusion are essential for trophoblast differentiation generating the multifunctional syncytiotrophoblast.

Genomic organization and alternative transcripts of the human Connexin40 gene.

The human Cx40 gene (NT_004434.5) was sorted out from the GenBank database and as a result of a BLAST homology search, two ESTs (BE784549 from a human lung database, and BE732411 from a human placenta database) overlapping with the coding exon 2 sequence and upstream regions of the gene were identified. These ESTs correspond to two transcripts 1A and 1B, which diverge from each other in their 5' regions. The transcript 1A corresponds to the only transcript previously identified for the mouse and rat Cx40 genes; whereas the transcript 1B is a new transcript. The human Cx40 gene therefore comprises three exons: exon 1A (100 bp), exon 1B (132 bp) and coding exon 2, with the exons 1A and 1B at 14 and 1.3 kb of the exon 2, respectively. The expression of these transcripts is cell-type specific. Transcript 1A is expressed in endothelial cells. Its expression was demonstrated in human umbilical vein endothelial cells (HUVEC). Transcript 1B is expressed in placental cytotrophoblasts. Its expression was demonstrated in malignant trophoblastic cells, BeWo, JAR and JEG-3, and purified cytotrophoblasts from human first trimester placental tissues. Interestingly, both transcripts 1A and 1B are expressed in the right atrial appendages (RAA), although the cell-type expression of the two transcripts in this particular tissue has not yet been determined. Both transcripts were found to be expressed in the various heart regions investigated, where transcript 1B was found to always occur rarely in comparison with transcript 1A. Transcripts 1A and 1B are both more abundant in the atria than in the ventricles. Luciferase reporter gene assays demonstrated that two genomic regions containing the exons 1A and 1B induced a cell-type specific expression. The 1.2 kb sequence, containing the exon 1A, induced an increase of the luciferase activity in HUVEC; whereas the 1.9 kb sequence, containing the exon 1B, induces an increase of expression of the luciferase activity in BeWo cells. The DNA sequence upstream of the exon 1A contains SP1 binding sites, but no TATA- or CAAT-box; whereas the region upstream of the exon 1B is preceded by three CAAT-boxes. Thus, in contrast to the mouse and rat Cx40 genes, the human Cx40 gene organized in three exons and generates two transcripts, which are cell-type specific.

Hormones and human trophoblast differentiation: a review.

In the human, fetal cytotrophoblastic cells play a key role in the implantation process and in placental development. With the progression of placentation, two pathways of differentiation lead to the formation of two distinct phenotypes. In the villous trophoblast (fusion phenotype), the trophblast differentiates from the fusion of mononuclear cytotrophoblastic cells into a syncytium, the syncytiotrophoblast. Bathing the maternal blood, the syncytiotrophoblast is involved in maternal-fetal exchanges and in placental endocrine functions. In the extravillous trophoblast (proliferative/invasive phenotype), the cytotrophoblastic cells proliferate and migrate into the decidua, remodeling the pregnant endometrium and its vasculature. This review summarizes our current knowledge of the key step of villous differentiation-the cell-cell fusion of the cytotrophoblastic cells--and on the invasion process of extravillous trophoblast. Experimental evidence demonstrates that the genetic differentiation/invasion programs of cytotrophoblastic cells could be modulated by their environment: oxygen, extracellular matrix, and soluble factors (cytokines, growth factors, and hormones). Cytotrophoblastic cells fusion and the functional differentiation of villous trophoblast are specifically stimulated by estradiol, glucocorticoids, and human chorionic gonadotropin (hCG) whereas progesterone is ineffective. Because these hormones are temporally secreted in large amounts and present at the fetomaternal interface, they are in good position to play a physiologic role in trophoblast differentiation. hCG may be important very early in pregnancy, when production of this glycoprotein is maximal, whereas estrogen increasingly produced by the fetoplacental unit and cortisol secreted from the fetal adrenal may be implicated in the end-stage maturation and aging of the trophoblast.