Marie-Benoîte Cohen

Factors regulating trophoblast invasion.

Implantation in the human is unique. This uniqueness is characterized on the maternal side by a spontaneous and massive decidualization of the endometrium and on the embryonic side by an almost unlimited invasive potential. Human embryos express an intrinsic invasive potential, which allows them to implant almost anywhere except in the endometrium because it protects itself from implantation. Human implantation is thus only possible during a limited period of time known as the implantation window. This mini review stresses the importance of studying trophoblast invasion into the endometrium as a model for human implantation. Cytotrophoblastic cells (CTB) can easily be isolated from first-trimester legal abortions and retain their invasive behavior when cultured in vitro. This model shows that matrix metalloproteinases (MMPs) are produced by CTB and are instrumental to their invasive behavior. Embryo implantation and tumor invasion use these same biochemical mediators for invasion. However, in contrast to tumor invasion, trophoblast invasion is limited both in time and space: it occurs during the first trimester of pregnancy and invasion does not go beyond the proximal third of the myometrium. Factors regulating MMP expression are of maternal and fetal origin.

Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells

BACKGROUND Feeder cells are frequently used for the early-stage of derivation and culture of human embryonic stem cell (hESC) lines. METHODS We established a conditionally immortalized human foreskin fibroblast line that secreted basic fibroblast growth factor (bFGF). These cells were used as feeder cells for hESC culture and induced pluripotent stem (iPS) cell derivation and expansion. This conditional immortalization was performed using lentiviral vector (LV) mediated transduction of Bmi-1 and human telomerase reverse transcriptase genes and the resulting cell line was further modified by LV-mediated transduction of a secreted form of bFGF gene product. Three different laboratories have tested whether this feeder cell line could support the maintenance of four different hESC lines. RESULTS Immortalized fibroblasts secreting stable amounts of bFGF supported the growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least 10 passages. Even at high passage (p56), these modified cells, when used as feeders, could support iPS derivation and propagation. Derived iPS cells expressed pluripotency markers, had hESC morphology and produced tissue components of the three germ layers when differentiated in vitro. CONCLUSION These modified fibroblasts are useful as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cells.

Hydroxychloroquine restores trophoblast fusion affected by antiphospholipid antibodies.

Obstetric antiphospholipid syndrome (APS) is defined by pregnancy complications associated with antiphospholipid antibodies (aPL). The mechanisms of the pathogenic effects of aPL in pregnancy are poorly understood. Toll‐like receptors (TLR) have been implicated previously in APS.

Glucose‐regulated protein 78: A new partner of p53 in trophoblast

Although wild‐type p53 protein is overexpressed in first trimester trophoblast, it is inactive towards its target genes Metalloproteinase 2 and 9. This seems to be due to a complex mechanism of inactivation and stabilization of p53 relying on the formation of protein complexes involving the N‐terminus of p53. To detect the proteins associated with this sequence, we incubated biotinylated p53 N‐terminal peptide in cytotrophoblastic cell medium 24 h before lysis of cells. We purified the proteins retained on biotinylated peptide using a neutravidin affinity column. Proteins were then identified by peptide mass finger printing followed or not by peptide fragmentation sequencing. Among these proteins, we identified glucose‐regulated protein 78 (GRP78) and verified its interaction with p53 in trophoblastic cells by immunoprecipitation and Western blot analysis. Moreover, the decreased expression of GRP78 induced by GRP78siRNA or versipelostatin decreased the formation of high molecular weight p53 complexes and p53 monomer and increased trophoblastic invasion. These results suggest that GRP78 is involved in inactivation and stabilization of p53 and in the regulation of trophoblastic invasion.

Heat shock proteins in ovarian cancer: A potential target for therapy

Ovarian cancer is the leading cause of death from gynecologic cancer in Western countries. Most of the patients are chemosensitive, and they will present a complete response after initial treatment, but most of them will relapse within 2 years. The resistance to standard chemotherapy represents a significant clinical challenge. Therefore, a better understanding of the molecular mechanisms involved in the drug resistance should allow to improve the treatment in this disease. Interestingly, recent data showed essential roles of heat shock proteins (HSPs) in various processes of carcinogenesis and their association with resistance to anticancer drugs. Here, we report the main investigations on HSPs in ovarian cancer with specific emphasis on clinical application.

Obstetrical Antiphospholipid Syndrome: From the Pathogenesis to the Clinical and Therapeutic Implications

Antiphospholipid syndrome (APS) is an acquired thrombophilia with clinical manifestations associated with the presence of antiphospholipid antibodies (aPL) in patient plasma. Obstetrical APS is a complex entity that may affect both mother and fetus throughout the entire pregnancy with high morbidity. Clinical complications are as various as recurrent fetal losses, stillbirth, intrauterine growth restriction (IUGR), and preeclampsia. Pathogenesis of aPL targets trophoblastic cells directly, mainly via proapoptotic, proinflammatory mechanisms, and uncontrolled immunomodulatory responses. Actual first-line treatment is limited to low-dose aspirin (LDA) and low-molecular weight heparin (LMWH) and still failed in 30% of the cases. APS pregnancies should be a major field in obstetrical research, and new therapeutics are still in progress.

Purified autoantibodies against glucose-regulated protein 78 (GRP78) promote apoptosis and decrease invasiveness of ovarian cancer cells

Circulating glucose-regulated protein 78 (GRP78) autoantibodies represent potential biomarker of epithelial ovarian cancer. However there is relatively limited identification of these antibodies in response to ovarian cancer. Here, we were interested in characterization of these antibodies and into their role in tumor development. We first purified GRP78 autoantibodies from sera of patients with ovarian cancer, and then tested them on SKOV-3 ovarian cancer cell line. A decrease of invasion and an increase of H(2)O(2)-induced apoptosis of SKOV-3 cells were observed, suggesting their protective role against ovarian cancer cells.

Regulation of MMP-9 by p53 in first trimester cytotrophoblastic cells

BACKGROUND The matrix metalloproteinase (MMP) family is known to play a key role in tissue remodelling during embryonic development and in pathological conditions, such as cardiovascular disease, arthritis and cancer metastasis. It has been shown previously that p53 regulates positively or negatively the expression of different MMPs. Because of p53 overexpression in trophoblastic cells, and its potential role in regulating MMP-2 and MMP-9 expression in different cell lines, we hypothesized that the expression of MMP-9 could also be regulated by p53 in first trimester cytotrophoblasts (CTB). METHODS and RESULTS Transfection experiments in CTB demonstrated that wild-type p53 down-regulates the −670 (P < 0.001) but not the −531 and −90 human MMP-9 promoter/CAT reporter plasmid activity, whereas p53 mutants partially lost this repressive activity. However, endogenous p53 is not able to regulate MMP-9 expression in CTB. The presence of high molecular weight complexes of p53 in CTB suggests a potential mechanism of inactivation of p53 transcriptional activity towards MMPs in these cells. CONCLUSIONS Although p53 is mutated in trophoblast, it is functionally incompetent towards MMPs in these cells.

GRP78 as a marker of pre-eclampsia: an exploratory study

Although the exact mechanisms that lead to shallow invasion or defective trophoblastic differentiation in pre-eclampsia are still unknown, it is widely admitted that the etiology of pre-eclampsia is a defect in trophoblast invasion of the uterine spiral arteries. We have previously observed that the status of a chaperone protein, glucose regulated protein 78 (GRP78) is associated with the invasive properties of cytotrophoblastic cells; we therefore hypothesized that circulating GRP78 could serve as a diagnostic tool in pre-eclampsia. In a prospective case-control study, we quantified GRP78 autoantibodies, complexes of GRP78 with autoantibodies and GRP78 (C-term fragment, N-term fragment and full-length GRP78) by ELISA. Plasma from women diagnosed with pre-eclampsia (n = 16), from women during the first trimester of pregnancy who subsequently developed pre-eclampsia (n = 10) and from healthy pregnant women (controls, n = 58 at term, n = 26 at first trimester) were analysed and compared. We observed no significant difference between pre-eclamptic and healthy pregnant women for autoantibodies-GRP78 complexes or total GRP78 at both first trimester and at delivery. In contrast, the ratio of C-terminal GRP78 over full length GRP78 was significantly different in plasma of pre-eclamptic patients as compared with controls both during first trimester (P < 0.004) and at term (P < 0.0001). Our findings suggest that circulating C-terminal GRP78 reflect the invasive properties of cells, and could be used as a predictive marker for pre-eclampsia early in pregnancy.

Involvement of Membrane GRP78 in Trophoblastic Cell Fusion

Background Glucose-regulated protein 78 (GRP78) is highly expressed in first trimester cytrophoblastic cells (CTBs), especially in syncytiotrophoblast (STB). However, the role of GRP78 in these cells has never been investigated. Methodology/Principal Findings In this study, we have examined the role of GRP78 in trophoblast fusion using the Bewo choriocarcinoma cell line as a model of cytotrophoblast fusion. Down regulation of GRP78 by siRNA or chemical inhibitors and use of antibodies against GRP78 in culture medium significantly decreased forskolin-induced fusion capacity of Bewo cells suggesting the involvement of membrane GRP78 in trophoblast fusion. GRP78 expression was also studied in preeclamptic (PE) CTBs which are known to have lower fusion capacity compared to control CTBs. Interestingly, despite the increase of GRP78 mRNA in PE CTBs, membrane GRP78 is significantly decreased in PE CTBs compared to control CTBs, suggesting that relocation of GRP78 from the endoplasmic reticulum to cell surface is probably altered in PE CTBs. Conclusions Our results imply that membrane GRP78 could play an important role in syncytialisation. They also suggest that deregulation of GRP78 expression or relocation at cell surface might be involved in pregnancy complication associated with defective syncytialisation, such as preeclampsia.