Alexandra Gellhaus

Analogous and Unique Functions of Connexins in Mouse and Human Placental Development

Here, we review the expression, localization and the possible role of the different connexin isoforms in placental function and development in mice and men. Connexin gene deletion in mice has shown that Cx26 is responsible for transplacental uptake of glucose in the labyrinth, and Cx31 as well as Cx31.1 for trophoblast cell lineage development. In the human placenta, it appears that Cx43 is required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast. Thus Cx26 and Cx43 serve different species-specific functions in the functionally analogous placental compartments, mouse labyrinth and human villous trophoblast. However, like Cx31 in the mouse, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblast cells invading the endometrium. Both connexin channels seem to have similar functions in analogous compartments of the placentas. Taken together, connexins are important in regulating trophoblast cell differentiation in both species. In mouse, connexin channels are specifically involved in passive transport of molecules across the placental barriers.

Regulation of the Matricellular Proteins CYR61 (CCN1) and NOV (CCN3) by Hypoxia-Inducible Factor-1α and Transforming-Growth Factor-β3 in the Human Trophoblast

It is known that a hypoxic environment is critical for trophoblast migration and invasion and is fundamental for appropriate placental perfusion. Because cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3) are expressed in the extravillous trophoblast and expression levels are deregulated in preeclampsia, we investigated their regulation properties in first-trimester placental explants and in JEG3 choriocarcinoma cells upon a physiological low oxygen tension of 1-3%. In placental explants, both proteins were expressed in the extravillous trophoblast cells and were increased upon hypoxia. JEG3 cells revealed a significant up-regulation of CYR61 and NOV intracellular as well as secreted protein upon hypoxic treatment accompanied by the stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha). Treatment with dimethyloxalylglycine to mimic hypoxia and silencing of HIF-1alpha using small interfering RNA revealed that only the increase in intracellular protein expression seems to be dependent on HIF-1alpha but obviously not the secretion process. Moreover, recombinant TGF-beta3 was able to further enhance the amount of intracellular CCN proteins as well as secreted CYR61 levels under hypoxia. These results indicate that low oxygen levels trigger elevation of intracellular as well as secreted CYR61 and NOV protein probably in two independent pathways. Addition of recombinant CYR61 and NOV proteins increases migration as well as invasion properties of JEG3 trophoblast cells, which strengthen their role in supporting trophoblast migration invasion properties. In summary, CYR61 and NOV are regulated by HIF-1alpha and TGF-beta3 in the trophoblast cell line JEG3, and their enhanced secretion could be implicated in appropriate placental invasion.

The Liver X Receptor (LXR) and its Target Gene ABCA1 are Regulated Upon Low Oxygen in Human Trophoblast Cells: A Reason for Alterations in Preeclampsia?

OBJECTIVES The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia. STUDY DESIGN We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists. MAIN OUTCOME MEASURES Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes. RESULTS In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317. CONCLUSIONS LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport.

The Molecular Role of Connexin 43 in Human Trophoblast Cell Fusion

ABSTRACT Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

The circulating proangiogenic factors CYR61 (CCN1) and NOV (CCN3) are significantly decreased in placentae and sera of preeclamptic patients.

It is thought that preeclampsia results from a shallow invasion of the extravillous trophoblast into the decidua and maternal vessels, which in turn leads to hypoxia and uteroplacental insufficiency. Here, the authors focus on the expression of the proangiogenic secreted molecules CYR61 (CCN1) and NOV (CCN3) in human placentae during normal pregnancy compared with preeclamptic placentae. CYR61 and NOV are strongly expressed in endothelial cells as well as in the extravillous trophoblast, with increasing levels during placental development. Interestingly, the authors found significantly decreased levels in early preeclamptic placentae compared with matched controls. Whereas both CYR61 and NOV proteins are present at constant high levels in the sera of nonpregnant and pregnant women, in the sera of patients with early-onset preeclampsia, levels were significantly reduced. The authors suggest that the reduction of both CCN molecules in preeclampsia could be 1 reason underlying the failure of uterine vascular remodeling. Moreover, their low maternal serum levels could serve as biomarkers for early diagnosis of this disease.

CCN3 regulates proliferation and migration properties in Jeg3 trophoblast cells via ERK1/2, Akt and Notch signalling

Previous studies showed that CCN3 is deregulated in early-onset pre-eclampsia (PE), a pregnancy disease associated with impaired trophoblast invasion, which leads to reduced fetal oxygen and nutrition support. Recently, we identified the glycosylated (g-CCN3) and the non-glycosylated (ng-CCN3) form of matricellular CCN3 as key factors in regulation of trophoblast proliferation and invasion. While Jeg3 cells revealed a decreased proliferation upon stimulation with both forms of CCN3, enhanced migration and invasion properties were only found for ng-CCN3. Here, we focused on the signalling cascades mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and Notch/p21 for mediating the dual function of CCN3 on trophoblast proliferation versus migration in Jeg3 cells upon stimulation with g- and ng-recombinant CCN3 (g/ng-rCCN3). Analysis of the CCN3-mediated signalling pathways showed that ng-rCCN3 stimulated migration properties by activating the Akt as well as the MAPK pathway. Moreover, cell migration stimulated by ng-rCCN3 was mediated via Akt and integrin α5β1 but not the antiproliferative effect of CCN3. There was evidence that the Notch pathway might contribute to the antiproliferative properties of both forms of CCN3 by an increase in Notch1 expression and its target gene, the cell cycle inhibitor p21. Our data showed that the presence of both forms of CCN3 is accompanied by a balance of trophoblast proliferation and migration/invasion properties, which are triggered by different signalling pathways. Thus, a deregulated expression of g/ng-CCN3 could lead to an imbalance in proliferation versus invasion, and might contribute to the shallow trophoblast invasion observed in PE.

Impact of CCN3 (NOV) glycosylation on migration/invasion properties and cell growth of the choriocarcinoma cell line Jeg3

BACKGROUND Recently we have shown that the matricellular CCN3 protein expressed in invasive extravillous trophoblast cells (EVTs) is decreased in early-onset pre-eclampsia and is regulated by oxygen tension. Pathogenesis of pre-eclampsia relies on a shallow invasion of EVTs into the spiral arteries, which leads to hypoxia accompanied by uteroplacental insufficiency. Here we investigated the function of glycosylated and non-glycosylated CCN3 protein on cell growth as well as migration and invasion properties of the malignant trophoblast cell line Jeg3 which is a widely used model for the invasive trophoblast. METHODS AND RESULTS Stable transfection of Jeg3 choriocarcinoma cells with full length CCN3 resulted in high expression of secreted glycosylated and cellular non-glycosylated CCN3. These cells revealed significantly reduced growth in cell numbers combined with a significantly increased migratory and invasive capacity. Matrix metalloprotease (MMP)-2 and MMP-9 activities were enhanced dependent on CCN3 expression, which could be confirmed by CCN3 knockdown studies. Using recombinant glycosylated and non-glycosylated CCN3, we revealed that CCN3 decreased growth in Jeg3 cell numbers independent of its glycosylation status, whereas only non-glycosylated CCN3 was able to enhance migration and invasion properties. CONCLUSIONS The present results suggest that CCN3 protein regulates the decrease in Jeg3 cell numbers independent of its glycosylation status, whereas migratory and invasive properties are influenced only by non-glycosylated CCN3. An impaired balance in the expression of glycosylated and non-glycosylated CCN3 could contribute to the shallow invasion of EVTs observed in pre-eclampsia.

HIF-1beta determines ABCA1 expression under hypoxia in human macrophages.

ATP-binding cassette transporter A1 plays (ABCA1) a major role in reverse cholesterol transport, a process closely related to atherogenesis. In the thickening atherosclerotic lesions lipid loaded macrophages are exposed to regions of local hypoxia that may influence reverse cholesterol transport. Here we studied the effect of hypoxia on ABCA1 regulation and cholesterol efflux in human macrophages. We found that the hypoxia-inducible factor 1 (HIF-1) specifically binds to the HIF-1 response element of the ABCA1 promoter and the HIF-1 complex increases ABCA1 promoter activity along with ABCA1 expression. Primary human macrophages exposed to hypoxia or expressing constitutively active HIF-1alpha responded with a potent change in ABCA1 expression, which showed a strong correlation with HIF-1beta expression (r: 0.95-0.91). Moreover, ABCA1-mediated cholesterol efflux was also found to be regulated by HIF-1beta under hypoxia. In vivo, in macrophages prepared from human atherosclerotic lesions ABCA1 levels showed a strong correlation with HIF-1beta expression. This in vivo regulatory mechanism was confirmed in human pre-eclamptic placentas, a clinical condition with severe local hypoxia. These results demonstrate that HIF-1beta availability determines ABCA1 expression and cholesterol efflux in macrophages under hypoxia and may contribute to the interpersonal variability of atherosclerotic lesion progression.

Ceramide-Enriched Membrane Domains in Infectious Biology and Development

Ceramide has been shown to be critically involved in multiple biological processes, for instance induction of apoptosis after ligation of death receptors or application of gamma-irradiation or UV-A light, respectively, regulation of cell differentiation, control of tumor cell growth, infection of mammalian cells with pathogenic bacteria and viruses or the control of embryo and organ development to name a few examples. Ceramide molecules form distinct large domains in the cell membrane, which may serve to re-organize cellular receptors and signalling molecules. Thus, in many conditions, ceramide may be involved in the spatial and temporal organisation of specific signalling pathways explaining the pleiotrophic effects of this lipid. Here, we focus on the role of ceramide and ceramide-enriched membrane domains, respectively, in bacterial infections, in particular of the lung, and sepsis. We describe the role of ceramide for infections with Neisseriae gonorhoeae, Staphylococcus aureus and Pseudomonas aeruginosa. Finally, we discuss newly emerging aspects of the cellular function of ceramide, i.e. its role in germ line and embryo development.

Connexin 31 (GJB3) Deficiency in Mouse Trophoblast Stem Cells Alters Giant Cell Differentiation and Leads to Loss of Oxygen Sensing

ABSTRACT The nonphysiological placental oxidative environment has been implicated in many complications during human pregnancy. Oxygen tension can influence a broad spectrum of molecular changes leading to alterations in trophoblast cell lineage development. In this study, we report that mouse wild-type trophoblast stem cells (TSCs) react to low oxygen (3%) with an enhanced differentiation into the giant cell pathway, indicated by a downregulation of the early stem cell markers Eomes and Cdx2 as well as by a significant upregulation of Tfap2c and the differentiation markers Tpbpa and Prl3d1. Here we demonstrated that connexin 31/GJB3-deficient TSCs failed to stabilize HIF-1A under low oxygen, resulting in nonresponsiveness of different marker genes, such as Cdx2 and Eomes and Tfap2c and Tpbpa. Moreover, connexin 31-deficient TSCs revealed a shift in giant cell differentiation from Prl3d1 expressing parietal giant cells to Ctsq, Prl3b1, and Prl2c2-positive giant cells, probably sinusoidal and canal lining trophoblast giant cells. Thus, loss of connexin 31 led to different giant cell subtypes which bypass the progenitor regulators Tfap2c and Tpbpa under low oxygen conditions.