S. Haider

Human Tumour Necrosis Factor: Physiological and Pathological Roles in Placenta and Endometrium

The cytokine tumour necrosis factor alpha (TNF) is a well known member of the TNF superfamily consisting of at least 18 ligands and 29 different receptors involved in numerous cellular processes. TNF signals through two distinct receptors TNFR1 and TNFR2 thereby controlling expression of cytokines, immune receptors, proteases, growth factors and cell cycle genes which in turn regulate inflammation, survival, apoptosis, cell migration, proliferation and differentiation. Since expression of TNF was discovered in amnion and placenta many studies demonstrated the presence of the cytokine and its receptors in the diverse human reproductive tissues. Whereas TNF has been implicated in ovulation, corpus luteum formation and luteolysis, this review focuses on the functions of TNF in human placental, endometrial and decidual cell types of normal tissues and also discusses its role in endometrial and gestational diseases. Physiological levels of the cytokine could be important for balancing cell fusion and apoptotic shedding of villous trophoblasts and to limit trophoblast invasion into maternal decidua. Regulation of the TNF/TNFR system by steroid hormones also suggests a role in uterine function including menstrual cycle-dependent destruction and regeneration of endometrial tissue. Aberrant levels of TNF, however, are associated with diverse reproductive diseases such as amniotic infections, recurrent spontaneous abortions, preeclampsia, preterm labour or endometriosis. Hence, concentrations, receptor distribution and length of stimulation determine whether TNF has beneficial or adverse effects on female reproduction and pregnancy.

The role of interleukin-1β in human trophoblast motility

The pleiotropic cytokine interleukin-1β (IL-1β) can promote physiological cell migration, as well as cancer cell invasion and metastasis. Its role in human trophoblast invasion, however, has not been satisfactorily answered since direct, indirect as well as no effects on trophoblast motility have been published. Therefore, the role of IL-1β has been re-evaluated by exclusively using human primary trophoblast model systems. Immunofluorescence of first trimester placentae indicated IL-1 receptor 1 (IL-1R1) protein expression in first trimester villous cytotrophoblasts (vCTB) and extravillous trophoblasts (EVT). The latter expressed higher mRNA levels of the receptor as shown by comparative gene chip data of vCTB and EVT. Similarly, Western blot analyses and immunofluorescence revealed a time- and differentiation-dependent increase of IL-1R1 in primary EVT seeded on fibronectin. IL-1β dose-dependently elevated migration of isolated first trimester EVT through fibronectin-coated transwells, which was inhibited in the presence of IL-1R antagonist (IL-1Ra), whereas proliferation of these cells was not affected. Similarly, the interleukin did not alter proliferation of vCTB and cell column trophoblasts in floating villi of early pregnancy, but promoted migration in villous explant cultures seeded on collagen I. Western blot analyses of supernatants of primary EVT and first trimester villous explant cultures revealed IL-1β induced secretion of urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1 and PAI-2, which was diminished upon combined IL-1β/IL-1Ra treatment. In conclusion, these data suggest that IL-1β directly promotes trophoblast motility of first trimester EVT involving the uPA/PAI system.

Expression, Regulation and Functional Characterization of Matrix Metalloproteinase-3 of Human Trophoblast

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1beta, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1beta-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1beta-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1beta failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.

Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts

Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.

Functional expression of the human neonatal Fc-receptor, hFcRn, in isolated cultured human syncytiotrophoblasts.

Materno-fetal IgG transfer in the mature human placenta involves transport across the syncytiotrophoblast (STB) and fetal endothelial cell layer. The MHC class I-related Fc gamma-receptor (hFcRn) localized in STB as well as in endothelial cells is involved in overall IgG transfer from the maternal into the fetal circulation. Functional hFcRn is a heterodimer of a transmembrane alpha-chain and beta2-microglobulin. To establish the basis for future studies to unravel the mechanism of IgG transport in STB, we investigated hFcRn alpha-chain and beta2-microglobulin expression in cytotrophoblasts (CTB) isolated from human term placentae and cultured in vitro under conditions where differentiation into multinuclear STB takes place (>or=48 h). Northern blot analysis demonstrated up-regulation of alpha-chain mRNA after 48 h of in vitro cultivation. Likewise, hFcRn alpha-chain and beta2-microglobulin were at the limit of detection by immunofluorescence microscopy in CTB immediately after isolation, but their expression increased upon STB formation. hFcRn alpha-chain co-localized with beta2-microglobulin in multinuclear STB and formed a functional, i.e. low pH IgG binding, receptor as shown by affinity isolation. The in vitro differentiated STB exhibited specific, low pH-dependent IgG binding to the plasma membrane. In conclusion, these cultures can now be applied to study the role of hFcRn in IgG transport and trafficking in STB cultures in vitro.

Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

Introduction Notch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations. Methods Localisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)+ and differentiated HLA-G+ CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage. Results In tissue sections Notch2 expression was higher in HLA-G+ distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G+ CTBs compared to EGFR+ CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility. Discussion Notch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell–cell contact of neighbouring dCCTs, could attenuate trophoblast migration.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast

INTRODUCTIONnTGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts.nnnMETHODSnExpression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium.nnnRESULTSnSmad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs.nnnDISCUSSIONnThe lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells.

Notch signalling in placental development and gestational diseases.

Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.