Monika Sundl

Expression of serum amyloid A transcripts in human trophoblast and fetal-derived trophoblast-like choriocarcinoma cells

The placenta comprises a highly specialized trophoblast layer, which arises from the embryo and differentiates during embryonic development to perform specialized functions, e.g., synthesis of pregnancy‐associated hormones, growth factors and cytokines. As there is no evidence of maternal acute‐phase protein transplacental transfer and trophoblast plays an important role in regulating immune responses at the feto‐maternal interface, the expression of acute‐phase serum amyloid A (A‐SAA) was investigated in human first trimester trophoblast and trophoblast‐like JAR and Jeg‐3 choriocarcinoma cells. We here show expression of cytokine receptors and cytokine‐dependent induction of A‐SAA in JAR and Jeg‐3 cells. While interleukin‐1α/β is a major agonist for A‐SAA expression in JAR, tumor necrosis factor‐α is the predominant agonist in Jeg‐3. First trimester trophoblast and JAR/Jeg‐3 cells further express the human homolog of SAA‐activating factor‐1, a transcription factor involved in cytokine‐mediated induction of A‐SAA genes. A‐SAA1 and A‐SAA2 transcripts were increased in first trimester trophoblast during pregnancy weeks 10 and 12 suggesting that A‐SAA plays a role during early fetal development.

Expression of serum amyloid A transcripts in human bone tissues, differentiated osteoblast‐like stem cells and human osteosarcoma cell lines

Although the liver is the primary site of cytokine‐mediated expression of acute‐phase serum amyloid A (SAA) protein, extrahepatic production has also been reported. Besides its role in amyloidosis and lipid homeostasis during the acute‐phase, SAA has recently been assumed to contribute to bone and cartilage destruction. However, expression of SAA in human osteogenic tissue has not been studied. Therefore, we first show that SAA1 (coding for the major SAA isoform) but not SAA2 transcripts are expressed in human trabecular and cortical bone fractions and bone marrow. Next, we show expression of (i) IL‐1, IL‐6, and TNF receptor transcripts; (ii) the human homolog of SAA‐activating factor‐1 (SAF‐1, a transcription factor involved in cytokine‐mediated induction of SAA genes); and (iii) SAA1/2 transcripts in non‐differentiated and, to a higher extent, in osteoblast‐like differentiated human mesenchymal stem cells. Third, we provide evidence that human osteoblast‐like cells of tumor origin (MG‐63 and SAOS‐2) express SAF‐1 under basal conditions. SAA1/2 transcripts are expressed under basal conditions (SAOS‐2) and cytokine‐mediated conditions (MG‐63 and SAOS‐2). RT‐PCR, Western blot analysis, and immunofluorescence technique confirmed cytokine‐mediated expression of SAA on RNA and protein level in osteosarcoma cell lines while SAA4, a protein of unknown function, is constitutively expressed in all osteogenic tissues investigated. J. Cell. Biochem. 103: 994–1004, 2008.

Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro

STUDY QUESTION How is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy? SUMMARY ANSWER Rather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space. WHAT IS KNOWN ALREADY Extravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells. STUDY DESIGN, SIZE, DURATION As well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis. PARTICIPANTS/MATERIALS, SETTING, METHODS First trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed. MAIN RESULTS AND THE ROLE OF CHANCE The earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation. LIMITATIONS, REASONS FOR CAUTION This is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations. WIDER IMPLICATIONS OF THE FINDINGS This study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

Placental fractalkine mediates adhesion of THP-1 monocytes to villous trophoblast

The chemokine fractalkine (CX3CL1) recently attracted increasing attention in the field of placenta research due to its dual nature, acting both as membrane-bound and soluble forms. While the membrane-bound form mediates flow-resistant adhesion of leukocytes to endothelial and epithelial cells via its corresponding receptor CX3CR1, the soluble form arises from metalloprotease-dependent shedding and bears chemoattractive activity for monocytes, natural killer cells and T cells. In human placenta, fractalkine is expressed at the apical microvillous plasma membrane of the syncytiotrophoblast, which may enable close physical contact with circulating maternal leukocytes. Based on these observations, we tested the hypothesis that fractalkine mediates adhesion of monocytes to the villous trophoblast. Forskolin-induced differentiation and syncytialization of the trophoblast cell line BeWo was accompanied with a substantial upregulation in fractalkine expression and led to increased adhesion of the monocyte cell line THP-1, which preferentially bound to syncytia. Blocking as well as silencing of the fractalkine receptor CX3CR1 proved involvement of the fractalkine/CX3CR1 system in adherence of THP-1 monocytes to villous trophoblast. Pre-incubation of THP-1 monocytes with human recombinant fractalkine as well as silencing of CX3CR1 expression in THP-1 monocytes significantly impaired their adherence to BeWo cells and primary term trophoblasts. The present study suggests fractalkine as another candidate among the panel of adhesion molecules enabling stable interaction between leukocytes and the syncytiotrophoblast.

Phospholipid scramblase 1 (PLSCR1) in villous trophoblast of the human placenta

A crucial factor for effective villous trophoblast fusion in the human placenta is the transient deregulation of plasma membrane phospholipid asymmetry leading to externalization of phosphatidylserine to the outer membrane leaflet. Screening of scramblase family members implicated in the collapse of phospholipid asymmetry revealed that phospholipid scramblase 1 (PLSCR1) is strongly expressed in villous trophoblast. Therefore, we assessed the putative role of PLSCR1 in villous trophoblast fusion. Spatio-temporal analysis in first trimester and term placenta showed abundant expression of PLSCR1 in syncytiotrophoblast, macrophages and endothelial cells, while it was virtually absent in villous cytotrophoblasts. For functional studies, BeWo cells, isolated primary term trophoblasts and first trimester villous explants were used. During forskolin-mediated BeWo cell differentiation, neither PLSCR1 mRNA nor protein levels showed significant changes. In contrast, when primary trophoblasts were stimulated with Br-cAMP, a decrease in PLSCR1 mRNA and protein expression was observed. To elucidate a role for PLSCR1 in syncytialization, we used RNA interference and a chemical scramblase inhibitor, R5421 (ethanedioic acid). Silencing of PLSCR1 using siRNA had no effects while inhibition of scramblase activity by R5421 increased GCM-1 mRNA expression, beta-hCG protein secretion and fusion rates of BeWo cells. In primary trophoblasts and villous explants, no effects of siRNA or R5421 treatment on fusion were detected. This study provides data on PLSCR1 localization and general expression in the human placenta. The data make it tempting to speculate on a role of PLSCR1 in negatively regulating trophoblast fusion.