Inhibiton of RET and JAK2 Signals and Upregulation of VEGFR3 Phosphorylation in Vitro by Galectin-1 in Trophoblast Tumor Cells BeWo
Introduction
The galectin family members are defined by a conserved amino acid sequence motif in the carbohydrate recognition domain (CRD) and an affinity for β-galactosides [1]. Although LacNAc is the basic ligand recognized by gal-1, the proto-type galectin binds with increased avidity to multiple Galβ1-4GlcNAc sequences presented on branched N-linked or on repeating LacNAc-residues on N- and O-linked glycans. Gal-1 having a single CRD forms a non-covalently associated homodimer to become functionally bivalent under physiological conditions. The bivalent nature of gal-1 facilitates glycan-mediated cell surface receptor cross-linking believed to be essential in inducing signalling events [2], [3]. Extracellularly, gal-1 exerts distinct biological effects in various tissues and on cells by the recognition of glycan ligands, including cell adhesion [4] metastasis [5], cell growth regulation [6], [7] immunosuppression [8] and apoptosis [2]. Furthermore, there is an evidence of a role for gal-1 in promoting fetomaternal tolerance in allogeneic matings, suggesting a potential approach for therapeutic intervention aimed at restoring immune cell homeostasis in failing pregnancies [9]. It has also been demonstrated that gal-1 was strongly expressed by syncytiotrophoblasts in term and first trimester placenta, whereas villous cytotrophoblasts were negative [10], [11]. The invading cytotrophoblast was weakly stained and BeWo cells expressed gal-1 particularly strongly [12]. We have demonstrated that this expression scheme is in striking conformity with the Thomsen-Friedenreich (TF)-antigen expression in the placenta. We have also shown that gal-1 binds to the TF-antigen, which is carried by Mucin-1, a receptor tyrosine kinase [13], [14]. In addition, it has been demonstrated that gal-1 induces cell differentiation processes in trophoblast cells [15]. Although multiple signalling pathways in trophoblast cells have been elucidated in the last years, including the members of the mitogen-activated protein family (MAPK) [16], [17] and the Janus kinase (JAK) and signal transducer and activator of transcription cytokine signal transducing pathway (STAT) (where several cytokines, including LIF, HGF, IL-6, IL-11 and GM–CSF are known to signal through) [18], [19], no information on the signal transduction processes by gal-1 in these cells is available so far. To understand the extracellular and intracellular roles of gal-1 in trophoblast cells, it is necessary to identify its interacting glycoconjugates and to study the signalling events. Because tyrosine phosphorylation is a major mechanism that controls multiple biological processes including cell differentiation, survival and proliferation [20], the aim of this study was to examine which receptor tyrosine kinases were influenced in trophoblast cells by gal-1. As BeWo chorionic carcinoma cell cultures reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype, they offer an attractive model to examine the effects of gal-1 on signal transduction in trophoblast cells.
Section snippets
Cells
The choriocarcinoma cell line BeWo was obtained from the European Collection of Cell Cultures (ECACC, UK). Cells were cultured in DMEM medium (Dulbecco's Modified Eagle Medium, Biochrom, Germany, 3.7 g/l NaHCO3, 4.5 g/l D-Glucose, 1.028 g/l stable glutamine, Na-Pyruvate) supplemented with 10% heat-inactivated FCS (fetal calf serum) without antibiotics and antimycotics.
RayBio® human RTK Phosphorylation Antibody Array 1
BeWo cells (40·106 cells in 15 ml supplemented DMEM medium) were incubated in 175 ml cell culture bottles for 24 h in the absence
The RayBio® Human RTK Phosphorylation Antibody Array 1
The attribution from the phosphorylation to the different human Receptor Tyrosine Kinases was obtained with Table 1, where 71 different human receptor tyrosine kinases (RTKs) were represented. Dots A1, B1, C1, D1, E1, F1, K13 and L13 were positive controls (pos) and A2, B2, C2, D2, K12 and L12 were negative controls (neg). Dots G8, H8, G10, H10, G13 and H13 showed a significant alteration of signal intensity after stimulation of the BeWo cells with gal-1.
As demonstrated in Fig. 1, Fig. 2,
Discussion
In contrast to tumor invasion, trophoblastic invasion during implantation and placentation is controlled in space and time. Furthermore, trophoblast cells are physiological cells that grow in a physiological setting and are capable to protect themselves from the maternal immune system [22]. Galectins are members of a family of soluble animal lectins defined by shared characteristic amino acid sequences and an affinity for β-galactosides [23].
Galectins have been shown to be involved in
Acknowledgement
We thank C. Kuhn and S. Schulze for excellent assistance with immunocytochemistry. The study was supported by the “Deutsche Forschungsgemeinschaft” (DFG).
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2013, PlacentaCitation Excerpt :In a recent study it was demonstrated that gal-1 induced a significant alteration of signal intensity in 3 of 71 different Receptor tyrosine kinases (RTKs), the RE arranged during Transfection [75], the Janus Kinase 2 (JAK2) and the Vascular endothelial growth factor receptor 3 (VEGFR3). These results showed the specificity of gal-1 binding to cell surface glycoproteins at the concentration used for this experiment [110]. In addition, gal-1 inhibits ERK1 signalling via phosphorylation of T202/Y204, ERK2T185/Y187, Akt-3 S472, Akt-pan S473,S474,S472, GSK-3α/βS21/S9 and up-regulates Elk1 signalling in BeWo cells [111].
The involvement of CD146 and its novel ligand galectin-1 in apoptotic regulation of endothelial cells
2013, Journal of Biological ChemistryAnti-GalNAcβ: A novel anti-glycan autoantibody associated with pregnancy loss in women with antiphospholipid syndrome and in a mouse experimental model
2012, Journal of AutoimmunityCitation Excerpt :Different members of this family have been shown to modulate several pathological processes such as allergic reactions, autoimmunity, and tumor invasion [45]. On JAR cells Galectin-1 induces phosphorylation of vascular endothelial growth factor receptor 3 and inhibits phosphorylation of rearranged during transfection and Janus Kinase (JAK)2 [46]. Experiments in mice showed that Galectin-1 prevents fetal loss and restored fetal tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which promoted the expansion of IL-10-secreting regulatory T cells in vivo [47].
The role of galectin-1 in trophoblast differentiation and signal transduction
2011, Journal of Reproductive ImmunologyCitation Excerpt :This points to a mechanism in which JAK2 and RET influence BeWo cells via the same signalling route, implicating STAT3. Further studies will elucidate whether this results in an inhibition of proliferation (Fischer et al., 2009). Gal-1 influences the immune system and modulates immune cell functions through several mechanisms.
Stimulation of syncytium formation in vitro in human trophoblast cells by Galectin-1
2010, PlacentaCitation Excerpt :It has also been demonstrated that gal-1 binds to BeWo cells, which form a syncytium in vitro, but does not bind to fresh isolated trophoblast cells and to choriocarcinoma cells Jeg-3, which cannot form a syncytium [10]. In a recent study we could demonstrate that the concentration of 60 μg/ml gal-1 induced a significant alteration of signal intensity in only 3 from 71 different Receptor tyrosine kinases, showing the specificity of gal-1 binding to cell surface glycoproteins at the concentration used for this experiment [15]. To demonstrate the effect of gal-1 on the syncytium formation in trophoblast cells, choriocarcinoma cell cultures BeWo, derived from human gestational choriocarcinoma and permanently established in 1968 by Pattillo et al. offer an attractive model because they reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype [16].