Elsevier

Placenta

Volume 30, Issue 12, December 2009, Pages 1078-1082
Placenta

Inhibiton of RET and JAK2 Signals and Upregulation of VEGFR3 Phosphorylation in Vitro by Galectin-1 in Trophoblast Tumor Cells BeWo

https://doi.org/10.1016/j.placenta.2009.10.003Get rights and content

Abstract

Background

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, binds to cell surface glycoproteins (Mucin-1) on trophoblast cells. Although it has been demonstrated that gal-1 induces cell differentiation processes in these cells, no information on its signal transduction processes is available so far. As tyrosine phosphorylation is a major mechanism that controls multiple biological processes including cell differentiation, survival and proliferation, the aim of this study was to examine which human receptor tyrosine kinases (RTKs) were phosphorylated in trophoblast cells by gal-1.

Materials and Methods

BeWo choriocarcinoma cells were incubated for 24 h in the absence (controls) and presence of 60 μg/ml galectin-1. With the RayBio® Human RTK Phosphorylation Antibody Array 1, the relative levels of phosphorylation of different human RTKs could be detected simultaneously. The signal intensities were compared and quantified with the Quantity One Version 4.5.2 program. Gal-1-treated and non-treated cells were incubated with antibodies against REarranged during Transfection (RET) and phosphorylated RETY905. Staining reaction was performed with the avidin-biotinylated peroxidase complex (ABC) reagent.

Results

We demonstrated that gal-1 inhibited RET and Janus Kinase 2 (JAK2) signals and upregulated Vascular endothelial growth factor receptor 3 (VEGFR3) signal in BeWo cells. We also showed the downregulation of phosphorylation on RET phosphotyrosine residue 905 in BeWo cells with phosphorylation specific antibodies and immunocytochemistry.

Conclusion

Out of a number of 71 different RTKs, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in only 3 RTKs: JAK2, RET and VEGFR3. Our data suggest that phosphorylation of these RTKs could be involved in cell differentiation processes that could be responsible for the already known effect of gal-1 on BeWo cells, the inhibition of proliferation.

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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