Human trophoblast requires galectin-3 for cell migration and invasion.

Invasive extravillous cytotrophoblast of the human placenta expresses galectins-1, -3, and -8 in vivo and in vitro. This study aimed to investigate the potential role of galectin-3 in cell migration and invasion, using recombinant human galectin-3 (rhgalectin-3), small molecule galectin inhibitor I47, and galectin-3 silencing. HTR-8/SVneo cell migration was stimulated by rhgalectin-3 and reduced by I47, which could be neutralised by rhgalectin-3. Inhibitor specificity and selectivity for the galectins expressed in extravillous trophoblast were validated in solid phase assays using recombinant galectin-1, -3, -8, confirming selectivity for galectin-3. HTR-8/SVneo cell migration and invasion, and invasion by isolated trophoblast cells in primary culture were significantly reduced in the presence of I47, which could be restored by rhgalectin-3. Upon HTR-8/SVneo cell treatment with galectin-3 siRNA both LGALS3 and galectin-3 protein were dramatically decreased. Silencing of galectin-3 induced significant reduction in cell migration and invasion, which was restored by rhgalectin-3. The influence on known mediators of cell invasion, MMP2 and -9, and integrins α1, α5, and ß1 was followed in silenced cells, showing lower levels of MMPs and a large reduction in integrin subunit ß1. These results show that galectin-3 acts as a pro-invasive autocrine/paracrine factor in trophoblast in vitro.

Carcinoembryonic antigen and related molecules in normal and transformed trophoblast.

Carcinoembryonic antigen (CEA, CD66e) and CEA-related cell adhesion molecules (CEACAMs) are important mediators in remodeling of diverse human tissues, and modulators of cell proliferation and differentiation. Expression by normal and transformed trophoblast of gestational trophoblastic diseases (GTDs), isolated cytotrophoblast and choriocarcinoma cell lines is presented here. Immunocyto/histochemistry of normal placenta (n=9), invasive mole (n=8), choriocarcinoma (n=7), a placental site trophoblastic tumor, cytotrophoblast in primary culture and JAr and JEG-3 cells was performed using polyclonal anti-CEA and specific monoclonal anti-CEA antibodies. Data were analyzed and scored using Mann-Whitney Test. CEA and CEA-related molecules were identified by Western blot and immunoaffinity chromatography in JAr and JEG-3 cells and extracts of 1st and 3rd trimester of pregnancy tissue and cytotrophoblast cell lysates. CEA is expressed throughout pregnancy, in first trimester predominantly in syncytiotrophoblast, but also in villous cytotrophoblast and extravillous trophoblast. Data presented here demonstrate that CEA is significantly increased in transformed trophoblast of GTDs (p<0.05). Both cytotrophoblast in primary culture and choriocarcinoma cell lines express CEA, with staining of granular deposits in JAr and cell membrane in JEG-3. The results suggest that CEA (CD66e) and other CEA-related protein(s) could be involved in trophoblast differentiation.

Pharmacological inhibition of MIF interferes with trophoblast cell migration and invasiveness.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) is expressed by villous and extravillous cytotrophoblast. This study was aimed to investigate functional relevance of MIF for human trophoblast. METHODS: MIF mRNA and protein were documented in cytotrophoblast (CT) and extravillous trophoblast cell line HTR-8/SVneo by RT-PCR, Western blot (WB), and immunocytochemistry. Recombinant human MIF (rhMIF), or its specific inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) were used in Wound healing migration and Matrigel invasion tests. Potential effectors, integrin subunits and matrix metalloproteinases (MMP) were studied using WB and gelatin zymography, respectively. RESULTS: Blocking endogenous MIF by ISO-1 decreased HTR-8/SVneo cell migration dose dependently, most significantly with 200 µg/ml to 65% of control. Supplementation with rhMIF induced a significant stimulation to 129% of control with 200 ng/ml. In CT cell invasion test, ISO-1 at 200 µg/ml reduced invasion to 59% of control, while rhMIF (200 ng/ml) induced stimulation to 159% of control. In HTR-8/SVneo cells, invasion was significantly inhibited by ISO-1 to 40%, and increased to 150% of control by rhMIF (200 ng/ml). Integrin α1 was reduced by ISO-1 in both cell types, while integrins α5 and ß1 were not changed. Addition of rhMIF increased integrin α1. In the presence of ISO-1, levels of MMP-2 and MMP-9 were reduced in CT and HTR-8/SVneo, while rhMIF stimulated MMP-2 in CT and MMP-9 in HTR-8/SVneo cells. CONCLUSION: Reported findings provide the first insight into the cellular effects of MIF in human trophoblast, which acts to promote cell migration and invasion.

Galectin-1 and galectin-3 in the trophoblast of the gestational trophoblastic disease.

While the presence and distribution of galectin-1 and galectin-3 in different normal trophoblast cell populations is known, no information is available regarding their occurrence in malignant trophoblast of gestational trophoblastic disease (GTD). Galectins-1 and -3 have, however, been implicated in malignancies of other tissues. Immunoreactivity for these galectins in the transformed trophoblast of invasive mole (n = 8), choriocarcinoma (n = 7) and one case of placental site trophoblastic tumor (PSTT) was compared to that of the invasive trophoblast of the normal first trimester of pregnancy implantation sites (n = 9). A large proportion of the transformed trophoblast cells of all GTD studied were positive for galectin-1 and galectin-3. Immunoreactivity was scored semiquantitatively to include both the prevalence among the trophoblast cells and the intensity of staining. Immunoreactivity for both galectin-1 and galectin-3 in gestational trophoblastic disease is increased (significant differences at p < 0.05, Mann-Whitney Rank Sum Test). This finding may suggest a possible implication of galectins-1 and -3 in the invasiveness of the transformed trophoblastic cell, although the exact physiological significance of this finding remains to be determined.

Expression of galectin-3 and carcinoembryonic antigen in the trophoblast of the gestational trophoblastic disease.

PURPOSE: Galectin-3, an endogenous beta-galactoside- binding protein, has been implicated in the regulation of many physiological and pathological cellular processes through specific interactions with complementary ligands. The level of galectin-3 expression has been correlated with metastatic potential in many tumor types. The present study was designed to investigate possible correlation of the expression of galectin-3 with carcinoembryonic antigen (CEA), one of the putative galectin-3 ligands, in the transformed trophoblast of the gestational trophoblastic diseases (GTDs) compared to the invasive trophoblast (interstitial and endovascular) of the normal first trimester of pregnancy placenta. MATERIALS AND METHODS: Double immunohistochemistry was used to examine paraffin sections of 9 specimens of normal placenta, 8 cases of invasive mole and 7 cases of choriocarcinoma. Immunoreactivity was scored semiquantitatively to include both the percent of trophoblast cells stained as well as the intensity of staining. RESULTS: Data presented here demonstrate similar patterns of expression for galectin-3 and CEA in transformed GTDs trophoblastic cells, as well as in normal invasive trophoblast. Upregulation of galectin-3 in transformed GTDs trophoblast was followed by high levels of CEA expression in the same tissue specimen (significant differences at < 0.05, Mann-Whitney Rank Sum Test). CONCLUSION: These results may point to the CEA as one of the ligands for galectin-3 in transformed trophoblast, which merits further investigation.

Galectin-8 is expressed by villous and extravillous trophoblast of the human placenta.

Galectins (gals) as multifunctional animal lectins are of great potential significance for establishment and function of the placenta, due to their capacity to modulate cellular functions including proliferation, adhesion, and invasion. Human trophoblast is known to express gal-1, gal-3 and gal-13 proteins, as well as mRNAs for gal-14, -16 and -17. This study was undertaken to establish cellular distribution of gal-8, not previously associated with trophoblast, since we have recently detected gal-8 RNA and protein in cytotrophoblast cell material. Results of immunolocalization showed that gal-8 was expressed by villous and extravillous cytotrophoblast.