Galectin-1 Is Part of Human Trophoblast Invasion Machinery - A Functional Study In Vitro

Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.These findings qualify gal-1 as a member of human trophoblast cell invasion machinery

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 mug/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 mug/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 alpha1 was reduced by ISO-1 in both cell types, while integrins alpha5 and beta1 were not changed. Addition of rhMIF increased integrin alpha1. 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

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-hydroxypheny1)-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 mu g/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 mu g/ml reduced invasion to 59\% of control, while rhMIF (200 ng/m1) 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 alpha 1 was reduced by ISO-1 in both cell types, while integrins alpha 5 and beta 1 were not changed. Addition of rhMIF increased integrin alpha 1. 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. (C) 2014 Elsevier Ltd. All rights reserved.Ministry of Education, Science and Technological Development, Republic of Serbia {[}173004]; {[}173013

Human trophoblast requires galectin-3 for cell migration and invasion

Abstract 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 β1 was followed in silenced cells, showing lower levels of MMPs and a large reduction in integrin subunit β1. These results show that galectin-3 acts as a pro-invasive autocrine/paracrine factor in trophoblast in vitro