Macrophage migration inhibitory factor is differentially expressed in normal and choriocarcinoma trophoblast cells.

Trophoblast cells are specific for placenta, the organ necessary for development of the fetus. Trophoblast derived choriocarcinoma is a rare cancer, with high metastatic potential, invading surrounding tissues and distant organs. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in a wide range of biological processes, which is increased in almost all human cancers. Expression of MIF in normal and choriocarcinoma trophoblast cells is investigated here, using normal extravillous trophoblast derived cell line HTR-8/SVneo, and choriocarcinoma cell lines JAR and JEG3. Expression of MIF and its receptors CD74 and CXCR2 was investigated at mRNA level using qPCR. Expression of MIF protein was studied using immunofluorescence and western blot, under reducing and native conditions, in whole cell lysates, subcellular fractions and conditioned media. The expression of MIF mRNA was similar in all three cell lines, while CD74 mRNA was more expressed in choriocarcinoma cells (14-fold for JAR, 12-fold for JEG3, p<0.01). CXCR2 mRNA was higher in JEG3 cell line compared to HTR-8/SVneo cells (6-fold, p<0.01). While the cellular level of MIF was similar, the level of secreted MIF was lower in JAR cell conditioned media compared to media of both HTR-8/SVneo (2.8-fold, p<0.01) and JEG3 cells (4.1-fold, p<0.001). Cellular distribution of MIF was similar between the studied cell types. MIF was predominantly cytoplasmic, but also detected in membrane, nuclear soluble and nuclear chromatin fraction. MIF appeared in high molecular weight complexes of >150 kDa under native conditions. A band of 140-145 kDa was consistently present in JEG3 cell lysates, while it was absent or very weak in other cell types. These results show that MIF/CD74 axis is shifted in choriocarcinoma, as previously shown for other cancers, and further justifies research towards the most effective MIF targeting therapeutics.

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.

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.

Prolactin stimulates cell migration and invasion by human trophoblast in vitro.

INTRODUCTION: Prolactin (PRL) is present in endometrium at the time of embryo implantation and throughout pregnancy. Extrapituitary PRL acts as a cytokine in cells expressing PRL receptor (PRLR). So far no specific function has been demonstrated for PRL in the trophoblast of early pregnancy. METHODS: PRLR in placental tissue and trophoblast cells was shown here immunochemically. The possibility that PRL could influence trophoblast cell migration and invasion was investigated in vitro using isolated cytotrophoblast of the first trimester of pregnancy placental tissue and HTR-8/SVneo cell line. Wound healing cell migration test was performed on HTR-8/SVneo cells, and both cell types were used in Matrigel invasion test. RESULTS: PRLR is expressed by extravillous cytotrophoblast of the cell column and the placental bed, as well as in isolated cytotrophoblast (CT) and HTR-8/SVneo cells. PRL (at 100 and 1000 ng/ml) stimulated HTR-8/SVneo cell migration and cell invasion in both cell types, which could be blocked by anti-PRLR. Integrins α1 and α5, and galectin-1 (gal-1) were variably increased in PRL treated CT and HTR-8/SVneo cells. DISCUSSION: To our knowledge this is the first study demonstrating that PRL stimulates trophoblast invasiveness through PRLR, which is accompanied by increased integrins and gal-1, not excluding change in other potential mediators. This finding further supports relevance of PRLR for invasive trophoblast. CONCLUSION: This report supports a possibility that PRL may have a role in trophoblast invasion in vivo.