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.

Mycophenolate mofetil inhibits differentiation, maturation and allostimulatory function of human monocyte-derived dendritic cells.

We have studied the effect of mycophenolate mofetil (MMF), a new drug used in prevention of transplant rejection, on differentiation, maturation and allostimulatory activity of human monocyte-derived dendritic cells (MDDC). MDDC were generated in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of MMF. MMF reduced the number of immature MDDC in culture, dose-dependently, by inducing apoptosis and inhibited their stimulatory activity on allogeneic lymphocytes. These changes correlated with down-regulation of co-stimulatory and adhesion molecules such as CD40, CD54, CD80 and CD86. No differences were observed in mannose receptor (MR)-mediated endocytosis, measured by the uptake of fluorescein isothiocyanate (FITC)-dextran. MDDC differentiated in the presence of MMF showed significantly reduced maturation upon stimulation with lipopolysaccharide, as judged by lower expresson of CD83 and co-stimulatory molecules, lower production of tumour necrosis factor (TNF)-alpha, IL-10, IL-12 and IL-18 as well as lower stimulation of alloreactive T cells including naive CD4+ CD45RA+ T cells. In contrast, MDDC matured in the presence of MMF showed a more marked decrease in the FITC-dextran uptake than mature MDDC cultivated without MMF and the phenomenon correlated with down-regulation of the MR expression. These results suggest that MMF impairs differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.