Macrophage migration inhibitory factor up-regulates alpha(v)beta(3) integrin and vascular endothelial growth factor expression in endometrial adenocarcinoma cell line Ishikawa
ABSTRACT Human endometrium undergoes a series of dynamic physiological changes during the menstrual cycle of reproductive age women. Many factors, including hormones, cytokines, growth factors, matrix metalloproteinases and integrins, are essential for the success of embryonic implantation into endometrial tissue. Herein, we used a well-differentiated endometrial adenocarcinoma cell line, Ishikawa, to investigate in vitro the role played by macrophage migration inhibitory factor (MIF) in the regulation of endometrial receptivity markers. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that MIF induced a slight increase in alpha(v) (alphav) mRNA integrin subunit expression during the first 12h, but reached a significant difference after 24h MIF treatment compared to control, whereas beta(3) (beta3) integrin subunit displayed significant increase in mRNA 2h following treatment. Immunocytofluorescence showed strong alphav and beta3 immunostaining at 25 ng/ml MIF, and Western blotting clearly indicated increased alphav and beta3 protein expression. MIF treatment significantly stimulated vascular endothelial growth factor (VEGF) mRNA expression in a dose- and time-dependent manner after 24 h treatment. Moreover, immunocytofluorescence revealed positive VEGF immunostaining compared to control, and analysis by ELISA of VEGF release in culture supernatants demonstrated that MIF (25 ng/ml) significantly induced VEGF secretion at 12 and 24 h. In conclusion, this study provides evidence that MIF directly up-regulates alphavbeta3 integrin and VEGF expression in human endometrial Ishikawa cells and may advance our understanding of factors involved in the establishment of endometrial receptivity and successful implantation.
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ABSTRACT: Galectin-3 (gal-3) is a beta-galactoside-binding protein which can be detected in endometrium. The study was designed to investigate synergism of gal-3 and integrinbeta3 in endometrial cell proliferation and adhesion in an in vitro model of endometrial receptivity. The RL95-2 cell line was employed as an in vitro model for receptive endometrium. Cells transfected with gal-3 siRNA or treated with exogenous gal-3 were incubated with or without function-blocking integrinbeta1/3 antibody for evaluating synergism of gal-3 and integrins on cell proliferation and adhesion. Proliferation was measured by BrdU incorporation, and adhesion to fibronectin (FN) was determined by an adhesion assay. Integrin expression was analyzed by Flow Cytometry and western blots. Bewo spheroids were co-cultured with the RL95-2 monolayer to mimic the blastocyst-endometrial interaction, and colocalization of gal-3, integrinbeta3 and FN at the interface was observed by confocal microscopy. The knock-down of gal-3 inhibited RL95-2 cell proliferation and adhesion. However, a reduction of proliferation and adhesion was also observed in presence of exogenous gal-3, and this was further reduced by a functional block to integrinbeta3. Moreover, gal-3 knock-down significantly increased integrinbeta3 expression, however, the colocalization of integrinbeta3 and FN was not increased. As expected, the colocalization of integrinbeta3 was decreased with the knock-down of gal-3. This study has provided an in vitro model for the complex interactions between gal-3 and integrinbeta3 in the regulation of endometrial cell proliferation and adhesion.Human Reproduction 08/2009; 24(11):2879-89. DOI:10.1093/humrep/dep250 · 4.59 Impact Factor
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ABSTRACT: Embryo implantation is the group of processes by which the developing blastocyst adheres to, and embeds into, the receptive endometrium. Changes in the expression of molecules on the cell surface have been seen in the conversion of the endometrial surface from a non-receptive to a receptive state. Cell adhesion molecules like integrins, or soluble factors such as Leukemia inhibitory factor (LIF) have been identified to play an important role during the implantation period. Studies have suggested that low-dose aspirin (<100 mg/d) treatment can improve implantation rate, ovarian responsiveness, and pregnant rates in IVF patients. The mechanism of this increased implantation rate is unclear, and in this study we hypothesised that the expression of integrin β3 or LIF may be altered, during implantation window, by treatment with low-dose aspirin. Female mice were treated with 0.9 mg/ml aspirin daily for 15 days and then were mated. The protein or mRNA levels of LIF and integrin β3 in the endmetrium were determined on the day 4 post-coitus using immuno-fluorescence and RT-PCR. We found the expression of LIF or integrin β3 was significantly increased in the endometrium of mice that were treated with low dose of aspirin by immuno-fluorescence. In addition, the mRNA level of LIF or integrin β3 on the endometrium of aspirin treated mice was also significantly increased compared to untreated mice. We conclude that low dose aspirin alters the expression of endometrial LIF and integrin β3 and that these changes may increase endometrial receptivity.Placenta 10/2010; 31(12):1101-5. DOI:10.1016/j.placenta.2010.10.002 · 3.29 Impact Factor
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ABSTRACT: To evaluate the role of macrophage migration inhibitory factor (MIF) in the wound healing process following severe chemical burns to the ocular surface. Chemical burning of the ocular surface was induced in mice (C57BL/6) via the application of 0.1 M NaOH. Macrophage migration inhibitory factor (MIF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) mRNA expression in the ocular surface and lacrimal gland was evaluated via real-time reverse transcription PCR on days 2, 7, and 30 after induction of the chemical burn. The expression of MIF protein in the ocular surface and lacrimal gland was evaluated via western blot analysis. Immunohistochemical staining was conducted to detect MIF and vasculoendothelial growth factor in the cornea during the wound healing process. The angiogenic role of MIF was further evaluated using an 8-0 polyglactin suture technique to induce corneal neovascularization. MIF, TNF-α, and IL-1β mRNA expression were elevated significantly in the ocular surface up to day 30 after chemical burn induction. TNF-α alone was elevated in the lacrimal gland. MIF protein elevation was confirmed via western blot analysis, and the spatial similarity of MIF and VEGF expression in the cornea was noted during the wound healing process. 8-0 polyglactin sutures soaked in MIF induced significantly higher numbers of new vessels on the mouse cornea after 7 days (p=0.003, Mann-Whitney test). These findings indicate that MIF performs a crucial role in wound healing on the ocular surface after the induction of chemical burns.Molecular vision 11/2010; 16:2402-11. · 2.25 Impact Factor