Signal transduction mechanism of cyclosporine A regulating the invasiveness and migration of human trophoblast cells: an in vitro experiment

Abstract

Objective To investigate the signal pathway by which cyclosporine A(CsA)improves the invasiveness and migration of the first trimester human trophoblast cells. Methods Human first trimester trophoblast cells obtained during artificial abortion and human chorioepithelioma cells of the line JAR were cultured and put into the upper chamber of Transwell cell invasion system.Dimethyl sulfoxide (DMSO)or CsA of the concentrations of 0.0001,0.001,0.01,0.1 and 1.0 μmol/L respectively were added into the upper chamber for 48 h. Some of the upper chambers underwent pretreatment of U0126,a specific inhibitor of MAPK/ERK1/2,for 20 min.The invasiveness and migratory capabilities of the human trophoblast cells was examined.ELISA was used to detect the MAP kinase(MAPK)activity of the human trophoblasts in response to CsA stimulation.JAR cells were inoculated in 96-well plate and co-transferred with p-nuclear factor of activated T cells(pNFAT)-luciferase and internal control plasmid pRL-sV40 for 24 h,and then stimulated with CsA,ionomycin,and phorbol myristate acetate for 4 h;luciferase reporter system was used to detect the activity of the luciferase so as to calculate the activity of NFAT.Results The invasiveness and migration of the human trophoblast cells were enhanced by CsA in a dose-dependent manner,peaked at the CsA concentration of 1.0μmol/L,and then decreased.Activation of MAPK/ERK1/2 in the trophoblasts was observed as early as 10 min after the stimulation of CsA(1.0 μmol/L), and peaked 30 min after the stimulation. Moreover,the CsA-induced ERK1/2 activation remained at a higher level for at least 1 h,and then decreased significantly 2 h following the CsA treatment.Pretreatment with U0126 inhibited the invasion and migration of the trophoblasts.Activation of Ca2+/calcineurin/NFAT by ionomycin inhibited the invasion and migration of human trophoblasts,which were restored by CsA as well as NFAT inhibitor.The promotion of the invasion and migration of human trophoblasts by CsA was stronger than that by the NFAT inhibitor,and no synergy was seen between these 2 factors.U0126 blocked the MAPK/ERK1/2 signal pathways,but not influenced the NFAT transcription activity.Conclusion CsA promotes the invasiveness and migration of human trophoblast cells by activating the MAPK/ERK1/2 and inhibitingCa2+/calcineurin/NFAT signal pathways independently. Key words: Cyclosporin A; Trophoblastic neoplansms; Neoplansms invasiveness