E-cadherin, as a negative regulator of invasive behavior of human trophoblast cells, is down-regulated by cyclosporin A via epidermal growth factor/extracellular signal-regulated protein kinase signaling pathway.
ABSTRACT Our previous study has demonstrated cyclosporin A (CsA) promotes the invasiveness of human first-trimester trophoblast cells. In the present study, we further investigated the intracellular signaling pathway responsible for the improvements in CsA-induced invasiveness of human trophoblast cells. We showed that CsA down-regulated E-cadherin transcription and translation in human primary cultured trophoblast cells and choriocarcinoma cell line JEG-3. U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK), attenuated the CsA-induced transcriptional repressor SNAI2 (also called Slug) expression and restored E-cadherin expression inhibited by CsA in JEG-3 cells. We further demonstrated that CsA amplified epidermal growth factor (EGF)-stimulated EGF receptor (EGFR) tyrosine phosphorylation in JEG-3 cells, and inhibition of EGFR tyrosine phosphorylation by AG1478, an EGFR tyrosine kinase inhibitor, abolished the down-regulation of E-cadherin by CsA through ERK signaling pathway. Moreover, our data showed that E-cadherin expression was negatively correlated to the invasiveness of JEG-3 cells, and CsA could reverse the decreased invasiveness of JEG-3 cells that resulted from E-cadherin overexpression. In conclusion, these observations indicate that CsA may decrease E-cadherin expression via EGFR/ERK signaling pathway and, ultimately, contribute to the invasiveness improvement of human trophoblast cells.
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ABSTRACT: Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-Bromo-2'-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells. Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478. CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.PLoS ONE 01/2012; 7(7):e38375. · 3.73 Impact Factor