CXCL12/CXCR4 axis triggers the activation of EGF receptor and ERK signaling pathway in CsA-induced proliferation of human trophoblast cells

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. CsA promotes CXCL12 secretion in primary human trophoblast cells and JEG-3 cells. A

: The mRNA expression of CXCL12 and CXCR4 in primary human trophoblast cells (labeled as PT) and JEG-3 cells was detected by RT-PCR (the upper panels). CXCR4 protein expression was detected by using western blotting (the lower panels). The GAPDH was used as a loading control. B: CXCL12 secretion in primary human trophoblast cells and JEG-3 cells was detected by ELISA. Human trophoblast cells were treated with 1 μM CsA in 1640 complete medium supplemented with 10% FBS for 48 h, and the culture medium was then collected and subjected to ELISA. Data are presented as mean ± SEM of three independent experiments. *P<0.05, compared to the control. Ctrl, control.

Figure 2. CXCL12 promotes the phosphorylation of EGFR in primary human trophoblast cells and JEG-3 cells. A

: Recombinant human CXCL12 (20 ng/ml) was used to treat the primary human trophoblast cells for 30, 60, 120 min, respectively. The levels of phosphorylated EGFR and total EGFR were detected by immunofluorescence analysis. Scale bar, 25 μm. B: Recombinant human CXCL12 (20 ng/ml) was used to treat JEG-3 cells for 30, 60, 120 min, respectively. The phosphorylated and total levels of EGFR were analyzed by western blotting. A typical blot (the lower panels) and densitometry analysis of the ratio of phosphorylated EGFR to total EGFR (the upper panels) are shown. Data are presented as mean ± SEM of three independent experiments. *P<0.05, compared to the control.

Figure 3. CsA promotes the phosphorylation of EGFR in a CXCL12/CXCR4 axis-dependent manner.

A: Primary human trophoblast cells were treated with neutralizing antibody against CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) for 48 h, and then treated with 1 μM CsA in 1640 complete medium supplemented with 10% FBS for 1 h and subjected to immunofluorescence analysis to analyze the levels of phosphorylated EGFR and total EGFR. Scale bar, 25 μm. B: JEG-3 cells were transfected with CXCR4 siRNA (125 nM), or CXCL12 siRNA (125 nM), or non-targeting siRNA control, or treated with neutralizing antibody against CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) for 48 h, and then treated with 1 μM CsA in 1640 complete medium supplemented with 10% FBS for 1 h, and subjected to western blotting to analyze the levels of phosphorylated EGFR and total EGFR. A typical blot (the lower panels) and densitometry analysis of the ratio of phosphorylated EGFR to total EGFR (the upper panels) are shown. Data are presented as mean ± SEM of three independent experiments. *P<0.05, compared to the control.

Figure 4. EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells.

A: Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or U0126 (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P<0.05, compared to the control. #P<0.05, compared to CsA treatment group. B: JEG-3 cells were pretreated with siRNA against CXCL12 or CXCR4, or non-targeting siRNA control, or AG1478 for 48 h, and treated with 1 μM CsA in 1640 complete medium supplemented with 10% FBS for 1 h, and then subjected to western blotting to analyze the levels of phosphorylated ERK and total ERK. A typical blot (the lower panels) and densitometry analysis of the ratio of phosphorylated ERK to total ERK (the upper panels) are shown. Data are presented as mean ± SEM of three independent experiments. *P<0.05, compared to the control. #P<0.05, compared to CsA treatment group.

Figure 5. Schematic representation of intracellular signaling pathways in the modulation of CsA on the invasion and proliferation of human trophoblast cells.

CsA promotes the invasion of human trophoblast cells via two independent signaling pathways, calcium/CaN/NFAT and EGFR/ERK. E-cadherin (labeled as E-cad), MMP-2, MMP-9, and titin may function as the downstream targets of EGFR/ERK signaling pathway in the CsA-induced invasion of human trophoblast cells. CsA may promote the activation of EGFR via CXCL12/CXCR4 axis. EGFR and its downstream ERK signaling pathway may also be involved in the CsA-induced proliferation of human trophoblast cells. Blue lines represent the signaling pathways proved to be involved in the regulation of CsA on the invasion of human trophoblast cells, and red lines represent the putative signaling pathways involved in the regulation of CsA on the proliferation of trophoblast cells.