Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways

Abstract

Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.

Keywords: Cyclosporine A; PCNA; migration; signal transduction pathway; trophoblast.

Figures

Figure 3

Blocking MAPK3/1 signaling inhibits CsA-induced PCNA expression and migration of trophoblasts. A: The expression of PCNA in trophoblasts was determined by flow cytometry. PCNA expression of human trophoblasts in CsA-treated group was significantly increased, and an addition of U0126 markedly inhibited the CsA-induced PCNA expression level. *P

Figure 5

CsA-mediated PCNA expression and migration of human trophoblasts is dependent on NF-κB activation. A: The expression of PCNA in trophoblasts was determined by flow cytometry. PCNA expression of human trophoblasts in CsA-treated group was significantly increased in vitro, and an addition of PTDC markedly inhibited the CsA-induced PCNA expression level (A). **P

Figure 1

CsA promotes the PCNA expression of human trophoblast in dose-related manner. The freshly isolated primary trophoblasts from humans in early pregnancy and the HTR8 cell line were treated with an increasing dose of CsA for 48 h. The cells were stained with PCNA and cytokeratin-7 and then analyzed using flow cytometry for the level of PCNA. The results were highly reproducible in 4 independent experiments (A), and the picture is a representative one (B). Data represent the mean ± standard error (SE) of 4 independent experiments with a total of 16 villi, performed in triplicate wells with 4 different samples. *P

Figure 2

CsA promotes human trophoblast migration in a dose-related manner. A: The wound-healing assay in HTR8/SVneo cells treated with different concentrations of CsA. An open furrow was generated by scratching cells at 90% confluent by using a pipette tip. Treatment with different concentrations of CsA speeded up the confluency. The distance between the furrow edges in the control or CsA-treated cells in 3 independent experiments was measured and presented graphically as percentage of the initial distance (0 h). Data represent the mean ± standard error (SE) of 3 independent experiments with a total of 15 villi, performed in triplicate wells with 3 different samples. *P

Figure 4

CsA activates NF-κB in human trophoblasts via MAPK3/1 signaling. A: Human trophoblast cells were serum starved overnight and then treated with 1.0 μM CsA at a different time. Western blot was used to analyze the total and phosphorylation levels of NF-κB p65 and I-κB. CsA induces NF-κB transactivation in human trophoblasts in time-related manner. B: Human trophoblast cells were serum starved overnight and then treated with vehicle, 1.0 μM CsA for 90 min. Some well were pre-added with 30 μM U0126 for 30 min. Protein expression were analyzed by Western blot. Blockade of MAPK3/1 inhibited the CsA-induced NF-κB transactivation. These pictures are representatives of 3 individual experiments with 16 villi.