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. 2014 Mar 18;9(3):e92252.
doi: 10.1371/journal.pone.0092252. eCollection 2014.

A small physiological electric field mediated responses of extravillous trophoblasts derived from HTR8/SVneo cells: involvement of activation of focal adhesion kinase signaling

Juan Zhang  1 , Rongmei Ren  1 , Xuefeng Luo  1 , Ping Fan  1 , Xinghui Liu  2 , Shanshan Liang  1 , Lei Ma  1 , Ping Yu  3 , Huai Bai  1
Affiliations

A small physiological electric field mediated responses of extravillous trophoblasts derived from HTR8/SVneo cells: involvement of activation of focal adhesion kinase signaling

Juan Zhang et al. PLoS One. .

Abstract

Moderate invasion of trophoblast cells into endometrium is essential for the placental development and normal pregnancy. Electric field (EF)-induced effects on cellular behaviors have been observed in many cell types. This study was to investigate the effect of physiological direct current EF (dc EF) on cellular responses such as elongation, orientation and motility of trophoblast cells. Immortalized first trimester extravillous trophoblast cells (HTR-8/SVneo) were exposed to the dc EF at physiological magnitude. Cell images were recorded and analyzed by image analyzer. Cell lysates were used to detect protein expression by Western blot. Cultured in the dc EFs the cells showed elongation, orientation and enhanced migration rate compared with non-EF stimulated cells at field strengths of 100 mV/mm to 200 mV/mm. EF exposure increased focal adhesion kinase (FAK) phosphorylation in a time-dependent manner and increased expression levels of MMP-2. Pharmacological inhibition of FAK impaired the EF-induced responses including motility and abrogated the elevation of MMP-2 expression. However, the expression levels of integrins like integrin α1, α5, αV and β1 were not affected by EF stimulation. Our results demonstrate the importance of FAK activation in migration/motility of trophobalst cells driven by EFs. In addition, it raises the feasibility of using applied EFs to promote placentation through effects on trophoblast cells.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Motility enhancement of trophoblast cells in a small physiological EF.
Trophoblast cells in culture exposed to an EF of 150/mm (over 5 hours) showed enhanced migration rate compared with non-EF stimulated cells (0 mV) at field strengths of 100 mV/mm and 200 mV/mm (**p<0.01, respectively).
Figure 2
Figure 2. Trophoblast cells elongated and orientated perpendicularly in the electric field.
Trophoblast cells exposed to small, applied EF (150 mV/mm) showed elongation and perpendicular orientation (Fig 2B), while control cells that were not subjected to EF showed no such responsiveness (Fig 2A). The morphology of control and EF treated cells stained by fluorescein isothiocyanate labeled Phalloidin (for staining F actin) showed in Fig 2C and D, respectively. Enhanced elongation and orientation compared with non-EF stimulated cells (control) at field strength of 150 mV/mm (Fig 2E and F, respectively). The error bars represent the S.E.*p<0.05 and **p<0.0001. Bar = 50 μm
Figure 3
Figure 3. A small EF induces activation of FAK in trophoblast cells.
A, FAK protein expression; B, quantification of FAK protein level. Trophoblast cells were stimulated by an EF of 150 mV/mm for 0, 5, 10, 30 and 60 min, respectively. Cell lysates were prepared after treatment of the cells with EF and Western blot was done for the FAK expression and activation as mentioned in Materials and Methods. FAK protein phosphorylations were increased significantly at the time points 10 to 60 min (compared with 0 h control), whereas FAK total protein levels showed no significant changes. The error bars represent the S.E. (*p<0.05, and **p<0.01).
Figure 4
Figure 4. Effect of EF on the expression of MMP-2 and TIMP-2.
EF stimulation enhanced the expression of MMP-2 (A), but not its endogenous inhibiting molecule TIMP-2 (B). Cell lysates were prepared after treatment of HTR-8/SVneo cells with EFs (150 mV/mm) for 5 and 10 h, respectively, and Western blot was done for the expression of MMP-2 and TIMP-2 as mentioned in Materials and Methods. A and B are representative of MMP-2 and TIMP-2 protein signal respectively, and house-keeping gene GAPDH as internal controls. Histograms depicting respective MMP-2 and TIMP-2 protein levels normalized to GAPDH. The error bars represent the S.E. (*p<0.05).
Figure 5
Figure 5. Effect of EF on expression of integrins.
EF stimulation did not alter the expression of integrins α1, α5, αV and β1 in trophoblast cells (A, B, C and D, respectively). Cell lysates were prepared after treatment of HTR-8/SVneo cells with EFs (150 mV/mm) for 5 and 10 h, respectively, and Western blot was done for the expression of α1, α5, αV and β1 as mentioned in Materials and Methods.
Figure 6
Figure 6. Inhibiting of FAK activation abrogates the EFs mediated cellular responses including migration/motility and MMP-2 expression.
A, EF stimulated cells without FAK inhibitor treatment; B and C, EF stimulated cells pretreated with 10 μM FAK inhibitor for 5 h and 10 h,respectively. D, Trophoblast cells were pretreated with FAK inhibitor (10 μM) for 1 hour, and then the cells were stimulated by an EF of 150 mV/mm for 5 h and 10 h, respectively. Cells cultured in EF that had not been subjected to inhibitor treatment was used as the control. Cell lysates were prepared after treatment of the cells with EFs and Western blot was done for the expression of MMP-2. Histogram depicting MMP-2 protein levels normalized to GAPDH. The error bars represent the S.E. (**p<0.01). Bar = 50 μm.
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Grants and funding

This work was supported by grants from the National Natural Science Foundation of China 30872774 (HB) and 81271733 (HB), Sichuan Province Science and Research foundation of China 2012SZ0011 (HB), National Basic Research Program of China (973 program, No. 2007CB948101), Program for Changjiang Scholars and Innovative Research Team in University IRT0935, and Research Seed Fund from West China Second University Hospital of Sichuan University (HB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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