Laboratorio de Canales Iónicos, Instituto de Investigaciones Médicas A. Lanari, IDIM-CONICET, Universidad de Buenos Aires, C. de Malvinas 3150, 1427 Buenos Aires, Argentina.
Laboratorio de Canales Iónicos, Instituto de Investigaciones Médicas A. Lanari, IDIM-CONICET, Universidad de Buenos Aires, C. de Malvinas 3150, 1427 Buenos Aires, Argentina.
Placenta. 2014 Feb;35(2):92-8. doi: 10.1016/j.placenta.2013.12.004. Epub 2013 Dec 21.
ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior.
CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method.
The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 μM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTR(inh)-172 (2 μM) for its inhibition. The conductances in the presence of CFTR(inh)-172 plus forskolin (16.0 ± 0.7 pS/pF and 32.6 ± 1.5 pS/pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/pF). The conductance of CFTR(inh)-172 inhibited currents was 14.9 ± 0.7 pS/pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0% of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTR(inh)-172, respectively. Proliferation was not changed by any of the treatments.
Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.
ENaC 和 CFTR 在细胞上皮中共同表达，并具有正或负的功能相互作用。此外，ENaC 和 CFTR 分别促进胎盘滋养层细胞和人气道细胞的迁移。在这里，我们检测了 CFTR 是否在胎盘滋养层细胞系 BeWo 细胞中具有功能性表达，以及它是否参与其迁移行为的想法。
通过 RT-PCR、生物素化和 Western blot 研究 BeWo 细胞中的 CFTR 表达。用膜片钳分析离子电流，用划痕愈合法分析细胞迁移。
存在成熟的 CFTR 160kDa 带，并且证实其位于细胞膜表面。用腺苷酸环化酶激活剂 forskolin（20μM）激活通道，然后用 CFTR(inh)-172（2μM）抑制。CFTR(inh)-172 加 forskolin存在时的电导（16.0±0.7 pS/pF 和 32.6±1.5 pS/pF）明显低于仅存在 forskolin时的电导（29.7±0.9 和 47.0±2.0 pS/pF）。CFTR(inh)-172 抑制电流的电导为 14.9±0.7 pS/pF，具有线性 I-V 关系，表明 CFTR 的非整流特性。测量细胞迁移，当细胞在对照、 forskolin和 forskolin 加 CFTR(inh)-172 下培养时，覆盖伤口的百分比分别为 11.2±0.4%、24.0±1.7%和 13.9±1.0%。增殖不受任何处理的影响。
我们的结果表明，BeWo 细胞功能性表达 CFTR，它在增加细胞迁移过程的伤口愈合中起作用。
