Nakajima Ken-Ichi, Tatsumi Makiko, Zhao Min
Department of Dermatology, University of California at Davis, Sacramento, CA, USA,
Department of Dermatology, University of California at Davis, Sacramento, CA, USA.
Cell Physiol Biochem. 2019;52(2):198-211. doi: 10.33594/000000014. Epub 2019 Feb 28.
BACKGROUND/AIMS: Directional migration of corneal epithelial cells is essential for healing of corneal wounds, which is a robust response mediated by biochemical and bioelectrical cues. Naturally occurring electric fields at corneal wounds provide a powerful guidance cue for directional cell migration, as does extracellular ATP. Our recent large-scale siRNA library screening identified a role for purinergic signaling in the electric field-guided migration (galvanotaxis/electrotaxis) of human corneal epithelial (hTCEpi) cells.
We examined the effect of extracellular ATP on galvanotaxis of hTCEpi cells. Galvanotactic cell migration was recorded by video microscopy, and directedness and migration speed was calculated. The role of purinergic receptors in galvanotaxis regulation was evaluated by pharmacological inhibition or knocking down of P2X and P2Y receptors.
Addition of ATP enhanced galvanotaxis, and most remarkably sensitized galvanotaxis response to very low level of electric fields in the physiological range (10-30 mV/mm). The stimulatory effect of extracellular ATP was diminished by apyrase treatment. Importantly, cells stimulated with extracellular ATP migrated with significantly increased directedness and speed, which were diminished by knocking down or pharmacological inhibition of P2X and P2Y receptors. Inhibition of pannexin-1 (ATP permeable channel) significantly impaired galvanotaxis. Moreover, pharmacological inhibition of ectoATPase enhanced galvanotaxis.
Extracellular ATP and physiological electric fields synergistically enhanced the galvanotaxis response of hTCEpi cells. hTCEpi cells are likely to secrete ATP actively, and purinergic signaling is down-regulated by ecto-ATPases. Both P2X and P2Y receptors coordinately play a role for galvanotaxis of hTCEpi cells.
背景/目的：角膜上皮细胞的定向迁移对于角膜伤口愈合至关重要，这是一种由生化和生物电信号介导的强烈反应。角膜伤口处自然产生的电场为细胞定向迁移提供了强大的引导信号，细胞外ATP也有此作用。我们最近通过大规模siRNA文库筛选确定了嘌呤能信号在人角膜上皮（hTCEpi）细胞电场引导迁移（趋电性/电趋性）中的作用。
我们研究了细胞外ATP对hTCEpi细胞趋电性的影响。通过视频显微镜记录趋电性细胞迁移，并计算方向性和迁移速度。通过药理学抑制或敲低P2X和P2Y受体来评估嘌呤能受体在趋电性调节中的作用。
添加ATP增强了趋电性，最显著的是使趋电性反应对生理范围内的极低电场水平（10 - 30 mV/mm）敏感。用腺苷三磷酸双磷酸酶处理可减弱细胞外ATP的刺激作用。重要的是，用细胞外ATP刺激的细胞迁移时方向性和速度显著增加，而敲低或药理学抑制P2X和P2Y受体可使其减弱。抑制泛素连接蛋白1（ATP通透通道）显著损害趋电性。此外，药理学抑制胞外ATP酶增强了趋电性。
细胞外ATP和生理电场协同增强了hTCEpi细胞的趋电性反应。hTCEpi细胞可能会主动分泌ATP，并且嘌呤能信号通过胞外ATP酶下调。P2X和P2Y受体在hTCEpi细胞趋电性中协同发挥作用。
