Pullar C E, Isseroff R R, Nuccitelli R
Department of Dermatology, University of California, Davis, CA 95616, USA.
Cell Motil Cytoskeleton. 2001 Dec;50(4):207-17. doi: 10.1002/cm.10009.
Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.
皮肤伤口愈合需要上皮细胞迁移以实现再上皮化、伤口闭合和正常功能的重建。我们认为，启动伤口愈合的最早信号之一是伤口电流产生的侧向电场。在生理强度为100 mV/mm的直流电中，正常人表皮角质形成细胞会朝着负极迁移，负极代表伤口中心。对于这些细胞用于感知内源性电场的信号转导机制，人们几乎一无所知。为了阐明蛋白激酶（PK）可能参与这一过程，我们使用了PK抑制剂。本文描述了两项重要发现。首先，添加50 nM的PKA抑制剂KT5720后，角质形成细胞在电场中的定向反应降低了53%，而对细胞的总体运动性没有显著影响。这种降低是剂量依赖性的，从5 nM到50 nM，定向反应逐渐降低。其次，添加1 μM的肌球蛋白轻链激酶抑制剂ML-7后，细胞迁移的距离大约减少了31%，但不影响定向迁移。4 μM的PKC抑制剂GF109203X、20 μM的H-7以及CaM激酶抑制剂W-7，虽然都会使定向迁移略有降低，但均未显著改变定向迁移或细胞迁移。15 μM的PKC抑制剂D-赤藓鞘氨醇对迁移距离或定向迁移几乎没有影响。这些发现表明，不同的激酶信号通路调节细胞的总体运动性和持续的定向迁移，并突出了所涉及的信号转导机制的复杂性。本文所述的抑制剂研究表明，PKA在调节对施加电场的定向迁移反应即趋电性中发挥作用。
