Keratinocyte Galvanotaxis and Wound Healing

描述(由申请人提供)有越来越多的证据表明 内生电场在组织修复和重塑中的重要性。我们的 研究已经扩展了内生电场与皮肤伤口愈合的生理相关性，证明了产生如此大小的直流场 一旦受伤，立即提供角质形成细胞迁移的指导信号。 角质形成细胞在生理直流电场中向阴极迁移 强度，而且由于场的阴极点位于 创伤，角质形成细胞的定向迁移被引导以促进创伤 再上皮化。我们现在已经开发了工具和资源来剖析 伤口愈合的这一重要方面所涉及的机制， 它的应用可能会导致新的治疗方法。其中一个 我们的成果的亮点是演示了一项要求 表皮生长因子受体(EGFR)激酶的激活及其意义 重新定位到细胞的阴极面。这促使我们提议 有两个具体的问题。首先，表皮生长因子受体是如何定位于阴极的？ 牢房的脸？我们将确定直流电场应用是否会直接 携带EGF受体的囊泡的局部阴极胞吐作用。本地化 胞吐作用会在细胞的阴极面增加膜，从而 板膜的伸展和定向运动，我们将检查细胞 电池阴阳极两端的膜张力以解决这一问题 问题。第二，由于趋电反应依赖于EGF受体激酶的活性，我们将剖析其上游通路。 直流场中的局部激活。由于活性氧物种(ROS)可以 激活EGFR，我们将确定DC场是否产生极化到 阴极细胞膜。ROS上调或下调对血管内皮细胞生长的影响 定向运动性和EGFR磷酸化将被研究。此外， 我们的初步数据表明G蛋白偶联的β2-肾上腺素能受体 (B2AR)，在角质形成细胞迁移中，因此通过DC反式激活EGFR 我们将研究电场诱导的B2AR的激活。定向调解 B2AR与GTP结合的GI或Gs选择性结合的迁移 蛋白质将被测定。我们认为，多条路径可能汇聚在 EGFR响应于DC场启动定向迁移。 对趋化电流通路的剖析可能会提供一种 理解定向迁移的基本细胞功能的范式 趋化性，并最终将指导我们进行潜在的治疗干预 以促进伤口愈合。

DESCRIPTION (provided by applicant) There is increasing evidence of the importance of endogenous electric fields in tissue repair and remodeling. Our work has extended the physiological relevance of endogenous electric fields to skin wound healing, demonstrating that DC fields of the magnitude generated immediately upon wounding provide guidance cues for keratinocyte migration. Keratinocytes migrate toward the cathode in DC electric fields of physiological strength, and since the cathodal pole of the field is at the center of the wound, directional migration of keratinocytes is guided to facilitate wound re-epithelialization. We now have developed the tools and resources to dissect the mechanisms involved in this important aspect of wound healing, the application of which may lead to novel therapeutic approaches. One of the highlights of our fmdings has been the demonstration of a requirement for epidermal growth factor receptor (EGFR) kinase activation and its re-localization to the cathodal face of the cell. This prompts us to propose two specific issues. First, how is the EGF receptor localized to the cathodal face of the cell? We will determine if DC electric field application directs localized cathodal exocytosis of EGF receptor-carrying vesicles. Localized exocytosis would add membrane at the cathodal face of the cell, allowing lamellipodial extension and directional movement, and we will examine cell membrane tension at cathodal and anodal facing ends of the cell to address this question. Second, because the galvanotaxis response is dependent on EGF receptor kinase activity, we will dissect the upstream pathways for its localized activation in DC fields. Since reactive oxygen species (ROS) can activate EGFR, we will determine if DC fields generate ROS polarized to the cathodal cell membrane. The effects of up or down-regulation of ROS on directional motility and EGFR phosphorylation will be investigated. Moreover, our pilot data implicate the G protein-coupled beta2-adrenergic receptor (B2AR), in keratinocyte migration, so transactivation of the EGFR through DC field-induced activation of the B2AR will be examined. Mediation of directional migration by selective association of B2AR with either the GTP-binding Gi or Gs proteins will be determined. We submit that multiple pathways may converge upon the EGFR to initiate directional migration in response to the DC field. Dissection of the pathways governing galvanotaxis will likely provide a paradigm for understanding the basic cell function of directional migration in chemotaxis and will ultimately direct us to potential therapeutic interventions to enhance wound healing.