Li Min, Firth James D, Putnins Edward E
Laboratory of Periodontal Biology, Department of Oral Biological and Medical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
J Dermatol Sci. 2009 Mar;53(3):182-91. doi: 10.1016/j.jdermsci.2008.10.008. Epub 2008 Dec 25.
KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood.
We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism.
Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors.
Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%).
Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.
角质形成细胞生长因子受体（KGFR）仅在上皮细胞中表达，在伤口愈合中起重要作用。然而，在伤口愈合过程中KGFR激活和信号传导的机制尚不清楚。
我们利用体外机械损伤模型来研究非配体依赖性KGFR激活、其受活性氧（ROS）的调节以及这种激活机制的功能意义。
对汇合的HaCaT细胞系培养物进行机械损伤，使用共聚焦显微镜免疫染色和蛋白质印迹免疫沉淀法检测KGFR内化和磷酸化。检测损伤诱导的活性氧生成和KGFR的非配体依赖性激活。此外，在存在和不存在ROS及通路特异性抑制剂的情况下，检测其相关分子FRS2和c-Src的磷酸化。在存在和不存在这些抑制剂的情况下，还检测了这种激活过程对细胞迁移的重要性。
机械损伤诱导非配体依赖性KGFR激活和内化。KGFR内化和磷酸化与伤口边缘的ROS生成有关，用NAC清除ROS可抑制KGFR磷酸化。在细胞内，c-Src因损伤而磷酸化，但其抑制剂PP1显著抑制KGFR激活和相关的FRS2磷酸化。机械损伤诱导伤口边缘迁移，选择性受体和通路抑制剂PP1（82.7%）、KGFR抑制剂SU5402（70%）和MAPK抑制剂PD98059（57%）可显著减少这种迁移。
机械损伤在伤口边缘诱导大量ROS生成，进而通过c-Src激酶信号传导诱导非配体依赖性KGFR和FRS2激活。在功能上，下游MAPK信号传导诱导伤口边缘细胞迁移。
