Kimura Kazuhiro, Teranishi Shinichiro, Yamauchi Junji, Nishida Teruo
Department of Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, 111 Minami-Kogushi, Ube City, Yamaguchi, Japan.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):125-32. doi: 10.1167/iovs.07-0725.
Migration of corneal epithelial cells plays an important role in healing of corneal epithelial wounds. The role of c-Jun NH(2)-terminal kinase (JNK), a member of the family of mitogen-activated protein kinases, in the intracellular signaling responsible for the migration of corneal epithelial cells during wound closure was examined.
Scratch wounds were introduced into cultured monolayers of simian virus 40-transformed human corneal epithelial (HCE) cells in the absence or presence of the JNK inhibitor SP600125. The phosphorylation and localization of JNK and paxillin during wound closure were examined by immunoblot and immunofluorescence analyses. The effects of a small interfering RNA (siRNA) specific for JNK and of a mutant form of paxillin on HCE cell migration were determined by transfection.
SP600125 inhibited wound healing in a time- and concentration-dependent manner. Immunoblot analysis showed that wounding increased the phosphorylation of JNK and of paxillin on serine (Ser) 178 in a manner sensitive to SP600125. Immunofluorescence staining revealed that phosphorylated JNK colocalized with paxillin at focal adhesions formed by HCE cells at the wound margin and that SP600125 inhibited the formation of such adhesions. Expression of JNK siRNA or of a paxillin mutant in which Ser178 is replaced by alanine inhibited HCE cell migration during wound closure.
JNK regulates HCE cell migration by modulating the phosphorylation of paxillin and the consequent formation of focal adhesions. A JNK-paxillin signaling pathway may thus play an important role in corneal epithelial wound healing in vivo.
角膜上皮细胞迁移在角膜上皮伤口愈合中起重要作用。本研究检测了丝裂原活化蛋白激酶家族成员c-Jun氨基末端激酶（JNK）在伤口闭合过程中负责角膜上皮细胞迁移的细胞内信号传导中的作用。
在不存在或存在JNK抑制剂SP600125的情况下，在猿猴病毒40转化的人角膜上皮（HCE）细胞的培养单层中制造划痕伤口。通过免疫印迹和免疫荧光分析检测伤口闭合过程中JNK和桩蛋白的磷酸化及定位。通过转染确定针对JNK的小干扰RNA（siRNA）和桩蛋白突变体对HCE细胞迁移的影响。
SP600125以时间和浓度依赖性方式抑制伤口愈合。免疫印迹分析表明，创伤以对SP600125敏感的方式增加了JNK和丝氨酸（Ser）178处桩蛋白的磷酸化。免疫荧光染色显示，磷酸化的JNK与桩蛋白在伤口边缘的HCE细胞形成的粘着斑处共定位，并且SP600125抑制了此类粘着斑的形成。JNK siRNA或Ser178被丙氨酸取代的桩蛋白突变体的表达在伤口闭合期间抑制了HCE细胞迁移。
JNK通过调节桩蛋白的磷酸化和随后粘着斑的形成来调节HCE细胞迁移。因此，JNK-桩蛋白信号通路可能在体内角膜上皮伤口愈合中起重要作用。
