胶质细胞源性神经营养因子（GDNF）诱导角膜上皮细胞的迁移和信号转导。

Glial cell-derived neurotrophic factor (GDNF)-induced migration and signal transduction in corneal epithelial cells.

作者信息

You L, Ebner S, Kruse F E

机构信息

Department of Ophthalmology, University of Heidelberg Medical School, Germany.

出版信息

Invest Ophthalmol Vis Sci. 2001 Oct;42(11):2496-504.

PMID:11581189

Abstract

PURPOSE

To identify signal-transduction pathways induced by glial cell-derived neurotrophic factor (GDNF) in corneal epithelial cells and to characterize its effect on cell migration.

METHODS

Expression of GDNF receptor (GFR) alpha-1 in human corneal epithelium was detected by RT-PCR and Western blot analysis. Expression and phosphorylation of Ret, activation of focal adhesion kinase (FAK) and mitogen-associated protein kinase (MAPK) signaling pathways, and phosphorylation of paxillin by GDNF were investigated by immunoprecipitation and Western blot analysis in primary human corneal epithelial cells and a corneal epithelial cell line. The tyrosine kinase inhibitor herbimycin A and Ras farnesyltransferase inhibitor manumycin were used to specifically inhibit GDNF-induced signaling pathways. In vitro wound-healing assays and modified Boyden chamber analysis were performed to investigate the effect of GDNF on epithelial cell migration.

RESULTS

Expression of GFRalpha-1 was detected in normal and transformed human corneal epithelium. GDNF induced tyrosine phosphorylation of Ret. Furthermore, tyrosine phosphorylation of FAK and phosphotyrosine kinase (Pyk) 2; serine phosphorylation of c-Raf, MEK1, and Elk 1; and tyrosine-threonine phosphorylation of Erk-1 and -2 were time-dependently activated in the presence of GDNF. Tyrosine phosphorylation of paxillin was also induced by GDNF. Migration of corneal epithelial cells was significantly stimulated by GDNF. Herbimycin A strongly inhibited the activation of Ret, FAK, c-Raf, and Erk-1 and -2; the phosphorylation of paxillin; and corneal epithelial cell migration. More specifically, the Ras inhibitor manumycin inhibited phosphorylation of c-Raf, MEK 1, Erk-1 and -2, and Elk 1, but not that of FAK.

CONCLUSIONS

Corneal epithelial cells express receptors specific for GDNF that are used by GDNF to induce intracellular signaling. FAK and MAPK pathways seem to be activated by GDNF to modulate gene transcription and cell migration. FAK seems to be an upstream regulator of the MAPK cascade for GDNF signal transduction. As an inducer of FAK-dependent corneal epithelial migration, GDNF may play an important role in corneal regeneration and wound healing.

摘要

目的

确定胶质细胞源性神经营养因子（GDNF）在角膜上皮细胞中诱导的信号转导途径，并表征其对细胞迁移的影响。

方法

通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测人角膜上皮中GDNF受体（GFR）α-1的表达。在原代人角膜上皮细胞和角膜上皮细胞系中，通过免疫沉淀和蛋白质免疫印迹分析研究Ret的表达和磷酸化、粘着斑激酶（FAK）和丝裂原活化蛋白激酶（MAPK）信号通路的激活以及GDNF对桩蛋白的磷酸化作用。使用酪氨酸激酶抑制剂赫伯霉素A和Ras法尼基转移酶抑制剂曼诺霉素特异性抑制GDNF诱导的信号通路。进行体外伤口愈合试验和改良的博伊登小室分析以研究GDNF对上皮细胞迁移的影响。

结果

在正常和转化的人角膜上皮中检测到GFRα-1的表达。GDNF诱导Ret的酪氨酸磷酸化。此外，在GDNF存在下，FAK和磷酸酪氨酸激酶（Pyk）2的酪氨酸磷酸化；c-Raf、MEK1和Elk 1的丝氨酸磷酸化；以及Erk-1和-2的酪氨酸-苏氨酸磷酸化呈时间依赖性激活。GDNF也诱导桩蛋白的酪氨酸磷酸化。GDNF显著刺激角膜上皮细胞的迁移。赫伯霉素A强烈抑制Ret、FAK、c-Raf和Erk-1及-2的激活；桩蛋白的磷酸化；以及角膜上皮细胞迁移。更具体地说，Ras抑制剂曼诺霉素抑制c-Raf、MEK 1、Erk-1和-2以及Elk 1的磷酸化，但不抑制FAK的磷酸化。