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Role of integrin-linked kinaae in neurite outgrowth and axon guidance on neuronal repair

整合素连接的激酶在神经突生长和轴突引导神经元修复中的作用

基本信息

批准号：
14560242
负责人：
ISHII Toshiaki
金额：
$ 2.18万
依托单位：
Obihiro University of Agriculture and Veterinary Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560242/
关键词：
ILK Integrin N1E-115 cell p38 MAP kinase GSK-3β Tau Neurite Differentiation LK neurite p38MAP kinase N1E-115 神経分化神経突起

项目摘要

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against β1 integrin inhibits neurite outgrowth. Thus, β1 integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of β1 integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild-type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whe … More reas that in the DN-ILK transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinases kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK expressed cells. The tune course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells. Moreover, ILK also controls tau phosphorylation via regulation of glycogen synthase kinase-3β (GSK-3β) activity in N1E-115 cells. Stable transfection of DN-ILK resulted in aberrant tau phosphorylation in N1E-115 cells at sites recognized by the Tau-1 antibody, which are identical to some of the phosphorylation sites in paired helical filaments, (PHF)-tau, in brains of patients with Alzheimer's disease. The tau phosphorylation levels in the DN-ILK-expressing cells are constant under normal and differentiating conditions. On the other hand, aberrant tau phosphorylation was not observed in the parental control cells. ILK inactivation resulted in an increase in the active form but a decrease in the inactive form of GSK-3β, which is a candidate kinase involved in PHF-tau formation. Moreover, inhibition of GSK-3β with lithium prevented aberrant tau phosphorylation in the DN-ILK-expressing cells. These results suggest that ILK inactivation results in aberrant tau phosphorylation via sustained activation of GSK-3β in N1E-115 Cells. ILK directly phosphorylates GSK-3β and inhibits its activity. Therefore, endogenous ILK protects against GSK-3β-induced aberrant tau phosphorylation via inhibition of GSK-3β activity in N1E-115 cells. Less

在层粘连蛋白基质上生长的小鼠N1E-115细胞在血清缺失的情况下表现出神经突的生长。用抗β1整合素抗体处理细胞可抑制神经突生长。因此，β1整合素参与了层粘连蛋白基质上N1E-115细胞的神经生成。整合素连接激酶（integrin -linked kinase， ILK）是最近发现的一种细胞质丝氨酸/苏氨酸蛋白激酶，它与β1整合素的细胞质结构域结合，在整合素的跨膜信号转导中起重要作用。我们报道ILK在N1E-115细胞中表达，其表达水平在正常和分化条件下都是恒定的。稳定转染ILK激酶缺陷突变体（DN-ILK）可抑制在层粘连蛋白上生长的血清饥饿的N1E-115细胞的神经突生长。另一方面，野生型ILK的短暂表达刺激了神经突的生长。亲本细胞在层粘连蛋白基质上播种后，ILK活性瞬间被激活，而转染DN-ILK的细胞则没有。这些结果表明，血清饥饿的N1E-115细胞在层粘连蛋白作用下的神经突生长需要ILK的短暂激活。在相同的条件下，p38丝裂原活化蛋白（MAP）激酶，而不是MAP激酶/细胞外信号调节激酶（MEK）和细胞外信号调节激酶（ERK），在N1E-115细胞附着于层粘连蛋白后被短暂激活，但在表达DN-ILK的细胞中没有。p38 MAP激酶的激活过程与ILK的激活过程非常相似。此外，p38 MAP激酶抑制剂SB203580显著阻断了神经突的生长。因此，p38 MAP激酶的激活参与了ilk介导的信号转导，导致N1E-115细胞中整合素依赖性的神经突生长。此外，ILK还通过调节N1E-115细胞中的糖原合成酶激酶-3β (GSK-3β)活性来控制tau磷酸化。稳定转染n - ilk导致N1E-115细胞中tau -1抗体识别位点的异常磷酸化，这些位点与阿尔茨海默病患者大脑中配对螺旋丝(PHF)-tau中的一些磷酸化位点相同。在正常和分化条件下，表达dn - ilk的细胞中的tau磷酸化水平是恒定的。另一方面，在亲代对照细胞中未观察到异常的tau磷酸化。ILK失活导致GSK-3β的活性形式增加，而非活性形式减少，GSK-3β是参与PHF-tau形成的候选激酶。此外，用锂抑制GSK-3β可以阻止表达dn - ilk的细胞中异常的tau磷酸化。这些结果表明，在N1E-115细胞中，ILK失活通过GSK-3β的持续激活导致了异常的tau磷酸化。ILK直接磷酸化GSK-3β并抑制其活性。因此，内源性ILK通过抑制N1E-115细胞中GSK-3β的活性来防止GSK-3β诱导的异常tau磷酸化。少