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

生长在层粘连蛋白基质上的小鼠N1E-115细胞在血清剥夺的情况下表现出突起生长。用β-1整合素抗体处理细胞可抑制轴突生长。因此，β1整合素参与了层粘连蛋白基质上N1E-115细胞的神经发生。整合素连接蛋白激酶(ILK)是新近发现的一种胞质丝氨酸/苏氨酸蛋白激酶，与β-1整合素的胞浆结构域结合，在整合素跨膜信号转导中发挥重要作用。我们报道了ILK在N1E-115细胞中的表达，其表达水平在正常和分化条件下都是恒定的。稳定地转染ILK的激酶缺陷突变体(DN-ILK)可以抑制生长在层粘连蛋白上的血清饥饿的N1E-115细胞的突起生长。另一方面，野生型ILK的瞬时表达刺激了神经突起的生长。在层粘连蛋白基质Whe…上种植后，亲代细胞中的ILK活性被瞬时激活更多的原因是在dN-ilk转染组细胞中没有。这些结果表明，在血清饥饿的N1E-115细胞中，ILK的瞬时激活是层粘连蛋白上突起生长所必需的。在相同条件下，N1E-115细胞与层粘连蛋白黏附后，p38丝裂原活化蛋白(MAP)激酶，而不是MAP/细胞外信号调节蛋白激酶(MEK)和细胞外信号调节蛋白激酶(ERK)被瞬时激活，而在表达DN-ILK的细胞中不被激活。P38MAPK的激活过程与ILK的激活过程非常相似。此外，p38MAPK抑制剂SB203580显著抑制了神经突起的生长。因此，p38MAPK的激活参与了ILK介导的信号转导，导致N1E-115细胞中整合素依赖的轴突生长。此外，ILK还通过调节N1E-115细胞中糖原合成酶激酶-3β(GSK-3β)的活性来控制tau的磷酸化。在N1E-115细胞中，稳定地转导dN-ILK导致Tau-1抗体识别的位置发生异常的tau磷酸化，这与阿尔茨海默病患者脑中成对螺旋细丝中的一些磷酸化位点(Phf)-tau相同。在正常和分化条件下，表达dN-ilk的细胞中tau的磷酸化水平是恒定的。另一方面，在亲本对照细胞中未观察到tau的异常磷酸化。ILK失活导致参与PHF-tau形成的候选激酶GSK-3β的活性增加而非活性减少。此外，锂抑制GSK-3β可阻止表达dN-ILK的细胞中tau的异常磷酸化。这些结果表明，ILK失活通过持续激活GSK-3β导致N1E-115细胞tau蛋白异常磷酸化。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β活性，对GSK-3 DNA诱导的tau异常磷酸化具有保护作用。较少

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