| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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
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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
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