Tan Jacqueline, Digicaylioglu Murat, Wang Stacy X J, Dresselhuis Jonathan, Dedhar Shoukat, Mills Julia
Department of Biology, Trinity Western University, Langley, British Columbia, Canada.
Departments of Neurosurgery and Physiology, Scripps Research, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Heliyon. 2019 Aug 16;5(8):e02294. doi: 10.1016/j.heliyon.2019.e02294. eCollection 2019 Aug.
Insulin promotes neuronal survival by activating a phosphatidylinositol 3-kinase (PI 3-kinase)/AKT-dependent signaling pathway and reducing caspase activation. We investigated a role for integrin-linked kinase (ILK) in insulin-mediated cell survival in cultured neurons and differentiated R28 cells. We used a serum and depolarization withdrawal model to induce apoptosis in cerebellar granule neurons and a serum withdrawal model to induce apoptosis in differentiated R28 cells. ILK knock-out decreased insulin-mediated protection as did the addition of pharmacological inhibitors of ILK, KP-392 or QLT-0267. Prosurvival effects of insulin were rescued by Boc-Asp (O-methyl)-CHF (BAF), a pancaspase inhibitor, in the presence of KP-392. Insulin and IGF-1 decreased caspase-3 activation, an effect that was inhibited by KP-392 and QLT-0267. Western blot analysis indicates that insulin-induced stimulation of AKT Ser-473 phosphorylation was decreased after the ILK gene was conditionally knocked-out, following overexpression of AKT-DN or in the presence of QLT-0267. Insulin and IGF-1 stimulated ILK kinase activity in primary neurons and this was inhibited following ILK-DN overexpression. Western blot analysis indicates that insulin exposure upregulated the expression of the cellular inhibitor of apoptosis protein c-IAP2 in an extracellular matrix-dependent manner, an effect blocked by KP-392. These results indicate that ILK is an important effector in insulin-mediated neuroprotection.
胰岛素通过激活磷脂酰肌醇3激酶（PI 3激酶）/AKT依赖性信号通路并减少半胱天冬酶激活来促进神经元存活。我们研究了整合素连接激酶（ILK）在培养的神经元和分化的R28细胞中胰岛素介导的细胞存活中的作用。我们使用血清和去极化撤除模型诱导小脑颗粒神经元凋亡，以及血清撤除模型诱导分化的R28细胞凋亡。ILK基因敲除降低了胰岛素介导的保护作用，添加ILK的药理抑制剂KP-392或QLT-0267也有同样效果。在存在KP-392的情况下，泛半胱天冬酶抑制剂Boc-Asp（O-甲基）-CHF（BAF）挽救了胰岛素的促存活作用。胰岛素和IGF-1降低了半胱天冬酶-3的激活，这种作用被KP-392和QLT-0267抑制。蛋白质印迹分析表明，在ILK基因被条件性敲除后、AKT-DN过表达后或存在QLT-0267的情况下，胰岛素诱导的AKT Ser-473磷酸化刺激作用降低。胰岛素和IGF-1刺激原代神经元中的ILK激酶活性，而在ILK-DN过表达后这种活性受到抑制。蛋白质印迹分析表明，胰岛素暴露以细胞外基质依赖性方式上调细胞凋亡抑制蛋白c-IAP2的表达，这种作用被KP-392阻断。这些结果表明，ILK是胰岛素介导的神经保护中的重要效应分子。
