Regulation of cell death and neurite outgrowth by overexpressed focal adhesion kinase in rat pheochrome cytoma PC 12

我们建立了高表达粘着斑激酶(FAK)或其突变基因的细胞命名为Y397F、K454R或Y925F。在用神经生长因子(NGF)诱导细胞分化后，我们评估了每个细胞对氧化应激或内质网应激诱导的细胞死亡的敏感性。(1)在过量表达的细胞中，氧化应激(鱼藤酮、多巴胺、H_2O_2)或内质网应激(布雷菲尔丁A、thapsigargin)可明显抑制细胞死亡。鱼藤酮诱导的caspase-2或caspase-3的激活也被抑制。(2)FAK的过表达倾向于抵抗氧化应激所致的细胞死亡。(3)在过表达Y397F突变的细胞中，与载体细胞相比，氧化应激诱导的细胞死亡更多。然而，K454R在催化部位的过表达可抑制细胞死亡。此外，鱼藤酮诱导的caspase-2或-3活性在Y397F细胞中也被增强，而在K454R细胞中被明显抑制。在C末端高表达Y925F的细胞中，细胞死亡的敏感性没有改变。(4)在高表达Y397F的细胞中，作为生存信号的一部分Akt的表达比载体细胞低。抗凋亡蛋白Bclxl的表达无明显变化。在K454R细胞中，这两种蛋白的表达没有改变。上述突变Y397F和K454R能够抑制FAK依赖的信号通路，但对细胞死亡的敏感性有所不同。有必要阐明FAK激活后信号的变化。(5)在过表达任何基因的细胞中，NGF诱导的轴突生长与载体细胞相似。

We established cells overexpressed focal adhesion kinase (FAK) or its mutated genes named Y397F, K454R or Y925F. After cells were differentiated with nerve growth factor (NGF), we estimated the sensitivity of each cell against cell death induced by oxidative stress or ER stress. We also determined the sensitivity of cells overexpressed antiapoptotic protein Bcl-2 or Bcl-xL.(1) In the cells overexpressed Bcl-2 or Bcl-xL, cell death was clearly suppressed induced by oxidative stress (rotenone, dopamine, H2O2) or ER stress (brefeldin A, thapsigargin). Activation of caspase-2 or 3 induced by rotenone was also inhibited.(2) Overexpression of FAK tended to resist against cell death by oxidative stress.(3) In the cells overexpressed Y397F mutation which is autophosphorylation site, cell death induced by oxidative stress was increased compared with vector cells. However, overexpression of K454R at catalytic site induced suppression of cell death. Moreover, rotenone-induced caspase-2 or -3 activity was also enhanced in Y397F cells, but clearly inhibited in K454R cells. In the cells overexpressed Y925F at C-terminus, sensitivity of cell death was not altered.(4) In the cells overexpressed Y397F, expression of Akt which is a part of survival signal, was decreased than vector cells. Antiapoptotic protein Bcl-xL expression was not altered. In the K454R cells, expression of both proteins were not altered. The above mutations, Y397F and K454R, are able to suppress FAK dependent signalling pathway, however, there is a difference against the sensitivity of cell death. It is necessary to clarify the varies of signalling after FAK activation.(5) In the cells overexpressed any genes, NGF-induced neurite outgrowth was similar to vector cells.