Reactive oxygen species (ROS) is needed for insulin-mediated signaling in SK-N-BE(2) human nueroblastoma cell line

다국어 초록 (Multilingual Abstract)

In multicellular organisms, the behavior of individual cells such as their growth, differentiation, and migration is tightly controlled by their environment. Two major classes of controlling components are diffusible peptide growth factors and insoluble extracellular matrix(ECM) proteins. These molecules act independently and cooperatively on the cells. This study focused on the signal transduction pathway activated by peptide growth factors in the human neuroblastoma cell line, SK-N-BE(2) [BE(2)].
A previous report in my laboratory demonstrated that BE(2) cells did not respond normally to the peptide growth factor such as insulin and NGF. To figure out why these cells did not respond to these growth factors, I examined insulin signaling in these cells. Tyrosine phosphorylation induced by insulin in BE(2) cells was much less than in PC12 cells although the tyrosine phosphorylation of insulin receptor was increased. The phosphorylation of IRS-1 in BE(2) cells was also increased less than in PC12 cells, and neither Grb2 nor GAP that regulates Ras activity interacted with the phosphorylated IRS-1. Moreover, MAP kinase, the downstream of Ras, was rarely activated by insulin in BE(2) cells. PI3-kinase bound to IRS-1 and the activity of PI3-kinase was increased by insulin in BE(2) cells. These results indicate that these cells may have some defects in the insulin-mediated Ras-MAP kinase activation.
To find out whether Ras-MAP kinase pathway in BE(2) cells has defects, BE(2) cells were treated with pervanadate, a PTPase inhibitor. The tyrosine phosphorylation of many proteins, interaction of IRS-1 with Grb2, and MAP kinase activity were observed. These results suggest that BE(2) cells have defects not in Ras-MAP kinase cascade but in upstream of Ras-MAP kinase pathway activated by insulin.
Recently it has been demonstrated that reactive oxygen species(ROS) including H₂O₂ and superoxide anion(O₂^(-)) is produced by cytokines and growth factors and may have a general role in mediating tyrosine phosphorylation through the inhibition of protein tyrosine phosphatases(PTPases). Insulin decreased ROS in BE(2) cells. To examine the possibility that the decrease of ROS by insulin could inactivate Ras-MAP kinase pathway, I applied H₂O₂ to BE(2) cells by adding glucose oxidase to the culeutr medium then treated the cells with insulin. Insulin enhanced the tyrosine phosphorylation of many proteins, interaction of IRS-1 with Grb2, and MAP kinase activity. To confirm the role of ROS in Ras-MAP kinase pathway, PC12 cells which respond normally to insulin were treated with N-acetylcysteine(NAC), a ROS scavenger, and the treatment of insulin was followed. Those results represented the opposite effects: NAC reduced the total tyrosine phosphorylation, interaction of IRS-1 with Grb2 and MAP kinase activity by insulin. These data represent that insulin-induced Ras-MAP kinase pathway requires an increase in intracellular H₂O₂.
To identify a signaling molecule responsible for the defect of BE(2) cells in ROS generation, Rac1 gene from BE(2) cells was sequenced because Rac1 plays an important role in generating ROS in both phagocytic and nonphagocytic cells. The sequence of Rac1 in BE(2) cells has three substitutions in nucleic acids resulting amino acid changes. From these results, I concluded that the unresponsiveness of BE(2) cells to insulin is likely due to the abnormality of a ROS generation system that regulates Ras-MAP kinase pathway, and that abnormal Rac1 protein is the cause of this defect.