Evolutional investigation of the insulin/IGF receptor-mediated signal transduction systems using proteomic and transcriptomic analysis

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Vertebrates possess in addition to the insulin receptor, the insulin-like growth factor (IGF) receptors, which are involved in the regulation of various biological processes including mitogenesis, invasion, metastasis, and apoptosis inhibition of cancer cells. Moreover, the insulin/IGF signaling system also exists in the oldest metazoan such as sponge and hydra, invertebrates including nematodes and insects, and vertebrates, and regulates growth and aging. In this study, in order to investigate the molecular basis of the difference in the metabolic and mitogenic functions of the signal transduction systems regulated by insulin and IGF molecules, we analyzed cultured cells expressing the for insulin/IGF receptors derived from human, hydra and fly using transcriptomic and proteomic approaches. We constructed the expressing vectors of chimeric proteins consisting of the extracellular domain of TrkC, a human neurotrophin receptor, and the transmembrane and intracellular regions of the huma … More n insulin receptor, the human IGF-1 receptor, the hydra insulin/IGF receptor, or the Drosophila insulin/IGF receptor (InR), and the stable HepG2 clones overexpressing these chimeric receptors were established. Then, the gene expression profile in response to neurotrophin-3 (NT3), the ligand for TrkC was analyzed using microarray. In addition, the NT3-induced protein expression profile was examined by 2D-DIGE. In HepG2 cells over expressing the chemeric receptors stimulated with NT3, the receptor autophosphorylation and the gene expression of early growth response-1 were detected indicating that these chimeric receptors are functional. Although 2D-DIGE analysis detected no significant protein expression change in response to NT3, microarray analysis verified that gene expressions of the metallothionein family were significantly different among HepG2 cells overexpressing TrkC chimera receptors containing insulin/IGF receptor-related proteins, suggesting the presence of evolutional difference in stress responses among insulin/IGF receptor-related proteins.【結果】NT3刺激によるキメラ受容体の自己リン酸化、インスリン応答性のEGR1遺伝子発現量増加がキメラ遺伝子発現細胞でNT3刺激でも起こることを確認した。Microarrayによる解析では3時間刺激により最多の遺伝子の発現変動が見られ、キメラ受容体間の比較では、ヒドラ・インスリン様受容体細胞内ドメイン発現にてメタロチオネイン遺伝子群の発現増加が認められた。2D-DIGE法での検討では、各細胞にてNT3刺激による有意な蛋白変動はほとんど認められなかった。【考察】インスリン/IGF受容体キメラ蛋白を発現した肝細胞での遺伝子発現プロファイル比較でメタロチオネインに差異があったことから、これらの受容体のストレス反応性に差異があるものと考えられる。 Less