細胞激素調控細胞增殖及分化訊息傳遞路徑之研究
Signal transduction pathways involved in the regulation of cell
proliferation and differentiation by cytokines
中文摘要 Part I:
Activin A 對於細胞的分化及細胞凋亡扮演重要角色。其中包括骨髓中的紅血球 分化。Basic fibroblast growth factor(bFGF)是由 bone marrow stromal cell 所產生， 它在造血幹細胞中，可以維持 primitive erythroid cell 的增生。然而，activin A 及 bFGF 以何種機制來使細胞分化或增生並不是很清楚。本篇論文中我們探討了 bFGF 可以抑制 activin A 誘導紅血球的分化。此外，此篇論文也探討了 activin A 及 bFGF 調控 K562 細胞分化時，不同的 mitogen-activated protein kinase 所扮演 的角色。在西方墨點法分析中顯示出：當細胞加入 activin A 後，p38 的磷酸化程 度增加，而 MAPK 及 JNK 則被抑制。另外，當細胞加入 bFGF 後，三種 MAPK 蛋白質磷酸化都被抑制。細胞若同時加入 activin A 及 bFGF，則 p38 磷酸化被抑 制。此外，activin A 誘導紅血球分化過程中，若以 U0126 抑制 MAPK 的活性， 發現紅血球分化程度增加，若以 SB203580 抑制 p38 的活性，則會抑制 hemoglobin 的產生。綜合以上結果得知， activin A 正調控 p38 磷酸化，而 bFGF 負調控 p38 磷酸化使紅血球分化。 PartⅡ: 細胞激素可以調節 hematopoietic cells 的生長、增生、分化及抑制細胞凋亡。當 細胞激素將訊息由細胞膜傳入到細胞核後，會改變基因，最後影響細胞的活性。 在細胞激素調控 hematopoiesis 時，Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway 扮演重要角色。大部份的細胞激素可以活化兩 個或兩個以上的 JAKs。當 IL-3/IL-5/GM-CSF 和其接受子結合後會活化 JAK1 及 JAK2。IL-3、IL-5 及 GM-CSF 接受子各自含有自己的α次單元，並共用β次單 元。然而，JAKs 以哪一部位和 IL-3 接受子結合並不清楚。先前實驗室已將 IL-3 接受子α次單元的 intracellular cDNA 及βc 的 intracellular cDNA 接到 GST 形成 融合基因，此融合基因可以產生融合蛋白。本篇論文中已得知 IL-3Rα次單元可 以和 JAK2 結合而 IL-3Rβc 次單元可以和 JAK1 結合。此結果和 IL-5 system 相 同(Blood 91:2264-71, 1998)，因此，我建構一系列去除某區域的 JAK1 蛋白質和 JAK2 蛋白質。接下來將以 GST-pull down assay 來探討 JAK1 和 JAK2 以哪一部 位和 IL-3 接受子結合，並且研究此區域對 IL-3 接受子訊號傳遞及 IL-3 依賴型細 胞生長活性的影響。
英文摘要 Part I:
Activin A is essential for cell differentiation and apoptosis including erythroid cell differentiation in the bone marrow. Basic fibroblast growth factor (bFGF) is produced by bone marrow stromal cells and by hematopoietic cells that can maintain
proliferation of primitive erythroid cells. However, the mechanism by which activin A and bFGF exert their differentiation or proliferation activity is still unknown. In this study, I have investigated the bFGF inhibit the effect of activin A-induced erythroid differentiation. Furthermore, this study examined the role of different
mitogen-activated protein kinase signal transduction pathways in activin A and bFGF-modulated differentiation of K562 cells. Western blot analysis of a panel of phosphorylated proteins revealed that the phosphorylation of p38 is increased and the phosphorylation of MAPK and JNK are inhibited after the cells of treatment with activin A. In addition, the phosphorylation of three MAPKs are inhibited after the cells of treatment with bFGF. The phosphorylation of p38 is inhibited after the cells of treatment with activin A and bFGF. Moreover, inhibition of MAPK activity by U0126 induced erythroid differentiation, whereas inhibition of p38 activity by SB203580 inhibited induction of hemoglobin production by activin A. These results suggest that the phosphorylation of p38 is positively regulated by activin A and is negatively regulated by bFGF in erythroid differentiation.
PartⅡ:
Cytokines regulate the growth, proliferation, differentiation and apoptosis prevention of hematopoietic cells. Cytokine signaling from cell membrane into the nucleus in which the genes are altered, and eventually cell activity are affected. Janus
kinase/signal transducer and activator of transcription (JAK/STAT) pathway play a very important role among the cytokine-regulated hematopoiesis. Most cytokines can activate two or more than two JAKs. When IL-3/IL-5/GM-CSF bind to their receptors, initiating activation of JAK1 and JAK2. The receptors for IL-3, IL-5 and GM-CSF each consist of a cytokine specific a chain, and a common b-chain (bc). However, which regions of JAKs interact with IL-3 receptor are now unclear. In previous study, the intracellular cDNA of IL-3 receptor a subunit and the intracellular cDNA of bc were fused with GST form fusion genes. These fusion genes can produce fusion proteins. In this study, the data show that IL-3R a chain interacts with JAK2 and IL-3R bc interacts with JAK1. These results consistent with the published data for IL-5 system (Blood 91: 2264-2271, 1998). Hence, I constructed a series of deletion mutants for JAK1 and JAK2. In the future, the regions of JAK1 and JAK2 interact with receptors by GST-pull down assay and the effect of these regions on IL-3
receptor signal transduction as well as survival activity in IL-3-dependent cells will be investigated.
