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

張庭維(Ting-Wei Chang)

指導教授 ： 黃惠美

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2003年)

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。

關鍵字

細胞激素

並列摘要

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.

參考文獻

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