SIGNAL TRANSDUCTION PATHWAYS IN HEMATOPOIESIS

造血中的信号转导途径

• 批准号: 6184783
• 负责人: BERNARD MATHEY-PREVOT
• 金额: $ 29.18万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-10 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6184783
• 关键词: Drosophilidae; JAK kinase; biological signal transduction; blood cells; cell proliferation; cell transformation; erythropoietin; genetic transcription; genetically modified animals; growth factor receptors; guanosine triphosphate; hematopoiesis; hematopoietic growth factor; molecular cloning; mutant; neoplastic transformation; nuclear factor kappa beta; phenotype; phosphorylation; protein structure function; protein tyrosine kinase; receptor expression; suppressor mutations; transcription factor; transfection

We postulate that there is extensive crosstalk between hematopoietic signal transduction pathways. We propose that this crosstalk helps to regulate the activation and termination of signals emanating from the surface of activated hematopoietic cells. To begin to address this hypothesis, two constitutively active receptors (cEpoR and CD8-cEyk), known to activate the JAK/STAT pathway, were introduced into murine hematopoietic (Ba/F3) cells. Both caused factor independence and transformation, as a result of activation of the JAK/STAT and Ras/Raf pathways. However, signaling events initiated by cEpoR and CD8-cEyk were clearly different. To further evaluate these differences, we have developed an in vivo assay in Drosophila blood cells. We take advantage of the fact that signaling pathways are remarkably conserved among metazoans. cDNAs for both receptors have been placed under the control of Gal4 transcriptional regulatory elements, and injected into Drosophila embryos. Transgenic flies were isolated and crossed with Drosophila lines expressing Gal4 protein under the control of a heat shock promoter. Upon heat shock, expression of the vertebrate receptors is induced, resulting in leukemia-like proliferation of blood cells and formation of melanotic tumors. This offers a powerful, novel assay to probe signal transduction circuits in vivo. We will further refine this assay, and determine the participation of the JAK/STAT, Ras/Raf, NF-kappaB and JNK pathways in hemocyte proliferation and tumorigenesis in genetic experiments. We will test whether loss of function mutants in these pathways can suppress the melanotic tumor phenotype. In addition, we will use this system to characterize a hyperphosphorylated STAT5 mutant that we have recently generated. We have shown that this STAT5 mutant is resistant to dephosphorylation. We expect to find that it confers a gain of function phenotype. Finally, we will exploit a genetic screen in Drosophila to isolate novel genes that interact with known signal transduction pathways to suppress the melanotic tumor phenotype. Viable and fertile flies, which all show melanotic tumors, will be crossed with mutant stocks carrying chromosomal aberrations, P-element insertions, or chemically- induced point mutations. Suppression of the melanotic tumor phenotype in the F1 generation will serve as the criterion to identify accessory factors that modulate known hematopoietic signal transduction pathways. This series of experiments should produce new insights into hematopoietic signal transduction that could not be obtained from standard biochemical experiments, or from gene manipulations in mice.

我们推测造血信号转导通路之间存在广泛的串扰。 我们提出，这种串扰有助于调节从活化的造血细胞表面发出的信号的激活和终止。 为了开始解决该假设，将已知激活JAK/STAT途径的两种组成型活性受体（cEpoR和CD 8-cEyk）引入鼠造血（Ba/F3）细胞中。 由于JAK/STAT和Ras/Raf通路的激活，两者都引起了因子独立性和转化。 然而，由cEpoR和CD 8-cEyk引发的信号传导事件明显不同。 为了进一步评估这些差异，我们开发了一种果蝇血细胞的体内试验。 我们利用的事实，即信号通路是显着保守的后生动物。 这两种受体的cDNA已置于Gal 4转录调控元件的控制下，并注射到果蝇胚胎中。 分离转基因果蝇，并与在热激启动子控制下表达Gal 4蛋白的果蝇品系杂交。 在热休克时，诱导脊椎动物受体的表达，导致血细胞的白血病样增殖和黑色素瘤的形成。这提供了一个强大的，新的测定探针在体内的信号转导电路。 我们将进一步完善该检测方法，并在遗传实验中确定JAK/STAT、Ras/Raf、NF-κ B和JNK通路在血细胞增殖和肿瘤发生中的参与。 我们将测试这些通路中的功能缺失突变体是否可以抑制黑色素瘤表型。 此外，我们将使用这个系统来表征我们最近产生的高度磷酸化的STAT 5突变体。 我们已经表明，这种STAT 5突变体是抗去磷酸化。 我们期望发现它赋予功能表型的增益。 最后，我们将利用果蝇的遗传筛选来分离新的基因，这些基因与已知的信号转导途径相互作用来抑制黑色素瘤表型。将所有显示黑色素瘤的活的和能生育的果蝇与携带染色体畸变、P元件插入或化学诱导的点突变的突变体原种杂交。F1代中黑色素瘤表型的抑制将作为鉴定调节已知造血信号转导途径的辅助因子的标准。 这一系列的实验应该产生新的见解造血信号转导，不能从标准的生化实验，或从小鼠基因操作。