SIGNAL TRANDCUTION PATHWAYS IN HEMATOPOIESIS

造血信号转导途径

• 批准号: 2834181
• 负责人: BERNARD MATHEY-PREVOT
• 金额: $ 29.79万
• 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/2834181
• 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，在造血信号转导通路之间存在广泛的串扰。我们认为这种串扰有助于调节从激活的造血细胞表面发出的信号的激活和终止。为了解释这一假说，两种已知激活JAK/STAT通路的结构性活性受体(cEpoR和CD8-cEyk)被引入小鼠造血细胞(BA/F3)。由于JAK/STAT和RAS/Raf通路的激活，这两种途径都导致了因子独立和转化。然而，cEpoR和CD8-cEyk启动的信号事件明显不同。为了进一步评估这些差异，我们开发了一种在果蝇血细胞中的体内试验。我们利用了这样一个事实，即信号通路在后生动物中非常保守。这两种受体的cDNA都被置于Gal4转录调控元件的控制下，并注射到果蝇胚胎中。在热休克启动子的控制下，分离转基因果蝇并与表达Gal4蛋白的果蝇品系杂交。在热休克时，脊椎动物受体的表达被诱导，导致血细胞白血病样增殖和黑色素瘤的形成。这为探测体内的信号转导通路提供了一种强有力的、新颖的方法。我们将进一步完善这一检测方法，并在遗传学实验中确定JAK/STAT、RAS/Raf、NF-kappaB和JNK通路在血细胞增殖和肿瘤发生中的作用。我们将测试这些途径中功能突变的缺失是否可以抑制黑色素瘤的表型。此外，我们将使用这个系统来表征我们最近产生的一个过度磷酸化的STAT5突变体。我们已经证明这个STAT5突变体对去磷酸化是抵抗的。我们期望发现，它赋予了功能表型的获得。最后，我们将利用果蝇的遗传筛选来分离与已知信号转导途径相互作用的新基因，以抑制黑色素瘤的表型。存活和可生育的果蝇都显示出黑色素瘤，它们将与携带染色体异常、P元素插入或化学诱导的点突变的突变种群杂交。F1代黑色素瘤表型的抑制将作为识别调节已知的造血信号转导途径的辅助因素的标准。这一系列实验应该会对造血信号转导产生新的见解，这是从标准的生化实验或小鼠的基因操作中无法获得的。