Control of cytokine function by modified signaling molecules

我们研究了RAS和STAT5的信号机制，RAS和STAT5是由IL-3/GM-CSF等造血细胞因子激活的主要信号分子，并试图通过修饰信号分子来调节这些通路。RAS是防止细胞因子诱导造血细胞凋亡所必需的。通过使用部分活性的RAS突变体，我们发现RAS通过激活RAF/MAP激酶级联以及P13激酶途径来防止细胞凋亡。由于细胞因子耗尽诱导细胞凋亡的机制尚不清楚，我们测试了Caspase是否参与了细胞凋亡过程。我们发现，Caspase-3在缺乏细胞因子的情况下被激活，而Caspase-3的激活是细胞凋亡所必需的。虽然STAT5被包括IL-3/GM-CSF在内的各种细胞因子激活，但STAT5在细胞因子功能中的作用尚不清楚。为了揭示STAT5的作用，我们试图分离STAT5的靶基因。在这些基因中，我们获得了一个新的SH-2蛋白--顺肿瘤抑制素M，它是IL-6家族细胞因子的一员。顺式调节蛋白通过STAT5被细胞因子诱导，并通过与酪氨酸磷酸化的信号分子结合来抑制信号转导。OSM在主动脉/性腺/中肾(AGM)区域表达，在那里被认为是最终的造血发生。在AGM细胞的体外培养中，OSM刺激造血细胞和内皮细胞的发育。这提出了一种可能性，即假定的造血细胞和内皮细胞的共同前体可能是OSM的靶标。我们产生了一种显性的阴性(DN)形式的STAT5，并证明了dnSTAT5的表达阻断了IL-3诱导的BaF3的增殖，提示STAT5可能参与了增殖。我们还发现STAT5参与了促红细胞生成素诱导的红系细胞系SKT6的成熟。

We investigated the signaling mechanisms of RAS and STAT5, which are major signaling molecules activated by hematopoietic cytokines such as IL-3/GM-CSF.We also attempted to develop means to regulate these pathways by modification of signaling molecules.RAS is required for prevention of apoptosis by cytokines in hematopoietic cells. By using partially active RAS mutants, we found that RAS prevents apoptosis through both activation of the RAF/MAP kinase cascade as well as the P13 kinase pathway. As'the mechanism of apoptosis induced by cytokine depletion has remained uncovered, we tested if Caspases are involved in the apoptotic process. We found that Caspase-3 is activated in the absence of a cytokine in hematopoietic cells and the activation of Caspase-3 is required for the apoptosis.While STAT5 is activated by various cytokines including IL-3/GM-CSF,the role of STAT5 in cytokine functions was unknown. To uncover the role of STAT5, we attempted to isolate STAT5 target genes. Among such genes we obtained a novel SH-2 protein CIS Oncostatin M,a member of IL-6 family cytokines. CIS is induced by cytokines through STAT5 and inhibits signaling by binding to a tyrosine phosphorylated signaling moleculea. OSM is expressed in the aorta/gonad/mesonephros (AGM) region where the definitive hematopoiesis is believed to emerge. OSM stimulates the development of hematopoietic cells as well as endothelial cells in the in vitro culture of AGM cells. This raised a possibility that the putative common precursor of hematopoietic cells and endothelial cells may be a target of OSM.We generated a dominant negative (dn) form of STAT5 and demonstrated that expression of dnSTAT5 blocks IL-3-induced proliferation of BaF3, suggesting that STAT5 may be involved in proliferation. We also showed that STAT5 is involved in erythropoietininduced maturation of the erythroid cell line, SKT6.