Control of cytokine function by modified signaling molecules

我们研究了造血细胞因子（如IL-3/GM-CSF）激活的主要信号分子RAS和STAT 5的信号转导机制，并试图通过修饰这些信号分子来调控这些信号通路。通过使用部分激活的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。由于细胞因子耗竭诱导细胞凋亡的机制尚未揭示，我们测试了Caspase是否参与细胞凋亡过程。我们发现，Caspase-3在造血细胞缺乏细胞因子的情况下被激活，Caspase-3的激活是造血细胞凋亡所必需的，而STAT 5则被多种细胞因子激活，包括IL-3/GM-CSF，但STAT 5在细胞因子功能中的作用尚不清楚。为了揭示STAT 5的作用，我们尝试分离STAT 5靶基因。在这些基因中，我们获得了一个新的SH-2蛋白CIS Oncostatin M，IL-6家族细胞因子的成员。CIS由细胞因子通过STAT 5诱导，并通过与酪氨酸磷酸化的信号分子结合来抑制信号传导。OSM在主动脉/性腺/中肾（AGM）区域中表达，在该区域中，确定的造血被认为出现。在体外培养AGM细胞时，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的靶点。我们产生了显性负性（dn）形式的STAT 5，并证明dnSTAT 5的表达阻断IL-3诱导的BaF 3增殖，表明STAT 5可能参与增殖。我们还发现STAT 5参与了红系细胞系SKT 6的促红细胞生成素诱导的成熟。

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.