JAK-3 AND MYELOID CELL DIFFERENTIATION

描述：(改编自调查人员摘要)Janus Kinase (JAK)是一个蛋白酪氨酸激酶家族(包括JAK1，JAK2， 和TYK2)，在信号转导过程中起着关键作用 由细胞因子和干扰素介导。Jak Kinase不同于其他 胞质激活酶的SH2和SH3结构域的缺失以及 存在两个激活区。越来越明显的是， 粒细胞集落刺激因子(G-CSF)等某些细胞因子 可以传递细胞增殖和分化的信号。 目前还不清楚这两个信号是否都被传输了 通过相同的JAK激酶，或者是这样的一整个家族是 参与了这一过程。可以假设，在一个不成熟的人 髓系细胞，一种细胞因子，如G-CSF，可诱导 一种JAK激酶，随着细胞因子的持续暴露，新的 JAK激酶被诱导，它传递一组新的信号，导致 诱导与细胞生长停滞和终止相关的基因 差异化。首席调查员最近发现了一种新的 JAK激酶家族的成员，命名为JAK3。JAK3是选择性的 在髓样细胞和淋巴样细胞中表达，似乎是原发的 G-CSF和IL-6诱导的应答基因。JAK3的表达水平非常低 在未成熟的造血细胞中的水平，并随着终末水平的升高而上调 差异化。在初步研究中，申请人已证明 JAK3在小鼠髓系细胞系(32Dcl3)中的强制表达 加速G-CSF诱导的分化程序。因此，这一点 研究人员假设，当JAK1和JAK2激酶结合到 G-CSF受体在传递G-CSF中起关键作用 有丝分裂信号，JAK3与提供促进 末端分化。此应用程序中建议的实验 旨在了解JAK-3在终端中的作用 髓系细胞沿粒细胞方向的分化过程 单核细胞途径。目标包括：1)扩展以前的研究 32Dcl3细胞(只能分化为粒细胞)为M1 细胞系(能对IL6作出反应而向单核细胞分化)； 由于JAK激酶已知通过以下途径发挥其生物学效应 STATS的磷酸化，将确定JAK3是否相互作用 结合并磷酸化在32Dc13和m1细胞中表达的任何STATS 分别暴露于G-CSF或IL-6；2)确定JAK3是否 正常小鼠骨髓细胞对IL-6、G-CSF、 和GMCSF，并确定JAK3的诱导是否由于新的 转录或转录后机制；3)确定如何 JAK-3表达对髓系细胞分化的影响 使用反义寡核苷酸抑制；此外，初步 结果表明，v-abl、v-myb、EGR-1和Hox-2.4基因抑制 粒细胞集落刺激因子诱导32Dc13细胞向粒细胞分化的能力 细胞系由于抑制了JAK-3的诱导表达。vbl.使用 这些模型系统，将决定是否构成表达式 使用外源启动子的JAK3将挽救32D/v-ABL、32D/v-MYB、 32D/EGR和32D/Hox2.4细胞株从阻断到粒细胞 G-CSF刺激后的分化；4)确定如何 JAK3通过细胞周期停滞促进末端分化；5) 对JAK3启动子/增强子区域进行详细分析，以 检查在细胞因子中起关键作用的序列元件 响应性；以及6)识别受JAK3途径调控的基因。

DESCRIPTION: (Adapted from investigator's abstract) The Janus Kinases (JAK) are a family of protein tyrosine kinases (which include JAK1, JAK2, and TYK2) that play a pivotal role in the signal transduction process mediated by cytokines and interferons. JAK kinases differ from other cytoplasmic kinases by their lack of SH2 and SH3 domains and by the presence of two kinase domains. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (G-CSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. It can be hypothesized that in an immature myeloid cell, a cytokine such as G-CSF may induce the phosphorylation of a JAK kinase, and with continued exposure to cytokines, expression of new JAK kinases are induced which transmit a new set of signals resulting in the induction of genes associated with cell growth arrest and terminal differentiation. The principal investigator has recently identified a new member of the JAK kinase family, named JAK3. JAK3 is selectively expressed in myeloid and lymphoid cells and appears to be a primary response gene induced by G-CSF and IL6. JAK3 is expressed at very low levels in immature hematopoietic cells and is up-regulated with terminal differentiation. In preliminary studies, the applicant has shown that forced expression of JAK3 in a murine myeloid cell line (32Dcl3) accelerates the G-CSF-induced differentiation program. Thus, this investigator hypothesizes that while the JAK1 and JAK2 kinases bind to the G-CSF receptor and play a critical role in delivering the G-CSF mitogenic signal, JAK3 is associated with providing signals that promote terminal differentiation. The experiments proposed in this application are aimed at understanding the role of JAK-3 in the terminal differentiation process of myeloid cells along the granulocytic and monocytic pathways. The aims include: 1) to extend previous studies with 32Dcl3 cells (capable of differentiating only to granulocytes) to the M1 cell line (capable of monocytic differentiation in response to IL6); since JAK kinases are known to exert their biological effects via phosphorylation of STATS, it will be determined whether JAK3 interacts with and phosphorylates any STATs expressed in 32Dc13 and M1 cells exposed to G-CSF or and IL-6 respectively; 2) to determine if JAK3 is expressed in normal murine bone marrow cells in response to IL6, G-CSF, and GMCSF, and to determine whether induction of JAK3 is due to new transcription or post-transcriptional mechanisms; 3) to determine how myeloid cell differentiation is affected when JAK-3 expression is inhibited using anti-sense oligonucleotides; in addition, preliminary results suggest that the v-abl, v-myb, EGR-1 and HOX-2.4 genes inhibit the ability of G-CSF to induce granulocytic differentiation of the 32Dc13 cell line due to inhibition of the induction of JAK-3 expression. Using these model systems, it will be determined if constitutive expression of JAK3 using exogenous promoters will rescue the 32D/v-abl, 32D/v-myb, 32D/EGR and 32D/Hox2.4 cell lines from the block to granulocytic differentiation following stimulation with G-CSF; 4) to determine how JAK3 promotes terminal differentiation through cell cycle arrest; 5) to carry out detailed analysis of the promoter/enhancer region of JAK3 to examine sequence elements that play a crucial role in cytokine responsiveness; and 6) to identify genes regulated by the JAK3 pathway.