JAK-3 AND MYELOID CELL DIFFERENTIATION

描述：（改编自研究者摘要）Janus激酶 (JAK)是蛋白酪氨酸激酶家族（包括JAK 1，JAK 2， 和TYK 2），在信号转导过程中发挥关键作用 由细胞因子和干扰素介导。JAK激酶不同于其他 细胞质激酶缺乏SH 2和SH 3结构域， 存在两个激酶结构域。越来越明显的是 某些细胞因子如粒细胞集落刺激因子（G-CSF） 可以传递细胞增殖和分化的信号。 目前尚不清楚这两种信号是否都是 是否由相同的JAK激酶或是否整个家族的这种激酶是 参与这个过程。可以假设，在一个不成熟的 在骨髓细胞中，细胞因子如G-CSF可以诱导 a JAK激酶，并且随着持续暴露于细胞因子， JAK激酶被诱导，其传递一组新的信号， 诱导与细胞生长停滞和终末相关的基因 分化首席研究员最近发现了一种新的 JAK激酶家族的成员，称为JAK 3。JAK 3选择性地 在骨髓和淋巴样细胞中表达，似乎是一种原发性 G-CSF和IL 6诱导的应答基因。JAK 3的表达水平非常低， 在未成熟的造血细胞水平，并上调与终端 分化在初步研究中，申请人表明， JAK 3在鼠骨髓细胞系（32 Dcl 3）中的强制表达 加速G-CSF诱导的分化程序。因此， 研究人员假设，虽然JAK 1和JAK 2激酶结合到 G-CSF受体，并在递送G-CSF中发挥关键作用 促有丝分裂信号，JAK 3与提供促进有丝分裂的信号有关。 终末分化本申请中提出的实验 旨在了解JAK-3在终端中的作用， 髓样细胞沿着粒细胞的分化过程， 单核细胞通路。目的包括：1）扩展以前的研究， 32 Dcl 3细胞（仅能分化为粒细胞）向M1 细胞系（能够响应IL 6而分化为单核细胞）; 由于已知JAK激酶通过以下途径发挥其生物学效应 通过STATs的磷酸化，将确定JAK 3是否与STATs相互作用。 与32 Dc 13和M1细胞中表达的任何STAT结合并磷酸化 分别暴露于G-CSF或IL-6; 2）确定JAK 3是否是 在正常鼠骨髓细胞中表达，以响应IL 6，G-CSF， 和GMCSF，并确定JAK 3的诱导是否是由于新的 转录或转录后机制; 3）确定如何 当JAK-3表达被抑制时， 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基因抑制了 G-CSF诱导32 Dc 13粒细胞分化的能力 细胞系中，由于抑制JAK-3表达的诱导。使用 这些模型系统，它将被确定，如果本构表达的 使用外源启动子的JAK 3将拯救32 D/v-abl，32 D/v-myb， 32 D/EGR和32 D/Hox 2.4细胞系从阻滞到粒细胞 用G-CSF刺激后的分化; 4）确定如何 JAK 3通过细胞周期阻滞促进终末分化; 5） 对JAK 3的启动子/增强子区域进行详细分析， 检查在细胞因子中起关键作用的序列元件， 反应性;和6）鉴定由JAK 3途径调节的基因。

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.