Regulation of B Cell Development by Ras and Raf

描述(由申请人提供)：B细胞在建立针对细菌和病毒的功能性免疫反应中起着至关重要的作用。因此，B细胞的发育过程必须产生一个成熟的B细胞群体，既有足够多样化的抗原受体，又有足够数量的细胞，以便对大量潜在的感染性病原体建立有效的防御。Res/Raf途径携带的信号在最早的Pre-Pro-B阶段调节早期B细胞的分化，本质上决定了沿着这条发育途径前进的细胞的数量。然而，该途径的上游激活物和下游效应物仍未确定。我们的假设是，Ras/Raf通路是由IL-7或flk2/flt3受体激活的，它通过改变转录调节因子的表达来影响下游反应，特别是ID和ETS基因家族的成员。因此，这项资助的目的是：(1)确定IL-7R或flk2/flt3是否通过RAS激活参与早期B细胞的发育；(2)确定依赖RAS/Raf的早期B细胞分化是否涉及ID或ETS家族蛋白的调节；以及(3)确定参与调节前亲B细胞分化的依赖RAS/Raf的转录因子。将利用若干战略来实现这些目标。首先，将使用生化方法来确定IL-7或flt3L是否能够激活前B细胞系或体外前B细胞中的RAS/Raf通路。其次，利用转基因小鼠表达活性形式的Raf(Raf-CAAX)的遗传方法将被用来确定强制激活Raf是否可以挽救IL-7R-orflk2/Flt3缺陷小鼠的B细胞发育。第三，在发育中的B细胞中表达显性阴性RAS或固有活性Raf的转基因小鼠将被用来确定这一途径是否调节ID-或ETS-家族成员的表达。这些实验将使用可诱导的方法在体内激活Raf途径来扩展。具体地说，这涉及到建立转基因小鼠，在其中激活形式的Raf(Raf-CAAX)的表达由阿霉素调节的启动子控制。最后，基因微阵列技术将与上述小鼠突变体相结合，以确定参与前B细胞分化的RAS/Raf途径的新靶点。这些研究有助于阐明RAS途径调节B细胞分化的分子机制。最近有观察到骨髓移植后B细胞重建进展缓慢，导致患者对许多细菌病原体易感，这突显了澄清这一过程的重要性。因此，对早期B细胞发育的更好理解应该被证明对未来优化骨髓移植方案是有用的。

DESCRIPTION (provided by the applicant): B cells play an essential role in the establishment of a functional immune response against bacteria and viruses. Therefore, the process of B cell development must produce a population of mature B cells with both sufficient diversity of antigen receptors and sufficient numbers of cells, in order to mount an effective defense against the vast array of potential infectious agents. Signals entrained by the Res/Raf pathway regulate early B cell differentiation at the earliest pre-pro-B stage, in essence determining the number of cells that proceed down this developmental pathway. However, the upstream activators and down stream effectors of this pathway remain undefined. Our hypothesis is that the Ras/Raf pathway is activated by the interleukin-7 orflk2/flt3 receptors, and that it affects downstream responses by altering the expression of transcriptional regulators, specifically members of the Id- and Ets-gene families. Thus the aims of this grant are: (1) To determine whether the IL-7R or flk2/flt3 entrain early B cell development via Ras activation, (2) To determine whether Ras/Raf-dependent early B cell differentiation involves regulation of Id- or Ets-family proteins, and (3) To identify Ras/Raf-dependent transcription factors involved in regulating pre-pro-B cell differentiation. A number of strategies will be utilized to achieve these goals. First, biochemical approaches will be used to determine whether either IL-7 or flt3L can activate the Ras/Raf pathway in pro-B cell lines or in ex vivo pre-pro-B cells. Second, genetic approaches that involve transgenic mice which express constitutively active forms of Raf (Raf-CAAX) will be used to determine whether forced Raf activation can rescue B cell development in IL-7R- orflk2/flt3-deficient mice. Third, transgenic mice which express either dominant negative Ras or constitutively active Raf in developing B cells will be utilized to determine whether this pathway regulates expression of Id- or Ets-family members. These experiments will be extended using inducible approaches for activating the Raf pathway in vivo. Specifically, this involves generating transgenic mice in which the expression of an activated form of Raf (Raf-CAAX) is controlled by adoxycycline-regulated promoter. Finally, gene microarray technology will be applied, in conjunction with the mouse mutants described above, to identify novel targets of the Ras/Raf pathway involved in pre-pro-B cell differentiation. These studies should help to illuminate the molecular mechanisms by which the Ras pathway regulates B cell differentiation. The importance of clarifying this process has been highlighted recently by the observation that B cell reconstitution following bone marrow transplantation frequently proceeds poorly, resulting in patient susceptibility to a number of bacterial pathogens. Thus a better understanding of early B cell development should prove useful for optimizing bone marrow transplantation protocols in the future.