Regulation of B Cell Development by Ras and Raf

描述（由申请方提供）：B细胞在建立针对细菌和病毒的功能性免疫应答中发挥重要作用。因此，B细胞发育的过程必须产生既具有足够多样性的抗原受体又具有足够数量的细胞的成熟B细胞群，以便对大量潜在的感染因子进行有效的防御。由Res/Raf途径携带的信号在最早的前前B阶段调节早期B细胞分化，实质上决定了沿着该发育途径进行的细胞数量。然而，该途径的上游激活物和下游效应物仍然不确定。我们的假设是Ras/Raf通路被白细胞介素-7 orflk 2/flt 3受体激活，并且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，本基金的目的是：（1）确定IL-7 R或flk 2/flt 3是否通过Ras活化来诱导早期B细胞发育，（2）确定Ras/Raf-dependent早期B细胞分化是否涉及Id-或Ets-家族蛋白的调节，和（3）鉴定Ras/Raf-dependent转录因子参与调节前原B细胞分化。为实现这些目标，将采用若干战略。首先，将使用生物化学方法来确定IL-7或flt 3 L是否可以在前-B细胞系或离体前-前-B细胞中激活Ras/Raf途径。第二，涉及表达组成型活性形式Raf的转基因小鼠（Raf-CAAX）的遗传方法将用于确定强制Raf活化是否可以拯救IL-7 R-orflk 2/flt 3缺陷小鼠中的B细胞发育。第三，利用在发育中的B细胞中表达显性负性Ras或组成型活性Raf的转基因小鼠来确定该途径是否调节Id或Ets家族成员的表达。这些实验将使用诱导型方法在体内激活Raf途径进行扩展。具体地，这涉及产生其中Raf的活化形式（Raf-CAAX）的表达由adoxycycline调节的启动子控制的转基因小鼠。最后，基因微阵列技术将与上述小鼠突变体一起应用，以鉴定参与前原B细胞分化的Ras/Raf途径的新靶点。这些研究将有助于阐明Ras通路调控B细胞分化的分子机制。最近，骨髓移植后B细胞重建经常进展不良，导致患者对许多细菌病原体的易感性，这一观察结果强调了阐明这一过程的重要性。因此，更好地了解早期B细胞的发展应证明是有益的优化骨髓移植方案在未来。

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.