Regulation of cyclin D3 in B lymphocyte development

描述(申请人提供)：B细胞发育是一个高度有序和受调控的过程，在这个过程中，血统承诺的前B细胞产生不同的外周克隆性B淋巴细胞群体，显示出广泛的抗原特异性。在B淋巴细胞生成的早期，IgM与代理轻链和IgA/Igb组装形成前BCR，该前BCR首先扩大带有一个框内重链重排的前B细胞群体，然后启动轻链重排。在以前的研究中，我们证明了细胞周期蛋白D3是克隆扩增前B细胞所必需的(NAT免疫7：489)。细胞周期蛋白D3的积累和细胞周期的启动受到GC依赖信号的协调调节，这些信号增强Ccnd3的mRNA，并通过前BCR增加核周期蛋白D3的可获得性。在初步结果和正在进行的工作(NAT免疫)中，我们证明了Ras/MEK/ERK信号通路的前BCR依赖激活协调了Ccnd3转录的终止和启动IgK重组。在ERK下游，特定的信号效应器调节每个离散的发育事件。IgK转录是由ERK介导的抑制ID蛋白和去抑制E2a诱导的，而诱导Aiolos沉默Ccnd3导致细胞周期退出。除了确定协调B细胞发育的中心途径外，这些发现还为理解BCR前信号和IL-7依赖信号之间的相互作用提供了一个框架。具体地说，我们证明了Igk转录是由内含子Igk增强子(Eki)上E2A和STAT 5之间的竞争控制的。相比之下，细胞周期蛋白D3蛋白受PI-3蛋白的调节，通过一种新的淋巴特异性机制，控制细胞周期蛋白D3相对于抑制因子p27kip1和CDK4/Rb效应复合体的亚核定位。这些观察结果唤起了一种模型，在该模型中，最初的前B细胞增殖是通过STAT5和PI-3k的协同激活来调节的。STAT5促进Ccnd3转录，抑制Igk重组，而PI-3k则促进细胞周期蛋白D3/CDK4/Rb复合体的形成。随后的IgK重组既需要逃离STAT 5，也需要通过前BCR激活ERK。该模型的中心预测将在以下特定目标中进行验证：目的1.确定E2A和STAT 5如何整合来调节Igk转录。目的2.研究PI-3激酶对细胞周期蛋白D3与CDK4、Rb和p27kip1的核定位和组装的调节作用。目的3：在体内确定为什么细胞周期蛋白D3对淋巴细胞发育有独特的贡献。 公共卫生相关性：B细胞在骨髓中的发育由骨髓中存在的细胞因子和淋巴细胞前体上B细胞前抗原受体(Pre-BCR)的表达决定。在这项资助申请中，我们将确定通过BCR前受体和细胞因子受体传递的细胞内信号如何整合到指导复杂的发育过程。这些研究将使人们更好地理解导致白血病的免疫系统的产生过程。

DESCRIPTION (provided by applicant): B cell development is a highly ordered and regulated process during which lineage-committed pro-B cells give rise to diverse peripheral populations of clonal B lymphocytes displaying a broad repertoire of antigen specificities. Early in B lymphopoiesis, Igm assembles with surrogate light chain and Iga/Igb to form a pre-BCR that first expands pre-B cell populations bearing one in-frame heavy chain rearrangements and then initiates light chain rearrangement. In previous studies, we demonstrated that cyclin D3 is uniquely required for clonal expansion of pre-B cells (Nat Immunol 7:489). The accumulation of cyclin D3, and the initiation of cell cycle, is coordinately regulated by gc-dependent signals that enhance Ccnd3 mRNA and signals through the pre-BCR that increase the availability of nuclear cyclin D3. In Preliminary Results, and in work now in press (Nat Immunol), we demonstrate that pre-BCR dependent activation of the Ras/MEK/ERK signaling pathway coordinates the termination of Ccnd3 transcription with the initiation Igk recombination. Downstream of ERK, specific signaling effectors mediate each discrete developmental event. Igk transcription is induced by ERK- mediated suppression of the Id proteins, and derepression of E2A, while induction of Aiolos silences Ccnd3 leading to cell cycle exit. In addition to identifying a central pathway coordinating B cell development, these findings provide a framework for understanding the interplay between pre-BCR and IL-7 dependent signals. Specifically, we demonstrate that Igk transcription is controlled by competition between E2A and STAT 5 at the intronic Igk enhancer (Eki). In contrast, cyclin D3 protein is regulated by PI-3 kinase that, through a novel, lymphoid specific mechanism, controls the subnuclear location of cyclin D3 relative to the inhibitor p27kip1 and the CDK4/Rb effector complex. These observations evoke a model in which initial pre-B cell proliferation is mediated by the coordinated activation of STAT 5 and PI-3k. STAT 5 enhances Ccnd3 transcription and suppresses Igk recombination while PI-3k drives the formation of productive cyclin D3/CDK4/Rb complexes. Subsequent Igk recombination requires both escape from STAT 5 and activation of ERK by the pre-BCR. The central predictions of this model will be tested in the following Specific Aims: Aim 1. To determine how E2A and STAT 5 integrate to regulate Igk transcription. Aim 2. To determine how PI-3 kinase regulates cyclin D3 nuclear localization and assembly with CDK4, Rb and p27kip1. Aim 3: To determine, in vivo, why cyclin D3 makes a unique contribution to lymphocyte development. PUBLIC HEALTH RELEVANCE: The development of B cells in the bone marrow is determined by both cytokines present in the bone marrow and by expression of the pre-B cell antigen receptor (pre-BCR) on lymphocyte precursors destined to become B cells. In this grant application, we will determine how intracellular signals, delivered through the pre-BCR and cytokine receptors, integrate to direct complex developmental processes. These studies will lead to a greater understanding of both the generation of the immune system of those processes that lead to leukemia.