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.
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.
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