Retinoic Acid Regulated Cell Differentiation: BLR1 Rcptr

这是一个研究机制的一个方面的建议， 其中视黄酸（RA）调节细胞生长和分化。作为饮食因子维生素A的代谢物，维生素A是正常幼年发育所必需的激素原，RA是胚胎发生期间的发育形态原，也是可将未成熟的肿瘤转化细胞转化为成熟分化表型的癌症化学治疗剂。拟议的研究利用未成熟的未定型前体细胞HL-60人成髓细胞白血病细胞，当分别用RA或维生素D3（D3）处理时，其经历GO停滞和髓样或单核细胞分化。RA引起MAPK信号传导的激活和一系列渐进的基因表达变化，最终在成熟的髓样分化细胞中停滞。RA诱导的MAPK活化是不寻常的，偏离原型肽激素诱导的促有丝分裂RAF/MEK/-ERK信号传导。这是一种类固醇样因子的结果。激活的开始是缓慢的，但一旦激活是持久的。存在MEK依赖性ERK激活，RAF激活令人惊讶地在ERK激活之后。结果是GO逮捕和分化。对RA的早期反应是BLR1/CXCRC5蛇形受体的诱导表达，其直到RA治疗才表达。BLR 1引起MAPK信号传导，并且过表达加速RA诱导的髓样以及133诱导的单核细胞分化和GO停滞。我们已经创建了HL-60亚系，其中BLR1通过稳定的转染异位表达，或通过同源重组敲除，从而排除RA诱导的表达。具体目标是：1）确定BLR1对RA诱导的MAPK信号传导的贡献，特别是信号的强度和持续时间; 2）鉴定RA处理的细胞中由BLR1信号传导激活/失活的候选转录因子; 3）确定RA调节的蛋白中由BLR1调节的下游蛋白靶; 4）鉴定依赖于BLR1的RA诱导的分化的特征，并鉴定BLR1调节的蛋白质对它们的影响。我们以前的研究已经确定了RA是如何通过一个由成对的GT盒组成的新的5 'RARE来调节BLR1表达的。现在的目的是跟踪RA的作用，从其调节BLR1受体信号传导，诱导转录因子激活，并最终改变基因表达。这是一个范例的原型，其中RA诱导受体的表达， 然后发出信号，激活转录因子，并引起基因表达的变化，最终导致RA诱导的细胞分化的特定特征。这意味着RA的最终作用可以通过相关的生长因子通过其同源受体信号传导来控制。

This is a proposal to study one aspect of the mechanism by which retinoic acid (RA) regulates cell growth and differentiation. A metabolite of the dietary factor vitamin A, a prohormone necessary for proper juvenile development, RA is a developmental morphogen during embryogenesis and a cancer chemolherapeutic agent that can convert immature neoplastically transformed cells to a mature differentiated phenotype. The proposed studies utilize an immature, uncommitted precursor cell, HL-60 human myeloblastic leukemia cells, which undergoes GO arrest arid either myeloid or monocytic differentiation when treated with RA or vitamin D3 (D3) respectively. RA causes activation of MAPK signaling and a panoply of progressive gene expression changes culminating in the (JO arrested mature myeloid differentiated cell. RA-induced MAPK activation is unusual, diverging from prototypical peptide hormone induced mitogenic RAF/MEK/-ERK signaling. It is a consequence of a steroid-like factor. Onset of activation is slow, but once activated is persistent. There is MEK de-pendent ERK activation with RAF activation surprisingly following ERK activation. And the outcome is GO arrest and differentiation. An early response to RA is the induced expression of the BLR1/CXCRC5 serpentine receptor, which is not expressed until RA treatment. BLR 1 causes MAPK signaling, and over expression accelerates RA-induced myeloid, as well as 133-induced monocytic, differentiation, and GO arrest. We have created HL-60 sublines where BLR1 is ectopically expressed by stable transfaction, or is knocked out by homologous recombination thereby precluding RA-induced expression. The specific aims are to: 1) determine the contribution of BLR1 to RA-induced MAPK signaling, in particular the intensity and duration of the signal; 2) identify candidate transcription factors activated/deactivated by BLR1 signaling in RA-treated cells; 3) determine the downstream protein targets regulated by BLR1 among RA-regulated proteins; 4) identify features of RA-induced differentiation that are dependent on BLR1 and identify BLR1 regulated proteins seminal to them. Our previous studies have established how RA regulates BLR1 expression through a novel 5' RARE consisting of paired GT boxes. The intention is to now track the action of RA from its regulation of BLR1 to receptor signaling, induced transcription factor activation, and finally changes in gene expression. This is the prototype of a paradigm where RA induces the expression of a receptor which then signals, activates transcription factors, and causes changes in gene expression culminating in specific features of RA-induced cellular differentiation. It has the implication that the ultimate effects of RA can be governed by relevant growth factors signaling through their cognate receptors.