Retinoic acid and regulation of BMP4 in development

描述(由申请人提供)：长期目标是了解维甲酸(RA；维生素A的主要生物活性代谢物)和骨形态发生蛋白4(BMP4)如何相互作用来调节发育。总体而言，RA通过与其核激素受体RARs结合来调节基因转录；BMP4是一种分泌蛋白，与细胞表面受体结合，启动涉及SMAD激活的信号级联反应。虽然骨中主要的BMP4启动子(1A启动子)是由RA诱导的，但我们发现前面描述的一个次要启动子(1B)和第二内含子(I2)中的一个新的启动子(I2)被PA抑制。I2启动子在发育中的内耳中高表达，而1A启动子则不表达。BMP4和PA都是已知的对正常内耳发育至关重要的物质。BMP4的拮抗作用可抑制半规管的发育。胚胎暴露于外源性RA会导致类似的表型，提示PA可能抑制内耳中的BMP4，有助于在空间和时间上适当地限制BMP4的表达。事实上，RA对新的i2启动子的抑制既发生在内耳来源的细胞系中，也发生在小鼠胚胎内耳的体内。此外，重组BMP4(在鸡体内)克服了RA抑制半规管发育的能力。这项建议旨在解决三个具体目标：1)确定PA下调BMP4转录的机制；2)表征BMP4 1B和I2启动子在发育小鼠胚胎中的时空表达；3)确定破坏BMP4 1B和I2启动子的突变在小鼠体内的影响。第一个目的通常与RA的作用有关，因为PA抑制靶基因的机制(S)还不是很清楚(与基因诱导相反)，而且令人感兴趣的是，PA通过另一个不同的启动子，即1A启动子上调骨骼中的骨形成蛋白4。第二个和第三个目标将确定体内Ib和I2启动子在内耳发育中的相对作用，并将研究这些启动子是否可能在内耳之外发挥重要作用。实现这些目标将依赖于经典的分子工具来研究细胞培养和无细胞系统中的基因调控，以及转基因和基因靶向的小鼠来研究BMP41B和i2启动子的体内功能。这些研究与了解1)维生素A过量或缺乏如何影响发育；2)导致内耳正常或异常发育的分子事件；以及3)PA抑制基因的分子途径有关。

DESCRIPTION (provided by applicant): The long term goal is to understand how retinoic acid (RA; the major biologically active metabolite of vitamin A) and bone morphogenetic protein 4 (BMP4) interact to regulate development. In general terms, RA regulates gene transcription by binding to its nuclear hormone receptors, the RARs; and BMP4 is a secreted protein that binds to cell surface receptors to initiate a signaling cascade that involves SMAD activation. Although the major BMP4 promoter in bone (1A promoter) is induced by RA, we have found that a previously described minor promoter (1B) and a novel promoter in intron 2 (i2) are repressed by PA. The i2 promoter is highly expressed in developing inner ear, whereas the 1A promoter is not. BMP4 and PA are both known to be essential for normal inner ear development. Antagonism of BMP4 inhibits development of the semicircular canals. Embryonic exposure to exogenous RA results in a similar phenotype, suggesting that PA may repress BMP4 in the inner ear, helping to restrict BMP4 expression appropriately in space and time. Indeed, repression of the novel i2 promoter by RA occurs both in an inner ear derived cell line and in vivo in mouse embryonic inner ear. Furthermore, the ability of RA to inhibit semicircular canal development is overcome by recombinant BMP4 (in chicks). This proposal seeks to address three Specific Aims: 1) Determine the mechanism by which PA down-regulates BMP4 transcription; 2) Characterize expression from the BMP4 1B and i2 promoters in space and time in developing mouse embryos; and 3) Determine the in vivo effects (in mice) of mutations that destroy the BMP4 1B and i2 promoters. The first aim is relevant to RA action in general in that the mechanism(s) of repression of target genes by PA is not well understood (in contrast to gene induction), and is of interest because PA up-regulates BMP4 in bone via a different promoter, the 1A promoter. The 2nd and 3rd Aims will establish the relative roles of the IB and i2 promoters in vivo in inner ear development, and will examine whether these promoters may play important roles outside the inner ear. Accomplishing these goals will rely on classical molecular tools to study gene regulation in cell culture and in cell free systems, as well as transgenic and gene-targeted mice to investigate the in vivo functions of the BMP4 1B and i2 promoters. The studies are relevant to understanding 1) how states of vitamin A excess or deficiency affect development; 2) molecular events that lead to normal or abnormal inner ear development; and 3) the molecular pathway of gene repression by PA.