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）如何相互作用以调节发育。一般来说，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，它与细胞表面受体结合，启动涉及SMAD激活的信号级联反应。尽管骨中的主要BMP4启动子（1A启动子）由RA诱导，但我们发现先前描述的次要启动子（1B）和内含子2中的新启动子（i2）受到PA的抑制。i2启动子在发育中的内耳中高度表达，而1A启动子则不是。众所周知，BMP4和PA对于正常的内耳发育至关重要。BMP4的拮抗抑制半规管的发育。胚胎暴露于外源性RA导致类似的表型，表明PA可能抑制内耳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i2启动子的抑制既发生在内耳衍生细胞系中，也发生在小鼠胚胎内耳的体内。此外，重组BMP4（在雏鸡中）克服了RA抑制半规管发育的能力。本提案旨在解决三个具体目标：1)确定PA下调BMP4转录的机制；2)在发育的小鼠胚胎中表征BMP4 1B和i2启动子在空间和时间上的表达；3)确定破坏BMP4 1B和i2启动子的突变在体内（小鼠）的影响。第一个目标与RA的作用有关，因为PA抑制靶基因的机制尚不清楚（与基因诱导相反），这是因为PA通过不同的启动子1A启动子上调骨中的BMP4。第2和第3个目标将确定IB和i2启动子在体内内耳发育中的相对作用，并研究这些启动子是否在内耳外发挥重要作用。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小鼠来研究BMP4 1B和i2启动子的体内功能。这些研究与了解1)维生素A过量或缺乏的状态如何影响发育有关；2)导致内耳发育正常或异常的分子事件；3) PA基因抑制的分子途径。

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.