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产物）和骨形态发生蛋白4（BMP 4）如何相互作用以调节发育。一般而言，RA通过与其核激素受体RAR结合来调节基因转录; BMP 4是一种分泌蛋白，其与细胞表面受体结合以启动涉及SMAD激活的信号级联。尽管骨中的主要BMP 4启动子（1A启动子）被RA诱导，但我们发现先前描述的次要启动子（1B）和内含子2中的新型启动子（i2）被PA抑制。i2启动子在发育中的内耳中高度表达，而1A启动子则不表达。BMP 4和PA都是内耳正常发育所必需的。BMP 4的拮抗作用抑制半规管的发育。胚胎暴露于外源性RA导致类似的表型，这表明PA可能抑制内耳中的BMP 4，有助于在空间和时间上适当地限制BMP 4表达。事实上，RA对新i2启动子的抑制发生在内耳来源的细胞系和小鼠胚胎内耳体内。此外，RA抑制半规管发育的能力被重组BMP 4克服（在小鸡中）。该提案旨在解决三个具体目标：1）确定PA下调BMP 4转录的机制; 2）表征发育中小鼠胚胎中BMP 4 1B和i2启动子在空间和时间上的表达; 3）确定破坏BMP 4 1B和i2启动子的突变的体内效应（在小鼠中）。第一个目的通常与RA作用相关，因为PA抑制靶基因的机制尚未得到很好的理解（与基因诱导相反），并且由于PA通过不同的启动子（1A启动子）上调骨中的BMP 4而引起关注。第二和第三个目的将建立IB和i2启动子在内耳发育中的体内相对作用，并将检查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BMP 4 1B和i2启动子的体内功能。这些研究与理解1）维生素A过量或缺乏的状态如何影响发育有关; 2）导致正常或异常内耳发育的分子事件; 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.