Role of Cited2 in Lung Development

描述（由申请人提供）：Cited2 [CBP/p300-与谷氨酸(E)和天冬氨酸(D)-富尾2相互作用的transactivators]是一个新的转录激活剂家族的创始成员之一。我的实验室克隆了Cited2，发现Cited2在不同细胞类型中受到细胞因子、血清和脂多糖（LPS）等多种生物刺激诱导，过表达时为转化基因。Cited2作为转录调节剂，通过与DNA结合蛋白（Tfap2、Smad2/3、Lhx2、PPAR和PPAR）相互作用来调节其相应靶基因的转录。它还作为HIF-1的负调节因子，与HIF-1竞争，结合CBP/p300的第一个富含半胱氨酸-组氨酸（CH1）区域，抑制HIF-1介导的信号传导。缺乏Cited2的小鼠在妊娠后期死亡，并伴有许多发育缺陷。最近，我们观察到E17.5至E18.5 Cited2-/-肺不成熟，由于I型和II型肺泡细胞分化停止，肺泡空间明显减少，肺泡细胞凋亡数量减少。与表型一致，与肺泡细胞分化相关的基因在野生型和Cited2-/-胚胎肺中表达差异。这些结果提示Cited2可能是呼吸窘迫综合征的潜在候选基因。我们进一步证明，在Cited2-/-胚胎肺中，气道上皮成熟的关键调节因子之一C/EBP的表达显著降低。有趣的是，在cited2缺失的胚胎肺中，许多表型与肺特异性缺失C/EBP和CBP CH1结构域缺失的小鼠相似。在瞬时转染实验中，Tfap2c在HeLa和A549细胞中作为C/EBP基因启动子的转录抑制因子，而这种抑制作用通过共转染Cited2而被消除。因此，我们假设Cited2-Tfap2c-CBP复合物通过调节C/EBP表达来控制肺成熟。为了验证这一假设，我们将(1)研究Cited2对C/EBP表达的调控机制，(2)利用遗传学方法证明C/EBP与Cited2-缺陷胚胎肺发育过程中的肺不成熟有关，(3)研究Cited2缺陷胚胎肺肺泡细胞存活和分化停滞的分子机制。该研究的完成不仅将阐明肺发育的分子机制，也将为呼吸窘迫综合征的分子发病机制提供新的线索，并有助于确定特异性治疗靶点。

DESCRIPTION (provided by applicant): Cited2 [CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is one of the founding members of a new family of transcriptional activators. My laboratory cloned Cited2 and showed that Cited2 is induced by many biological stimuli such as cytokines, serum and lipopolysaccharide (LPS) in different cell types and it is a transforming gene when overexpressed. Cited2 acts as a transcriptional modulator by interacting with DNA binding proteins (Tfap2, Smad2/3, Lhx2, PPAR, and PPAR) to regulate the transcription of their corresponding target genes. It also functions as a negative regulator of HIF-1 by competing with HIF-1 in binding to the first cysteine-histidine-rich (CH1) region of CBP/p300 to inhibit HIF-1 mediated signaling. Mice lacking Cited2 die at late gestation with numerous developmental defects. Recently, we observed pulmonary immaturity in E17.5 to E18.5 Cited2-/- lungs, which display significantly reduced alveolar space due to an arrest in the type I and type II alveolar cell differentiation and decreased number of apoptotic alveolar cells. Consistent with the phenotype, genes associated with alveolar cell differentiation were differentially expressed between wild-type and Cited2-/- embryonic lungs. These results suggest that Cited2 may be a potential candidate for respiratory distress syndrome. We further demonstrated that expression of C/EBP, one of the key regulators of airway epithelial maturation, was significantly decreased in Cited2-/- embryonic lungs. Interestingly, many of the phenotypes in Cited2-null embryonic lungs were similar to those in mice bearing lung-specific deletion of C/EBP and deletion in the CH1 domain of CBP. In transient transfection assays, Tfap2c functioned as a transcriptional repressor of the C/EBP gene promoter in HeLa and A549 cells and this repressive effect was abolished by co-transfection of Cited2. Thus, we hypothesize that Cited2-Tfap2c-CBP complex controls lung maturation by regulating C/EBP expression. To test this hypothesis, we will (1) study the regulatory mechanism of Cited2 on C/EBP expression, (2) use genetic approaches to show that C/EBP is responsible for the pulmonary immaturity in Cited2- deficient embryonic lung during development, and (3) study the molecular mechanism of alveolar cell survival and differentiation arrest in Cited2-deficient embryonic lung. The accomplishment of the study will not only elucidate molecular mechanisms of lung development but also provide new clues to the molecular pathogenesis of respiratory distress syndrome and help identify targets for specific therapies.