Role of Cited2 in Lung Development

描述（由申请人提供）：Cited 2 [CBP/p300-interacting transactivators with glutamic acid（E）and aspartate acid（D）-rich tail 2]是一个新的转录激活因子家族的创始成员之一。本实验室克隆了Cited 2基因，发现Cited 2基因在不同的细胞类型中受多种生物刺激物如细胞因子、血清和脂多糖（LPS）的诱导，并且当过表达时，它是一个转化基因。Cited 2通过与DNA结合蛋白（Tfap 2、Smad 2/3、Lhx 2、PPAR和PPAR）相互作用来调节其相应靶基因的转录，从而充当转录调节剂。它还通过与HIF-1竞争结合CBP/p300的第一个富含半胱氨酸-组氨酸（CH 1）的区域来抑制HIF-1介导的信号传导，从而作为HIF-1的负调节剂发挥作用。缺乏Cited 2的小鼠在妊娠晚期死亡，并伴有许多发育缺陷。最近，我们在E17.5至E18.5 Cited 2-/-肺中观察到肺不成熟，由于I型和II型肺泡细胞分化停滞和凋亡肺泡细胞数量减少，这些肺显示肺泡空间显著减少。与表型一致，与肺泡细胞分化相关的基因在野生型和Cited 2-/-胚胎肺之间差异表达。这些结果表明，Cited 2可能是呼吸窘迫综合征的潜在候选者。我们进一步证实了C/EBP的表达，气道上皮成熟的关键调节因子之一，在Cited 2-/-胚胎肺中显著降低。有趣的是，Cited 2缺失的胚胎肺中的许多表型与携带肺特异性C/EBP缺失和CBP CH 1结构域缺失的小鼠中的表型相似。在瞬时转染实验中，Tfap 2c在HeLa和A549细胞中作为C/EBP基因启动子的转录抑制因子发挥作用，并且这种抑制作用通过共转染Cited 2而被消除。因此，我们假设Cited 2-Tfap 2c-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Cited 2对C/EBP表达的调控机制，（2）用遗传学方法证明C/EBP与Cited 2缺陷的胚胎肺发育过程中的肺不成熟有关，（3）研究Cited 2缺陷的胚胎肺中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，还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的靶点。

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.