应用斑马鱼模型研究CFTR调控Wnt信号影响造血作用的分子机制

Mutation of cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF) with symptom of inflammation and anemia; recently, we observed angiogenesis in tumorigenesis promoted by CFTR, suggesting that CFTR functions in hematopoiesis. However, the underlying molecular mechanism of hematopoiesis regulated by CFTR remains elusive. At present, we have found the decreased expression of hematopoiesis marker genes in the CFTR knockdown embryos. The phenotype of CFTR knockdown embryo was similar to the deficiency of Wnt3 with curved axis and shorten tail. Furthermore, CFTR located downstream of Wnt3 and controlled the expression of Dvl. The results above suggest the effect of CFTR on hematopoiesis through regulation of Wnt signaling. Based on the results above, we plan to identify the position of CFTR in Wnt3 - Dvl pathway first. Given the fact that CFTR is a transmembrane protein and both CFTR and Dvl contain PDZ domain, we propose that detection of protein binding between them will be the key point. Finally, the detailed molecular mechanism of positive regulation of Dvl by CFTR to transduce Wnt signaling will be uncovered through ubiquitination assays. Thus, our research focus on the regulation of Wnt signaling by CFTR to promote hematopoiesis, which will contribute to understanding of the etiological factor for cystic fibrosis from the point of hematopoiesis and uncover new molecular mechanism of hematopoiesis.

CFTR基因突变导致囊性纤维化，患者具有炎症、贫血等症状；我们近期研究发现CFTR促进肿瘤生成的过程中伴随新生血管形成,提示CFTR具有调控造血的作用，但其中分子机制有待阐明。我们已发现在斑马鱼早期发育的胚胎中CFTR的表达被抑制后造血标记基因的表达明显减弱；CFTR表达被抑制后胚胎出现与Wnt3缺失相似的体轴缩短和尾部卷曲表型；CFTR位于Wnt3的下游且正相关调控Dvl的表达。以上结果提示CFTR对造血的影响与其调控Wnt信号通路有关。本研究以此切入首先明确CFTR与Wnt3、Dvl等基因间的上下游关系；鉴于CFTR是跨膜蛋白以及CFTR和Dvl同时具有PDZ结构域的特点，将重点探讨CFTR与Dvl蛋白间的结合关系；从蛋白酶体等途径阐明CFTR正向调控Dvl进而影响Wnt信号转导的分子机制。本项目可从CFTR调控Wnt信号影响造血的角度了解囊性纤维化病因，同时揭示新的造血分子机理。

囊性纤维化跨膜传导因子(CFTR)基因的突变导致囊性纤维化疾病的发生，患者具有多种临床症状。其中一些患者表现出严重的贫血症状，提示CFTR在调控造血方面具有重要作用，但其中分子机制有待阐明。我们利用斑马鱼模型，使用TALEN技术获得了CFTR突变体，在该突变体胚胎中原始造血和之后的定向造血都表现出缺陷，并伴随着Wnt信号的减弱。在进一步的研究中，我们发现CFTR在体内与造血前体细胞关键调控信号Wnt的关键蛋白分子Dishevelled (Dvl)具有结合关系，而且，他们的结合作用是通过PDZ结构域实现的。CFTR对Dvl的结合能保护Dvl不遭受Dapper1 (Dpr1)蛋白诱导的溶酶体途径降解。CFTR突变体胚胎中的造血缺陷和减弱的Wnt 信号可以被野生型CFTR蛋白以及通道功能缺失的G551D突变蛋白恢复。G551D突变蛋白虽然离子通道功能缺失，但含有完整的PDZ结构域。此外，我们根据在斑马鱼上的发现，在人类B细胞急性淋巴白血病病人中检测到异常表达升高的CFTR和Wnt信号。在白血病细胞株中干扰CFTR可以诱导Dvl2和β-catenin蛋白的表达下降，并且削弱细胞的增殖。综上，我们的发现表明CFTR通过结合Dvl2，让Dvl2避免遭受溶酶体途径降解，从而维持Wnt信号的转导，最后诱导造血细胞的生成。本研究从CFTR调控Wnt信号影响造血的角度探讨了囊性纤维化病因，同时揭示了新的造血分子机理，并且表明CFTR可以作为新的治疗白血病的潜在靶点。