Nucleoredoxin is required for the maintenance of Wnt/beta-catenin signaling in mice embryo

We previously showed that nucleoredoxin (NRX) suppresses Wnt/beta-catenin signaling through its binding to Dishevelled (Dvl) (Nat Cell Biol 8, 501-508 (2006)). To clarify the in vivo role of NRX in mammals, we here generate NRX gene-knockout mice (NRX-/- mice) by homologous recombination. NRX-/- mice die around birth. Therefore, we performed microarray analyses with NRX+/+ and NRX-/- embryos of E9.5 and E11.5 stages. Surprisingly, in the genes commonly upregulated at both stages, we could not observe Wnt/beta-catenin targets. Rather, several target genes for Wnt/beta-catenin pathway, such as Frizzled2 and Occludin, are downregulated in NRX-/- whole embryos. Frizzled2 is a gene reportedly expressed in developmental heart. Indeed, by RT-PCR analyses we confirmed that the expression of Frizzled2, as well as other Wnt/beta-catenin target genes, was downregulated in embryonic heart of NRX-/- mice. We also found that the amount of unphosphorylated (i.e. activated) form of beta-catenin was downregulated in NRX-/- embryonic heart. These results reveal that NRX plays another role which was unidentified in culture cell studies; it is required for the maintenance of Wnt/beta-catenin signaling activity. Total RNAs were extracted from E9.5 and E11.5 embryos derived from NRX+/+ and NRX-/- C57BL/6J mice using RNeasy extraction kit (Qiagen). Two dye-swapped experiments were performed by hybridizing complimentary RNA (cRNA) labeled with either Cyanine (Cy) -3 or Cy-5 (Perkin-Elmer) onto Whole Mouse Genome Oligo Microarray (G4122A; Agilent Technologies). The signature genes with mean fold changes > +2.0 or < -2.0 at both stages were subjected for further evaluation.我们此前已有研究证实，核氧化还原蛋白（nucleoredoxin, NRX）可通过结合散乱蛋白（Dishevelled, Dvl）抑制Wnt/β-连环蛋白信号通路（《自然·细胞生物学》, Nat Cell Biol, 8, 501-508, 2006）。为阐明核氧化还原蛋白在哺乳动物体内的生理功能，本研究通过同源重组技术构建了核氧化还原蛋白基因敲除小鼠（NRX敲除小鼠，NRX-/-）。NRX-/-小鼠在围产期死亡，因此我们对E9.5及E11.5阶段的野生型（NRX+/+）与基因敲除型（NRX-/-）小鼠胚胎进行了基因芯片分析。令人意外的是，在两个阶段均上调的基因中，我们并未检测到Wnt/β-连环蛋白通路的靶基因；相反，包括卷曲蛋白2（Frizzled2）与闭合蛋白（Occludin）在内的若干Wnt/β-连环蛋白通路靶基因在NRX-/-全胚胎中呈现表达下调。卷曲蛋白2是一种在发育中心脏中特异性表达的基因。随后我们通过逆转录聚合酶链反应（RT-PCR）分析证实，NRX-/-小鼠胚胎心脏中，卷曲蛋白2及其他Wnt/β-连环蛋白通路靶基因的表达均出现下调。此外，我们还发现NRX-/-小鼠胚胎心脏中，未磷酸化（即活化形式）的β-连环蛋白含量显著下调。上述研究结果揭示了核氧化还原蛋白此前未在细胞培养实验中被发现的另一功能：其对维持Wnt/β-连环蛋白信号通路的活性至关重要。本研究使用RNeasy提取试剂盒（Qiagen公司），从NRX+/+与NRX-/- C57BL/6J小鼠的E9.5及E11.5胚胎中提取总RNA。通过将分别标记有菁染料（Cyanine）-3与Cy-5（Perkin-Elmer公司）的互补RNA（cRNA）杂交至全小鼠基因组寡核苷酸芯片（G4122A，安捷伦科技公司），我们完成了两次染料互换实验。将两个阶段中平均表达倍数变化大于+2.0或小于-2.0的特征基因用于后续验证分析。