Recombinant R-spondin2 and Wnt3a Up- and Down-Regulate Novel Target Genes in C57MG Mouse Mammary Epithelial Cells
Recombinant R-spondin2 and Wnt3a Up- and Down-Regulate Novel Target Genes in C57MG Mouse Mammary Epithelial Cells
复制标题
DOI：
10.1371/journal.pone.0029455
复制
发表时间：
2012-01-04
期刊：
影响因子：
3.7
通讯作者：
Rubin, Jeffrey S.
中科院分区：
文献类型：
--
作者：
Baljinnyam, Bolormaa;Klauzinska, Malgorzata;Rubin, Jeffrey S.
R-spondins（Rspos）包括四种分泌蛋白的家族，其在细胞增殖、细胞命运决定和器官发生中具有重要作用。Rspos通常通过增强Wnt/β-连环蛋白信号传导途径发挥其作用。为了系统地研究Rspo/Wnt对基因表达的影响，我们使用重组Rspo 2和/或Wnt 3a处理的C57 MG小鼠乳腺上皮细胞进行了微阵列分析。我们观察到了几个以前未鉴定的靶基因的上调和下调，包括编码参与免疫反应的蛋白质的基因、其他生长因子信号通路的效应子和转录因子。通过定量真实的时间RT-PCR验证了数十种这些变化。时程实验表明，Rspo 2通常在3或6小时对Wnt依赖性基因表达几乎没有影响，但在24小时增强表达，这与表明Rspo 2主要用于维持而不是急剧增加Wnt途径活化的生化数据一致。通过用Dickkopf 1（Wnt/β-连环蛋白通路拮抗剂）预处理和通过siRNA敲低β-连环蛋白表达来抑制基因表达的上调。虽然Dickkopf 1阻断Rspo 2/Wnt 3a依赖性下调，但许多下调的基因不受β-连环蛋白敲低的影响，这表明在这些情况下，下调是由β-连环蛋白非依赖性机制介导的。
R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/beta-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/beta-catenin pathway antagonist, and by siRNA knockdown of beta-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by beta-catenin knockdown, suggesting that in these instances down-regulation was mediated by a beta-catenin-independent mechanism.