Functional analysis of DAN and DA41 proteins in early embryogenesis

(A)DANNorthern印迹分析显示，DAN在人骨肉瘤细胞系SAOS-2中的表达显著下调。克隆形成实验表明，DAN在SAOS-2中的过表达导致大量的生长抑制。N端截短型的DAN没有表现出生长抑制活性，说明分泌型的DAN是活性的。报告基因分析表明，DAN抑制了小鼠胚胎癌细胞P19的BMP4信号转导。这些观察结果表明，在FOG胚胎和哺乳动物细胞中，DAN以及Cerberus和Gremlin可能是BMP的拮抗剂。(B)在V-Ha-ras转化的3Y1细胞(ras-3Y1)中，DA41异位过表达大鼠DA41导致显著的生长抑制，这与CDK2活性显著降低有关。荧光原位杂交显示DA41被定位在人染色体9q21.2-q21.3上，该位置与膀胱癌候选抑癌基因重叠。在数据库中搜索DA41相关蛋白(S)，鉴定出小鼠PLIC-1、PLIC-2、青蛙XDRP1和酵母菌DSK2。结果表明，XDRP1对细胞周期蛋白A的降解有抑制作用，并能抑制细胞分裂。这些观察结果表明，DA41和XDRP1在细胞周期调控中可能具有相似的功能。

(A) Functional analysis of DANNorthern blot analysis revealed that the expression of DAN was significantly down-regulated in human osteosarcoma cell line SAOS-2. Overexpression of DAN in SAOS-2 resulted in a massive growth suppression as demonstrated by colony formation assays. N-terminal truncated form of DAN did not exhibit the growth suppressive activity, indicating that the secreted form of DAN is active. Reporter gene assay demonstrated that DAN inhibited the BMP4 signaling in mouse embryonic carcinoma P19 cells. These observations suggest that DAN, as well as Cerberus and Gremlin, may function as an antagonist against BMP in fog embryos and mammalian cells.(B) Characterization of DA41Ectopic overexpression of rat DA41 in V-Ha-ras-transformed 3Y1 cells (ras-3Y1) resulted in a significant growth suppression, which was associated with a remarkable reduction of CDK2 activity. Fluorescence in situ hybridization revealed that DA41 was mapped to human chromosome 9q21.2-q21.3, a position overlapping the candidate tumor suppressor locus for bladder cancer. Data base search for DA41-related proteins(s) identified mouse PLIC-1, PLIC-2, frog XDRP1, and yeast DSK2. It has been shown that XDRP1 inhibited the degradation of cyclin A and blocked the cell division. These observations suggest that DA41 and XDRP1 could share the similar function in the cell cycle regulation.