Functional analysis of DAN and DA41 proteins in early embryogenesis

(A) DAN功能分析northern blot分析显示，在人骨肉瘤细胞系SAOS-2中，DAN的表达明显下调。在SAOS-2中，DAN的过表达导致了大量的生长抑制，这一点在集落形成实验中得到了证实。n端截断形式的DAN没有表现出生长抑制活性，说明分泌形式的DAN是有活性的。报告基因实验表明，DAN抑制小鼠胚胎癌P19细胞BMP4信号通路。这些观察结果表明，DAN以及Cerberus和Gremlin可能在雾胚和哺乳动物细胞中作为BMP的拮抗剂。大鼠DA41在v - ha -ras-转化的3Y1细胞（ras-3Y1）中的异位过表达导致显著的生长抑制，这与CDK2活性的显著降低有关。荧光原位杂交显示，DA41定位于人类染色体9q21.2-q21.3，与膀胱癌候选肿瘤抑制基因位点重叠。对da41相关蛋白进行数据库检索，鉴定出小鼠PLIC-1、PLIC-2、青蛙XDRP1和酵母DSK2。研究表明，XDRP1能够抑制细胞周期蛋白A的降解，阻断细胞分裂。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.