Genetos Damian C, Wong Alice, Weber Thomas J, Karin Norman J, Yellowley Clare E
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA, United States of America.
Systems Toxicology, Pacific Northwest National Laboratory, Richland, WA, United States of America.
PLoS One. 2014 Sep 15;9(9):e107482. doi: 10.1371/journal.pone.0107482. eCollection 2014.
Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles.
膜联蛋白是一类钙结合蛋白，在脂筏调节、炎症、纤维蛋白溶解、转录编程和离子运输中具有多种功能。在骨骼中，它们作为矿化基质小泡的成分已得到充分表征，尽管关于它们在骨生成过程中的功能知之甚少。我们利用短发夹RNA（shRNA）构建了膜联蛋白A2（AnxA2）或膜联蛋白A5（AnxA5）敲低的前成骨细胞，并与pSiren（Si）对照相比，确定敲低细胞中的增殖或成骨分化是否发生改变。我们报告称，作为增殖标志物的DNA含量在AnxA2和AnxA5敲低细胞中均显著降低。培养14天后，AnxA2或AnxA5敲低细胞中的碱性磷酸酶表达和活性也受到抑制。敲低细胞中成骨基因表达模式发生改变，与pSiren相比，Col1a1在敲低细胞中表达更快。相反，培养14天后，Runx2、Ibsp和Bglap均显示表达下降。与pSiren对照相比，在AnxA2和AnxA5敲低细胞中，白细胞介素诱导的STAT6信号均明显减弱。这些数据表明，AnxA2和AnxA5除了在基质小泡中已被充分研究的功能外，还可通过调节骨祖细胞的增殖、分化和对细胞因子的反应来影响骨形成。
