Reems Jo-Anna, Wang Wenjing, Tsubata Ken, Abdurrahman Najla, Sundell Birgitta, Tijssen Marloes R, van der Schoot Ellen, Di Summa Franca, Patel-Hett Sunita, Italiano Joseph, Gilligan Diana M
Northwest Tissue Services/Puget Sound Blood Center, Department of Medicine, Hematology Division, University of Washington, Seattle, WA 98104, USA.
Exp Hematol. 2008 Dec;36(12):1714-27. doi: 10.1016/j.exphem.2008.08.010.
High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of <or=0.001 with a fold change cutoff of three or more in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2+/-2.1 (mean+/-standard deviation). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2, and DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time quantitative polymerase chain reaction confirmed that DNM3 increased by 20.7-+/-3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in proplatelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs, with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from culture-derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Overexpression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony-forming cells and colony-forming unit-megakaryocytes, and a concomitant increase in the expression of nuclear factor erythroid 2 (NF-E2) and beta-tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.
高密度寡核苷酸微阵列用于比较未培养的CD34+/CD38lo细胞和培养来源的巨核细胞（MKs）的基因表达谱。如先前发表的那样，使用Rosetta Resolver软件程序分析了来自三个不同器官供体骨髓来源的三个重复微阵列数据集。在设定严格的p值<或=0.001且表达水平的变化倍数截止值为三倍或更高后，发现发动蛋白3（DNM3）在MK发育过程中差异表达，平均变化倍数为8.2±2.1（平均值±标准差）。DNM3是机械化学酶家族（DNM1、DNM2和DNM3）的成员，该家族以通过水解核苷酸将细胞膜与肌动蛋白细胞骨架连接来参与膜动力学而闻名。实时定量聚合酶链反应证实，在巨核细胞生成过程中DNM3增加了20.7±3.4倍（n = 4，p = 0.09），蛋白质印迹分析表明DNM3蛋白在人MKs中表达。共聚焦显微镜显示，DNM3在MKs的细胞质中弥漫分布，在前血小板过程中呈点状外观。免疫金电子显微镜也显示DNM3广泛分布于MKs的细胞质中，未明显定位于特定细胞器。DNM3的开放阅读框从培养来源的人MKs中克隆出来，确定与Ensemble数据库中公布的DNM3转录变体ENST00000367731编码的蛋白质100%相同。在脐带血CD3 +细胞中过表达DNM3导致有核细胞总数增加、总集落形成细胞和集落形成单位-巨核细胞扩增，以及核因子红细胞2（NF-E2）和β-微管蛋白表达随之增加。这些发现共同提供了首个证据，证明机械化学酶发动蛋白家族的一个成员存在于人MKs中，并表明DNM3是在介导MK/血小板发育过程中发生的细胞骨架和膜变化中发挥重要作用的极佳候选者。
