Lapidot-Lifson Y, Patinkin D, Prody C A, Ehrlich G, Seidman S, Ben-Aziz R, Benseler F, Eckstein F, Zakut H, Soreq H
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):579-83. doi: 10.1073/pnas.89.2.579.
Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.
尽管有几条证据表明胆碱能信号与造血分化之间存在关联，但触发多能骨髓干细胞向单核巨噬细胞或多核巨核细胞等分化谱系定向分化的机制仍不清楚。我们现在展示了一种编码CHED（胆碱酯酶相关细胞分裂控制器）的cDNA的克隆，它是粟酒裂殖酵母细胞分裂周期2（cdc2）样激酶的人类同源物，是有丝分裂细胞周期的通用控制器。文库筛选、RNA印迹杂交以及从细胞mRNA逆转录的cDNA的直接PCR扩增表明，CHED mRNA在包括骨髓在内的多种组织中表达。CHED蛋白包含激酶特有的共有ATP结合和磷酸化结构域，与其他cdc2相关激酶具有34 - 42%的相同氨基酸序列，并且在其氨基和羧基末端长得多。一种设计用于中断CHED表达的反义寡脱氧核苷酸（AS - CHED）在添加到培养物后1小时内显著降低了CHED mRNA与肌动蛋白mRNA之间的比例，这种降低持续了4天。AS - CHED处理选择性地抑制了小鼠骨髓培养物中的巨核细胞发育，但没有阻止其他造血途径，单核细胞数量增加证明了这一点。一种阻断乙酰胆碱水解酶丁酰胆碱酯酶产生的寡脱氧核苷酸对巨核细胞生成表现出类似的抑制作用。相反，一种阻断人类2Hs cdc2同源物产生的寡脱氧核苷酸干扰了细胞增殖，但没有改变这些培养物的细胞类型组成。因此，这些发现加强了胆碱能信号与造血过程中细胞分裂控制之间的联系，并表明CHED和胆碱酯酶都参与了这一分化过程。
