Physiologic role of signal transaction pathways in primary cultured human erythroid progenitor cells.

红细胞的发育是一个由多种细胞因子调控的复杂过程。集落实验揭示了每种细胞因子的生理重要性。然而，从CD34+细胞体外产生的高度纯化的人集落形成单位-红系（CFU-E）的生化研究直到最近才开始。我们和其他人的研究表明，原代红系细胞的信号转导可能与细胞系或其他物种的原代细胞有很大的不同。我们已经开发了一种方法来产生纯化的红细胞祖细胞，从人外周血中的CD34+细胞扩增到足够数量的红细胞祖细胞，以允许检查EPO或凋亡的早期信号。利用这些细胞，我们发现SCF可以保护纯化的人糖蛋白a阳性(GPA^+)/c-kit^+细胞免于凋亡，并提示介导的Src激酶激活参与Akt激活和细胞存活。此外，我们还发现SCF可以通过src家族酪氨酸激酶的活性来阻止fas诱导的红系祖细胞凋亡。我们还在红系细胞中发现了活跃的Lyn。这些数据表明SCF在红细胞发育中存在一种新的src家族/AKT依赖功能。同时，我们鉴定出一个新的Kruppel关联盒（KRAB）锌指基因ZNF317。ZNF317编码了一个由13个克虏伯样锌指和一个克虏伯结构域组成的蛋白质。RT-PCR分析显示4个选择性剪接产物（ZNF317-1 ~ ZNF317-4）。ZNF317基因有7个外显子，位于人类染色体19p13上。ZNF317-1和ZNF317-2转录本普遍表达，而ZNF317-3和ZNF317-4转录本仅在淋巴细胞、脾脏和肺中检测到。ZNF317 mRNA的表达在红系成熟过程中显著降低。在淋巴细胞中，ZNF317的表达在有丝分裂刺激下降低。我们认为ZNF317可能在红细胞成熟和淋巴细胞增殖中起重要作用。少

Development of erythrocytes is a comlex process governed by multiple cytokines. Colony assay revealed the physiologic importance of each of the cytokines. However, biochemical studies of highly purified human colony forming unit-erythroid (CFU-E) generated in vitro from CD34+ cells have only recently begun. Studies from our groups and others suggested that signal transduction in the primary erythroid cells may differ considerably from that in cell lines or the primary cells from other species. We have developed a method to generate purified erythroid progenitors, which are expanded from CD34+ cells in human peripheral blood in a sufficient number to allow for examination of early signaling by EPO or apoptosis. Using these cells, we have found that SCF protected purified human glycophorin A-positive (GPA^+)/c-kit^+ cells from apoptosis, and suggeswted that mediated Src kinase activation is involved in Akt activation and cell survival. Further, we have suggested that SCF prevents Fas-med … More iated apoptosis of erythroid progenitor cells in a manner dependent on the activity of Src-family tyrosine kinases. We also identified active Lyn in erythroid cells. These data suggest the presence of a novel Src-family/AKT dependent function of SCF in the development of erythrocytes. At the same time, we identified a novel Kruppel associated box (KRAB) zinc finger gene, ZNF317. ZNF317 encoded a protein comprised of thirteen Kruppel-like zinc fingers and a KRAB domain. RT-PCR analysis revealed four alternatively splicing products (ZNF317-1 through ZNF317-4). The ZNF317 gene has seven exons and is located in human chromosome 19p13. ZNF317-1 and ZNF317-2 transcripts were ubiquitously expressed, whereas ZNF317-3 and ZNF317-4 transcripts were detected only in lymphocytes, spleen and lung. The expression of ZNF317 mRNA significantly decreased during erythroid maturation. In lymphocytes, ZNF317 expression was reduced in response to mitogenic stimulation. We propose that ZNF317 may play an important role in erythroid maturation and lymphoid proliferation. Less