Frigon N L, Shao L, Young A L, Maderazo L, Yu J
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Blood. 1992 Feb 1;79(3):765-72.
Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot-blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.
最近的研究表明，一种纯化的蛋白质——激活素A，属于转化生长因子β（TGF-β）超家族。与TGF-β相似，激活素A根据靶组织的不同可具有不同的生物学活性。我们使用重组激活素A来证明这种蛋白质在调节人红系细胞系K562中的人类红系分化方面可能具有的调节作用。使用包含α、β、γ和ε珠蛋白第二个外显子的基因组探针，通过S1核酸酶分析检测未处理和激活素处理的K562细胞中各种类型珠蛋白转录本的相对丰度。尽管各种珠蛋白序列之间存在相当高的同源性，但在分析中这些珠蛋白探针对其独特的mRNA种类具有高度特异性。结果显示，在用激活素A处理的K562细胞中，受S1消化保护的γ和ε珠蛋白（209个核苷酸）以及α珠蛋白（180个核苷酸）的特异性珠蛋白探针片段的丰度增加了许多倍。相比之下，在对照细胞或激活素处理的细胞中均未检测到β珠蛋白的特异性转录本。胎儿和胚胎β样及α珠蛋白转录本水平的增加也证实了早期使用珠蛋白cDNA作为探针的Northern印迹和狭缝印迹分析研究。此外，使用分离细胞核的核转录延伸分析表明，激活素处理后珠蛋白转录本的大部分增加可归因于珠蛋白基因转录速率的刺激。瞬时转染分析也提供了证据，表明激活素A显著刺激了K562细胞中ε珠蛋白启动子的转录活性，但在非红系中国仓鼠卵巢细胞中则没有。因此，得出的结论是，激活素A在红系细胞的转录水平上对珠蛋白基因表达发挥作用。
