Nomiyama H, Hieshima K, Hirokawa K, Hattori T, Takatsuki K, Miura R
Department of Biochemistry, Kumamoto University Medical School, Japan.
Mol Cell Biol. 1993 May;13(5):2787-801. doi: 10.1128/mcb.13.5.2787-2801.1993.
Cytokine LD78 is a human counterpart of the mouse macrophage inflammatory protein 1 alpha/hematopoietic stem cell inhibitor. Promoters of the LD78 alpha and LD78 beta genes showed similar inducible activities in two leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the LD78 alpha promoter indicated that multiple positive and negative regulatory elements are present, some of which are differentially required for induction and repression of the promoter activity in different cells. One of the negative regulatory elements, ICK-1, functioned in both cell lines in the absence and presence of stimulation and was shown to be a recognition site for positive and negative transcriptional factors. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic growth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 and Jurkat cells formed several protein-DNA complexes with the LD78 alpha ICK-1 element, one of which was also observed with the IL-3 and GM-CSF ICK-1 elements. Results from in vivo and in vitro analyses suggested that the protein forming this complex functions as a negative factor. The binding affinity of this protein, ICK-1A, to the LD78 alpha ICK-1 element was low and was significantly affected by the incubation temperature and the salt concentration in the binding buffer. ICK-1B, another protein bound specifically by the LD78 alpha ICK-1 element, was shown to be a positive factor important for induction of the promoter. These results suggested that ICK-1A plays an important role in balanced expression of LD78, IL-3, and GM-CSF during hematopoietic cell growth and differentiation.
细胞因子LD78是小鼠巨噬细胞炎性蛋白1α/造血干细胞抑制剂的人类对应物。LD78α和LD78β基因的启动子在两种白血病细胞系K562和Jurkat中表现出相似的诱导活性，但两种细胞系的诱导机制不同。对LD78α启动子的进一步表征表明，存在多个正调控元件和负调控元件，其中一些元件在不同细胞中对启动子活性的诱导和抑制有不同的需求。负调控元件之一ICK-1在有无刺激的情况下均在两种细胞系中起作用，并且被证明是正负转录因子的识别位点。该ICK-1元件包含一个直接重复序列，在造血生长因子白细胞介素-3（IL-3）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）基因启动子的负调控元件中也发现了类似的重复序列。K562和Jurkat细胞的核提取物与LD78α ICK-1元件形成了几种蛋白质-DNA复合物，其中一种也在IL-3和GM-CSF ICK-1元件中观察到。体内和体外分析结果表明，形成这种复合物的蛋白质起负因子的作用。这种蛋白质ICK-1A与LD78α ICK-1元件的结合亲和力较低，并且受到结合缓冲液中孵育温度和盐浓度的显著影响。ICK-1B是另一种与LD78α ICK-1元件特异性结合的蛋白质，被证明是启动子诱导的重要正因子。这些结果表明，ICK-1A在造血细胞生长和分化过程中LD78、IL-3和GM-CSF的平衡表达中起重要作用。
