Celada A, Borràs F E, Soler C, Lloberas J, Klemsz M, van Beveren C, McKercher S, Maki R A
Departament de Fisiologia (Immunologia), Facultat de Biologia, Universitat de Barcelona, Spain.
J Exp Med. 1996 Jul 1;184(1):61-9. doi: 10.1084/jem.184.1.61.
PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.
PU.1是一种组织特异性转录因子，在造血谱系细胞中表达，包括巨噬细胞、粒细胞和B淋巴细胞。用反义PU.1表达构建体转染或用反义寡核苷酸处理的骨髓来源巨噬细胞与对照相比，增殖减少。相反，用正义PU.1表达构建体转染的骨髓巨噬细胞表现出增强的巨噬细胞集落刺激因子（M-CSF）依赖性增殖。有趣的是，PU.1的正义或反义构建体对不依赖M-CSF的细胞系的增殖没有影响，这表明该反应是M-CSF依赖性的。巨噬细胞用正义或反义PU.1构建体转染分别显示M-CSF受体表面表达水平升高或降低，这一发现进一步支持了上述观点。增殖增强似乎对PU.1具有选择性，因为用ets家族的其他几个成员（包括ets-2和fli-1）进行转染没有效果。还测试了PU.1的各种突变体影响巨噬细胞增殖的能力。当用表达PU.1 DNA结合结构域的构建体转染细胞时，发现巨噬细胞增殖减少。当将不含PEST结构域的PU.1表达构建体转染到骨髓来源的巨噬细胞中时，发现PU.1蛋白的PEST（富含脯氨酸、谷氨酸、丝氨酸和苏氨酸的区域）序列对PU.1增强的巨噬细胞增殖没有影响，而PEST序列在B细胞中是蛋白质-蛋白质相互作用的重要结构域。在体内，PU.1在几个丝氨酸残基上被磷酸化。用五个丝氨酸各自发生突变的含PU.1质粒进行转染表明，只有第41和45位对增强巨噬细胞增殖至关重要。我们得出结论，PU.1是巨噬细胞M-CSF依赖性增殖所必需的。该转录因子与增殖相关的靶标之一可能是M-CSF受体。
