顆粒球コロニー刺激因子受容体を介する情報伝達機構の解析

为探讨粒细胞集落刺激因子受体在增殖分化信号转导机制中的作用，将粒细胞集落刺激因子受体基因导入小鼠髓系前体细胞L-GM中表达，并在G-CSF刺激下向中性粒细胞分化。构建了几个突变的G-CSF受体cDNA，编码一系列缺失受体或受体，其中每个酪氨酸残基都被苯丙氨酸取代。通过分析这些突变对G-CSF诱导的信号转导的影响，发现p54(Shc)的酪氨酸磷酸化需要受体的第四个酪氨酸残基，而第一和第二个酪氨酸残基是诱导中性粒细胞分化表型所必需的，包括核分叶、生长抑制和髓过氧化物酶基因表达。在G-CSF刺激下，JAK1、JAK2、STAT3和Shc的酪氨酸残基发生了磷酸化。JAK1和JAK2的磷酸化需要受体的Box1和Box2区域，而STAT3的磷酸化不仅需要Box1和Box2区域，还需要第一酪氨酸残基附近的区域。此外，将中性粒细胞中特异表达的髓过氧化物酶(MPO)基因的上游启动子及其截短区域连接到报告基因，并检测其对G-CSF的依赖表达。在转录起始点上游约800bp处发现了一个顺式调控元件，负责G-CSF依赖基因的表达。通过凝胶移位实验鉴定了转录因子NF/G-CSF与该元件结合。用寡核苷酸亲和层析法纯化了核因子/粒细胞集落刺激因子，并测定了其N端氨基酸序列。获得的序列与已知的转录因子NF-Y的序列相同。因此，NF-Y可能参与了G-CSF依赖的MPO基因的表达。

To investigate proliferation and differentiation signal transduction mechanisms through granulocyte-colony stimulating factor (G-CSF) receptor, the receptor was expressed in murine myeloid precursor cell, L-GM,by transfecting its cDNA.When the obtained transformant was stimulated by G-CSF,it was differentiated to neutrophil. Several mutant G-CSF receptor cDNAs were constructed that encoded either a series of deletion receptors or the receptors where each tyrosine residue was replaced with phenylalanine. By analyzing the effects of these mutations on the G-CSF-induced signal transduction, tyrosine-phosphorylaion of p54 (Shc) was found to require the 4th tyrosine residue of the receptor and 1st and 2nd tyrosine residues were indispensable for inducing the neutrophil-differentiation phenotypes, including nuclear lobulation, growth suppression and myeloperoxidase gene expression. JAK1, JAK2, STAT3 and Shc were found to phosphorylated on their tyrosine residues upon G-CSF stimulation. Phosphorylation of JAK1 and JAK2 required the Box1 and Box2 region of the receptor, while STAT3 phosphorylation need not only the Box1 and Box2 but the region around the 1st tyrosine residue.Furthermore, the upstream promoter and its truncated regions of myeloperoxidase (MPO) gene, which is expressed specifically in neutrophils, was connected to the reporter gene, and its G-CSF dependent expression was examined. A cis-regulatory element was identified at about 800bp upstream from the transcription start site that was responsible for the G-CSF dependent gene expression. A transcription factor, NF/G-CSF,was identified to bind to the element by gel-shift assay. Using oligonucleotide affinity chromatography, NF/G-CSF was purified and its N-terminal amino acid sequence was determined. The obtained sequence was identical to that of a known transcription factor, NF-Y.Therefore, NF-Y appears to be involved in the G-CSF dependent MPO gene expression.