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

粒细胞集落刺激因子受体介导的信息传递机制分析

• 批准号: 07680702
• 负责人: MURAKAMI Hiroshi
• 金额: $ 1.28万
• 依托单位: Okayama University (1996)Osaka Bioscience Institute (1995)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680702/
• 关键词: granulocyte; gene-expression; receptor; signal-transduction; proliferation; differentiation; tyrosine-kinase; colony-stimulating-factor

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.

为了研究粒细胞集落刺激因子(G-CSF)受体的增殖和分化信号转导机制，通过转染小鼠骨髓前体细胞L-GM的cDNA，使该受体在小鼠骨髓前体细胞L-GM中表达。当获得的转化体受到G-CSF刺激时，分化为中性粒细胞。构建了几种突变型G-CSF受体cDNA，其编码一系列缺失受体或每个酪氨酸残基被苯丙氨酸取代的受体。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对于诱导中性粒细胞分化是必不可少的表型，包括核分叶、生长抑制和髓过氧化物酶基因表达。发现 JAK1、JAK2、STAT3 和 Shc 在 G-CSF 刺激后其酪氨酸残基发生磷酸化。 JAK1和JAK2的磷酸化需要受体的Box1和Box2区域，而STAT3磷酸化不仅需要Box1和Box2，还需要第1个酪氨酸残基周围的区域。此外，髓过氧化物酶(MPO)基因的上游启动子及其截短区域，将其在中性粒细胞中特异性表达，与报告基因连接，并检查其 G-CSF 依赖性表达。在转录起始位点上游约 800bp 处鉴定出一个顺式调控元件，该元件负责 G-CSF 依赖性基因表达。通过凝胶迁移测定，鉴定出转录因子 NF/G-CSF 与该元件结合。使用寡核苷酸亲和层析纯化 NF/G-CSF 并测定其 N 末端氨基酸序列。获得的序列与已知转录因子NF-Y的序列相同。因此，NF-Y似乎参与G-CSF依赖性MPO基因表达。

项目成果

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