Hermans Mirjam H A, van de Geijn Gert-Jan, Antonissen Claudia, Gits Judith, van Leeuwen Daphne, Ward Alister C, Touw Ivo P
Institute of Hematology, Erasmus University of Rotterdam, The Netherlands.
Blood. 2003 Apr 1;101(7):2584-90. doi: 10.1182/blood-2002-07-2062. Epub 2002 Dec 5.
Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.
粒细胞集落刺激因子（G-CSF）是中性粒细胞生成的主要调节因子。细胞系研究表明，G-CSF受体（G-CSF-R）胞质结构域内保守的酪氨酸残基Tyr704、Tyr729、Tyr744、Tyr764对G-CSF诱导的增殖、分化及细胞存活有显著贡献。然而，在更接近生理的条件下这些酪氨酸残基是否同等重要尚不清楚。在此，我们研究了单个G-CSF-R酪氨酸残基如何影响原代髓系祖细胞的G-CSF反应。我们构建了G-CSF-R缺陷小鼠，并用酪氨酸“缺失”突变体（m0）、单个酪氨酸“回补”突变体或野生型（WT）受体转导其骨髓细胞。在原代集落分析、连续再接种及悬浮培养中测定G-CSF诱导的反应。我们发现去除所有酪氨酸对原代集落生长无重大影响。然而，回补Tyr764强烈增强增殖反应，这可通过抑制ERK活性逆转。我们发现Tyr729与细胞因子信号转导抑制因子SOCS3相关，对集落形成有负面影响。重复再接种后，与WT相比，表达m0的细胞的克隆形成能力逐渐下降。参与信号转导和转录激活因子3（STAT3）激活的Tyr729以及Tyr704和Tyr744的存在进一步降低再接种效率。相反，Tyr764极大提高了髓系祖细胞的克隆形成能力，在第五次再接种后集落形成细胞比m0增加超过10^4倍。这些发现表明，G-CSF-R胞质结构域中的酪氨酸残基虽然对G-CSF诱导的集落生长并非必需，但可募集调节髓系祖细胞维持和增殖的信号传导机制。
