Lévesque Jean-Pierre, Hendy Jean, Winkler Ingrid G, Takamatsu Yasushi, Simmons Paul J
Stem Cell Laboratory, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 3000, Australia.
Exp Hematol. 2003 Feb;31(2):109-17. doi: 10.1016/s0301-472x(02)01028-7.
Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic progenitor and stem cells from the bone marrow into the peripheral blood. Although the mechanisms leading to the mobilization of primitive hematopoietic cells is not fully understood, it has been noted that the yield of mobilization in humans is correlated to the down-regulation of c-KIT/CD117 expression on mobilized cells. We sought to determine the mechanisms responsible for the reduced expression of c-KIT on mobilized hematopoietic progenitor cells.
Mice were mobilized with G-CSF and primitive hematopoietic cells were collected from bone marrow and blood to analyze c-KIT expression. Using cell lines expressing mouse and human c-KIT and a recombinant protein comprising the entire extracellular domain of human c-KIT, we analyzed by flow cytometry and immunoblotting the proteolytic cleavage of c-KIT by proteases released in bone marrow extracellular fluids extracted from mobilized mice.
Administration of G-CSF into mice results in the reduction of c-KIT expression on primitive hematopoietic cells in bone marrow and peripheral blood. Bone marrow extracellular fluids isolated from G-CSF-mobilized mice contain serine proteases that cleave c-KIT into discrete fragments. Proteases capable of cleaving c-KIT include neutrophil elastase, cathepsin G, proteinase-3 and matrix metalloproteinase-9.
In addition to transcriptional controls, exocytosis, and ligand-induced internalization, the direct proteolytic cleavage of c-KIT by neutrophil and macrophage proteases represents a novel pathway to regulate the levels of c-KIT expression at the surface of hematopoietic cells and may be responsible in part for the down-regulation of c-KIT expression on mobilized hematopoietic progenitors in vivo.
给予粒细胞集落刺激因子（G-CSF）可促使造血祖细胞和干细胞从骨髓动员至外周血。尽管导致原始造血细胞动员的机制尚未完全明确，但已注意到人类的动员产量与动员细胞上c-KIT/CD117表达的下调相关。我们试图确定导致动员的造血祖细胞上c-KIT表达降低的机制。
用G-CSF动员小鼠，从骨髓和血液中收集原始造血细胞以分析c-KIT表达。使用表达小鼠和人类c-KIT的细胞系以及包含人类c-KIT整个胞外域的重组蛋白，我们通过流式细胞术和免疫印迹分析了从动员小鼠提取的骨髓细胞外液中释放的蛋白酶对c-KIT的蛋白水解切割。
给小鼠注射G-CSF导致骨髓和外周血中原始造血细胞上c-KIT表达降低。从G-CSF动员的小鼠分离的骨髓细胞外液含有将c-KIT切割成离散片段的丝氨酸蛋白酶。能够切割c-KIT的蛋白酶包括中性粒细胞弹性蛋白酶、组织蛋白酶G、蛋白酶-3和基质金属蛋白酶-9。
除了转录调控、胞吐作用和配体诱导的内化作用外，中性粒细胞和巨噬细胞蛋白酶对c-KIT的直接蛋白水解切割代表了一种调节造血细胞表面c-KIT表达水平的新途径，并且可能部分负责体内动员的造血祖细胞上c-KIT表达的下调。
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