Yokota T, Oritani K, Mitsui H, Aoyama K, Ishikawa J, Sugahara H, Matsumura I, Tsai S, Tomiyama Y, Kanakura Y, Matsuzawa Y
Second Department of Internal Medicine and the Department of Hematology/Oncology, Osaka University Medical School, Osaka, Japan.
Blood. 1998 May 1;91(9):3263-72.
Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34(+)/VLA4(bright)/VLA5(dull) hematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor-deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34(+) cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34(+) cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.
纤连蛋白（FN）被认为通过与极晚期抗原4（VLA4）和VLA5结合在造血的各个方面发挥重要作用。然而，FN对造血干细胞的影响在很大程度上尚不清楚。为了确定FN是否对造血干细胞具有生长支持活性，在血清和生长因子缺乏的培养基中，对人CD34(+)/VLA4(明亮)/VLA5(暗淡)造血干细胞和小鼠干细胞因子（SCF）依赖性多能细胞系EML-C1进行有无FN的处理，然后在造血生长因子存在的情况下进行集落形成测定。用FN预处理CD34(+)细胞可导致粒细胞-巨噬细胞集落形成单位（CFU-GM）、红系爆式集落形成单位和混合红系-髓系集落形成单位的数量显著增加。此外，分别用SCF以及SCF和白细胞介素-3组合培养EML-C1细胞后形成的原始细胞集落形成单位和CFU-GM的数量，通过FN预处理得到了增加。FN增强的集落形成被CS1片段的添加完全消除，但GRGDSP肽的添加则没有，这表明FN-VLA4相互作用在FN的作用中起重要作用。此外，测试了由含RGDS的细胞结合结构域（CBD）、肝素结合结构域（HBD）和/或CS1部分组成的各种FN片段对CD34(+)细胞集落形成生长的影响。CBD-CS1和CBD-HBD-CS1片段诱导了集落形成的增加，但缺乏CBD或CS1结构域的其他片段则没有，这表明FN的CS1和CBD对于体外增强造血干/祖细胞的集落形成生长都是必需的。除了体外作用外，还发现将CBD-CS1片段体内给予小鼠可剂量依赖性地增加骨髓和脾脏中造血祖细胞的数量。因此，FN可能在体外和体内作为造血干/祖细胞的生长支持因子发挥作用。
