Sugahara H, Kanakura Y, Furitsu T, Ishihara K, Oritani K, Ikeda H, Kitayama H, Ishikawa J, Hashimoto K, Kanayama Y
Second Department of Internal Medicine, Osaka University Medical School, Japan.
J Exp Med. 1994 Jun 1;179(6):1757-66. doi: 10.1084/jem.179.6.1757.
Extracellular matrix (ECM) molecules such as fibronectin (FN), collagens, and laminin have important roles in hematopoiesis. However, little is known about the precise mechanisms by which ECM molecules regulate proliferation of human hematopoietic progenitor cells. In this study, we have investigated the effects of ECM molecules, particularly of FN, on the proliferation of a myeloid leukemia cell line, M07E, which proliferates in response to either human granulocyte/macrophage colony-stimulating factor (GM-CSF) or stem cell factor (SCF). The [3H]thymidine incorporation and cell enumeration assays showed that FN strikingly inhibited GM-CSF- or SCF-induced proliferation of M07E cells in a dose-dependent manner, whereas little or no inhibition was induced by collagen types I and IV. The growth suppression of M07E cells was not due to the inhibitory effect of FN on ligand binding or very early events in the signal transduction pathways from the GM-CSF or SCF receptors. DNA content analysis using flow cytometry after staining with propidium iodide revealed that the treatment of M07E cells with FN did not block the entry of the cells into the cell cycle after stimulation with GM-CSF or SCF, whereas the treatment resulted in the appearance of subdiploid peak. Furthermore, FN was found to induce oligonucleosomal DNA fragmentation and chromatin condensation in the cells even in the presence of GM-CSF or SCF, suggesting the involvement of programmed cell death (apoptosis) in the FN-induced growth suppression. The growth suppression or apoptosis induced by FN was rescued by the addition of either anti-FN antibody, anti-very late antigen 5 monoclonal antibody (anti-VLA5 mAb), or GRGDSP peptide, but not by that of anti-VLA4 mAb or GRGESP peptide, suggesting that the FN effects on M07E cells were mediated through VLA5. In addition, the FN-induced apoptosis was detectable in VLA5-positive human hematopoietic cell lines other than M07E cells, but not in any of the VLA5-negative cell lines. These results suggest that FN is capable of inducing apoptosis via its interaction with VLA5, and also raise the possibility that the FN-VLA5 interaction may contribute, at least in part, to negative regulation of hematopoiesis.
细胞外基质（ECM）分子，如纤连蛋白（FN）、胶原蛋白和层粘连蛋白，在造血过程中发挥着重要作用。然而，关于ECM分子调节人类造血祖细胞增殖的精确机制，人们知之甚少。在本研究中，我们研究了ECM分子，特别是FN，对髓系白血病细胞系M07E增殖的影响，该细胞系可响应人类粒细胞/巨噬细胞集落刺激因子（GM-CSF）或干细胞因子（SCF）而增殖。[3H]胸苷掺入和细胞计数分析表明，FN以剂量依赖的方式显著抑制GM-CSF或SCF诱导的M07E细胞增殖，而I型和IV型胶原蛋白几乎没有或没有诱导抑制作用。M07E细胞的生长抑制并非由于FN对配体结合的抑制作用或GM-CSF或SCF受体信号转导途径中非常早期事件的抑制作用。用碘化丙啶染色后通过流式细胞术进行的DNA含量分析表明，用FN处理M07E细胞不会阻止细胞在GM-CSF或SCF刺激后进入细胞周期，而该处理导致亚二倍体峰的出现。此外，即使在存在GM-CSF或SCF的情况下，也发现FN会诱导细胞中的寡核小体DNA片段化和染色质浓缩，这表明程序性细胞死亡（凋亡）参与了FN诱导的生长抑制。通过添加抗FN抗体、抗极晚期抗原5单克隆抗体（抗VLA5 mAb）或GRGDSP肽可挽救FN诱导的生长抑制或凋亡，但抗VLA4 mAb或GRGESP肽则不能，这表明FN对M07E细胞的作用是通过VLA5介导的。此外，在除M07E细胞外的VLA5阳性人类造血细胞系中可检测到FN诱导的凋亡，但在任何VLA5阴性细胞系中均未检测到。这些结果表明，FN能够通过与VLA5相互作用诱导凋亡，也增加了FN-VLA5相互作用可能至少部分有助于造血负调控的可能性。
