Takahira H, Gotoh A, Ritchie A, Broxmeyer H E
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Blood. 1997 Mar 1;89(5):1574-84.
Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin beta 1 and combination of anti-alpha 4 with anti-alpha 5 antibodies, indicating alpha 4 beta 1 and alpha 5 beta 1 integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10(-7) mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10(-7) mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-alpha 4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK through activation of integrin ("inside-out" signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.
造血祖细胞与骨髓基质细胞外基质成分之间由整合素介导的相互作用在造血过程中至关重要。粘着斑激酶（pp125FAK）在通过整合素受体进行的信号转导中起核心作用。我们研究了人生长因子依赖性细胞系TF-1中的基质-整合素相互作用及后续信号传导。未受刺激的TF-1细胞与纤连蛋白包被的孔的粘附被抗整合素β1以及抗α4与抗α5抗体的组合所阻断，表明α4β1和α5β1整合素介导了粘附。干细胞因子（SLF）以剂量依赖性方式增加TF-1与纤连蛋白的粘附，而10^(-7) mol/L渥曼青霉素抑制SLF诱导的粘附。用抗磷酸酪氨酸抗体进行免疫沉淀和免疫印迹显示，在没有细胞因子的情况下，TF-1细胞与纤连蛋白的粘附导致了几种被鉴定为pp125FAK和桩蛋白的蛋白质的酪氨酸磷酸化。SLF诱导粘附的TF-1细胞铺展，并以剂量依赖性方式增强pp125FAK和桩蛋白的酪氨酸磷酸化。在未铺在纤连蛋白上的情况下用SLF处理不会诱导pp125FAK的酪氨酸磷酸化。10^(-7) mol/L的渥曼青霉素完全消除了SLF诱导的pp125FAK酪氨酸磷酸化增强，而c-kit自身磷酸化不受影响。这表明pp125FAK酪氨酸磷酸化的增加是通过渥曼青霉素敏感途径介导的，而不是通过对c-kit酪氨酸激酶的直接作用。用RGDS肽加抗α4抗体处理粘附的TF-1细胞也抑制了SLF诱导的pp125FAK酪氨酸磷酸化增强，而TF-1细胞未脱离。这些数据表明，SLF通过激活整合素（“由内向外”信号传导）以及随后的整合素占据来增强整合素-纤连蛋白依赖性的pp125FAK酪氨酸磷酸化。这在c-kit/SLF途径与整合素纤连蛋白信号传导之间建立了一种新的联系。
