Shimizu M, Minakuchi K, Tsuda A, Hiroi T, Tanaka N, Koga J, Kiyono H
Department of Mucosal Immunology, Osaka University, Suita, Osaka, 565-0871, Japan.
Exp Cell Res. 2001 Jun 10;266(2):311-22. doi: 10.1006/excr.2001.5221.
The interaction of stem cell factor (SCF) and c-kit is considered to be an important signaling event for the homeostasis of the epithelial barrier function in the intestinal tract. This study was designed to investigate the role of the SCF and c-kit signaling pathway in adhesion of intestinal epithelial cells (IECs) to fibronectin (FN) using primary cells. Fetal murine IECs were prepared from the small intestine of mouse fetus. The mRNAs coding for SCF in mesenchymes and c-kit in IECs were detected by reverse transcription-PCR. The expression of FN receptor VLA-5 on IECs was examined by flow cytometry. A cell adhesion assay showed that the stimulation of IECs with SCF increased the number of cells adhering to FN. Experiments using specific antibody against SCF indicated that this increase in cell adhesion was SCF-dependent. On the other hand, SCF did not influence the expression of VLA-5 on IECs. The IEC adhesion to FN was inhibited by specific antibody against the FN receptor (VLA-5), as well as competitive Arg-Gly-Asp (RGD) peptide. When alteration of intracellular signal transduction induced by SCF was examined, it was found that SCF stimulated a tyrosine-specific c-kit autophosphorylation cascade of IECs. Further, preincubation of IECs with an optimal concentration of genistein resulted in the inhibition of SCF-induced c-kit phosphorylation and adhesion of IECs to FN. These results suggested that adhesion of immature IECs to FN is regulated by activation of RGD-dependent VLA-5 through the SCF and c-kit signal transduction pathway. SCF, which may be produced by mesenchymes locally, is an important regulatory factor for the adhesion of immature IECs to basement membrane matrix via VLA-5 and FN interaction. This cytokine-regulated interaction between VLA-5 and FN may play an important role in the development and wound repair of the intestinal tract.
干细胞因子（SCF）与c-kit的相互作用被认为是肠道上皮屏障功能稳态的重要信号事件。本研究旨在利用原代细胞研究SCF和c-kit信号通路在肠道上皮细胞（IECs）与纤连蛋白（FN）黏附中的作用。从小鼠胎儿小肠制备胎鼠IECs。通过逆转录聚合酶链反应检测间充质中编码SCF和IECs中c-kit的mRNA。通过流式细胞术检测IECs上FN受体VLA-5的表达。细胞黏附试验表明，用SCF刺激IECs可增加黏附于FN的细胞数量。使用抗SCF特异性抗体的实验表明，这种细胞黏附的增加依赖于SCF。另一方面，SCF不影响IECs上VLA-5的表达。抗FN受体（VLA-5）特异性抗体以及竞争性精氨酸-甘氨酸-天冬氨酸（RGD）肽可抑制IECs与FN的黏附。当检测SCF诱导的细胞内信号转导变化时，发现SCF刺激了IECs的酪氨酸特异性c-kit自磷酸化级联反应。此外，用最佳浓度的染料木黄酮预孵育IECs可抑制SCF诱导的c-kit磷酸化以及IECs与FN的黏附。这些结果表明，未成熟IECs与FN的黏附通过SCF和c-kit信号转导通路激活RGD依赖的VLA-5来调节。可能由间充质局部产生的SCF是未成熟IECs通过VLA-5与FN相互作用黏附于基底膜基质的重要调节因子。这种细胞因子调节的VLA-5与FN之间的相互作用可能在肠道发育和伤口修复中起重要作用。
