Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Cytokine. 2012 Oct;60(1):162-70. doi: 10.1016/j.cyto.2012.06.237. Epub 2012 Jul 17.
Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains.
纤连蛋白（FN）已知具有四个 DNA 结合域，尽管其生理意义尚不清楚。已经表明，与鼠巨噬细胞样细胞系相比，原代鼠腹膜巨噬细胞对富含 CpG 基序的质粒 DNA（pDNA）、Toll 样受体 9（TLR9）配体的反应性明显降低。本研究旨在探讨是否具有 DNA 结合域的 FN 参与了这一现象。FN 在原代巨噬细胞中的表达明显高于巨噬细胞样细胞系 RAW264.7，这表明丰富的 FN 可能抑制原代巨噬细胞的反应性。然而，电泳分析表明，在生理浓度的二价阳离子存在下，FN 不会与 pDNA 结合。令人惊讶的是，外源性添加 FN 以浓度依赖的方式显著降低了 CpG DNA 刺激后鼠巨噬细胞产生的肿瘤坏死因子-α（TNF-α），但 BSA、层粘连蛋白或胶原蛋白则没有。FN 不影响细胞对明显 pDNA 的摄取。此外，FN 降低了由 polyI:C（TLR3 配体）和咪喹莫特（TLR7 配体）诱导的 TNF-α产生，但不降低由 LPS（TLR4 配体）或非 CpG pDNA/阳离子脂质体复合物诱导的 TNF-α产生。共聚焦显微镜研究表明，在 RAW264.7 中，pDNA 与 FN 共定位于同一细胞内隔室，提示 FN 通过内体/溶酶体隔室抑制细胞因子信号转导。总之，本研究首次揭示了 FN 的一种新作用，即该糖蛋白通过 CpG-DNA/TLR9 相互作用调节巨噬细胞中的细胞因子信号转导，而无需通过其假定的 DNA 结合域直接与 DNA 结合。
