Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
J Cell Biochem. 2012 Jan;113(1):80-92. doi: 10.1002/jcb.23330.
Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.
细菌感染后，脂多糖（LPS）会诱导单核细胞/巨噬细胞迁移到受感染区域，并产生促炎介质。我们研究了 LPS 刺激迁移的机制，发现 100ng/ml 的 LPS 可诱导巨噬样 J774 细胞快速伸长和皱襞。划痕实验表明，LPS 还激活了定向细胞运动，随后产生 TNF-α。LPS 的 CD14 和 TLR4 受体转位到极化细胞的前缘，在那里它们短暂地共定位，触发肌动蛋白丝和磷脂酰肌醇 4,5-二磷酸的局部积累。Triton X-100 细胞裂解物的分级分离表明，LPS 通过 50%诱导细胞骨架肌动蛋白丝的聚合，这与细胞迁移的峰值一致。该微丝群体的出现是以刺激前细胞骨架中组成质膜的短丝和占细胞肌动蛋白约 60%的肌动蛋白单体为代价的。与肌动蛋白聚合同时，LPS 刺激两个肌动蛋白调节蛋白的磷酸化，paxillin 在酪氨酸 118 上磷酸化 80%，N-WASP 在丝氨酸 484/485 上磷酸化 20%，这些事件发生在 NF-κB 激活之前。LPS 诱导的蛋白磷酸化和肌动蛋白细胞骨架的重组被 PP2 抑制，PP2 是一种影响 Src 家族酪氨酸激酶活性的药物。这些数据表明，paxillin 和 N-WASP 参与了 LPS 刺激细胞迁移的肌动蛋白细胞骨架的重组。细胞松弛素 D 引起的肌动蛋白组织紊乱并没有抑制 TNF-α 的产生，这表明 LPS 诱导的细胞迁移对于 TNF-α 的释放不是必需的。
