Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
Department of Ear, Nose and Throat Diseases, University Medicine Greifswald, Greifswald, Germany.
Sci Rep. 2017 Aug 30;7(1):9916. doi: 10.1038/s41598-017-10116-4.
Glomerular hypertension causes glomerulosclerosis via the loss of podocytes, which are challenged by increased mechanical load. We have demonstrated that podocytes are mechanosensitive. However, the response of podocytes to mechanical stretching remains incompletely understood. Here we demonstrate that the actin-bundling protein fascin-1 plays an important role in podocytes that are exposed to mechanical stress. Immunofluorescence staining revealed colocalization of fascin-1 and nephrin in mouse kidney sections. In cultured mouse podocytes fascin-1 was localized along actin fibers and filopodia in stretched and unstretched podocytes. The mRNA and protein levels of fascin-1 were not affected by mechanical stress. By Western blot and 2D-gelelectrophoresis we observed that phospho-fascin-1 was significantly downregulated after mechanical stretching. It is known that phosphorylation at serine 39 (S39) regulates the bundling activity of fascin-1, e.g. required for filopodia formation. Podocytes expressing wild type GFP-fascin-1 and non-phosphorylatable GFP-fascin-1-S39A showed marked filopodia formation, being absent in podocytes expressing phosphomimetic GFP-fascin-1-S39D. Finally, the immunofluorescence signal of phosphorylated fascin-1 was strongly reduced in glomeruli of patients with diabetic nephropathy compared to healthy controls. In summary, mechanical stress dephosphorylates fascin-1 in podocytes in vitro and in vivo thereby fascin-1 may play an important role in the adaptation of podocytes to mechanical forces.
肾小球高血压通过 podocyte 的丢失导致肾小球硬化，而 podocyte 受到机械负荷增加的挑战。我们已经证明 podocyte 是机械敏感的。然而，podocyte 对机械拉伸的反应仍不完全清楚。在这里，我们证明了肌动蛋白束蛋白 fascin-1 在暴露于机械应激的 podocyte 中发挥重要作用。免疫荧光染色显示 fascin-1 和 nephrin 在小鼠肾切片中存在共定位。在培养的小鼠足细胞中，fascin-1 定位于拉伸和未拉伸足细胞中的肌动蛋白纤维和丝状伪足。fascin-1 的 mRNA 和蛋白水平不受机械应激的影响。通过 Western blot 和 2D 凝胶电泳，我们观察到机械拉伸后磷酸化 fascin-1 显著下调。已知丝氨酸 39 位（S39）的磷酸化调节 fascin-1 的束状活性，例如丝状伪足形成所必需的。表达野生型 GFP-fascin-1 和非磷酸化 GFP-fascin-1-S39A 的足细胞显示出明显的丝状伪足形成，而表达磷酸模拟 GFP-fascin-1-S39D 的足细胞则没有。最后，与健康对照组相比，糖尿病肾病患者肾小球中磷酸化 fascin-1 的免疫荧光信号明显减少。总之，机械应激在体外和体内使足细胞中的 fascin-1 去磷酸化，因此 fascin-1 可能在足细胞对机械力的适应中发挥重要作用。
