Department of Anatomy and Cell Biology, University Medical School Göttingen, Göttingen, Germany.
Sci Rep. 2019 Mar 18;9(1):4739. doi: 10.1038/s41598-019-41299-7.
Development of lymphatics takes place during embryogenesis, wound healing, inflammation, and cancer. We previously showed that Wnt5a is an essential regulator of lymphatic development in the dermis of mice, however, the mechanisms of action remained unclear. Here, whole-mount immunostaining shows that embryonic day (ED) 18.5 Wnt5a-null mice possess non-functional, cyst-like and often blood-filled lymphatics, in contrast to slender, interconnected lymphatic networks of Wnt5a and wild-type (wt) mice. We then compared lymphatic endothelial cell (LEC) proliferation during ED 12.5, 14.5, 16.5 and 18.5 between Wnt5a, Wnt5a and wt-mice. We did not observe any differences, clearly showing that Wnt5a acts independently of proliferation. Transmission electron microscopy revealed multiple defects of LECs in Wnt5a-null mice, such as malformed inter-endothelial junctions, ruffled cell membrane, intra-luminal bulging of nuclei and cytoplasmic processes. Application of WNT5A protein to ex vivo cultures of dorsal thoracic dermis from ED 15.5 Wnt5a-null mice induced flow-independent development of slender, elongated lymphatic networks after 2 days, in contrast to controls showing an immature lymphatic plexus. Reversely, the application of the WNT-secretion inhibitor LGK974 on ED 15.5 wt-mouse dermis significantly prevented lymphatic network elongation. Correspondingly, tube formation assays with human dermal LECs in vitro revealed increased tube length after WNT5A application. To study the intracellular signaling of WNT5A we used LEC scratch assays. Thereby, inhibition of autocrine WNTs suppressed horizontal migration, whereas application of WNT5A to inhibitor-treated LECs promoted migration. Inhibition of the RHO-GTPase RAC, or the c-Jun N-terminal kinase JNK significantly reduced migration, whereas inhibitors of the protein kinase ROCK did not. WNT5A induced transient phosphorylation of JNK in LECs, which could be inhibited by RAC- and JNK-inhibitors. Our data show that WNT5A induces formation of elongated lymphatic networks through proliferation-independent WNT-signaling via RAC and JNK. Non-canonical WNT-signaling is a major mechanism of extension lymphangiogenesis, and also controls differentiation of lymphatics.
淋巴管的发育发生在胚胎发生、创伤愈合、炎症和癌症过程中。我们之前表明，Wnt5a 是小鼠真皮中淋巴管发育的必需调节剂，然而，其作用机制尚不清楚。在这里，整体免疫染色显示，与 Wnt5a 和野生型（wt）小鼠的细长、相互连接的淋巴管相比，胚胎期（ED）18.5 期 Wnt5a 缺失的小鼠具有无功能的、囊性的、常常充满血液的淋巴管。然后，我们比较了 Wnt5a、Wnt5a 和 wt- 小鼠在 ED12.5、14.5、16.5 和 18.5 期间淋巴管内皮细胞（LEC）的增殖。我们没有观察到任何差异，这清楚地表明 Wnt5a 独立于增殖而发挥作用。透射电子显微镜显示 Wnt5a 缺失的小鼠 LEC 存在多种缺陷，例如内皮细胞间连接异常、细胞膜起皱、核内和细胞质突起腔内膨出。将 WNT5A 蛋白应用于 ED15.5 期 Wnt5a 缺失的小鼠背部胸皮的离体培养物中，在 2 天后诱导出无流依赖性的细长、伸长的淋巴管网络，而对照显示出未成熟的淋巴管丛。相反，将 WNT 分泌抑制剂 LGK974 应用于 ED15.5wt- 小鼠的真皮中，显著阻止了淋巴管网络的伸长。相应地，在体外用人真皮 LEC 进行的管形成测定显示，应用 WNT5A 后管长度增加。为了研究 WNT5A 的细胞内信号，我们使用了 LEC 划痕测定。由此，自分泌 WNTs 的抑制抑制了横向迁移，而将 WNT5A 应用于抑制剂处理的 LEC 促进了迁移。RHO-GTPase RAC 的抑制或 c-Jun N 末端激酶 JNK 的抑制显著减少了迁移，而 ROCK 蛋白激酶抑制剂则没有。WNT5A 诱导 LEC 中 JNK 的瞬时磷酸化，该磷酸化可被 RAC 和 JNK 抑制剂抑制。我们的数据表明，WNT5A 通过非经典 WNT 信号转导通过 RAC 和 JNK 诱导细长的淋巴管网络形成，该信号转导不依赖于增殖。非经典 WNT 信号转导是淋巴管延伸发育的主要机制，也控制淋巴管的分化。
