Non-canonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling

Abstract

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.

Regeneration of spinal motor axons: Negative regulation by Celsr2 implicating Cdc42/Rac1 and JNK/c-Jun signaling

“…Previous studies outlined a role of JNK in PCP and noncanonical Wnt signaling, implicating, among others, the Wnt5a ligand and frizzled receptor Fzd6, a close relative of Fzd3 (Cheyette et al, 2002;Lutze et al, 2019;Yamanaka et al, 2002;Yang & Mlodzik, 2015). Our data are a first indication that Celsr2 acts via the same pathway.…”

WNT Signaling in Neuroblastoma

“…PCP signaling is initiated by binding of WNT5A or WNT11 to ROR1, ROR2 or RYK receptor tyrosine kinases. Wnt5a knock-out mice show severe craniofacial defects and malformation of dermal lymphatics [97] (reviewed in [10]). A milder phenotype is caused by the loss of Ror2, whereas Ror1/2 double knock-out mice recapitulate the phenotype of Wnt5a-deficient mice.…”

Development of A New Mouse Model for Intrahepatic Cholangiocellular Carcinoma: Accelerating Functions of Pecam-1

“…In murine ICC, we observed upregulation of the non-canonical WNT-ligand Wnt5a. The importance of Wnt5a for angiogenesis and lymphangiogenesis has clearly been shown [30,31], and in other tumor entities, Wnt5a has been characterized as a motility-promoting factor [32]. Such functions may be preserved in ICC, and, in sum, the features described above characterize our mouse model as highly similar to the human ICC [33].…”