Lysophosphatidic acid–mediated NF-κB activation promotes FOXC2 expression essential for lymphatic valve development
Daisuke Yasuda, Nana Sato, Keisuke Yanagida, Tomomi Hashidate-Yoshida, Tomohiro Shiiya, Hideo Shindou, Atsuki Taira, Takashi Ebihara, Takao Shimizu, Masanori Hirashima, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii
Daisuke Yasuda, Nana Sato, Keisuke Yanagida, Tomomi Hashidate-Yoshida, Tomohiro Shiiya, Hideo Shindou, Atsuki Taira, Takashi Ebihara, Takao Shimizu, Masanori Hirashima, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii
View: Text | PDF
Research Article Cell biology Development Vascular biology

Lysophosphatidic acid–mediated NF-κB activation promotes FOXC2 expression essential for lymphatic valve development

Abstract

The lymphatic system maintains tissue fluid balance, and FOXC2 mutations cause lymphoedema-distichiasis syndrome, which is characterized by lymphatic valve defects. Although oscillatory shear stress regulates FOXC2 expression, other extracellular regulators remain unclear. In this study, we identified LPA4 and LPA6, two Gα12/Gα13-coupled receptors for the bioactive lipid lysophosphatidic acid (LPA), as key regulators of FOXC2 expression and lymphatic valve development. Lymphatic endothelial cell–specific (LEC-specific) Lpa4 Lpa6–deficient mice exhibited impaired lymphatic valve formation and maintenance, which resembled phenotypes of LEC-specific Foxc2-deficient mice, including abnormal lymphatic vessel patterning. Mechanistically, lymphatic endothelial Lpa4/Lpa6 ablation reduced FOXC2 expression in vitro and in vivo. NF-κB was found to be essential for LPA-induced FOXC2 expression through the LPA4/LPA6-Gα12/Gα13-Rho kinase signaling axis. Accordingly, pharmacological inhibition of NF-κB and Rho kinase impaired lymphatic valve maintenance in mice. These results suggested that lymphatic endothelial LPA4 and LPA6 synergistically regulate FOXC2 expression through NF-κB activation and play an important role in lymphatic valve formation and maintenance. Our findings provide a molecular basis for lymphatic vessel development with a therapeutic potential for targeting lymphatic system–associated diseases.

Authors

Daisuke Yasuda, Nana Sato, Keisuke Yanagida, Tomomi Hashidate-Yoshida, Tomohiro Shiiya, Hideo Shindou, Atsuki Taira, Takashi Ebihara, Takao Shimizu, Masanori Hirashima, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii

×

Figure 2

Lymphatic endothelial LPA4/LPA6 are essential for lymphatic valve formation and maintenance.

Options: View larger image (or click on image) Download as PowerPoint
Lymphatic endothelial LPA4/LPA6 are essential for lymphatic valve format...
(A) Schematic diagram of Lpa4 and Lpa6 ablation in Lpa4 Lpa6iΔLEC mice and tamoxifen injection procedure for analysis at E15.5. (B) Gross morphology of control and Lpa4 Lpa6iΔLEC littermate embryos, displaying severe edema (arrowheads) and hemorrhage. Scale bar: 10 mm. (C and D) Ratios of edema (C) and hemorrhage (D) in control and Lpa4 Lpa6iΔLEC embryos, with the number of affected embryos and total numbers of embryos analyzed shown above each bar. (E) Representative hematoxylin-counterstained transverse sections of the jugular area in littermate embryos. LECs are immunostained for VEGFR3. Lymph sacs are remarkably enlarged in Lpa4 Lpa6iΔLEC embryos compared with controls. LS, lymph sac; V, vein; A, aorta. Scale bars: 200 μm. (F) Quantification of lymph sac area (μm2) (n = 6–9 embryos). (G) Schematic diagram of Lpa4 and Lpa6 ablation in Lpa4 Lpa6iΔLEC mice and tamoxifen injection procedure for analysis at E17.5. (H) Representative confocal images of dorsal skin lymphatic vascular networks, showcasing PROX1 immunostaining. White arrowheads indicate putative lymphatic valve–forming PROX1hi LEC clusters. Scale bars: 200 μm. (I and J) Quantification of vessel width (I) and the number of PROX1hi LEC clusters (J) in control and Lpa4 Lpa6iΔLEC embryos (n = 9 embryos). (K) Schematic diagram of Lpa4 and Lpa6 ablation in Lpa4 Lpa6iΔLEC mice and tamoxifen injection procedure for analysis at E18.5. (L) Representative confocal images of LYVE1+ lymphatic vessels covered with αSMA+ cells in the dorsal skin of control and Lpa4 Lpa6iΔLEC littermate embryos (n = 4 embryos). Areas in yellow boxes are magnified in the bottom panels. Scale bars: 200 μm. (M) Schematic diagram of Lpa4 and Lpa6 ablation in Lpa4 Lpa6iΔLEC mice and tamoxifen injection procedure for analysis at P7. (N) Representative confocal images of mesenteric lymphatic vessels in control and Lpa4 Lpa6iΔLEC littermates, showing PROX1 immunostaining. White arrowheads indicate lymphatic valves. Scale bars: 200 μm. (O) Quantification of lymphatic valve number (n = 11–12 mice). **P < 0.01, ****P < 0.0001; 2-tailed unpaired Student’s t test.