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 1

Mice deficient in lymphatic endothelial Lpa4 and Lpa6 exhibit severe edema, hemorrhage, and impaired lymphatic valve formation.

Options: View larger image (or click on image) Download as PowerPoint
Mice deficient in lymphatic endothelial Lpa4 and Lpa6 exhibit severe ede...
(A) LPA receptor mRNA expression in mouse LECs detected via qRT-PCR (n = 3 independent LEC preparations from WT mice). (B) Impaired survival of Lpa4 Lpa6ΔEC mice. Schematic diagram of Lpa4 and Lpa6 ablation in Lpa4 Lpa6ΔEC mice is shown on the left. The numbers of mice at P7 are indicated in the bars. Detailed data are shown in Supplemental Figure 5B. Note that Lpa4 is located on the X chromosome. (C and D) Growth retardation, severe edema (arrowheads), and hemorrhage observed in Lpa4 Lpa6ΔEC littermate embryos at E15.5 (C) and E18.5 (D). Scale bars: 10 mm. (E) H&E-stained transverse sections of littermate embryos at E18.5, showing severe edema in Lpa4 Lpa6ΔEC embryos (arrowheads). Scale bars: 10 mm. (F–H). Ratios of growth retardation (F), edema (G), and hemorrhage (H) in control and Lpa4Lpa6ΔEC embryos at E15.5 and E18.5. The numbers of affected embryos and total number of embryos analyzed are shown above each bar. (I) Representative confocal images of lymphatic vascular networks in the dorsal skin at E16.5, showing PROX1 immunostaining. White arrowheads indicate putative lymphatic valve-forming PROX1hi LEC clusters. Scale bars: 200 μm. (J and K) Quantification of vessel width (J) and number of PROX1hi LEC clusters (K) in control and Lpa4 Lpa6ΔEC embryos (n = 8–9 embryos). (L) Representative confocal images of LYVE1+ lymphatic vessels covered with αSMA+ cells in the dorsal skin of control and Lpa4 Lpa6ΔEC littermate embryos at E18.5 (n = 4–5 embryos). Areas in yellow boxes are magnified in the bottom panels. LECs display lower CD31 expression, while BECs express high CD31 levels. Scale bars: 200 μm. (M) Body weights of control and Lpa4 Lpa6ΔEC mice at P7 (n = 5–12 mice). (N) Representative confocal images of mesenteric lymphatic vessels in control and Lpa4 Lpa6ΔEC mice at P7, showing PROX1 immunostaining. White arrowheads indicate lymphatic valves. Scale bars: 200 μm. (O) Quantification of lymphatic valve numbers in control and Lpa4 Lpa6ΔEC mice at P7 (n = 5–11 mice). **P < 0.01, ***P < 0.001; 2-tailed unpaired Student’s t test.