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
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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

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Figure 7

LPA4/LPA6 signaling activates NF-κB through ROCK in LECs.

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LPA4/LPA6 signaling activates NF-κB through ROCK in LECs. (A) Increased ...
(A) Increased NF-κB reporter activity in response to LPA (10 μM, 6 hours) was attenuated by Lpa4/Lpa6 deletion in serum-starved mouse lung LECs. Increase in the NF-κB reporter activity by TNF-α (50 ng/mL, 6 hours, positive control) remained unaffected by Lpa4/Lpa6 deletion. Data are presented as mean ± SEM of triplicates. *P < 0.05, **P < 0.01, ****P < 0.0001; 1-way ANOVA followed by Tukey’s multiple-comparison test. (B) Increased NF-κB reporter activity in response to alkyl-OMPT (10 μM, 6 hours) was attenuated by Y27632 (10 μM, 1-hour pretreatment), Bay 11-7082 (5 μM, 1-hour pretreatment), CAPE (30 μM, 1-hour pretreatment), and SC75741 (10 μM, 1-hour pretreatment) in serum-starved HMVECs-dNeo. Data are presented as mean ± SEM of triplicates. *P < 0.05, ***P < 0.001; 1-way ANOVA followed by Dunnett’s test. (C) Phosphorylation of IκBα and RelA induced by alkyl-OMPT (10 μM, 30 minutes) was blocked by Y27632 (10 μM, 1-hour pretreatment) in serum-starved HMVECs-dNeo. Immunoblotting was performed using phosphorylated (P) and total (T) primary antibodies. TNF-α (50 ng/mL, 30 minutes) was used as a positive control. Unprocessed Western blot scans are shown in Supplemental Figure 27. (D and E) Nuclear translocation of RelA in response to alkyl-OMPT (10 μM, 1 hour) was blocked by Y27632 (10 μM, 1-hour pretreatment) in serum-starved HMVECs-dNeo. TNF-α (50 ng/mL, 1 hour) was used as a positive control. Representative confocal images (D) and corresponding quantification of RelA intracellular localization (E) (n = 76–101 cells). Scale bars: 100 μm.