Lysophosphatidic acid–induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4
Daisuke Yasuda, … , Satoru Takahashi, Satoshi Ishii
Daisuke Yasuda, … , Satoru Takahashi, Satoshi Ishii
Published October 1, 2019; First published July 23, 2019
Citation Information: J Clin Invest. 2019;129(10):4332-4349. https://doi.org/10.1172/JCI121955.
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Research Article Angiogenesis

Lysophosphatidic acid–induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4

Abstract

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein–coupled receptors (GPCRs), LPA1–LPA6. Previous studies have demonstrated that LPA–Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double-knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell–specific (EC-specific) Lpa4;Lpa6-DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6–mediated Gα12/Gα13–Rho–ROCK signaling in ECs. YAP/TAZ knockdown increased endothelial expression of the Notch ligand delta-like ligand 4 (DLL4) that was mediated by β-catenin and Notch intracellular domain (NICD). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Notably, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6-DKO mice. Overall, these results suggest that the Gα12/Gα13–coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ–mediated developmental angiogenesis. Our findings provide insight into the biology of GPCR-activated YAP/TAZ.

Authors

Daisuke Yasuda, Daiki Kobayashi, Noriyuki Akahoshi, Takayo Ohto-Nakanishi, Kazuaki Yoshioka, Yoh Takuwa, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii

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Figure 3

LPA4 and LPA6 in HUVECs mediate LPA-induced Gα12/Gα13 activation.

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LPA4 and LPA6 in HUVECs mediate LPA-induced Gα12/Gα13 activation. (A) Ex...
(A) Expression of mRNA for LPA receptors was detected by RT-qPCR. (B) SRF-RE-Luc reporter assay was performed to detect Gα12/Gα13–Rho activation. LPA (10 μM, 6 hours) increased the reporter activity, which was attenuated by the ROCK inhibitor Y27632 (10 μM, 10 minutes pretreatment) but not by the LPA1/LPA3 antagonist Ki16425 (10 μM, 10 minutes pretreatment). (C) LPA-induced SRF-RE-Luc activity was attenuated by LPA4/LPA6 siRNAs (12.5 nM each, 48 hours pretreatment). (D) Intracellular calcium influx assay was performed to detect Gαq activation. A representative trace of changes in intracellular Ca2+ concentration ([Ca2+]i) is shown on the right. HUVECs were unresponsive to LPA (10 μM). Adenosine 5′-triphosphate (ATP; 10 μM) was used as a positive control, because ATP evokes calcium influx via P2Y receptors that predominantly couple with Gαq. (E) cAMP assay was performed to detect Gαs activation. No significant increase in cAMP level was observed in HUVECs treated with LPA (10 μM). Forskolin (20 μM) was used as a positive control. (F) cAMP assay was performed to detect Gαi activation. Inhibitory effect of LPA (10 μM) on cAMP accumulation stimulated by forskolin (20 μM) was not observed in HUVECs. Sphingosine-1-phosphate (S1P; 100 nM) was used as a positive control. Data are mean ± SEM of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Tukey’s multiple-comparisons test.