c-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow
Bochuan Li, … , Yi Zhu, Ding Ai
Bochuan Li, … , Yi Zhu, Ding Ai
Published January 10, 2019
Citation Information: J Clin Invest. 2019;129(3):1167-1179. https://doi.org/10.1172/JCI122440.
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c-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow

Abstract

Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin α5β1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin α5β1–blocking peptide (ATN161) in Apoe–/– mice. Activation of integrin α5β1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin α5β1 with ATN161 abolished the phosphorylation of YAP at Y357 induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin α5β1–induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and YAPY357 was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques versus normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of YAPY357 and the development of atherosclerosis in Apoe–/– mice. The c-Abl/YAPY357 pathway serves as a mechanism for the activation of integrin α5β1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.

Authors

Bochuan Li, Jinlong He, Huizhen Lv, Yajin Liu, Xue Lv, Chenghu Zhang, Yi Zhu, Ding Ai

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Figure 1

Oscillatory shear stress (OSS) increased Yes-associated protein (YAP) nuclear translocation via integrin α5β1.

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Oscillatory shear stress (OSS) increased Yes-associated protein (YAP) nu...
(A and B) Human umbilical vein endothelial cells (HUVECs) were exposed to OSS (0.5 ± 4 dyn/cm2) for the indicated times. Cells with static treatment (ST) were a control. After treatment, cells underwent immunofluorescence staining with YAP (green), phalloidin (red), and DAPI (blue). Percentage of nuclear YAP was quantified. Scale bar: 20 μm. Data are mean ± SEM, *P < 0.05 versus ST (1-way ANOVA with Bonferroni multiple comparison post hoc test), n = 5. (C and D) HUVECs were exposed to OSS for 6 hours. Western blot analysis of YAP subcellular distribution in nucleus and cytoplasm. Data are mean ± SEM, *P < 0.05 (Student’s t test), n = 6. (E–H) HUVECs were seeded in dishes coated with collagen (Col) or fibronectin (FN) (10 μg/ml) for 6 hours. (E) Immunofluorescence staining for YAP (red), Act-α5 (green), and DAPI (blue). Scale bars: 20 μm. (F) Ratio of nuclear to cytoplasmic fraction of YAP and fluorescent intensity of Act-α5 in panel E. Data are mean ± SEM, *P < 0.05 (Student’s t test), n = 8. (G) Western blot analysis of nuclear and cytoplasmic protein to detect YAP expression. (H) Quantification of t-YAP in panel G. Data are mean ± SEM, *P < 0.05 (Student’s t test), n = 3. (I) HUVECs were exposed to OSS or ST for 6 hours with or without pretreatment with ATN161 (10 μmol/l). Immunofluorescence staining for YAP (red) and DAPI (blue). Scale bars: 20 μm. (J) Ratio of nuclear to cytoplasmic fraction of YAP in panel I. Data are mean ± SEM, *P < 0.05 (2-way ANOVA with Bonferroni multiple comparison post hoc test), n = 6. (K) En face immunostaining of YAP (red), CD31 (green), and DAPI (blue) in the inner and outer curvature of the aortic arch (AA) and thoracic aorta (TA) from WT (α5+/+) and Itga5+/– (α5+/− ) mice (8 weeks old, n = 5). Scale bars: 20 μm.