Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation
Young-June Jin, … , Nina Wettschureck, Stefan Offermanns
Young-June Jin, … , Nina Wettschureck, Stefan Offermanns
Published September 9, 2021
Citation Information: J Clin Invest. 2021;131(21):e145734. https://doi.org/10.1172/JCI145734.
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Research Article Cell biology Vascular biology

Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation

Abstract

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

Authors

Young-June Jin, Ramesh Chennupati, Rui Li, Guozheng Liang, ShengPeng Wang, András Iring, Johannes Graumann, Nina Wettschureck, Stefan Offermanns

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Figure 7

Endothelial PKN2 deficiency results in loss of flow-induced vasodilation and arterial hypertension.

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Endothelial PKN2 deficiency results in loss of flow-induced vasodilation...
(A) Immunoblot analysis of PKN1 and PKN2 expression in mouse lung endothelial cells (MLECs) from 8-week-old WT and induced EC-Pkn2–KO mice. (B) Effect of acetylcholine (ACh) on the tone of mesenteric artery stripes from WT (n = 7) and EC-Pkn2–KO animals (n = 7) was determined after precontraction with 10 μM phenylephrine. (C) Mesenteric arteries isolated from tamoxifen-treated WT or EC-Pkn2–KO mice were precontracted with 100 nM of the thromboxane A2 analog U46619 and were then exposed to stepwise increases in perfusion flow. Flow-induced vasorelaxation is shown as a percentage of the passive vessel diameter (n = 7 WT and EC-Pkn2–KO mice). (D) Blood pressure in WT (n = 12), EC-Pkn1–KO mice (n = 8), and EC-Pkn2–KO (n = 12) animals before, during, and after tamoxifen treatment (induction). Average blood pressure 3 days before treatment was set at 100 %. Graphs show systolic and diastolic arterial blood pressure 4 days before tamoxifen treatment and in the second week after treatment. (E) eNOS phosphorylation at S1176 and S1178 in lysates from mesenteric arteries prepared from tamoxifen-treated WT and EC-Pkn2–KO mice (n = 3 animals). (F) Plasma nitrate and NOx levels in WT (n = 7) and EC-Pkn2–KO mice (n = 7) 10 days after treatment. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by 2-way ANOVA with Bonferroni’s post hoc test (B and C) and paired, 2-tailed Student’s t test (D) or unpaired, 2-tailed Student’s t test (F).