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 1

Flow induces eNOS phosphorylation at serines 1177 and 1179.

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Flow induces eNOS phosphorylation at serines 1177 and 1179. (A) eNOS ami...
(A) eNOS amino acid sequence in different species. (B) Phosphorylation of eNOS serine residues of BAECs exposed to laminar flow (15 dynes/cm2) determined by LC-MS/MS. Shown are the fold changes of the corrected ratios of flow versus static conditions (n = 3). (C and D) BAECs (C) and HUAECs (D) were exposed to laminar flow (15 dynes/cm2), and total and p-eNOS levels were determined by immunoblotting. Graphs show the densitometric evaluation (n = 3 independent experiments). (E) HEK293 cells expressing the indicated mutants of human eNOS were lysed and analyzed by immunoblotting using phosphosite-specific antibodies. (F and G) WT or mutant human eNOS was expressed by lentiviral transduction after siRNA-mediated eNOS knockdown in HUAECs, and the NOx concentration in the cell culture medium was determined in the absence (F and G) or presence (G) of flow. Expression of eNOS was analyzed by immunoblotting (F and G, lower panels). n = 3 independent experiments. (H) eNOS–/– mice were infected with AAV2-QuadYF virus, which transduces WT or mutant eNOS or EGFP. Seven days later, carotid arteries were isolated, and flow-induced vasorelaxation was analyzed. The immunoblot demonstrates equal expression levels of different eNOS mutants. (I) HUAECs transfected with control siRNA or siRNAs directed against AKT1 were exposed to laminar flow (15 dynes/cm2). Total AKT and total and p-eNOS levels were determined by immunoblotting. Graphs show the densitometric evaluation of blots (n = 3 independent experiments). 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 (C, D, G, and H; P values in H describe the difference compared with eNOS WT) and 1-way ANOVA, with Tukey’s post hoc test (F and I). P values in 1c and d describe difference compared with time point zero.