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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.

SUBMITTER: Jin YJ

PROVIDER: S-EPMC8553558 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsHemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKC?)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair.ResultsBidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1?±?3 dyne·cm-2 at 1?Hz) upregulated VEGFR-dependent PKC? expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23?±?8 dyne·cm-2 at 1?Hz) in human aortic endothelial cells (p?<?0.05, n?=?3). PSS and OSS further upregulated PKC?-dependent PFKFB3 expression for glycolysis (p?<?0.05, n?=?4). Constitutively active PKC? increased, whereas dominant-negative PKC? reduced both basal and maximal extracellular acidification rates for glycolytic flux (p?<?0.01, n?=?4). Metabolomic analysis demonstrated an increase in PKC?-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p?<?0.05 vs. control, n?=?6). In a New Zealand White rabbit model, both PKC? and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p?<?0.01, n?=?20), which was restored with PKC? messenger RNA (mRNA) rescue (p?<?0.05, n?=?5). As a corollary, siPKC? inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKC?-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair. Antioxid. Redox Signal. 28, 31-43.

Project description:We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that endothelial integrity is essential to the maintenance of erectile function.

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

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