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PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells.

ABSTRACT: OBJECTIVE:Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells. Approach and Results: In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT. CONCLUSIONS:These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.

SUBMITTER: Panicker SR

PROVIDER: S-EPMC7316612 | 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.

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PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells.

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