Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine

2001 ◽  
Vol 281 (1) ◽  
pp. H266-H274 ◽  
Author(s):  
Yoshiyuki Rikitake ◽  
Seinosuke Kawashima ◽  
Tomosaburo Takahashi ◽  
Tomomi Ueyama ◽  
Satoshi Ishido ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Phorbol Esters ◽  
Vascular Endothelial Cells ◽  
Biological Effects ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Vascular Endothelial ◽  
Acetoxymethyl Ester

Lysophosphatidylcholine (LPC), a component of oxidized low-density lipoprotein, exerts various biological effects on vascular endothelial cells. However, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipitation and Western blotting assays. Treatment of cells with LPC promoted a rapid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phosphorylation was inhibited by calcium chelators, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phosphorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine phosphorylation, and Grb2 binding to Shc and induced Ras activation. These results provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LPC-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Shc/Grb2 signaling.

scholarly journals Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells

2003 ◽  
Vol 14 (9) ◽  
pp. 3553-3564 ◽  
Author(s):  
Naoko Kogata ◽  
Michitaka Masuda ◽  
Yuji Kamioka ◽  
Akiko Yamagishi ◽  
Akira Endo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecule ◽  
Intracellular Signaling ◽  
Vascular Endothelial Cells ◽  
Expression Cloning ◽  
Vascular Endothelial ◽  
Cell Contacts ◽  
Cell Cell

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

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

2020 ◽  
Vol 40 (7) ◽  
pp. 1748-1762
Author(s):  
Sumith R. Panicker ◽  
Indranil Biswas ◽  
Hemant Giri ◽  
Xiaofeng Cai ◽  
Alireza R. Rezaie
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Signaling ◽  
Vascular Endothelial Cells ◽  
Dominant Negative ◽  
Vascular Endothelial ◽  
Kinase C ◽  
Pkc Protein Kinase C ◽  
Anti Inflammatory

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.

scholarly journals 17β-Estradiol Increases APE1/Ref-1 Secretion in Vascular Endothelial Cells and Ovariectomized Mice: Involvement of Calcium-Dependent Exosome Pathway

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 1040
Author(s):  
Yu-Ran Lee ◽  
Hee-Kyoung Joo ◽  
Eun-Ok Lee ◽  
Sungmin Kim ◽  
Hao Jin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Vascular Inflammation ◽  
Vascular Endothelial ◽  
Endogenous Hormones ◽  
Imaging Data ◽  
Acetoxymethyl Ester ◽  
Multifunctional Protein ◽  
Ovariectomized Mice ◽  
17Β Estradiol

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted, and recently suggested as new biomarker for vascular inflammation. However, the endogenous hormones for APE1/Ref-1 secretion and its underlying mechanisms are not defined. Here, the effect of twelve endogenous hormones on APE1/Ref-1 secretion was screened in cultured vascular endothelial cells. The endogenous hormones that significantly increased APE1/Ref-1 secretion was 17β-estradiol (E2), 5?-dihydrotestosterone, progesterone, insulin, and insulin-like growth factor. The most potent hormone inducing APE1/Ref-1 secretion was E2, which in cultured endothelial cells, E2 for 24 h increased APE1/Ref-1 secretion level of 4.56 ± 1.16 ng/mL, compared to a basal secretion level of 0.09 ± 0.02 ng/mL. Among the estrogens, only E2 increased APE1/Ref-1 secretion, not estrone and estriol. Blood APE1/Ref-1 concentrations decreased in ovariectomized (OVX) mice but were significantly increased by the replacement of E2 (0.39 ± 0.09 ng/mL for OVX vs. 4.67 ± 0.53 ng/mL for OVX + E2). E2-induced APE1/Ref-1secretion was remarkably suppressed by the estrogen receptor (ER) blocker fulvestrant and intracellular Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), suggesting E2-induced APE1/Ref-1 secretion was dependent on ER and intracellular calcium. E2-induced APE1/Ref-1 secretion was significantly inhibited by exosome inhibitor GW4869. Furthermore, APE1/Ref-1 level in CD63-positive exosome were increased by E2. Finally, fluorescence imaging data showed that APE1/Ref-1 co-localized with CD63-labled exosome in the cytoplasm of cells upon E2 treatment. Taken together, E2 was the most potent hormone for APE1/Ref-1 secretion, which appeared to occur through exosomes that were dependent on ER and intracellular Ca2+. Furthermore, hormonal effects should be considered when analyzing biomarkers for vascular inflammation.

scholarly journals Gi-mediated Cas Tyrosine Phosphorylation in Vascular Endothelial Cells Stimulated with Sphingosine 1-Phosphate

2000 ◽  
Vol 276 (7) ◽  
pp. 5274-5280 ◽  
Author(s):  
Tsukasa Ohmori ◽  
Yutaka Yatomi ◽  
Hirotaka Okamoto ◽  
Yoshie Miura ◽  
Ge Rile ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Sphingosine 1 Phosphate

scholarly journals Mechanism of temporal gradients in shear-induced ERK1/2 activation and proliferation in endothelial cells

2001 ◽  
Vol 281 (1) ◽  
pp. H22-H29 ◽  
Author(s):  
Xuping Bao ◽  
Chuanyi Lu ◽  
John A. Frangos
Keyword(s):  
Endothelial Cells ◽  
Intracellular Signaling ◽  
Proliferative Response ◽  
Vascular Endothelial Cells ◽  
Inhibitory Effect ◽  
Steady Shear ◽  
Endothelial Cell Proliferation ◽  
Vascular Endothelial ◽  
Extracellular Signal ◽  
Intracellular Signaling Cascade

The aim of the current study was to investigate the intracellular signaling cascade that leads to temporal gradients in shear (TGS)-induced endothelial cell proliferation, with a focus on the involvement of extracellular signal-regulated kinases 1 and 2 (ERK1/2). With the use of well-defined pulsatile, impulse, step, and ramp laminar flow profiles, we found that TGS (impulse flow and pulsatile flow) induced an enhanced and sustained (>30 min) phosphorylation of ERK1/2 relative to step flow (which contains a step increase in shear followed by steady shear), whereas steady shear (ramp flow) alone downregulated activated ERK1/2. Nitric oxide (NO) was found to mediate both the stimulatory effect of TGS and the inhibitory effect of steady shear on endothelial ERK1/2 phosphorylation. Reactive oxygen species (ROS) were also demonstrated to be associated with TGS-induced ERK1/2 phosphorylation. Both Gq/11 and Gi3 were necessary for the activation of ERK1/2 by TGS. Finally, the TGS-induced endothelial proliferative response was abolished by ERK1/2 inhibition. Our study demonstrated the essential role of G proteins, NO, and ROS in TGS-dependent ERK1/2 activation and proliferative response in vascular endothelial cells.

scholarly journals Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures

2006 ◽  
Vol 173 (4) ◽  
pp. 627-637 ◽  
Author(s):  
Nicolas Doulet ◽  
Emmanuel Donnadieu ◽  
Marie-Pierre Laran-Chich ◽  
Florence Niedergang ◽  
Xavier Nassif ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neisseria Meningitidis ◽  
Vascular Endothelial Cells ◽  
Bacterial Pathogen ◽  
Cell Interaction ◽  
Dominant Negative ◽  
Vascular Endothelial ◽  
Endothelial Monolayers ◽  
Membrane Protrusions ◽  
Novel Strategy

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte–endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH2-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte–endothelial cell interaction.

scholarly journals Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2805-2814 ◽  
Author(s):  
RR Schumann ◽  
D Pfeil ◽  
N Lamping ◽  
C Kirschning ◽  
G Scherzinger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitors ◽  
Vascular Endothelial Cells ◽  
Mitogen Activated Protein Kinase ◽  
Vascular Endothelial ◽  
Protein Tyrosine Phosphorylation ◽  
Soluble Cd14 ◽  
Protein Tyrosine ◽  
Mitogen Activated Protein

Human vascular endothelial cells (HUVECs), which do not display the lipopolysaccharide (LPS) receptor CD14, were examined for protein tyrosine phosphorylation after LPS stimulation in the presence and absence of soluble CD14 (sCD14). By phosphotyrosine Western blotting and immunocomplex kinase assays we show that LPS was capable of inducing in these cells rapid protein tyrosine phosphorylation and kinase activation of two members of the mitogen-activated protein kinase (MAPK) family erk-1 and the newly discovered p38, requiring the presence of sCD14. LPS-induced tyrosine phosphorylation of MAPK was associated with increased transcript- and surface protein expression of intracellular adhesion molecule-1 by HUVECs. MAPK phosphorylation and activation was induced by LPS in concentrations as little as 30 ng/mL and as early as 15 minutes after stimulation. Furthermore, tyrosine kinase inhibitors such as Genistein partially inhibited this effect. These results show that LPS triggers similar signaling events in both CD14+ myelo-monocytic cells and cells lacking the putative LPS-receptor CD14, suggesting the presence of a common, yet unidentified element in LPS-signaling in both cell types.

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