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Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation.

ABSTRACT: Spleen tyrosine kinase (Syk), expressed in endothelial cells, has been implicated in migration and proliferation and in vasculogenesis. This study was conducted to determine the contribution of Syk and the underlying mechanism to the angiogenic effect of ANG II and VEGF. Angiogenesis was determined by tube formation from the endothelial cell line EA.hy926 (EA) and human umbilical vein endothelial cells (HUVECs) and microvessel sprouting in rat aortic rings. ANG II (10 nM), EGF (30 ng/ml), and VEGF (50 ng/ml) stimulated EA cells and HUVECs to form tubular networks and increased aortic sprouting; these effects were blocked by VEGF receptor-1 and Flt-1 antibody (Flt-1/Fc) but not by the VEGF receptor-2 (Flk-1) antagonist SU-1498. ANG II increased the phosphorylation of Flt-1 but not Flk-1, whereas VEGF increased the phosphorylation of both receptors in EA cells and HUVECs. VEGF expression elicited by ANG II was not altered by Flt-1/Fc or SU-1498. EGF stimulated tube formation from EA cells and HUVECs and Flt-1 phosphorylation and aortic sprouting, which were blocked by the EGF receptor antagonist AG-1478 and Flt-1/Fc but not by SU-1498. ANG II-, EGF-, and VEGF-induced tube formation and aortic sprouting were attenuated by the Syk inhibitor piceatannol and by Syk short hairpin interfering (sh)RNA and small interfering RNA, respectively. ANG II, EGF, and VEGF increased Syk phosphorylation, which was inhibited by piceatannol and Syk shRNA in EA cells and HUVECs. Neither piceatannol nor Syk shRNA altered ANG II-, EGF-, or VEGF-induced phosphorylation of Flt-1. These data suggest that ANG II stimulates angiogenesis via transactivation of the EGF receptor, which promotes the phosphorylation of Flt-1 and activation of Syk independent of VEGF expression.

SUBMITTER: Buharalioglu CK

PROVIDER: S-EPMC3191073 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation.

Buharalioglu Cuneyt K CK Song Chi Young CY Yaghini Fariborz A FA Ghafoor Hafiz U B HU Motiwala Mustafa M Adris Tusita T Estes Anne M AM Malik Kafait U KU

American journal of physiology. Heart and circulatory physiology 20110603 3

Spleen tyrosine kinase (Syk), expressed in endothelial cells, has been implicated in migration and proliferation and in vasculogenesis. This study was conducted to determine the contribution of Syk and the underlying mechanism to the angiogenic effect of ANG II and VEGF. Angiogenesis was determined by tube formation from the endothelial cell line EA.hy926 (EA) and human umbilical vein endothelial cells (HUVECs) and microvessel sprouting in rat aortic rings. ANG II (10 nM), EGF (30 ng/ml), and VE ...[more]

PMID: 21642504

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Project description:The aim of this study was to test whether c-Src tyrosine kinase mediates connexin-43 (Cx43) reduction and sudden cardiac death in a transgenic mouse model of cardiac-restricted overexpression of angiotensin-converting enzyme (ACE8/8 mice).Renin-angiotensin system activation is associated with an increased risk for arrhythmia and sudden cardiac death, but the mechanism is not well understood. The up-regulation of c-Src by angiotensin II may result in the reduction of Cx43, which impairs gap junction function and provides a substrate for arrhythmia.Wild-type and ACE8/8 mice with and without treatment with the c-Src inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) were studied. Telemetry monitoring, in vivo electrophysiologic studies, Western blot analyses for total and phosphorylated c-Src and Cx43, immunohistochemistry staining for Cx43, and functional assessment of Cx43 with fluorescent dye diffusion were performed.The majority of the arrhythmic deaths resulted from ventricular tachycardia degenerating to ventricular fibrillation (83%). Levels of total and phosphorylated c-Src were increased and Cx43 reduced in ACE8/8 mice. PP1 reduced total and phosphorylated c-Src levels, increased Cx43 level by 2.1-fold (p < 0.005), increased Cx43 at the gap junctions (immunostaining), improved gap junctional communication (dye spread), and reduced ventricular tachycardia inducibility and sudden cardiac death. The survival rate increased from 11% to 86% with 4 weeks of PP1 treatment (p < 0.005). Treatment with an inactive analog did not change survival or Cx43 levels.Renin-angiotensin system activation is associated with c-Src up-regulation, Cx43 loss, reduced myocyte coupling, and arrhythmic sudden death, which can be prevented by c-Src inhibition. This suggests that an increase in c-Src activity may help mediate renin-angiotensin system-induced arrhythmias and that c-Src inhibitors might exert antiarrhythmic activity.

Project description:Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.

Project description:The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here, we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGFβ to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGFβ-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy-related 7 (ATG7) or pharmacologic inhibition of SYK activity with fostamatinib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, this study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells. SIGNIFICANCE: These findings present inhibition of spleen tyrosine kinase as a therapeutic option to limit breast cancer metastasis by promoting systemic tumor dormancy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/1831/F1.large.jpg.See related commentary by Farrington and Narla, p. 1756.

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Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation.

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Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation.

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