Christina M Warboys1, Nan Chen1, Qiuping Zhang1, Yasin Shaifta2, Genevieve Vanderslott1, Gabriella Passacquale1, Yanhua Hu1, Qingbo Xu1, Jeremy P T Ward2, Albert Ferro3. 1. Cardiovascular Division, Department of Clinical Pharmacology, British Heart Foundation Centre of Research Excellence, King's College London, 3.07 Franklin-Wilkins Building, 150 Stamford Street, London, UK. 2. Division of Asthma, Allergy, and Lung Biology, King's College London, London, UK. 3. Cardiovascular Division, Department of Clinical Pharmacology, British Heart Foundation Centre of Research Excellence, King's College London, 3.07 Franklin-Wilkins Building, 150 Stamford Street, London, UK albert.ferro@kcl.ac.uk.
Abstract
AIMS: β-catenin has been shown to be regulated by inducible nitric oxide synthase (NOS) in endothelial cells. We investigated here whether β-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes β-catenin signalling. METHODS AND RESULTS: We identified β-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of β-catenin and eNOS immunoprecipitates. This was confirmed by in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in β-catenin(-/-) mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation (using adenosine, salbutamol, thrombin, or histamine), or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of β-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of β-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in β-catenin(-/-) MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing β-catenin(-/-) MPECs exhibited reduced angiogenesis compared with plugs containing wild-type MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel. CONCLUSION: These data reveal bidirectional cross-talk and regulation between the NO-cGMP and β-catenin signalling pathways. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: β-catenin has been shown to be regulated by inducible nitric oxide synthase (NOS) in endothelial cells. We investigated here whether β-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes β-catenin signalling. METHODS AND RESULTS: We identified β-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of β-catenin and eNOS immunoprecipitates. This was confirmed by in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in β-catenin(-/-) mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation (using adenosine, salbutamol, thrombin, or histamine), or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of β-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of β-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in β-catenin(-/-) MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing β-catenin(-/-) MPECs exhibited reduced angiogenesis compared with plugs containing wild-type MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel. CONCLUSION: These data reveal bidirectional cross-talk and regulation between the NO-cGMP and β-catenin signalling pathways. Published on behalf of the European Society of Cardiology. All rights reserved.
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