Abstract
Aimsβ-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 resultsWe 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.ConclusionThese data reveal bidirectional cross-talk and regulation between the NO-cGMP and β-catenin signalling pathways.
Highlights
Nitric oxide (NO), generated by the vascular endothelium through the action of endothelial NO synthase, plays an essential part in vascular homeostasis, principally through causing vasodilation and inhibiting platelet function, thrombogenesis, vascular smooth muscle cell proliferation, and progression of atherosclerosis.[1,2] Classically, eNOS is activated through an increase in cytosolic Ca2+ and consequent binding of Ca2+calmodulin.[3]
We identified b-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass and results spectroscopy and western blot analyses of b-catenin and eNOS immunoprecipitates
We found that b-catenin2/2 mouse pulmonary endothelial cells (MPECs) exhibited significantly higher levels of eNOS phosphorylation under non-stimulated conditions, which were only partially reduced by the Akt inhibitor, and that stimulation of these cells with adenosine or histamine did not produce any further rise in eNOS phosphorylation (Figure 2B)
Summary
Nitric oxide (NO), generated by the vascular endothelium through the action of endothelial NO synthase (eNOS), plays an essential part in vascular homeostasis, principally through causing vasodilation and inhibiting platelet function, thrombogenesis, vascular smooth muscle cell proliferation, and progression of atherosclerosis.[1,2] Classically, eNOS is activated through an increase in cytosolic Ca2+ and consequent binding of Ca2+calmodulin.[3]. Junction, in endothelial cells.[6] in MLO-Y4 osteocytes, NO has been shown to activate the b-catenin signalling pathway;[7] and a recent study reported that NO increases endothelial permeability through S-nitrosylation of b-catenin in response to vascular endothelial growth factor (VEGF).[8]. Activity of the transcription factor c-jun is inhibited by NO,[17] and NF-kB activity is down-regulated due to NO-induced S-nitrosylation.[18] A potential link between transcriptional regulation by NO and b-catenin has been revealed in conditionally immortalized murine colonic epithelial cells, where the NO donor NOR-1 was reported to induce b-catenin association with LEF in the nucleus.[19]
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