Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C9
Nitric oxide induces nuclear translocation of β-catenin in human umbilical vein endothelial cells
N. Chen1, Y. Shaifta2, Q. Zhang3, J. Ward2, A. Ferro1
1. Department of Clinical Pharmacology, Cardiovascular Divsion, King's College London, London, United Kingdom. 2. Division of Asthma, Allergy and Lung Biology, King’s College London, London, United Kingdom. 3. Cardiovascular Divsion, King’s College London, London, United Kingdom.
Introduction: Endothelium-derived nitric oxide (NO), generated from L-arginine by the action of NO synthase type 3 (NOS-3), has been implicated in increased endothelial permeability through modulating β-catenin function in the adherens junction. Interaction between NOS-3 and associated proteins (e.g. heat shock protein-90, β-actin) is important in NOS-3 regulation. Here we investigated the hypotheses that endothelial cell NOS-3 directly interacts with β-catenin, and that NOS-3 activation with consequent endothelial NO generation results in β-catenin translocation to the nucleus, with resultant effects on gene transcription. Methods and results: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured to confluence at passage 3 from umbilical cords obtained with consent following healthy uncomplicated pregnancies. In HUVEC lysates, using co-immunoprecipitation, we found a direct association between NOS-3 and β-catenin, and the level of this association was increased in response to the NOS-3 agonists histamine (100μM), thrombin (1U/ml), salbutamol (1μM) and adenosine (100μM)). Immunocytochemistry revealed that, following NOS-3 activation, β-catenin translocated to the nucleus, and this was confirmed by western blotting of nuclear extracts for β-catenin. However, whereas histamine and thrombin (which activate NOS-3 in a Ca2+-sensitive manner) induced β-catenin nuclear translocation both in the absence and presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100µM), adenosine and salbutamol (which induce Ca2+-insensitive NOS-3 activation) did so in the absence but not the presence of L-NAME. The Ca2+ ionophore ionomycin similarly induced β-catenin nuclear translocation in an L-NAME-insensitive manner, as did the NO donor spermine NONOate (10 µM) and the stable cGMP analogue 8-bromo-cGMP (10 µM). Salbutamol-induced nuclear translocation of β-catenin was also inhibited by L-lysine (a competitive inhibitor of L-arginine transport; 1 mM) and ODQ (a specific inhibitor of soluble guanylyl cyclase; 10 µM). Furthermore, in both HUVEC and HEK-293 cells, spermine NONOate, 8-bromo-cGMP and sildenafil (a phosphodiesterase type 5 inhibitor; 100nM) all resulted in T cell factor / lymphocyte enhancing factor transcriptional stimulation, an index of β-catenin-mediated transcriptional activation. Conclusions: NOS-3 is associated with β-catenin in HUVEC, and this association increases in response to NOS-3 activation. Both NOS-3 stimulation or activation of the NO-cGMP pathway by other means induces nuclear translocation of β-catenin, with resultant transcriptional activation. This phenomenon may contribute importantly to the known effects of NO on gene transcriptional regulation.
Where applicable, experiments conform with Society ethical requirements