Nitric oxide synthase type 3 interacts with beta-catenin in human umbilical vein endothelial cells

Nan Chen1, Colleen Hamid1, Silke Becker1, Vladimir Snetkov2, Yasin Shaifta2, Jeremy Ward2, Albert Ferro1. 1Cardiovascular Division, King’s College London, London, United Kingdom, 2Division of Asthma, Allergy and Lung Biology, King’s College London, London, United Kingdom.

Introduction: Endothelium-derived nitric oxide is an important vasorelaxant and anti-atherogenic mediator, generated from L-arginine by the action of nitric oxide synthase type 3 (NOS-3). NOS-3 can undergo activation classically through the Ca 2+-calmodulin pathway, or in a Ca 2+-independent fashion through phosphorylation of key residues as well as by association with a number of regulatory proteins. Beta-catenin is an integrin located in endothelial cell junctions, which can undergo nuclear translocation and hence modulate transcription of a number of genes, following activation of the Wnt signalling pathway. The aim of this study was to determine whether beta-catenin associates with NOS-3 in endothelial cells, and whether such association gives rise to nuclear translocation of beta-catenin.

Methods: HHuman umbilical vein endothelial cells (HUVEC), isolated from healthy uncomplicated pregnancies, were cultured to confluence at passage 3. Cells were treated with histamine (100μM), thrombin (1U/ml), salbutamol (1μM), adenosine (100μM), and corresponding vehicle (Krebs solution), for 2 min at 37℃. Cells were lysed and placed on ice. Lysates were co-incubated with specific antibodies (to NOS-3, or beta catenin respectively), and immuno-complexes were isolated by binding to Protein G Sepharose. Immunoprecipitates were separated by SDS-PAGE and probed for both NOS-3 and beta-catenin. In separate experiments, beta-catenin localisation was visualised in HUVEC in response to the same stimuli by immunofluorescence staining; and nuclear extracts of HUVEC treated as above were prepared using cell fractionation, separated by SDS-PAGE and probed for beta-catenin or lamin A/C (a nuclear marker, used for normalising beta-catenin expression in nuclear extracts). Statistical analysis was done using one way ANOVA with post-hoc Dunnett’s test.

Results: Western blotting demonstrated that immunoprecipitation (IP) of beta-catenin resulted in co-precipitation of NOS-3, and similarly NOS-3 IP resulted in co-precipitation of beta-catenin, thereby confirming the co-association of these two proteins (n=3). The degree of co-association between beta-catenin and NOS-3 was significantly increased by histamine (208±114%), salbutamol (222±110%), adenosine (207±113%) and thrombin (211±112%) (p<0.01 for each, n=6). Each of these agents also elicited translocation of beta catenin from the cytosol to the nucleus, as visualised by immunofluorescence (n=2). In addition, immunoblotting of nuclear extracts confirmed an increase in nuclear beta-catenin in response to the NOS activators tested (n=5).

Conclusion: Beta-catenin associates with NOS-3 in HUVEC, and the degree of interaction is increased by a number of NOS-3 activators. These activators also cause translocation of beta-catenin from the cytosol to the nucleus. These data suggest that association with NOS-3 may have important effects on functions such as gene transcription mediated by beta-catenin in the nuclei of endothelial cells, but this needs to be further investigated. The role of beta-catenin binding in the activation of NOS-3 also needs further exploration.