Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues

Abstract

Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

Figures

FIGURE 1. VE-cadherin protein expression in mouse adult tissues

Panel A: Twenty μg of tissue lysate proteins were analyzed by SDS-PAGE and Western blotted with the anti VE-cadherin antibody. Panel B: Films from three Western blots were semiquantified by densitometry for evaluation of variations in VE-cadherin content among tissues. Results are expressed as mean percentages (+/− SEM) of lung VE-cadherin content. Panel C: VE-cadherin immunoprecipitates (1 mg of tissue proteins) were analyzed by Western blot first with the anti-phosphotyrosine (P-tyr) antibody and second with the anti-VE-cadherin antibody after stripping.

FIGURE 2. Effect of peroxovanadate treatment on tyrosine phosphorylation pattern in adult tissues

Panel A: The pattern of tyrosine phosphorylated proteins were analyzed in mice treated with peroxovanadate or vehicle alone. After 5 min of treatment, the animals were sacrificed and tissue proteins were extracted. Samples containing 10 μg of total proteins were analyzed by Western blot with the anti-phosphotyrosine antibody. This analysis was performed for each treated mouse to check that the treatment was successful. Panel B: Evaluation of VE-cadherin phosphorylation in peroxovanadate-treated adult tissues. VE-cadherin immunoprecipitates were Western blotted with the anti VE-cadherin antibody and the anti-phosphotyrosine antibody, sequentially. Lung extracts were analyzed separately because of the much higher VE-cadherin content of this organ. The anti-phosphotyrosine antibody only labeled the full-length VE-cadherin (arrow) and not the truncated form below, devoid of the cytoplasmic domain.

FIGURE 3. VE-cadherin tyrosine phosphorylation in hormone-stimulated ovary and uterus

Panel A: Female mice were stimulated with PMSG/hCG and treated with peroxovanadate (see “Materials and Methods”). Tyrosine phosphorylated proteins (10 μg) were analyzed by Western blot with the anti-phosphotyrosine antibody. Panel B: VE-cadherin was immunoprecipitated from 1 mg of proteins from ovary, uterus and lung extracts. Immunoprecipitates were immunoblotted with the anti-phosphotyrosine antibody, stripped and re-probed with anti-VE-cadherin antibody. Panel C: Western blots were semiquantified by densitometry for calculation of the ratio of the phosphorylated versus total VE-cadherin in the immunoprecipitates. Data are presented as fold induction by hormone treatment. This experiment is representative of two additional experiments.

FIGURE 4. Flk1/VE-cadherin protein association in mouse ovary and uterus stimulated by hormones

Panel A: Mice were treated with PMSG/hCG or vehicle alone, and peroxovanadate. Flk1/VE-cadherin association was revealed by immunoprecipitation of VE-cadherin from 1 mg of tissue protein lysates, immunoblotting with the anti-Flk1 and the anti-VE-cadherin antibodies. This experiment is representative of two additional experiments. Panel B: Flk1-VE-cadherin association was confirmed by immunoprecipitation of uterus protein extracts (1 mg) with the anti-Flk1 antibody and immunoblotting with the anti-Flk1 and the anti-VE-cadherin antibodies. The lower band (50 kDa) is the Ig-heavy chain.

FIGURE 5. Src/VE-cadherin association and Src phosphorylation state in mouse ovary and uterus

Panel A: Mice were treated by PMSG/hCG or vehicle alone, and peroxovanadate. Src/VE-cadherin association was analyzed in VE-cadherin immunoprecipitates from protein lysates (0.5 mg for lung and 1 mg for ovary and uterus) and immunoblotting with the anti-Src antibody. The membrane was stripped and reprobed with the anti-phosphotyrosine antibody. The lower band (50 kDa) is the Ig-heavy chain. Panel B: To confirm Src/VE-cadherin association, uterus extracts (1 mg) were immunoprecipitated with anti-Src antibody. Proteins were then Western blotted with the anti-VE-cadherin and the anti-Src antibodies. This experiment is representative of three additional experiments.

Figure 6. Src inhibitors impaired VEGF-induced VE-cadherin phosphorylation in endothelial cells but not VE-cadherin-Src association

Panel A: Confluent cultures of HUVEC were serum starved (1% serum) for 6 h and incubated with 50 ng/ml VEGF for 0 to 30 min. Proteins were immunoprecipitated with anti-VE-cadherin and Western blotted with anti-P-tyr and anti-VE-cadherin antibodies. Panel B: Src inhibitors prevented VEGF-induced VE-cadherin phosphorylation. VE-cadherin was immunoprecipitated from VEGF-stimulated HUVEC treated with increasing concentrations of SU6656 or PP2, as indicated. The presence of P-tyr- VE-cadherin was detected as above. Panel C: Src inhibitor did not disrupt VE-cadherin-Src association. VE-cadherin was immunoprecipitated from HUVEC treated with VEGF and increasing concentrations of SU6656. VE-cadherin and Src were revealed by Western blotting. Panel D: Immunolocalization of Src and VE-cadherin in confluent HUVEC. Double immunofluorescence staining (Src, green; VE-cadherin, red) showed protein colocalization at cell-cell junctions (yellow staining in merged images).