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Visualization of CD146 dimerization and its regulation in living cells

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2007; 1773(4):513-20. DOI: 10.1016/j.bbamcr.2007.01.009
Source: PubMed

ABSTRACT Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescence-tagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.

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Available from: Xun Shen, Jan 23, 2014
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    • "This transmembrane glycoprotein has important functions in early and late development and it has been suggested to play an important role in cancer, angiogenesis, cardiovascular diseases and placentation [30]. Moreover, a significant number of studies in the field of cancer research have assigned to CD146 a critical role in tumor growth and metastasis, as well as in tumor angiogenesis [31], [32]. At the core of an efficient cross-talk between endothelial cells, mural cells and ECM, are thought to be a diversity of proteins, namely PDGF, TGF, VEGF, Collagen, laminin and others, both soluble or trapped on the ECM [33]–[35]. "
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    • "Results CD146 dimerization is required for VEGF-induced tube formation and signal transduction Our group has previously found that CD146 dimerization in endothelial cells is inducible and regulatable by the NF-κB pathway in tumor-induced angiogenesis [16]. To study tumor-induced angiogenesis , VEGF, an important tumor-secreted cytokine, was used to stimulate human endothelial cells. "
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    ABSTRACT: CD146 dimerization plays an important role in tumor-induced angiogenesis. Stimulation of target cells with vascular endothelial growth factor (VEGF), a major angiogenic factor produced by tumor cells, elicits a burst of reactive oxygen species (ROS) that enhances angiogenesis. However, the molecular mechanism coupling CD146 dimerization with the VEGF-related oxidant-generating apparatus has not been elucidated. Here, we show that CD146 dimerization is induced by VEGF and is significantly diminished by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidase, suggesting a potential role for NADPH oxidase (NOX) in VEGF-induced CD146 dimerization. Importantly, we found that overexpression of NADPH oxidase 4 (NOX4), which is the predominant NOX expressed in endothelial cells, significantly enhances VEGF-induced ROS generation and CD146 dimerization. By contrast, these VEGF effects were dramatically attenuated after transfection with siRNA to reduce NOX4 expression. Furthermore, expression of Rac1 N17, a dominant negative mutant of Rac1, a member of the Rho family of small GTPases, suppressed VEGF-induced ROS generation and CD146 dimerization. These studies show for the first time that VEGF alteration of CD146 dimerization is mediated via a NOX4-dependent pathway and provide novel insight into the significant role of NOX in redox regulation of the dimerization of cell adhesion molecules.
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    • "Previously, we have reported the dimerization of CD146 in living cells using the method of FRET (Bu et al., 2007). We postulated that dimerization may be related to the role of CD146 in tumor angiogenesis . "
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