Molekulare Mechanismen für die Interaktion zwischen CEACAM1 und VEGFR-3 /Prox1 in Lymphendothelzellen
Zusammenfassung der Projektergebnisse
Based on our published results, that have demonstrated an important role of CEACAM1 on lymphangiogenesis but also as a key regulator during lymphatic endothelial differentiation, the current project should clarify the mechanisms involved in CEACAM1 regulation and CECAM1 functions in lymphatic endothelial cells (LECS). Thus, the objective of this project was to elucidate the molecular mechanisms of CEACAM1 interaction with lymphatic transcription factor Prox1 and with VEGFR3 as the main signal transducing VEGF receptor for lymphangiogenesis. Furthermore we planned to analyse the relevance of these interactions in an in vivo lymphangiogenesis model. Regarding the transcriptional regulation of CEACAM1 in lymphatic endothelial cells we could demonstrate that Prox1 overexpression lead to up-regulation of CEACAM1 in these cells, whereas transfection with the dominant negative form Prox1M did not affect its expression, suggesting an essential role of amino acids asparagine (N625) and arginine (R627) in the transcriptional activation of CEACAM1 promoter. Surprisingly, these results could not be corroborated in the luciferase reporter assay. We are currently performing ChIP assay in order to clarify whether Prox1 directly binds to CEACAM1 promoter. A direct interaction between CEACAM1 and the lymphangiogenesis receptor VEGFR-3 could be shown using immunoprecipitation experiments in the endothelial cell line HMEC1. In primary lymphatic endothelial cells we observed co-localization of CEACAM1 and VEGFR-3 receptors using immunoflourescence. Interestingly, after stimulation with VEGF-C we detected internalization of CEACAM1 and co-localization of both receptors in the cytosolic space of LECs. This data let us assume that CEACAM1 is directly interacting with VEGFR-3 and is further involved in VEGF-C-induced signaling. Moreover, our preliminary results suggest that CEACAM1 presence in LECs plays an important role in VEGFR-3-mediated signaling pathways, since CEACAM1 knock-down in LECs using siRNAs affect phosphorylation of downstream effectors of VEGFR-3. To corroborate these preliminary results, further analyses are currently ongoing on CECAM1-silenced LECs and additionally on endothelial cells transfected with a CEACAM1 mutated form (CEACAM1/y--), which has been previously described to fail CEACAM1-mediated signaling activation. In summary, we have achieved very interesting and novel results and could confirm some of the hypothesis proposed in our project proposal. To our opinion further experiments, that are currently being performed, are necessary to enhance the scientific quality of this study. Specially, the in vivo experiments are essential in order to corroborate our observations of in vitro systems.
