PROX1 Utilizes Distinct Mechanisms to Induce Two Key Lymphatic Growth Factor Receptors:Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) and Fibroblast Growth Factor Receptor-3 (FGFR-3)

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Eshraghi, Mehdi
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The lymphatic vasculature is a network of unidirectional capillaries and ducts, which serve to return extracellular fluid and macromolecules to the systemic blood circulation. In addition, the lymphatic vessels have important roles in immune surveillance and fat absorption. Dysfunction of lymphatic vessels has profound physiological consequences. Insufficient lymph uptake results in lymphedema, a chronic disabling condition that currently has no cure. Lymphedemcanoccureither due to developmental defects (primary lymphedema) or due to injuries of existing lymphatic vessels (secondary lymphedema). The significance of lymphangiogenesis in tumour metastasis is demonstrated by the finding that increased lymphangiogenesis is associated with a higher rate of metastasis and poorer prognosis in cancer patients. Prox1, a homeoboxgene, regulates the development of the lymphatic vasculature by upregulating the expression of lymphatic endothelial markers and simultaneously repressing the expression of blood endothelial markers. To explore the mechanisms by which Prox1 establishes lymphatic cell fate, we compared Prox1 mediated activation oftwo key lymphatic cell surface receptors: theVascular Endothelial Growth Factor Receptor-3 (VEGFR-3) and theFibroblast Growth Factor Receptor-3 (FGFR-3) genes. Using a combination of luciferase gene reporter assays and immunoblotting, we compared the ability of different Prox1 constructs to activate either VEGFR-3 or FGFR-3 at both the mRNA and protein levels, respectively. Furthermore, we tested whether recombinant PROX1 protein was able to bind to the proximal promoter regions of VEGFR-3 and FGFR-3usingelectrophoretic mobility shift assays (EMSA). DNA binding deficient Prox1 versions did not activate the FGFR-3 promoter. In contrast, these versions of Prox1still efficiently activated transcription of the VEGFR-3 promoter. In agreement with our luciferase reporter gene assays, immunoblotting of HUVECs demonstrated that only infection with wt Prox1adenovirus increased expression of the FGFR-3 protein. Infection of HUVECs with adenoviral vectors encoding eitherwt Prox1orHDPD∆ Prox1was sufficient to induce a significant increase in VEGFR-3 protein levels. Surprisingly, our EMSA results with recombinant PROX1 demonstrated that PROX1 can bind to the promoter region of both VEGFR-3 and FGFR-3 genes via its DNA binding domain. We showed that PROX1 potentially binds to the promoter region of the VEGFR-3 gene via a consensus Prospero binding site (CGCCTCGGC). Our data demonstrates that, in endothelial cells, PROX1 utilizes distinct mechanisms to activate these two key endothelial growth factor receptors.
lymphangiogenesis, Prox1, VEGFR3, FGFR3