Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the <em>Vegfr3</em> Gene in Lymphatic Endothelial Cells

<div><p>Modulation of VEGFR-3 expression is important for altering lymphatic endothelial cell (LEC) characteristics during the lymphangiogenic processes that occur under developmental, physiological, and pathological conditions. However, the mechanisms underlying the modulation of <em>Vegfr3</em> gene expression remain largely unknown. Using genetically engineered mice and LECs, we demonstrated previously that Ras signaling is involved not only in VEGFR-3-induced signal transduction but also in <em>Vegfr3</em> gene expression. Here, we investigated the roles of the transcription factor Ets and the histone acetyltransferase p300 in LECs in Ras-mediated transcriptional regulation of <em>Vegfr3</em>. Ras activates Ets proteins via MAPK-induced phosphorylation. Ets knockdown, similar to Ras knockdown, resulted in a decrease in both <em>Vegfr3</em> transcript levels and acetylated histone H3 on the <em>Vegfr3</em> gene. <em>Vegfr3</em> knockdown results in altered LEC phenotypes, such as aberrant cell proliferation and network formation, and Ets knockdown led to milder but similar phenotypic changes. We identified evolutionarily conserved, non-coding regulatory elements within the <em>Vegfr3</em> gene that harbor Ets-binding motifs and have enhancer activities in LECs. Chromatin immunoprecipitation (ChIP) assays revealed that acetylated histone H3 on the regulatory elements of the <em>Vegfr3</em> gene was decreased following Ras and Ets knockdown, and that activated Ets proteins, together with p300, were associated with these regulatory elements, consistent with a reduction in <em>Vegfr3</em> gene expression in p300-knockdown LECs. Our findings demonstrate a link between Ras signaling and Ets- and p300-mediated transcriptional regulation of <em>Vegfr3</em>, and provide a potential mechanism by which VEGFR-3 expression levels may be modulated during lymphangiogenesis.</p>