Mechanisms of PROX1 mediated regulation of the lymphatic endothelial cell cycle
Baxter, Shannon A.
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The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.