STAT5a/b proteins bind to the PROX1 proximal promoter and mediate IL3Rα-induced PROX1 upregulation.

<p>(A) Adenoviral overexpression of IL3Rα increased phosphorylation of STAT5a/b (p-STAT5a/b) in BECs and HUVECs, but not LECs. AdCTR, control adenovirus; AdIL3Rα, IL3Rα-expressing adenovirus. The relative intensity ratio of the p-STAT5a/b band over the total STAT5a/b band is shown below the p-STAT5a/b western blot result. (B) The constitutively active (CA), but not wild type (WT) or dominant negative (DN), form of STAT5b induced PROX1 upregulation in BECs and HUVECs, but not in LECs. Comparable expression of FLAG-tagged rat STAT5b proteins (Flag) was confirmed by western blot assays. CTR, a control vector. (C) Inhibition of STAT5a/b with siRNA-mediated knockdown abrogated the IL3Rα-induced PROX1 upregulation in BECs and HUVECs. siCTR, siRNA for luciferase gene; siSTAT5a/b, siRNA for STAT5a/b. (D) STAT5a/b ChIP assay against the PROX1 promoter sequences revealed an increased binding of STAT5a/b proteins to the two newly identified putative STAT5 binding sites (E1 and E2) upon adenoviral IL3Rα-overexpression in BECs and HUVECs. Relative locations of E1, two PROX1 transcriptional start sites (TS1 based on the PROX1 Ensemble exon number ENSE00001936642 and TS2 based on ENSE00001443122), E2 and the PROX1 initiation codon (ATG) are illustrated. (E) EMSA showing that nuclear fractions isolated from BECs contain binding activity for the E1 and E2 sites and that this binding activity was increased upon IL3Rα-overexpression. (F) The binding activity to the E1 and E2 sites could be inhibited by an anti-STAT5a/b antibody, indicating that the binding activity was caused by STAT5a/b proteins. Arrow-pointed bands in E and F indicate STAT5a/b protein complex with the isotope-labeled E1 and E2 DNA probes. Asterisks point a protein/probe complex that did not respond to the anti-STAT5a/b antibody and thus was likely caused by an unrelated factor.