Identification and functional role of endothelial isoforms of the transcription factor ERG

The ETS transcription factor ERG is highly expressed in endothelial cells (EC) and has a crucial role in controlling homeostasis and angiogenesis. ERG drives expression of genes which promote EC identity, monolayer integrity, survival and angiogenesis. Studies in non-EC have identified multiple ERG isoforms that display functional differences. Notably, some ERG isoforms are phosphorylated through the MAPK pathway, which is downstream of pro-angiogenic growth factors, such as VEGF. The objective of this study was to identify the expression pattern and activity of ERG isoforms in EC and to define their functional role during angiogenesis and in the maintenance of vascular quiescence. Using a PCR screen of human umbilical vein EC (HUVEC) we identify multiple ERG isoforms which differ in the use of alternative transcription and translation initiation sites, and in alternative splicing of a highly evolutionary conserved 24 amino acid domain encoded by exon 7b (ex7b). Isoforms containing ex7b were found to be predominantly expressed in EC. Molecular tools such as ERG isoform specific siRNA and over expression plasmids were designed and validated to investigate the functional role of endothelial ERG isoforms in HUVEC. Selective siRNA targeting of ERG isoforms in HUVEC followed by in vitro Matrigel tube formation assays and propidium iodide staining/flow cytometry demon strated an enhanced role for ex7b-containing isoforms in the regulation of EC tube formation and cell survival. PCR screening of HUVEC treated with ERG isoform-specific siRNAs identified genes regulating cell survival and the cell cycle as differentially regulated. Furthermore, we demonstrate that VEGF stimulation increased endothelial ERG phosphorylation at S215 in an ERK2 and ex7b-dependent manner; leading to enhanced EC proliferation, tube formation and increased expression of multiple ERG target genes. Thus, we show that the responses to VEGF are dependent on the presence of ex7b-containing ERG isoforms, suggesting that alternative splicing of ERG ex7b may provide a mechanism to regulate ERG's transcriptional activity in response to growth factors.