CD13/APN transcription is induced by RAS/MAPK-mediated phosphorylation of Ets-2 in activated endothelial cells

CD13/aminopeptidase N (CD13/APN) is a potent regulator of angiogenesis both in vitro and in vivo and transcription of CD13/APN in endothelial cells is induced by angiogenic growth factors via the RAS/MAPK pathway. We have explored the nuclear effectors downstream of this pathway that are responsible for CD13/APN induction. The response to serum/angiogenic growth factors mapped to a 38-bp region of the CD13/APN promoter containing an Ets-core motif that specifically binds a protein complex from nuclear lysates from activated endothelial cells. This motif and the proteins that target it are functionally relevant because mutation of this sequence abrogates CD13/APN transcription. Analysis of endothelial Ets family members showed that Ets-2, and to a lesser extent Ets-1, transactivate CD13/APN promoter activity via the Ets-core motif, whereas Fli, Erg, and NERF are ineffective. We investigated the possibility that the induction of CD13/APN is mediated by phosphorylation of Ets-2 via RAS/MAPK. A phosphorylation-defective Ets-2 mutant, T72A, failed to transactivate CD13/APN, suggesting that Ets-2 phosphorylation is obligatory for CD13/APN induction. To confirm a role for endogenous Ets-2 in CD13/APN expression, we specifically abrogated Ets-2 mRNA and protein by siRNA knockdown that significantly inhibited CD13/APN transcription. Finally, to assess the relevance of Ets-2 in endothelial cell function, we induced endothelial cells containing Ets-2 siRNA oligonucleotides to form capillary networks. Cells containing the Ets-2 inhibitory small interfering RNAs were completely incapable of forming the organized networks characteristic of endothelial morphogenesis. Thus, the phosphorylation of Ets-2 by RAS/MAPK is a prerequisite for CD13/APN endothelial induction and Ets-2 and its targets play essential roles in endothelial cell function.