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J. Biol. Chem., Vol. 278, Issue 49, 49358-49368, December 5, 2003

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CD13/APN Transcription Is Induced by RAS/MAPK-mediated Phosphorylation of Ets-2 in Activated Endothelial Cells^*

Nenad Petrovic, Shripad V. Bhagwat, William J. Ratzan, Michael C. Ostrowski¶, and Linda H. Shapiro||

From the Center for Vascular Biology, Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030, OSI Pharmaceuticals Inc., Farmingdale, New York 11735, and the ^¶Department of Molecular Genetics and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210

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.

Received for publication, July 24, 2003

^* This work was supported by National Institutes of Health Grants R01 CA 85714 and R01 HL 69442 (to L. H. S.) and R01 CA53271 (to M. C. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Center for Vascular Biology MC3501, Dept. of Physiology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. Tel.: 860-679-4373; Fax: 860-679-1201; E-mail: lshapiro{at}neuron.uchc.edu.

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