gms | German Medical Science

27. Deutscher Krebskongress

Deutsche Krebsgesellschaft e. V.

22. - 26.03.2006, Berlin

Interaction of Sp1 and Sp3 with an upstream GC-rich enhancer is essential for the transcriptional control of the human Angiopoietin-2 Gene (ang-2) in Endothelial Cells

Meeting Abstract

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  • corresponding author presenting/speaker Jonas Wenke - Labor für Angiogenese und Metastasierung, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Deutschland
  • Johannes Hertel - Labor für Angiogenese und Metastasierung, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum
  • Mathias Z. Strowski - Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum
  • Michael Höcker - Labor für Angiogenese und Metastasierung, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum

27. Deutscher Krebskongress. Berlin, 22.-26.03.2006. Düsseldorf, Köln: German Medical Science; 2006. DocPO492

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Veröffentlicht: 20. März 2006

© 2006 Wenke et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen ( Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.



Introduction: Angiopoietin-2 (ang-2), predominantly expressed by endothelial cells has been identified as important regulator of angiogenesis. As a ligand of the endothelial receptor tyrosine kinase Tie-2 it acts as a naturally occurring antagonist of Ang-1. Despite the accumulating evidence confirming the involvement of Ang-2 in tumor-related angiogenesis, the molecular mechanisms controlling ang-2 expression are unclear.

Methods: Following genomic cloning of 3kB of human ang-2 5’ flanking DNA, regulatory promoter elements were identified by functional 5’ deletion analysis. Identification of transcription initiation site (TIS) was done by rapid amplification of cDNA ends (RACE). Nuclear proteins binding to the ang-2 core promoter were identified by standard EMSAs and functionally confirmed by ectopic expression of wild type and dominant-negative factor mutants along with ang-2 reporter constructs. The influence of promoter methylation was studied employing demethylating agent Aza-dC and subsequent real-time PCR analysis or reporter gene assays.

Results: We identified the promoter region -105 to -56 relative to TIS as sufficient and necessary for the ang-2 gene transcription. EMSA analysis identified the zinc finger proteins Sp1 and Sp3 as dominant nuclear protein binding to the ang-2 promoter. Moreover, the region spanning -78/-74 was identified as essential Sp1 site regulating ang-2 expression. In contrast, transcription factors of the Ets-family as well as Ap2, Egr and GATA were not detected at the ang-2 gene promoter. Ectopic expression of Sp1 and Sp3 in Sp-factor deficient SL-2 cells revealed potent transactivation (15-25 fold) of the ang-2 promoter. 5-Aza-dC treatment resulted in potent up-regulation of ang-2 mRNA levels and promoter activity.

Conclusion: Our results reveal new insights of ang-2 regulation and strongly suggest that Sp1/Sp3- dependent activation of an upstream enhancer at -105 to -56 is crucial for the regulation of ang-2 expression. Promoter methylation represents an important control mechanism regulating the expression of the human ang-2 gene in endothelial cells. Further investigations are intended to reveal physiological and pathophysiological context of ang-2 regulation.