Artikel
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
Suche in Medline nach
Autoren
Veröffentlicht: | 20. März 2006 |
---|
Gliederung
Text
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.