Identification of a critical Sp1 site within the endoglin promoter and itsinvolvement in the transforming growth factor-beta stimulation

Endoglin, a component of the transforming growth factor-beta (TGF-beta) rec eptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as h ereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is th e underlying mechanism for hereditary hemorrhagic telangiectasia type 1, un derstanding the regulation of endoglin gene expression appears to be a cruc ial step to correct the disease. In this study we have identified an Sp1 si te at -37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF- beta and this stimulation is located at the Spl-containing proximal region, we have investigated the possible involvement of Spl in the TGF-beta -medi ated induction. Mutation of the Sp1-binding sequence, or addition of the Sp l inhibitor WP631, abolished both the basal transcription activity and the TGF-beta responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region -50/-29 was evidenced by electrophoretic mo bility shift assays and DNA affinity precipitation studies. Furthermore, sy nergistic cooperation on the promoter activity between Sp1 and TGF-beta or Smad3 could be demonstrated by co-transfection experiments of reporter prom oter constructs. The molecular mechanism underlying this cooperation appear s to involve a direct physical interaction between Sp1 and Smad3/Smad4.