Abstract. Endoglin is a homodimeric membrane glycoprotein which can bind the 131 and 133 isoforms of transforming growth factor-13 (TGF-13). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-13 itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L-or S-endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-t31 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-132 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-131, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule i (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-131 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-131 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.

Endoglin, a component of the transforming growth factor-␤ (TGF-␤) receptor 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 hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at ؊37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-␤ and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-␤-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-␤ responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region ؊50/؊29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-␤ or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4.

It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.