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.

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Endoglin is a transforming growth factor-␤ (TGF-␤) co-receptor expressed mainly on endothelial cells and involved in cardiovascular development, angiogenesis, and vascular remodeling. This is illustrated by the fact that mutations in the endoglin gene give rise to hereditary hemorrhagic telangiectasia type 1, a dominant vascular disease with clinical manifestations that originate by a mechanism of haploinsufficiency. Thus, studies on the regulated expression of endoglin are crucial to devising therapeutic strategies for hereditary hemorrhagic telangiectasia type 1. Endoglin is highly expressed in the neovasculature associated with hypoxia such as ischemic tissues and tumors, but the molecular mechanism of this up-regulation is unknown. Here, we have investigated the possible regulation of endoglin expression by hypoxia. Surface protein, transcript, and promoter activity levels of endoglin were found to be up-regulated by hypoxia, indicating that the regulation takes place at the transcriptional level. A hypoxia-responsive element downstream of the main transcription start site of the endoglin gene was functionally characterized. Whereas hypoxia alone moderately stimulated endoglin transcription, addition of TGF-␤ under hypoxic conditions resulted in transcriptional cooperation between both signaling pathways, leading to marked stimulation of endoglin expression. Because basal endoglin transcription is sustained by Sp1, and TGF-␤ and hypoxia signaling pathways are mediated by Smad proteins and hypoxia-inducible factor-1 (HIF-1), respectively, the involvement of these transcription factors was analyzed. Functional and co-immunoprecipitation experiments demonstrated the existence of a multiprotein complex (Sp1⅐Smad3⅐HIF-1) on the endoglin promoter, mediating the cooperation between the hypoxia and TGF-␤ pathways. Within this multiprotein complex, Smad3 appears to function not only as a coactivator factor, but also as an adaptor between HIF-1 and Sp1. We propose that basal endoglin transcription (highly dependent on Sp1) may switch from a constitutive to an inducible state through Sp1 interaction with HIF-1 and Smad transcription factors, induced by hypoxia and TGF-␤, respectively.