Abstract
Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.
Highlights
Endoglin (CD105) is a homodimeric transmembrane glycoprotein expressed on all types of endothelial cells [1] and in
One of these receptor-regulated Smads combines with the common Smad4, 1 The abbreviations used are: HHT, hereditary hemorrhagic telangiectasia; TGF, transforming growth factor ; ENG, endoglin gene; E3, ubiquitin-protein isopeptide ligase; MEEC, murine embryonic endothelial cells; TRII, TGF type II receptor; ALK, activin receptor-like kinase; ACVRL1, activin receptor-like kinase 1 gene; FBS, fetal bovine serum; pAb, polyclonal antibody; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PECAM, platelet/endothelial cell adhesion molecule
Eng Null Endothelial Cells Show Increased Proliferation and Growth Inhibitory Response to TGF—To better understand the role of endoglin in TGF-mediated signaling in endothelial cells, we generated endothelial cell lines from murine embryos and yolk sacs (E9.0) prior to onset of the lethal phenotype in Eng null mice as described under “Experimental Procedures.”
Summary
Derivation of MEEC—Engϩ/ϩ and EngϪ/Ϫ murine embryos and yolk sacs were isolated at E9.0 of gestation (N4 generation, C57BL/6 mice), prior to onset of the lethal phenotype in the Eng null mice as previously described [38]. For inhibition of proliferation by TGF1 (R&D Systems), cells were left overnight in complete medium containing 15% FBS and 15 g/ml endothelial mitogen, starved for 2 h, and incubated with increasing concentrations of TGF1 ranging from 0 to 100 pM in medium containing 1% FBS and 15 g/ml endothelial mitogen for 48 h, including an 8-h pulse with [3H]thymidine (1 Ci/ well) These conditions were optimized for cell density, growth factor concentration, and time in culture with both matched sets of lines to obtain maximal inhibition of proliferation by TGF1. Smad2/3 Nuclear Translocation Assay—15,000 cells of each MEEC line were seeded per well of 96-well plates and after 24 h were rinsed, starved for 2 h in serum-free MCDB/131 medium, and treated with TGF1 in medium containing 0.2% FBS for the times and concentrations indicated. Pixels were converted to cpm by comparing to known amounts of 125I-TGF1 that were spotted onto filters and exposed along with gels and quantified as above
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