ABSTRACT
OBJECTIVE: To determine if patients with hereditary hemorrhagic telangiectasia (HHT) show alterations in transforming growth factor (TGF)-beta and its pathways. METHODS: Blood samples were obtained from HHT patients and controls, while endothelial cells were derived from umbilical veins of newborns (HUVEC) from HHT families. TGF-beta1 in plasma, or secreted by HUVEC, and plasma endoglin levels were measured by ELISA. Cellular levels of endoglin and receptor Smad proteins were tested by metabolic labeling and immunoprecipitation, mRNA levels for endoglin and TGF-beta1 by real-time PCR, and receptor Smad phosphorylation by Western blotting. RESULTS: TGF-beta1 and endoglin plasma levels analyzed in 197 individuals showed an inverse correlation with age. Circulating levels of TGF-beta1 were reduced in HHT1 patients (with Endoglin mutations) compared to control, but not in HHT2 patients (with ALK1 mutations). Endoglin levels were unchanged in plasma but decreased in activated monocytes and HUVEC with an HHT1 genotype. These HUVEC also expressed reduced levels of endoglin and TGF-beta1 mRNA, secreted less TGF-beta1, and showed normal receptor Smad expression and phosphorylation. CONCLUSIONS: Decreased plasma TGF-beta1 levels in HHT1 patients correlate with reduced production by endothelial cells. The lower endoglin expression in these cells may alter the regulation of TGF-beta1 via Smad-independent pathways.
Subject(s)
Endothelium, Vascular/metabolism , Telangiectasia, Hereditary Hemorrhagic/metabolism , Transforming Growth Factor beta/metabolism , Antigens, CD , Case-Control Studies , Cells, Cultured , Endoglin , Humans , Immunoprecipitation , Infant, Newborn , Mutation , RNA, Messenger/analysis , Receptors, Cell Surface , Reverse Transcriptase Polymerase Chain Reaction , Smad Proteins, Receptor-Regulated/analysis , Smad Proteins, Receptor-Regulated/genetics , Statistics, Nonparametric , Transforming Growth Factor beta/blood , Transforming Growth Factor beta/genetics , Umbilical Veins , Vascular Cell Adhesion Molecule-1/blood , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
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.
Subject(s)
Endothelium, Vascular/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Transforming Growth Factor beta/metabolism , Active Transport, Cell Nucleus , Activin Receptors, Type I/metabolism , Activin Receptors, Type II , Animals , Binding Sites , Biotinylation , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cell Proliferation , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Endoglin , Enzyme Activation , Flow Cytometry , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/physiology , Kinetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neovascularization, Pathologic , Phosphorylation , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein Transport , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad2 Protein , Smad3 Protein , Time Factors , Trans-Activators/metabolismABSTRACT
The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.