Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

The interaction of endoglin with beta-arrestin2 regulates transforming growth factor-beta-mediated ERK activation and migration in endothelial cells

In endothelial cells, transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation and migration, the ALK-1/Smads 1/5/8 pathway and the ALK-5/Smads 2/3 pathway. TGF-beta signaling through these pathways is further regulated in endothelial cells by the endothelial specific TGF-beta superfamily co-receptor, endoglin. The importance of endoglin, ALK-1, and ALK-5 in endothelial biology is underscored by the embryonic lethal phenotypes of knock-outs in mice due to defects in angiogenesis, and by the presence of disease-causing mutations in these genes in human vascular diseases. However, the mechanism of action of endoglin is not well defined. Here we define a novel interaction between endoglin and the scaffolding protein beta-arrestin2. Both co-immunoprecipitation and fluorescence confocal studies demonstrate the specific interaction between endoglin and beta-arrestin2 in endothelial cells, enhanced by ALK-1 and to a lesser extent by the type II TGF-beta receptor. The endoglin/beta-arrestin2 interaction results in endoglin internalization and co-accumulation of endoglin and beta-arrestin2 in endocytic vesicles. Whereas endoglin did not have a direct impact on either Smad 2/3 or Smad 1/5/8 activation, endoglin antagonized TGF-beta-mediated ERK signaling, altered the subcellular distribution of activated ERK, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with beta-arrestin2. Reciprocally, small interfering RNA-mediated silencing of endogenous beta-arrestin2 expression restored TGF-beta-mediated ERK activation and increased endothelial cell migration in an endoglin-dependent manner. These studies define a novel function for endoglin, and further expand the roles mediated by the ubiquitous scaffolding protein beta-arrestin2.     

Assignment of transforming growth factor beta1 and beta3 and a third new ligand to the type I receptor ALK-1

Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF-beta binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF-beta responses. The initial interactions on the cell surface between endoglin and TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co-expressed and is associated with betaglycan, a TGF-beta accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand-dependent or a ligand-independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF-beta receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF-beta signalling by interacting with both betaglycan and the TGF-beta signalling receptors at physiological receptor concentrations and ratios.     

Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II

Endoglin is an auxiliary component of the transforming growth factor-beta (TGF-beta) receptor system, able to associate with the signaling receptor types I (TbetaRI) and II (TbetaRII) in the presence of ligand and to modulate the cellular responses to TGF-beta1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TbetaRI or TbetaRII. This study shows that full-length endoglin interacts with both TbetaRI and TbetaRII, independently of their kinase activation state or the presence of exogenous TGF-beta1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TbetaRI interaction was different from that of endoglin/TbetaRII. TbetaRII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas TbetaRI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both TbetaRI and TbetaRII interact with the cytoplasmic domain of endoglin, but TbetaRI only interacts when the kinase domain is inactive, whereas TbetaRII remains associated in its active and inactive forms. Upon association, TbetaRI and TbetaRII phosphorylate the endoglin cytoplasmic domain, and then TbetaRI, but not TbetaRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TbetaRII, TbetaRI, and downstream Smad proteins as well as a modulation of TGF-beta signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-beta responses.     

Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells.

Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.     

Endoglin is expressed in the chicken vasculature and is involved in angiogenesis.

Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex, highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant vascular disorder caused by a haploinsufficiency mechanism. Vascular lesions (telangiectasia and arteriovenous malformations) in HHT1 are associated with loss of the capillary network, suggesting the involvement of endoglin in vascular repair processes. Using the chick chorioallantoic membrane (CAM) as an angiogenic model, we have analyzed the expression and function of chicken endoglin. A pan-specific polyclonal antibody (pAb) recognized chicken endoglin as demonstrated by immunostaining and Western blot analysis. In ovo treatment of chicken embryos with this pAb resulted in a significantly increased area of CAM. This effect was likely mediated by modulation of the ligand binding to endoglin as this pAb was able to inhibit TGF-beta1 binding. These results support the involvement of endoglin in the angiogenic process.     

Identification of endoglin in rat hepatic stellate cells: new insights into transforming growth factor beta receptor signaling

Transforming growth factor beta (TGF-beta) signaling is mediated by the cell surface TGF-beta type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, TbetaRII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M(r) 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-beta receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-beta receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-beta. In conclusion, we hypothesize that TGF-beta signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.     

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene targeting. Mice lacking TbetaRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TbetaRI is crucial for the function of TGF-beta during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-beta signaling via TbetaRI.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.