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.     

Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.

Characterization of the three mammalian transforming growth factor-beta (TGF-beta) isoforms, TGF-beta 1, -beta 2, and -beta 3, indicates that TGF-beta 3 is somewhat more potent (ED50 = 0.5 pM versus 2 pM) than TGF-beta 1 and TGF-beta 2 as a growth inhibitor of the Mv1Lu mink lung epithelial cell line. In the fetal bovine heart endothelial (FBHE) cell line, however, TGF-beta 1 and -beta 3 are at least 50-fold more potent than TGF-beta 2 which is a very weak growth inhibitor (ED50 greater than or equal to 0.5 nM). Thus, as growth inhibitors, TGF-beta 1 and -beta 3 resemble each other more than TGF-beta 2. The presence of serum alpha 2-macroglobulin in the FBHE cell assays decreases the biological potency of TGF-beta s, in particular TGF-beta 2. This effect of alpha 2-macroglobulin, however, is not sufficient to explain the low responsiveness of FBHE cells to TGF-beta 2. Evaluation of the role of TGF-beta receptors as determinants of cell-specific responsiveness to TGF-beta isoforms indicates that TGF-beta 1, -beta 2, and -beta 3 have similar affinity for the membrane proteoglycan, betaglycan. They differ, however, in their ability to bind to receptor types I and II which are implicated in TGF-beta signal transduction. TGF-beta 1 is similar, albeit not identical, to TGF-beta 3 and much more potent than TGF-beta 2 as a competitor for binding to the overall population of receptors I and II in all cell lines tested. A subset of receptors I and II has been identified in Mv1Lu cells which has high affinity for TGF-beta 2 (KD approximately 10 pM) and binds this factor at concentrations that are biologically active in Mv1Lu cells. This receptor subset could not be detected in FBHE cells, suggesting that cell-specific differences in the level of high affinity of TGF-beta 2 receptors may lead to cell-specific differences in responsiveness to this isoform. Thus, despite their structural and biological similarities, TGF-beta 1, -beta 2, and -beta 3 diverge in their ability to bind to receptors in a manner that correlates with their potency as growth inhibitors.     

Characterization of endoglin on mouse uterine stromal cells

During the oestrous cycle and early pregnancy, the uterus undergoes a variety of morphological and physiological modifications involving uterine cell proliferation and differentiation as well as extensive tissue remodelling. Transforming growth factor beta (TGF-beta) has powerful effects on these events and thus is thought to have a critical role in uterine physiology. Endoglin is a transmembrane glycoprotein that binds TGF-beta 1 and -beta 3 and interacts with TGF-beta signalling receptors to modulate many effects of this growth factor in different types of cell. Studies in mice revealed the highest concentrations of endoglin in the reproductive tract, notably on stromal cells of cyclic and pregnant uteri. The objective of the present study was to investigate the role of endoglin expressed on uterine stromal cells in binding TGF-beta and in the cellular responses induced by this growth factor. Highly purified populations of uterine stromal cells were isolated by cell affinity to the monoclonal antibody MJ7/18, which is specific to mouse endoglin. Affinity labelling of these cells with 125I-labelled TGF-beta followed by immunoprecipitation with endoglin-specific polyclonal 1256:4b antiserum indicated that endoglin expressed at the surface of uterine stromal cells binds TGF-beta 1 and interacts with TGF-beta signalling receptors. Treatment of uterine stromal cells with different concentrations of TGF-beta 1 induced a biphasic proliferative response and addition of MJ7/18 as well as neutralizing TGF-beta antibodies showed endoglin to be a modulator of TGF-beta-induced stromal cell proliferation. Given the importance of TGF-beta in the regulation of uterine physiology, these results indicate a role for endoglin during uterine tissue remodelling and decidualization.     

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.     

Isoform-specific transforming growth factor-beta binding proteins with membrane attachments sensitive to phosphatidylinositol-specific phospholipase C.

We report the identification of cell surface glycoproteins that bind transforming growth factor-beta (TGF-beta) in an isoform-specific manner, and are distinct from TGF-beta receptors I and II or the TGF-beta binding proteoglycan beta-glycan. The novel TGF-beta binding proteins have been identified in various cell lines including fetal bovine heart endothelial cells and MG-63 human osteosarcoma cells. They include proteins of 90-100 and 180 kDa that preferentially bind TGF-beta 1 (KD 0.1-0.2 nM) and proteins of 60 and 140 kDa that preferentially bind TGF-beta 2 (KD 0.5-1 nM). The 180-kDa TGF-beta 1 binding protein and the 60- and 140-kDa TGF-beta 2 binding proteins can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that these proteins are attached to the plasma membrane through a phosphatidylinositol anchor. The expression of these three proteins as well as their sensitivity to phosphatidylinositol-specific phospholipase C is cell line-dependent. The 90-100-kDa TGF-beta 1 binding proteins are components of a 190-kDa disulfide-linked complex. The structural properties of these proteins and their high affinity and selectivity for different TGF-beta isoforms defines them as a novel class of cell surface TGF-beta binding proteins.     

Transforming growth factor-beta (TGF-beta) binding to the extracellular domain of the type II TGF-beta receptor: receptor capture on a biosensor surface using a new coiled-coil capture system demonstrates that avidity contributes significantly to high affinity binding

Mature TGF-beta isoforms, which are covalent dimers, signal by binding to three types of cell surface receptors, the type I, II and III TGF-beta receptors. A complex composed of the TGF-beta ligand and the type I and II receptors is required for signaling. The type II receptor is responsible for recruiting TGF-beta into the heteromeric ligand/type I receptor/type II receptor complex. The purpose of this study was to test for the extent that avidity contributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain of the type II receptor (TbetaRIIED) at the biosensor surface in an oriented and stable manner by using a de novo designed coiled-coil (E/K coil) heterodimerizing system. We characterized the kinetics of binding of three TGF-beta isoforms to this immobilized TbetaRIIED. The results demonstrate that the stoichiometry of TGF-beta binding to TbetaRIIED was one dimeric ligand to two receptors. All three TGF-beta isoforms had rapid and similar association rates, but different dissociation rates, which resulted in the equilibrium dissociation constants being approximately 5pM for the TGF-beta1 and -beta3 isoforms, and 5nM for the TGF-beta2 isoform. Since these apparent affinities are at least four orders of magnitude higher than those determined when TGF-beta was immobilized, and are close to those determined for TbetaRII at the cell surface, we suggest that avidity contributes significantly to high affinity receptor binding both at the biosensor and cell surfaces. Finally, we demonstrated that the coiled-coil immobilization approach does not require the purification of the captured protein, making it an attractive tool for the rapid study of any protein-protein interaction.     

Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity.

Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.     

Endoglin modulates cellular responses to TGF-beta 1

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta 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-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S- endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF- beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (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-beta 1 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-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

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.     

Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-beta receptor complex

Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.     

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.     

Growth factor-binding sequence in human alpha2-macroglobulin targets the receptor-binding site in transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) binds transforming growth factor-beta1 (TGF-beta1) and TGF-beta2, forcing these growth factors into a state of latency. The mechanism by which this occurs remains unclear. In this paper, we demonstrate that peptides, derived from the structure of human alpha(2)M (amino acids 714-729), bind directly to TGF-beta1 and block the binding of TGF-beta1 to the type I and II TGF-beta receptors. The alpha(2)M-derived peptides are notable for hydrophobic tripeptide sequences (WIW or VVV) and acidic residues (Glu(714) and Asp(719) in the mature alpha(2)M subunit), which may function analogously to the structural elements that mediate TGF-beta-binding in the type II receptor. Mutating Glu(714) and Asp(719) in the alpha(2)M-peptide-GST fusion protein, FP3, which contains the putative growth factor-binding site, significantly decreased the binding affinity of FP3 for TGF-beta1. The alpha(2)M-derived peptides, which bind TGF-beta1, inhibited the interaction of TGF-beta1 with its receptors in fetal bovine heart endothelial cells. The same peptides also inhibited the activity of TGF-beta1 in endothelial cell proliferation assays. These results demonstrate that alpha(2)M-derived peptides target the receptor-binding sequence in TGF-beta.     

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, an ancillary TGFbeta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development

Endoglin (CD105) is expressed on the surface of endothelial and haematopoietic cells in mammals and binds TGFbeta isoforms 1 and 3 in combination with the signaling complex of TGFbeta receptors types I and II. Endoglin expression increases during angiogenesis, wound healing, and inflammation, all of which are associated with TGFbeta signaling and alterations in vascular structure. The importance of endoglin for normal vascular architecture is further indicated by the association of mutations in the endoglin gene with the inherited disorder Hereditary Haemorrhagic Telangiectasia Type 1 (HHT1), a disease characterised by bleeding from vascular malformations. In order to study the role of endoglin in vivo in more detail and to work toward developing an animal model of HHT1, we have derived mice that carry a targeted nonsense mutation in the endoglin gene. Studies on these mice have revealed that endoglin is essential for early development. Embryos homozygous for the endoglin mutation fail to progress beyond 10.5 days postcoitum and fail to form mature blood vessels in the yolk sac. This phenotype is remarkably similar to that of the TGFbeta1 and the TGFbeta receptor II knockout mice, indicating that endoglin is needed in vivo for TGFbeta1 signaling during extraembryonic vascular development. In addition, we have observed cardiac defects in homozygous endoglin-deficient embryos, suggesting endoglin also plays a role in cardiogenesis. We anticipate that heterozygous mice will ultimately serve as a useful disease model for HHT1, as some individuals have dilated and fragile blood vessels similar to vascular malformations seen in HHT patients.     

Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites.

Transforming growth factor-beta (TGF-beta) is a bifunctional, dose-dependent regulator of endothelial cell proliferation induced in vitro by heparin-binding growth factor 1 (HBGF-1, acidic FGF). Here we have examined the relationship between endothelial cell growth and the expression of cell surface binding sites for TGF-beta and HBGF-1. Fetal bovine heart endothelial cell (FBHEC) growth was stimulated by low concentrations of TGF-beta and inhibited by high concentrations of TGF-beta while expressing two distinct classes of TGF-beta binding sites with binding constants of 24 pM (6300 sites/cell) and 900 pM (12,000 sites/cell). In contrast, human umbilical vein endothelial cells (HUVEC), whose growth was slightly promoted by TGF-beta, exhibited a single class of high-affinity TGF-beta binding sites (Kd = 45 pM, 4500 sites/cell). Affinity crosslinking using [125I]TGF-beta showed that FBHEC expressed two distinct low molecular weight TGF-beta binding sites (Mr 85,000 and 58,000), while HUVEC expressed a single type of low molecular weight TGF-beta binding site (Mr 85,000). As detected by binding of [125I]HBGF-1, preincubation of FBHEC with high concentrations of TGF-beta transmodulated the expression of high-affinity HBGF-1 receptors. In contrast, no transmodulation of HBGF-1 receptors occurred in FBHEC during preincubation with low concentrations of TGF-beta. Furthermore, preincubation of HUVEC with TGF-beta did not transmodulate the expression of HBGF-1 receptors. The data suggest that the ability of TGF-beta to stimulate or inhibit endothelial cell proliferation in a dose-dependent manner correlated with the expression of specific TGF-beta binding site subtypes and involved the transmodulation of HBGF-1 receptors.     

Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites

Betaglycan, also known as the TGF-beta type III receptor, is a membrane- anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.     

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.