Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts

10 p.-9 fig.-1 tab.Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type ß (TGF-ß) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of α5ß1 integrin and of an activation epitope of ß1 integrin were unchanged, a polyclonal antibody to α5ß1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation.This work has been supported by grants from Camisión Interministerial de Ciencia yTecnología (CICYT-SAF97-0034 to C. Bernabéu, and CICYT-SAF97-0064-C03-02 to A. García-Pardo), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-CT95-0995 to C. Bernabéu).Peer reviewe

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

14 p.-10 fig.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.This work was supported by grants from Ministerio de Ciencia y Tecnologı́a (SAF2000-0132) and Comunidad Autónoma de Madrid (CAM).Peer reviewe

Role of endoglin in cellular responses to transforming growth factor-beta. A comparative study with betaglycan

10 p.-7 fig.Endoglin (CD105) is the target gene for the hereditary hemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. It shares with betaglycan a limited amino acid sequence homology and being components of the membrane transforming growth factor-beta (TGF-beta) receptor complex. Using rat myoblasts as a model system, we found that overexpression of endoglin led to a decreased TGF-beta response to cellular growth inhibition and plasminogen activator inhibitor-1 synthesis, whereas overexpression of betaglycan resulted in an enhanced response to inhibition of cellular proliferation and plasminogen activator inhibitor-1 induced expression in the presence of TGF-beta. The regulation by endoglin of TGF-beta responses seems to reside on the extracellular domain, as evidenced by the functional analysis of two chimeric proteins containing different combinations of endoglin and betaglycan domains. Binding followed by cross-linking with 125I-TGF-beta1 demonstrated that betaglycan expressing cells displayed a clear increase (about 3. 5-fold), whereas endoglin expressing cells only displayed an slight increment (about 1.6-fold) in ligand binding with respect to mock transfectants. SDS-polyacrylamide gel electrophoresis analysis of radiolabeled receptors demonstrated that expression of endoglin or betaglycan is associated with an increased TGF-beta binding to the signaling receptor complex; however, while endoglin increased binding to types I and II receptors, betaglycan increased the binding to the type II receptor. Conversely, we found that TGF-beta binding to endoglin required the presence of receptor type II as evidenced by transient transfections experiments in COS cells. These findings suggest a role for endoglin in TGF-beta responses distinct from that of betaglycan.This work was supported by Comisión Interministerial de Ciencia y Tecnologı́a Grant CICYT-SAF97-0034, Comunidad Autónoma de Madrid (CAM), and Biomed Program of the European Community Grant BMH4-CT95-0995 (to C. B.)Peer reviewe

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2-dependent signalling

13 páginas, 8 figuras -- PAGS nros. 456-468The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF-β) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium-derived nitric oxide in endoglin+/− (Eng+/−) mice and cultured endothelial cells. Here, we find that TGF-β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/− mice, suggesting the involvement of endoglin in the TGF-β regulated vascular homeostasis. The endoglin-dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF-β receptor ALK5 and its downstream substrate Smad2. In addition, Smad2-specific signaling is upregulated in endoglin-induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin-dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin-inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome-dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF-β signaling, and leading to an increased eNOS expression in endothelial cells. J. Cell. Physiol. 210: 456–468, 2007. © 2006 Wiley-Liss, Inc.ALK, activin receptor-like kinase; Dox, doxycycline; eNOS, endothelial nitric oxide synthase; MEFs, murine embryonic fibroblasts; Pp, perfusion pressure; siRNA, small interference RNA; TGF-β, transforming growth factor-β; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; HHT, hereditary hemorrhagic telangiectasia. The endothelial nitric oxide synthase (eNOS or NOS3) is a critical regulator of cardiovascular homeostasis, vascular remodeling, and angiogenesis, and whose dysregulation leads to different types of vascular pathology (Kawashima and Yokoyama, 2004; Sessa, 2004; Tai et al., 2004). eNOS-derived NO is an endogenous vasodilatory molecule that regulates the tone of blood vessels and maintains an anti-thrombotic, anti-proliferative, and anti-apoptotic environment in the vessel wall (Sessa, 2004). Elucidation of the mechanisms and factors determining the expression and activity of eNOS under different physiological and pathophysiological conditions has long been considered central in order to understand the alterations in vascular NO production (Tai et al., 2004). The expression of eNOS is regulated by extracellular stimuli such as shear stress, estrogens, hypoxia, or growth factors (Davis et al., 2001; Simoncini et al., 2002; Tai et al., 2004). Among these, TGF-β1 has been shown to increase bovine aortic endothelial cell (BAEC) and human umbilical vein endothelial cell (HUVEC) steady-state eNOS mRNA expression (Inoue et al., 1995). Interestingly, eNOS expression is modulated not only by transcriptional, but also by post-transcriptional mechanisms (Sessa, 2004; Tai et al., 2004). At the transcriptional level, the eNOS promoter lacks a TATA box, but exhibits proximal elements such as Sp1 and GATA motifs, which are common characteristics of constitutively expressed genes (Zhang et al., 1995; Tai et al., 2004). The 5′-flanking region also contains many putative sites for further transcriptional regulation of eNOS. However, only a few of these sites have been formally shown to regulate eNOS transcription, including PEA3 (Cieslik et al., 1998) and AP-1 binding sites (Navarro-Antolin et al., 2000). Also, a region of the eNOS promoter extending from −1269 and −935 that contains Smad binding sites and mediates TGF-β induction of eNOS transcription, has been identified (Saura et al.,2002)Endoglin (CD105), is a 180-kDa homodimeric membrane glycoprotein which is strongly expressed by human endothelial cells and is involved in angiogenesis, cardiovascular development, and vascular homeostasis (Gougos and Letarte, 1990; Duff et al., 2003; Lebrin et al., 2005). The gene encoding endoglin has been identified as the target for the dominant vascular disorder known as hereditary hemorrhagic telangiectasia type 1 (HHT1) (Guttmacher et al., 1995). HHT is a highly penetrant autosomal dominant vascular dysplasia associated with frequent epistaxis, gastrointestinal bleedings, telangiectases, and arteriovenous malformations in brain, lung, and liver (Shovlin and Letarte, 1999; Marchuk and Lux, 2001). The mechanistic role of endoglin in angiogenesis and vascular remodeling is not known, but it is likely related to the transforming growth factor-β (TGF-β) system, as endoglin is a functional component of the membrane TGF-β receptor complex (Lastres et al., 1996) and is a substrate for TGF-β receptor-mediated phosphorylation (Guerrero-Esteo et al., 2002; Koleva et al., 2006). Signaling induction by TGF-β and related members of this superfamily regulate a variety of human diseases including cancer, fibrosis, developmental disorders, or cardiovascular pathology (Blobe et al., 2000; Siegel and Massague, 2003; Waite and Eng, 2003; Lebrin et al., 2005). TGF-β signaling is initiated when ligand induces formation of heteromeric complexes of type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively), which in turn activate and induce translocation of the Smad family of proteins to the nucleus (Attisano and Wrana, 2002; Miyazawa et al., 2002; Shi and Massague, 2003; Feng and Derynck, 2005). The association of endoglin with TβRII and TβRI (ALK1 and ALK5) regulates Smad signaling and cellular responses to TGF-β (Lastres et al., 1996; Letamendia et al., 1998; Li et al., 2000; Guerrero-Esteo et al., 2002). Endoglin regulates signal transduction of TGF-β1 by potentiating ALK1/Smad1 pathway and repressing the ALK5/Smad3 (Lebrin et al., 2004; Blanco et al., 2005), whereas it enhances ALK5/Smad2 signaling (Guerrero-Esteo et al., 2002). Conversely, in endoglin-deficient mice it has been shown that TGF-β/ALK5 signaling from endothelial cells to adjacent mesothelial cells is defective, as evidenced by reduced phosphorylation of Smad2 (Carvalho et al., 2004).Recently, it has been shown that endoglin regulates nitric oxide-dependent vasodilatation, as well as eNOS expression and activity (Jerkic et al., 2004; Toporsian et al., 2005). In addition, eNOS derived NO seems to play a major role in endoglin-dependent angiogenesis (Jerkic et al., 2006a) and COX-2 regulated expression (Jerkic et al., 2006b). The role of endoglin in the control of vascular tone was examined by measuring NO-dependent vasodilation in haploinsufficient mice (Eng+/−) and their Eng+/+ littermates (Jerkic et al., 2004). Urinary and plasma concentrations of nitrites were lower in Eng+/− than in Eng+/+ mice. The levels of eNOS in kidneys and femoral arteries were about half in Eng+/− than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/− compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively (Jerkic et al., 2004). However, the mechanisms by which endoglin regulates eNOS expression are poorly understood. In the present work we show that endoglin enhances eNOS expression by increasing Smad2 protein levels and thereby enhancing TGF-β-dependent induction of eNOS in endothelial cellsThis work was supported by Ministerio de Educacion y Ciencia, Spain; grant numbers: SAF2004-01390 to C.B. and BFU2004-00285/BFI to J.M.L.-N.; Fondo de Investigaciœn Sanitaria, Spain; grant number: PI020200 to C.B.; COBRE Program, National Center for Research Resources, NIH, USA; grant number: P20-RR15555 to C.P.H.V.; Fondo Nacional de Ciencia y Tecnologia, Chile; grant number: FONDECYT-1050476 to J.F.S.; HHT Foundation International, Inc.; grant number: 3 to C.B.; Canadian Institute of Health Research (CIHR) to L.A.Peer reviewe