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

Structural and Functional Insights into Endoglin Ligand Recognition and Binding

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

SPARC prevents endoglin association with αV integrin, which blocks the activation of TGF-β signaling and promotes pericyte migration to nascent blood vessels

Identificación de proteínas que interaccionan con los dominios extracelular e intracelular de endoglina

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-10-200

Endoglin regulates cytoskeletal organization through binding to ZRP-1, a member of the LIM family of proteins

11 p.-7 fig.Endoglin is a component of the transforming growth factor-β receptor complex abundantly expressed at the surface of endothelial cells and plays an important role in cardiovascular development and vascular remodeling. By using the cytoplasmic domain of endoglin as a bait for screening protein interactors, we have identified ZRP-1 (zyxin-related protein 1), a 476-amino acid member that belongs to a family of LIM containing proteins that includes zyxin and lipoma-preferred partner. The endoglin interacting region was mapped within the three double zinc finger LIM domains of the ZRP-1 C terminus. Analysis of the subcellular distribution of ZRP-1 demonstrated that in the absence of endoglin, ZRP-1 mainly localizes to focal adhesion sites, whereas in the presence of endoglin ZRP-1 is found along actin stress fibers. Because the LIM family of proteins has been shown to associate with the actin cytoskeleton, we investigated the possibility of a regulatory role for endoglin with regard to this structure. Expression of endoglin resulted in a dramatic reorganization of the actin cytoskeleton. In the absence of endoglin, F-actin was localized to dense aggregates of bundles, whereas in the presence of endoglin, expressed in endothelial cells, F-actin was in stress fibers and colocalized with ZRP-1. Furthermore, small interfering RNA-mediated suppression of endoglin or ZRP-1, or clustering of endoglin in endothelial cells, led to mislocalization of F-actin fibers. These results suggest a regulatory role for endoglin, via its interaction with ZRP-1, in the actin cytoskeletal organization.This work was supported by Ministerio de Ciencia y Tecnologia, Fondo de Investigación Sanitaria Grant PI020200, Comunidad Autonóma de Madrid (to C. B.), and National Center for Research Resources Grant P20 15555 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer Reviewe

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

11 páginas, 8 figuras -- PAGS nros. 574-584Transforming growth factor-β (TGF-β) 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-β 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-β receptor endoglin interact with ALK-1 (a type I TGF-β receptor). In addition, endoglin potentiates TGF-β/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-β/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-β. © 2005 Wiley-Liss, Inc. ALK, activin receptor-like kinase; Dox, doxycycline; HA, hemagglutinin epitope; HHT, hereditary hemorrhagic telangiectasia; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; siRNA, small interference RNA; TGF-β, transforming growth factor-β; TβRI, TGF-β receptor type I; TβRII, TGF-β receptor type II. Transforming growth factor-β (TGF-β) receptors are essential for angiogenesis in development and vascular remodeling in the adult (Oshima et al., 1996; Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000; Oh et al., 2000; Larsson et al., 2001; Srinivasan et al., 2003). The TGF-β superfamily signals through binding to a heteromeric complex containing two different transmembrane serine/threonine kinases known as type I (TβRI) and type II (TβRII) signaling receptors (Miyazono et al., 2000; Shi and Massague, 2003). Upon ligand binding to the TβRI/TβRII complex, activated TβRI propagates intracellular signal to the nucleus by phosphorylating members of the Smad family of proteins (Mehra and Wrana, 2002; Derynck and Zhang, 2003). In endothelial cells, the existence of two different types of TβRI, named ALK-1 and ALK-5, has been proposed to alternatively regulate the switch between proliferation and quiescence, respectively (Goumans et al., 2002, 2003a), and to explain the pro- or anti-angiogenic effects of TGF-β (Pepper, 1997; Goumans et al., 2003b). ALK-1 is specifically expressed in endothelial cells where the TGF-β receptor complex also contains an auxiliary co-receptor named endoglin (Gougos and Letarte, 1990; Duff et al., 2003). Endoglin binds different members of the TGF-β superfamily in the presence of the signaling receptors types I and II (Cheifetz et al., 1992; Yamashita et al., 1994; Letamendia et al., 1998; Barbara et al., 1999), and modulates TGF-β1-dependent cellular responses (Lastres et al., 1996; Letamendia et al., 1998; Li et al., 2000; Guerrero-Esteo et al., 2002). Ectopic expression of endoglin counteracts the TGF-β-induced growth inhibition in monocytes (Lastres et al., 1996), myoblasts (Letamendia et al., 1998), and endothelial cells (Li et al., 2000), in agreement with the high endoglin levels found in proliferating versus resting endothelial cells (Miller et al., 1999; Duff et al., 2003). Interestingly, adenoviral expression of a constitutively active form of ALK-1 leads to increased endothelial cell proliferation (Goumans et al., 2002). Both endoglin and ALK-1 are expressed (Gougos and Letarte, 1990; Duff et al., 2003;Seki et al., 2003) and can be co-immunoprecipitated (Abdalla et al., 2000) on endothelial cells, and play important roles in cardiovascular development and vascular remodeling. Genetic inactivation of endoglin or ALK-1 in the mouse shows that embryos homozygous for these genes die at 10–10.5 days postcoitum due to vascular and cardiac anomalies (Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000; Oh et al., 2000). Furthermore, genes encoding endoglin and ALK-1 are targets for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia (McAllister et al., 1994; Johnson et al., 1996). In spite of the above data suggesting the physical interaction and functional collaboration between endoglin and ALK-1 within the endothelial TGF-β receptor pathway, the molecular basis for this association is not understood. In this study, we have analyzed the interaction between endoglin and ALK-1, as well as the role of endoglin in the ALK-1 mediated TGF-β signalingThis work was supported by Fondo de Investigacion Sanitaria grant PI020200, Ministerio de Educacion y Ciencia grant SAF2004-01390 (to C.B.), National Center for Research Resources Grant P20 15555 from the National Institutes of Health (to C.P.H.V.), and Ministerio de Educacion Cultura y Deporte postdoctoral fellowship to J.F.S.Peer reviewe