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Caveolin-1 Interacts and Cooperates with the Transforming Growth Factor-B Type I Receptor ALK1 in Endothelial Caveolae

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Caveolin-1 Interacts and Cooperates with the Transforming Growth Factor-B Type I Receptor ALK1 in Endothelial Caveolae Cardiovascular Research (2008) 77, 791–799 doi:10.1093/cvr/cvm097 Caveolin-1 interacts and cooperates with the transforming growth factor-b type I receptor ALK1 in endothelial caveolae Juan F. Santibanez1,†,‡,FranciscoJ.Blanco1,‡, Eva M. Garrido-Martin1, Francisco Sanz-Rodriguez1,2, Miguel A. del Pozo3, and Carmelo Bernabeu1* Downloaded from https://academic.oup.com/cardiovascres/article/77/4/791/335299 by guest on 24 September 2021 1Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas (CSIC), and Center for Biomedical Research on Rare Diseases (CIBERER), Ramiro de Maeztu 9, 28040 Madrid, Spain; 2Departamento de Biologia, Universidad Autonoma, Madrid, Spain; and 3Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Received 24 May 2007; revised 17 November 2007; accepted 30 November 2007; online publish-ahead-of-print 8 December 2007 Time for primary review: 27 days KEYWORDS Aims Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-b type I membrane Angiogenesis; receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and Caveolae; angiogenesis. Previous reports have shown that the ubiquitous TGF-b type I receptor ALK5 and the Growth factors; type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of Endothelial receptors; this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveo- Signal transduction lin-1 on the TGF-b/ALK1 signalling pathway. Methods and results The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-b signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-12/2mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol- depleting agents such as nystatin or methyl-b-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this asso- ciation. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-b/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway. Conclusion ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-b pathway in angiogenesis. 1. Introduction growth factor-b (TGF-b).2 TGF-b triggers the signal to the nucleus through the heterodimeric association of two differ- Angiogenesis is the process by which new blood vessels are 1 ent receptors (types I and II) with cytoplasmic Ser/ formed from pre-existing ones. Upon angiogenic stimuli, Thr-kinase activity.3 Activin receptor-like kinase (ALK)5 quiescent endothelial cells (ECs) proliferate and migrate in and ALK1 are TGF-b type I receptors expressed in cultured response to different cytokines such as the transforming ECs and involved in angiogenesis.3–5 The almost endothelial- restricted ALK16 has been related to the activation phase of * Corresponding author. Tel: þ34 34 91 8373112 ext. 4246; fax: þ34 91 angiogenesis, promoting cellular proliferation and migration 5360432. and basal membrane degradation.4,5 In contrast, the ubiqui- E-mail address: [email protected] tous ALK5 has been linked to the resolution phase of angio- †Present address. Laboratorio de Biologia Celular, Instituto de Nutricio´n y Tecnologia de los Alimentos, INTA, Universidad de Chile. genesis, involving inhibition of proliferation and migration as 3 ‡These authors contributed equally to this work. well as activation of the extracellular matrix synthesis. Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007. For permissions please email: [email protected]. 792 J.F. Santibanez et al. ALK5 and ALK1 propagate the TGF-b signalling by phosphor- 2.1 Cell culture ylating Smad proteins. Whereas Smad2/3 are the targets for The human microvascular EC line HMEC-1 (provided by Dr Edwin Ades, ALK5 activity, Smad1/5/8 are phosphorylated by ALK1. Once Centers for Disease Control and Prevention, Atlanta, USA) was cul- phosphorylated, these Smad proteins associate with the tured as described.27 The human epithelial cell line HEK 293T (Amer- common partner Smad4 and translocate into the nucleus ican Type Culture Collection, CRL-11268), the rat L6E9 myoblasts to regulate gene expression. Within this TGF-b pathway, (provided by Dr Joan Massague, Memorial Sloan-Kettering Cancer several components, including ALK1, have been identified Center, NY, USA), and the mouse embryonic fibroblasts (MEFs) from as critical mediators of the angiogenic process.3 WT and Cav12/2 mice23 were grown in DMEM medium plus 10% FCS. The ACVRL1 gene codes for ALK1 protein and mutations in For caveolae disruption assays, cells were incubated with this gene are responsible for an autosomal dominant methyl-b-cyclodextrin (MbCD) or nystatin (Sigma) for 1 h in the vascular dysplasia termed Hereditary Haemorrhagic Telan- absence of FCS. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Insti- giectasia (HHT) type 2,7 characterized by recurrent haemor- tutes of Health (NIH Publication No. 85–23, revised 1996). Downloaded from https://academic.oup.com/cardiovascres/article/77/4/791/335299 by guest on 24 September 2021 rhages and cerebral, hepatic and pulmonary arteriovenous 8 malformations. The pathogenic mechanism of this vascular 2.2 Plasmids and transfections disorder appears to be the haploinsufficiency of ALK1,8 a hypothesis supported by the fact that heterozygous knock- Expression vectors encoding HA-tagged full-length ALK1 wild-type out mice for ALK1 (Acvrl1þ/2) show an HHT phenotype.8,9 (WT) and kinase death (KR, Lys229Arg) in pcDNA3-HASL plasmid In addition, genetic inactivation in mice shows that were provided by Dr Kohei Miyazono (University of Tokyo). ALK1-WT was used as a template in site-directed mutagenesis embryos homozygous for ALK1 (Acvrl12/2) die at assays to generate ALK1-001 (Trp406Ala) and ALK1-011 (Phe401Gly 10–10.5 days postcoitum due to vascular and cardiac 10 and Trp406Ala) mutants. The myc-tagged caveolin-1 expression anomalies. vectors WT and mutated in the scaffolding domain (SDmut, Caveolae are plasma membrane microdomains enriched in Phe92Ala, and Val94Ala), and the caveolin-1 deleted in the scaffold- cholesterol and glycosphingolipids closely related to lipid ing domain (D82–101) have been described.28,29 For RNA interfer- rafts but characterized by the presence of a protein family ence (siRNA) studies, the pRNA-U6/cav-1 vector, that suppresses named caveolins.11,12 Caveolin-1 is the representative and the rat caveolin-1 gene,30 was used. Cell transfections were most abundant member of this family. It is a small integral carried out with SuperFect Reagent (Qiagen). membrane protein, and the principal component of caveo- lae in many cell types, with particularly high levels in 2.3 Antibodies 11–13 ECs. The region comprised between Asp82 and Arg101 ALK1 was detected with a rabbit31 or goat (R&D Systems) polyclonal is termed scaffolding domain and is implicated in protein– antibody (pAb) and caveolin-1 was recognized with a rabbit pAb protein interactions.14 This domain can recognize a set of (sc-894; Santa Cruz). Proteins with HA or myc epitopes were binding motifs with the consensus sequences FXFXXXXF, detected with 12CA5 (Boehringer Mannheim) or 9E10 (sc-40, Santa FXXXXFXX,orFXFXXXXFXXF, where F represents an aro- Cruz) monoclonal antibody (mAb), respectively. a-tubulin was matic residue and X any other.14 Caveolin-1 has been impli- revealed with a mouse mAb (Sigma). cated in the regulation of diverse biological functions such as angiogenesis,15 cellular adhesion and migration,16–18 2.4 Immunofluorescence microscopy 19 13,20 receptor signalling and tumour suppression. HMEC-1 were fixed and permeabilized as described.27 Samples were Caveolin-1 lacking mice, Cav12/2, show a caveolae defec- incubated with anti-ALK1 or anti-caveolin-1 antibodies, followed by tive phenotype. The absence of these organelles impairs incubations with Alexa-488 green-conjugated anti-goat IgG or nitric oxide synthesis and calcium signalling in the cardiovas- Alexa-647 red-conjugated anti-rabbit IgG antibodies (Molecular cular system, causing aberrations in endothelium-dependent Probes), respectively. Nuclei were visualized by incubation with 0 relaxation, contractility, and maintenance of myogenic 4 -6-diamidino-2-phenylindole, DAPI (Sigma). Samples were mounted with ProLong Gold (Invitrogen) and observed with a spec- tone.21,22 In addition, the lungs of Cav12/2 animals tral confocal microscope (Leica Microsystems). display thickening of alveolar
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