MINIREVIEW Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction Eva Faurobert and Corinne Albiges-Rizo ´ Centre de recherche, INSERM U823-CNRS ERL 3148, Universite J Fourier, Grenoble, France Keywords angiogenesis; blood brain barrier; cadherin; CCM; cytoskeleton; endothelial cell; HEG; hemorrhage; integrin; Krit1 Correspondence E Faurobert, Centre de recherche, INSERM ´ U823-CNRS ERL 3148, Universite J Fourier, ´ Site sante La tronche, BP170 38042, Grenoble, France Fax: +33 476 54 94 25 Tel: +33 476 54 94 74 E-mail: eva.faurobert@ujf-grenoble.fr (Received August 2009, revised November 2009, accepted 25 November 2009) doi:10.1111/j.1742-4658.2009.07537.x Cerebral cavernous malformations (CCM) are common vascular malformations with an unpredictable risk of hemorrhage, the consequences of which range from headache to stroke or death Three genes, CCM1, CCM2 and CCM3, have been linked to the disease The encoded CCM proteins interact with each other within a large protein complex Within the past years, a plethora of new data has emerged on the signaling pathways in which CCM proteins are involved CCM proteins regulate diverse aspects of endothelial cell morphogenesis and blood vessel stability such as cell–cell junctions, cell shape and polarity, or cell adhesion to the extracellular matrix Although fascinating, a global picture is hard to depict because little is known about how these pathways coordinate to orchestrate angiogenesis Here we present what is known about the structural domain organization of CCM proteins, their association as a ternary complex and their subcellular localization Numerous CCM partners have been identified using two-hybrid screens, genetic analyses or proteomic studies We focus on the best-characterized partners and review data on the signaling pathways they regulate as a step towards a better understanding of the etiology of CCM disease Introduction Cerebral cavernous malformations (CCM) are common vascular malformations with a prevalence of in every 200–250 individuals Leakage of blood can be detected by magnetic resonance imaging around each lesion and individuals with these vascular lesions are subject to an unpredictable risk of hemorrhage in midlife Although lesions have been described in a variety of vascular beds, clinical manifestations are most common in the central nervous system where the consequences may be stroke, seizure or any kind of neurological disorder, and can lead to death [1] The lesions consist of densely packed, grossly dilated, capillary-like sinusoids lined by a single endothelial layer embedded in a thick collagen matrix Importantly, these lesions lack the components of organized mature vessels such as pericytes, astrocytic foot processes and intact endothelial cell–cell junctions [2] Both sporadic and familial forms of CCM have been identified The genetics of the disease is developed in a minireview by Riant et al [3] Briefly, three loci have been mapped and the genes responsible for the disease, CCM1 to CCM3, have been identified in these loci Within the Abbreviations CCM, cerebral cavernous malformation; FERM, band 4.1 ezrin radixin moesin; FN, fibronectin; HEG1, heart of glass 1; ICAP-1, integrin cytoplasmic adaptor protein-1; Krit1, K-Rev interaction trapped 1; MAPK, mitogen-activated protein kinase; MEKK3, mitogen-activated protein kinase kinase kinase 3; MKK, mitogen-activated protein kinase kinase; MST4, mammalian sterile twenty-like 4; OSM, osmosensing scaffold for MEKK3; PTB, phosphotyrosine binding; STRIPAK, striatin interacting phosphatase and kinase; STK, serine ⁄ threonine kinase; vEGF, vascular epidermal growth factor 1084 FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works E Faurobert and C Albiges-Rizo past two years, fascinating data have emerged on the signaling pathways regulated by the products of these three genes However, a global picture is hard to depict because not much is known about how these signaling pathways coordinate Many advances have been made in describing the core complex formed by the association of these three proteins and in the identification of numerous CCM partners In this minireview, we focus on partners that open new avenues for CCM research and discuss recent insights into their role in cytoskeletal remodeling, regulation of cell matrix adhesion and cell–cell junction homeostasis Structural domain organization of CCM1 ⁄ Krit1, CCM2 ⁄ OSM ⁄ MGC4607 and CCM3 ⁄ PDCD10 proteins CCM1 encodes a protein also named K-Rev interaction trapped (Krit1) Krit1 was first identified in Emerging signaling pathways regulated by CCM proteins 1997 as a partner of the small G-protein Krev-1 ⁄ Rap1 from a yeast two-hybrid screen [4] Two years later, the CCM1 locus was mapped to the gene encoding Krit1 [5,6] Krit1 is an 84 kDa scaffold protein with no catalytic activity which contains several distinct domains involved in protein–protein interaction (Fig 1) Remarkably, Krit1 possesses a C-terminal band 4.1 ezrin radixin moesin (FERM) domain, a signature of membrane binding proteins like talin, ezrin, radixin or moesin FERM domains are composed of three subdomains, F1–F3, arranged in cloverleaf shape The F3 subdomain has a phosphotyrosine binding (PTB) fold PTB domains recognize a canonical NPXY ⁄ F motif often found on the cytoplasmic tail of transmembrane receptors Recruitment of PTB or FERM proteins to transmembrane receptors is a conserved mechanism used by cells to build intracellular signaling hubs Remarkably, in addition to its FERM domain, Krit1 possesses three N-terminal NPXY ⁄ F Fig Structural domains of CCM proteins Krit1 ⁄ CCM1 bears a C-terminal FERM (band 4.1 erzin radixin moesin) domain and three N-terminal NPXY ⁄ F motifs allowing either the folding of the protein on itself or its interaction with ICAP-1 and CCM2 ANK, ankyrin domain; MT, microtubules; NLS, nuclear localization signal The phosphotyrosine binding (PTB) domain of CCM2 ⁄ OSM interacts with a Krit1 NPXY ⁄ F motif L198R and F217A mutations prevent CCM2 interaction with Krit1 CCM3 has no homology with any known domain Its N-terminal fragment (L33 to K50) interacts with MST4, STK24 and STK25 Ser39 and Thr43 are the substrate of phosphorylation by STK25 HEG1 is a heavily glycosylated ( ) transmembrane protein carrying two extracellular EGF-like repeats and a C-terminal NPXY ⁄ F motif which interacts with Krit1 Its extracellular ligand is not known ICAP-1 has a Ser ⁄ Thr riche N-terminus containing a NLS and sites of phosphorylation by calmodulin-dependent kinase II, protein kinase A and protein kinase C Reported interactions with b1 integrin, Krit1, Rho-associated kinase I-kinase and NM23 FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works 1085 Emerging signaling pathways regulated by CCM proteins motifs (Fig 1) This peculiar structural organization allows the N- and C-terminal halves of Krit1 to interact with each other in a glutathione S-transferase pulldown [7] or a yeast two-hybrid interaction assay [8], suggesting that Krit1 may adopt a closed and open conformation in vivo, resulting from either intramolecular folding or dimerization The first [7] or third [8] NPXY ⁄ F motif may be involved in this interaction Systematic mutagenesis of each of the three motifs should help to determine the contribution made by each of them Three ankyrin repeats are present between the NPXY ⁄ F motifs and the FERM domain (Fig 1) Although ankyrin repeats are found in thousands of proteins and support interaction with many diverse proteins, no partner interacting with Krit1 ankyrin repeats has been found Compared with Krit1, CCM2 and CCM3 have a much simpler structural domain organization CCM2 encodes a scaffold protein of 51 kDa also containing a PTB domain [9,10], but no other known domain (Fig 1) It was identified in a yeast two-hybrid screen using mitogen-activated protein kinase kinase kinase (MEKK3) as bait to identify proteins involved in the cell response to hyperosmotic shock [11] and was named osmosensing scaffold for MEKK3 (OSM) The last mutated gene CCM3 or PDCD10 has been identified more recently [12] and is upregulated in fibroblasts exposed to specific apoptosis inducers, such as staurosporine, cycloheximide and tumor necrosis factor-a [13] Apoptotic or, by contrast, proliferative functions have been attributed to CCM3 [14,15] No homology with any known domain is found on CCM3 but it has been suggested that this small protein (25 kDa) folds as one stable domain [16] (Fig 1) CCM complexes and their subcellular localizations Interactions within the CCM1, -2, -3 complex Consistent with their involvement in the same pathology, Krit1 ⁄ CCM1, CCM2 and CCM3 are able to interact Co-immunoprecipitations, glutathione S-transferase pull-downs and mutagenesis have allowed us to identify the interaction sites between the three proteins in this complex Endogenous or overexpressed Krit1 and CCM2 interact with each other [17,18] Mutations in the PTB domain of CCM2 on conserved residues critical for the NPXY ⁄ F motif binding (Fig 1) are deleterious for the Krit1–CCM2 interaction One, L198R, a single missense mutation was found in a CCM patient [10], the other, F217A, was engineered based on homology with 1086 E Faurobert and C Albiges-Rizo a known PTB domain [17] The N-terminus of CCM2 also takes part in this interaction because a lack of amino acid residues 11–68, an inframe deletion observed in patients [19], prevents the interaction of CCM2 with Krit1 [20] Conversely, the binding domain for CCM2 on Krit1 remains uncertain Because their interaction involves the CCM2 PTB domain, it is likely that the counterpart on Krit1 is one of its three NPXY ⁄ F motifs Indeed, a yeast twohybrid assay using small fragments of CCM2 centered on NPXY ⁄ F2 and -3 have identified these motifs as CCM2 interacting sites [18] However, single amino acid substitution in each of these motifs has no effect on the binding of Krit1 to CCM2 [17] Additional mutagenesis on residues immediately N- or C-terminal of the NPXY ⁄ F might be required to significantly reduce the affinity CCM2 interacts with CCM3 [16,20] but their respective interaction sites are not known None of the three CCM2 mutations cited above impairs its binding to CCM3 [20] showing that CCM2 binding domains for Krit1 and CCM3 are not redundant Indeed, the three overexpressed proteins form a complex CCM2 is the linker protein that brings together Krit1 and CCM3, which otherwise have no affinity for each other [16,20,21] Remarkably, this ternary complex was detected using proteomic approaches [21,22] However, CCM3 was also identified by proteomic analysis as a component of another large complex named striatininteracting phosphatase and kinase (STRIPAK) which assembles phosphatases and kinases arranged around a protein phosphatase 2A core [22] Interestingly, neither Krit1 nor CCM2 was detected in the STRIPAK complex, but small amounts of CCM3 could be pulleddown along with Krit1 on CCM2 beads This suggests that CCM3 associates with (at least) two different complexes; in substoichiometric amounts with the Krit1–CCM2 complex and in large amounts with the striatin-interacting phosphatase and kinase complex Shuttling of CCM proteins between the membrane and nucleus The in vitro data suggest that the three CCM proteins associate in a ternary complex in vivo, but they are also very likely engaged in several other complexes having different localizations (Fig 2) As such, Krit1 associates with the b1 integrin regulator integrin cytoplasmic adaptor protein-1 (ICAP-1; as discussed below) and this complex can shuttle between the cytosol and the nucleus Both Krit1 and ICAP-1 have a nuclear localization signal motif in their N-terminus and both localize in a nuclear localization signal- FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works E Faurobert and C Albiges-Rizo A Golgi Emerging signaling pathways regulated by CCM proteins B Microtubules Adherens junction formation and stabilization Cell polarity P ERM MST4 MST4 ? CCM3 ? Lkb1 p120 -catenin Krit1 AF6 Cell polarity Cadherin Rap1 Actin polymerization Membrane ruffles CCM2 RhoA degradation Adherens junction formation and stability Endothelial cell permeability p38MAPK Rac ? Actin stress fiber RhoA MEKK3 MKK3 RhoA CCM2 CCM2 Krit1 HEG Smurf1 Myocardiac cells distribution along endocardial-myocardial axis Cell-cell junctions CCM2 ? Krit1 Rap1 ? ICAP-1 Integrin Cdc42 Cell-matrix adhesion Cell migration ECM remodeling Tubulogenesis ECM mural cell recruitment Intracellular compartment Extracellular compartment Cell polarity Lumen Formation Fig Emerging signaling pathways and vascular processes controlled by the CCM proteins (A) Cadherins, HEG1 and integrins are three transmembrane receptors connected to CCM proteins or functions All three receptors are known to have roles in different steps of vessel morphogenesis Possible cross-talk between their dependent signaling pathways through CCM proteins are represented by arrows (B) CCM2 is a scaffold for small GTPases of the Rho family and for p38MAPK kinase It is involved in actin cytoskeleton remodeling through scaffolding of Rac, activation of the p38 MAPK kinase pathway and proteosomal degradation of RhoA CCM2 may also be involved directly or indirectly in Cdc42 activation As a result, cell–cell junctions, cell polarity and lumen formation are likely to be dependent on CCM2 signaling dependent manner to the nucleus of transfected cells [8,17] Interestingly, it has been shown that during cell spreading, ICAP-1 shuttles from the plasma membrane to the nucleus where it stimulates transcription and cellular proliferation [23] However, binding of CCM2 to Krit1 inhibits nuclear translocation of the Krit1– ICAP-1 complex Indeed, cotransfection of CCM2 with Krit1 and ICAP-1 induces the formation of a ternary complex between the three proteins that sequesters Krit1–ICAP-1 in the cytosol [8,17] The association of CCM2 with Krit1–ICAP-1 may therefore be a key event and the target of upstream signaling pathways to control Krit1–ICAP-1 transcriptional regulatory functions Transport along microtubules may be a way for Krit1 and its partners to shuttle between the cytoplasm and the nucleus Interestingly, a- and b-tubulins have been identified using proteomic analysis of proteins coimmunoprecipitating with flagged CCM2 in stably transfected macrophages [21] The presence of tubulin subunits in the pulled-down complex depended on CCM2–Krit1 interaction because a functional PTB domain was required on CCM2, suggesting that Krit1 is the direct partner of tubulin In fact, Krit1 has been shown to co-sediment with in vitro polymerized microtubules [7], and to co-localize with microtubules in bovine aortic endothelial cells [24] Two binding sites for microtubules have been mapped on Krit1: one which contributes the most to the binding overlaps with the nuclear localization signal sequence, the other lies in its last 50 amino acids PTB and FERM domains have structural features enabling their interaction with phosphoinositides in membranes As such, Krit1, CCM2 and CCM3 bind to phosphoinositides [7,21] Purified Krit1 binds to liposomes only when supplemented with phosphoinositides [7] Modeling of the Krit1 FERM domain using known structures has highlighted a basic cleft between the F1 and F3 subdomains which may interact with the negative charges of phosphate groups CCM2 and CCM3 also bind directly to phospholipids, as shown by overlay experiments on phosphatidylinositol phosphate arrays [21] CCM2 most likely interacts via its PTB domain The CCM3 lipid-interacting domain is not yet known CCM2 binds preferentially to monoover biphosphorylated phosphatidylinositols, a result also observed for Krit1 (our unpublished data) Conversely, CCM3 has a higher affinity for bi- and triphosphorylated phosphatidylinositols, an additional argument suggesting that Krit1 together with CCM2 might localize to different membrane compartments than CCM3 FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works 1087 Emerging signaling pathways regulated by CCM proteins New partners for the CCM proteins: what they tell us on putative regulated signaling pathways CCM proteins are expressed in many different cell types Thus, a crucial and intriguing question about the etiology of the cavernous malformations in blood vessels is to ask what is unique to endothelial cells Indeed, depletion of CCM2 targeted to the endothelium and not to the surrounding tissue results in vascular defects in mouse embryos [25,26] (see also Chan et al [27]) One possibility is that specific subsets of interactions occur in endothelial cells Even though many studies have not been conducted in endothelial cells, they have been very helpful in identifying new partners for CCM proteins As such, proteomic studies performed in macrophages and astrocytes have helped identify no fewer than 114 proteins interacting with CCM2 [21] Here, we review only the best-characterized partners which may give clues to the function of CCM proteins in vascular integrity An increasing amount of data indicates that CCM proteins are connected to the plasma membrane and regulate cell–cell adhesion, cell shape and polarity, and most likely cell adhesion to the extracellular matrix (Fig 2) This makes sense with regard to the phenotype of CCM lesions in which endothelial cells are joined loosely to each other, mural cells (i.e pericytes and astrocytes) are absent, and the basal lamina surrounding the endothelium is abnormal [2] Both cell– cell adhesion and cell polarity require the assembly of two specialized intercellular adhesion structures that regulate vascular permeability Adherens junctions initiate and maintain strong contacts between endothelial cells and promote tight junction assembly Tight junctions are specialized for the passage of ions and solutes through the paracellular route They may also act as a physical barrier along the cell surface allowing the asymmetrical distribution of proteins and lipids between apical and basolateral domains, a phenomenon known as cell polarization Cell adhesion to the extracellular matrix requires integrins clustered in highly dynamic adhesive structures which regulate cytoskeleton rigidity, extracellular matrix remodeling and probably cell–cell junctions Rap1, the master regulator of cell–cell and cell–extracellular matrix adhesion It has previously been established that the Ras family small G protein Rap1 stimulates cell adhesion to the extracellular matrix by activating integrins and cell–cell 1088 E Faurobert and C Albiges-Rizo adhesion by stimulating the formation and maintenance of adherens junctions It does so by activating a large number of effectors most of which are involved in regulating actin dynamics [28,29] Rap1 was the first reported Krit1 partner and was used as the bait to clone Krit1 in a yeast two-hybrid screen [4] This interaction was questioned until 2007 when two groups used biochemical in vitro assays [7] and functional studies [30] to confirm that Krit1 is a Rap1 effector However, Rap1 is not found in the CCM complex defined by proteomic analysis, suggesting that Rap1– Krit1 may form an independent complex Interestingly, Rap1a and -1b knockout mice show defective angiogenesis, characterized by delayed perinatal retinal vascularization, reduced microvessel sprouting from aortic rings in response to angiogenic factors or reduced neovascularization of ischemic hind limbs [31–33] Reduction of the function of Rap1b using morpholinos in zebrafish embryos disrupts endothelial junctions and provokes intracranial hemorrhage Importantly, a minor reduction in Rap1b, in combination with a similar reduction in Krit1 results in a high incidence of intracranial hemorrhage, whereas injection of each morpholino independently has almost no effect [34] This indicates that Rap1 and Krit1 act in a common molecular pathway Indeed, Glading et al [30] showed that small interfering RNA depletion of Krit1 blocks the ability of Rap1 to stabilize endothelial cell–cell junctions in culture cells [30] CCM partners in cell–cell junctions Proteins of adherens junctions Endogenous Krit1 localizes to cell–cell junctions on a bovine aortic endothelial cell confluent monolayer and co-immunoprecipitates with the Rap1 effector AF-6 ⁄ afadin, b-catenin and p120-catenin This localization requires a Krit1 FERM domain and is dependent upon activation of Rap1 [30] It has consistently been shown that in vitro Rap1 binding to the Krit1 FERM domain enhances the association of Krit1 with liposomes, most likely by inducing a conformational change in its basic pocket which gives Krit1 a better affinity for phosphoinositides [7] Depletion of Krit1 by small interfering RNA leads to disruption of b-catenin localization to adherens junctions and increases the permeability of the monolayer barrier [30], a phenotype reminiscent of that observed in human lesions Therefore, by localizing b-catenin to adherens junction, Krit1 is likely to be involved in the formation and maintenance of the endothelial barrier (Fig 2A) However, it is not yet known whether the Krit1–b-catenin interaction is direct FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works E Faurobert and C Albiges-Rizo The transmembrane glycosylated protein heart of glass Heart of glass (HEG1) is a transmembrane protein of unknown function bearing a large extracellular domain with two epidermal growth factor-like domains, a transmembrane segment and a short cytoplasmic tail (100 amino acids) with a conserved C-terminal NPXY ⁄ F motif (Fig 1) Its extracellular domain is predicted to be highly glycosylated It is expressed specifically in the endothelium and the endocardium No extracellular ligand is known HEG1 is the mammalian homolog of the zebrafish heart of glass Zebrafish heart of glass mutants show enlarged cardiac chambers resulting from improper distribution of myocardiac cells along the endocardial-to-myocardial axis [35] Two other genes, santa and valentine, functioning in the same molecular pathways, were identified and found to be Krit1 and CCM2, respectively They display the same phenotype as heart of glass when disrupted in zebrafish or when a combination of lowdose morpholinos against the three proteins is injected [36] Recently, HEG1 and CCM2 were also shown to interact genetically in the mouse [37] Indeed, Heg1) ⁄ );Ccm2lacZ ⁄ + [37] like Ccm2) ⁄ ) mice [25,26] have severe cardiovascular defects and die early in development owing to a failure of nascent endothelial cells to form patent vessels Both mice displayed shortened endothelial junctions compared with control littermates [37] More details can be found in the accompanying minireview on animal models of CCM disease [27] In addition, the ternary complex between HEG1, Krit1 and CCM2 has been demonstrated biochemically [37] (Fig 2A) A CCM2 mutant unable to bind Krit1 is not recruited in the HEG1–Krit1 complex, suggesting that Krit1 is the adaptor connecting CCM2 to the transmembrane receptor It is very likely that the association of HEG1 with Krit1 requires HEG1 NPXY ⁄ F motif and Krit1 FERM domain but this remains to be tested As a hint toward its function, HEG1 is evolutionary related to mucin 13 [38] Mucins are either secreted or inserted as transmembrane glycoproteins in polarized epithelia Transmembrane mucin can associate with fibroblast growth factor receptor [39] and b-catenin to activate b-catenin-driven transcription of Wnt target genes [40,41] Interestingly, an emerging idea concerning mucin function is that loss of polarity through a breach in the cell layer could enable growth factor receptors and mucins to associate and engage in signaling, which would activate gene transcription designed to repair the breach and re-establish cell polarity [42] This signaling pathway Emerging signaling pathways regulated by CCM proteins would make sense with regard to loss of the integrity of the endothelial barrier and a putative dysfunction of repair mechanisms in CCM lesions Consistent with this, Liebner et al [43] have shown that Wnt ⁄ bcatenin signaling is required for the endothelial cell expression of proteins necessary for the development of the blood–brain barrier [43] Therefore, under the control of HEG1, Krit1 and b-catenin may be involved in the dual role of stabilizing cell–cell junctions and regulating the expression of blood–brain barrier-specific players Partners in cell-shape remodeling and polarity Along with a role for Krit1 in cell–cell adhesion, a network of data identifies the CCM complex as a scaffold for the Rho family GTPases RhoA, Rac and Cdc42, and for mitogen-activated protein kinase (MAPK) and Ser ⁄ Thr kinases These proteins regulate endothelial cell shape and polarity How RhoA, Rac and Cdc42 interplay to orchestrate cell–cell junction formation and polarity is still under active investigation, and is reviewed in Iden & Collard [44] Nevertheless, emerging data suggest that CCM proteins are involved in the spatiotemporal tuning of these small GTPases and consequently are able to remodel the actin cytoskeleton (Fig 2B) CCM2 as a scaffold of actin cytoskeleton machinery CCM2 ⁄ OSM was first identified by two-hybrid screening as a scaffold for the MEKK3 ⁄ mitogen-activated protein kinase kinase (MKK)3 complex [11] which is needed to restore cell volume and shape in response to hyperosmotic shock p38 MAPK is a downstream substrate of MEKK3 MAPKs are ubiquitously expressed and contribute to a wide variety of cell responses to very diverse stimuli MAPKs are the terminal kinases in a three-kinase phospho-relay module, in which MAPKs are phosphorylated and activated by MKKs, which are themselves phosphorylated and activated by mitogen-activated protein kinase kinase kinase like MEKK3 [45] p38 MAPK is a critical kinase for long-term cellular adaptation to prolonged hyperosmotic exposure It regulates gene transcription and actin remodeling This pathway is conserved from yeast to mammals and in multiple tissues, suggesting its importance in cellular physiology beyond that of hyperosmolarity responses Indeed, the p38 MAPK pathway has also been shown to play an important role in angiogenesis Deletion of MEKK3 causes severe vascular defects [46], and defective angiogenesis in Rap1b-deficient mice FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works 1089 Emerging signaling pathways regulated by CCM proteins is associated with an impaired p38 MAPK signaling pathway [32] Moreover, p38 MAPK is required for the effect of vascular epidermal growth factor (vEGF) on actin remodeling in human vein umbilical endothelial cells [47] The p38 MAPK signaling pathway leads to the activation of heat shock protein 27, an F-actin cap-binding protein which in turn activates actin polymerization and stabilization It is proposed that CCM2 exists in a stable complex with MEKK3 Upon hyperosmotic stress, CCM2 and MEKK3 are recruited to membrane ruffles through direct interaction of CCM2 with Rac, where they co-localize with F-actin [11] Therefore, CCM2 may serve as a scaffold for the actin polymerization machinery (Fig 2B) A link between CCM2, Rac and MEKK3 has been confirmed by proteomic analysis of the CCM complex [21] Control of RhoA degradation and actin stress fibers formation by CCM2 More recently, the effects of the depletion of CCM2 on endothelial cell cytoskeletal architecture and signaling have been studied [26] CCM2 depletion by small interfering RNA leads to an increased number of actin stress fibers and enhanced permeability of the endothelial layer, a phenotype also observed upon depletion of Krit1 [30] In addition to Rac1, CCM2 also coimmunoprecipitates with RhoA CCM2 depletion leads to increased activated RhoA, whereas it has no effect on Rac1 activation [26] By contrast to hyperosmotic shock, CCM2 depletion does not affect p38 MAPK signaling but rather another MAPK module, i.e the c-Jun N-terminal kinase, MKK4, MKK7 pathway [26] c-Jun N-terminal kinase activation is blocked by the Rho-associated kinase inhibitor Y-27632 suggesting that CCM2 loss activates the c-Jun N-terminal kinase pathway through RhoA Therefore, a physiological function of CCM2 may be to limit RhoA activation Crose et al [48] recently gave a molecular explanation for this inhibitory effect by identifying the E3 ubiquitin ligase Smurf1 as a new CCM2 partner They showed by co-immunoprecipitation on overexpressed proteins that Smurf1 interacts with CCM2 through a PTB ⁄ NPXY interaction and that this interaction leads to loss of RhoA (Fig 2B) Proteosomal degradation is one of the modes used by cells to spatially restrict small G-protein signaling In particular, localized degradation of RhoA has already been involved in the control of cell polarity or migration [49,50] Importantly, HEG1, expressed only in endothelial cells, may be a long sought after piece of the puzzle 1090 E Faurobert and C Albiges-Rizo which gives the CCM pathway its endothelial-specific nature Interaction of Krit1 with HEG1 and VE-cadherin in the endothelial monolayer might create a physical link between these receptors to negatively control RhoA-dependent stress fiber formation and promote a Rac-dependent cell–cell junction Putative regulation of lumenogenesis by CCM2 via Cdc42 activation By contrast to Rac and RhoA, no interaction has been observed between CCM2 and Cdc42 However, depletion of CCM2 leads to less basal-activated Cdc42, implying that CCM2 is somehow involved in activating Cdc42 [26] In addition to its role in actin filament bundling during filopodia formation and cell migration, Cdc42 has a conserved role in regulating cell polarity in many eukaryotic cells, mainly by interaction with the polarity complex PAR (PAR6–PAR3– aPKC) Cdc42 affects cell–cell junction formation and the polarized trafficking of proteins to the apical and basal domains [51] Concomitant with a decrease in the level of activated Cdc42 [26], knockdown of CCM2 in human vein umbilical endothelial cells has been reported to decrease lumen formation in 3D in vitro culture [26,37] (Fig 2B) This is consistent with the previously described role of Cdc42 in lumenogenesis During capillary formation, endothelial cells assemble into chains, polarize and generate apical membrane vesicles via pinocytosis The intracellular vesicles then coalesce into an elongated vacuole-like structure spanning the length of the cell, which fuses with the plasma membrane to open to the exterior and establish luminal continuity with the next cell in the chain [52] Cdc42 and Rac1 are both required for lumenogenesis by involving Pak2, Pak4 and the PAR complex [53] Consistent with this, in CCM2-depleted mice or zebrafish, endothelial cells failed to organize in lumenized vessels However, endothelial vacuole-like structures form normally in the intersegmental vessels of zebrafish embryos lacking CCM2, as visualized using green fluorescent protein–Cdc42 to label these vacuoles [37] By contrast, CCM2-deficient human vein umbilical endothelial cells showed a strong decrease in vacuoles and lumen formation in a 3D in vitro culture [26] Whereas it is proposed in Kleaveland et al [37] that steps downstream of vacuole formation might be affected by the loss of CCM2 and lead to the absence of a lumen, the quantification of intracellular vacuoles in Whitehead et al [26] pinpoints a default at the level of vacuole formation Further experiments are needed to solve the discrepancy between these results FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works E Faurobert and C Albiges-Rizo Putative control of cell polarization by CCM3 through germinal center kinase III kinases Using yeast two-hybrid screen and proteomic analysis, serine ⁄ threonine kinase (STK)24, STK25 and mammalian sterile twenty-like (MST4) were identified as partners of CCM3 [15,16,22] These STKs belong to the germinal center kinase III (GCKIII) subfamily, and are related to the yeast protein kinase sterile 20 (Ste20) STK25 and MST4 bind at the N-terminus of CCM3 between Leu33 and Lys50 [54], a region removed by an inframe deletion of exon in a family of patients [12] CCM3 is phosphorylated by STK25 at Ser39 and Thr43 [54], but the role of this phosphorylation is not yet known Both STK25 and MST4 localize to the Golgi apparatus in unpolarized cells and regulate cell migration and polarity [55] Interestingly, MST1, a germinal center kinase kinase which interacts with the Rap1 effector RAPL, translocates from the Golgi on vesicles moving along microtubules aimed at assembling specialized plasma membrane domains such as leading edge during T-cell polarization [56] The recent connection of MST4 with Lkb1 function in cell polarity might help in understanding the role of CCM3 Lkb1 is a tumor suppressor gene responsible for Peutz–Jeghers syndrome, a cancer predisposition disorder characterized by gastrointestinal polyps Lkb1 regulates cell polarity in epithelial cells in a cell autonomous fashion ten Klooster et al [57] recently showed that, upon Lkb1 activation, MST4 translocates from the Golgi to the subapical domain of the epithelial cell near the brush border where it phosphorylates ezrin, a membrane–actin microfilaments linker necessary for normal microvilli Whereas Lkb1 seems to control MST4 subcellular localization, CCM3 might regulate MST4 kinase activity (Fig 2A) Indeed, it has been shown that CCM3 enhances MST4 activity in vitro [15] It would therefore be very interesting to place CCM3 in the newly described Lkb1 pathway and to check whether it also applies to endothelial polarization by regulating the function of ezrin radixin moesin proteins Interestingly, phosphorylated ezrin is localized to the cell–cell junction in endothelial cells and regulates junction formation and stability [58] Importantly, conditional Lkb1 deletion targeted to endothelial cells leads to embryonic death with loss of vascular smooth muscle cells (vSMCs) around the vessels and vascular disruption [59], a phenotype also observed in CCM lesions This phenotype is attributed to a loss of transforming growth factor-b production in endothelial cells and blocking of subsequent signaling to adjacent differenciating vSMCs Emerging signaling pathways regulated by CCM proteins Partners in cell–extracellular matrix adhesion The most recent articles strongly emphasize the role of CCM proteins on the formation of cell–cell junctions However, we think that a control of the interaction of endothelial cells with their surrounding environment should not be ruled out Indeed, ultrastructural analyses of CCM lesions clearly demonstrated the absence of perivascular ensheating cells or astrocytic foot processes around the vessel, and the presence of a thicker and multilayered collagenous matrix [2] Moreover and strikingly, no defects in cell junctions between endothelial cells was observed in zebrafish CCM1 and CCM2 mutants, but rather increased spreading of endothelial cell around dilated vessels [60] Finally, the first chronologically identified CCM partner, ICAP-1 is involved in regulating cell adhesion to the extracellular matrix ICAP-1 was identified as a Krit1 partner in a yeast two-hybrid screen and their interaction confirmed by co-immunoprecipitation [61,62] ICAP-1 is present in the CCM complex identified by proteomic analysis [21] Like CCM2, ICAP-1 has a C-terminal PTB domain linked to a short N-terminal moiety (60 amino acids) containing several consensus sites for kinases (Fig 1) The ICAP-1 PTB domain interacts with the first NPXY ⁄ F motif of Krit1 Importantly, a ternary complex can form between ICAP-1, Krit1 and CCM2 [17], suggesting that Krit1 may act as a scaffold for ICAP-1- and CCM2-dependent signaling pathways ICAP-1 inhibits b1 integrin activation and focal adhesion assembly Although its role in the CCM complex is not known, ICAP-1 has been well characterized as inhibitor of b1 integrin activation by talin ICAP-1 binds specifically to the b1 integrin cytoplasmic tail [63] Its overexpression in cells leads to disruption of b1 integrin focal adhesions, subsequent decreased cell adhesion to fibronectin and increased cell migration [64,65] ICAP-1 competes in vitro with talin for binding to b1 integrin Consistent with this, live cell imaging performed in Icap-1-deficient mouse embryonic fibroblasts confirmed that ICAP-1 inhibits the b1 integrin high-affinity state favored by talin, slows down the rate of focal adhesion assembly and controls matrix sensing [66] In addition, ICAP-1 interacts with Rho-associated kinase and recruits it to b1 integrin in the lamellipodia [67] The most evident phenotype of ICAP-1-deficient mice is their smaller size and weight, their craniofacial abnormalities and a general skeletal defect because of a reduced proliferation and differentiation defect in FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works 1091 Emerging signaling pathways regulated by CCM proteins osteoblast cells [68] In addition, C57Bl6 ICAP-1-deficient mice display a high rate of perinatal mortality (D Bouvard & R Fassler, personal communication) ă Whether ICAP-1-decient mice suffer from vascular defects is not yet known Importantly, depletion of Krit1 by small interfering RNA leads to the depletion of ICAP-1 in HeLa or human vein umbilical endothelial cells [69] This reduced level of ICAP-1 is not because of a downregulation of its mRNA [69], implying that ICAP-1 is stabilized upon its association with Krit1 This observation suggests that ICAP-1 might also be reduced in patients with a mutated CCM1 gene b1 Integrin regulates vascular morphogenesis: a target for CCM proteins? Ligand-activated integrins are essential to control intracellular actin cytoskeleton organization [70] and extracellular matrix remodeling [71] Mouse models have been very valuable in highlighting the role of b1 integrin in blood vessel morphogenesis Indeed, conditional deletion of b1 integrin in endothelial cells induces general vascular defects, including reduced branching and sprouting and is embryonic lethal [72–74] Interestingly, blood vessels are frequently discontinuous [73], cranial vessels are dilated [73,74] and sporadic large cerebral hemagiomas can be seen [74] Moreover, the staining of fibronectin (FN), a ligand of a5b1 integrin, is reduced and more diffused in mutant embryo basement membranes around the vessels [73] b1 integrin regulates several processes involved in vascular morphogenesis such as extracellular matrix remodeling and growth factor delivery, lumen formation and the recruitment of mural cells [75–77] Three-dimensional in vitro culture experiments and chorioallantoic membrane assays in chicken embryos have shown that FN fibrillogenesis is required for endothelial cell tubulogenesis [78] In vivo, FN fibrillogenesis is likely to be a a5b1 integrin-driven process resulting in extracellular FN organization in fibrils [71,79] which modulates environment rigidity Remarkably, at identical substrate densities, plating endothelial cells on rigid surfaces promotes cell–extracellular matrix interactions and endothelial cell dispersion, whereas plating endothelial cells on softer surfaces promotes cell–cell interactions and network formation [80] In addition, FN fibrillogenesis organizes the deposition of collagen [81] This regulates cell contractility and migration and might be crucial for proper tubulogenesis Moreover, organized matrix can tether soluble growth factors like vEGF or transforming growth factor-b and generate gradients that elicit endothelial 1092 E Faurobert and C Albiges-Rizo chemotactic responses It has been shown that matrixbound vEGF induces capillary sprouting with a small lumen, whereas soluble vEGF induces capillary hyperplasia and lumen enlargement [82] The major dilation observed in CCM lesions in humans may be a consequence of an incorrect growth factor gradient Lumenogenesis per se is another process possibly involving the b1 integrin family It is proposed that integrins signal to Rac and Cdc42 to activate vacuolization [76,83] Finally, b1 integrin promotes blood vessel maturation by stimulating the adhesion of mural cells to endothelial cells For example, a4b1 integrin on endothelial cells can interact with vascular cell adhesion molecule1, a transmembrane adhesion receptor present on mural cells to mediate apposition of the two cell types [84] Conversely, b1 integrin in pericytes is necessary for their correct spreading along the vessels [85,86] The defect in coverage with mural cells in CCM lesions might be a consequence of b1 integrin dysfunction either in endothelial or mural cells Because ICAP-1 regulates b1 integrin function, CCM proteins may regulate processes involving b1 integrin (Fig 2A) Interestingly, it has been reported using yeast two-hybrid assays that Krit1 can compete with b1 integrin for binding to ICAP-1 [62], suggesting that Krit1 may regulate the ICAP-1 inhibitory effect on b1 integrin Conversely, b1 integrin and ICAP-1 may regulate Krit1 functions on cell–cell adhesion These intriguing hypotheses need further work to be tested What about HEG1? The numerous HEG1 glycosylated moieties might bind to galactoside-binding lectins, named galectins, as mucins Upon binding to galectin-3, epithelial cell MUC1 clusters on the cell surface, possibly unraveling adhesion sites, and this leads to epithelial cell to endothelial cell binding [87] Moreover, galectin-3 has been reported to regulate a2b1 binding to collagen I and collagen IV [88] Consistent with this, early adhesion of cells to the extracellular matrix involving receptors other than integrins, for example proteoglycan or hyaluronan receptors, was reported to precede the formation of adhesive structures driven by integrins [89] Therefore, HEG1, together with integrins, may participate in a temporally regulated adhesion process to either extracellular matrix or mural cells Perspectives The last two years have been extraordinarily rewarding in that new avenues have opened for the comprehension FEBS Journal 277 (2010) 1084–1096 Journal compilation ª 2010 FEBS No claim to original French government works E Faurobert and C Albiges-Rizo of CCM protein physiology Although many hints about various signaling pathways have been collected, numerous gaps in the jigsaw puzzle persist, making it difficult to catch sight of the whole In future, effort will be needed to describe the cross-talk between these different pathways What stands out for now is that HEG1 may ignite endothelial-specific pathways involving CCM proteins necessary for the morphogenesis of blood vessels Putative molecular links between HEG1 and adherens junctions, on the one hand, and integrins, on the other hand, deserve to be thoroughly explored If molecular links between the two types of cell adhesion are found to involve CCM partners, they may lift the veil on the long known but poorly understood cross-talk between integrins and cadherins Emerging signaling pathways regulated by CCM proteins Acknowledgements We thank Olivier Destaing, Daniel Bouvard, Sophie ´ Beraud Dufour, and Mireille Faurobert for 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