ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis.     

Structural basis of the junctional anchorage of the cerebral cavernous malformations complex

The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K_d = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr^1,380-Phe^1,381), thus defining a new mode of FERM domain–membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM–membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.     

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3–0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function.In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells.Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.     

Structural Basis for the Disruption of the Cerebral Cavernous Malformations 2 (CCM2) Interaction with Krev Interaction Trapped 1 (KRIT1) by Disease-associated Mutations

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1^NPX(Y/F)3, revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1^NPX(Y/F)3. We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.     

VEGF signalling enhances lesion burden in KRIT1 deficient mice

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial‐specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.     

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.     

Cancer-secreted exosomal miR-21-5p induces angiogenesis and vascular permeability by targeting KRIT1

Cancer-secreted exosomes are critical mediators of cancer-host crosstalk. In the present study, we showed the delivery of miR-21-5p from colorectal cancer (CRC) cells to endothelial cells via exosomes increased the amount of miR-21-5p in recipient cells. MiR-21-5p suppressed Krev interaction trapped protein 1 (KRIT1) in recipient HUVECs and subsequently activated β-catenin signaling pathway and increased their downstream targets VEGFa and Ccnd1, which consequently promoted angiogenesis and vascular permeability in CRC. A strong inverse correlation between miR-21-5p and KRIT1 expression levels was observed in CRC-adjacent vessels. Furthermore, miR-21-5p expression in circulating exosomes was markedly higher in CRC patients than in healthy donors. Thus, our data suggest that exosomal miR-21-5p is involved in angiogenesis and vascular permeability in CRC and may be used as a potential new therapeutic target.Subject terms: Cancer microenvironment, Colon cancer     

KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling

Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1^+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.     

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed.Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms.We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner.As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.     

Sorting nexin 17, a non-self-assembling and a PtdIns(3)P high class affinity protein, interacts with the cerebral cavernous malformation related protein KRIT1

The mammalian sorting nexin (SNX) proteins are involved in the endocytosis and the sorting machinery of transmembrane proteins. Additionally to the family defining phox homology (PX) domain, SNX17 is the only member with a truncated FERM (4.1, ezrin, radixin, and moesin) domain and a unique C-terminal region (together designated as FC unit). By gel filtration and lipid overlay assays we show that SNX17 is a non-self-assembling and a PtdIns(3)P high class affinity protein. A SNX17 affinity to any other phosphoinositides was not detected. By yeast two-hybrid- and GST-trapping assays we identified KRIT1 (krev1 interaction trapped 1) as a new specific interaction partner of the FC unit of SNX17. KRIT1 binds SNX17 by its N-terminal region like the known interaction partner ICAP1alpha (integrin cytoplasmic domain-associated protein-1). The interaction was also detected in HEK 293 cells transiently expressing GFP-tagged KRIT1 and Xpress-tagged SNX17. KRIT1 mutations cause cerebral cavernous malformation (CCM1). Our finding suggests a SNX17 involvement in the indicated KRIT1 function in cell adhesion processes by integrin signaling.     

Proteomic identification of the cerebral cavernous malformation signaling complex

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.     

一中国脑海绵状血管瘤家系中发现krit1基因新的缺失突变

脑海绵状血管瘤（CCM）是多定位于中枢神经系统的一种脑部血管异常,少数在皮肤和视网膜处有并发症。依据致病基因在染色体上的不同位置分为CCM1、CCM2和CCM33种类型。目前,CCM1、CCM2和CCM3的致病基因已经被克隆,分别为krit1、MGC4607和细胞程序性死亡10基因（PDCD10）。利用连锁分析发现内蒙古的一个家系属于CCM1,突变检测发现患者CCM1基因（krit1）第9内含子和第10外显子拼接位点处存在一“GTA”缺失,该突变导致终止密码子提前出现,产生截短蛋白。实验结果支持krit1为CCM1致病基因。     

MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.     

Amplification of protease-activated receptors signaling in sporadic cerebral cavernous malformation endothelial cells

In the central nervous system, thrombin-mediated activation of protease-activated receptors (PARs) results in neuroinflammation and increased vascular permeability. These events have been linked to cancer and neurodegeneration. Endothelial cells (ECs) isolated from sporadic cerebral cavernous malformation (CCM) specimens showed dysregulation of genes involved in “thrombin-mediated PAR-1 activation” signaling. CCM is a vascular disease involving brain capillaries. In CCM, ECs show defective cell junctions. Oxidative stress and neuroinflammation play a key role in disease onset and progression. In order to confirm the possible role of thrombin pathway in sporadic CCM pathogenesis, we evaluated PARs expression in CCM-ECs. We found that sporadic CCM-ECs overexpress PAR1, PAR3 and PAR4, together with other coagulation factor encoding genes. Moreover, we investigated about expression of the three familial CCM genes (KRIT1, CCM2 and PDCD10) in human cerebral microvascular ECs, following thrombin exposure, as well as protein level. Thrombin exposure affects EC viability and results in dysregulation of CCM gene expression and, then, in decreased protein level. Our results confirm amplification of PAR pathway in CCM suggesting, for the first time, the possible role of PAR1-mediated thrombin signaling in sporadic CCM. Thrombin-mediated PARs over activation results in increased blood-brain barrier permeability due to loss of cell junction integrity and, in this context, also the three familial CCM genes may be involved.     

Structural basis for small G protein effector interaction of Ras-related protein 1 (Rap1) and adaptor protein Krev interaction trapped 1 (KRIT1)

Cerebral cavernous malformations (CCMs) affect 0.1–0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Å co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain “modules.”     

Signalling through cerebral cavernous malformation protein networks

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to haemorrhages, stroke, epilepsy and focal neurological deficits. CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2 or CCM3. These widely expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and (either alone or in complex) modulate signalling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. Recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease. Here, we review current knowledge of CCM protein signalling with a focus on three pathways which have generated the most interest—the RhoA–ROCK, MEKK3–MEK5–ERK5–KLF2/4 and cell junctional signalling pathways—but also consider ICAP1-β1 integrin and cdc42 signalling. We discuss emerging links between these pathways and the processes that drive disease pathology and highlight important open questions—key among them is the role of subcellular localization in the control of CCM protein activity.