Latrophilin 2 controls vascular morphogenesis and function by inhibiting endothelial cell adhesion and YAP/TAZ mechanosignaling
Chiara CamilloNicola FacchinelloGiulia VillariDafne GaysNoemi GioelliChiara SandriMarco AreseLuca TamagnoneDonatella ValdembriMassimo SantoroGuido Serini
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Dynamic modulation of endothelial cell (EC) adhesion to extracellular matrix (ECM) in response to mechanostimuli is essential for blood vessel patterning and functioning. Yet, the molecular mechanisms involved in this biological process are far to be completely deciphered. Here, we identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of vascular morphogenesis and endothelial barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM adhesions, signals through cAMP/Rap1, and, via its fibronectin-leucine-rich transmembrane (FLRT)-binding domain, negatively regulates ECM-elicited haptotaxis. ECs also express endogenous FLRT2 ligand that promotes cAMP/Rap1 signaling and hinders haptotaxis in a LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive Hippo mechanosensing pathway, and lack proper intercellular junctions. Indeed, intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.Keywords:
Rap1
Angiogenesis depends on specific molecular interactions between vascular cells and components of the extracellular matrix (ECM). This Perspective focuses on the functional role of integrins in angiogenesis and neovascularization. Specifically, we discuss the mechanism by which antagonists of αv integrins disrupt angiogenesis in vivo and how they may impact patients with cancer and inflammatory disease. Role of ECM and integrins during angiogenesis and vasculogenesis. Angiogenesis depends not only on growth factors and their receptors but is also influenced by receptors for ECM proteins. In general, cell adhesion to the ECM is mediated by integrins, heterodimeric transmembrane proteins that comprise a diverse family of over 15 α and 8 β subunits. Integrin subunits can heterodimerize in over 20 combinations. Different integrin combinations may recognize a single ECM ligand, while others bind several different ECM proteins. Integrin-mediated adhesion leads to intracellular signaling events that regulate cell survival, proliferation, and migration (1). These signals include elevation in intracellular pH and calcium, inositol lipid synthesis, and the tyrosine phosphorylation of a wide range of nonreceptor tyrosine kinases such as focal adhesion kinase and Src kinases, as well as adaptor proteins such as Shc, p130 CAS, and Crk II. These signaling events trigger a number of downstream signals, including activation of the Ras/mitogen-activated protein (MAP) kinase pathway (1).
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The extracellular matrix (ECM) is the non-cellular constituent of the tissues that, far from being an inert structural scaffold, provides biochemical and biomechanical cues that impact on cell behavior. Several reports have focused on the molecular systems by which the ECM interaction impacts on the Hippo signaling pathway to regulate YAP nuclear shuttling and its consequent co-transcriptional activity1,2.In the present work, we describe the mechanism by which the mechanotransducer YAP directly controls through its transcriptional activity both the deposition of extracellular matrix components and the assembly of the inner apparatus of cell-ECM interaction.In fact, by exploiting ChIP-seq technology and YAP mutants obtained by CRISPR/Cas9 targeted approach, we unveil a number of targets of YAP-DNA binding activity that lead to the formation of membrane complexes devoted to the interaction with ECM including various integrin subunits like ITGA1, ITGA4, ITGAVand ITGB1, talin2, cadherins and catenins. At the same time, YAP binds DNA elements connected to the activation of genes encoding for ECM structural proteins like versican, collagens, laminins, fibronectin and osteonectin or involved in the processing of ECM components, like hyaluronan synthase 3, connective tissue growth factor (CTGF) and metallopeptidases.As expected, YAP mutant clones underwent a substantial switch in the expression of genes involved in structural ECM composition and remodeling, thus leading to the complete absence of focal adhesions. As a consequence, cells failed to spread, invade and migrate through the surrounding matrix, when challenged in 2D and 3D assays and lose the ability to spread and acquire the given shape, develop tension through the cytoskeleton and exert force against the surrounding ECM.Consistent with the model of YAP acting as a master of cell-ECM interaction, cell biophysical parameters were partially recovered by the re-expression of ITGAV integrin subunit in conjunction with ITGB3 subunit, two of the proteins being more affected in YAP-defective cells.In conclusion, YAP functions as an important regulator of the cell-matrix interface, being able to control the expression of crucial genes involved in the compositionand arrangement of the extracellular environment, together with key components of cell mechanosome. Moreover, these results pave the way for the design of novel biomaterials controlling cell adhesion and ECM deposition by tuning YAP expression.References:1. D. Mosqueira, S. Pagliari, K. Uto et al. (2014), ACS Nano. 8(3):2033-472 NG. Kim and BM. Gumbiner (2015), J Cell Biol. 210(3): 503–515
Versican
Hippo signaling pathway
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Endothelial cells line blood vessels and provide a dynamic interface between the blood and tissues. They remodel to allow leukocytes, fluid and small molecules to enter tissues during inflammation and infections. Here we compare the signaling networks that contribute to endothelial permeability and leukocyte transendothelial migration, focusing particularly on signals mediated by small GTPases that regulate cell adhesion and the actin cytoskeleton. Rho and Rap GTPase signaling is important for both processes, but they differ in that signals are activated locally under leukocytes, whereas endothelial permeability is a wider event that affects the whole cell. Some molecules play a unique role in one of the two processes, and could therefore be targeted to selectively alter either endothelial permeability or leukocyte transendothelial migration.
Vascular permeability
Small GTPase
Cell Signaling
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Cell migration and adhesion to the extracellular matrix (ECM) are crucial in many biological and pathological processes such as morphogenesis, tissue repair, inflammatory responses, survival, and cancer. Cell-matrix adhesion is mediated by the integrin family of transmembrane receptors, which not only anchor cells to their surroundings, but also transmit bidirectional signalling at the cell surface and couple the ECM to the cytoskeleton. Another group of adhesion receptors are the syndecan proteoglycans, which engage the ECM and possess signalling activity in response to a variety of ligands. Cell migration is a complex process that requires spatial and temporal coordination of adhesion, cell contractility, intracellular traffic of integrins, and matrix turnover by matrix metalloproteinases (MMPs). Thus, integrins and syndecans, as well as MMPs, play essential roles in cancer cell migration and invasion. The understanding of the cooperation of syndecans and integrins was broadened in this thesis study. The results reveal that syndecan-1 functions in concert with α2β1 integrin in cell adhesion to collagen, whereas syndecan-4 is essential in α2β1 integrin-mediated matrix contraction. Finally, oncogenic K-Ras was shown to regulate α2β1 integrin, membrane-type 1 MMP, and syndecan-1 and -4 expression and their cooperation in cell invasion. Epithelial-mesenchymal transition (EMT) is fundamental during embryogenesis and organ development. Activation of EMT processes, including the upregulation of mesenchymal intermediate filament protein vimentin, has also been implicated in the acquisition of a malignant phenotype by epithelial cancer cells. Members of the protein kinase C (PKC) superfamily are involved in cell migration and various integrindependent cellular functions. One aim of this work was to shed light on the role of vimentin in the regulation of integrin traffic and cell motility. In addition, the mechanism by which vimentin participates in EMT was investigated. The results show that integrin recycling and motility are dependent on the PKCe–mediated phosphorylation of vimentin. In addition, vimentin was found to be a positive regulator of EMT and regulate the expression of several migratory genes. Specifically, vimentin governs the expression of receptor tyrosine kinase Axl, which is implicated in tumour growth and metastasis. Taken together, the findings described in this thesis reveal novel aspects of the complex interplay between distinct cellular components: integrins, syndecans, and the vimentin cytoskeleton, which all contribute to the regulation of human cancer cell adhesion, migration, and invasion.
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The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell-cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions. The vasculature is a dynamic organ that is constantly exposed to a variety of signaling stimuli and mechanical stresses. In embryogenesis, nascent blood vessels form via a process termed vasculogenesis, wherein mesodermally derived endothelial precursor cells aggregate into cords, which subsequently form a lumen that permits trafficking of plasma and erythrocytes. (1)(,) (2) Angiogenesis occurs after establishment of this primitive vascular network, where new vessels sprout from existing vessels, migrate into newly expanded tissues, and anastomose to form a functional and complex circulatory network. (1)(,) (2) In the mouse, this process occurs through the second half of embryogenesis and into postnatal development in some tissues, such as the developing retinal vasculature. (3) Further, angiogenesis occurs in a variety of pathological conditions, such as diabetic retinopathy, age-related macular degeneration, inflammatory diseases such as rheumatoid arthritis, wound healing, and tumor growth. (1)(,) (2)(,) (4) Both vasculogenesis and angiogenesis are driven through signaling by vascular endothelial growth factor (VEGF), and therapeutic agents targeting this pathway have shown efficacy in a number of diseases. (5)(-) (9) Blood vessels must have a sufficient degree of integrity so as to not allow indiscriminate leak of plasma proteins and blood cells into the underlying tissue. However, vessels must be able to sense their environment, respond to local conditions, and mediate the regulated passage of protein, fluid, and cells. For example, endothelial cells are the primary point of attachment for immune cells leaving the blood stream and entering tissue, and leukocytes subsequently migrate either through the endothelial cell body itself (the transcellular route), or through transient disassembly of cell-cell junctions (the paracellular route). (10) Precise regulation of endothelial junctions is critical to the proper maintenance of vascular integrity and related processes, and disruption of vascular cell-cell contacts is an underlying cause or contributor to numerous pathologies such as cerebral cavernous malformations (CCM) and hereditary hemorrhagic telangiectasia (HHT). (11)(-) (13) Understanding the basic mechanisms of endothelial junction formation and maintenance will therefore lead to a greater chance of success of therapeutic intervention in these pathologic conditions, especially in instances where targeting of VEGF signaling is insufficient to resolve vascular abnormalities.
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Acquisition of new genes encoding for extracellular matrix (ECM) proteins and their cognate integrin adhesive receptors, as well as secreted pro- and anti-angiogenic factors, proved to be essential for the development of functional vascular networks in the vertebrate embryo. There is now clear evidence that post-natal, pathological tissue neo-vascularization is crucial for cancer growth and therapy as well. Integrins are major ECM receptors that can exist in different functional states with respect to their affinity for ECM proteins. Regulation of integrin activation is crucial for their biological functions. In the embryo, the development of a properly patterned network of blood vessels relies upon the fine modulation of integrin activation by chemoattractant and chemorepulsive cues, such as angiogenic growth factors and semaphorins. Such a fine-tuning of endothelial integrin function is likely to be disrupted in cancer. Here, the vasculature is structurally and functionally abnormal and therefore inadequate for an efficient drug and oxygen delivery, which is a mandatory pre-requisite for successful chemotherapy and radiotherapy. It is thus important to identify the molecular mechanisms that regulate integrin function in normal ECs and which are altered in tumor ECs.
Lymphangiogenesis
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Cell Signaling
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In the current review, we summarize recent progress on vasculature-specific function and regulation of integrins and integrin-associated proteins, including advances in our understanding of inside-out integrin activation. The studies on regulation of integrin activation received new impulse in 2009 with the identification of kindlin protein family members as crucial mediators of integrin inside-out signaling. In the current review, we outline the recent findings on the role of kindlins in the vascular system, as well as new studies that have begun shaping the mechanistic model of kindlins' function.Several tissue-specific knockout models for integrins and genes associated with the integrin functions have been recently presented, including smooth muscle-specific integrin-linked kinase and endothelial-specific focal adhesion kinase and talin-1 ablation. In the heterozygous animal knockout model, kindlin-2 has been demonstrated as a crucial modulator of angiogenesis and vascular permeability. As a number of articles have advanced our understanding of kindlin function, they are reviewed and discussed in further detail. New findings include an additional lipid-binding site within the kindlin molecule and preferential binding of the nonphosphorylated form of β-integrins.The role of integrins in angiogenesis has been demonstrated to include, in addition to cell adhesion and mechanotransduction, specific signaling functions. The importance of integrin inside-out pathway in vascular physiology has been unequivocally proven, and endothelial permeability is directly regulated by this process. Inhibition of kindlin-dependent steps in the inside-out pathway as an approach to block platelet aggregation should be paralog-specific, as it may have adverse effects on vascular permeability.
Mechanotransduction
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Adherens junction
Crosstalk
VE-cadherin
Barrier function
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Cell–cell interaction
Cell Signaling
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