Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5ß1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling.

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

ALCAM/CD166 Is Involved in the Binding and Uptake of Cancer-Derived Extracellular Vesicles.

Colorectal cancer (CRC) and ovarian cancer (OvC) patients frequently develop peritoneal metastasis, a condition associated with a very poor prognosis. In these cancers, tumor-derived extracellular vesicles (EVs) cause immunosuppression, facilitate the direct attachment and invasion of cancer cells through the mesothelium, induce the conversion of peritoneal mesothelial cells (PMCs) into cancer-associated fibroblasts (CAFs) and transfer a more aggressive phenotype amongst cancer cells. Although the promoting role of EVs in CRC and OvC peritoneal metastasis is well established, the specific molecules that mediate the interactions between tumor-derived EVs and immune and non-immune target cells remain elusive. Here, we employed the SKOV-3 (ovarian adenocarcinoma) and Colo-320 (colorectal adenocarcinoma) human cell lines as model systems to study the interactions and uptake of EVs produced by ovarian carcinoma and colorectal carcinoma cells, respectively. We established that the adhesion molecule ALCAM/CD166 is involved in the interaction of cancer-derived EVs with recipient cancer cells (a process termed "EV binding" or "EV docking") and in their subsequent uptake by these cells. The identification of ALCAM/CD166 as a molecule mediating the docking and uptake of CRC and OvC-derived EVs may be potentially exploited to block the peritoneal metastasis cascade promoted by EVs in CRC and OvC patients.

Mesothelial-to-Mesenchymal Transition and Exosomes in Peritoneal Metastasis of Ovarian Cancer.

Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.

Cellular Integrin α5ß1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells.

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size-exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5ß1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

Structural mechanism of laminin recognition by integrin.

Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6ß1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin ß1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the ß-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.

Phagocytic Integrins: Activation and Signaling.

Phagocytic integrins are endowed with the ability to engulf and dispose of particles of different natures. Evolutionarily conserved from worms to humans, they are involved in pathogen elimination and apoptotic and tumoral cell clearance. Research in the field of integrin-mediated phagocytosis has shed light on the molecular events controlling integrin activation and their effector functions. However, there are still some aspects of the regulation of the phagocytic process that need to be clarified. Here, we have revised the molecular events controlling phagocytic integrin activation and the downstream signaling driving particle engulfment, and we have focused particularly on αMß2/CR3, αXß2/CR4, and a brief mention of αVß5/αVß3integrins.

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway.

Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.

Development of a quantitative method to measure EV uptake.

The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.

CD9 Controls Integrin α5ß1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17.

Integrin α5ß1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5ß1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5ß1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5ß1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5ß1-mediated cell adhesion to its ligands fibronectin and ADAM17.

Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression.

Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs) that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and disease.

Tetraspanin CD9: A Key Regulator of Cell Adhesion in the Immune System.

The tetraspanin CD9 is expressed by all the major subsets of leukocytes (B cells, CD4+ T cells, CD8+ T cells, natural killer cells, granulocytes, monocytes and macrophages, and immature and mature dendritic cells) and also at a high level by endothelial cells. As a typical member of the tetraspanin superfamily, a prominent feature of CD9 is its propensity to engage in a multitude of interactions with other tetraspanins as well as with different transmembrane and intracellular proteins within the context of defined membranal domains termed tetraspanin-enriched microdomains (TEMs). Through these associations, CD9 influences many cellular activities in the different subtypes of leukocytes and in endothelial cells, including intracellular signaling, proliferation, activation, survival, migration, invasion, adhesion, and diapedesis. Several excellent reviews have already covered the topic of how tetraspanins, including CD9, regulate these cellular processes in the different cells of the immune system. In this mini-review, however, we will focus particularly on describing and discussing the regulatory effects exerted by CD9 on different adhesion molecules that play pivotal roles in the physiology of leukocytes and endothelial cells, with a particular emphasis in the regulation of adhesion molecules of the integrin and immunoglobulin superfamilies.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4ß1.

Stimulation by chemokines of integrin α4ß1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4ß1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4ß1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4ß1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4ß1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4ß1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4ß1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4ß1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9.

The tetraspanin CD9 has been shown to interact with different members of the ß1 and ß3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the ß2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation.

Carcinoma-associated fibroblasts derive from mesothelial cells via mesothelial-to-mesenchymal transition in peritoneal metastasis.

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a ß1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization.

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis.

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2'-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration.

Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5ß1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5ß1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.