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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., Ministry of Science and Innovation/Fondo Europeo de Desarrollo Regional (MICINN/FEDER), grant numbers PID2019-110132RB-I00/AEI/10.13039/501100011033 (to M.L.-C.) and SAF2016-77096-R (to C.C.), and by Consejo Superior de Investigaciones Científicas (CSIC), grant number 2020AEP018 (to C.C.)

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 α6β1 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 β1 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 β-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., Recognition of laminin by integrin receptors mediates epithelial cell adhesion to basement membrane. Here, the structures of the α6β1 integrin alone and in complex with three-chain laminin-511 fragment reveal the laminin-integrin interface in molecular detail.

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.

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.

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., German Research Foundation (Deutsche Forschungsgemeinschaft, DFG FL 696/3-1 for LF and DFG Project Number 125440785-SFB877 (A4) for KR). Johannes Strunk gratefully acknowledges financial support from the Max Planck Graduate Center with the University of Mainz

The phagocytic integrins and complement receptors &alpha, M&beta, 2/CR3 and &alpha, X&beta, 2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of &beta, 2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

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., Ministerio Español de Economía y Competitividad (MINECO) and by a grant from Fundación Ramón Areces “Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014” to MY-M; and by SAF2016-77096-R from MINECO

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3∂-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ϵ sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells., Ministry of Economy and Competitiveness (MINECO) with research grants SAF2012-33218 and SAF2016-75656-P. The later from the Agencia Estatal de investigación cofounded by “fondo Europeo de desarrollo regional (FEDER)

© 2015 Elsevier B.V. The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 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 β2 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., SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO

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., This work has been supported by grant SAF2016-77096-R from Ministerio Español de Economía y Competitividad (MINECO), awarded to CC. BC salary has been supported by a grant from Fundación Ramón Areces (Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014) and by the grant SAF2016-77096-R from MINECO. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 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, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-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 α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation., This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.

We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1. On the surface of K562 cells, α5β1 is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains. ![][1] Thermodynamic analysis of the three integrin α5β1 conformation states offers a model for signaling regulation via an interplay between extracellular and transmembrane/intracellular domains in a large class of receptors built from multiple domains. [1]: /embed/graphic-1.gif

Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories, This work was supported by grants BFU2014-55478-R from Ministerio de Economía y Competitividad, and from Fundación Ramón Areces to MY-M; grants PI13/00050 from the “Fondo de Investigación Sanitaria” (FISISCIII), 2014SGR804 from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la“Generalitat de Catalunya” and REDinREN (16/0009 Feder Funds) to FEB; BFU2014-54181-P from Ministerio de Economía y Competitividad to JLC, and by SAF2016-77096-R from Ministerio de Economía y Competitividad to CC. H.S. was supported by a FPI-UAM and GEIVEX Mobility fellowship; AGV is sponsored by a Grant (482/U/2014) from Fundació La Marató TV3; FEB is sponsored by the “Researchers Stabilization Program” from the Spanish “Sistema Nacional de Salud” (SNS-ISCIII) and “Direcció d’Estratègia i Coordinació”, Catalan Health Dept. (CES07/015) and MY-M by Ramón y Cajal contract RYC-2012-11025

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 β1-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. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., Ministerio de Economıa y Competitividad (SAF2010–21249); Comunidad Autonoma de Madrid (S2010/BMD-2321); Ministerio de Economıa y Competitividad (SAF2010-18733); Instituto de Salud Carlos III (PI10/00101); Fondo de Investigaciones Sanitarias (FIS); Fundacion Mutua Madrilena; Digna-Biotech; Asociacion Espanola Contra el Cancer; Fundación Ramón Areces

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naive T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

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 endoglincoated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5β1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5β1 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. © 2013 by The American Society of Hematology., Ministerio de Economia y Competitividad of Spain (SAF2010-61 827, SAF2010-15 881); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER); Red de Investigacion Cooperativa en Enfermedades Renales (REDINDEN)

Whether β integrin ectodomains visit conformational states similarly to β and β integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α β ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α or β legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swingout. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α β is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α β on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms., NIH Grant HL-108248

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.

TEMs (tetraspanin-enriched microdomains) are specialized platforms in the plasma membrane that include adhesion receptors and enzymes. Insertion into TEMs dictates the local concentration of these molecules, regulates their internalization rate, their interaction and cross-talk with other receptors at the plasma membrane and provides links with certain signalling pathways. We focus on the associations described for tetraspanins with membrane proteases and their substrates, reviewing the emerging evidence in the literature that suggests that TEMs might be essential platforms for regulating protein shedding, RIP (regulated intramembrane proteolysis) and matrix degradation and assembly.

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-a and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-a and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17, s. This work was supported by grants BFU2007-66443/BMC and BFU2010-19144/BMC from Ministerio de Ciencia e Innovación, a grant from Fundación de Investigación Médica Mutua Madrileña and by RETICS Program RD08/0075-RIER (Red de Inflamacio´n y Enfermedades Reumáticas) from Instituto de Salud Carlos III (to C.C.), a grant from Fundación de Investigación Médica Mutua Madrileña (to M.D.G.L.), and grants PI080794 from Instituto de Salud Carlos III (to M.Y-M) and SAF2007-60578 from Ministerio de Ciencia e Innovación (to E.M.L.). M.D.G.L. was supported by a contract associated to grant SAF2004-01715 from Ministerio de Ciencia e Innovacio´n. S.O. was supported by an I3P predoctoral Fellowship from Consejo Superior de Investigaciones Científicas (CSIC) and by a contract associated to grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovacio´n. A.G. has been supported by a predoctoral Fellowship from Instituto de Salud Carlos III and by grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovación.

The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Resumen Las tetraspaninas son moleculas de la superficie celular de amplia distribucion en los organismos eucarioticos. Poseen como caracteristica estructural peculiar cuatro dominios transmembranales, regiones N- y C-terminales intracitoplasmicas, y dos lazos extracelulares de distinto tamano. Tambien poseen un motivo de secuencia CCG en el lazo extracelular mayor, asi como residuos polares conservados en los dominios transmembranales. Las celulas sanguineas de los mamiferos expresan combinaciones peculiares de distintas tetraspaninas, incluyendo los antigenos de diferenciacion CD9, CD37, CD53, CD81/TAPA-1, CD82, CD151/PETA-3 y CD231/TALLA1. En este trabajo se resumen la estructura y las interacciones de sus regiones citoplasmicas con proteinas del citoesqueleto y senalizadoras, como la proteina cinasa C (PKC) o la Fosfatidil-Inositol 4-cinasa (PI4-K). Sus interacciones especificas con otras tetraspaninas, con integrinas, antigenos de histocompatibilidad, y miembros de la superfamilia de las inmunoglobulinas son tambien revisadas. Las tetraspaninas son proteinas “adaptadoras” o “facilitadoras”. Al formar parte de complejos moleculares, modulan funciones celulares clave que incluyen la fusion celular, la adhesion, la migracion, la diferenciacion y la transduccion de senales. Las tetraspaninas se organizan en una red con distintos niveles de asociacion, determinados por su resistencia a la solubilizacion por detergentes. En concreto, se analizan las tetraspaninas como reguladoras del Sistema Inmunitario gracias a sus interacciones con los receptores de antigeno de los linfocitos T y B, las moleculas de histocompatibilidad de clase I y clase II, y los co-receptores CD2, CD4, CD5, CD8 y CD19. Por ultimo, se revisa detalladamente el papel de la tetraspanina CD9 en la funcion de las celulas linfoides y mieloides, su relevancia en infecciones como el HIV, y la importancia de su asociacion con integrinas en la progresion cancerosa.

11 páginas, 8 figuras -- PAGS nros. 746-756, α4β1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. α4β1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti- β1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of α4β1 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, α-CD3 mAb or the chemokine SDF-1α followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (α4β1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the α4β1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of α4β1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements

Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9–large extracellular loop (LEL)–glutathione S–transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.

OBJECTIVE: The purpose of this study was to examine the relationship between alpha4beta1-integrin state of activation on CD4+ T-cell subsets and their adhesive interaction to VCAM-1 under flow. METHODS: Human CD4+ memory and naive T-cells were freshly isolated and effector-helper T-cell subsets. Th1 and Th2 cells, were differentiated in vitro from CD4+ naive T-cells. The expression of activation/ligand induced epitopes on beta1-integrins of each T-cell subset was assessed using mAb HUTS21 and mAb 15/7. T-cell subsets attachment and rolling on VCAM-1 was determined under defined flow conditions and the rates of attachment (ka), accumulation, and instantaneous rolling velocities were correlated to their beta1-integrin activation epitope expression. RESULTS: A subset of memory T-cells constitutively express activation/ligand induced epitopes on beta1-integrins recognized by mAb HUTS21 and 15/7, whereas expression levels on naive T-cells is low or not detectable. Consistent with an activated phenotype, memory T-cells exhibit significantly higher rates of attachment and accumulation on VCAM-1 under flow as compared to naive T-cells. Interestingly, the expression of activation/ligand induced epitopes on beta1-integrins on Th2 cells and the ability of these cells to interact with VCAM-1 are comparable to memory T-cells. In contrast, Th1 cells did not interact as efficiently with VCAM-1, which correlated with lower expression of activation/ligand induced epitopes on these cells. VCAM-1 interactions are inhibited completely by pretreatment of the T-cells with blocking mAb to alpha4-integrins or beta1-integrins, indicating that alpha4beta1 is the predominant T-cell integrin involved. CONCLUSIONS: Memory T-cells express constitutively active alpha4beta1-integrins, as compared to naive T-cells, which mediate high rates of initial attachment and sustained high-affinity adhesive interactions with VCAM-1 under flow conditions in vitro. Similarly, in vitro differentiated Th2 cells but not Th1 cells, which also express elevated levels of activated alpha4beta1-integrins, are capable of sustaining high-affinity adhesive interactions with VCAM-1. The differences observed in beta1-integrin activation on T-cell subsets may underlie selective recruitment patterns of T-cell subsets in vivo.

The relationship between activation-dependent changes in beta1 integrin conformation, T cell adhesion to immobilized fibronectin, and T cell migration in vitro was analyzed in this study. Stimulation of Jurkat T cells and peripheral T cells with Mn(2+), the activating beta1 integrin-specific monoclonal antibody (mAb) TS2 /16, CD2, or CD28 stimulation led to increased adhesion, soluble fibronectin (FN) binding and expression of the activation epitope defined by the beta1 integrin mAb HUTS-21. Phorbol 12-myristate 13-acetate treatment increased adhesion, but not soluble FN binding or HUTS-21 epitope expression. In peripheral T cells, CD3 or CD7 stimulation also led to increased adhesion, soluble FN binding and HUTS-21 epitope expression. Soluble FN blocked peripheral T cell adhesion induced by Mn(2+) or TS2/16, but had no effect on adhesion induced by the other integrin-activating signals. In contrast, migration induced by TS2/16, CD2, CD3, CD7 or CD28 stimulation was blocked by excess soluble FN. Phosphoinositide 3-OH kinase (PI 3-K) inhibitors blocked receptor-mediated increases in cell adhesion, but not soluble FN binding or HUTS-21 expression. Migration was similarly unaffected by PI 3-K inhibitors, with the exception of CD7- and CD28-induced migration, which was specifically blocked by LY294,002. These results suggest that activation-dependent changes in beta1 integrin conformation are PI 3-K-independent and are involved in T cell migration but not adhesion.

Natural killer (NK) cell activation is the result of a balance between positive and negative signals triggered by specific membrane receptors. We report here the activation of NK cells induced through the transmembrane glycoprotein CD43 (leukosialin, sialophorin). Engagement of CD43 by specific antibodies stimulated the secretion of the chemokines RANTES, macrophage inflammatory protein (MIP)-1, and MIP-1β, which was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, signaling through CD43 increased the cytotoxic activity of NK cells and stimulated an increase in the tyrosine kinase activity in antiphosphotyrosine immune complexes of NK cell lysates. PYK-2 was identified among the tyrosine kinase proteins that become activated. Hence, PYK-2 activation was observed after 20 minutes of CD43 stimulation, reached a maximum after 45 to 60 minutes, and decreased to almost basal levels after 120 minutes of treatment. Together, these results demonstrate the role of CD43 as an activation molecule able to transduce positive activation signals in NK cells, including the regulation of chemokine synthesis, killing activity, and tyrosine kinase activation.

Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex, highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant vascular disorder caused by a haploinsufficiency mechanism. Vascular lesions (telangiectasia and arteriovenous malformations) in HHT1 are associated with loss of the capillary network, suggesting the involvement of endoglin in vascular repair processes. Using the chick chorioallantoic membrane (CAM) as an angiogenic model, we have analyzed the expression and function of chicken endoglin. A pan-specific polyclonal antibody (pAb) recognized chicken endoglin as demonstrated by immunostaining and Western blot analysis. In ovo treatment of chicken embryos with this pAb resulted in a significantly increased area of CAM. This effect was likely mediated by modulation of the ligand binding to endoglin as this pAb was able to inhibit TGF-beta1 binding. These results support the involvement of endoglin in the angiogenic process.

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic α4β1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial α4β1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of β1integrins, particularly those associated with α5 integrins.Activation of β1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated β1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated β1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis. J. Clin. Invest. 103:613–625 (1999)

Objective To study the effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the adhesion of peripheral blood lymphocytes (PBL) to activated human umbilical vein endothelial cells (HUVEC) under conditions that resemble blood flow. Methods Assays of adhesion of PBL to HUVEC or recombinant vascular cell adhesion molecule 1 (rVCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin were performed under continuous rotation at 37°C. The phenotype of PBL subpopulations attached was characterized by flow cytometry. Lymphocytes were pretreated with different doses (5-100 µg/ml) of aceclofenac, diclofenac, indomethacin, or piroxicam or with inhibitory monoclonal antibodies (MAb) prior to the adhesion assays. The effect of NSAIDs on lymphocyte adhesion molecules was assessed by flow cytometry. To determine whether NSAIDs interfere with the affinity state of very late activation antigen 4 (VLA-4) integrin, we studied the effect of these drugs on the appearance of a β1 activation-dependent epitope recognized by the HUTS21 MAb both on human T lymphoblasts and on synovial fluid lymphocytes (SFL). Results In the flow-resembling model, PBL-HUVEC adhesion was mainly mediated by the VLA-4/VCAM-1 adhesion pathway. The major PBL subset attached was the CD3+, CD45RO+ memory T cell, with CD49dhigh expression. Aceclofenac, diclofenac, and indomethacin, but not piroxicam, were able to inhibit PBL adhesion to HUVEC or rVCAM-1. However, the quantitative expression of VLA-4 was not affected by treatment of PBL with any of the NSAIDs studied. On T lymphoblasts and SFL, mostly CD45RO+ cells, the expression of the β1 activation-dependent epitope detected by HUTS21 MAb was significantly decreased by aceclofenac, diclofenac, and indomethacin. Conclusion Some NSAIDs are able to inhibit the adhesion of PBL to HUVEC under conditions that resemble blood flow by interfering with the conformational change in VLA-4 that increases its affinity for VCAM-1.

Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

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. © Springer Basel AG 2012., MICINN BFU2010-19144/BMC y SAF2012-34561; Fundación Médica Mutua Madrileña; the RETICS Program RD08/0075-RIER from Instituto de Salud Carlos III; Fundación MAPFRE

It is known that the chemokine receptor CXCR7 (RDC1) can be engaged by both chemokines CXCL12 (SDF-1) and CXCL11 (I-TAC), but the exact expression pattern and function of CXCR7 is controversial. CXCR7 expression seems to be enhanced during pathological inflammation and tumor development, and emerging data suggest this receptor is an attractive therapeutic target for autoimmune diseases and cancer. CXCR7/CXCR4 heterodimerization, β-arrestin-mediated signaling, and modulation of CXCL12 responsiveness by CXCR7 suggest that the monogamous CXCR4/CXCL12 signaling axis is an oversimplified model that needs to be revisited. Consequently, research into CXCR7 biology is of great interest and further studies are warranted. This review summarizes recent findings about the CXCR7 receptor and analyses its impact on understanding the roles of CXCL12 biology in health and disease. © 2012 Elsevier Ltd., Ministerio de Ciencia e Innovacion (BFU2010-19144); Instituto de Salud Carlos III-RedesTematicas de Investigacion Cooperativa en Salud (ISCIII-RETICS) RD08/0075/0002

Cells communicate with their environment through several kinds of cell surface receptor. One of the most important families of cell adhesion receptors are the integrins, which include receptors that mediate cell-cell as well as cell-extracellular matrix interactions. A distinctive feature of integrins is their variable adhesive competence that is reversibly modified depending on the state of cell differentiation and/or activation or in response to environmental signals. The acquisition of adhesive function by integrins may be a consequence of conformational changes in these receptors that result in an increased ligand binding affinity. In addition, cells can control integrin-mediated adhesion through other mechanisms, including receptor clustering and association to cytoskeleton, phenomena that regulate the avidity of integrins for ligand molecules without altering their monovalent affinity. These phenomena have collectively been designated as 'post-receptor occupancy events'. These two interesting aspects of the regulation of integrin function are reviewed.