Olga Barreiro

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes

Membrane lipids and proteins are non-randomly distributed and are unable to diffuse freely in the plane of the membrane. This is because of multiple constraints imposed both by the cortical cytoskeleton and by the preference of lipids and proteins to cluster into diverse and specialized membrane domains, including tetraspanin-enriched microdomains, glycosylphosphatidyl inositol-linked proteins nanodomains and caveolae, among others. Recent biophysical characterization of tetraspanin-enriched microdomains suggests that they might be specially suited for the regulation of avidity of adhesion receptors and the compartmentalization of enzymatic activities. Moreover, modulation by tetraspanins of the function of adhesion receptors involved in inflammation, lymphocyte activation, cancer and pathogen infection suggests potential as therapeutic targets. This review explores this emerging picture of tetraspanin microdomains and discusses the implications for cell adhesion, proteolysis and pathogenesis.

Tetraspanins CD9 and CD81 Modulate HIV-1-Induced Membrane Fusion

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction–associated proteins and promote occludin retention in the endoplasmic reticulum

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ‐associated proteins occludin, claudin‐1, and Zonula Occludens protein‐1 (ZO‐1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ‐associated function, measured as FITC‐dextran paracellular permeability, of genomic replicon‐containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon‐α (IFN‐α) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ‐associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ‐associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co‐localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co‐immunoprecipitation and pull‐down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot‐like accumulation of occludin in infected Huh7 cells. Conclusion: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ‐associated proteins, which may provide new insights for HCV‐related pathogenesis. (HEPATOLOGY 2008.)

Functional insights on the polarized redistribution of leukocyte integrins and their ligands during leukocyte migration and immune interactions

Summary:  Cell–cell and cell–matrix interactions are of critical importance in immunobiology. Leukocytes make extensive use of a specialized repertoire of receptors to mediate such processes. Among these receptors, integrins are known to be of crucial importance. This review deals with the central role of integrins and their counterreceptors during the establishment of leukocyte–endothelium contacts, interstitial migration, and final encounter with antigen‐presenting cells to develop an appropriate immune response. Particularly, we have addressed the molecular events occurring during these sequential processes, leading to the dynamic subcellular redistribution of adhesion receptors and the reorganization of the actin cytoskeleton, which is reflected in changes in cytoarchitecture, including leukocyte polarization, endothelial docking structure formation, or immune synapse organization. The roles of signaling and structural actin cytoskeleton‐associated proteins and organized membrane microdomains in the regulation of receptor adhesiveness are also discussed.

MT1-MMP collagenolytic activity is regulated through association with tetraspanin CD151 in primary endothelial cells

MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.

A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting.

Intercellular adhesion molecule 3 (ICAM-3) is a leukocyte-specific receptor involved in primary immune responses. We have investigated the interaction between ICAM-3 and ezrin/radixin/moesin (ERM) proteins and its role in LFA-1-induced cell-cell interactions and membrane positioning of ICAM-3 in polarized migrating lymphocytes. Protein-protein binding assays demonstrated a phosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3 and the amino-terminal domain of ERM proteins. This interaction was not essential for the binding of ICAM-3 to LFA-1. Dynamic fluorescence videomicroscopy studies of cells demonstrated that moesin and ICAM-3 coordinately redistribute on the plasma membrane during lymphocyte migration. Furthermore, overexpression of the amino-terminal domain of moesin, which lacks the consensus moesin actin-binding site, caused the subcellular mislocalization of ICAM-3. A CD4 chimerical protein containing the cytoplasmic tail of ICAM-3 was targeted to the trailing edge. Point mutation of Ser487, Ser489, and Ser496 to alanine in the juxtamembrane region of ICAM-3 significantly impaired both ERM binding and polarization of ICAM-3. ERM-directed polarization of ICAM-3 was also impaired by phosphorylation-like mutation of Ser487 and Ser489, but not of Ser496. Our results underscore the key role of specific serine residues within the cytoplasmic region of ICAM-3 for its ERM-directed positioning at the trailing edge of motile lymphocytes.

The RhoA Effector mDia Is Induced During T Cell Activation and Regulates Actin Polymerization and Cell Migration in T Lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.

G protein‐coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein‐coupled receptors (GPCRs). We find that increased levels of G protein‐coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin‐mediated cell migration involves the paracrine/autocrine activation of a sphingosine‐1‐phosphate (S1P) Gi‐coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation‐dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR‐directed epithelial cell migration.

The Rho Exchange Factors Vav2 and Vav3 Control a Lung Metastasis–Specific Transcriptional Program in Breast Cancer Cells

Two Vav isoforms could be targeted to prevent breast tumors from metastasizing to the lung. Metastatic Route to the Lung Many individuals with cancer die from secondary tumors or metastases that spread through blood or lymph vessels to other tissues from the primary tumor site. The members of the Rho family of guanosine triphosphatases (GTPases) promote tumor growth and metastasis and are activated by guanine nucleotide exchange factors (GEFs). Rho GEFs are attractive pharmacological targets because they have potentially druggable catalytic activities and more restricted distribution patterns than Rho proteins. Citterio et al. found that the mRNA abundance of the GEFs Vav2 and Vav3 was increased in certain breast cancer subtypes in patient samples. Mice implanted with breast cancer cells in which Vav2 and Vav3 had been silenced developed slowly growing breast tumors and did not develop lung metastases. Vav2- and Vav3-deficient breast cancer cells showed an altered transcriptional profile, leading the authors to further analyze the role of select target genes encoding proteins that could be pharmacologically inhibited, such as the enzyme cyclooxygenase-2. When implanted into mice, breast cancer cells with deficiencies in individual Vav target genes showed defects in proliferation, angiogenesis, the ability to enter or exit blood vessels during metastasis, and the ability to colonize the lung. When applied to human breast cancer data sets, the changes in the abundance of a subset of mRNAs from the Vav transcriptome generated a gene signature that accurately predicted if patients survived and were free of detectable lung metastasis. These results identify possible targets for treating breast cancer and preventing secondary lung metastases and provide a potential prognostic tool for clinicians. The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, and many of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs. Microarray analyses revealed that Vav2 and Vav3 controlled a vast transcriptional program in breast cancer cells through mechanisms that were shared between the two proteins, isoform-specific or synergistic. Furthermore, the abundance of Vav-regulated transcripts was modulated by Rac1-dependent and Rac1-independent pathways. This transcriptome encoded therapeutically targetable proteins that played nonredundant roles in primary tumorigenesis and lung-specific metastasis, such as integrin-linked kinase (Ilk), the transforming growth factor–β family ligand inhibin βA, cyclooxygenase-2, and the epithelial cell adhesion molecule Tacstd2. It also contained gene signatures that predicted disease outcome in breast cancer patients. These results identify possible targets for treating breast cancer and lung metastases and provide a potential diagnostic tool for clinical use.