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Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2–ShcD signaling axis in glioma cell invasion, which may be of clinical significance. Implications: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular structures known as invadopodia, the function of which depends upon soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated vesicular transport of cellular cargo. Recently, it has been shown the SNARE Syntaxin4 (Stx4) mediates trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) to invadopodia, and that Stx4 is regulated by Munc18c in this context. Here, it is observed that expression of a construct derived from the N-terminus of Stx4, which interferes with Stx4-Munc18c interaction, leads to perturbed trafficking of MT1-MMP, and reduced invadopodium-based invasion in vitro, in models of triple-negative breast cancer (TNBC). Expression of Stx4 N-terminus also led to increased survival and markedly reduced metastatic burden in multiple TNBC models in vivo. The findings are the first demonstration that disrupting Stx4-Munc18c interaction can dramatically alter metastatic progression in vivo, and suggest that this interaction warrants further investigation as a potential therapeutic target. Implications: Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.

Malignant cancer cells can invade extracellular matrix (ECM) through the formation of F-actin-rich subcellular structures termed invadopodia. ECM degradation at invadopodia is mediated by matrix metalloproteinases (MMPs), and recent findings indicate that membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a primary role in this process. Maintenance of an invasive phenotype is dependent on internalization of MT1-MMP from the plasma membrane and its recycling to sites of ECM remodeling. Internalization of MT1-MMP is dependent on its phosphorylation, and here we examine the role of β1 integrin-mediated signaling in this process. Activation of β1 integrin using the antibody P4G11 induced phosphorylation and internalization of MT1-MMP and resulted in increased cellular invasiveness and invadopodium formation in vitro. We also observed phosphorylation of Src and epidermal growth factor receptor (EGFR) and an increase in their association in response to β1 integrin activation, and determined that Src and EGFR promote phosphorylation of MT1-MMP on Thr567. These results suggest that MT1-MMP phosphorylation is regulated by a β1 integrin-Src-EGFR signaling pathway that promotes recycling of MT1-MMP to sites of invadopodia formation during cancer cell invasion.

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell–extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro. Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

The SNAREs SNAP23, Syntaxin4, and VAMP7 associate to target the delivery of MT1-MMP to sites of invadopodium formation in breast tumor cells. The interaction of these SNAREs correlates with decreased phosphorylation of Syntaxin4. The targeted delivery of MT1-MMP is required for efficient ECM degradation and cell invasion., Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.

1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Detection of early carotid vascular disease in patients with systemic lupus erythematosus (SLE) is considered mandatory for evaluation of subclinical atherosclerosis (ATS), and various ultrasonographic (US) parameters have been proposed. In the present investigation, 33 SLE and 33 healthy age-matched females have been studied by colour-coded sonography of the common carotid artery, assessing intima-media thickness (IMT), vascular strain (VS), vascular distensibility (VD), vascular stiffness (VSf) and pressure-strain elastic modulus (PSEM) as possible markers of early ATS. Patients with SLE, despite equivalent exposure to “traditional” cardiovascular risk factors, presented a higher mean IMT of the common carotid artery than healthy subjects (0.7 ± 0.2 mm vs 0.5 ± 0.1 mm – P

Alexithymia is conceptualized as a disorder of emotion regulation mechanisms, which involves a dissociation of emotional and physic responses to life events and bodily sensations. Our results might suggest a possible relationship between the alexithymic construct and TNF levels in RA patients. These preliminary findings corroborate the integrated bidirectional interactions between neuropsychological mechanisms and the neuroendocrine-immune system in patients affected by autoimmune diseases and contribute to finding a common biological pathway linking alexithymia and autoimmune-inflammatory diseases.

Objective To evaluate the metabolic effect of buccal spray insulin compared with subcutaneous regular insulin in patients with type 1 diabetes. Research Design and Methods This study compared plasma glucose, insulin, and C-peptide levels in 18 patients with type 1 diabetes treated with subcutaneous regular or buccal spray insulin on 2 consecutive mornings. On day 1, patients were treated with their usual subcutaneous regular insulin regimens. On day 2, patients received buccal spray insulin. In the morning of both days 1 and 2, patients received a standard meal of 630 kJ. No intermediate or long-acting insulin was administered to patients on the morning of the test. Blood samples were collected for up to 4 hours for biokinetic analysis. In a subset of 3 patients, premeal buccal spray insulin was administered for 2 entire consecutive days. In these patients, glucose levels were monitored using the glucose sensor monitoring system. Results Overall, there were no statistically significant differences in glucose, insulin, or C-peptide levels measured after administration of subcutaneous vs buccal spray insulin. However, at 90 and 120 minutes after subcutaneous regular insulin administration, significantly higher insulin levels and more prolonged hypoglycemic effect were detected compared with buccal spray insulin administration. In the 3 patients who received 1 day of regular and 2 entire days of buccal spray insulin, no significant differences were observed in glucose levels during the 3 days of glucose sensor monitoring. Conclusions Insulin administered via the buccal spray formulation is as effective as the subcutaneous route in lowering blood glucose levels.

Acquisition of an invasive phenotype is prerequisite for tumor metastasis. Degradation of the extracellular matrix (ECM), and subsequent invasion by tumor cells, is mediated, in part, through subcellular structures called invadopodia. Src-dependent cytoskeletal rearrangements are required to form invadopodia, and here we identify an association between Src, epidermal growth factor receptor (EGFR), and β1 integrin that facilitates invadopodia formation. The association of Src, EGFR and β1 integrin is dependent upon membrane traffic that is mediated by syntaxin13 (officially known as STX12) and SNAP23; a similar dependence on these two SNARE proteins was observed for invadopodium-based matrix degradation and cell invasion. Inhibition of SNARE function impaired the delivery of Src and EGFR to developing invadopodia, as well as the β1-integrin-dependent activation of Src and phosphorylation of EGFR on Tyr residue 845. We also identified an association between SNAP23 and β1 integrin, and inhibition of β1 integrin increased this association, whereas the interaction between syntaxin13 and SNAP23 was reduced. The results suggest that SNARE-dependent trafficking is regulated, in part, by β1 integrin and is required for the delivery of Src and EGFR to sites of invadopodia formation in order to support tumor cell invasion.

The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin–ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research.

Objective.To study concentrations of adipokines in patients with systemic lupus erythematosus (SLE) and the relationship among adipokines, the metabolic syndrome (MeS), and cardiovascular disease (CVD) risk factors.Methods.We enrolled 50 SLE patients and 26 controls, all women. Leptin, resistin, visfatin, and adiponectin were measured by commercial ELISA kits.Results.MeS prevalence was increased among subjects with SLE. Leptin levels were higher in patients with SLE than controls. Among SLE patients, independent determinants of leptin were insulin levels (p < 0.0001), triglycerides (p = 0.03), body mass index (p = 0.02), corticosteroid dosage (p = 0.02), and SLE Disease Activity Index (p = 0.005). Other adipokines did not differ between SLE patients and controls.Conclusion.Leptin was increased in SLE patients and could play a role in SLE-related cardiovascular diseases.

The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. We used a two-hybrid screen to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encoded a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. We report here that this integrin-linked kinase (ILK) phosphorylated a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitated with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity was reduced in response to fibronectin. Overexpression of p59ILK disrupted epithelial cell architecture and inhibited adhesion to integrin substrates, while inducing anchorage-independent growth. We propose that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction.

Site-directed mutagenesis was used to develop deletion and point mutants of human 5-lipoxygenase-activating protein (FLAP), which were then expressed in COS-7 cells. Membrane preparations from these cells were analyzed in a radioligand binding assay. Binding of leukotriene biosynthesis inhibitors to FLAP mutants containing deletions of 2 to 6 amino acids within the region from residue 48–61 was undetectable. This finding is consistent with previous studies which suggest that residues amino-terminal to the proposed second transmembrane of FLAP are critical for inhibitor binding. The present study also defines residues of FLAP a) amino-terminal to residue 48, b) between the proposed second and third transmembrane regions and c) in the C-terminal region of the protein which are not involved in inhibitor binding.

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.

Neuropsychiatric involvement in patients with Systemic Lupus Erythematosus (SLE), first mentioned by Kaposi more than 100 years ago, still remains one of the main challenge facing rheumatologist and other physicians. The diagnosis of neuropsychiatric SLE (NPSLE) is complex not only because of the considerable prevalence variation (14-80%) but also because of the wide spectrum of NP manifestations. They vary from overt neurologic alterations (seizure, psychosis), to subtle abnormalities (neurocognitive dysfunctions). Different NP manifestations result from a variety of mechanisms including antibodies, vasculitis, thrombosis, hemorrhages and cytokine-mediated damages. Of note, despite the dramatic clinical manifestations, too often changes at the morphological neuroimaging techniques are minimal and non specific. There is no one diagnostic tool specific for NPSLE and diagnosis must be based on the combinated use of immunoserological tests, functional and anatomical neuroimaging and standardized specific criteria. Symptomatic, immunosuppressive and anticoagulant therapies are the main strategies available in the management of these patients. Therapy for CNS lupus should be adjusted according to the needs of the individual patients. The coming years promise to be an important time for the development of new neuroimaging techniques and for the study of disease mechanism. An early and objective identification of brain involvement will allow for appropriate treatment to avoid severe complications.

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α(4)β(1) and α(5)β(1), facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca(2+) ions. Interestingly, the Ca(2+)-activated K(+)-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

Cellular remodeling of the extracellular matrix (ECM), an essential component of many physiological and pathological processes, is dependent on the trafficking and secretion of matrix metalloproteinases (MMPs). Soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic has documented roles in cell-ECM interactions and the present study specifically examines SNARE function in the trafficking of MMPs during ECM degradation. Using the invasive human fibrosarcoma cell line HT-1080, we demonstrate that a plasma membrane SNARE, SNAP23, and an endosomal v-SNARE, VAMP3 (also known as cellubrevin), partly colocalize with MMP2 and MMP9, and that inhibition of these SNAREs using dominant-negative SNARE mutants impaired secretion of the MMPs. Inhibition of VAMP3, SNAP23 or syntaxin-13 using dominant-negative SNARES, RNA interference or tetanus toxin impaired trafficking of membrane type 1 MMP to the cell surface. Consistent with these observations, we found that blocking the function of these SNAREs reduced the ability of HT-1080 cells to degrade a gelatin substrate in situ and impaired invasion of HT-1080 cells in vitro. The results reveal the importance of VAMP3, syntaxin-13 and SNAP23 in the trafficking of MMP during degradation of ECM substrates and subsequent cellular invasion.

The antiphospholipid syndrome (APS) is an autoimmune disorder, characterized by a wide spectrum of clinical manifestations. Thromboembolic events, with a greater involvement of extremities veins, are the most common features, and obstruction of abdominal vessels are sporadically reported. We present a singular case of a patient with primary APS (PAPS) that developed a spontaneous splenorenal shunt, secondary to a total portal, mesenteric and splenic vein thrombosis. Spontaneous splenorenal shunt, an uncommon circumstance reported in cirrhotic disease, to the best of our knowledge, has not been previously described in PAPS.

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcγ receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.

As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.

Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells1,2. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation3. It has been shown that calreticulin associates with the cytoplasmic domains of integrin α-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix4,5. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibro-blasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.

An 18-kDa leukocyte membrane protein, termed 5-lipoxygenase-activating protein (FLAP), has recently been shown to be the target of two structurally distinct classes of leukotriene biosynthesis inhibitors. These classes of inhibitors are based on indole and quinoline structures and are represented by MK-886 and L-674,573, respectively. A novel class of hybrid structure based on the indole and quinoline classes of inhibitors, termed quindoles, has recently been developed. These compounds, exemplified by L-689,037, are potent inhibitors of leukotriene biosynthesis, both in vitro and in vivo. In the present study, we have developed and characterized a potent radioiodinated photoaffinity analogue of L-689,037, termed [125I]L-691,678. This compound was used in immunoprecipitation studies with FLAP antisera to show that the quindole series of leukotriene biosynthesis inhibitors interact directly with FLAP. In addition, we show that MK-886, L-674,573, and L-689,037 specifically compete, in a concentration-dependent manner, with both [125I]L-691,678 and [125I]L-669,083, a photoaffinity analogue of MK-886, for binding to FLAP. These results suggest that these three classes of leukotriene biosynthesis inhibitors share a common binding site on FLAP, providing further evidence that FLAP represents a suitable target for structurally diverse classes of leukotriene biosynthesis inhibitors.

Objective: Aim of this study was to evaluate the prevalence of alexithymia in patients affected by SLE or RA and to investigate the correlation between alexithymia and immunoendocrine parameters (PRL, hGH, IL-6 and TNF-alfa). Methods: Twenty-five patients (12 and 13 affected by SLE and RA, respectively) were enrolled into the study. The Toronto Alexithymia Scale-20 (TAS-20) was administered. PRL, hGH, IL-6 and TNF-alfa levels were measured by commercially available ELISA kits. Results: Alexithymia prevalence (TAS-20≥51) was 54% in RA and 42% in SLE patients. hGH serum levels were 3.1±4.2 and 1.1±0.9 IU/ml in SLE and RA, respectively. PRL concentration was 18.4±6.5 ng/ml and 14.2±4.0 ng/ml in SLE and RA patients, respectively (p=0.03). In RA group, TNF-alpha was 20±36.2 whereas in SLE it was 4.9±12.8 pg/ml (p=0.03); IL-6 serum concentrations were 24.4±25.1 and 2.9±5.4 pg/ml, in RA and SLE respectively (p=0.004). The serum level of hGH showed slight increase in alexithymic group (A) compared to non alexithymic group (NA) in both SLE and RA patients. PRL serum levels in SLE-A patients was 26.7±17.3 ng/ml while in SLE-NA patients was 12.4±3.3 ng/ml (p=0.04). In RA patients increased values of IL-6 and TNF-alpha were present in the A group compared to NA group (IL-6: 35.3±28 pg/mL vs 3.5±3.9 pg/mL, p=0.01; TNF-alpha: 34.7±39 pg/mL vs 3.1±3.4 pg/mL, p=0.01). Conclusions: In this preliminary results we found an high prevalence of alexithymia and a correlation between immunoendocrine parameters and alexhytimic features in SLE and RA, suggesting that an immunomodulatory pathway could influence this cognitive style in patients with autoimmune disorders. Other studies should contribute to find a common biological pathway linking alexithymia and autoimmunity.

Background Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling. Results We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling. Conclusions Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.