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Stable T98G cells expressing ShcD-WT show increased invasion but fail to exhibit ShcD-Tie2 synergy.

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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 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.

During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells’ invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types.

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.

All mammalian cells release small endosome-derived exosomes that function in intercellular communication, but the secretion process is poorly understood. Verweij et al. developed a live-imaging approach and demonstrate that external cues can trigger exosome release from a subpopulation of multivesicular bodies by phosphorylating the target membrane SNARE SNAP23 at serine residue 110., 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.

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.

Membrane ruffles are actin-rich protrusions of the plasma membrane that can be observed on the surface of many cell types. Phase contrast and fluorescent microscopy are widely used in the analysis of ruffles, which are commonly identified in cells stained withfluores-cently labeled phalloidin. Currently, comparison of cellular ruffle formation under different experimental conditions is generally qualitative or semiquantitative. Ruffle structures are often defined using manual tracing and thresholding methods. Here, we report the rapid and accurate segmentation of ruffles from two-dimensional confocal projections of cells using an automated method based on well-established image processing and analysis methods. Line-shaped ruffles were detected using line detectors and were then separated from the filtered images. Automated categorizing of the segmented line structures enabled accurate quantification of the ruffles. This automated approach is efficient and reliable and hence can serve as a powerful tool in studies of the mechanism of ruffle formation.

In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of alpha5beta1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.

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.

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.

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.

Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion,soluble NSF attachment proteinreceptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N -ethylmaleimide-sensitivefactor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcγ receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.

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.

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.

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.

We have previously demonstrated an interaction between the highly conserved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domains and calreticulin. Since this highly conserved sequence motif has been implicated in the regulation of the integrin affinity state, we wanted to determine whether the calreticulin-integrin interaction also depended on the integrin affinity state, and whether calreticulin occupation of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state. We now demonstrate that anti-integrin antibody- or phorbol 12-myristate 13-acetate (PMA)-induced activation of the alpha 2 beta 1 integrin on Jurkat cells, as determined by stimulation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha 2 beta 1 activation with either anti-beta 1 or anti-alpha 2 monoclonal antibodies resulted in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abrogated the calreticulin-integrin interaction. A correlation was also found between PMA-induced alpha 2 beta 1 activation and the amount of calreticulin bound to this integrin. Furthermore, pretreatment of streptolysin O-permeablized Jurkat cells with an anti-calreticulin antibody resulted in a significant and specific inhibition of the adhesion to collagen type I that could be induced by antibodies to alpha 2 beta 1 or by PMA. These data suggest that the active, high affinity form of alpha 2 beta 1 binds calreticulin and that calreticulin binding to the alpha 2 cytoplasmic domain may be required for stabilizing the high affinity state of this integrin. The data presented here also demonstrate, for the first time, an inducible interaction of an integrin with an intracellular protein that occurs via the alpha subunit of the integrin.

Malignant transformation of fibroblast and epithelial cells is accompanied by increased beta 1-6 N-acetylglucosaminyltransferase V (GlcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GlcNAc-TV in Mv1Lu cells, an immortalized lung epithelial cell line results in loss of contact-inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme alpha-mannosidase II. In serum-deprived and high density monolayer cultures, the GlcNAc-TV transfectants formed foci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GlcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GlcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface alpha 5 beta 1 or alpha v beta 3 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins alpha 5, alpha v and beta 1 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that beta 1-6GlcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells.

Background Previous studies on pain prevalence among systemic sclerosis (SSc) patients confirms its prominent role in disease outcomes 1 . Although pain perception is receiving more importance among patient-reported outcomes in SSc research, still its interface with mood disorders and immune system need to be further elucidated. The prevalence of clinically significant symptoms of depression or anxiety in SSc varies between 36% and 65% according to the evaluation used, and are rather associated with increased disability and altered HRQoL, than with disease-specific organ manifestations. An endocrine influence on pain perception and threshold is supported by robust evidences, more specifically related to sex, adrenal and thyroid hormones. Objectives To analyze the association between pain threshold, depression and cortisol serum levels in systemic sclerosis patients, major depression disorder patients and healthy controls. Methods Thirty SSc patients, 30 major depressive disorder (MDD) patients and 30 healthy controls completed the study. All participants underwent pressure pain threshold (PPT) assessment through an algometer in three anatomical surfaces, the ungula bed (UPPT) and the metacarpophalangeal joint (MPPT) of the second finger of the right hand and the anterior portion of the quadriceps muscle (QPPT), and depression evaluation, assessed by patient health questionnaire 9 (PHQ-9). Serum cortisol was measured in SSc and MDD patients for further analysis. Patients under treatment with corticosteroids were excluded. Results PHQ-9 values showed that 15% of SSc patients had no signs of depression, while 30% had mild and another 30% moderate signs of depression, 18% moderate/severe and 9% severe signs. Among MDD patients, PHQ-9 12% had mild depression, 45% moderate, 15 moderate to severe and 28% severe depression. PHQ-9 scores were significantly higher in SSc and MDD compared to controls (p Conclusions The interface between pain, depression and cortisol could represent an additional complex aspect of care of patients with either SSc or MDD. References Schieir O, Thombs BD, Hudson M, Boivin JF, Steele R, Bernatsky S, Hanley J, Baron M; Canadian Scleroderma Research Group. Prevalence, severity, and clinical correlates of pain in patients with systemic sclerosis. Arthritis Care Res (Hoboken). 2010 Mar;62(3):409–17. Disclosure of Interest None declared

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.

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK–Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.

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.

Background To compare tumor necrosis in hepatoma induced in rats by a single percutaneous injection of ethanol (PEI) or acetic acid (PAI). Methods BW7756 hepatomas of 1 mm3 were implanted in the liver of 40 male healthy rats. After 14 days, the 36 surviving rats were treated, in a single session, by ultrasound-guided injection of 300 μl of 95% ethanol (n = 17) or 100 μl of 50% acetic acid (n = 19). They were sacrificed 14 days after treatment and explanted tumoral livers were examined. The same PAI procedure was repeated on 13 additional rats to exclude a suspected occurrence of technical failures during the experiment, due to a surprisingly high rate of deaths within 30 minutes after PAI. Results Four rats died within four days after tumor implantation; after PEI, 1/17 (6%) died, whereas after PAI 9/19 (47%) died. The remaining 26 rats, after 14 days post-percutaneous ablation, were sacrificed. Gross and microscopic examinations showed that the hepatoma's nodules treated with PEI had 45.3 ± 19.4% tumor necrosis compared to 49 ± 23.3% (P = NS) for those treated with PAI. Complete tumor necrosis was not found in any animal. Peritoneal invasion was present in 4/16 (25%) and 2/10 (20%) rats treated with PEI or PAI, respectively (P = NS). Autopsy was performed in the 5 additional rats that died within 30 minutes after PAI. Conclusion Our results show that there is no significant difference in the percentage of tumor necrosis between two local ablation methods in spite of the different dosages used. However, mortality in the PAI-treated group was greater than in PEI-treated group, presumably due to greater acetic acid systemic diffusion and its metabolic side effects. In human subjects, HCC occurs in the setting of cirrhosis, where the non-tumoral tissue is firmer than the tumor structure, with consequent reduction of drug diffusion. This could be the reason why some human studies have concluded similar or even better safety and efficacy with PAI compared to PEI.

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.

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP(2) from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcgamma receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP(2) and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP(2) levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.

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.