Publications by authors named "Vladimir Leksa"

20 Publications

  • Page 1 of 1

The Role of CX3CL1 and ADAM17 in Pathogenesis of Diffuse Parenchymal Lung Diseases.

Diagnostics (Basel) 2021 Jun 11;11(6). Epub 2021 Jun 11.

Institute of Immunology, Faculty of Medicine Comenius University, 811 08 Bratislava, Slovakia.

Fractalkine (CX3CL1) is a unique chemokine that functions as a chemoattractant for effector cytotoxic lymphocytes and macrophages expressing fractalkine receptor CX3CR1. CX3CL1 exists in two forms-a soluble and a membrane-bound form. The soluble CX3CL1 is released from cell membranes by proteolysis by the TNF-α-converting enzyme/disintegrin-like metalloproteinase 17 (TACE/ADAM17) and ADAM10. In this study, we evaluated the diagnostic relevance and potential roles of CX3CL1 and ADAM17 in the pathogenesis of diffuse parenchymal lung diseases (DPLDs) in the human population. The concentration of CX3CL1 and ADAM17 was measured by the enzyme-linked immunosorbent assay (ELISA) test in bronchoalveolar lavage fluids of patients suffering from different DPLDs. The concentration of CX3CL1 was significantly higher in patients suffering from idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis patients compared to the control group. A significantly higher concentration of CX3CL1 was measured in fibrotic DPLDs compared to non-fibrotic DLPD patients. We found a positive correlation of CX3CL1 levels with the number of CD8+ T cells, and a negative correlation with CD4+ T cells in BALF and diffusion capacity for carbon monoxide. The concentration of ADAM17 was significantly lower in the IPF group compared to the other DPLD groups. We noticed a significantly higher CX3CL1/ADAM17 ratio in the IPF group compared to the other DPLD groups. We suggest that CX3CL1 has a distinctive role in the pathogenesis of DPLDs. The level of CX3CL1 strongly correlates with the severity of lung parenchyma impairment. The results suggest that high values of CX3CL1/ADAM17 could be diagnostic markers for IPF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/diagnostics11061074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230701PMC
June 2021

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

Eur J Immunol 2021 04 25;51(4):848-863. Epub 2021 Jan 25.

Division of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4 T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL T cells are naïve and MAL T cells memory subtypes. Further, resting MAL T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.202048603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248321PMC
April 2021

The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis.

J Leukoc Biol 2019 03 18;105(3):519-530. Epub 2019 Jan 18.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Centre for Pathophysiology, Infectiology & Immunology, Medical University of Vienna, Vienna, Austria.

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/JLB.1AB0417-160RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392118PMC
March 2019

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

Front Immunol 2018 27;9:852. Epub 2018 Apr 27.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβCD39CD73 Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.00852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946032PMC
June 2019

Lactoferrin is a natural inhibitor of plasminogen activation.

J Biol Chem 2018 06 18;293(22):8600-8613. Epub 2018 Apr 18.

From the Institute for Hygiene and Applied Immunology and

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes ( tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species ( or ) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion and also plasminogen activation driven by Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.003145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986228PMC
June 2018

Biotin-Chasing Assay to Evaluate uPAR Stability and Cleavage on the Surface of Cells.

Methods Mol Biol 2018 ;1731:39-47

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Centre for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

The plasminogen activation system, i.e., the fibrinolytic system, is one of the major plasma proteolytic pathways. The proteolytic conversion of the zymogen plasminogen to the active serine protease plasmin is on the cell surface catalyzed by the serine protease urokinase-type plasminogen activator (urokinase, uPA). Upon binding to the urokinase receptor (uPAR, CD87), single-chain pro-uPA is processed to double-chain uPA which in turn specifically converts cell-bound plasminogen to plasmin. Plasmin is harnessed in many physiological processes, e.g., blood clots' resolution, or proteolytic activation of growth factors. Plasmin is essential also for migratory cells, for instance, activated immune cells; however, malignant cells hijack plasmin for invasion as well. The activation of plasminogen to plasmin is thus at the physiological level tightly controlled. One of the negative regulators of plasminogen activation has been identified in the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CIMPR, CD222). M6P/IGF2R is a multifunctional receptor involved in protein sorting, internalization, and degradation, being considered a tumor suppressor. M6P/IGF2R binds both plasminogen and uPAR and facilitates in this way the proteolytic cleavage of uPAR resulting in the loss of the uPA binding on the cell surface. Hence, this molecular device contributes to the negative feedback loop in regulation of pericellular plasminogen activation and cell invasion.In this chapter, we describe the experimental approach, i.e., biotin-chasing assay, to evaluate uPAR stability and cleavage on the surface of cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7595-2_4DOI Listing
January 2019

Unravelling novel functions of the endosomal transporter mannose 6-phosphate/insulin-like growth factor receptor (CD222) in health and disease: An emerging regulator of the immune system.

Immunol Lett 2017 10 16;190:194-200. Epub 2017 Aug 16.

Centre for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria.

Properly balanced cellular responses require both the mutual interactions of soluble factors with cell surface receptors and the crosstalk of intracellular molecules. In particular, immune cells exposed unceasingly to an array of positive and negative stimuli must distinguish between what has to be tolerated and attacked. Protein trafficking is one of crucial pathways involved in this labour. The approximately >270-kDa protein transporter called mannose 6- phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222) is a type I transmembrane glycoprotein present largely intracellularly in the Golgi apparatus and endosomal compartments, but also at the cell surface. It is expressed ubiquitously in a vast majority of higher eukaryotic cell types. Through binding and trafficking multiple unrelated extracellular and intracellular ligands, CD222 is involved in the regulation of a plethora of functions, and thus implicated in many physiological but also pathophysiological conditions. This review describes, first, general features of CD222, such as its evolution, genomic structure and regulation, protein structure and ligands; and second, its specific functions with a special focus on the immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.imlet.2017.08.011DOI Listing
October 2017

Folate Receptor β Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen.

J Immunol 2016 09 17;197(6):2229-38. Epub 2016 Aug 17.

Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria;

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ(+) macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRβ(-) counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1501878DOI Listing
September 2016

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

J Immunol 2016 Feb 4;196(3):1387-99. Epub 2016 Jan 4.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, 1090 Vienna, Austria;

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1501889DOI Listing
February 2016

The mannose-6-phosphate analogue, PXS64, inhibits fibrosis via TGF-β1 pathway in human lung fibroblasts.

Immunol Lett 2015 Jun 27;165(2):90-101. Epub 2015 Apr 27.

Drug Discovery, Pharmaxis, Australia; School of Medical & Molecular Biosciences, University of Technology Sydney, Australia.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-β1 to induce increases in fibrotic markers. rhTGF-β1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-β3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-β1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-β1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.imlet.2015.04.003DOI Listing
June 2015

The late endosomal transporter CD222 directs the spatial distribution and activity of Lck.

J Immunol 2014 Sep 15;193(6):2718-32. Epub 2014 Aug 15.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria; Laboratory of Molecular Immunology, Institute of Molecular Biology, Slovak Academy of Sciences, 84551 Bratislava, Slovak Republic

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1303349DOI Listing
September 2014

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells.

J Biol Chem 2012 Jun 21;287(27):22450-62. Epub 2012 May 21.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.339663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391135PMC
June 2012

Soluble M6P/IGF2R released by TACE controls angiogenesis via blocking plasminogen activation.

Circ Res 2011 Mar 27;108(6):676-85. Epub 2011 Jan 27.

Molecular Immunology Unit, Pathophysiology, Infectiology & Immunology, Medical University of Vienna, Austria.

Rationale: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R).

Objective: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system.

Methods And Results: By using specific inhibitors and RNA interference, we show that the tumor necrosis factor α convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo.

Conclusions: The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.110.234732DOI Listing
March 2011

Sequential cooperation of CD2 and CD48 in the buildup of the early TCR signalosome.

J Immunol 2009 Jun;182(12):7672-80

Department of Molecular Immunology, Centre for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.

The buildup of TCR signaling microclusters containing adaptor proteins and kinases is prerequisite for T cell activation. One hallmark in this process is association of the TCR with lipid raft microdomains enriched in GPI-proteins that have potential to act as accessory molecules for TCR signaling. In this study, we show that GPI-anchored CD48 but not CD59 was recruited to the immobilized TCR/CD3 complex upon activation of T cells. CD48 reorganization was vital for T cell IL-2 production by mediating lateral association of the early signaling component linker for activated T cells (LAT) to the TCR/CD3 complex. Furthermore, we identified CD2 as an adaptor linking the Src protein tyrosine kinase Lck and the CD48/LAT complex to TCR/CD3: CD2 associated with TCR/CD3 upon T cell activation irrespective of CD48 expression, while association of CD48 and LAT with the TCR/CD3 complex depended on CD2. Consequently, our data indicate that CD2 and CD48 cooperate hierarchically in the buildup of the early TCR signalosome; CD2 functions as the master switch recruiting CD48 and Lck. CD48 in turn shuttles the transmembrane adapter molecule LAT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.0800691DOI Listing
June 2009

The mannose 6-phosphate/insulin-like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells.

Int J Cancer 2009 Jun;124(11):2559-67

Institute of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R-deficient SCC-VII murine squamous cell carcinoma cells secrete large amounts of pro-invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC-VII cells. To assess the link between M6P/IGF2R status and tumour invasion, we have now generated SCC-VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC-VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC-VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC-VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.24236DOI Listing
June 2009

Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor.

Mol Biol Cell 2009 Feb 26;20(3):745-56. Epub 2008 Nov 26.

Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.e08-06-0569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633380PMC
February 2009

LFA-1-mediated leukocyte adhesion regulated by interaction of CD43 with LFA-1 and CD147.

Mol Immunol 2008 Mar 9;45(6):1703-11. Epub 2007 Nov 9.

Department of Molecular Immunology, Center for Physiology, Pathophysiology & Immunology, Medical University of Vienna, Vienna, Austria.

The activity of the lymphocyte-function associated antigen 1 (LFA-1; CD11a/CD18) must be tightly controlled during the onset of cellular immunity. It is well known that the sialoglycoprotein CD43 can influence LFA-1-mediated cell adhesion in an either anti- or pro-adhesive manner through mechanisms not well understood. By using a yeast-2-hybrid screen and co-immunoprecipitation we identified physical association of CD43 with two novel partners, LFA-1 itself and the Ig-family member CD147 (EMMPRIN, basigin), and characterized how these interactions are involved in LFA-1-mediated cell adhesion. Monoclonal antibodies (mAbs) to both CD43 and CD147 induced similar homotypic cell aggregation and adhesion of Jurkat T cells and U937 myeloid cells. Both CD43 and CD147 mAbs induced dynamic co-capping of LFA-1 together with the CD43 and the CD147 molecule to cell contact zones. However, in contrast to CD43, we were not able to co-immunoprecipitate LFA-1 with CD147, which indicates that CD43 interacts with CD147 and LFA-1 in two distinct but similarly reorganized complexes. Co-transfection of CD43 interfered with the CD147-induced cell adhesion and aggregation, and siRNA-mediated knock down of CD43 in human T cells resulted in enhanced LFA-1 activation induced via CD147 and also the T cell antigen receptor. These results indicate that triggering CD43 and the underlying signaling pathways enhance LFA-1 adhesiveness while CD43 also negatively regulates LFA-1 induction via other receptors by dynamic interaction with either LFA-1 or CD147.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2007.09.032DOI Listing
March 2008

TGF-beta-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen.

J Cell Sci 2005 Oct;118(Pt 19):4577-86

BMT, BioMolecular Therapeutics, Brunner Strasse 59, 1235 Vienna, Austria.

Transforming growth factor-beta (TGF-beta), a key modulator of endothelial cell apoptosis, must be activated from the latent form (LTGF-beta) to induce biological responses. In the present study, we report activation of TGF-beta by functional and physical co-operation of the mannose-6-phosphate/insulin-like-growth-factor-II receptor (CD222) and the urokinase-type plasminogen activator receptor (CD87). We show that endothelial cells express CD222 and CD87 in a membrane complex and demonstrate that the association of these two receptors is essential for the release of active TGF-beta in the transduced mouse fibroblast used as model cells. By contrast, smooth-muscle cells, which express CD222 and CD87 at similar density to endothelial cells but not in complexed form, do not activate TGF-beta. We also have found that mini-plasminogen is a high-affinity ligand for CD222 and is essential for the activation of TGF-beta by the CD87-CD222 complex to induce apoptosis in endothelial cells. This specific mechanism of TGF-beta-mediated apoptosis in endothelial cells is thus a potential novel target to be considered for treatment of pathological vascular disorders (e.g. tumor angiogenesis).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.02587DOI Listing
October 2005

Engagement of Na,K-ATPase beta3 subunit by a specific mAb suppresses T and B lymphocyte activation.

Int Immunol 2002 Dec;14(12):1407-14

Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

In order to identify new molecules involved in regulation of T cell proliferation, we generated various mAb by immunization of mice with the T cell line Molt4. We found one mAb (termed P-3E10) that down-regulated the in vitro T cell proliferation induced by CD3-specific OKT3 mAb. The P-3E10 mAb was also able to inhibit IFN-gamma, IL-2, IL-4 and IL-10 production of OKT3-activated T cells. The antigen recognized by P-3E10 mAb is broadly expressed on all hematopoietic as well as on all non-hematopoietic cell lines tested so far. Within peripheral blood leukocytes, the P-3E10 antigen was detected on lymphocytes, monocytes and granulocytes. Human umbilical vein endothelial cells (HUVEC) also scored positively. By evaluating the effect of P-3E10 mAb on these cell types we found that it also inhibited anti-IgM-induced B cell proliferation. However, it did not block growth factor-mediated proliferation of HUVEC, and spontaneous proliferation of SupT-1, Jurkat, Molt4 and U937 cell lines. Moreover, it did not influence phagocytosis of human blood monocytes and granulocytes. Biochemical analysis revealed that the P-3E10 antigen is a protein with a mol. wt of 45-50 kDa under non-reducing and 50-55 kDa under reducing conditions. By using a retroviral cloning system, the P-3E10 antigen was cloned. Sequence analysis revealed the P-3E10 antigen to be identical to the beta3 subunit of the Na,K-ATPase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/intimm/dxf112DOI Listing
December 2002

The N terminus of mannose 6-phosphate/insulin-like growth factor 2 receptor in regulation of fibrinolysis and cell migration.

J Biol Chem 2002 Oct 19;277(43):40575-82. Epub 2002 Aug 19.

Institute of Immunology, Vienna International Research Cooperation Center at Novartis Forschungs-institut, University of Vienna, Vienna A-1235, Austria.

Leukocyte migration to sites of inflammation is a multistep process involving transient adhesion to the endothelium followed by cell surface-controlled proteolysis for transmigration through the vessel wall and chemotactic movement within tissues. One of the key players in this machinery appears to be the urokinase-type plasminogen activator (uPA)/uPA receptor system. The role of uPA and its receptor (CD87) in plasminogen (Plg) activation, cell adhesion, and chemotaxis is well established; however, less is known of how these activities are regulated. Here we provide evidence that the mannose 6-phosphate/insulin-like growth factor 2 receptor (CD222) controls CD87-mediated functions. Expression of human CD222 in CD222-/- mouse fibroblasts down-regulated Plg activation, cell adhesion, and chemotaxis induced by the uPA/CD87 system. In addition, we demonstrate that the N-terminal region of CD222, which is similar to the Plg-binding site of streptokinase, plays a crucial role in binding of CD87 and Plg. A peptide derived from this region in CD222 is able to disrupt the physical interaction of CD222 with CD87 and, furthermore, mimics the inhibitory effects of CD222 on CD87 functions. Taken together, our results indicate a novel role for CD222 in regulation of fibrinolysis, cell adhesion, and migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M207979200DOI Listing
October 2002
-->