Ewa Pocheć

Tetraspanin CD151 regulates glycosylation of α3β1 integrin

The tetraspanin CD151 forms a stoichiometric complex with integrin α3β1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of α3β1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. α6β1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucα1–2Gal and bisecting GlcNAc-β(1→4) linkage on N-glycans of the α3 integrin subunit. The modulatory activity of CD151 toward α3β1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of α3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward α3β1. We also found that glycosylation of CD151 is also critical; Asn159 → Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or α3β1 but negated its modulatory function. Changes in the glycosylation pattern of α3β1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of α3β1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.

Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma.

The new face of nucleolin in human melanoma

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin.

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors - integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.

L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.

Octylphenol affects morphology and steroidogenesis in mouse tumor Leydig cells

Exposure to xenoestrogens has been shown to cause adverse effects on reproductive function of various animal species. However, direct effects of xenoestrogens on Leydig cell function remain scarcely known. The aim of our study was to demonstrate the effects of 4-tert-Octylphenol (OP) on the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and androgen receptor (AR) in MA-10 Leydig cells. Cells were treated for 3 and 12h with OP (10(-4)-10(-8)M). In cell cultures treated with high OP concentrations for 3 and 12h morphological alterations were observed. Oil Red O staining revealed differences in lipid droplet size and distribution when compared to controls. Immunoreactive 3β-HSD was found in the cytoplasm, whereas immunoreactive AR was detected in the nuclei of both control and OP-exposed Leydig cells. A dose-related decrease in the expression of both proteins in OP-exposed cells was found. Qualitative results by immunocytochemistry and Western blot were confirmed by further quantitative analyses. Radioimmunological analysis revealed time-dependent secretion of progesterone (P(4)) by control and OP-exposed Leydig cells. In 3 and 12h OP-treated Leydig cells a significant decrease in P(4) level was found. Our study demonstrated a dose- and time-dependent action of OP on both morphology and steroidogenic function of MA-10 cells. It seems likely that OP besides acting via ER may mediate its action through AR modulating Leydig cell function.

Anti-Inflammatory Properties of Irisin, Mediator of Physical Activity, Are Connected with TLR4/MyD88 Signaling Pathway Activation

Irisin, an adipomiokine known as a mediator of physical activity, induces the browning of adipose tissue and it has potentially protective properties in the development of obesity-related states, such as insulin resistance, arteriosclerosis, and type 2 diabetes. Despite numerous studies conducted on this factor, still little is known about its impact on the functioning of immunocompetent cells, but its potential anti-inflammatory properties were previously suggested. In the current study we investigated the role of irisin (0-100 nM) in the downstream pathway activation of Toll-like receptor 4 (TLR4) in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS; 100 ng/mL). The results have shown that irisin in high concentrations (50, 100 nM) significantly decreased the TLR4 and MyD88 protein levels, as well as the phosphorylation of nuclear factor-κB (NF-κB), consequently leading to the reduction in the release of crucial pro-inflammatory cytokines. The above was confirmed for interleukin 1β (IL-1β), tumor necrosis factor α (TNFα), interleukin 6 (IL-6), keratinocyte chemoattractant (KC), monocyte chemotactic protein 1 (MCP-1), as well as for high mobility group box 1 (HMGB1). Moreover, our results indicate that this effect is connected with irisins impact on the phosphorylation of mitogen-activated protein kinases (MAPKs), where a significant reduction in p-JNK and p-ERK but not p-p38 was observed. In conclusion, these data suggest that irisin has potentially anti-inflammatory properties connected with the downregulation of downstream pathways of TLR4/MyD88.

Vitamin B2 deficiency enhances the pro-inflammatory activity of adipocyte, consequences for insulin resistance and metabolic syndrome development

Aims: Adipose tissue is an endocrine organ important for regulation of such physiological processes as energy metabolism or lipids homeostasis. In an obesity state, it participates in the induction of chronic systemic inflammation accompanied by pro‐inflammatory cytokines and fatty acid elevation. For this reasons, adipose tissue is involved in, e.g., insulin resistance, type 2 diabetes or hyperlipidemia development. In our previous study, we have shown that riboflavin deficiency induces a pathological pro‐inflammatory response of macrophages, the main component of adipose tissue. Therefore, in the current study, we investigated the alteration of the pro‐inflammatory activity of adipocytes. Main methods: The study was conducted on mouse 3T3 L1 preadipocytes differentiated to adipocyte and culture in the state of riboflavin deficiency (3.1 nM) or control condition (10.4 nM). The cell viability, adiposity and glucose uptake was assessed. Moreover, mRNA expression, as well as crucial pro‐inflammatory cytokines (TNF&agr;, IL‐6) and adipokines (adiponectin, leptin, resistin) release and NF&kgr;B activation, were evaluated. Key findings: Results showed that riboflavin deprivation induced a significant elevation in adipocyte lipolysis and enhance obesity‐related apoptosis of adipocytes. The generation of reactive oxygen species was enhanced in riboflavin‐deficient adipocytes by 43%. Moreover, NF&kgr;B phosphorylation and the expression and release of both TNF&agr;, IL‐6 as well as leptin were elevated in a deficient group what was accompanied by a reduction of adiponectin level. Conclusion: Our study shows that riboflavin deficiency can promote the intensification of pro‐inflammatory activity of adipocyte cells, leading consequently to the severity of chronic inflammation that accompanies obesity state.

On the trail of the glycan codes stored in cancer-related cell adhesion proteins.

Changes in the profile of protein glycosylation are a hallmark of ongoing neoplastic transformation. A unique set of tumor-associated carbohydrate antigens expressed on the surface of malignant cells may serve as powerful diagnostic and therapeutic targets. Cell-surface proteins with altered glycosylation affect the growth, proliferation and survival of those cells, and contribute to their acquisition of the ability to migrate and invade. They may also facilitate tumor-induced immunosuppression and the formation of distant metastases. Deciphering the information encoded in these particular glycan portions of glycoconjugates may shed light on the mechanisms of cancer progression and metastasis. A majority of the related review papers have focused on overall changes in the patterns of cell-surface glycans in various cancers, without pinpointing the molecular carriers of these glycan structures. The present review highlights the ways in which particular tumor-associated glycan(s) coupled with a given membrane-bound protein influence neoplastic cell behavior during the development and progression of cancer. We focus on altered glycosylated cell-adhesion molecules belonging to the cadherin, integrin and immunoglobulin-like superfamilies, examined in the context of molecular interactions.