RESUMO
Drug resistance is a major obstacle to successful cancer treatment. Therefore, it is essential to understand the molecular mechanisms underlying drug resistance to develop successful therapeutic strategies. α6ß4 integrin confers resistance to apoptosis and regulates the survival of cancer cells; however, it remains unclear whether α6ß4 integrin is directly involved in chemoresistance. Here, we show that α6ß4 integrin promotes doxorubicin resistance by decreasing caspase-3-mediated apoptosis. We found that the overexpression of α6ß4 integrin by the ß4 integrin gene rendered MDA-MB435S and Panc-1 cells more resistant to doxorubicin than control cells. The acquired resistance to doxorubicin by α6ß4 integrin expression was abolished by the deletion of the cytoplasmic signal domain in ß4 integrin. Similar results were found in MDA-MB435S and Panc-1 cells when N-glycan-defective ß4 integrin mutants were overexpressed or bisecting GlcNAc residues were increased on ß4 integrin by the co-expression of N-acetylglucosaminyltransferase III with ß4 integrin. The abrogation of α6ß4 integrin-mediated resistance to doxorubicin was accompanied by reduced cell viability and an increased caspase-3 activation. Taken together, our results clearly suggest that α6ß4 integrin signaling plays a key role in the doxorubicin resistance of cancer cells, and N-glycans on ß4 integrin are involved in the regulation of cancer cells.
Assuntos
Integrina alfa6beta4 , Neoplasias , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Integrina beta4/genética , Transdução de Sinais , Apoptose/fisiologiaRESUMO
Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility.
Assuntos
Movimento Celular , Neoplasias/metabolismo , Neoplasias/patologia , Osteopontina/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Invasividade Neoplásica , FosforilaçãoRESUMO
Epithelial-mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvß3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvß3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvß3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-ß-independent fashion. Additionally, TGF-ß1 promoted EMT progression synergistically with αvß3 integrin, while a TGF-ß signaling inhibitor showed no effect on αvß3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvß3 integrin-induced partial EMT by suppressing αvß3 integrin cell surface expression. These findings indicate that αvß3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.
Assuntos
Transição Epitelial-Mesenquimal/genética , Integrina alfaVbeta3/genética , Fator de Crescimento Transformador beta1/genética , Animais , Feminino , Integrina alfaVbeta3/metabolismo , Camundongos , Camundongos Nus , Fator de Crescimento Transformador beta1/metabolismoRESUMO
α6ß4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased ß1,6GlcNAc-branched N-glycans on ß4 integrin suppress ß4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of ß1,6GlcNAc-branched N-glycans with ß4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that ß1,6GlcNAc-branched N-glycans are upregulated on ß4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of ß1,6GlcNAc-branched N-glycans on ß4 integrins as well as ß4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of ß1,6GlcNAc-branched N-glycans to ß4 integrin compared with normal skin samples. These findings suggest that the addition of ß1,6GlcNAc-branched N-glycans onto ß4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of ß1,6GlcNAc-branched N-glycans on ß4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression.
Assuntos
Carcinoma de Células Escamosas/química , Integrina beta4/análise , Polissacarídeos/análise , Neoplasias Cutâneas/química , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Pele/químicaRESUMO
BACKGROUND: Mucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid. RESULTS: Using a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories. CONCLUSIONS: Our findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.
Assuntos
Moléculas de Adesão Celular/imunologia , Imunoensaio/métodos , Mucosa/metabolismo , Penfigoide Mucomembranoso Benigno/imunologia , Penfigoide Mucomembranoso Benigno/metabolismo , Autoanticorpos/análise , Autoanticorpos/imunologia , Autoantígenos/análise , Autoantígenos/imunologia , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Humanos , CalininaRESUMO
Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvß3 and ß1 integrins, as well as αvß3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.
Assuntos
Adesão Celular , Mutação , Neoplasias/patologia , Osteopontina/metabolismo , Sequência de Aminoácidos , Glicosilação , Humanos , Integrina alfaVbeta3/metabolismo , Mutagênese Sítio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Osteopontina/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Células Tumorais CultivadasRESUMO
Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. ß4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of ß4-integrin contributes to the activation of signaling pathways that promote ß4-dependent tumor development and progression. Increased expression of ß1,6GlcNAc-branched N-glycans was found to be colocalized with ß4-integrin in human cutaneous squamous cell carcinoma tissues, and that the ß1,6GlcNAc residue was abundant on ß4-integrin in transformed keratinocytes. Interruption of ß1,6GlcNAc-branching formation on ß4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on ß4-integrin impaired ß4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of ß4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to ß4-integrin via ß1,6GlcNAc-branched N-glycans promoted ß4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated ß4-integrin N-glycan-mediated PI3K activation and inhibited the ability of ß4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed ß4-integrin N-glycan-mediated tumorigenesis. These findings provide a novel role for N-glycosylation of ß4-integrin in tumor development and progression, and the regulatory mechanism for ß4-integrin/PI3K signaling via the galectin-3-N-glycan complex.Implications:N-Glycosylation of ß4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3-N-glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR.
Assuntos
Galectina 3/metabolismo , Integrina beta4/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Proteínas Sanguíneas , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Galectinas , Humanos , Camundongos , Camundongos Nus , Transdução de SinaisRESUMO
Although quotient of alpha2 macroglobulin (Qα2MG) was previously reported to be useful for the evaluation of blood-brain barrier (BBB) function, it is not commonly used. We therefore evaluated BBB function among the various subsets of neuropsychiatric systemic lupus erythematosus (NPSLE) using quotient Q α2MG. Furthermore, we determined the correlation between Q α2MG and cerebrospinal (CSF) interleukin (IL)-6 level and quotient complement component 3 (Q C3). To determine intrathecal production of C3, the C3 index (Q C3/Q α2MG) was also calculated. Fifty-six patients with SLE were included in this study. Of these, 48 were diagnosed with NPSLE, consisting of 30 diffuse NPSLE patients (acute confusional state (ACS): n = 14, non-ACS: n = 16) and 18 patients with focal NPSLE. CSF IL-6 concentration, and paired serum and CSF levels of α2MG and C3, were measured by enzyme-linked immuno solvent assay (ELISA). The Q α2MG, Q C3, and C3 index were then calculated. Q α2MG, Q C3, and IL-6 concentrations in the CSF were significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, significant increases in Q α2MG, CSF IL-6, and Q C3 were observed in ACS compared with non-ACS or focal NPSLE. There was a positive correlation between CSF IL-6 level and Q α2MG, as well as between Q C3 and Q α2MG, in diffuse NPSLE. There were no significant differences in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. Our study suggests that BBB disruption is present in ACS, and elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, might result from their entry to the CSF from the systemic circulation through the damaged BBB, as well as increased intrathecal production. Furthermore, Q α2MG might be useful for the evaluation of BBB integrity.
Assuntos
Barreira Hematoencefálica/metabolismo , Complemento C3/líquido cefalorraquidiano , Interleucina-6/líquido cefalorraquidiano , Vasculite Associada ao Lúpus do Sistema Nervoso Central/líquido cefalorraquidiano , alfa-Macroglobulinas/líquido cefalorraquidiano , Adulto , Estudos de Casos e Controles , Feminino , Glucose/líquido cefalorraquidiano , Humanos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/sangue , MasculinoRESUMO
Malignant transformation is accompanied with aberrant glycosylation of proteins. Such changes in glycan structure also occur in the integrins, which are a large family of cell surface receptors for the extracellular matrix and play key roles in tumor progression. There is now increasing evidence that glycosylation of integrins affects cellular signaling and interaction with the extracellular matrix, receptor tyrosine kinases, and galectins, thereby regulating cell adhesion, motility, growth, and survival. Integrin α6ß4 is a receptor for laminin-332 and the increased expression level is correlated with malignant progression and poor survival in various types of cancers. Recent studies have revealed that integrin α6ß4 plays central roles in tumorigenesis and the metastatic process. In this review, we summarize our current understanding of the molecular mechanisms of tumor progression driven by integrin α6ß4 and also discuss the modification of glycans on integrin ß4 subunit to address the important roles of glycan in integrin-mediated tumor progression.
RESUMO
Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribonuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDAMB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDAMB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDAMB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase3/7.
Assuntos
Proteínas de Anfíbios/farmacologia , Neoplasias da Mama/metabolismo , Caspase 7/metabolismo , Lectinas/farmacologia , Rana catesbeiana/metabolismo , Ribonucleases/farmacologia , Serpinas/metabolismo , Proteínas de Anfíbios/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7 , Mutação , Fosforilação/efeitos dos fármacos , Rana catesbeiana/genética , Ribonucleases/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OPN (osteopontin) is a multiphosphorylated extracellular glycoprotein, which has important roles in bone remodelling, inflammation and cancer metastasis. OPN regulates cell spreading and adhesion primarily through its association with several integrins such as αvß3, and its phosphorylation affects these processes. However, the mechanism by which OPN O-glycosylation affects these processes is not completely understood. In the present study, we demonstrated that OPN O-glycosylation self-regulates its biological activities and also affects its phosphorylation status. We prepared two recombinant OPNs, WT (wild-type)-OPN and mutant OPN (ΔO-OPN), which lacks five O-glycosylation sites at a threonine/proline-rich region. O-glycan defects in OPN increased its phosphorylation level, as observed by dephosphorylation assays. Moreover, compared with WT-OPN, ΔO-OPN exhibited enhanced cell spreading and adhesion activities and decreased associations with ß1 integrins. This suggested that defects in O-glycans in OPN altered these activities, and that ß1 integrins have a less important role in adhesion to ΔO-OPN. The cell-adhesion activity of dephosphorylated ΔO-OPN was higher than the cell-adhesion activities of ΔO-OPN and dephosphorylated WT-OPN. This suggested that some of the phosphorylation in ΔO-OPN caused by O-glycan defects and O-glycans of OPN suppressed the OPN cell-adhesion activity. Thus functional activities of OPN can be determined by the combined glycosylation and phosphorylation statuses and not by either status alone.
Assuntos
Integrina alfaVbeta3/metabolismo , Osteopontina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Humanos , Integrina alfaVbeta3/genética , Osteopontina/genética , Fosforilação/fisiologiaRESUMO
We developed a high-throughput Enzyme-linked immunosorbent assay (ELISA) for measuring α2,6-sialylated transferrin (Tf), based on inhibition of anti-Tf antibody binding to α2,6-sialylated Tf by a lectin, Sambucus sieboldiana Agglutinin (SSA). The inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform specific. This finding was applied to an automated latex-agglutination immunoassay, using SSA lectin as an inhibitor (SSA-ALI). The concentration of α2,6-sialylated Tf measured by SSA-ALI in human cerebrospinal fluid was correlated with that of ELISA (r2 = 0.8554), previously developed for measuring α2,6-sialylated Tf.
Assuntos
Reações Antígeno-Anticorpo/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/líquido cefalorraquidiano , Lectinas de Plantas/química , Proteínas Inativadoras de Ribossomos/química , Transferrina/líquido cefalorraquidiano , Animais , Ligação Competitiva , Glicosilação , Cabras , Ensaios de Triagem em Larga Escala , Humanos , Neuraminidase/química , Lectinas de Plantas/imunologia , Ligação Proteica , Coelhos , Proteínas Inativadoras de Ribossomos/imunologiaRESUMO
The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins; the adhesion receptors, the cell-extracellular matrix (ECM) proteins, and the cytoplasmic plaque/peripheral membrane proteins. The cell adhesion receptors are usually transmembrane glycoproteins (for example E-cadherin and integrin) that mediate binding at the extracellular surface and determine the specificity of cell-cell and cell-ECM recognition. E-cadherin-mediated cell-cell adhesion can be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. On the other hand, integrin-mediated cell-ECM interactions play important roles in cytoskeleton organization and in the transduction of intracellular signals to regulate various processes such as proliferation, differentiation and cell migration. ECM proteins are typically large glycoproteins, including the collagens, fibronectins, laminins, and proteoglycans that assemble into fibrils or other complex macromolecular arrays. The most of these adhesive proteins are glycosylated. Here, we focus mainly on the modification of N-glycans of integrins and laminin-332, and a mutual regulation between cell adhesion and bisected N-glycan expression, to address the important roles of N-glycans in cell adhesion.
Assuntos
Adesão Celular , Glicosiltransferases/metabolismo , Animais , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Integrinas/genética , Integrinas/metabolismo , Polissacarídeos/metabolismoRESUMO
Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.
Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Queratinócitos/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Matriz Extracelular/ultraestrutura , Células HEK293 , Humanos , Immunoblotting , Integrinas/metabolismo , Queratinócitos/citologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neoplasias/metabolismo , Neoplasias/patologia , Polímeros/metabolismo , Ligação Proteica , CalininaRESUMO
α6ß4 integrin is an essential component of hemidesmosomes and modulates cell migration in wound healing and cancer invasion. To elucidate the role of N-glycosylation on ß4 integrin, we investigated keratinocyte adhesion and migration through the re-expression of wild-type or N-glycosylation-defective ß4 integrin (ΔNß4) in ß4 integrin null keratinocytes. N-glycosylation of ß4 integrin was not essential for the heterodimer formation of ß4 integrin with α6 integrin and its expression on a cell surface, but N-glycosylation was required for integrin-mediated cell adhesion and migration. Concomitantly with the reduction of ß4 integrin in the membrane microdomain, the intracellular signals of Akt and ERK activation were decreased in cells expressing ΔNß4 integrin. Forced cross-linking of ß4 integrin rescued the decreased ERK activation in ΔNß4 integrin-expressing cells to a similar extent in wild-type ß4 integrin-expressing cells. Surprisingly, compared with cells expressing wild-type ß4 integrin, an alternation in N-glycan structures expressed on epidermal growth factor receptor (EGFR), and the induction of a stronger association between EGFR and ß4 integrin were observed in ΔNß4 integrin-expressing cells. These results clearly demonstrated that N-glycosylation on ß4 integrin plays an essential role in keratinocyte cellular function by allowing the appropriate complex formation on cell surfaces.
Assuntos
Adesão Celular , Integrina beta4/metabolismo , Queratinócitos/citologia , Movimento Celular , Citometria de Fluxo , Glicosilação , Humanos , Laminina/metabolismo , Microscopia de FluorescênciaRESUMO
Laminins present in the basement membranes (BM) of blood vessels are involved in angiogenesis and other vascular functions that are critical for tumor growth and metastasis. Two major vascular laminins, the α4 (laminin-411/421) and α5 (laminin-511/521) types, have been well characterized. We recently found a third type of vascular laminin, laminin-3B11, consisting of the α3B, ß1 and γ1 chains, and revealed its biological activity. Laminin-3B11 potently stimulates vascular endothelial cells to extend lamellipodial protrusions. To understand the roles of laminin-3B11 in blood vessel functions and tumor growth, we examined localization of the laminin α3B chain in normal mammary glands and breast cancers, in comparison with the α4 and α5 laminins. In the immunohistochemical analysis, the α3B laminin was co-localized with the α4 and α5 laminins in the BM of venules and capillaries of normal breast tissues, but α3B was scarcely detected in vessels near invasive breast carcinoma cells. In contrast, the α4 laminin was overexpressed in capillaries of invasive carcinomas, where a large number of macrophages were found. The α5 laminin appeared to be weakly downregulated in cancer tissues, especially in capillary vessels. Furthermore, our in vitro analysis indicated that TNF-α significantly suppressed the laminin α3B expression in vascular endothelial cells, while it, as well as IL-1ß and TGF-α, upregulated the α4 expression. These results suggest that Lm3B11/3B21 may be required for normal mature vessels and interfere with tumor angiogenesis.
Assuntos
Membrana Basal/metabolismo , Neoplasias da Mama/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Laminina/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In previous studies, we reported that N-acetylglucosaminyltransferase III (GnT-III) activity and the enzyme product, bisected N-glycans, both were induced in cells cultured under dense conditions in an E-cadherin-dependent manner (Iijima, J., Zhao, Y., Isaji, T., Kameyama, A., Nakaya, S., Wang, X., Ihara, H., Cheng, X., Nakagawa, T., Miyoshi, E., Kondo, A., Narimatsu, H., Taniguchi, N., and Gu, J. (2006) J. Biol. Chem. 281, 13038-13046). Furthermore, we found that α-catenin, a component of the E-cadherin-catenin complex, was also required for this induction (Akama, R., Sato, Y., Kariya, Y., Isaji, T., Fukuda, T., Lu, L., Taniguchi, N., Ozawa, M., and Gu, J. (2008) Proteomics 8, 3221-3228). To further explore the molecular mechanism of this regulation, the roles of ß-catenin, an essential molecule in both cadherin-mediated cell adhesion and canonical Wnt signaling, were investigated. Unexpectedly, shRNA knockdown of ß-catenin resulted in a dramatic increase in GnT-III expression and its product, the bisected N-glycans, which was confirmed by RT-PCR and GnT-III activity and by E4-PHA lectin blot analysis. The induction of GnT-III expression increased bisecting GlcNAc residues on ß1 integrin, which led to down-regulation of integrin-mediated cell adhesion and cell migration. Immunostaining showed that nuclear localization of ß-catenin was greatly suppressed; intriguingly, the knockdown of ß-catenin in the nuclei was more effective than that in cell-cell contacts in the knockdown cells, which was also confirmed by Western blot analysis. Stimulation of the Wnt signaling pathway by the addition of exogenous Wnt3a or BIO, a GSK-3ß inhibitor, consistently and significantly inhibited GnT-III expression and its products. Conversely, the inhibition of ß-catenin translocation into the nuclei increased GnT-III activation. Taken together, the results of the present study are the first to clearly demonstrate that GnT-III expression may be precisely regulated by the interplay of E-cadherin-catenin complex-mediated cell-cell adhesion and Wnt/ß-catenin signaling, which are both crucial in the process of epithelial-mesenchymal transitions in physiological and pathological conditions.
Assuntos
Núcleo Celular/metabolismo , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , N-Acetilglucosaminiltransferases/biossíntese , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Ativação Enzimática/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genéticaRESUMO
N-acetylglucosaminyltransferase III (GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to core mannose with a beta1,4 linkage, so-called bisecting GlcNAc, in N-glycans. The bisecting GlcNAc is found in various hybrid and complex N-glycans. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of a bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by other GlcNAc transferases to form branching structures, such as N-acetylglucosaminyltransferase V (GnT-V), since GnT-V cannot utilize the bisected oligosaccharide as a substrate. Considering that expression of the enzyme leads to a remarkable structural alteration of the N-glycans on cell surface, it has been postulated that the enzyme is associated with various biological events such as cell adhesion, migration, cell growth, cell differentiation, and tumor invasion. Integrin is a major carrier of N-glycans. In fact, overexpression of GnT-III reduced the beta1,6 GlcNAc branching structures, in conjunction with the increase in the bisected N-glycans on integrins, and resulted in an inhibition of integrin-mediated cell spreading and migration, and the cellular phosphorylation levels. Conversely, knockdown of endogenous GnT-III expression resulted in increased cell migration, concomitant with an increase in beta1,6 GlcNAc-branched N-glycans on integrins. Thus, N-glycan could be considered as either a positive or negative regulator for biological functions of integrin.
Assuntos
Acetilglucosamina/fisiologia , Fenômenos Fisiológicos Celulares , Integrinas/fisiologia , Acetilglucosamina/metabolismo , Animais , Sequência de Carboidratos , Adesão Celular/genética , Adesão Celular/fisiologia , Fenômenos Fisiológicos Celulares/genética , Técnicas Genéticas , Glicosilação , Humanos , Integrinas/metabolismo , Modelos Biológicos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Neoplasias/genética , Neoplasias/metabolismoRESUMO
The basement membrane (BM) proteins laminins, which consist of α, ß, and γ chains, support tissue structures and cellular functions. To date only α4 and α5 types of laminins have been identified in the BMs of blood vessels. Our recent study suggested the presence of novel α3B-containing laminins in vascular BMs. Here we identified and characterized the third member of vascular laminins, laminin-3B11 (Lm3B11). RT-PCR analysis showed that microvascular endothelial (MVE) cells and umbilical vein endothelial cells expressed the messages for the α3B, ß1, ß2, and γ1 chains. In the culture of MVE cells, α3B was associated with ß1 and γ1, producing Lm3B11. Recombinant Lm3B11 was overexpressed by introducing the cDNAs of the three chains into HEK-293 cells and purified to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both α3ß1 and α6ß1 integrins. Most characteristically, Lm3B11 strongly stimulated MVE cells to extend many lamellipodial protrusions. This pseudopodial branching was blocked by an inhibitor for Src or phosphatidylinositol 3-kinase. Consistently, Lm3B11 stimulated the phosphorylation of Src and Akt more strongly than other laminins, suggesting that the integrin-derived signaling is mediated by these factors. The unique activity of Lm3B11 appears to be favorable to the branching of capillaries and venules.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Endoteliais/metabolismo , Microvasos/metabolismo , Pseudópodes/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Endoteliais/citologia , Humanos , Microvasos/citologia , Proteína Oncogênica pp60(v-src)/genética , Proteína Oncogênica pp60(v-src)/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , CalininaRESUMO
Laminin-332 (Lm332; formerly laminin-5) is a basement membrane protein in the skin, which promotes cell motility in wound healing and cancer invasion. In a previous study, we reported that the introduction of bisecting GlcNAc into Lm332 (GnT-III-Lm332), catalyzed by N-acetylglucosaminyltransferase III (GnT-III), reduced cell migration (Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008) J. Biol. Chem. 283, 33036-33045). However, the underlying molecular mechanism by which GnT-III-Lm332 suppresses the normal biological functions of Lm332 remains to be elucidated. In this study, we show that galectin-3, which is a beta-galactoside-binding protein, strongly bound to unmodified Lm332 but not to GnT-III-Lm332 and that binding of galectin-3 was completely blocked by lactose. Exogenous galectin-3 significantly enhanced keratinocyte cell motility on control Lm332 but not on GnT-III-Lm332. A functional blocking antibody against galectin-3 inhibited Lm332-induced alpha3beta1 and alpha6beta4 integrin clustering and focal contact formation. Co-immunoprecipitation revealed that galectin-3 associated with both beta4 integrin and epidermal growth factor receptor, thereby cross-linking the two molecules. The associations were inhibited by either the presence of lactose or expression of GnT-III. Moreover, galectin-3 consistently enhanced ERK activation. Taken together, the results of this study are the first to clearly identify the molecular mechanism responsible for the inhibitory effects of GnT-III on extracellular matrix-integrin-meditated cell adhesion, migration, and signal transduction. The findings presented herein shed light on the importance of N-glycosylation-mediated supramolecular complex formation on the cell surface.