RESUMO
Anoikis is a process of programmed cell death induced by the loss of cell/matrix interactions. In previous work, we have shown that the acquisition of anoikis resistance upregulates syndecan-4 (SDC4) expression in endothelial cells. In addition, SDC4 gene silencing by microRNA interference reverses the transformed phenotype of anoikis-resistant endothelial cells. Due to this role of SDC4 in regulating the behavior of anoikis-resistant endothelial cells, we have evaluated that the functional consequences of SDC4 silencing in the extracellular matrix (ECM) remodeling in anoikis-resistant rabbit aortic endothelial cells submitted to SDC4 gene silencing (miR-Syn4-Adh-1-EC). For this, we evaluated the expression of adhesive proteins, ECM receptors, nonreceptor protein-tyrosine kinases, and ECM-degrading enzymes and their inhibitors. Altered cell behavior was monitored by adhesion, migration, and tube formation assays. We found that SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells; this was accompanied by an increase in adhesion to fibronectin. Furthermore, after SDC4 silencing, we observed an increase in the expression of fibronectin, collagen IV, and vitronectin, and a decrease in the expression of integrin α5ß1 and αvß3, besides that, silenced cells show an increase in Src and FAK expression. Quantitative polymerase chain reaction and Western blot analysis demonstrated that SDC4 silencing leads to altered gene and protein expression of MMP2, MMP9, and HSPE. Compared with parental cells, SDC4 silenced cells showed a decrease in nitric oxide production and eNOS expression. In conclusion, these data demonstrate that SDC4 plays an important role in ECM remodeling. In addition, our findings represent an important step toward understanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.
Assuntos
Anoikis , Células Endoteliais , Matriz Extracelular , Inativação Gênica , Sindecana-4 , Sindecana-4/metabolismo , Sindecana-4/genética , Animais , Matriz Extracelular/metabolismo , Células Endoteliais/metabolismo , Coelhos , Adesão Celular , Movimento Celular , Fibronectinas/metabolismo , Células CultivadasRESUMO
Prostate cancer (PCa) is the leading cause of cancer-associated mortality in men, and new biomarkers are still needed. The expression pattern and protein tissue localization of proteoglycans of the syndecan family (SDC 1-4) and syntenin-1 (SDCBP) were determined in normal and prostatic tumor tissue from two genetically engineered mouse models and human prostate tumors. Studies were validated using SDC 1-4 and SDCBP mRNA levels and patient survival data from The Cancer Genome Atlas and CamCAP databases. RNAseq showed increased expression of Sdc1 in Pb-Cre4/Ptenf/f mouse Pca and upregulation of Sdc3 expression and downregulation of Sdc2 and Sdc4 when compared to the normal prostatic tissue in Pb-Cre4/Trp53f/f-;Rb1f/f mouse tumors. These changes were confirmed by immunohistochemistry. In human PCa, SDC 1-4 and SDCBP immunostaining showed variable localization. Furthermore, Kaplan-Meier analysis showed that patients expressing SDC3 had shorter prostate-specific survival than those without SDC3 expression (log-rank test, p = 0.0047). Analysis of the MSKCC-derived expression showed that SDC1 and SDC3 overexpression is predictive of decreased biochemical recurrence-free survival (p = 0.0099 and p = 0.045, respectively), and SDC4 overexpression is predictive of increased biochemical recurrence-free survival (p = 0.035). SDC4 overexpression was associated with a better prognosis, while SDC1 and SDC3 were associated with more aggressive tumors and a worse prognosis.
Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias da Próstata/patologia , Sindecana-1/genética , Sindecana-3/genética , Sindecana-4/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Transplante de Neoplasias , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Análise Serial de Proteínas , Análise de Sequência de RNA , Análise de Sobrevida , Sindecana-1/metabolismo , Sindecana-3/metabolismo , Sindecana-4/metabolismo , Sinteninas/genética , Sinteninas/metabolismoRESUMO
The cell's resistance to cell death by adhesion loss to extracellular matrix (anoikis), contributes to tumor progression and metastasis. Various adhesion molecules are involved in the anoikis resistance, including the syndecan-4 (SDC4), a heparan sulfate proteoglycan (HSPG) present on the cell surface. Changes in the expression of SDC4 have been observed in tumor and transformed cells, indicating its involvement in cancer. In previous works, we demonstrated that acquisition of anoikis resistance resistance by blocking adhesion to the substrate up-regulates syndecan-4 expression in endothelial cells. This study investigates the role of SDC4 in the transformed phenotype of anoikis resistant endothelial cells. Anoikis-resistant endothelial cells (Adh1-EC) were transfected with micro RNA interference (miR RNAi) targeted against syndecan-4. The effect of SDC4 silencing was analyzed by real-time PCR, western blotting and immunofluorescence. Transfection with miRNA-SDC4 resulted in a sequence-specific decrease in syndecan-4 mRNA and protein levels. Furthermore, we observed a reduction in the number of heparan and chondroitin sulfate chains in the cell extract and culture medium. The SDC4 silencing led to downregulation of proliferative and invasive capacity and angiogenic abilities of anoikis-resistant endothelial cells. Compared with the parental cells (Adh1-EC), SDC4 silenced cells (SDC4 miR-Syn-4-1-Adh1-EC e miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to collagen and laminin and also in the apoptosis rate. Moreover, transfection with miRNA-SDC4 caused a decrease in the number of cells in the S phase of the cell cycle. This is accompanied by an increase in the heparan sulfate synthesis after 12 h of simulation with fetal calf serum (FCS). SDC4 silencing cells are more dependent of growth factors present in the FCS to synthesize heparan sulfate than parental cells. Similar data were obtained for the wild-type cell line (EC). Our results indicated that downregulation of SDC4 expression reverses the transformed phenotype of anoikis resistant endothelial cells. These and other findings suggest that syndecan-4 is suitable for pharmacological intervention, making it an attractive target for cancer therapy.
Assuntos
Anoikis , Células Endoteliais/metabolismo , MicroRNAs/metabolismo , Interferência de RNA , Sindecana-4/biossíntese , Animais , MicroRNAs/genética , Coelhos , Sindecana-4/genéticaRESUMO
Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.
Assuntos
Aorta/citologia , Transformação Celular Neoplásica/genética , Sindecana-4/genética , Sindecana-4/metabolismo , Animais , Anoikis , Bromodesoxiuridina/farmacologia , Adesão Celular , Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Coelhos , Regulação para CimaRESUMO
Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/ß-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/ß-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/ß-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling.
Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sindecana-4/metabolismo , Trombospondinas/metabolismo , beta Catenina/metabolismo , Animais , Técnicas de Cultura de Células , Fibronectinas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Sindecana-4/genética , Trombospondinas/genética , Transfecção , Via de Sinalização WntRESUMO
Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2(Lp) compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2(Lp) mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2(Lp/+) enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2(Lp/+) embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2(Lp/Lp)) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.
Assuntos
Polaridade Celular , Embrião de Mamíferos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/citologia , Sindecana-4/metabolismo , Via de Sinalização Wnt , Animais , Embrião de Mamíferos/citologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Células Ciliadas Auditivas/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Tubo Neural/metabolismo , Defeitos do Tubo Neural/metabolismo , Sindecana-4/genética , Cicatrização , XenopusRESUMO
Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis, and ventricular diameter in hearts of mice with chronic Chagas disease. Here we investigated the transcriptomic recovery induced by BMC therapy by comparing the heart transcriptomes of control, chagasic, and BMC transplanted mice. Out of the 9390 unique genes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 96% of the alterations detected in infected hearts were restored to normal levels, although an additional 109 genes were altered by treatment. Transcriptomic recovery, a new measure that considers both resotrative and side effects of treatment, was remarkably high (84%). Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. In conclusion, by using large-scale gene profiling for unbiased assessment of therapeutic efficacy we demonstrate immunomodulatory effects of BMC therapy in chronic chagasic cardiomyopathy and identify potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets.
Assuntos
Transplante de Medula Óssea/patologia , Cardiomiopatia Chagásica/genética , Regulação da Expressão Gênica/imunologia , Miocárdio/imunologia , Miocárdio/patologia , Trypanosoma cruzi/imunologia , Animais , Transplante de Medula Óssea/imunologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/terapia , Doença Crônica , Modelos Animais de Doenças , Feminino , Fibrose , Galectina 3/genética , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Sindecana-4/genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/patogenicidade , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: Changes in the proteoglycans glypican and syndecan-4 have been reported in several pathological conditions, but little is known about their expression in the heart during diabetes. The aim of this study was to investigate in vivo heart function changes and alterations in mRNA expression and protein levels of glypican-1 and syndecan-4 in cardiac and skeletal muscles during streptozotocin (STZ)-induced diabetes. METHODS: Diabetes was induced in male Wistar rats by STZ administration. The rats were assigned to one of the following groups: control (sham injection), after 24 hours, 10 days, or 30 days of STZ administration. Echocardiography was performed in the control and STZ 10-day groups. Western and Northern blots were used to quantify protein and mRNA levels in all groups. Immunohistochemistry was performed in the control and 30-day groups to correlate the observed mRNA changes to the protein expression. RESULTS: In vivo cardiac functional analysis performed using echocardiography in the 10-day group showed diastolic dysfunction with alterations in the peak velocity of early (E) diastolic filling and isovolumic relaxation time (IVRT) indices. These functional alterations observed in the STZ 10-day group correlated with the concomitant increase in syndecan-4 and glypican-1 protein expression. Cardiac glypican-1 mRNA and skeletal syndecan-4 mRNA and protein levels increased in the STZ 30-day group. On the other hand, the amount of glypican in skeletal muscle was lower than that in the control group. The same results were obtained from immunohistochemistry analysis. CONCLUSION: Our data suggest that membrane proteoglycans participate in the sequence of events triggered by diabetes and inflicted on cardiac and skeletal muscles.
Assuntos
Diabetes Mellitus Experimental/complicações , Glipicanas/metabolismo , Miocárdio/metabolismo , Sindecana-4/metabolismo , Disfunção Ventricular Esquerda/etiologia , Função Ventricular Esquerda , Animais , Northern Blotting , Western Blotting , Diabetes Mellitus Experimental/diagnóstico por imagem , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diástole , Glipicanas/genética , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Imuno-Histoquímica , Masculino , Músculo Esquelético/metabolismo , Miocárdio/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sindecana-4/genética , Fatores de Tempo , Ultrassonografia , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Dynamic regulation of cell adhesion receptors is required for proper cell migration in embryogenesis, tissue repair, and cancer. Integrins and Syndecan4 (SDC4) are the main cell adhesion receptors involved in focal adhesion formation and are required for cell migration. SDC4 interacts biochemically and functionally with components of the Wnt pathway such as Frizzled7 and Dishevelled. Non-canonical Wnt signaling, particularly components of the planar cell polarity branch, controls cell adhesion and migration in embryogenesis and metastatic events. Here, we evaluate the effect of this pathway on SDC4. We have found that Wnt5a reduces cell surface levels and promotes ubiquitination and degradation of SDC4 in cell lines and dorsal mesodermal cells from Xenopus gastrulae. Gain- and loss-of-function experiments demonstrate that Dsh plays a key role in regulating SDC4 steady-state levels. Moreover, a SDC4 deletion construct that interacts inefficiently with Dsh is resistant to Wnt5a-induced degradation. Non-canonical Wnt signaling promotes monoubiquitination of the variable region of SDC4 cytoplasmic domain. Mutation of these specific residues abrogates ubiquitination and results in increased SDC4 steady-state levels. This is the first example of a cell surface protein ubiquitinated and degraded in a Wnt/Dsh-dependent manner.
Assuntos
Sindecana-4/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Proteínas Desgrenhadas , Eletroforese em Gel de Poliacrilamida , Gástrula/metabolismo , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sindecana-4/genética , Ubiquitinação , Proteínas Wnt/genética , Proteína Wnt-5a , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Syndecan-4 and integrins are involved in the cell migration and adhesion processes in several cell types. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is associated to focal adhesions in adherent cells and has been described as a marker of satellite cells in skeletal muscle. In this tissue, beta1 integrin forms heterodimers with alpha5 and alpha6 during myoblast differentiation and with alpha7 in adult muscle. Here, we show that the levels of these two cell surface membrane molecules are regulated by spontaneous electrical activity during the differentiation of rat primary myoblasts. Syndecan-4 and beta1 integrin protein levels decrease after the inhibition of electrical activity using tetrodotoxin (TTX). Syndecan-4 also decreases substantially in denervated rat tibialis anterior muscle. Indirect immunofluorescence analysis shows that syndecan-4 and beta1 integrin co-localize with vinculin, a molecular marker of costameres in skeletal muscle myofibers. Co-localization is lost in inactive myotubes adopting a diffuse pattern, suggesting that the costameric organization is disrupted in TTX-treated myotubes. Moreover, the inhibition of spontaneous electrical activity decreases myotube cell adhesion. In summary, this work shows that syndecan-4 and beta1 integrin protein levels and their localization in costameric structures are regulated by electrical activity and suggests that this regulatory mechanism influences the adhesion properties of skeletal myotubes during differentiation.