RESUMEN
The metastatic potential of malignant tumor has been shown to be correlated with the increased expression of tri- and tetra-antennary ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) N-glycans. In this study, We found that GnT-V expression was negatively correlated with receptor protein tyrosine phosphatase type µ(RPTPµ) in human glioma tissues. To study whether RPTPµ is a novel substance of GnT-V which further affect RPTPµ's downstream dephosphorylation function, we preform lentiviral infection with GnT-V gene to construct stably transfected GnT-V glial cell lines. We found RPTPµ undergone severer cleavage in GnT-V transfected glioma cells compare to Mock cells. RPTPµ intracellular domain fragments increased while ß1,6-GlcNAc-branched N-glycans increased, in consistent with the decrease of RPTPµ's catalytic activity. The results showed that abnormal glycosylation could decrease the phosphorylation activity of PTP µ, and affect PLCγ-PKC pathways. Both protease inhibitor Furin and N-glycan biosynthesis inhibitor swainsonine could decrease cell mobility in GnT-V-U87 transfectants and other glioma cell lines. All results above suggest increased post-translational modification of RPTPµ N-glycans by GnT-V attenuates its tyrosine phosphatase activity and promotes glioma cell migration through PLCγ-PKC pathways, and that the ß1,6-GlcNAc-branched N-glycans of RPTPµ play a crucial role in glioma invasivity.
Asunto(s)
Glioma/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , beta-Glucanos/metabolismo , Movimiento Celular , Adhesiones Focales , Glioma/fisiopatología , Glicosilación , Humanos , Fosfolipasa C gamma/metabolismo , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
Receptor-like protein tyrosine phosphatase alpha (RPTPα or PTPα), a type I transmembrane glycoprotein with complex N-glycans, executes multifunction roles on cell behaviors. However, its effect on tumorigenesis and metastasis remains controversial. In this study, PTPα is identified as a novel substrate of N-Acetylglucosaminyltransferase V (GnT-V). Immunofluorescence results showed that addition of ß1,6 GlcNAc branches on PTPα enhanced PTPα's cytomembrane assemble in GnT-V-MCF-7 compared with Mock-MCF-7 (MCF7 cells transfected with the vector pcDNA3). Then we found the alleviating degradation of PTPα was observed in GnT-V-MCF-7 while PTPα in Mock-MCF-7 was prone to quick degradation. Increased cell-surface retention subsequently enhanced PTPα's catalytic activity on the dephosphorylation of Src kinase at Tyr529 and promoted focal adhesion formation and mature. Therefore, our findings could provide an insight into the molecular mechanism of how GnT-V promoted cell migration, suggesting that PTPα could be one of factors regulating promote migration of breast cancer cell.
Asunto(s)
Adhesiones Focales/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Neoplasias de la Mama/metabolismo , Carcinogénesis , Catálisis , Línea Celular Tumoral , Movimiento Celular , Femenino , Glicosilación , Humanos , Integrina beta1/metabolismo , Lectinas/química , Células MCF-7 , Metástasis de la Neoplasia , Fosforilación , Plásmidos/metabolismo , Polisacáridos/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Cadherin is crucial for cell-cell adhesion and N-glycosylation of N-cadherin has been implicated in the process of mammary, renal, and ovarian carcinogenesis. However, whether N-glycosylation of N-cadherin plays a role in glioma remains unknown. Previous studies had indicated that N-glycosylation could occur at three asparagine residues of N-cadherin. By generating and over-expressing N-glycosylation-deficient N-cadherin mutants in the human glioma cell lines SHG66 and U87, we found that mutation of N402 but not of the other potentially N-glycosylated residues destabilized N-cadherin and led to its ubiquitylation and subsequent proteasomal degradation. Furthermore, destabilized N-cadherin inhibited cadherin-mediated cell-cell adhesion and promoted cell migration. Our findings reveal that N-glycosylation controls N-cadherin stability and plays a role in glioma migration. J. Cell. Biochem. 118: 1423-1431, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , Asparagina/química , Cadherinas/química , Cadherinas/metabolismo , Glioma/metabolismo , Antígenos CD/genética , Asparagina/genética , Cadherinas/genética , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Glioma/genética , Glicosilación , Humanos , Mutación , N-Acetilglucosaminiltransferasas/metabolismo , Estabilidad ProteicaRESUMEN
AIM: Maternal dietary protein restriction reduces nephron number in offspring and increases the risk of cardiovascular and chronic kidney diseases. Perlecan is the major basement membrane/extracellular matrix heparan sulfate proteoglycan (HSPG) that plays a crucial role in nephron formation. This study was to determine whether maternal dietary protein restriction during pregnancy leads to an abnormal perlecan expression pattern during kidney development and a correlation with aberrant cell proliferation and apoptosis. METHODS: Pregnant Sprague-Dawley rats were divided into two groups, maintained on either a low-protein diet (MLP group) or a normal-protein diet (MNP group). Kidneys were dissected from embryos of different kidney development stages. Real-time PCR and immunohistochemistry were performed to detect the transcript level of rHSPG2, the coding gene of perlecan, and its protein expression pattern. Apoptosis and proliferation cell were detected by TUNEL system and Ki67 marker. RESULTS: Embryonic weights and nephron number were significantly affected by maternal low protein diets. The transcript level of rHSPG2 in the MLP group was significantly lower at embryonic day 18 and the neonatal period. Immunohistochemistry study was consistent with the RT-PCR results. The proliferation level of the MLP group was significantly lower than the MNP group at E18 and more apoptotic cells was detected in MLP newborn. CONCLUSION: Maternal protein restriction reduced the expression of perlecan and lead aberrant cell proliferation and apoptosis during mid-metanephrogenesis in offspring. This data may provide new evidence to understand the mechanism of reduced nephron number due to maternal protein restriction and enlighten solution.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales , Dieta con Restricción de Proteínas , Proteoglicanos de Heparán Sulfato/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Nefronas/metabolismo , Animales , Apoptosis , Proliferación Celular , Regulación hacia Abajo , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Proteoglicanos de Heparán Sulfato/genética , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Nefronas/embriología , Estado Nutricional , Organogénesis , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Our previous study shows that Calpain 6 (CAPN6) expression is regulated by PI3K-Akt in liver cancer through POU2F1 and CAPN6 which promote cell proliferation and inhibit apoptosis of liver cancer cells. microRNAs (miRNAs) plays important roles in regulation of gene expression. However, whether miRNAs regulates CAPN6 expression and its cellular function is still unknown. This study aims to investigate how miRNAs regulate liver cancer apoptosis through POU2F1-CAPN6. It was verified that POU2F1 could promote cell proliferation and inhibit apoptosis through CAPN6. Using methods of bioinformatics, miR-449a was predicted as a potential regulator of both CAPN6 and POU2F1. It was verified that CAPN6 and POU2F1 were the target genes of miR-449a by luciferase assay. CAPN6 and POU2F1 protein and mRNA levels decreased in liver cancer cells with miR-449a overexpression using western blot and real time RT-PCR assays. miR-449a expression was lower in liver cancer tissues compared with their normal ones, so did the cells. Overexpression of miR-449a inhibited cell proliferation, induced G1 phase arrest and cell apoptosis in liver cancer. Further research demonstrated that miR-449a inhibited cancer cell proliferation and induced apoptosis via suppressing both POU2F1 and CAPN6. The study indicated that miR-449a functions as a tumor inhibitor in liver cancer by decreasing POU2F1 and CAPN6 expression in liver cancer.
Asunto(s)
Calpaína/metabolismo , Carcinoma Hepatocelular/secundario , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Calpaína/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Proliferación Celular , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Factor 1 de Transcripción de Unión a Octámeros/genética , Pronóstico , Transducción de Señal , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
BACKGROUND: Podocyte detachment and apoptosis are two risk factors causing podocyte loss, F-actin rearrangement is involved in detachment and apoptosis. However, the nature of events that promote detachment and apoptosis of podocytes and whether detachment occurred simultaneously with apoptosis are still unclear. Previously, it was found that angiopoietin-like3 (Angptl3) induces F-actin rearrangement in podocytes. In this study we investigate whether Angptl3 influences podocyte loss (detachment and apoptosis) and the process through which Angptl3 exactly influenced the podocyte loss. METHODS: In conditionally immortalized mice podocytes, recombinant mice Angptl3 protein (rm-Angptl3) was used to mimic Angptl3 overexpression model and transfection with small interfering RNA (siRNA) to knockdown the expression of Angptl3. Both flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were used to detect apoptosis. Rearrangement of F-actin was assessed using confocal microscopy. Western blot assay was used to measure levels of Angptl3, integrin α3ß1, integrin-linked kinase (ILK), p53, caspase 3, and phosphorylation of integrin ß1. RESULTS: In a puromycin aminonucleoside (PAN)-induced podocyte injury model, rm-Angptl3 accelerated the loss of podocytes, both detachment and apoptosis occurred, and F-actin rearrangement is involved in the process. However, knockdown of Angptl3 by siRNA markedly ameliorated these injuries. Observed effects were partially correlated with the altered integrin α3ß1, ILK and p53, rather than caspase 3. CONCLUSIONS: Angptl3 is a novel factor involved in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro. This study helps to deepen the understanding of the mechanisms of podocyte loss and lays the foundation for developing a new successful therapy for podocyte injury via lower expression of Angptl3.
Asunto(s)
Angiopoyetinas/metabolismo , Apoptosis/efectos de los fármacos , Podocitos/metabolismo , Puromicina Aminonucleósido/farmacología , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/genética , Animales , Biomarcadores/análisis , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Noqueados , Podocitos/citología , Podocitos/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Rol , Sensibilidad y Especificidad , TransfecciónRESUMEN
BACKGROUND: Angiopoietin-like-3 (ANGPTL3) expression is increased in glomerular podocytes of nephrotic syndrome. We hypothesize whether ANGPTL3 plays an important role in podocyte injury and promoting proteinuria. METHODS: Angptl3(+/+) and Angptl3(-/-) female mice on B6;129S5 gene background were injected with adriamycin by tail vein at the dose of 25 mg/Kg to produce nephropathy. Proteinuira was measured and podocytes ultrastructure was observed by electron microscopy. The interaction between ANGPTL3 and intergrin ß3 was analyzed by CO-IP and confocal immunofluorescence. The relative gene and protein expression were analyzed by RT-PCR and western blot. RESULTS: The deletion of ANGPTL3 tremendously attenuates proteinuria (more than a fivefold decrease in albuminuria) and protects podocytes from injury in a mouse model of adriamycin-induced nephropathy. We further demonstrate that ANGPTL3 interacts with and activates podocyte-expressed integrin ß3 and regulate expression of α-actinin-4, which may result in the cytoskeletal rearrangement of podocytes. Additionally, we identify the activation of the ANGPTL3-integrin ß3 signaling pathway in patients with nephrotic syndrome. CONCLUSION: ANGPTL3 might play a crucial role in podocyte injury. Either decreasing ANGPTL3 expression or interfering with the ANGPTL3-integrin ß3 interaction might be benefit for podocyte protection and decrease proteinuira.
Asunto(s)
Angiopoyetinas/metabolismo , Integrina beta3/metabolismo , Síndrome Nefrótico/metabolismo , Podocitos/ultraestructura , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/genética , Animales , Western Blotting , Doxorrubicina/efectos adversos , Femenino , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Síndrome Nefrótico/inducido químicamente , Podocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cystathionine-γ-lyase (CSE) is a major endogenous enzyme producing H2S which, as a third gasotransmitter, plays important roles in many physiological and pathological processes. The mechanism of regulating CSE gene expression is unclear and the roles of CSE/H2S in tumor also have not got a profound understanding, especially in colon cancer. Our study demonstrated that CSE gene expression was regulated by the Wnt pathway on transcriptional level. Activating the Wnt pathway by either Wnt3a or LiCl increased CSE mRNA and protein levels, while siRNA-mediated silence of ß-catenin decreased CSE mRNA and protein levels. XAV939 treatment which accelerated ß-catenin degradation could reduce CSE protein level. To reveal the mechanism, two TCF/LEF binding sites were found in CSE promoter whose activity had a positive response to ß-catenin overexpression in 293T cells. Mutations of TCF/LEF binding sites led to an increase of the promoter activity. It indicated that TCF/LEF likely acted as a repressor to CSE gene transcription, and Wnt signal contributed to free ß-catenin accumulation to possibly relieve the repression. Either knockdown of CSE by shRNA (shCSE) or its inhibition by PAG decreased SW480 cell proliferation, migration, and tumor xenograft growth in nude mice. In conclusion, we have demonstrated that the Wnt pathway regulates CSE gene expression on transcriptional level and CSE/H2S plays important roles in colon cancer.
Asunto(s)
Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Cistationina gamma-Liasa/genética , Transcripción Genética , Vía de Señalización Wnt , Alquinos/farmacología , Animales , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Cistationina gamma-Liasa/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Ratones Desnudos , Mutación/genética , Regiones Promotoras Genéticas , Factores de Transcripción TCF/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549-GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549-GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Células Madre Neoplásicas/efectos de los fármacos , TransfecciónRESUMEN
The change of oligosaccharide structure has been revealed to be crucial for glycoproteins' biological functions and cell biological characteristics. N-acetylglucosaminy transferase V (GnT-V), a key enzyme catalysing the reaction of adding ß1, 6-N-acetylglucosamine (GlcNAc) on asparagine-linked oligosaccharides of cell proteins, has been implicated to a metastastic-promoting oncoprotein in some carcinomas. However, this correlation might not be subjected to all types of cancers, for example, in non-small cell lung cancers, low level of GnT-V expression is associated with relatively short survival time and poor prognosis. To explain the role of GnT-V in lung cancer progression, we studied the association of GnT-V expression with lung cancer EMT behaviour. We found that GnT-V expression was correlated with epithelial marker positively and mesenchymal marker negatively. GnT-V levels, as well as ß1,6-GlcNAc branched N-glycans, were strongly reduced in TGF-ß1-induced EMT of human lung adenocarcinoma A549 cells. Further studies showed that suppression of ß1,6-GlcNAc branched N-glycans by inhibitor or GnT-V silencing in A549 cells could promote TGF-ß1-induced EMT-like changes, cell migration and invasion. Meanwhile, overexpression of GnT-V impaired TGF-ß1-induced EMT, migration and invasion. It suggests that GnT-V suppresses the EMT process of lung cancer cells through inhibiting the TGF-ß/Smad signalling and its downstream transcription factors in a GnT-V catalytic activity-dependent manner. Taken together, the present study reveals a novel mechanism of GnT-V as a suppressor of both EMT and invasion in human lung cancer cells, which may be useful for fully understanding N-glycan's biological roles in lung cancer progression.
Asunto(s)
Acetilglucosamina/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares/patología , Polisacáridos/farmacología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Acetilglucosamina/genética , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mesodermo/metabolismo , Mesodermo/patología , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Receptor-like protein tyrosine phosphatases (RPTPs) are type I transmembrane glycoproteins with N-glycans whose catalytic activities are regulated by dimerization. However, the intrinsic mechanism involved in dimerizing processes remains obscure. In this study, receptor protein tyrosine phosphatase rho (PTPRT) is identified as a novel substrate of N-Acetylglucosaminyltransferase V (GnT-V). We show that addition of ß1,6 GlcNAc branches on PTPRT prolongs PTPRT's cell-surface retention time. GnT-V overexpression enhances galectin-3's cell-surface retention and promotes PTPRT's dimerization mediated by galectin-3. Increased dimerization subsequently reduces PTPRT's catalytic activity on the dephosphorylation of signal transducer and activator of transcription 3 (STAT3) at tyrosine residue 705 (pY705 STAT3), then the accumulated pY705 STAT3 translocates into the nucleus. Collectively, these findings provide an insight into the molecular mechanism by which GnT-V promotes cell migration, suggesting that accumulation of ß1,6 GlcNAc branched N-glycans promotes PTPRT's dimerization and decreases its catalytic activity, resulting in enhanced cell migratory capacity.
Asunto(s)
Movimiento Celular , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Línea Celular , Movimiento Celular/genética , Activación Enzimática , Galectina 3/metabolismo , Técnicas de Silenciamiento del Gen , Glicosilación/efectos de los fármacos , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Polisacáridos/metabolismo , Polisacáridos/farmacología , Unión Proteica , Multimerización de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Glomerular podocytes are highly differentiated cells whose foot processes, which are mainly maintained by the architecture of actin filaments, have a unique morphology. A rearrangement of F-actin in podocytes causes changes in their motility that involve foot process effacement and proteinuria in glomerular diseases. Members of the Rho family small GTPases, especially RhoA, Rac1, and Cdc42, are key molecules in the regulation of actin cytoskeleton rearrangement. Our previous study showed that angiopoietin-like 3 (Angptl3) can increase the motility of podocytes in vitro. In this study, we found that recombinant Angptl3 treatment, together with the activation of Rac1, could cause F-actin rearrangement in podocytes. We also found that these effects could be blocked by an integrin α(V)ß3 inhibitor, implicating integrin α(V)ß3 as the Angptl3 receptor in its effects on actin cytoskeleton rearrangement. In addition, we studied the molecular pathway for this process. Our results showed that in podocytes, Angptl3 could induce actin filament rearrangement, mainly in lamellipodia formation, and that this process was mediated by integrin α(V)ß3-mediated FAK and PI3K phosphorylation and Rac1 activation. Our results might provide a new explanation for the effect of Angptl3 on increasing podocyte motility.
Asunto(s)
Actinas/metabolismo , Angiopoyetinas/farmacología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfaVbeta3/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Podocitos/enzimología , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Activación Enzimática/efectos de los fármacos , Integrina alfaVbeta3/antagonistas & inhibidores , Ratones , Fosforilación/efectos de los fármacos , Podocitos/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Mutations in canonical transient receptor potential channel 6 (TRPC6) have been identified as responsible for the development of focal segmental glomerulosclerosis, a proteinuric disease with steroid resistance and poor prognosis. This study explores the prevalence of TRPC6 variants in Chinese children with idiopathic nephrotic syndrome (INS), the genotype/phenotype correlation of TRPC6 variants, the therapeutic response, and the underlying molecular mechanism. METHODS: Fifty-one children with sporadic INS were enrolled: 23 steroid-sensitive cases and 28 steroid-resistant cases Polymerase chain reaction was used to amplify 13 exons and the promoter sequences of TRPC6 before sequencing. The expression of TRPC6 in renal tissues was illustrated by immunohistochemistry staining. The transcriptional activity of variants in TRPC6 promoter was measured by the luciferase assay. RESULTS: Three variants (-254C>G, rs3824934; +43C/T, rs3802829; and 240 G>A, rs17096918) were identified. The allele frequency of the -254C>G single-nucleotide polymorphism (SNP) in the steroid-resistant nephrotic syndrome (SRNS) patients (40.5%) was higher than that in the steroid-sensitive nephrotic syndrome subjects (27.1%; P = 0.046). The -254C>G SNP enhanced transcription from TRPC6 promoter in vitro and was associated with increased TRPC6 expression in renal tissues of SRNS patients. CONCLUSION: -254C>G, a SNP underlying enhanced TRPC6 transcription and expression, may be correlated with the development of steroid resistance in Chinese children with INS.
Asunto(s)
Pueblo Asiatico/genética , Síndrome Nefrótico/congénito , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Canales Catiónicos TRPC/genética , Transcripción Genética/genética , Secuencia de Bases , Niño , Exones/genética , Frecuencia de los Genes , Humanos , Inmunohistoquímica , Riñón/metabolismo , Luciferasas , Datos de Secuencia Molecular , Síndrome Nefrótico/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Canal Catiónico TRPC6RESUMEN
Adaptor proteins are involved in the assembly of various intracellular complexes and the regulation of cellular functions. Membrane-associated guanylate kinase inverted 2 (MAGI2), also known as synaptic scaffolding molecule (S-SCAM), plays a critical role in signal transduction by assembling and anchoring its ligands. However, the role of MAGI2 in mediating apoptosis remains largely unknown. In the present study, BEL-7404 human hepatocellular carcinoma cells were transfected with a plasmid containing myc-MAGI2 or an empty plasmid and cell viability was then determined using the Cell Counting kit-8. Apoptosis was also detected using an Annexin V apoptosis assay. The cells were then treated with various doses of staurosporine (STS) for different periods of time. The overexpression of myc-MAGI2 was found to sensitize the BEL-7404 cells to apoptosis in response to STS in a time- and dose-dependent manner. Our results demonstrated that MAGI2 enhanced STS-induced apoptosis by increasing the protein expression of cytoplasmic phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and decreasing its protein degradation. The apoptotic sensitivity of the cells caused by the overexpression of myc-MAGI2 was reversed by the silencing of PTEN expression by PTEN siRNA, thus revealing a momentous role of PTEN in the enhancement of the sensitivity of cancer cells to STS-induced apoptosis by MAGI2. Finally, we observed that the MAGI-PTEN complex triggered by MAGI2 overexpression reduced the phosphorylation levels of AKT. These results suggest that MAGI2 overexpression enhances the sensitivity of cancer cells harboring ectopic PTEN to STS-induced apoptosis.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas Portadoras/genética , Resistencia a Antineoplásicos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Estaurosporina/farmacología , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática , Estabilidad de Enzimas/efectos de los fármacos , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Guanilato-Quinasas , Humanos , Fosfohidrolasa PTEN/genética , Unión Proteica , Transporte de Proteínas , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Factores de Tiempo , TransfecciónRESUMEN
Abnormal DNA methylation is one of the important characteristics in tumor cells. Apoptosis plays an essential role in cell survival and processing. It is not clear whether DNA methyltransferases (DNMTs) change in apoptosis and how DNMTs are regulated in apoptosis. In this study, we found that SMMC-7721 or BEL-7404 cells were induced to apoptosis by STS, meanwhile the DNMT3B protein and mRNA level were decreased. To explore the mechanism of DNMT3B down-regulation, we found that the mRNA decay was not changed and core promoter activity of DNMT3B gene was decreased in STS-induced apoptosis. In order to figure out the signal molecule involved in transcriptional regulation of DNMT3B gene by STS, p-JNK, p-ERK, and p-p38 were examined. In STS-induced apoptosis p-JNK level was increased, and p-ERK and p-p38 were decreased. Furthermore, the inhibitor of p-JNK significantly alleviated the decline of DNMT3B protein. We also found that the siRNA of DNMT3B strengthened the cleavage of PARP and pro-caspase-3 as well as up-regulated the p16 gene expression in STS-treated cells. We concluded here that STS-regulated DNMT3B gene expression via p-JNK and down-regulation of DNMT3B-mediated STS-induced apoptosis through the up-regulation p16 expression.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Estaurosporina/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes p16 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MAP Quinasa Quinasa 4/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Interferente Pequeño , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , ADN Metiltransferasa 3BRESUMEN
BACKGROUND: Nucleation of cholesterol monohydrate crystals following the aggregation and fusion of cholesterol-enriched vesicles is a critical procedure in the formation of cholesterol gallstone. Biliary proteins play important roles in the process. It is inefficient to screen pro-nucleating or anti-nucleating proteins with routine physiochemical techniques, by which we discovered several pro-nucleating proteins. METHODOLOGY/PRINCIPAL FINDINGS: Based on comparative proteomic technologies, we investigated the differentially expressed proteins between the cholesterol gallstone and control groups, and between the vesicular phase and micellar phase. There are 401±75 spots detected on the cholesterol gallstone group and 389±94 spots on the control group gels, 120±24 spots detected on vesicular phase and 198±37 on micellar phase gels, and accordingly 22 and 8 differentially expressed proteins were identified successfully, respectively. Three of them, HSA, Profilin and Retinol Binding Protein, were validated by Western blot. CONCLUSION/SIGNIFICANCE: Some of the identified proteins are in good agreement with proteins reported to be involved in the gallstone formation before. The information from this study might provide some important clues to uncover the key proteins involved in the formation of cholesterol gallstone.
Asunto(s)
Colelitiasis/metabolismo , Vesícula Biliar , Cálculos Biliares , Proteoma/metabolismo , Adulto , Bilis/metabolismo , Colelitiasis/química , Colelitiasis/patología , Colesterol/química , Colesterol/metabolismo , Cristalización , Femenino , Vesícula Biliar/metabolismo , Vesícula Biliar/patología , Cálculos Biliares/metabolismo , Cálculos Biliares/patología , Humanos , Masculino , Micelas , Persona de Mediana Edad , Proteoma/análisisRESUMEN
AIM: Glucocorticoid therapy has been used in childhood nephrotic syndrome since the 1950s, where the characteristic change is effacement of the actin-rich foot process of glomerular podocytes. Recent studies have shown that glucocorticoids, in addition to their general immunosuppressive and anti-inflammatory effects, have a direct effect on podocytes, regulate some apoptotic factors, and increase the stability of actin filaments. However, the precise mechanism(s) underlying the protective effects of glucocorticoids on podocytes remain unclear. It is known that adriamycin (ADR) can induce podocyte foot process effacement and trigger massive proteinuria in rodent models. However, few reports have examined the direct role of ADR in podocyte actin rearrangement in vitro. In this study, we investigated how ADR directly induced podocyte actin cytoskeleton rearrangement and further analyzed how dexamethasone prevented such injury. METHODS: We used confocal microscopy to assess podocyte actin rearrangement. Western blot analysis and real-time polymerase chain reaction were performed to measure the protein and mRNA levels of α-actinin-4. RESULTS: We demonstrated that there was a time-dependent ADR-induced podocyte actin rearrangement with less than 12 h of ADR treatment in cultured podocytes. Dexamethasone could protect podocytes from ADR-induced injury and also stabilize the expression of α-actinin-4. CONCLUSION: This study showed that dexamethasone had direct effects on podocytes: α-actinin-4 may be one of the potential target molecules.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Actinina/metabolismo , Antibióticos Antineoplásicos/toxicidad , Dexametasona/farmacología , Doxorrubicina/toxicidad , Podocitos/efectos de los fármacos , Sustancias Protectoras/farmacología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Actinina/genética , Animales , Western Blotting , Células Cultivadas , Citoprotección , Ratones , Microscopía Confocal , Podocitos/metabolismo , Podocitos/patología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de TiempoRESUMEN
Hydrogen sulfide (H(2)S) has been found to play an important role as a novel gasotransmitter involved in many biological processes. The regulatory role of endogenous H(2)S-producing enzyme on cancer cell survival is complex and unclear. According to the data that cystathionine γ-lyase (CSE) gene, catalyzed H(2)S production in trans-sulfuration pathway, was upregulated in Akt stably transformed mouse embryonic fibroblast cells, the mechanisms that elevated CSE expression by PI3K/Akt signaling pathway and its biological functions in cell survival were studied. In the present study, firstly, the results showed that PI3K/Akt positively correlated with CSE expression levels in human hepatocellular carcinoma cell lines. CSE expression was decreased by the PI3K inhibitor or Akt deletion, while upregulated with the activating of Akt. Based on dual-luciferase reporter assay, the -592/+139 gene fragment represented the CSE core promoter, and the PI3K/Akt pathway regulated CSE expression on transcriptional level. Sp1 was the critical transcription factor in regulation of CSE expression via the mutation of transcription factor binding sites on the promoter. Furthermore, we proved that Sp1 could directly bind to CSE promoter by ChIP assay. In addition, we explored that the endogenous H(2)S production was connected with the regulated CSE expression, and CSE/H(2)S promoted human hepatocellular carcinoma cell proliferation via cell cycle progression regulation. In summary, we have, for the first time, demonstrated that PI3K/Akt pathway regulates the CSE expression via Sp1, which is particularly important to understand the effect of PI3K/Akt and CSE on the tumorigenesis.
Asunto(s)
Carcinoma Hepatocelular/genética , Cistationina gamma-Liasa/genética , Neoplasias Hepáticas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción Sp1/metabolismo , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Cistationina gamma-Liasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Factor de Transcripción Sp1/genética , Sulfitos/metabolismoRESUMEN
Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5ß1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5ß1. These results suggest that the insufficient fibronectin caused by the integrin α5ß1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin ß1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5ß1, and suggested a new target for breast cancer therapy.
Asunto(s)
Actinas/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Inhibidores Enzimáticos/farmacología , Integrina beta1/metabolismo , Profilinas/fisiología , Estaurosporina/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: This study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model. MATERIAL/METHODS: The nucleation role of osteopontin was determined in patients' and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined. RESULTS: Osteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends. CONCLUSIONS: Our results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.