RESUMEN
Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.
Asunto(s)
Antineoplásicos/administración & dosificación , Ácidos Cafeicos/administración & dosificación , Cisplatino/administración & dosificación , Endopeptidasas/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Neoplasias Ováricas/enzimología , Alcohol Feniletílico/análogos & derivados , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/biosíntesis , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Alcohol Feniletílico/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: The tumor susceptibility gene 101 (Tsg101), a component of the endosomal sorting complex required for transport (ESCRT) complex I, is involved in multiple biological processes involving endomembranous structures and the plasma membrane. The role of Tsg101 in the uterine epithelium was investigated in Tsg101 floxed mice crossed with Lactoferrin-iCre mice (Tsg101d/d). METHODS: Tsg101d/d mice were bred with stud male mice and the status of pregnancy was examined on days 4 and 6. Histological analyses were performed to examine the uterine architecture. Immunofluorescence staining of several markers was examined by confocal microscopy. Uterine epithelial cells (UECs) were isolated from Tsg101f/f and Tsg101d/d mice, and the expression of necroptosis effectors was examined by RT-PCR, western blotting, and immunofluorescence staining. UECs were also subjected to RNA expression profiling. RESULTS: Tsg101d/d female mice were subfertile with implantation failure, showing unattached blastocysts on day 6 of pregnancy. Histological and marker analyses revealed that some Tsg101d/d day 4 pregnant uteri showed a disintegrated uterine epithelial structure. Tsg101d/d UECs began to degenerate within 18 h of culture. In UECs, expression of necroptosis effectors, such as RIPK1, RIPK3, and MLKL were first confirmed. UECs responded to a stimulus to activate necroptosis and showed increased cell death. CONCLUSIONS: Tsg101 deficiency in the uterine epithelium causes implantation failure, which may be caused by epithelial defects. This study provides evidence that UECs harbor a necroptotic machinery that responds to death-inducing signals. Thus, Tsg101 expression in the uterine epithelium is required for normal pregnancy in mice.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Implantación del Embrión/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Células Epiteliales/metabolismo , Factores de Transcripción/biosíntesis , Útero/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células Epiteliales/patología , Femenino , Masculino , Ratones , Ratones Transgénicos , Embarazo , Factores de Transcripción/genética , Útero/patologíaRESUMEN
Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with currently no cure. These two diseases share a clinical continuum with overlapping genetic causes. Mutations in the CHMP2B gene are found in patients with ALS, FTD and ALS-FTD. To highlight deregulated mechanisms occurring in ALS-FTD linked to the CHMP2B gene, we performed a whole transcriptomic study on lumbar spinal cord from CHMP2Bintron5 mice, a model that develops progressive motor alterations associated with dementia symptoms reminiscent of both ALS and FTD. To gain insight into the transcriptomic changes taking place during disease progression this study was performed at three stages: asymptomatic, symptomatic and end stage. We showed that before appearance of motor symptoms, the major disrupted mechanisms were linked with the immune system/inflammatory response and lipid metabolism. These processes were progressively replaced by alterations of neuronal electric activity as motor symptoms appeared, alterations that could lead to motor neuron dysfunction. To investigate overlapping alterations in gene expression between two ALS-causing genes, we then compared the transcriptome of symptomatic CHMP2Bintron5 mice with the one of symptomatic SOD1G86R mice and found the same families deregulated providing further insights into common underlying dysfunction of biological pathways, disrupted or disturbed in ALS. Altogether, this study provides a database to explore potential new candidate genes involved in the CHMP2Bintron5-based pathogenesis of ALS, and provides molecular clues to further understand the functional consequences that diseased neurons expressing CHMP2B mutant may have on their neighbor cells.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Demencia Frontotemporal/genética , Proteínas del Tejido Nervioso/genética , Superóxido Dismutasa-1/genética , Transcriptoma/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value. METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome. RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009). CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.
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Biomarcadores de Tumor/biosíntesis , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Vesículas Extracelulares/metabolismo , Carioferinas/biosíntesis , Neoplasias de la Próstata/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Anciano , Vesículas Extracelulares/patología , Acido Graso Sintasa Tipo I/biosíntesis , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias de la Próstata/patología , Análisis de Matrices Tisulares , Proteína Exportina 1RESUMEN
CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Proteínas de Unión al ADN/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Factores de Transcripción/biosíntesis , Enzimas Ubiquitina-Conjugadoras/biosíntesis , Ubiquitina-Proteína Ligasas/biosíntesis , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Humanos , Proteínas Proto-Oncogénicas c-mdm2/genética , Factores de Transcripción/genética , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the É4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-ß and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral É3 allele we show that the É4 allele, whether homozygous or heterozygous with an É3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-ß precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.
Asunto(s)
Apolipoproteína E4/biosíntesis , Encéfalo/metabolismo , Exosomas/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Alelos , Animales , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Proteínas de Unión al ADN/biosíntesis , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Exosomas/ultraestructura , Espacio Extracelular/metabolismo , Femenino , Genotipo , Humanos , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Factores de Transcripción/biosíntesis , Proteínas de Unión al GTP rab/biosíntesisRESUMEN
BACKGROUND Cervical cancer is one of the most common female malignancies in the world. The ubiquitin-specific protease 8 (USP8) functions by removing ubiquitin from protein substrates, and its potential role in cancer development was recently uncovered in lung cancer. The aim of this study was to investigate the expression and function of USP8 in cervical squamous cell carcinoma (CSCC). MATERIAL AND METHODS Immunohistochemical staining and quantitative PCR were performed to explore the expression of USP8 in both CSCC tissues and adjacent normal cervical tissues. Univariate and multivariate analyses were conducted to evaluate the clinical significance of USP8 in CSCC. Proliferation, migration, and invasion abilities of 2 CSCC cell lines were assessed after overexpression or silencing USP8, respectively. RESULTS Both the RNA and protein levels of USP8 were upregulated in CSCC tissues compared to normal cervical tissues. High expression of USP8 was correlated with advanced tumor stage and high recurrence risk. Moreover, USP8 was identified as a novel independent prognostic factor for CSCC patients. Cellular studies showed that USP8 can enhance the proliferation, migration, and invasion abilities of CSCC cells, thereby promoting tumor progression. CONCLUSIONS High expression of USP8 is frequent in CSCC tissues, which promotes tumor proliferation and invasion, and is correlated with a poor overall survival. Targeting USP8 may be a novel direction for drug development for CSCC therapy.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Endopeptidasas/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Ubiquitina Tiolesterasa/biosíntesis , Neoplasias del Cuello Uterino/enzimología , Adulto , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Humanos , Inmunohistoquímica/métodos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/fisiopatología , Pronóstico , Estudios Retrospectivos , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Regulación hacia Arriba , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Degradation of most integral membrane proteins is directed by the endosomal sorting complex required for transport (ESCRT) machinery, which selectively targets ubiquitin-modified cargoes into intralumenal vesicles (ILVs) within multivesicular endosomes (MVEs). To better understand the mechanisms underlying ESCRT-mediated formation of ILVs, we exploited the rapid, de novo biogenesis of MVEs during the oocyte-to-embryo transition in C. elegans. In contrast to previous models suggesting that ILVs form individually, we demonstrate that they remain tethered to one another subsequent to internalization, arguing that they bud continuously from stable subdomains. In addition, we show that membrane bending and ILV formation are directed specifically by the ESCRT-III complex in vivo in a manner regulated by Ist1, which promotes ESCRT-III assembly and inhibits the incorporation of upstream ESCRT components into ILVs. Our findings underscore essential actions for ESCRT-III in membrane remodeling, cargo selection, and cargo retention, which act repetitively to maximize the rate of ILV formation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Oocitos/crecimiento & desarrollo , Transporte de Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The current study aims to investigate the biological roles and clinical significance of HCRP1 in human gastric cancer. The expression pattern of HCRP1 in gastric cancer tissue and adjacent non-cancerous tissue was detected by immunohistochemistry. HCRP1 downregulation was found in 57 of 137 human gastric cancer samples and correlated with advanced TNM stage, positive nodal status, and relapse. Log-rank test showed that HCRP1 downregulation also correlated with poor overall survival and reduced relapse-free survival. In addition, we found that HCRP1 overexpression inhibited proliferation, colony formation, and invasion in HGC-27 cells. On the other hand, HCRP1 depletion by small interfering RNA promoted proliferation, colony formation, and invasion in SGC-7901 cells. We also treated gastric cancer cells with cisplatin. MTT and Annexin V/PI analysis were carried out to examine change of chemoresistance. We found that HCRP1 overexpression sensitized HGC-27 cells to cisplatin while its depletion reduced sensitivity in SGC-7901 cells. Moreover, we found that HCRP1 overexpression negatively regulated cyclin D1, MMP-2, p-EGFR, p-ERK, and p-AKT. HCRP1 depletion showed the opposite effects. In conclusion, our results suggest that HCRP1 downregulation might serve as an indicator for poor prognosis in gastric cancer patients. HCRP1 reduces drug resistance through regulation of EGFR-AKT signaling.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Regulación Neoplásica de la Expresión Génica/fisiología , Transducción de Señal , Neoplasias Gástricas/patología , Adulto , Anciano , Supervivencia sin Enfermedad , Regulación hacia Abajo , Receptores ErbB/metabolismo , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Gástricas/mortalidadRESUMEN
AIM: To determine the regulation and function of the neural precursor cell expressed developmentally down regulated protein 4 (NEDD4) in PDAC and to determine its dependency on phosphatase and tensin homolog (PTEN) and PI3K/AKT signaling. METHODS: We investigated the expression of NEDD4 and the tumor suppressor PTEN in normal immortalized human pancreatic duct epithelial cell line and pancreatic adenocarcinoma (PDAC) cell lines. We further evaluated whether RNAi-mediated depletion of NEDD4 can attenuate PDAC cell proliferation and migration. We subsequently determined the crosstalk between NEDD4 expression and the PTEN/PI3K/AKT signaling pathway. Finally, we determined the mechanism behind differential NEDD4 protein expression in pancreatic cancer. RESULTS: The expression of NEDD4 was heterogeneous in PDAC cells, but was significantly higher compared to normal pancreatic ductal epithelial cells. Analogically, PTEN was decreased in the PDAC cells. A combination of MTT assay, wound healing migration assay, and transwell invasion assays confirmed that depletion of NEDD4 decreased the proliferation and migration ability of PDAC cells. Western blot and immunofluorescence results revealed that NEDD4 could affect PTEN/PI3K/AKT signaling pathway in PDAC cells. Polysomal profiling revealed that higher NEDD4 protein expression in PDAC cells was due to undefined mechanism involving translational activation. CONCLUSIONS: Our results reveal a novel mechanism of upregulation of NEDD4 expression in PDAC. Our findings indicate that NEDD4 potentially plays a critical role in activating the PI3K/AKT signaling pathway by negatively regulating PTEN levels in PDAC cells, which promotes pancreatic cancer cell proliferation and metastasis. Therefore, NEDD4 may be a potential therapeutic target in PDAC.
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Adenocarcinoma/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Pancreáticas/patología , Ubiquitina-Proteína Ligasas/biosíntesis , Adenocarcinoma/metabolismo , Western Blotting , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Ubiquitina-Proteína Ligasas Nedd4 , Fosfohidrolasa PTEN/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación Transcripcional/fisiología , Transcriptoma , Regulación hacia ArribaRESUMEN
AIMS: Previous study has indicated that inhibition of proteasome function ameliorates the development of pulmonary arterial hypertension (PAH), while its underlying mechanisms are still unclear. This study was performed to address these issues. MATERIAL AND METHODS: Male Sprague-Dawley (SD) rats were divided into five groups: control group, PAH group, vehicle treated PAH group, MG-132 treated PAH group and bortezomib treated PAH group. PAH model was established by a single intraperitoneal injection of monocrotaline (MCT). MG-132 and bortezomib were administered to inhibit proteasome function. The right ventricular systolic pressure (RVSP), the right ventricle hypertrophy index (RVHI) and the percentage of medial wall thickness (%MT) were used to evaluate the development of PAH. Hematoxylin and eosin staining was performed to measure vascular remodeling. Immunoblotting was used to determine Akt phosphorylation, expression of PTEN and NEDD4, and the level of ubiquitinated-PTEN protein. KEY FINDINGS: MCT increased RVSP, RVHI and %MT in rats, while these changes were suppressed by treatment of PAH rats with MG-132 or bortezomib. In PAH model, expression of PTEN was decreased and phosphorylation of Akt was increased, these were accompanied by an elevation of NEDD4 protein level. Treatment of PAH model with MG-132 or bortezomib increased PTEN expression and accumulation of ubiquitinated-PTEN protein and decreased Akt phosphorylation, while didn't change NEDD4 expression. SIGNIFICANCE: Inhibition of proteasome function ameliorates pulmonary arterial remodeling by suppressing UPS-mediated PTEN degradation and subsequent inhibition of PI3K/Akt pathway, indicating that UPS might be a novel target for prevention of PAH.
Asunto(s)
Bortezomib/farmacología , Hipertensión Pulmonar , Leupeptinas/farmacología , Monocrotalina/toxicidad , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Arteria Pulmonar , Remodelación Vascular/efectos de los fármacos , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/fisiopatología , Masculino , Ubiquitina-Proteína Ligasas Nedd4 , Fosfohidrolasa PTEN/biosíntesis , Arteria Pulmonar/enzimología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Ratas , Ratas Sprague-Dawley , Ubiquitina-Proteína Ligasas/biosíntesisRESUMEN
PURPOSE: NEDD4-like ubiquitin protein ligase 2 (NEDL2) plays an important role in many physiological and pathological processes. NEDL2 is a positive regulator of GDNF/Ret signaling during enteric neurogenesis. Mice lacking NEDL2 exhibit decreased numbers of enteric neurons, progressive bowel dysmotility and intestinal hypoganglionosis. We designed this study to investigate the expression of NEDL2 in the normal human colon and in HSCR. METHODS: HSCR tissue specimens (n=10) were collected at the time of pull-through surgery and divided into aganglionic and ganglionic segments. Colonic control samples (n=10) were obtained from patients with imperforate anus at the time of colostomy closure. Immunolabeling of NEDL2 was visualized using confocal microscopy to assess protein distribution, while Western blot analysis was undertaken to quantify NEDL2 protein expression. RESULTS: Confocal microscopy revealed that NEDL2-immunoreactivity colocalized with ICCs and neurons within the submucosa, myenteric plexus and smooth muscle in controls and ganglionic specimens, with markedly reduced NEDL2-immunoreactivity in aganglionic specimens. Western blotting revealed high levels of the NEDL2 protein in normal controls and the ganglionic region of HSCR, while there was a marked decrease in NEDL2 protein expression in the aganglionic region of HSCR. CONCLUSION: We report, for the first time, the expression of NEDL2 in the human colon. The decreased expression of NEDL2 in the aganglionic colon suggests that NEDL2 may play a role in the pathophysiology of HSCR.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Enfermedad de Hirschsprung/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Western Blotting , Femenino , Enfermedad de Hirschsprung/patología , Humanos , Lactante , Masculino , Microscopía Confocal , Plexo Mientérico/metabolismo , Plexo Mientérico/patología , Ubiquitina-Proteína Ligasas Nedd4 , Plexo Submucoso/metabolismo , Plexo Submucoso/patologíaRESUMEN
Mutations in the charged multivesicular body protein 2B (CHMP2B) are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and with a mixed ALS-FTD syndrome. To model this syndrome, we generated a transgenic mouse line expressing the human CHMP2Bintron5 mutant in a neuron-specific manner. These mice developed a dose-dependent disease phenotype. A longitudinal study revealed progressive gait abnormalities, reduced muscle strength and decreased motor coordination. CHMP2Bintron5 mice died due to generalized paralysis. When paralyzed, signs of denervation were present as attested by altered electromyographic profiles, by decreased number of fully innervated neuromuscular junctions, by reduction in size of motor endplates and by a decrease of sciatic nerve axons area. However, spinal motor neurons cell bodies were preserved until death. In addition to the motor dysfunctions, CHMP2Bintron5 mice progressively developed FTD-relevant behavioural modifications such as disinhibition, stereotypies, decrease in social interactions, compulsivity and change in dietary preferences. Furthermore, neurons in the affected spinal cord and brain regions showed accumulation of p62-positive cytoplasmic inclusions associated or not with ubiquitin and CHMP2Bintron5 As observed in FTD3 patients, these inclusions were negative for TDP-43 and FUS. Moreover, astrogliosis and microgliosis developed with age. Altogether, these data indicate that the neuronal expression of human CHMP2Bintron5 in areas involved in motor and cognitive functions induces progressive motor alterations associated with dementia symptoms and with histopathological hallmarks reminiscent of both ALS and FTD.
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Esclerosis Amiotrófica Lateral/metabolismo , Conducta Animal , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Demencia Frontotemporal/metabolismo , Regulación de la Expresión Génica , Intrones , Mutación , Neuronas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Axones/metabolismo , Axones/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Demencia Frontotemporal/fisiopatología , Humanos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Nervio Ciático/metabolismo , Nervio Ciático/patología , Nervio Ciático/fisiopatologíaRESUMEN
The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-ß (TGF-ß)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-ß-mediated EMT in lung cancer cells. miR-93 binds directly to the 3'-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-ß signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-ß treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-ß-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Factor de Crecimiento Transformador beta/genética , Ubiquitina-Proteína Ligasas/genética , Carcinogénesis/genética , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Masculino , MicroARNs/biosíntesis , Ubiquitina-Proteína Ligasas Nedd4 , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteína Smad2/biosíntesis , Ubiquitina-Proteína Ligasas/biosíntesisRESUMEN
The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation-correlating with Pax7 ubiquitination and decline-and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a drop in Pax7 levels and precocious muscle differentiation. Consequently, we propose that Nedd4 functions as a novel Pax7 regulator, which activity is temporally and spatially controlled to modulate the Pax7 protein levels and therefore satellite cell fate.
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Diferenciación Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Desarrollo de Músculos , Factor de Transcripción PAX7/biosíntesis , Células Satélite del Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Proliferación Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Ubiquitina-Proteína Ligasas Nedd4 , Factor de Transcripción PAX7/genética , Complejo de la Endopetidasa Proteasomal/genética , Células Satélite del Músculo Esquelético/citología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
OBJECTIVE: The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. METHODS: NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. RESULTS: Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. CONCLUSIONS: This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway.
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Neoplasias Endometriales/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Ubiquitina-Proteína Ligasas/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma Endometrioide/enzimología , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Activación Enzimática , Femenino , Humanos , Inmunohistoquímica , Ubiquitina-Proteína Ligasas Nedd4 , Oncogenes , Receptor IGF Tipo 1/biosíntesis , Análisis de Matrices Tisulares , Ubiquitina-Proteína Ligasas/biosíntesisRESUMEN
Charged multivesicular body protein 4B (CHMP4B), a subunit of the endosomal sorting complex required for transport (ESCRT)-III complex, plays an important part in cytokinetic membrane abscission and the late stage of mitotic cell division. In this study, we explored the prognostic significance of CHMP4B in human hepatocellular carcinoma (HCC) and its impact on the physiology of HCC cells. Western blot and immunohistochemistrical analyses showed that CHMP4B was significantly upregulated in HCC tissues, compared with adjacent non-tumorous tissues. Meanwhile, clinicopathological analysis revealed that high CHMP4B expression was correlated with multiple clinicopathological variables, including AFP, cirrhosis, AJCC stage, Ki-67 expression, and poor prognosis. More importantly, univariate and multivariate survival analyses demonstrated that CHMP4B served as an independent prognostic factor for survival of HCC patients. Using HCC cell cultures, we found that the expression of CHMP4B was progressively upregulated after the release from serum starvation. To verify whether CHMP4B could regulate the proliferation of HCC cells, CHMP4B was knocked down through the transfection of CHMP4B-siRNA oligos. Flow cytometry and CCK-8 assays indicated that interference of CHMP4B led to cell cycle arrest and proliferative impairment of HCC cells. Additionally, depletion of CHMP4B expression could increase the sensitivity to doxorubicin in HepG2 and Huh7 cells. Taken together, our results implied that CHMP4B could be a promising prognostic biomarker as well as a potential therapeutic target of HCC.
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Biomarcadores de Tumor/biosíntesis , Carcinoma Hepatocelular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Neoplasias Hepáticas/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Pronóstico , ARN Interferente PequeñoRESUMEN
The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation of tight syndecan clusters that are recruited by the multivalent adaptor syntenin to the ALIX-ESCRT sorting machinery at endosomes.
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Proteínas de Unión al Calcio/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Glucuronidasa/genética , Sindecanos/biosíntesis , Sinteninas/biosíntesis , HumanosRESUMEN
Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.
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Proteínas de Unión al Calcio/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Glucuronidasa/genética , Sindecanos/biosíntesis , Sinteninas/biosíntesis , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Exosomas/genética , Exosomas/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucuronidasa/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Células MCF-7 , Proteínas de la Membrana/biosíntesis , Transducción de Señal , Sindecanos/genética , Sinteninas/genéticaRESUMEN
Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.