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Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , N-Metilaspartato , Proteínas do Tecido Nervoso , Animais , Proteína 4 Homóloga a Disks-Large , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptor A1 de Adenosina , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical for understanding such heterogeneity. Here, we propose an approach for detecting communities from a genetic network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities were partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights, even for a large, sparse dataset, in the single-cell analysis field.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , HumanosRESUMO
Epithelial ovarian cancer is classified into four major histological subtypes: serous, clear cell, endometrioid and mucinous. Ovarian clear cell carcinoma (OCCC) responds poorly to conventional chemotherapies and shows poor prognosis. Thus, there is a need to develop new drugs for the treatment of OCCC. In this study, we performed CRISPR/Cas9 screens against OCCC cell lines and identified candidate genes important for their proliferation. We found that quite different genes are required for the growth of ARID1A and PIK3CA mutant and wild-type OCCC cell lines, respectively. Furthermore, we found that the epigenetic regulator KDM2A and the translation regulator PAIP1 may play important roles in the growth of ARID1A and PIK3CA mutant, but not wild-type, OCCC cells. The results of our CRISPR/Cas9 screening may be useful in elucidating the molecular mechanism of OCCC tumorigenesis and in developing OCCC-targeted drugs.
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Ovarian cancer is the fifth most common cause of cancer-related death in women. Ovarian clear cell carcinoma (OCCC) is a chemotherapy-resistant epithelial ovarian cancer with poor prognosis. As a basis for the development of therapeutic agents that could improve the prognosis of OCCC, we performed a screen for proteins critical for the tumorigenicity of OCCC using the CRISPR/Cas9 system. Here we show that knockdown of the phosphate exporter XPR1/SLC53A1 induces the growth arrest and apoptosis of OCCC cells in vitro. Moreover, we show that knockdown of XPR1/SLC53A1 inhibits the proliferation of OCCC cells xenografted into immunocompromised mice. These results suggest that XPR1/SLC53A1 plays a critical role in the tumorigenesis of OCCC cells. We speculate that XPR1/SLC53A1 might be a promising molecular target for the therapeutic treatment of OCCC.
Assuntos
Adenocarcinoma de Células Claras , Neoplasias Ovarianas , Adenocarcinoma de Células Claras/patologia , Animais , Carcinoma Epitelial do Ovário/genética , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Fosfatos/uso terapêutico , PrognósticoRESUMO
Long non-coding RNAs (lncRNAs) are aberrantly expressed in many disease conditions, including cancer. Accumulating evidence indicates that some lncRNAs may play critical roles in cancer progression and metastasis. Here, we identify a set of lncRNAs that are upregulated in metastatic subpopulations isolated from colon cancer HCT116 cells in vivo and show that one of these lncRNAs, which we name CALIC, is required for the metastatic activity of colon cancer cells. We show that CALIC associates with the RNA-binding protein hnRNP-L and imparts specificity to hnRNP-L-mediated gene expression. Furthermore, we demonstrate that the CALIC/hnRNP-L complex upregulates the tyrosine kinase receptor AXL and that knockdown of CALIC or AXL using shRNA in colon cancer cells attenuates their ability to form metastases in mice. These results suggest that the CALIC/hnRNP-L complex enhances the metastatic potential of colon cancer cells.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/genética , Receptores Proteína Tirosina Quinases/genética , Ribonucleoproteínas/genética , Animais , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Progressão da Doença , Feminino , Células HCT116 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Dysbiosis of oral microbiota is implicated not only in oral inflammatory lesions, but also in a variety of extraoral diseases. The etiology of palmoplantar pustulosis (PPP) remains unclear; however, it has been suggested that chronic inflammation caused by periodontopathic bacterial infection may play a role. OBJECTIVES/METHODS: To determine whether patients with PPP have altered diversity and composition of oral microbiota, we conducted the 16S rDNA analysis using saliva samples collected from 21 outpatients with PPP and 10 healthy individuals. RESULTS: We found that the proportion of bacteria in the phylum Proteobacteria was significantly lower in PPP patients (p = 0.025). At the genus level, patients with PPP had a significantly lower abundance of Neisseria (p = 0.014), which best accounted for the observed decrease in Proteobacteria. We also identified multiple minor genera and species that were represented at a significantly higher level in the PPP group, several of which have been associated with periodontal diseases. CONCLUSION: Our results suggest a possible link between PPP and dysbiosis of oral microbiota, particularly the lower abundance of Neisseria, the most predominant genus of Proteobacteria in healthy oral microbiota. Probiotics that improves oral dysbiosis may be beneficial for patients with PPP as an adjunctive therapy.
Assuntos
Disbiose/epidemiologia , Microbiota , Boca/microbiologia , Psoríase/microbiologia , Estudos de Casos e Controles , Disbiose/diagnóstico , Humanos , Saliva/microbiologiaRESUMO
BACKGROUND: The etiology of ulcerative colitis (UC) remains elusive even though many genetic and environmental pathogenic factors have been reported. Aberrant inflammatory responses mediated by specific subsets of T cells have been observed in ulcerative lesions of UC patients. OBJECTIVES: To elucidate the involvement of a delayed-type hypersensitivity reaction in UC, we focused on dental metal hypersensitivity, a T cell-mediated, delayed-type allergic reaction that causes oral contact mucositis and systemic cutaneous inflammation. METHOD: We recruited 65 Japanese UC patients and 22 healthy controls (HC) and used the in vitro lymphocyte stimulation test to quantify their sensitivity to zinc, gold, nickel, and palladium - the metals that have been widely used in dentistry. All subjects were users of metallic dental implants and/or prostheses containing zinc, gold, nickel, and/or palladium as major constituents. RESULTS: Sixty percent of the UC patients were hypersensitive to at least one metal species, whereas 32% of the HC were hypersensitive to only a single metal species. The overall incidence of metal hypersensitivity was significantly higher for UC patients than for HC. Furthermore, a significantly greater proportion of UC patients were hypersensitive to nickel or palladium. The severity of the sensitivity to nickel and palladium was also significantly greater for UC patients than for HC. CONCLUSIONS: This pilot study demonstrates that UC patients have a significantly higher incidence of hypersensitivity to nickel and palladium, suggesting the possible involvement of dental metal hypersensitivity in UC pathogenesis.
Assuntos
Colite Ulcerativa/imunologia , Materiais Dentários/efeitos adversos , Hipersensibilidade Tardia/complicações , Níquel/imunologia , Paládio/imunologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Feminino , Ouro/efeitos adversos , Ouro/imunologia , Humanos , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/diagnóstico , Hipersensibilidade Tardia/epidemiologia , Incidência , Masculino , Pessoa de Meia-Idade , Níquel/efeitos adversos , Paládio/efeitos adversos , Projetos Piloto , Prevalência , Adulto Jovem , Zinco/efeitos adversos , Zinco/imunologiaRESUMO
Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Sirtuína 2/metabolismo , Proteína Tumoral p73/metabolismo , Acetilação , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Furanos/farmacologia , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Lisina/metabolismo , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Quinolinas/farmacologia , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/genética , Células Tumorais Cultivadas , Proteína Tumoral p73/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth.
Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Proteína Coatomer/genética , Anormalidades Craniofaciais/genética , Mutação , Adulto , Proteína Coatomer/metabolismo , Colágeno/metabolismo , Estresse do Retículo Endoplasmático , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , SíndromeRESUMO
While most asthma can be treated with steroids, about 10%, called severe asthma, is refractory to steroids. It has recently been shown that in a subgroup of severe asthma cases, neutrophils that infiltrate into the airways play an important role in inflammation. However, the mechanisms underlying this increased neutrophil infiltration are not well understood. Here, using a mouse model of steroid-resistant neutrophilic inflammation, we show that mice deficient for the RNA-binding protein Mex-3B have significantly less neutrophil infiltration in the airways than wild-type mice. We further demonstrate that Mex-3B post-transcriptionally upregulates CXCL2, a chemokine that induces neutrophil chemotaxis and migration. Moreover, we show that treatment with either anti-CXCL2 antibody or anti-Mex-3B antisense oligonucleotide suppresses neutrophilic allergic airway inflammation. These results suggest that Mex-3B-mediated induction of CXCL2 is crucial for steroid-resistant neutrophilic allergic airway inflammation. Our findings suggest new strategies for therapeutic intervention in steroid-resistant severe asthma.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Esteroides/farmacologia , Animais , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Asma/tratamento farmacológico , Asma/metabolismo , Quimiocina CXCL2/imunologia , Feminino , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/metabolismo , Oligonucleotídeos/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidoresRESUMO
Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Boro/metabolismo , Regulação da Expressão Gênica de Plantas , Fases de Leitura Aberta/genética , Estabilidade de RNA/fisiologia , Ribossomos/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fases de Leitura Aberta/fisiologia , Estabilidade de RNA/genética , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Wnt/ß-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that ß-catenin directly activates the transcription of the long noncoding RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. ASBEL interacts with and recruits TCF3 to the activating transcription factor 3 (ATF3) locus, where it represses the expression of ATF3. Furthermore, we demonstrate that ASBEL-TCF3-mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/ß-catenin-mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/ß-catenin signaling.
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Many long noncoding RNAs (lncRNAs) are reported to be dysregulated in human cancers and play critical roles in tumor development and progression. Furthermore, it has been reported that many lncRNAs regulate gene expression by recruiting chromatin remodeling complexes to specific genomic loci or by controlling transcriptional or posttranscriptional processes. Here we show that an lncRNA termed UPAT [ubiquitin-like plant homeodomain (PHD) and really interesting new gene (RING) finger domain-containing protein 1 (UHRF1) Protein Associated Transcript] is required for the survival and tumorigenicity of colorectal cancer cells. UPAT interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its ß-transducin repeat-containing protein (TrCP)-mediated ubiquitination. Furthermore, we demonstrate that UHRF1 up-regulates Stearoyl-CoA desaturase 1 and Sprouty 4, which are required for the survival of colon tumor cells. Our study provides evidence for an lncRNA that regulates protein ubiquitination and degradation and thereby plays a critical role in the survival and tumorigenicity of tumor cells. Our results suggest that UPAT and UHRF1 may be promising molecular targets for the therapy of colon cancer.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias do Colo/genética , RNA Longo não Codificante/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/química , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitinação , Regulação para CimaRESUMO
Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-ß receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-ß receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-ß receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proliferação de Células , Biologia Computacional/métodos , Meios de Cultura/farmacologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Fosforilação , Receptor do Fator de Crescimento Transformador beta Tipo II , Soro , Transdução de SinaisRESUMO
Lineage development is a stepwise process, governed by stage-specific regulatory factors and associated markers. Astrocytes are one of the principle cell types in the CNS and the stages associated with their development remain very poorly defined. To identify these stages, we performed gene-expression profiling on astrocyte precursor populations in the spinal cord, identifying distinct patterns of gene induction during their development that are strongly correlated with human astrocytes. Validation studies identified a new cohort of astrocyte-associated genes during development and demonstrated their expression in reactive astrocytes in human white matter injury (WMI). Functional studies on one of these genes revealed that mice lacking Asef exhibited impaired astrocyte differentiation during development and repair after WMI, coupled with compromised blood-brain barrier integrity in the adult CNS. These studies have identified distinct stages of astrocyte lineage development associated with human WMI and, together with our functional analysis of Asef, highlight the parallels between astrocyte development and their reactive counterparts associated with injury. SIGNIFICANCE STATEMENT: Astrocytes play a central role in CNS function and associated diseases. Yet the mechanisms that control their development remain poorly defined. Using the developing mouse spinal cord as a model system, we identify molecular changes that occur in developing astrocytes. These molecular signatures are strongly correlated with human astrocyte expression profiles and validation in mouse spinal cord identifies a host of new genes associated with the astrocyte lineage. These genes are present in reactive astrocytes in human white matter injury, and functional studies reveal that one of these genes, Asef, contributes to reactive astrocyte responses after injury. These studies identify distinct stages of astrocyte lineage development and highlight the parallels between astrocyte development and their reactive counterparts associated with injury.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Regeneração da Medula Espinal/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Troca de Nucleotídeo Guanina Rho , Fatores de TempoRESUMO
BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in Japan. Smoking and drinking alcohol are environmental risk factors for ESCC, whereas single nucleotide polymorphisms in ADH1B and ALDH2, which increase harmful intermediates produced by drinking alcohol, are genetic risk factors. We conducted a large-scale genomic analysis of ESCCs from patients in Japan to determine the mutational landscape of this cancer. METHODS: We performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCC who underwent surgery at 5 hospitals in Japan. We also performed single-nucleotide polymorphism array-based copy number profile and germline genotype analyses of polymorphisms in ADH1B and ALDH2. Polymorphisms in CYP2A6, which increase harmful effects of smoking, were analyzed. Functions of TET2 mutants were evaluated in KYSE410 and HEK293FT cells. RESULTS: A high proportion of mutations in the 144 tumor samples were C to T substitution in CpG dinucleotides (called the CpG signature) and C to G/T substitutions with a flanking 5' thymine (called the APOBEC signature). Based on mutational signatures, patients were assigned to 3 groups, which associated with environmental (drinking and smoking) and genetic (polymorphisms in ALDH2 and CYP2A6) factors. Many tumors contained mutations in genes that regulate the cell cycle (TP53, CCND1, CDKN2A, FBXW7); epigenetic processes (MLL2, EP300, CREBBP, TET2); and the NOTCH (NOTCH1, NOTCH3), WNT (FAT1, YAP1, AJUBA) and receptor-tyrosine kinase-phosphoinositide 3-kinase signaling pathways (PIK3CA, EGFR, ERBB2). Mutations in EP300 and TET2 correlated with shorter survival times, and mutations in ZNF750 associated with an increased number of mutations of the APOBEC signature. Expression of mutant forms of TET2 did not increase cellular levels of 5-hydroxymethylcytosine in HEK293FT cells, whereas knockdown of TET2 increased the invasive activity of KYSE410 ESCC cells. Computational analyses associated the mutations in NFE2L2 we identified with transcriptional activation of its target genes. CONCLUSIONS: We associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genômica , Mutação , Polimorfismo de Nucleotídeo Único , Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Povo Asiático/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/etnologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Ilhas de CpG , Citocromo P-450 CYP2A6/genética , Análise Mutacional de DNA , Neoplasias Esofágicas/etnologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Exoma , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Interação Gene-Ambiente , Estudos de Associação Genética , Predisposição Genética para Doença , Genômica/métodos , Células HEK293 , Humanos , Japão/epidemiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fatores de Risco , TransfecçãoRESUMO
Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.
Assuntos
Citoesqueleto de Actina/metabolismo , Permeabilidade da Membrana Celular , Endotélio Vascular/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Artéria Pulmonar/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Western Blotting , Células Cultivadas , Endotélio Vascular/citologia , Imunofluorescência , Humanos , Imunoprecipitação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/genética , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens.
Assuntos
Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Inflamação/patologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Upon binding of a Wnt ligand to the frizzled (FZD)-low density lipoprotein receptor related protein 5/6 (LRP5/6) receptor complex, the ß-catenin destruction complex, composed of Axin1, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), is immediately inactivated, which causes ß-catenin stabilization. However, the molecular mechanism of signal transduction from the receptor complex to the ß-catenin destruction complex is controversial. Here we show that Wnt3a treatment promotes the dissociation of the Axin1-APC complex in glioblastoma cells cultured in serum-free medium. Experiments with the GSK3 inhibitor BIO suggest that Axin1-APC dissociation was controlled by phosphorylation. Introduction of a phosphomimetic mutation into Thr160 of Axin1, located in the APC-binding region RGS, abrogated the interaction of Axin1 with APC. Consistent with these observations, the Axin1 phosphomimetic mutant lost the ability to reduce ß-catenin stability and to repress ß-catenin/TCF-dependent transcription. Taken together, our results suggest a novel mechanism of Wnt signaling through the dissociation of the ß-catenin destruction complex by Axin1 Thr160 modification.
Assuntos
Proteína Axina/química , Proteína Axina/metabolismo , Complexo de Sinalização da Axina/química , Complexo de Sinalização da Axina/metabolismo , Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína Axina/genética , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Quinase 3 da Glicogênio Sintase/química , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Estabilidade Proteica , RNA Interferente Pequeno/genética , Treonina/química , Via de Sinalização Wnt , beta Catenina/química , beta Catenina/metabolismoRESUMO
Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer.