RESUMO
Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have been recognized to have an etiopathogenetic role in the development and progression of precursor melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor/MET (HGF/MET) axis is emerging as a critical player not only in the tumor itself but also in the immune microenvironment in which the tumor grows and advances in its development. Moreover, the activation of this pathway has emerged as a paradigm of tumor resistance to modern targeted therapies, and the assessment of its expression in patients' samples may be a valuable biomarker of tumor progression and response to targeted therapy. Here we summarize our current understanding of this important receptor tyrosine kinase in normal melanocyte proliferation/motility, in tumor progression and metastasis, its genetic alterations in certain subtype of melanocytic lesions, and how its pathway has been explored for the development of selective inhibitors.
Assuntos
Melanócitos/patologia , Melanoma/genética , Proteínas Proto-Oncogênicas c-met/fisiologia , Neoplasias Cutâneas/genética , Animais , Movimento Celular/genética , Proliferação de Células/genética , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Melanócitos/metabolismo , Melanoma/patologia , Transdução de Sinais/genética , Neoplasias Cutâneas/patologia , Microambiente Tumoral/genética , Melanoma Maligno CutâneoRESUMO
The frontal sinus is an airspace behind the brow ridge in the skull and can affect the accuracy of the regional cerebral oxygen saturation measurements. We evaluated the optimal location for placement of a cerebral oximeter probe while avoiding the frontal sinus in pediatric patients. This retrospective observational study included 203 pediatric patients aged 3-17 years who had undergone brain computed tomography from November 2010 to December 2015. The patients were divided into five subgroups based on their age. The frontal sinus height was measured from the superior orbital rim. Pneumatization of the frontal sinus was not visible in 78% (3-5 years) and 22% (6-8 years) of the patients. The mean (SD) of the frontal sinus height was 5.9 (3.4), 9.5 (4.1), 14.0 (6.2) 18.6 (8.4), and 21.1 (7.9) mm in the 3-5, 6-8, 9-11, 12-14, and 15-17 year age-groups, respectively. Age was positively correlated with the frontal sinus height (r = 0.61, P < 0.001, 95% confidence interval [CI] 0.513-0.688). A frontal sinus height shorter than 1, 2, and 3 cm were seen in 10 of 11 (91%), 69 of 74 (94%), and 108 of 118 (90%) patients aged 3-5, 6-10, and 11-17 years, respectively. When oximeter probes are applied in pediatric patients, placement based on age can help avoid the frontal sinus.
Assuntos
Seio Frontal , Monitorização Hemodinâmica/métodos , Oximetria/métodos , Adolescente , Circulação Cerebrovascular , Criança , Pré-Escolar , Feminino , Seio Frontal/anatomia & histologia , Seio Frontal/diagnóstico por imagem , Monitorização Hemodinâmica/efeitos adversos , Monitorização Hemodinâmica/instrumentação , Humanos , Masculino , Oximetria/efeitos adversos , Oximetria/instrumentação , Oxigênio/sangue , Estudos Retrospectivos , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Tomografia Computadorizada por Raios XRESUMO
Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5'-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576-584, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regiões 5' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Imunoprecipitação , Camundongos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Receptores Opioides mu/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Spitz and Spitzoid lesions represent one of the most challenging melanocytic neoplasms in dermatopathology. Nosologic classification has been more recently improved by the discovery of novel molecular drivers, particularly translocations. In the current study, we aimed to use an unbiased approach to explore the gene expression profile of a group of melanocytic Spitz and Spitzoid melanocytic lesions ranging from benign lesions to melanoma, including intermediate lesions such as SPARK nevi and atypical Spitz tumors/melanocytomas. Using unsupervised analysis of gene expression data, we found some distinct hierarchical clusters of lesions, including groups characterized by ALK and NTRK translocations. Few non-ALK translocated tumors demonstrated increased ALK expression, confirmed by immunohistochemistry. Spitz tumors with overlapping features of dysplastic nevi, so-called SPARK nevi, appear to have a common gene expression profile by hierarchical clustering. Finally, weighted gene correlation network analysis identified gene modules variably regulated in subtypes of these cases. Thus, gene expression profiling of Spitz and Spitzoid lesions represents a viable instrument for the characterization of these lesions.
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The opioid receptors are among the most highly studied members of the superfamily of G-protein coupled receptors. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Expression of opioid receptor proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. Here, we demonstrate for the first time the role of vimentin as a post-transcriptional repressor in MOR gene regulation. To identify potential regulators of the mouse MOR gene, we performed affinity column chromatography using 5'-UTR-specific RNA oligonucleotides using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified an intermediate filament protein, vimentin, which bound specifically to the region between -175 and -150 (175-150) of the MOR 5'-UTR. Binding was confirmed by western blot analysis and RNA supershift assay. Furthermore, a cotransfection study demonstrated that the presence of vimentin resulted in reduced expression of the mouse MOR. Our data suggest that vimentin functions as a repressor of MOR translation, dependent on 175-150 of the MOR 5'-UTR.
Assuntos
Regiões 5' não Traduzidas , Processamento Pós-Transcricional do RNA , Receptores Opioides mu/metabolismo , Transcrição Gênica , Vimentina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Motivos de Nucleotídeos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Opioides mu/genética , Deleção de SequênciaRESUMO
Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 5'-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 5'-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71 bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as post-transcriptional repressors in MOR gene regulation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/fisiologia , Receptores Opioides mu/metabolismo , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , Receptores Opioides mu/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Melanoma is the leading cause of death from cutaneous malignancy. While targeted therapy and immunotherapy with checkpoint inhibitors have significantly decreased the mortality rate of this disease, advanced melanoma remains a therapeutic challenge. Here, we confirmed that interferon-gamma (IFN-γ)-induced PD-L1 expression in melanoma cell lines. This increased expression was down-regulated by the reduction in phosphorylated STAT3 signaling via MET tyrosine kinase inhibitor treatment. Furthermore, immunoprecipitation and confocal immunofluorescence microscopy analysis reveals MET and PD-L1 protein-protein interaction and colocalization on the cell surface membrane of melanoma cells. Together, these findings demonstrate that the IFN-γ-induced PD-L1 expression in melanoma cells is negatively regulated by MET inhibition through the JAK/STAT3 signaling pathway and establish the colocalization and interaction between an RTK and a checkpoint protein in melanoma cells.
RESUMO
The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs.
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Tumor heterogeneity is a relevant hallmark of melanoma due to the high mutation burden and immunogenicity commonly encountered. Heterogeneity at the histologic level frequently corresponds to heterogeneity at the molecular level. A better understanding of this feature of malignancy can help refine the development of predictive biomarkers and to define more effective targeted therapies. Here, we describe a case of melanoma displaying a dual phenotype: a DPN-like/plexiform portion in conjunction with a conventional epithelioid morphology. Molecular studies revealed shared BRAF and PTEN mutations in both components but a CTNNB1 mutation was exclusively found in the DPN-like area of the tumor, consistent with the distinct morphology observed. There was considerable heterogeneity in sequence variants identified in the two regions. Gene expression analysis highlighted differentially regulated genes between the two histologies, including a relevant cluster of genes in the receptor tyrosine kinase (RTK) family and related signaling pathways upregulated in the DPN-like/plexiform area.
Assuntos
Melanoma , Nevo , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Mutação/genética , Nevo/patologia , Receptores Proteína Tirosina Quinases/genética , Neoplasias Cutâneas/patologiaRESUMO
BAP-1 (BRCA1-associated protein 1) inactivated melanocytic lesions are a group of familial or sporadic lesions with unique histology and molecular features. They are of great clinical interest, at least in part due to the potential for malignant transformation and association with a familial cancer predisposition syndrome. Here, we describe a patient with multiple spatially and temporally distinct melanocytic lesions with loss of BAP1 expression by immunohistochemistry. RNA sequencing was performed on three independent lesions spanning the morphologic spectrum: a benign nevus, an atypical tumor, and a melanoma arising from a pre-existing BAP1-inactivated nevus. The three lesions demonstrated largely distinct gene expression and mutational profiles. Gene expression analysis revealed that genes involved in receptor protein kinase pathways were progressively upregulated from nevus to melanoma. Moreover, a clear enrichment of genes regulated in response to UV radiation was found in the melanoma from this patient, as well as upregulation of MAPK pathway-related genes and several transcription factors related to melanomagenesis.
Assuntos
Expressão Gênica/genética , Melanócitos/patologia , Melanoma/genética , Mutação/genética , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adulto , Biomarcadores Tumorais/genética , Humanos , Masculino , Melanoma/patologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Neoplasias Cutâneas/patologiaRESUMO
Mu opioid receptor (MOR) is the main site of interaction for major clinical analgesics, particularly morphine. MOR expression is regulated at the transcriptional and post-transcriptional levels. However, the protein expression of the MOR gene is relatively low and the translational control of MOR gene has not been well studied. The 5'-untranslated region (UTR) of the human MOR (OPRM1) mRNA contains four upstream AUG codons (uAUG) preceding the main translation initiation site. We mutated the four uAUGs individually and in combination. Mutations of the third uAUG, containing the same open reading frame, had the strongest inhibitory effect. The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays. Toeprinting results showed that OPRM1 ribosomes initiated efficiently at the first uAUG, and subsequently re-initiated at the in-frame #3 uAUG and the physiological AUG site. This re-initiation resulted in negative expression of OPRM1 under normal conditions. These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.
Assuntos
Códon de Iniciação/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas/genética , Receptores Opioides mu/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Ribossomos/metabolismoRESUMO
The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis- and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5'-aza-2'-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.
Assuntos
Epigênese Genética , Regiões Promotoras Genéticas/genética , Receptores Opioides mu/genética , Animais , Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Imunoprecipitação da Cromatina , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Modelos Biológicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Opioides mu/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The pharmacological effects of morphine and morphine-like drugs are mediated primarily through the micro opioid receptor. Here we show that differential use of an in-frame translational start codon in the 5'-untranslated region of the OPRM1 generates different translational products in vivo and in vitro. The 5'-end of the OPRM1 gene is necessary for initiating the alternate form and for subsequent degradation of the protein. Initiation of OPRM1 at the upstream site decreases the initiation at the main AUG site. However, alternative initiation of the long form of OPRM1 produces a protein with a short half-life, resulting from degradation mediated by the ubiquitin-proteasome pathway. Reporter and degradation assays showed that mutations of this long form at the second and third lysines reduce ubiquitin-dependent proteasome degradation, stabilizing the protein. The data suggest that MOP expression is controlled in part by initiation of the long form of MOP at the alternate site.
Assuntos
Códon de Iniciação , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Receptores Opioides mu/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Opioides mu/metabolismoRESUMO
The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the mu opioid receptor. Previously a single strand DNA element of the mouse mu opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, alpha-complex proteins (alphaCP) alphaCP1, alphaCP2, alphaCP2-KL, and alphaCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, alphaCP1, alphaCP2, and alphaCP2-KL activated the Oprm1 promoter activity, whereas alphaCP3 acted as a repressor. Ectopic expression of hnRNP K, alphaCP1, alphaCP2, and alphaCP2-KL also led to activation of the endogenous Oprm1 transcripts, and alphaCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and alphaCP binding to the single strand DNA element.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/análise , Receptores Opioides mu/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , ProteômicaRESUMO
The proto-oncogene MET, the hepatocyte growth factor (HGF) receptor, is a transmembrane receptor tyrosine kinase (RTK) with a prominent role in tumor metastasis and resistance to anti-cancer therapies. Melanoma demonstrates relatively frequent MET aberrations, including MET gene amplification. Concurrently, programmed death-ligand 1 (PD-L1), with its ability to evade anti-tumor immune responses, has emerged as a prominent therapeutic target in melanoma and other malignancies and its expression is used as a predictive biomarker of response to immunotherapy. We performed immunohistochemistry analysis of MET and PD-L1 in 18 human melanoma cell lines derived from both primary and metastatic lesions, and in a human melanoma tissue microarray containing one hundreds melanocytic lesions, including primary cutaneous melanomas, primary mucosal melanomas, metastatic melanomas and benign melanocytic nevi as controls. After color deconvolution, each core was segmented to isolate staining and calculate the percentage of positive cells. Overall, MET expression was higher in tumors with increased PD-L1 expression. Moreover, a robust correlation between MET and PD-L1 expression was found in samples from metastatic melanoma and not in primary cutaneous or mucosal melanoma. These data suggest that relative expression levels of these proteins in combination is a marker of advanced disease and testing for expression of these markers should be considered in patients with melanoma.
RESUMO
The pharmacological action of morphine as a pain medication is mediated primarily through the mu-opioid receptor (MOR). With few exceptions, MOR is expressed in brain regions where opioid actions take place. The basis for this unique spatial expression of MOR remains undetermined. Recently, we reported that DNA methylation of the MOR promoter plays an important role in regulating MOR in P19 cells. In this study, we show that the differential expression of MOR in microdissected mouse brain regions coincides with DNA methylation and histone modifications. MOR expression could be induced by a demethylating agent or a histone deacetylase inhibitor in MOR-negative cells, suggesting that the MOR gene can be silenced under epigenetic control. Increases in the in vivo interaction of methyl-CpG-binding protein 2 (MeCP2) were observed in the cerebellum, in which the MOR promoter was hypermethylated and MOR expression was the lowest among all brain regions tested. MeCP2 is associated closely with Rett syndrome, a neurodevelopmental disorder. We also established novel evidence for a functional role for MeCP2's association with the chromatin-remodelling factor Brg1 and DNA methyltransferase Dnmt1, suggesting a possible role for MeCP2 in chromatin remodelling during MOR gene regulation. We conclude that MOR gene expression is epigenetically programmed in various brain regions and that MeCP2 assists the epigenetic program during DNA methylation and chromatin remodelling of the MOR promoter.
Assuntos
Encéfalo/metabolismo , Cromatina/metabolismo , DNA Helicases/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteínas Nucleares/metabolismo , Receptores Opioides mu/genética , Fatores de Transcrição/metabolismo , Animais , Encéfalo/patologia , Ilhas de CpG , Metilação de DNA , Primers do DNA/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras GenéticasRESUMO
The neuron-restrictive silencer factor (NRSF) functions as a transcriptional repressor of neuronal genes in nonneuronal cells. However, it is expressed in certain mature neurons in adults, suggesting that it might have complex and novel roles depending on its cellular and physiological context. Overexpression of NRSF led to both increased opioid ligand-binding activity of the endogenous MOR and MOR-GFP fusion protein expression. In RNA immunoprecipitation and gel-shift assays, NRSF specifically interacted with the NRSE sequence of MOR mRNA. When MOR and NRSF genes were coexpressed, the specific ligand-binding activity of MOR was increased in neuroblastoma NMB cells, but decreased in PC12 cells result from its localization. Indeed, after overexpressing NRSF in NMB cells, the target RNA moved to the translationally active polysomal fraction. Overexpression of NRSF also led to enhanced phosphorylation of eIF4G. In contrast, knockdown of NRSF by siRNA transfection significantly decreased eIF4G phosphorylation. These findings indicate that NRSF may deliver the target MOR transcripts to the polyribosomal complex and activate eIF4G phosphorylation, resulting in translational activation. We report here a novel function of NRSF that enhance the translation of the mu opioid receptor (MOR) gene through its RNA binding sequence, the neuron-restrictive silencer element (NRSE).
Assuntos
Regulação da Expressão Gênica/genética , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Citoplasma/metabolismo , Genes Reporter/genética , Humanos , Ligantes , Fosforilação , Biossíntese de Proteínas , RNA Mensageiro/genética , Ratos , Receptores Opioides mu/metabolismo , Proteínas Repressoras/genéticaRESUMO
Poly(C)-binding proteins (PCBPs) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). They can be divided into two groups: hnRNP K and PCBP1-4. These proteins are involved mainly in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). In this review, we summarize and discuss how PCBPs act as transcriptional regulators by binding to specific elements in gene promoters that interact with the RNA polymerase II transcription machinery. Transcriptional regulation of PCBPs might itself be regulated by their localization within the cell. For example, activation by p21-activated kinase 1 induces increased nuclear retention of PCBP1, as well as increased promoter activity. PCBPs can function as a signal-dependent and coordinated regulator of transcription in eukaryotic cells. We address the molecular mechanisms by which PCBPs binding to single- and double-stranded DNA mediates gene expression.
Assuntos
Regulação da Expressão Gênica , Poli C/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/química , Fatores de Transcrição/químicaRESUMO
Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5'-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions.
Assuntos
Regulação da Expressão Gênica , Fases de Leitura Aberta , Biossíntese de Proteínas , Receptores Opioides mu/genética , Regiões 5' não Traduzidas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Códon de Iniciação , Humanos , Camundongos , Dados de Sequência Molecular , Receptores Opioides mu/biossíntese , Ribossomos/metabolismoRESUMO
The alpha-complex proteins (alphaCP) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). These proteins are mainly involved in various post-transcriptional regulations (e.g., mRNA stabilization or translational activation/silencing). Here we report a novel function of alphaCP3, a member of the alphaCP family. alphaCP3 bound to the double-stranded poly(C) element essential for the mu opioid receptor (MOR) promoter and repressed the promoter activity at the transcriptional level. We identified alphaCP3 using affinity column chromatography containing the double-stranded poly(C) element and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. AlphaCP3 binding to the poly(C) sequence of the MOR gene was sequence specific, as confirmed by the supershift assay. In cotransfection studies, alphaCP3 repressed the MOR promoter only when the poly(C) sequence was intact. Ectopic expression of alphaCP3 led to repression of the endogenous MOR transcripts in NS20Y cells. When alphaCP3 was disrupted using small interfering RNA (siRNA) in NS20Y cells, the transcription of the endogenous target MOR gene was increased significantly. Our data suggest that alphaCP3 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of alphaCP3 as a transcriptional repressor in MOR gene regulation.