RESUMEN
Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.
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Proteínas Bacterianas/metabolismo , Fibrosis Quística/microbiología , Gluconatos/metabolismo , Metaboloma , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Fibrosis Quística/patología , Farmacorresistencia Bacteriana/fisiología , Femenino , Humanos , Masculino , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.
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Empalme Alternativo/genética , Intrones/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Animales , Dendritas , Hipocampo/citología , Neuronas , ARN Mensajero , RatasRESUMEN
High single-channel conductance K+ channels, which respond jointly to membrane depolarization and micromolar concentrations of intracellular Ca2+ ions, arise from extensive cell-specific alternative splicing of pore-forming alpha-subunit mRNAs. Here, we report the discovery of an endogenous BK(Ca) channel alpha-subunit intron-containing mRNA in the cytoplasm of hippocampal neurons. This partially processed mRNA, which comprises approximately 10% of the total BK(Ca) channel alpha-subunit mRNAs, is distributed in a gradient throughout the somatodendritic space. We selectively reduced endogenous cytoplasmic levels of this intron-containing transcript by RNA interference without altering levels of the mature splice forms of the BK(Ca) channel mRNAs. In doing so, we could demonstrate that changes in a unique BK(Ca) channel alpha-subunit intron-containing splice variant mRNA can greatly impact the distribution of the BK(Ca) channel protein to dendritic spines and intrinsic firing properties of hippocampal neurons. These data suggest a new regulatory mechanism for modulating the membrane properties and ion channel gradients of hippocampal neurons.
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Hipocampo/fisiología , Intrones , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Neuronas/fisiología , ARN Mensajero/genética , Potenciales de Acción , Animales , Células Cultivadas , Dendritas , Hipocampo/citología , Hipocampo/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Neuronas/metabolismo , ARN Interferente Pequeño , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares/metabolismoRESUMEN
Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.
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Comunicación Celular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neurofarmacología , Fracciones Subcelulares/efectos de los fármacos , Animales , Sistemas de Liberación de Medicamentos , HumanosRESUMEN
Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.
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Hialuronano Sintasas/metabolismo , Ácido Hialurónico/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/virología , alfa-Globulinas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Gripe Humana/metabolismo , Macrófagos/inmunología , Ratones , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Pruebas de Función RespiratoriaRESUMEN
N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.
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Empalme Alternativo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Ganglios Espinales/metabolismo , Dolor/fisiopatología , Animales , Canales de Calcio Tipo N/metabolismo , Capsaicina/farmacología , Conductividad Eléctrica , Exones , Ganglios Espinales/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Dolor/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.
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Encéfalo/metabolismo , Transcriptoma , Adulto , Anciano , Encéfalo/citología , Células Cultivadas , Femenino , Humanos , Masculino , MicroARNs/metabolismo , Microglía/citología , Microglía/metabolismo , Persona de Mediana Edad , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Análisis de Componente Principal , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Adulto JovenRESUMEN
OBJECTIVES: Biofield treatments have been used for pain control in patients with cancer and chronic pain. However, research on the effect of biofield treatment on specific somatic disorders is lacking. This study intends to investigate the effect of oscillating biofield therapy (OBFT) on symptoms of carpal tunnel syndrome. DESIGN: Randomized, placebo-controlled, double-blind study. PARTICIPANTS: Thirty patients with chronic carpal tunnel syndrome participated in the study. INTERVENTION: Patients were randomly assigned to active or placebo treatment groups. Those in the treatment group received six sessions of OBFT with intention to treat during a period of 2 weeks. Patients in the placebo group had the same number of treatment sessions with mock OBFT treatment. OUTCOME MEASURE: The Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire; Symptom Severity Scale (SSS); and Functional Status Scale (FSS) were used for outcome assessment. RESULTS: Both clinically and statistically significant changes in intensity of pain with activity (95% confidence interval [CI], 2.5-4.2; p = 0.000), night pain (p = 0.000, 95% CI, 3.2-5.7), DASH questionnaire (95% CI, 12.0-21.9; p = 0.000), SSS (95% CI, 0.64-1.15; p = 0.003), and FSS (95% CI, 0.41-0.97; p = 0.029) were found between the treatment and placebo groups. Statistically significant reduction in number of patients with positive results on the Phalen test (87%; p = 0.000), Tinel sign (73%; p = 0.000), and hand paresthesia (80%; p = 0.000) was noted in the treatment group. During 6-month follow-up, 86% of patients in the treatment group remained pain free and had no functional limitations. CONCLUSION: OBFT can be a viable and effective treatment for improving symptoms and functional limitations associated with chronic carpal tunnel syndrome.
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Síndrome del Túnel Carpiano/fisiopatología , Síndrome del Túnel Carpiano/terapia , Manejo del Dolor/métodos , Modalidades de Fisioterapia , Tacto Terapéutico/métodos , Adulto , Anciano , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor/fisiopatologíaRESUMEN
The sensitivity of new transcriptomic techniques is rapidly improving to the point that single-cell molecular analysis is now becoming commonplace. However to obtain accurate transcriptome data, the initial experimental steps must strive to maintain the natural environment of cell and always get set in motion under in vivo conditions. Achieving these critical experimental parameters is technically challenging for investigators and currently the most frequently used molecular techniques experimentally commence with tissues or cells in artificial environments or under in vitro conditions. Here we review an innovative experimental approach that is called transcriptome in vivo analysis (TIVA) that was designed to overcome theses well-known limitations. The TIVA methods permit cell-specific transcriptome capture from viable intact heterogeneous tissues. Cell-penetrating peptides (CPPs) are used to deliver multifunctional transcriptome-capture tags (TIVA tags) to the cytoplasm of the cell under in vivo conditions. The TIVA capture tag enables investigators to target and isolate cell-specific transcriptomes in their natural microenvironments. The combination of maintaining in vivo conditions and selective cell-specific transcriptome capture provides investigators with the opportunity to yield the most biologically accurate and informative transcriptome data hitherto.
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Perfilación de la Expresión Génica/métodos , Genómica/métodos , Análisis de la Célula Individual/métodos , Animales , Supervivencia Celular , Péptidos de Penetración Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Exones , Humanos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.
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Péptidos de Penetración Celular/metabolismo , Genómica/métodos , Ácidos Nucleicos de Péptidos/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Empalme Alternativo , Animales , Supervivencia Celular , Precipitación Química , Electroforesis en Gel de Poliacrilamida/métodos , Exones , Humanos , Espectrometría de Masas/métodos , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
Much of the biology surrounding macrophage functional specificity has arisen through examining inflammation-induced polarizing signals, but this also occurs in homeostasis, requiring tissue-specific environmental triggers that influence macrophage phenotype and function. The TAM receptor family of receptor tyrosine kinases (Tyro3, Axl and MerTK) mediates the non-inflammatory removal of apoptotic cells by phagocytes through the bridging phosphatidylserine-binding molecules growth arrest-specific 6 (Gas6) or Protein S. We show that one such TAM receptor (Axl) is exclusively expressed on mouse airway macrophages, but not interstitial macrophages and other lung leukocytes, under homeostatic conditions and is constitutively ligated to Gas6. Axl expression is potently induced by granulocyte-macrophage colony-stimulating factor expressed in the healthy and inflamed airway, and by type I interferon or Toll-like receptor-3 stimulation on human and mouse macrophages, indicating potential involvement of Axl in apoptotic cell removal under inflammatory conditions. Indeed, an absence of Axl does not cause sterile inflammation in health, but leads to exaggerated lung inflammatory disease upon influenza infection. These data imply that Axl allows specific identification of airway macrophages, and that its expression is critical for macrophage functional compartmentalization in the airspaces or lung interstitium. We propose that this may be a critical feature to prevent excessive inflammation because of secondary necrosis of apoptotic cells that have not been cleared by efferocytosis.
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Pulmón/inmunología , Macrófagos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Neumonía/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Pulmón/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Especificidad de Órganos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Neumonía/genética , Neumonía/patología , Proteína S/genética , Proteína S/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Tirosina Quinasa del Receptor AxlRESUMEN
Alveolar macrophages exist in a unique microenvironment and, despite historical evidence showing that they are in close contact with the respiratory epithelium, have until recently been investigated in isolation. The microenvironment of the airway lumen has a considerable influence on many aspects of alveolar macrophage phenotype, function and turnover. As the lungs adapt to environmental challenges, so too do alveolar macrophages adapt to accommodate the ever-changing needs of the tissue. In this Review, we discuss the unique characteristics of alveolar macrophages, the mechanisms that drive their adaptation and the direct and indirect influences of epithelial cells on them. We also highlight how airway luminal macrophages function as sentinels of a healthy state and how they do not respond in a pro-inflammatory manner to antigens that do not disrupt lung structure. The unique tissue location and function of alveolar macrophages distinguish them from other macrophage populations and suggest that it is important to classify macrophages according to the site that they occupy.
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Macrófagos Alveolares/fisiología , Animales , Antígenos de Superficie/fisiología , Microambiente Celular , Humanos , Lectinas Tipo C/fisiología , Activación de Macrófagos , Receptor de Manosa , Lectinas de Unión a Manosa/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Orexina , Especificidad de Órganos , PPAR gamma/fisiología , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Receptor Activador Expresado en Células Mieloides 1RESUMEN
This protocol describes how to amplify mRNA isolated from a single cell and then analyze its gene expression profile using polymerase chain reaction (PCR). Single-cell analysis is advantageous over studies of cell populations because it allows identification of a range of normal physiological states expressed by different cells of the same cell type without the confounding effects of averaging that result from measuring physiological states of cell populations. This is especially important when addressing questions of physiology in tissues, which comprises many different cell types. However, a single cell does not contain enough mRNA for all of the expressed transcripts to be detected or measured by any current molecular biology techniques. The antisense RNA (aRNA) amplification method was developed to amplify the picogram amounts of mRNA found within a single cell to microgram amounts of aRNA after three rounds of amplification. This aRNA can then easily be analyzed by microarray or next-generation sequencing. These methods allow identification of all expressed mRNA species within a single cell, including previously unknown mRNAs or those mRNAs specifically affected by a certain treatment. mRNA species of interest identified by these techniques can be further analyzed by designing primers targeting these species and performing PCR. cDNA synthesized from RNA at any stage in the aRNA amplification procedure, including material directly from collected unamplified cells, can be analyzed using PCR. Regardless of downstream applications, single-cell aRNA amplification is a powerful tool for studying single-cell physiological dynamics.
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Técnicas de Amplificación de Ácido Nucleico/métodos , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP-RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557-1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP-RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.
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Fraccionamiento Químico/métodos , Exones/genética , Ácidos Nucleicos de Péptidos/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Biotinilación , Supervivencia Celular , Cartilla de ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Magnetismo , Oligonucleótidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tinción con Nitrato de Plata , Estreptavidina/metabolismo , Especificidad por SustratoRESUMEN
RNA precursors give rise to mRNA after splicing of intronic sequences traditionally thought to occur in the nucleus. Here, we show that intron sequences are retained in a number of dendritically-targeted mRNAs, by using microarray and Illumina sequencing of isolated dendritic mRNA as well as in situ hybridization. Many of the retained introns contain ID elements, a class of SINE retrotransposon. A portion of these SINEs confers dendritic targeting to exogenous and endogenous transcripts showing the necessity of ID-mediated mechanisms for the targeting of different transcripts to dendrites. ID elements are capable of selectively altering the distribution of endogenous proteins, providing a link between intronic SINEs and protein function. As such, the ID element represents a common dendritic targeting element found across multiple RNAs. Retention of intronic sequence is a more general phenomenon than previously thought and plays a functional role in the biology of the neuron, partly mediated by co-opted repetitive sequences.
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Citoplasma/genética , Dendritas/genética , Intrones/genética , Elementos de Nucleótido Esparcido Corto/genética , Animales , Células Cultivadas , Citoplasma/metabolismo , Dendritas/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Hibridación in Situ , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasAsunto(s)
Bancos de Muestras Biológicas , Investigación Biomédica , Bancos de Muestras Biológicas/historia , Bancos de Muestras Biológicas/tendencias , Investigación Biomédica/historia , Investigación Biomédica/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Proyecto Genoma Humano , HumanosRESUMEN
Sensorineural hearing deficiencies result from the loss of auditory hair cells. This hearing loss is permanent in humans and mammals because hair cells are not spontaneously replaced. In other animals such as birds, this is not the case. Damage to the avian cochlea evokes proliferation of supporting cells and the generation of functionally competent replacement hair cells. Signal transduction pathways are clinically useful as potential therapeutic targets, so there is significant interest in identifying the key signal transduction pathways that regulate the formation of replacement hair cells. In a previous study from our lab, we showed that forskolin (FSK) treatment induces auditory supporting cell proliferation and formation of replacement hair cells in the absence of sound or aminoglycoside treatment. Here, we show that FSK-induced supporting cell proliferation is mediated by cell-specific accumulation of cyclic adenosine monophosphate (cAMP) in avian supporting cells and the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. By a combination of immunostaining and pharmacological analyses, we show that FSK treatment increases cAMP levels in avian auditory supporting cells and that several ERK MAP inhibitors effectively block FSK-induced supporting cell proliferation. Next, we demonstrate by Western blotting and immunostaining analyses the expression of several ERK MAPK signaling molecules in the avian auditory epithelium and the cell-specific expression of B-Raf in avian auditory supporting cells. Collectively, these data suggest that FSK-induced supporting cell proliferation in the avian auditory epithelium is mediated by increases of cAMP levels in supporting cells and the cell-specific expression of the ERK MAPK family member B-Raf in supporting cells.