RESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.
Asunto(s)
Perfilación de la Expresión Génica , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Regiones Promotoras Genéticas/genética , Testículo/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Exones/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células 3T3 Swiss , TransfecciónRESUMEN
Paclitaxel-induced painful peripheral neuropathy is a major dose-limiting factor. Recently, it has been reported that macrophages accumulated in the dorsal root ganglion of paclitaxel-treated rats, and their activation is suggested to contribute to generation and development of the neuropathy. However, the mechanism for macrophage activation is still unknown. In this study, to explore candidate genes involved in the mechanism for macrophage activation in the dorsal root ganglion of paclitaxel-treated rats, we developed model rats for paclitaxel-induced neuropathic pain and performed a microarray assay to analyze the changes of gene expressions in the dorsal root ganglion. Among the genes with changed expression levels, we focused on matrix metalloproteinase-3 (MMP-3, stromelysin-1) and CD163, a macrophage marker. By reverse transcription-polymerase chain reaction, the expression levels of MMP-3 and CD163 were markedly up-regulated in paclitaxel-treated dorsal root ganglion. As a result of immunohistochemical study, large ganglion neurons, but neither Schwann cells nor macrophages, predominantly expressed MMP-3. This MMP-3 up-regulation occurred prior to macrophage accumulation in the dorsal root ganglion. In addition, recombinant MMP-3 led to the activation of RAW264 macrophages in vitro. Taken together, the up-regulation of MMP-3 and following macrophage activation caused in the dorsal root ganglion might be a significant event to trigger a series of reactions developing paclitaxel-induced peripheral neuropathic pain.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ganglios Espinales/metabolismo , Metaloproteinasa 3 de la Matriz/biosíntesis , Paclitaxel/farmacología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Regulación hacia Arriba , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Expresión Génica , Ratas , Receptores de Superficie Celular/biosíntesisRESUMEN
Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein alphaB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of alphaB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length alphaB-crystallin cDNA. Overexpression of alphaB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and alphaB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity.