RESUMEN
Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs ß), and burs α-ß is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs ß are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.
Asunto(s)
Proteínas de Artrópodos/genética , Astacoidea/genética , Inmunidad Innata , Hormonas de Invertebrados/fisiología , Factores de Transcripción/genética , Animales , Péptidos Catiónicos Antimicrobianos/genética , Proteínas de Artrópodos/inmunología , Astacoidea/inmunología , Regulación de la Expresión Génica , Conformación Proteica , Transducción de Señal , Factores de Transcripción/inmunologíaRESUMEN
Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after ~10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.
Asunto(s)
Histamina/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Serotonina/farmacología , Trombospondina 1/fisiología , Animales , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Neovascularización Fisiológica/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Trombospondina 1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Effective dust removal has long been a challenge in the blasting mining of underground metal mine tunnels, and uncontrolled dust diffusion seriously endangers workers' respiratory systems and the underground space safety environment. However, the vast majority of existing numerical studies on dust diffusion are focused on coal mine fully mechanized mining, which is different from metal mine blasting excavation in terms of stope structure and dust properties. Furthermore, the mechanism by which the forced and exhaust ventilation modes affect the diffusion characteristics of inhalable particles is unclear. In this work, gas-solid flow characteristics for dust diffusion in a typical metal mine blasting tunnel were numerically investigated based on the Euler-Lagrange method, where the blasting face instantly released 6.37 × 107 particles with 100 different sizes, ranging from 0.8 to 200 µm. The interphase forces between airflow and dust particles are comprehensively modeled, and the particle diffusion effect caused by fluid turbulence is described by a discrete random walk model. Detailed information for airflow turbulence and particle migration was revealed, and dust removal efficiencies for inhalable particulate matter (PM10) by forced, exhaust, and hybrid ventilation were analyzed. Numerical results predict a complex airflow pattern in the working roadway, including the jet-flow region, return airflow core region, airflow disorder region, and secondary flow region. Dust diffusion temporal characteristics can be divided into three stages, namely, the initial stage of dust generation, the efficient ventilation and dust removal stage, and the later stage of dust diffusion. Dust diffusion spatial characteristics indicate that under the Coanda wall attachment effect, the dust concentration exhibits nonuniform distribution in both vertical and horizontal directions of the return air roadway. The dust removal efficiency of hybrid ventilation on inhalable particles above respiratory height is better than that of forced ventilation, especially in the return air roadway. The additional exhaust air duct based on forced ventilation can discharge more inhalable particles from the tunnel.
RESUMEN
Transforming growth factor (TGF)-ß family members exert strong effects on restoration of liver mass after injury. Bone morphogenetic proteins (BMPs) are members of the TGF-ß family and are found in the liver, suggesting that these proteins may play a role in liver regeneration. We examined BMP signaling in the liver during hepatectomy. We found that BMP4 is constitutively expressed in the peribiliary stroma and endothelial cells of the liver and that expression is decreased after hepatectomy. Mice driven to maintain BMP4 expression in the liver display inhibited hepatocyte proliferation and restoration of liver mass after hepatectomy, suggesting that reduced BMP4 is necessary for normal regeneration. Consistent with this finding, hepatocyte-specific deletion of the BMP receptor activin receptor-like kinase 3 (Alk3) enhances regeneration and reduces phosphorylation of SMAD1/5/8, a transducer of BMP signaling. In contrast to experiments in wild-type mice, maintaining BMP4 levels has no effect on liver regeneration in hepatocyte-specific Alk3 null mice, providing evidence that BMP4 signals through Alk3 to inhibit liver regeneration. Consistent with these findings, the BMP4 antagonist Noggin enhances regeneration. Furthermore, high-dose BMP4 inhibits proliferation of primary hepatocytes and HepG2 cells in culture. These findings elucidate a new, potentially clinically relevant paradigm in which a constitutively expressed paracrine inhibitory factor plays a critical role in liver regeneration.
Asunto(s)
Proteína Morfogenética Ósea 4/fisiología , Regeneración Hepática/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 4/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Dependovirus/fisiología , Células Hep G2 , Hepatectomía , Humanos , Ratones , Proteína Smad1/metabolismoRESUMEN
In this study, an Fe-Cr-Mo-Ni-B coating was prepared using plasma cladding on Cr5 steel substrate. The microstructure, phase evolution and tribological performance of the Fe-Cr-Mo-Ni-B coating were investigated. The microstructure is mainly composed of Mo2FeB2, Fe2B, α-Fe, γ-Fe and MoB. The process of phase evolution in the coating was observed in situ by HT-CLSM. The Mo2FeB2 phase with good thermodynamic stability can exist in the high-temperature liquid phase. It also has a phenomenon of connection and merging and turns into different morphology during the plasma cladding process. The hardness value of coating was much higher than the base metal, and the hardness value of Mo2FeB2 (785.5 HV) was higher than the eutectic matrix (693.2 HV). The wear mechanisms of the cladding under dry sliding were primarily caused by adhesive wear, accompanying slight oxidation wear. The Mo2FeB2 phase has an important effect on the wear resistance property.
RESUMEN
Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.
Asunto(s)
Movimiento Celular , Células Dendríticas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Western Blotting , Quimiotaxis , Citometría de Flujo , Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Quinasa 2 de Adhesión Focal/genética , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptores CCR5/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirfostinos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Oxytocin receptors in the nucleus accumbens have been implicated in the regulation of alloparental behavior and pair bond formation in the socially monogamous prairie vole. Oxytocin receptor density in the nucleus accumbens is positively correlated with alloparenting in juvenile and adult female prairie voles, and oxytocin receptor antagonist infused into the nucleus accumbens blocks this behavior. Furthermore, prairie voles have higher densities of oxytocin receptors in the accumbens than nonmonogamous rodent species, and blocking accumbal oxytocin receptors prevents mating-induced partner preference formation. Here we used adeno-associated viral vector gene transfer to examine the functional relationship between accumbal oxytocin receptor density and social behavior in prairie and meadow voles. Adult female prairie voles that overexpress oxytocin receptor in the nucleus accumbens displayed accelerated partner preference formation after cohabitation with a male, but did not display enhanced alloparental behavior. However, partner preference was not facilitated in nonmonogamous meadow voles by introducing oxytocin receptor into the nucleus accumbens. These data confirm a role for oxytocin receptor in the accumbens in the regulation of partner preferences in female prairie voles, and suggest that oxytocin receptor expression in the accumbens is not sufficient to promote partner preferences in nonmonogamous species. These data are the first to demonstrate a direct relationship between oxytocin receptor density in the nucleus accumbens and variation in social attachment behaviors. Thus, individual variation in oxytocin receptor expression in the striatum may contribute to natural diversity in social behaviors.
Asunto(s)
Arvicolinae/fisiología , Núcleo Accumbens/metabolismo , Receptores de Oxitocina/metabolismo , Conducta Sexual Animal/fisiología , Conducta Social , Animales , Femenino , Masculino , Preferencia en el Apareamiento Animal/fisiología , Núcleo Accumbens/química , Núcleo Accumbens/fisiología , Apareamiento , Receptores de Oxitocina/fisiologíaRESUMEN
The molecular mechanisms underlying the evolution of complex behaviour are poorly understood. The mammalian genus Microtus provides an excellent model for investigating the evolution of social behaviour. Prairie voles (Microtus ochrogaster) exhibit a monogamous social structure in nature, whereas closely related meadow voles (Microtus pennsylvanicus) are solitary and polygamous. In male prairie voles, both vasopressin and dopamine act in the ventral forebrain to regulate selective affiliation between adult mates, known as pair bond formation, as assessed by partner preference in the laboratory. The vasopressin V1a receptor (V1aR) is expressed at higher levels in the ventral forebrain of monogamous than in promiscuous vole species, whereas dopamine receptor distribution is relatively conserved between species. Here we substantially increase partner preference formation in the socially promiscuous meadow vole by using viral vector V1aR gene transfer into the ventral forebrain. We show that a change in the expression of a single gene in the larger context of pre-existing genetic and neural circuits can profoundly alter social behaviour, providing a potential molecular mechanism for the rapid evolution of complex social behaviour.
Asunto(s)
Arvicolinae/genética , Arvicolinae/fisiología , Prosencéfalo/metabolismo , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Conducta Sexual Animal/fisiología , Animales , Conducta de Elección/fisiología , Femenino , Masculino , Apareamiento , Receptores Dopaminérgicos/análisisRESUMEN
BACKGROUND: Due to the complex pathogenesis, the molecular mechanism of nonalcoholic steatohepatitis (NASH) remains unclear. In this study, we aimed to reveal the comprehensive metabolic and signaling pathways in the occurrence of NASH. METHODS: C57BL/6 mice were treated with high-fat diet for 4 months to mimic the NASH phenotype. After the treatment, the physiochemical parameters were evaluated, and the liver tissues were prepared for untargeted metabolomic analysis with ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Then, three relevant Gene Expression Omnibus (GEO) datasets were selected for integrative analysis of differentiated messenger RNA and metabolites. RESULTS: The levels of phosphatidylethanolamine (PE) (16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), oleic acid, and sphingomyelin (SM) (d18:0/12:0) were significantly increased, and the content of adenosine was severely reduced in NASH mice. The integrated interpretation of transcriptomic and metabolomic data indicated that the glycerophospholipid metabolism and necroptosis signaling were evidently affected in the development of NASH. The high level of SM (d18:0/12:0) may be related to the expression of acid sphingomyelinase (ASMase), and the elevated arachidonic acid was coordinated with the upregulation of cytosol phospholipase A2 (cPLA2) in the necroptosis pathway. CONCLUSIONS: In summary, the inflammatory response, necroptosis, and glycerophospholipid may serve as potential targets for mechanistic exploration and clinical practice in the treatment of NASH.
RESUMEN
Vasopressin modulates many social and nonsocial behaviors, including emotionality. We have previously reported that male mice with a null mutation in the V1a receptor (V1aR) exhibit a profound impairment in social recognition and changes in anxiety-like behavior. Using site-specific injections of a V1aR-specific antagonist, we demonstrate that the lateral septum, but not the medial amygdala, is critical for social recognition. Reexpressing V1aR in the lateral septum of V1aR knockout mice (V1aRKO) using a viral vector resulted in a complete rescue of social recognition. Furthermore, overexpression of the V1aR in the lateral septum of wild-type (wt) mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior. These results demonstrate that the V1aR in the lateral septum plays a critical role in the neural processing of social stimuli required for complex social behavior.
Asunto(s)
Conducta Animal/fisiología , Receptores de Vasopresinas/fisiología , Reconocimiento en Psicología/fisiología , Núcleos Septales/metabolismo , Conducta Social , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Trastornos de Ansiedad/genética , Femenino , Vectores Genéticos , Habituación Psicofisiológica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Receptores de Vasopresinas/genética , Núcleos Septales/efectos de los fármacos , Conducta Sexual Animal/fisiología , Olfato/genética , TransfecciónRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells. However, its short half-life, poor delivery, and TRAIL-resistant tumor cells have diminished its clinical efficacy. In this study, we explored whether novel delivery methods will represent new and effective ways to treat gliomas and if adjuvant therapy with the chemotherapeutic agent temozolomide would enhance the cytotoxic properties of TRAIL in glioma lines resistant to TRAIL monotherapy. We have engineered adeno-associated virus (AAV) vectors encoding recombinant secreted TRAIL (S-TRAIL) and bioluminescent-fluorescent marker fusion proteins and show that AAV-delivered S-TRAIL leads to varying degrees of killing in multiple glioma lines, which correspond with caspase-3/7 activation. In vivo, dual bioluminescent imaging revealed efficient delivery of therapeutic AAV vectors directly into the tumor mass, which induced marked attenuation of tumor progression. Treatment of glioma cells with the chemotherapeutic agent temozolomide alone lead to a significant accumulation of cells in G(2)-M phase, activated the cell cycle checkpoint protein Chk1, and increased death receptor expression in a time-dependent manner. Furthermore, combined treatment with AAV-S-TRAIL or neural stem cell-S-TRAIL and temozolomide induced cell killing and markedly up-regulated proapoptotic proteins in glioma cells least sensitive to TRAIL. This study elucidates novel means of delivering S-TRAIL to gliomas and suggests combination of clinically relevant temozolomide and S-TRAIL may represent a new therapeutic option with increased potency for glioblastoma patients.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Dacarbazina/análogos & derivados , Glioma/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Antineoplásicos Alquilantes/administración & dosificación , Línea Celular Tumoral , Dacarbazina/uso terapéutico , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Microscopía Fluorescente , Transducción de Señal , Trasplante de Células Madre , Células Madre/citología , Células Madre/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca(2+)(o), a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca(2+)(o) did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca(2+)(o) did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca(2+)(o)-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca(2+)(o) is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca(2+)(o) stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.
Asunto(s)
Osteoblastos/citología , Osteoblastos/fisiología , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Animales , Especificidad de Anticuerpos , Northern Blotting , Calcio/metabolismo , División Celular/fisiología , Ciclina D , Ciclinas/genética , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Expresión Génica/fisiología , Proteínas Inmediatas-Precoces/genética , Factor I del Crecimiento Similar a la Insulina/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores Sensibles al Calcio/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/citología , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclin-dependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Regeneración Hepática/fisiología , Proteínas Represoras/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas Portadoras/genética , Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Dependovirus/genética , Exorribonucleasas , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Hepatectomía , Hepatocitos/metabolismo , Hepatocitos/patología , Péptidos y Proteínas de Señalización Intracelular , Hígado/enzimología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Unión Proteica , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas , Transducción de Señal/genética , Proteínas Smad/metabolismo , Transcripción GenéticaRESUMEN
The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.
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Quimiocina CCL2/metabolismo , Quimiotaxis , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/citología , Neuronas/metabolismo , Receptores Sensibles al Calcio/fisiología , Animales , Calcio/fisiología , Recuento de Células , Células Cultivadas , Femenino , Ratones , Ratones Noqueados , Receptores CCR2 , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
By using pharmacological and molecular approaches, we previously showed that the G-protein-coupled, extracellular calcium (Ca2+(o))-sensing receptor (CaR) regulates a large-conductance (approximately 140 pS), Ca(2+)-activated K+ channel [IK(Ca); CAKC] in U87 astrocytoma cells. Here we show that elevated Ca2+(o) stimulates extracellular-signal-regulated kinase (ERK1/2) and p38 MAP kinase (MAPK). The effect of high Ca2+(o) on p38 MAPK but not ERK1/2 is CaR mediated, insofar as transduction with a dominant-negative CaR (R185Q) using recombinant adeno-associated virus (rAAV) attenuated the activation of p38 MAPK but not of ERK1/2. p38 MAPK activation by the CaR is likely to be protein kinase C (PKC) independent, in that the pan-PKC inhibitor GF109203X failed to abolish the high-Ca2+(o)-induced phosphorylation of p38 MAPK. Consistently with our data on the activation of this kinase, we observed that inhibiting p38 MAPK blocked the activation of the CAKC induced by the specific pharmacological CaR activator NPS R-467. In contrast, inhibiting MEK1 only transiently inhibited the activation of this K+ channel by NPS R-467, despite the continued presence of the antagonist. Similarly to the lack of any effect of the PKC inhibitor on the activation of ERK1/2 and p38 MAPK, inhibiting PKC had no effect on NPS R-467-induced activation of this channel. Therefore, our data show that the CaR, acting via p38 MAPK, regulates a large-conductance CAKC in U87 cells, a process that is PKC independent. Large-conductance CAKCs play an important role in the regulation of cellular volume, so our results have important implications for glioma cell volume regulation.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Receptores Sensibles al Calcio/metabolismo , Calcio/farmacología , Línea Celular Tumoral , Humanos , Canales de Potasio Calcio-Activados/agonistas , Receptores Sensibles al Calcio/agonistas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
This study explores the effects of enhancing vasopressin V1a receptor expression in the septum using viral vector-mediated gene transfer on social discrimination and social interactions. Bilateral infusion of an adeno-associated viral vector containing the prairie vole V1a receptor gene (V1aR-AAV) regulated by a neuron-specific enolase promoter resulted in a stable increase in V1a receptor binding density in the rat septum without affecting oxytocin receptor density. Control animals were infused with a vector expressing the lacZ gene. In a social discrimination paradigm, only V1aR-AAV-treated animals succeeded in discriminating a previously encountered from a novel juvenile after an interexposure interval (IEI) of more than 2 h, demonstrating the functional incorporation of the vole V1a receptor in the rat septal circuits underlying short-term memory processes. Microdialysis administration of synthetic vasopressin during the first juvenile exposure, used to mimic intraseptal release patterns of the neuropeptide, produced similar prolongations in recognition (up to an IEI of 24 h) in both V1aR-AAV and control animals. Septal microdialysis administration of a selective V1a, but not oxytocin, receptor antagonist in both groups prevented discrimination even after an IEI of as short as 0.5 h, confirming the specificity of the vole V1a receptor involvement in social discrimination abilities. In addition, active social interactions were found to be increased among V1aR-AAV rats compared to controls. Viral vector-mediated gene transfer provides a valuable tool for studies on the role of localized gene expression on behavioural parameters.
Asunto(s)
Técnicas de Transferencia de Gen , Receptores de Vasopresinas/genética , Tabique del Cerebro/fisiología , Conducta Social , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Arvicolinae , Autorradiografía , Dependovirus/genética , Discriminación en Psicología/efectos de los fármacos , Vectores Genéticos , Operón Lac , Masculino , Microdiálisis , Ratas , Ratas Wistar , Receptores de Oxitocina/antagonistas & inhibidores , Receptores de Oxitocina/metabolismo , Vasoconstrictores/administración & dosificación , Vasoconstrictores/farmacología , Vasopresinas/administración & dosificación , Vasopresinas/farmacologíaRESUMEN
Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.