RESUMEN
Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.
Asunto(s)
Betaína , Pseudomonas aeruginosa , Betaína/metabolismo , Pseudomonas aeruginosa/metabolismo , Esfingosina/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , Ceramidasas/metabolismoRESUMEN
Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida's detection of the animal-derived amine compound creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida's ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small, and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida's metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.
Asunto(s)
Pseudomonas putida , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Creatina/genética , Creatina/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Filogenia , Regiones Promotoras Genéticas , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMEN
Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.IMPORTANCEStenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.
Asunto(s)
Fibrosis Quística/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , Esputo/microbiología , Stenotrophomonas maltophilia/genética , Antibacterianos/farmacología , Genoma Bacteriano , Humanos , Filogenia , Especificidad de la Especie , Stenotrophomonas maltophilia/efectos de los fármacos , Stenotrophomonas maltophilia/metabolismoRESUMEN
The interactions between Klebsiella pneumoniae and the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response of K. pneumoniae MGH 78578 to purified pulmonary surfactant. This work revealed changes within the K. pneumoniae transcriptome that likely contribute to host colonization, adaptation, and virulence in vivo Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts to K. pneumoniae survival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators of K. pneumoniae survival within the lung and confirmed previous evidence for the importance of de novo leucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation in K. pneumoniae and identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring between K. pneumoniae and the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro.
Asunto(s)
Biopelículas/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Surfactantes Pulmonares/farmacología , Aminoácidos de Cadena Ramificada/biosíntesis , Animales , Poliaminas Biogénicas/fisiología , Fimbrias Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno , Klebsiella pneumoniae/patogenicidad , Klebsiella pneumoniae/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Virulencia/genéticaRESUMEN
Many Pseudomonas aeruginosa infections are derived from residential, recreational, or surface water sources; thus, these environments represent an important preinfection niche. To better understand P. aeruginosa biology in these environments, we quantified transcriptional changes by microarray after exposure to diluted LB, diluted R2B, potable tap water, and freshwater from a eutrophic pond. Quantitative reverse transcription-PCR (qRT-PCR) confirmed the conservation of these responses in other water sources, and competition experiments were used to test the importance of three implicated metabolic pathways. The global transcriptional responses in potable water and freshwater showed strong induction of genes involved in metabolism of the head groups and acyl tails of phospholipids, as well as nucleotide metabolism, with commensurate decreased transcript expression of genes encoding their synthetic pathways. These data suggest that phospholipids and nucleotides are part of the nutritional milieu of these two environments. A unique response in municipal-delivered potable water was to the metals in the piping system, particularly copper. To identify potential nutrient sources used by P. aeruginosa in these environments, we used competition assays between the wild-type and deletion mutant strains in three pathways induced under these conditions. For phospholipid head-group metabolism, ethanolamine utilization (eutB) was important for competition in potable water, while choline oxidation (betBA) was important for competition in freshwater. Nucleotide utilization, particularly pyrimidine metabolism (dht), showed a trend toward importance in freshwater but was not statistically significant. These findings provide new insights into the P. aeruginosa response to potable water and freshwater and led to the identification of potentially important nutrient sources in these environments.IMPORTANCE Much of our knowledge about Pseudomonas aeruginosa comes from the infection niche, and much less is known about its lifestyle in the environment. P. aeruginosa is an adaptable bacterium capable of growing in many environments but is particularly common in potable water systems and freshwater. We used the transcriptional responses of P. aeruginosa to these environments to identify important nutrient sources specific to either of these two environments. Additionally, these environments could provide experimental situations to understand gene function for the large number of transcripts with unknown functions induced under these conditions.
Asunto(s)
Agua Potable/análisis , Agua Dulce/análisis , Pseudomonas aeruginosa/fisiología , Transcripción Genética/fisiología , Pseudomonas aeruginosa/genéticaRESUMEN
The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σ(F), σ(E), σ(G), and σ(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σ(F), σ(E), σ(G), and σ(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σ(F)-dependent, 169 were σ(E)-dependent, 34 were σ(G)-dependent, and 31 were σ(K)-dependent. In contrast with B. subtilis, C. difficile σ(E) was dispensable for σ(G) activation, σ(G) was dispensable for σ(K) activation, and σ(F) was required for post-translationally activating σ(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.
Asunto(s)
Clostridioides difficile/genética , Diarrea/microbiología , Factor sigma/genética , Esporas Bacterianas/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Clostridioides difficile/crecimiento & desarrollo , Diarrea/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Humanos , Mutación , Análisis de Secuencia de ARN , Factor sigma/aislamiento & purificación , Factor sigma/metabolismo , Esporas Bacterianas/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Ralstonia insidiosa and Chryseobacterium gleum are bacterial species commonly found in potable water systems, and these two species contribute to the robustness of biofilm formation in a model six-species community from the International Space Station (ISS) potable water system. Here, we set about characterizing the interaction between these two ISS-derived strains and examining the extent to which this interaction extends to other strains and species in these two genera. The enhanced biofilm formation between the ISS strains of R. insidiosa and C. gleum is robust to starting inoculum and temperature and occurs in some but not all tested growth media, and evidence does not support a soluble mediator or coaggregation mechanism. These findings shed light on the ISS R. insidiosa and C. gleum interaction, though such enhancement is not common between these species based on our examination of other R. insidiosa and C. gleum strains, as well as other species of Ralstonia and Chryseobacterium. Thus, while the findings presented here increase our understanding of the ISS potable water model system, not all our findings are broadly extrapolatable to strains found outside of the ISS. IMPORTANCE Biofilms present in drinking water systems and terminal fixtures are important for human health, pipe corrosion, and water taste. Here, we examine the enhanced biofilm of cocultures for two very common bacteria from potable water systems: Ralstonia insidiosa and Chryseobacterium gleum. While strains originally isolated on the International Space Station show enhanced dual-species biofilm formation, terrestrial strains do not show the same interaction properties. This study contributes to our understanding of these two species in both dual-culture and monoculture biofilm formation.
RESUMEN
MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC(1) receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC(1)HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC(1)HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC(1)HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on G alpha(q)/phosphatidylinositol 3-kinase gamma activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase gamma-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC(1)HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.
Asunto(s)
Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Animales , Supervivencia Celular , Fosfatidilinositol 3-Quinasa Clase Ib , Endocitosis , Endosomas/metabolismo , Femenino , Isoenzimas/metabolismo , Fosforilación , Isoformas de Proteínas , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cytokines are upregulated in a variety of inflammatory conditions and cytokine/receptor interactions can activate JAK-STAT signaling. Previous studies demonstrated upregulation of numerous cytokines in the urinary bladder following cyclophosphamide (CYP)-induced cystitis. The role of JAK-STAT signaling in urinary bladder inflammation and referred somatic sensitivity has not been addressed. The contribution of JAK-STAT signaling pathways in CYP-induced bladder hyperreflexia and referred somatic hypersensitivity was determined in CYP-treated rats using a JAK2 inhibitor, AG490. Acute (4 h; 150 mg/kg ip), intermediate (48 h; 150 mg/kg ip), or chronic (75 mg/kg ip, once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Phosphorylation status of STAT-3 was increased in urinary bladder after CYP-induced cystitis (4 h, 48 h, chronic). Blockade of JAK2 with AG490 (5-15 mg/kg ip or intravesical) significantly (P < or = 0.05) reduced bladder hyperreflexia and hind paw sensitivity in CYP-treated rats. These studies demonstrate a potential role for JAK-STAT signaling pathways in bladder hyperreflexia and referred pain induced by CYP-induced bladder inflammation.
Asunto(s)
Cistitis/metabolismo , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Vejiga Urinaria/metabolismo , Animales , Antineoplásicos Alquilantes , Ciclofosfamida , Cistitis/inducido químicamente , Femenino , Janus Quinasa 2/antagonistas & inhibidores , Fosforilación , Ratas , Ratas Wistar , Estrés Fisiológico , TirfostinosRESUMEN
The high and preferential expression of the PAC(1)(short)HOP1 receptor in postganglionic sympathetic neurons facilitates microarray studies for mechanisms underlying PACAP-mediate neurotrophic signaling in a physiological context. Replicate primary sympathetic neuronal cultures were treated with 100 nM PACAP27 either acutely (9 h) or chronically (96 h) before RNA extraction and preparation for Affymetrix microarray analysis. Compared to untreated control cultures, acute PACAP treatment modulated significantly the expression of 147 transcripts of diverse functional groups, including peptides, growth factors/cytokines, transcriptional factors, receptors/signaling effectors and cell cycle regulators, that collectively appeared to facilitate neuronal plasticity, differentiation and/or regeneration processes. Some regulated transcripts, for example, were related to BDNF/TrkB, IL-6/Jak2/Socs2 and TGF/follistatin signaling; many transcripts affected bioactive peptide and polyamine biosynthesis. Although chronic PACAP treatments altered the expression of 109 sympathetic transcripts, only 43 transcripts were shared between the acute and chronic treatment data sets. The PACAP-mediated changes in transcript expression were corroborated independently by quantitative PCR measurement. The PACAP-regulated transcripts in sympathetic neurons did not bear strong resemblance to those in PACAP-treated pheochromocytoma cells. However, many PACAP-targeted sympathetic transcripts, especially those related to peptide plasticity and nerve regeneration processes, coincided significantly with genes altered after peripheral nerve injury. The ability for sympathetic PAC(1)(short)HOP1 receptors to engage multiple downstream signaling cascades appeared to be reflected in the number and diversity of genes targeted in a multifaceted strategy for comprehensive neurotrophic responses.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Factores de TiempoRESUMEN
The bacterial pathogen Pseudomonas aeruginosa undergoes adaptation and selection over the course of chronic respiratory tract infections which results in repeatedly-observed phenotypic changes that are proposed to enable its persistence. Two of the clinically significant P. aeruginosa phenotypic changes are loss of flagellar motility and modifications to LPS structure, including loss of O-antigen expression. The effect of loss of O-antigen, frequently described as conversion from smooth to rough LPS, and the combined effect of loss of motility and O-antigen on phagocytic susceptibility by immune cells remain unknown. To address this, we generated genetic deletion mutants of waaL, which encodes the O-antigen ligase responsible for linking O-antigen to lipid A-core oligosaccharide, in both motile and non-motile P. aeruginosa strains. With the use of these bacterial strains we provide the first demonstration that, despite a progressive selection for P. aeruginosa with rough LPS during chronic pulmonary infections, loss of the LPS O-antigen does not confer phagocytic resistance in vitro. However, use of the waaLmotABmotCD mutant revealed that loss of motility confers resistance to phagocytosis regardless of the smooth or rough LPS phenotype. These findings reveal how the O-antigen of P. aeruginosa can influence bacterial clearance during infection and expand our current knowledge about the impact of bacterial phenotypic changes during chronic infection.
Asunto(s)
Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , Enfermedades Pulmonares , Antígenos O , Fagocitosis , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Ligasas de Carbono-Oxígeno/genética , Ligasas de Carbono-Oxígeno/inmunología , Enfermedad Crónica , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/inmunología , Ratones , Antígenos O/genética , Antígenos O/inmunología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunologíaRESUMEN
Pituitary adenylate cyclase activating polypeptides (PACAP) and PAC1 receptor signaling have diverse roles in central and peripheral nervous system development and function. In recent microarray analyses for PACAP and PAC1 receptor modulation of neuronal transcripts, the mRNA of Homer 1a (H1a), which encodes the noncrosslinking and immediate early gene product isoform of Homer, was identified to be strongly upregulated in superior cervical ganglion (SCG) sympathetic neurons. Given the prominent roles of Homer in synaptogenesis, synaptic protein complex assembly and receptor/channel signaling, we have examined the ability for PACAP to induce H1a expression in sympathetic, cortical and hippocampal neurons to evaluate more comprehensively the roles of PACAP in synaptic function. In both central and peripheral neuronal cultures, PACAP peptides increased transiently H1a transcript levels approximately 3.5- to 6-fold. From real-time quantitative PCR measurements, the temporal patterns of PACAP-mediated H1a mRNA induction among the different neuronal cultures appeared similar although the onset of sympathetic H1a transcript expression appeared protracted. The increase in H1a transcripts was accompanied by increases in H1a protein levels. Comparative studies with VIP and PACAP(6-38) antagonist demonstrated that the PACAP effects reflected PAC1 receptor activation and signaling. The PAC1 receptor isoforms expressed in central and peripheral neurons can engage diverse intracellular second messenger systems, and studies using selective signaling pathway inhibitors demonstrated that the cyclic AMP/PKA and MEK/ERK cascades are principal mediators of the PACAP-mediated H1a induction response. In modulating H1a transcript and protein expression, these studies may implicate broad roles for PACAP and PAC1 receptor signaling in synaptic development and plasticity.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , ADN Complementario/genética , Proteínas de Andamiaje Homer , Análisis de Secuencia por Matrices de Oligonucleótidos , Nervios Periféricos/citología , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Transducción de Señal/efectos de los fármacos , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/efectos de los fármacos , Ganglio Cervical Superior/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Single-nucleotide polymorphisms (SNPs) in the genes for pituitary adenylyl cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with stress-related psychiatric disorders. Although, from recent work, we have argued that stress-induced PACAP expression in the bed nucleus of the stria terminalis (BNST) may mediate stress-related psychopathology, it is unclear whether stress-induced increases in BNST PACAP expression require acute or repeated stressor exposure and whether increased BNST PACAP expression is related to stress-induced increases in circulating glucocorticoids. In the current work, we have used real-time quantitative polymerase chain reaction (qPCR) to assess transcript expression in brain punches from rats after stressor exposure paradigms or corticosterone injection. BNST PACAP and PAC1 receptor transcript expression was increased only after 7 days of repeated stressor exposure; no changes in transcript levels were observed 2 or 24 hours after a single-restraint session. Moreover, repeated corticosterone treatment for 7 days was not sufficient to reliably increase BNST PACAP transcript levels, suggesting that stress-induced elevations in corticosterone may not be the primary drivers of BNST PACAP expression. These results may help clarify the mechanisms and temporal processes that underlie BNST PACAP induction for intervention in stress-related anxiety disorders.
Asunto(s)
Corticosterona/sangre , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Núcleos Septales/metabolismo , Estrés Psicológico/metabolismo , Animales , Masculino , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismoRESUMEN
Recent studies suggest that janus-activated kinases-signal transducer and activator of transcription signaling pathways contribute to increased voiding frequency and referred pain of cyclophosphamide (CYP)-induced cystitis in rats. Potential upstream chemical mediator(s) that may be activated by CYP-induced cystitis to stimulate JAK/STAT signaling are not known in detail. In these studies, members of the interleukin (IL)-6 family of cytokines including, leukemia inhibitory factor (LIF), IL-6, and ciliary neurotrophic factor (CNTF) and associated receptors, IL-6 receptor (R) α, LIFR, and gp130 were examined in the urinary bladder in control and CYP-treated rats. Cytokine and receptor transcript and protein expression and distribution were determined in urinary bladder after CYP-induced cystitis using quantitative, real-time polymerase chain reaction (Q-PCR), western blotting, and immunohistochemistry. Acute (4 h; 150 mg/kg; i.p.), intermediate (48 h; 150 mg/kg; i.p.), or chronic (75 mg/kg; i.p., once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Q-PCR analyses revealed significant (p ≤ 0.01) CYP duration- and tissue- (e.g., urothelium, detrusor) dependent increases in LIF, IL-6, IL-6Rα, LIFR, and gp130 mRNA expression. Western blotting demonstrated significant (p ≤ 0.01) increases in IL-6, LIF, and gp130 protein expression in whole urinary bladder with CYP treatment. CYP-induced cystitis significantly (p ≤ 0.01) increased LIF-immunoreactivity (IR) in urothelium, detrusor, and suburothelial plexus whereas increased gp130-IR was only observed in urothelium and detrusor. These studies suggest that IL-6 and LIF may be potential upstream chemical mediators that activate JAK/STAT signaling in urinary bladder pathways.
RESUMEN
Exposure to chronic stress has been argued to produce maladaptive anxiety-like behavioral states, and many of the brain regions associated with stressor responding also mediate anxiety-like behavior. Pituitary adenylate cyclase activating polypeptide (PACAP) and its specific G protein-coupled PAC(1) receptor have been associated with many of these stress- and anxiety-associated brain regions, and signaling via this peptidergic system may facilitate the neuroplasticity associated with pathological affective states. Here we investigated whether chronic stress increased transcript expression for PACAP, PAC(1) receptor, brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB) in several nuclei. In rats exposed to a 7 days chronic variate stress paradigm, chronic stress enhanced baseline startle responding induced by handling and exposure to bright lights. Following chronic stress, quantitative transcript assessments of brain regions demonstrated dramatic increases in PACAP and PAC(1) receptor, BDNF, and TrkB receptor mRNA expression selectively in the dorsal aspect of the anterolateral bed nucleus of the stria terminalis (dBNST). Related vasoactive intestinal peptide (VIP) and VPAC receptor, and other stress peptide transcript levels were not altered compared to controls. Moreover, acute PACAP38 infusion into the dBNST resulted in a robust dose-dependent anxiogenic response on baseline startle responding that persisted for 7 days. PACAP/PAC(1) receptor signaling has established trophic functions and its coordinate effects with chronic stress-induced dBNST BDNF and TrkB transcript expression may underlie the maladaptive BNST remodeling and plasticity associated with anxiety-like behavior.
Asunto(s)
Ansiedad/etiología , Factor Neurotrófico Derivado del Encéfalo/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Núcleos Septales/metabolismo , Estrés Psicológico/genética , Adaptación Psicológica/efectos de los fármacos , Animales , Ansiedad/inducido químicamente , Ansiedad/genética , Ansiedad/metabolismo , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Enfermedad Crónica , Masculino , Modelos Biológicos , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Núcleos Septales/efectos de los fármacos , Estrés Psicológico/inducido químicamente , Estrés Psicológico/metabolismo , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Regulation of the VEGF-VEGF receptor system was examined in the urinary bladder after acute (2-48 h) and chronic (10 days) cyclophosphamide (CYP)-induced cystitis. ELISAs demonstrated significant (P < or = 0.01) upregulation of VEGF in whole urinary bladder with acute and chronic CYP-induced cystitis; however, the magnitude of increase was greater after acute (2-4 h) cystitis. Immunohistochemistry for VEGF immunoreactivity revealed a significant (P < or = 0.05) increase in VEGF immunoreactivity in the urothelium, suburothelial vasculature, and detrusor smooth muscle with acute (4 and 48 h) CYP treatment. RT-PCR identified the isoform VEGF-164, the VEGF receptor VEGFR-2, and the VEGF co-receptors neuropilin (Npn)-1 and Npn-2 in the urinary bladder. Quantitative PCR demonstrated upregulation of VEGF-164 transcript with acute and chronic CYP-induced cystitis, but VEGFR-2, Npn-1, and Npn-2 transcripts were upregulated (P < or = 0.01) in whole bladder only with chronic CYP-induced cystitis. Additional studies demonstrated regulation of VEGF transcript expression in the urinary bladder by nerve growth factor (NGF) in a novel line of NGF-overexpressing mice. These studies demonstrated that urinary bladder inflammation and NGF regulate the VEGF-VEGF receptor system in the urinary bladder. Functional role(s) for the VEGF-VEGF receptor system in urinary bladder inflammation remain to be determined.
Asunto(s)
Cistitis/metabolismo , Neuropilinas/metabolismo , Vejiga Urinaria/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Ciclofosfamida/farmacología , Cistitis/inducido químicamente , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Proteínas de la Membrana/genética , Músculo Liso/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Vejiga Urinaria/irrigación sanguínea , Vejiga Urinaria/efectos de los fármacos , Uroplaquina II , Urotelio/metabolismoRESUMEN
Among bone morphogenetic proteins (BMPs), the decapentaplegic (Dpp; BMP2, BMP4) and glass bottom boat (Gbb/60A; BMP5, BMP6, BMP7) subgroups have well-described functions guiding autonomic and sensory neuronal development, fiber formation and neurophenotypic identities. Evaluation of rat superior cervical ganglia (SCG) post-ganglionic sympathetic neuron developmental regulators identified that selected BMPs of the transforming growth factor beta superfamily have reciprocal effects on neuronal pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) expression. Dpp and Gbb/60A BMPs rapidly down-regulated PACAP expression, while up-regulating other sympathetic neuropeptides, including PACAP-related VIP. The suppressive effects of BMP on PACAP mRNA and peptide expression were potent, efficacious and phosphorylated mothers against decapentaplegic homolog (Smad) signaling-dependent. Axotomy of SCG dramatically increases PACAP expression, and the possibility that abrogation of inhibitory retrograde target tissue BMP signaling may contribute to this up-regulation of sympathetic neuron PACAP was investigated. Replacement of BMP6 to SCG explant preparations significantly blunted the injury-induced elevated PACAP expression, with a concomitant decrease in sympathetic PACAP-immunoreactive neuron numbers. These studies suggested that BMPs modulate neuropeptide identity and diversity by stimulating or restricting the expression of specific peptidergic systems. Furthermore, the liberation of SCG neurons from target-derived BMP inhibition following axotomy may be one participating mechanism associated with injury-induced neuropeptidergic plasticity.