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1.
PLoS One ; 14(6): e0217519, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31163038

RESUMO

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor (SSRI), has been shown to exhibit other mechanisms of action in various cell types. Consequently, it might have unexpected adverse effects not related to its intended use, possibly in the endocrine regulation of reproduction. We show in the present report that after a 1-hour preincubation of MLTC-1 Leydig cells with FLX, the intracellular cyclic adenosine monophosphate (cAMP) responses to Luteinizing Hormone (LH) and forskolin (FSK) are reduced through AMPK-dependent and -independent pathways respectively. FLX at low concentrations (12.5µM and 25µM) induced this inhibition without triggering AMPK phosphorylation, while higher FLX concentrations (50µM and 100µM) induced AMPK phosphorylation and lowered ATP concentration similarly to Metformin. Pretreatment with the specific AMPK inhibitor Compound C (CpdC), significantly diminished the inhibition of cAMP synthesis caused by high concentration of FLX. Moreover, as expected FLX also caused a decline of steroidogenesis which is under the control of cAMP. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent and occurs in MLTC-1 cells through two mechanisms, AMPK-independent and AMPK-dependent, at low and high concentrations, respectively. FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells and mouse testicular Leydig cells, suggesting similar mechanisms in both cell types.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , AMP Cíclico/biossíntese , Fluoxetina/farmacologia , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/farmacologia , Animais , Linhagem Celular , Colforsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células Intersticiais do Testículo/citologia , Masculino , Metformina/farmacologia , Camundongos
2.
J Pharmacol Exp Ther ; 370(1): 104-110, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068382

RESUMO

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.


Assuntos
Adenilil Ciclases/metabolismo , Brônquios/citologia , AMP Cíclico/biossíntese , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/citologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Humanos , Isoproterenol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos
3.
Eur J Pharmacol ; 853: 308-315, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30980797

RESUMO

Microbiota produce short chain fatty acids (SCFAs), which are known to maintain gut homeostasis, by the fermentation of dietary fiber in the human colon. Among SCFAs, butyrate has been considered as the most physiologically effective SCFA in colorectal epithelial cells for growth and differentiation. Here we show that the E-type prostanoid 4 (EP4) receptor expression level is regulated by different concentrations of butyrate, but not by other SCFAs, in human colon cancer HCA-7 cells, through sodium-coupled monocarboxylate transporter-1 (SMCT-1)-mediated uptake followed by the activation of histone acetyltransferase: cAMP response element binding protein-binding protein/p300. Of particular interest, the prostanoid EP4 receptors are known to be expressed in normal colorectal crypt epithelial cells and maintain intestinal homeostasis by preserving mucosal integrity, while they are also known to be involved in the early stage of carcinogenesis. Thus, the links between butyrate and the expression of prostanoid EP4 receptors are both important factors for maintaining homeostasis. Based on in silico analysis, almost half of colorectal cancer tissues have lost the expression of SMCT-1 mRNA when compared with healthy corresponding tissues. Therefore, with the collapse of homeostasis systems such as a decrease in the concentration of butyrate in colorectal tissues, or reduced butyrate uptake, there is a possibility of early stage colorectal cancer development; the transformation of normal cells to the cancerous phenotype may be due to the overexpression of prostanoid EP4 receptors followed by excessive cyclooxygenase-2 induction, which are caused by a reduced amount of butyrate and/or its uptake, in/around colorectal epithelial cells.


Assuntos
Butiratos/metabolismo , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Proliferação de Células , AMP Cíclico/biossíntese , Proteína p300 Associada a E1A/metabolismo , Indução Enzimática , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Sialoglicoproteínas/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30844417

RESUMO

Epilepsy is marked by seizures that are a manifestation of excessive brain activity and is symptomatically treatable by anti-epileptic drugs (AEDs). Unfortunately, the older AEDs have many side effects, with cognitive impairment being a major side effect that affects the daily lives of people with epilepsy. Thus, this study aimed to determine if newer AEDs (Zonisamide, Levetiracetam, Perampanel, Lamotrigine and Valproic Acid) also cause cognitive impairment, using a zebrafish model. Acute seizures were induced in zebrafish using pentylenetetrazol (PTZ) and cognitive function was assessed using the T-maze test of learning and memory. Neurotransmitter and gene expression levels related to epilepsy as well as learning and memory were also studied to provide a better understanding of the underlying processes. Ultimately, impaired cognitive function was seen in AED treated zebrafish, regardless of whether seizures were induced. A highly significant decrease in γ-Aminobutyric Acid (GABA) and glutamate levels was also discovered, although acetylcholine levels were more variable. The gene expression levels of Brain-Derived Neurotrophic Factor (BDNF), Neuropeptide Y (NPY) and Cyclic Adenosine Monophosphate (CAMP) Responsive Element Binding Protein 1 (CREB-1) were not found to be significantly different in AED treated zebrafish. Based on the experimental results, a decrease in brain glutamate levels due to AED treatment appears to be at least one of the major factors behind the observed cognitive impairment in the treated zebrafish.


Assuntos
Anticonvulsivantes/efeitos adversos , Disfunção Cognitiva/induzido quimicamente , Pentilenotetrazol , Convulsões/tratamento farmacológico , Convulsões/psicologia , Peixe-Zebra/fisiologia , Acetilcolina/metabolismo , Animais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Disfunção Cognitiva/complicações , AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Interações de Medicamentos , Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Locomoção/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Neuropeptídeo Y/biossíntese , Convulsões/induzido quimicamente , Convulsões/complicações , Ácido gama-Aminobutírico/metabolismo
5.
Mol Cell Endocrinol ; 490: 15-20, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30922932

RESUMO

The steroidogenic acute regulatory protein (STAR) governs the rate-limiting step in steroidogenesis, and its expression varies depending on the needs of the specific tissue. It is well known that tight control of steroid production is essential for multiple processes involved in reproduction. We recently showed that Star is regulated at the posttranscriptional level in vitro by H19 and let-7. Here we demonstrate that this novel regulatory mechanism is functional in vivo, regulated by cAMP, and that loss of H19 not only disrupts ovarian STAR but also results in altered progesterone production in an H19KO mouse model. This work further strengthens the possibility that noncoding-RNA-mediated regulation of STAR may play an important role in the regulation of steroid hormone production, and contributes further to our understanding of the many ways in which this important gene is regulated.


Assuntos
Ovário/metabolismo , Fosfoproteínas/metabolismo , Progesterona/biossíntese , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular Tumoral , AMP Cíclico/biossíntese , Feminino , Humanos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética
6.
Anal Bioanal Chem ; 411(12): 2493-2509, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30911800

RESUMO

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.


Assuntos
Antraz/patologia , Antígenos de Bactérias/sangue , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Infecções Respiratórias/patologia , Espectrometria de Massas em Tandem/métodos , Toxemia/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antraz/sangue , Estudos de Casos e Controles , AMP Cíclico/biossíntese , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Humanos , Limite de Detecção , Macaca mulatta , Reação em Cadeia da Polimerase , Infecções Respiratórias/sangue , Toxemia/sangue , Toxemia/microbiologia
7.
Mol Cell Endocrinol ; 486: 96-104, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30853600

RESUMO

Severe inflammation in the islets is observed in obese patients with type 2 diabetes. Inflammation in the islets is caused by obesity-induced serum free fatty acids. Asprosin is a fasting-induced adipokine, which contributes to hepatic glucose production. However, the effects of asprosin on inflammation and cellular dysfunction in pancreatic ß-cells remain to be elucidated. Here, we demonstrated that treatment of mouse insulinoma MIN6 cells and human primary islets containing ß-cells with palmitate increased asprosin expression and secretion. Treatment of MIN6 cells and human primary islets with palmitate increased phosphorylation of the inflammatory marker nuclear factor-kappa B (NFκB) and the release of pro-inflammatory cytokines including TNF and MCP-1 and decreased glucose-stimulated insulin secretion and cell viability. However, siRNA-mediated suppression of asprosin reversed these changes. Recombinant asprosin treatment of MIN6 cells and human primary islets augmented the inflammation response, cellular dysfunction, and apoptosis in a dose-dependent manner. Asprosin induced toll-like receptor (TLR) 4 expression and JNK phosphorylation. siRNA for TLR4 or JNK mitigated the effects of asprosin on inflammation and cellular dysfunction. These results suggest that palmitate-derived asprosin secretion from ß-cells results in their inflammation and dysfunction through a TLR4/JNK-mediated pathway. This report suggests asprosin as a novel therapeutic target for the treatment of type 2 diabetes through preservation of ß-cell function.


Assuntos
Glucose/toxicidade , Inflamação/metabolismo , Inflamação/patologia , Secreção de Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Fragmentos de Peptídeos/metabolismo , Hormônios Peptídicos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/biossíntese , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Palmitatos/toxicidade , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Neuropharmacology ; 148: 347-357, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30710569

RESUMO

Spinal cord injury results in sensation dysfunction. This study explored miR-142-3p, which acts a critical role in sciatic nerve conditioning injury (SNCI) promoting the repair of the dorsal column injury and validated its function on primary sensory neuron(DRG). miR-142-3p expression increased greatly in the spinal cord dorsal column lesion (SDCL) group and increased slightly in the SNCI group. Subsequently, the expression of adenylate cyclase 9 (AC9), the target gene of miR-142-3p, declined sharply in the SDCL group and declined limitedly in the SNCI group. The expression trend of cAMP was opposite to that of miR-142-3p. MiR-142-3p inhibitor improved the axon length, upregulated the expression of AC9, cAMP, p-CREB, IL-6, and GAP43, and downregulated the expression of GTP-RhoA. miR-142-3p inhibitor combined with AC9 siRNA showed shorter axon length, the expression of AC9, cAMP, p-CREB, IL-6, and GAP43 was decreased, and the expression of GTP-RhoA was increased. H89 and AG490, inhibitors of cAMP/PKA pathway and IL6/STAT3/GAP43 axis, respectively, declined the enhanced axonal growth by miR-142-3p inhibitor and altered the expression level of the corresponding proteins. Thus, a substitution therapy using Sorafenib that downregulates the miR-142-3p expression for SNCI was investigated. The results showed the effect of Sorafenib was similar to that of miR-142-3p inhibitor and SNCI on both axon growth in vitro and sensory conduction function recovery in vivo. In conclusion, miR-142-3p acts a pivotal role in SNCI promoting the repair of dorsal column injury. Sorafenib mimics the treatment effect of SNCI via downregulation of miR-142-3p, subsequently, promoting sensory conduction function recovery post dorsal column injury.


Assuntos
Adenilil Ciclases/fisiologia , AMP Cíclico/fisiologia , MicroRNAs/fisiologia , Sensação/efeitos dos fármacos , Sorafenibe/farmacologia , Traumatismos da Medula Espinal/fisiopatologia , Adenilil Ciclases/biossíntese , Animais , AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Proteína GAP-43/biossíntese , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/biossíntese , Interleucina-6/biossíntese , Isoquinolinas/farmacologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Rodaminas , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismo , Sulfonamidas/farmacologia , Tirfostinas/farmacologia , Regulação para Cima/efeitos dos fármacos
9.
J Biol Chem ; 294(4): 1095-1103, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30559293

RESUMO

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.


Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/biossíntese , Ativação Enzimática/efeitos da radiação , Luminescência , Animais , Proliferação de Células , Células Cultivadas , Células HEK293 , Humanos , Luz , Luciferases/metabolismo , Ratos
10.
PLoS One ; 13(11): e0205195, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30383775

RESUMO

Treprostinil is applied for pulmonary arterial hypertension (PAH) therapy. However, the mechanism by which the drug achieves its beneficial effects in PAH vessels is not fully understood. This study investigated the effects of treprostinil on PDGF-BB induced remodelling parameters in isolated human pulmonary arterial smooth muscle cells (PASMC) of four PAH patients. The production of TGF-ß1, CTGF, collagen type-I and -IV, and of fibronectin were determined by ELISA and PCR. The role of cAMP was determined by ELISA and di-deoxyadenosine treatment. Proliferation was determined by direct cell count. Treprostinil increased cAMP levels dose and time dependently, which was not affected by PDGF-BB. Treprostinil significantly reduced PDGF-BB induced secretion of TGF-ß1 and CTGF, both was counteracted when cAMP generation was blocked. Similarly, the PDGF-BB induced proliferation of PASMC was dose dependently reduced by treprostinil through signalling via cAMP-C/EBP-α p42 -p21(WAf1/Cip1). In regards to extracellular matrix remodelling, treprostinil significantly reduced PDGF-BB-TGF-ß1-CTGF induced synthesis and deposition of collagen type I and fibronectin, in a cAMP sensitive manner. In contrast, the deposition of collagen IV was not affected. The data suggest that this action of treprostinil in vessel wall remodelling may benefit patients with PAH and may reduce arterial wall remodelling.


Assuntos
Becaplermina/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Epoprostenol/análogos & derivados , Hipertensão Pulmonar Primária Familiar/tratamento farmacológico , Fator de Crescimento Transformador beta1/genética , Adulto , Becaplermina/sangue , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo IV/genética , Fator de Crescimento do Tecido Conjuntivo/sangue , AMP Cíclico/biossíntese , Epoprostenol/administração & dosagem , Matriz Extracelular/efeitos dos fármacos , Hipertensão Pulmonar Primária Familiar/sangue , Hipertensão Pulmonar Primária Familiar/genética , Hipertensão Pulmonar Primária Familiar/patologia , Feminino , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/sangue , Remodelação Vascular/efeitos dos fármacos
11.
Biochem Biophys Res Commun ; 506(3): 543-547, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30366671

RESUMO

Adenylate cyclase 7 (AC7) has been reported to participate in various biological processes during cancer progression. However, the roles of AC7 in all-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) cells are still unknown. In this study, firstly, our results showed that AC7 affected intracellular cAMP level and influenced ATRA-induced differentiation of APL cells. Secondly, we revealed that miR-192 could directly target AC7 expression and knockdown of miR-192 promoted ATRA-induced APL cell differentiation by regulating AC7 expression. Furthermore, we found that AC7 expression was lower in patients with relapsed APL than that in patients with newly diagnosed APL, while miR-192 expression was relatively higher in patients with relapsed APL. Taken together, our results show that miR-192-mediated AC7 could play important roles in differentiation of APL cells, AC7 and miR-192 might be new biomarkers and therapeutic targets for patients with relapsed APL.


Assuntos
Adenilil Ciclases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , MicroRNAs/metabolismo , Tretinoína/farmacologia , Adenilil Ciclases/genética , Sequência de Bases , Linhagem Celular Tumoral , AMP Cíclico/biossíntese , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Leucemia Promielocítica Aguda/diagnóstico , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recidiva , Regulação para Cima/efeitos dos fármacos
12.
Front Immunol ; 9: 919, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29765373

RESUMO

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the ß-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.


Assuntos
Adenilil Ciclases/metabolismo , Bacillus anthracis/enzimologia , Bordetella pertussis/enzimologia , AMP Cíclico/biossíntese , Sinapses Imunológicas/metabolismo , Linfócitos T/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Compartimento Celular/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Ativação Linfocitária , Transdução de Sinais/imunologia
14.
J Cell Physiol ; 233(9): 7226-7238, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29574744

RESUMO

Numerous studies have demonstrated that Gram-positive microbiomes play an important role in the pathogenesis and in the way of treatment of chronic rhinosinusitis (CRS). Kinins are inflammatory mediators and one of their receptors, namely bradykinin receptor 1 (BKR1 or B1R), is believed to be induced and involved in inflammation in pathophysiological conditions. In the present study, we investigated the effect of peptidoglycan (PGN), a major cell wall component of G(+) bacteria, on BKR expression and its signaling pathway in nasal fibroblasts from CRS without nasal polyp (CRSsNP). The PGN induced increases in B1R mRNA and protein production. The induction was abolished by the NF-κB and protein kinase A inhibitor. In parallel, the PGN treatment directly activated IκB/NF-κB signaling and CREB phosphorylation. Interestingly, a further analysis suggested no involvement of cAMP/PKA/CREB pathway in this induction. The B1R expression and IκB/NF-κB signaling pathway could be attenuated by Toll-like receptor-2 (TLR2) blocking/neutralizing Ab. In a functional assay, the addition of B1R selective agonist (Des-Arg10 -kallidin) to the fibroblasts after PGN stimulation led to an increase in CXCL8 release and p38 MAPK and ERK1/2 phosphorylation, which could be inhibited by the B1R antagonist. Taken together, our results revealed for the first time that PGN can increase B1R expression in human nasal mucosa-derived fibroblasts through TLR2 activation and NF-κB signaling pathway. This induction functionally leads to MAPKs activation and CXCL8 release upon B1R stimulation. Our results also suggest that a major component of G(+) bacteria can participate in B1R upregulation in nasal mucosa during CRSsNP progression.


Assuntos
Fibroblastos/metabolismo , NF-kappa B/metabolismo , Mucosa Nasal/patologia , Peptidoglicano/farmacologia , Receptor B1 da Bradicinina/metabolismo , Rinite/patologia , Sinusite/patologia , Receptor 2 Toll-Like/metabolismo , Doença Crônica , AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor B1 da Bradicinina/genética , Transdução de Sinais/efeitos dos fármacos
15.
J Mol Endocrinol ; 60(3): 213-224, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29535183

RESUMO

Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4 Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Colo/metabolismo , AMP Cíclico/biossíntese , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Humanos , Insulina/genética , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo
16.
J Med Chem ; 61(7): 3218-3223, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29528634

RESUMO

Glucagon-like peptide 2 (GLP-2) is a hormone that has been shown to stimulate intestinal growth and attenuate intestinal inflammation. Despite being efficacious in a variety of animal models of disease, its therapeutic potential is hampered by the short half-life in vivo. We now describe a highly potent, stapled long-acting GLP-2 analog, peptide 10, that has a more than 10-fold longer half-life than teduglutide and improved intestinotrophic and anti-inflammatory effects in mouse models of DSS-induced colitis.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colite/tratamento farmacológico , Fármacos Gastrointestinais/farmacologia , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacocinética , Colite/induzido quimicamente , Reagentes para Ligações Cruzadas , AMP Cíclico/biossíntese , Sulfato de Dextrana , Desenho de Drogas , Feminino , Fármacos Gastrointestinais/síntese química , Fármacos Gastrointestinais/farmacocinética , Peptídeo 2 Semelhante ao Glucagon/síntese química , Peptídeo 2 Semelhante ao Glucagon/farmacocinética , Meia-Vida , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Peptídeos/farmacocinética , Peptídeos/farmacologia
17.
Arthritis Res Ther ; 20(1): 39, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490676

RESUMO

BACKGROUND: Prostaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD). METHODS: CR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA. RESULTS: CR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate. CONCLUSIONS: CR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Animais , Antirreumáticos/metabolismo , Artrite Reumatoide/metabolismo , AMP Cíclico/biossíntese , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Células HEK293 , Humanos , Macrófagos/metabolismo , Masculino , Metotrexato/uso terapêutico , Camundongos Endogâmicos DBA , Ensaio Radioligante/métodos , Ratos Endogâmicos Lew , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células THP-1 , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
J Neurochem ; 145(6): 436-448, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29500819

RESUMO

The indirect pathway striatal medium spiny projection neurons (iMSNs) are critical to motor and cognitive brain functions. These neurons express a high level of cAMP-increasing adenosine A2a receptors. However, the potential effects of cAMP production on iMSN spiking activity have not been established, and recording identified iMSNs in freely moving animals is challenging. Here, we show that in the transgenic mice expressing cAMP-producing G protein Gs -coupled designer receptor exclusively activated by designer drug (Gs-DREADD) in iMSNs, the baseline spike firing in MSNs is normal, indicating DREADD expression does not affect the normal physiology of these neurons. Intraperitoneal injection of the DREADD agonist clozapine-N-oxide (CNO; 2.5 mg/kg) increased the spike firing in 50% of the recorded MSNs. However, CNO did not affect MSN firing in Gs-DREADD-negative mice. We also found that CNO injection inhibited the spike firing of globus pallidus external segment (GPe) neurons in Gs-DREADD-positive mice, further indicating CNO excitation of iMSNs. Temporally coincident with these effects on spiking firing in the indirect pathway, CNO injection selectively inhibited locomotion in D2 Gs-DREADD mice. Taken together, our results strongly suggest that cAMP production in iMSNs can increase iMSN spiking activity and cause motor inhibition, thus addressing a long-standing question about the cellular functions of the cAMP-producing adenosine A2a receptors in iMSNs. Cover Image for this issue: doi: 10.1111/jnc.14181.


Assuntos
Corpo Estriado/metabolismo , AMP Cíclico/biossíntese , Globo Pálido/fisiologia , Vias Neurais/metabolismo , Neurônios/fisiologia , Núcleo Subtalâmico/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Clozapina/farmacologia , Corpo Estriado/citologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso
19.
Neuropsychopharmacology ; 43(7): 1481-1491, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29463911

RESUMO

Current antidepressant therapies meet with variable therapeutic success and there is increasing interest in therapeutic approaches not based on monoamine signaling. Histone deacetylase 6 (HDAC6), which also deacetylates α-tubulin shows altered expression in mood disorders and HDAC6 knockout mice mimic traditional antidepressant treatments. Nonetheless, a mechanistic understanding for HDAC6 inhibitors in the treatment of depression remains elusive. Previously, we have shown that sustained treatment of rats or glioma cells with several antidepressants translocates Gαs from lipid rafts toward increased association with adenylyl cyclase (AC). Concomitant with this is a sustained increase in cAMP production. While Gαs modifies microtubule dynamics, tubulin also acts as an anchor for Gαs in lipid-rafts. Since HDAC-6 inhibitors potentiate α-tubulin acetylation, we hypothesize that acetylation of α-tubulin disrupts tubulin-Gαs raft-anchoring, rendering Gαs free to activate AC. To test this, C6 Glioma (C6) cells were treated with the HDAC-6 inhibitor, tubastatin-A. Chronic treatment with tubastatin-A not only increased α-tubulin acetylation but also translocated Gαs from lipid-rafts, without changing total Gαs. Reciprocally, depletion of α-tubulin acetyl-transferase-1 ablated this phenomenon. While escitalopram and imipramine also disrupt Gαs/tubulin complexes and translocate Gαs from rafts, they evoke no change in tubulin acetylation. Finally, two indicators of downstream cAMP signaling, cAMP response element binding protein phosphorylation (pCREB) and expression of brain-derived-neurotrophic-factor (BDNF) were both elevated by tubastatin-A. These findings suggest HDAC6 inhibitors show a cellular profile resembling traditional antidepressants, but have a distinct mode of action. They also reinforce the validity of antidepressant-induced Gαs translocation from lipid-rafts as a biosignature for antidepressant response that may be useful in the development of new antidepressant compounds.


Assuntos
Antidepressivos Tricíclicos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Inibidores de Captação de Serotonina/farmacologia , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Linhagem Celular Tumoral , Citalopram/farmacologia , AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácidos Hidroxâmicos/farmacologia , Imipramina/farmacologia , Indóis/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos
20.
Mol Microbiol ; 108(3): 258-275, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29453849

RESUMO

Candida albicans is a major human fungal pathogen, causing superficial, as well as life-threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast-to-hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine-induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S-adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino-propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine-induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP-PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.


Assuntos
Candida albicans/metabolismo , Metionina/metabolismo , Adenilil Ciclases/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , AMP Cíclico/biossíntese , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Macrófagos/microbiologia , Morfogênese/fisiologia , Transdução de Sinais , Virulência/fisiologia , Fatores de Virulência/metabolismo
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