RESUMO
The formation of neurites is an important process affecting the cognitive abilities of an organism. Neurite growth requires the addition of new membranes, but the metabolic remodeling necessary to supply lipids for membrane expansion is poorly understood. Here, we show that synaptic activity, one of the most important inducers of neurite growth, transcriptionally regulates the expression of neuronal glucose transporter Glut3 and rate-limiting enzymes of glycolysis, resulting in enhanced glucose uptake and metabolism that is partly used for lipid synthesis. Mechanistically, CREB regulates the expression of Glut3 and Siah2, the latter and LDH activity promoting the normoxic stabilization of HIF-1α that regulates the expression of rate-limiting genes of glycolysis. The expression of dominant-negative HIF-1α or Glut3 knockdown blocks activity-dependent neurite growth in vitro while pharmacological inhibition of the glycolysis and specific ablation of HIF-1α in early postnatal mice impairs the neurite architecture. These results suggest that the manipulation of neuronal glucose metabolism could be used to treat some brain developmental disorders.
Assuntos
Estruturas da Membrana Celular/metabolismo , Neuritos/metabolismo , Sinapses/metabolismo , Animais , Estruturas da Membrana Celular/genética , Estruturas da Membrana Celular/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 3/biossíntese , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glicólise/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Neuritos/patologia , Ratos , Ratos Sprague-Dawley , Sinapses/genética , Sinapses/patologia , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Hipocampo/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dendritos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Cultura Primária de Células , RatosRESUMO
Bone-resorbing osteoclasts significantly contribute to osteoporosis, and understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss. Here, we report that POLR2A is upregulated during osteoclastogenesis. Functional analyses showed that the inhibition of POLR2A decreased osteoclastogenesis, whereas the overexpression of POLR2A had completely opposite effects in vitro. Notably, the osteoclast-specific deletion of POLR2A blocks bone resorption in vivo. Furthermore, POLR2A loss-of-function suppresses estrogen deficiency-induced bone resorption. Mechanistically, POLR2A regulates the assembly of CREB1 on the regulatory elements of its target genes. Collectively, using genetic, pharmacological, and disease mouse models, we have identified a previously undescribed protein that interacts with CREB1 to regulate osteoclastic bone resorption.
Assuntos
Reabsorção Óssea/prevenção & controle , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , RNA Polimerases Dirigidas por DNA/metabolismo , Osteoporose/prevenção & controle , Animais , Reabsorção Óssea/patologia , RNA Polimerases Dirigidas por DNA/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/citologia , Osteogênese/fisiologia , Osteoporose/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Transcrição Gênica/genéticaRESUMO
BACKGROUND: Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation. METHODS: To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed. RESULTS: LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF. CONCLUSION: Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células Epiteliais/patologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Lisofosfolipídeos/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Proteínas do Tecido Nervoso , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC2) in the spinal cords of rats with peripheral nerve injury (PNI) in the lumbar 4-lumbar 5 (L4-L5) section. In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4-L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4-L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU- and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV-KCC2 pathway.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Região Lombossacral , Neuralgia/terapia , Transdução de Sinais , Simportadores/biossíntese , Terapia por Ultrassom/métodos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Hiperalgesia/fisiopatologia , Hiperalgesia/terapia , Região Lombossacral/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Neuralgia/patologia , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/terapia , Estimulação Física , Ratos , Ratos Sprague-Dawley , Cotransportadores de K e Cl-RESUMO
We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Regulação da Expressão Gênica , Alcaloides/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Cicloeximida/farmacologia , Células HEK293 , Humanos , Leupeptinas/farmacologia , Proteínas/genética , Proteínas/metabolismoRESUMO
OBJECTIVE: Activity-dependent changes have been reported in animal models and in human epileptic specimens and could potentially be used as tissue biomarkers to evaluate the propensity of a tissue to generate seizure activity. In this context, cAMP-response element binding protein (CREB) activation was specifically reported in human epileptic foci and related mainly to interictal spike activity. To get further insights into CREB activation in human epilepsy, we analyzed pCREB expression on brain tissue samples from patients who underwent surgery for drug-resistant focal epilepsy, correlating this expression with intracranial stereo-electroencephalography (SEEG) recording in a subgroup. METHODS: Neocortical specimens from patients with neuropathological diagnosis of no lesion (cryptogenic), malformations of cortical development,mainly type II focal cortical dysplasia (FCD), and hippocampi with and without hippocampal sclerosis have been analyzed by immunohistochemistry. Peritumoral cortex from non-epileptic patients and autoptic samples were used as controls, whereas rat brains were used to test possible loss of pCREB antigenicity due to fixation procedures and postmortem delay. RESULTS: pCREB was consistently expressed in layer II neuronal nuclei in regions with normal cortical lamination both in epileptic and non-epileptic surgical tissues. In patients with SEEG recordings, this anatomical pattern was unrelated to the presence of interictal spike activity. Conversely, in the core of type II FCD, as well as in other developmental malformations, pCREB was scattered without any laminar specificity. Furthermore, quantitative data did not reveal significant differences between epileptic and non-epileptic tissues, except for an increased immunoreactivity in the core of type IIB FCD lesion related mainly to reactive glial and balloon cells. SIGNIFICANCE: The present data argue against the reliability of pCREB immunohistochemistry as a marker of epileptic focus but underscores its layer-related expression, suggesting a potential application in the study of malformations of cortical development, a wide range of diseases arising from perturbations of normal brain development.
Assuntos
Encéfalo/metabolismo , Encéfalo/cirurgia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Epilepsia Resistente a Medicamentos/metabolismo , Epilepsia Resistente a Medicamentos/cirurgia , Adolescente , Adulto , Idoso , Animais , Encéfalo/patologia , Pré-Escolar , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Epilepsia Resistente a Medicamentos/genética , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Técnicas EstereotáxicasRESUMO
The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.
Assuntos
Hormônio Liberador da Corticotropina/biossíntese , Hipófise/metabolismo , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Estresse Psicológico/metabolismo , Doença Aguda , Animais , Hormônio Liberador da Corticotropina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Psicológico/psicologiaRESUMO
In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3ß/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3ß, p-GSK-3ß, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3ß. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3ß, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3ß/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Glicogênio Sintase Quinase 3 beta/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Rifampina/farmacologia , Rotenona/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
CREB3 is an ER membrane-bound transcription factor; however, post-translational regulation of CREB3, including expression, processing, and activation, is not fully characterized. We therefore constructed several types of mouse CREB3 expression genes and elucidated their expression in Neuro2a cells by treatment with stimuli and co-transfection with genes associated with ER-Golgi homeostasis, such as mutant Sar1 [H79G], GRP78, and KDEL receptor 1 (KDELR1). Interestingly, treatment of Neuro2a cells expressing Flag-tagged full-length CREB3 with monensin and nigericin induced the expression of the approximately 50 kDa N-terminal fragment; however, its cleavage was not parallel to the levels of GADD153 and LC3-II. Co-transfection of full-length CREB3 together with Sar1 [H79G], GRP78, or KDELR1 showed that only Sar1 [H79G] induced expression of the cleaved form, and KDELR1 dramatically decreased the expression of the full-length form. Accordingly, Sar1 [H79G]- and KDELR1-overexpression influenced GAL4-CREB3-dependent luciferase activities. To understand the activation of CREB3 under more pathophysiological conditions, we focused on the effect of metal ions on CREB3 cleavage in Neuro2a cells. Among the six metal ions we tested, only copper ion stabilized full-length CREB3 expression. Copper ion also increased its N-terminal form and GAL4-CREB3-dependent luciferase activity, which was accompanied by the increase in the ubiquitinated proteins in Neuro2a cells. Taken together, CREB3 expression is regulated by multiple ER-Golgi resident factors in a post-translational manner, but its processing is not directly associated with ER stress and autophagic dysfunction. This finding is especially true for the unique action of the copper ion on CREB3 stabilization and processing in parallel to aberration of ubiquitin-proteasome system, which might provide new insights into understanding the mechanisms of intractable disorders.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Complexo de Golgi/genética , Complexo de Golgi/patologia , CamundongosRESUMO
Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Núcleo Inferior Caudal do Nervo Trigêmeo/efeitos dos fármacos , Gânglio Trigeminal/efeitos dos fármacos , Animais , Dura-Máter/efeitos dos fármacos , Masculino , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/fisiopatologia , Nitroglicerina/toxicidade , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Gânglio Trigeminal/metabolismo , Regulação para Cima , Vasodilatadores/toxicidadeRESUMO
BACKGROUND: About 2% of pregnant women receive non-obstetric surgery under general anesthesia each year. During pregnancy, general anesthetics may affect brain development of the fetus. This study aimed to investigate safe dosage range of isoflurane. METHODS: Forty-eight SpragueDawley (SD) pregnant rats were randomly divided into 3 groups and inhaled 1.3% isoflurane (the Iso1 group), 2.0% isoflurane (the Iso2 group) and 50% O2 alone (the control group) for 3 h, respectively. Their offsprings were subjected to Morris water maze at day 28 and day 90 after birth to evaluate learning and memory. The expression of cAMP-response element binding protein (CREB) and phosphorylated cAMP-response element binding protein (p-CREB) was detected in the hippocampus dentate gyrus. RESULTS: Less offsprings of Iso2 group were able to cross the platform than that of the control group (P < 0.05). Accordingly, the Iso2 offsprings expressed p-CREB mainly in the subgranular zone in contrast to the whole granular cell layer of hippocampus dentate gyrus as detected in the Iso1 and control offsprings; the expression level of pCREB was also lower in the Iso2 than Iso1 or control offsprings (P < 0.05). CONCLUSION: Inhalation of isoflurane at 1.3% during pregnancy has no significant influence on learning and memory of the offspring; exposure to isoflurane at 2.0% causes damage to spatial memory associated with inhibition of CREB phosphorylation in the granular cell layer of hippocampus dentate gyrus.
Assuntos
Isoflurano/efeitos adversos , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/psicologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Giro Denteado/metabolismo , Relação Dose-Resposta a Droga , Feminino , Masculino , Fosforilação/efeitos dos fármacos , Gravidez , RatosRESUMO
AIMS: Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes. METHODS AND RESULTS: We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. CONCLUSIONS: LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes.
Assuntos
Lipoproteínas LDL/sangue , Monócitos/metabolismo , Receptor 4 Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Citocinas/biossíntese , Citocinas/genética , Elafina/genética , Humanos , Lipoproteínas LDL/biossíntese , NF-kappa B/biossíntese , NF-kappa B/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
BACKGROUND: Tisp40, a transcription factor of the CREB/CREM family, is involved in cell proliferation, differentiation and other biological functions, but its role in renal tubulointerstitial fibrosis is unknown. METHODS: In our study, we investigated the effects of Tisp40 on extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT) and the underlying molecular mechanisms in transforming growth factor-ß (TGF-ß)-stimulated TCMK-1 cells by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence in vitro, and further explored the role of Tisp40 on renal fibrosis induced by ischemia-reperfusion (I/R) by qPCR, Western blot analysis, hydroxyproline analysis, Masson trichrome staining and immunohistochemistry staining in vivo. RESULTS: The data showed that Tisp40 was upregulated in a model of renal fibrosis induced by I/R injury (IRI). Upon IRI, Tisp40-deficient mice showed attenuated renal fibrosis compared with wild-type mice. Furthermore, the expression of α-smooth muscle actin, E-cadherin, fibronectin, and collagen I was suppressed. Tisp40 overexpression aggravated ECM accumulation and EMT in the TGF-ß-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfering RNA (siRNA) targeting Tisp40. Importantly, it is changes in the Smad pathway that attenuate renal fibrosis. CONCLUSION: These findings suggest that Tisp40 plays a critical role in the TGF-ß/ Smads pathway involved in this process. Hence, Tisp40 could be a useful therapeutic target in the fight against renal tubulointerstitial fibrosis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fibrose/genética , Nefrite Intersticial/genética , Fator de Crescimento Transformador beta/genética , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Transição Epitelial-Mesenquimal/genética , Fibrose/patologia , Regulação da Expressão Gênica/genética , Humanos , Rim/metabolismo , Rim/patologia , Camundongos , Nefrite Intersticial/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteínas Smad/biossíntese , Fator de Crescimento Transformador beta/metabolismoRESUMO
Breast cancer is the most frequent cancer among women worldwide. Tumor immunology suggests relationships between the immune system, chronic inflammation, and cancer. The immune system may either prevent or promote carcinogenesis. Here, we evaluated molecular signaling pathways common in inflammation and cancer and detected the microRNAs which play pivotal roles in mediating these pathways. Using bioinformatics assays, signaling pathways common in inflammation and cancer, and microRNAs mediating these pathways were identified. MiR-590 was selected and cloned into the pLenti-III-eGFP vector and transfected into the breast cancer cell lines. The expression level of microRNA and the candidate genes was evaluated by real-time quantitative reverse transcription polymerase chain reaction, and the apoptosis level in transfected cells was measured by Annexin V-7AAD assay. The cell migration was tested by real-time quantitative reverse transcription polymerase chain reaction for MMP2/MMP9. The expression levels of miR-590 and the selected genes (i.e. JAK2, PI3K, MAPK1, and CREB) were measured 72 h after transfection. While miR-590 showed an over-expression, the genes were significantly down-regulated. A significant increase was observed in apoptosis level in both cell lines and MMP2/MMP9 was significantly decreased in MDA-MB-231 cells. MiR-590 was selected as a microRNA which triggers and down-regulates critical genes of signaling pathways similar in cancer and inflammation. Following the miR-590 treatment, JAK2, PI3K, MAPK1, and CREB were down-regulated and the apoptosis level was increased in breast cancer cell lines. Apparently, some microRNAs can be good candidates for novel treatments of cancer. Although miR-590 showed good results in this study, further studies are required to investigate the role of miR-590 in breast cancer therapy.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Inflamação/genética , MicroRNAs/genética , Apoptose/genética , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/patologia , Janus Quinase 2/biossíntese , Células MCF-7 , MicroRNAs/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Transdução de Sinais , TransfecçãoRESUMO
Glioma is stemmed from the glial cells in the brain, which is accounted for about 45% of all intracranial tumors. The characteristic of glioma is invasive growth, as well as there is no obvious boundary between normal brain tissue and glioma tissue, so it is difficult to resect completely with worst prognosis. The metabolism of glioma is following the Warburg effect. Previous researches have shown that GLUT1, as a glucose transporter carrier, affected the Warburg effect, but the molecular mechanism is not very clear. CREB1 (cAMP responsive element-binding protein1) is involved in various biological processes, and relevant studies confirmed that CREB1 protein regulated the expression of GLUT1, thus mediating glucose transport in cells. Our experiments mainly reveal that the CREB1 could affect glucose transport in glioma cells by regulating the expression of GLUT1, which controlled the metabolism of glioma and affected the progression of glioma.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Glucose/metabolismo , Proteínas de Neoplasias/biossíntese , Linhagem Celular Tumoral , HumanosRESUMO
Spermatogenic cells express cell-specific molecules with the potential to be seen as "foreign" by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway-specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.
Assuntos
Proteínas I-kappa B/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , NF-kappa B/antagonistas & inibidores , Testículo/imunologia , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Inibidor de NF-kappaB alfa , Poli I-C , Ratos , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Testículo/citologia , Receptores Toll-Like/imunologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections, macrophages are an important source of IL-10; however, the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. In this study, bone marrow-derived macrophages exposed to excretory/secretory products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Moreover, skin-resident tissue macrophages, which encounter S. mansoni excretory/secretory products during infection, are the first monocytes to produce IL-10 in vivo early postinfection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site, and we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Interleucina-10/biossíntese , Macrófagos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteínas de Ligação a DNA/imunologia , Metabolismo Energético/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-10/genética , Subunidade p35 da Interleucina-12/biossíntese , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Calcineurina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Longevidade/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Inibidores de Calcineurina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Regulação para Baixo , Metabolismo Energético , Ativação Enzimática , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Longevidade/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/química , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
Recent studies focus on promoting neurite outgrowth to remodel the central nervous network after brain injury. Currently, however, there are few drugs treating brain diseases in the clinic by enhancing neurite outgrowth. In this study, we established an NGF-induced PC12 differentiation model to screen novel compounds that have the potential to induce neuronal differentiation, and further characterized 4,10-Aromadendranediol (ARDD) isolated from the dried twigs of the Baccharis gaudichaudiana plant, which exhibited the capability of promoting neurite outgrowth in neuronal cells in vitro. ARDD (1, 10 µmol/L) significantly enhanced neurite outgrowth in NGF-treated PC12 cells and N1E115 cells in a time-dependent manner. In cultured primary cortical neurons, ARDD (5, 10 µmol/L) not only significantly increased neurite outgrowth but also increased the number of neurites on the soma and the number of bifurcations. Further analyses showed that ARDD (10 µmol/L) significantly increased the phosphorylation of ERK1/2 and the downstream GSK-3ß, subsequently induced ß-catenin expression and up-regulated the gene expression of the Wnt ligands Fzd1 and Wnt3a in neuronal cells. The neurite outgrowth-promoting effect of ARDD in neuronal cells was abolished by pretreatment with the specific ERK1/2 inhibitor PD98059, but was partially reversed by XAV939, an inhibitor of the Wnt/ß-catenin pathway. ARDD also increased the expression of BDNF, CREB and GAP-43 in N1E115 cells, which was reversed by pretreatment with PD98059. In N1E115 cells subjected to oxygen and glucose deprivation (OGD), pretreatment with ARDD (1-10 µmol/L) significantly enhanced the phosphorylation of ERK1/2 and induced neurite outgrowth. These results demonstrated that the natural product ARDD exhibits neurite outgrowth-inducing activity in neurons via activation of the ERK signaling pathway, which may be beneficial to the treatment of brain diseases.