RESUMO
Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Canais de Cátion TRPM , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Ciclo-Oxigenase 2/genética , Canais de Cátion TRPM/genética , Leucócitos Mononucleares/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inflamação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Occupational and environmental exposure of various toxins or cigarette smoke causes non-small cell lung carcinoma (NSCLC); a devastating disease with a very low survival rate after metastasis. Increased activity of plasmin is a hallmark in NSCLC metastasis. It is accepted that metastatic cells exhibit higher plasmin activity than cells from primary tumors. Mechanisms behind this elevation, however, are barely understood. We compared plasmin activity and cell migration of A549 cells derived from a primary lung tumor with metastatic H1299 lung cells isolated from lymph nodes. Surprisingly, we found higher plasmin activity and migration for A549 cells. mRNA levels of the plasminogen activator inhibitor-1 (PAI-1) were higher in H1299 cells and activity of extracellular-regulated kinases-1/2 (ERK-1/2) was increased. An inhibitor of ERK-1/2 decreased PAI-1 mRNA levels and increased plasmin activity or cell migration in H1299 cells. Transforming growth factor-ß (TGF-ß) decreased plasmin activity and migration in A549 cells but enhanced both in H1299 cells. The cytokine massively increased PAI-1 and decreased urokinase plasminogen activator (uPA) levels in A549 cells but strongly induced uPA and only weakly PAI- 1 expression in H1299 cells. Consequently, TGF-ß enhanced plasmin activity and cell migration in H1299. Additionally, TGF-ß activated ERK-1/2 stronger in H1299 than in A549 cells. Accordingly, an ERK-1/2 inhibitor completely reversed the effects of TGF-ß on uPA expression, plasmin activity and migration in H1299 cells. Hence, we provide first data indicating TGF-ß-promoted increased plasmin activity and suggest that blocking TGF-ß-promoted ERK-1/2 activity might be a straightforward approach to inhibit NSCLC metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fibrinolisina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Neoplasias Pulmonares/patologia , Movimento Celular , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Magnesium is an essential mediator of a vast number of critical enzymatic cellular reactions in the human body. Some clinical epidemiological studies suggest that hypomagnesemia accounts for declines in insulin secretion in patients with type 2 diabetes (T2D); however, the results of various experimental studies do not support this notion. To address this discrepancy, we assessed the short- and long-term effects of hypomagnesemia on ß-cell function and insulin secretion in primary mouse islets of Langerhans and in a mouse model of hypomagnesemia known as Trpm6Δ17 /fl;Villin1-Cre mice. We found that lowering the extracellular Mg2+ concentration from 1.2 mM to either 0.6 or 0.1 mM remarkably increased glucose-induced insulin secretion (GIIS) in primary islets isolated from C57BL/6 mice. Similarly, both the plasma insulin levels and GIIS rose in isolated islets of Trpm6Δ17 /fl;Villin1-Cre mice. We attribute these rises to augmented increases in intracellular Ca2+ oscillations in pancreatic ß-cells. However, the glycemic metabolic profile was not impaired in Trpm6Δ17 /fl;Villin1-Cre mice, suggesting that chronic hypomagnesemia does not lead to insulin resistance. Collectively, the results of this study suggest that neither acute nor chronic Mg2+ deficiency suppresses glucose-induced rises in insulin secretion. Even though hypomagnesemia can be symptomatic of T2D, such deficiency may not account for declines in insulin release in this disease.
Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Humanos , Animais , Secreção de Insulina , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Endogâmicos C57BL , Insulina/metabolismo , Glucose/metabolismoRESUMO
Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.
Assuntos
Intolerância à Glucose , Canais de Cátion TRPM , Animais , Camundongos , Glucose , Insulina/metabolismo , Camundongos Knockout , Obesidade , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
TRPA1 (transient receptor potential ankyrin 1) is a nonselective Ca2+-permeable cation channel, which was originally cloned from human lung fibroblasts (HLFs). TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals, including allyl isothiocyanate (AITC), and a protective effect of TRPA1 activation in the development of cardiac fibrosis has been proposed. Yet the function of TRPA1 in TGF-ß1 (transforming growth factor-ß1)-driven fibroblast-to-myofibroblast differentiation and the development of pulmonary fibrosis remains elusive. TRPA1 expression and function were analyzed in cultured primary HLFs, and mRNA concentrations were significantly reduced after adding TGF-ß1. Expression of genes encoding fibrosis markers (e.g., ACTA2, SERPINE1 [plasminogen activator inhibitor 1], FN1 [fibronectin], COL1A1 [type I collagen]) was increased after siRNA-mediated downregulation of TRPA1 mRNA in HLFs. Moreover, AITC-induced Ca2+ entry in HLFs was decreased after TGF-ß1 treatment and by application of TRPA1 siRNAs, while AITC treatment alone did not reduce cell viability or enhance apoptosis. Most interestingly, AITC-induced TRPA1 activation augmented ERK1/2 (extracellular signal-regulated kinase 1/2) and SMAD2 linker phosphorylation, which might inhibit TGF-ß-receptor signaling. Our results suggest an inhibitory function of TRPA1 channels in TGF-ß1-driven fibroblast-to-myofibroblast differentiation. Therefore, activation of TRPA1 channels might be protective during the development of pulmonary fibrosis in patients.
Assuntos
Fibrose Pulmonar , Fator de Crescimento Transformador beta1 , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Fibrose Pulmonar/patologia , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Diferenciação Celular/fisiologia , Fibrose , RNA Mensageiro/genética , Células Cultivadas , Canal de Cátion TRPA1/metabolismoRESUMO
Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-ß1 (TGF-ß1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-ß1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-ß1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-ß1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-ß1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-ß1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-ß1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.
Assuntos
Fibrose Pulmonar , Canais de Cátion TRPM , Colágeno/antagonistas & inibidores , Colágeno/metabolismo , Fibrinolisina/metabolismo , Fibroblastos , Fibronectinas/efeitos adversos , Fibronectinas/antagonistas & inibidores , Fibronectinas/metabolismo , Fibrose , Humanos , Pulmão/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Serina-Treonina Quinases , Fibrose Pulmonar/induzido quimicamente , Canais de Cátion TRPM/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Glucose provides vital energy for cells and contributes to gene expression. The hypothalamus is key for metabolic homeostasis, but effects of glucose on hypothalamic gene expression have not yet been investigated in detail. Thus, herein, we monitored the glucose-dependent transcriptome in murine hypothalamic mHypoA-2/10 cells by total RNA-seq analysis. A total of 831 genes were up- and 1390 genes were downregulated by at least 50%. Key genes involved in the cholesterol biosynthesis pathway were upregulated, and total cellular cholesterol levels were significantly increased by glucose. Analysis of single genes involved in fundamental cellular signaling processes also suggested a significant impact of glucose. Thus, we chose ≈100 genes involved in signaling and validated the effects of glucose on mRNA levels by qRT-PCR. We identified Gnai1-3, Adyc6, Irs1, Igfr1, Hras, and Elk3 as new glucose-dependent genes. In line with this, cAMP measurements revealed enhanced noradrenalin-induced cAMP levels, and reporter gene assays elevated activity of the insulin-like growth factor at higher glucose levels. Key data of our studies were confirmed in a second hypothalamic cell line. Thus, our findings link extra cellular glucose levels with hypothalamic lipid synthesis and pivotal intracellular signaling processes, which might be of particular interest in situations of continuously increased glucose levels.
Assuntos
Glucose , Transcriptoma , Animais , Colesterol/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Glucose/metabolismo , Hipotálamo/metabolismo , Camundongos , Transdução de Sinais , Transcriptoma/genéticaRESUMO
Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.
Assuntos
AMP Cíclico/metabolismo , Citosol/metabolismo , Receptores Adrenérgicos beta 2/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Fatores de Transcrição ARNTL/metabolismo , Aminopiridinas/farmacologia , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Hipotálamo/fisiologia , Camundongos , Neurônios/fisiologia , Norepinefrina/farmacologia , Receptores Adrenérgicos beta 2/biossíntese , Receptores Adrenérgicos beta 3/biossíntese , Receptores Adrenérgicos beta 3/fisiologia , Fatores de Transcrição STAT/metabolismo , Xinafoato de Salmeterol/farmacologia , Transdução de Sinais/fisiologia , Temperatura , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismoAssuntos
Peptídeo 1 Semelhante ao Glucagon , Serotonina , Animais , Células Enteroendócrinas , Intestinos , Lipídeos , CamundongosRESUMO
Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.
Assuntos
Anafilaxia/etiologia , Anafilaxia/metabolismo , Canais de Cálcio/deficiência , Suscetibilidade a Doenças , Mastócitos/imunologia , Mastócitos/metabolismo , Biomarcadores , Sinalização do Cálcio , Degranulação Celular , Citocinas/metabolismo , Predisposição Genética para Doença , Histamina/metabolismo , Imunoglobulina E/imunologia , Mediadores da Inflamação/metabolismoRESUMO
Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.
Assuntos
Ar , Exposição por Inalação/efeitos adversos , Pulmão/citologia , Material Particulado/toxicidade , Gases/toxicidade , Humanos , Fumaça/efeitos adversosRESUMO
In vivo experiments are still widely used for the testing of lung toxicity but there is an ethical and legal obligation to replace, reduce and refine animal testing. Lung A549 cells could serve as an in vitro indicator for acute lung toxicity but little data about the correlation of the cytotoxicity in A549 cells and data leading to CLP classifications are available. We exposed A549 cells to 19 CLP-classified substances with doses of 25, 50, and 100⯵g/cm2 either under submerged (SME) condition or with aerosols at the air-liquid interface (ALIF) and determined accuracy, precision, sensitivity and the F1 score with the CLP classifications H330, H332, or H335. When data from both exposure methods were combined, we found accuracies of 0.84⯱â¯0.05, precisions of 0.74⯱â¯0.1, sensitivities of 0.93⯱â¯0.08 and F1 scores of 0.82⯱â¯0.04. Separated from each other, ALIF exposure was more sensitive at any dose but, at higher doses, also less accurate and precise compared to SME. Considering the 19 substances tested, our data suggest that cytotoxicity in A549 cells could be a reliable in vitro indicator for in vivo toxicity. Thus, we discuss how A549 could be integrated into validation test guidelines.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Alternativas aos Testes com Animais/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Neoplasias Pulmonares/patologia , Material Particulado/toxicidade , Células A549 , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Camundongos , Tamanho da Partícula , Pós , Reprodutibilidade dos Testes , Medição de RiscoRESUMO
The CULTEX® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100⯵g/cm2). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.
Assuntos
Poluentes Atmosféricos/toxicidade , Técnicas de Cultura de Células/métodos , Material Particulado/toxicidade , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Humanos , Exposição por Inalação , Reprodutibilidade dos TestesRESUMO
Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global "kinase-dead" point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+ Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.
Assuntos
Mucosa Intestinal/metabolismo , Minerais/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Inativação de Genes , Homeostase , Rim/metabolismo , Magnésio/metabolismo , Camundongos , Camundongos Knockout , Canais de Cátion TRPM/genética , Zinco/metabolismoRESUMO
Brain and muscle ARNT-like protein-1 (BMAL-1) is an important component of the cellular circadian clock. Proteins such as epidermal (EGF) or nerve growth factor (NGF) affect the cellular clock via extracellular signal-regulated kinases-1/2 (ERK-1/2) in NIH3T3 or neuronal stem cells, but no such data are available for the insulin-like growth factor-1 (IGF-1). The hypothalamus expresses receptors for all three growth factors, acts as a central circadian pacemaker, and releases hormones in a circadian fashion. However, little is known about growth factor-induced modulation of clock gene activity in hypothalamic cells. Here, we investigated effects of IGF-1, EGF, or NGF on the Bmal-1 promoter in two hypothalamic cell lines. We found that only IGF-1 but not EGF or NGF enhanced activity of the Bmal-1 promoter. Inhibition of ERK-1/2 activity did not affect IGF-1-induced Bmal-1 promoter activation and all three growth factors similarly phosphorylated ERK-1/2, questioning a role for ERK-1/2 in controlling BMAL-1 promoter activity. Of note, only IGF-1 induced sustained phosphorylation of glycogen synthase kinase-3ß (GSK-3ß). Moreover, the GSK-3ß inhibitor lithium or siRNA-mediated GSK-3ß knockdown diminished the effects of IGF-1 on the Bmal-1 promoter. When IGF-1 was used in the context of temperature cycles entraining hypothalamic clock gene expression to a 24-h rhythm, it shifted the phase of Bmal-1 promoter activity, indicating that IGF-1 functions as a zeitgeber for cellular hypothalamic circadian clocks. Our results reveal that IGF-1 regulates clock gene expression and that GSK-3ß but not ERK-1/2 is required for the IGF-1-mediated regulation of the Bmal-1 promoter in hypothalamic cells.
Assuntos
Relógios Circadianos , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipotálamo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Hipotálamo/enzimologia , Camundongos , Células NIH 3T3 , Fosforilação , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca2+ handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca2+ handling. Therefore, intracellular Ca2+ transporters are lead candidate structures for novel and safer antiarrhythmic therapies. Mitochondria and mitochondrial Ca2+ transport proteins are important regulators of cardiac Ca2+ handling. Here we evaluated the potential of pharmacological activation of mitochondrial Ca2+ uptake for the treatment of cardiac arrhythmia. To this aim,we tested substances that enhance mitochondrial Ca2+ uptake for their ability to suppress arrhythmia in a murine model for ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT) in vitro and in vivo and in induced pluripotent stem cell-derived cardiomyocytes from a CPVT patient. In freshly isolated cardiomyocytes of RyR2R4496C/WT mice efsevin, a synthetic agonist of the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane, prevented the formation of diastolic Ca2+ waves and spontaneous action potentials. The antiarrhythmic effect of efsevin was abolished by blockade of the mitochondrial Ca2+ uniporter (MCU), but could be reproduced using the natural MCU activator kaempferol. Both mitochondrial Ca2+ uptake enhancers (MiCUps), efsevin and kaempferol, significantly reduced episodes of stress-induced ventricular tachycardia in RyR2R4496C/WT mice in vivo and abolished diastolic, arrhythmogenic Ca2+ events in human iPSC-derived cardiomyocytes.
RESUMO
Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipotálamo/metabolismo , Receptor Tipo 4 de Melanocortina/genética , Animais , Linhagem Celular , Ingestão de Alimentos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hipotálamo/citologia , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Receptor Tipo 4 de Melanocortina/metabolismoRESUMO
Serotonin 5-HT2C receptors (5-HT2CR) activate Gq proteins and are expressed in the central nervous system (CNS). 5-HT2CR regulate emotion, feeding, reward, or cognition and may serve as promising drug targets to treat psychiatric disorders or obesity. Owing to technical difficulties in isolating cells from the CNS and the lack of suitable cell lines endogenously expressing 5-HT2CR, our knowledge about this receptor subtype in native environments is rather limited. The hypothalamic mHypoA-2/10 cell line was recently established and resembles appetite-regulating hypothalamic neurons of the paraventricular nucleus (PVN), where 5-HT2CR have been detected in vivo. Therefore, we tested mHypoA-2/10 cells for endogenous 5-HT2CR expression. Serotonin or the 5-HT2CR preferential agonist WAY-161,503 initiated cAMP response element (CRE)-dependent gene transcription with EC50 values of 15.5 ± 9.8 and 1.1 ± 0.9 nM, respectively. Both responses were blocked by two unrelated 5-HT2CR-selective antagonists (SB-242,084, RS-102,221) but not by a 5-HT2AR (EMD-281,014) or 5-HT2BR (RS-127,455) antagonists. By single-cell calcium imaging, we found that serotonin and WAY-161,503 induced robust calcium transients, which were also blunted by both 5-HT2CR antagonists. Additionally we revealed, first, that 5-HT2CR induced CRE activation via protein kinase C (PKC)-mediated engagement of extracellular-regulated kinases-1/2 and, second, that intrinsic activity of WAY-161,503 was in the range of 0.3-0.5 compared with serotonin, defining the frequently used 5-HT2CR agonist as a partial agonist of endogenous 5-HT2CR. In conclusion, we have shown that hypothalamic mHypoA-2/10 cells endogenously express 5-HT2CR and thus are the first cell line in which to analyze 5-HT2CR pharmacology, signaling, and regulation in its natural environment.
Assuntos
AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteína Quinase C/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Fosforilação , Pirazinas/farmacologia , Piridinas/farmacologia , Quinoxalinas/farmacologia , Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Glucose/farmacologia , Hipotálamo/metabolismo , Hormônios Estimuladores de Melanócitos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Western Blotting , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ensaio de Imunoadsorção Enzimática , Hipotálamo/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Besides its capacity to inhibit the 1,4,5-trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co-localize with the proton pumping V-ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co-localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and/or the final fertilization process.