RESUMEN
The ß2AR is a prototypical G protein-coupled receptor (GPCR) known to orchestrate different cellular responses by the stimulation of specific signaling pathways. The best-established signaling pathways for the ß2AR are the canonical Gs pathway and the alternative ß arrestin 2 (ßarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the ß2AR with its cognate transducers, Gαs and ßarr2, using short linkers as a novel approach for restricting the conformation of the receptor and preferentially activating one of its two signaling pathways. We characterized the behavior of our fusion proteins ß2AR-Gαs and ß2AR-ßarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacological modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the ß2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by ß2AR ligands. Based on these characteristics, we further explored the possibility of a gain-of-function mechanism in the human lung non-tumorigenic epithelial cell line, BEAS-2B cells. This immortalized human bronchial epithelial cell line has immunomodulatory properties through cytokine release mediated by ß2AR stimulation. Our findings suggest that each signaling pathway of the ß2AR is biased toward either the Th1 or Th2 inflammatory response suggesting a role in regulating the immune phenotype of respiratory diseases. Our data imply that our fusion proteins can be used as tools to isolate the function elicited by a single signaling pathway in physiologically relevant cell types.
RESUMEN
Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on ß-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of ß3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.
RESUMEN
Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas de la Membrana , Transporte de Proteínas/fisiología , Espectrometría de Fluorescencia/métodos , COVID-19 , Desarrollo de Medicamentos/métodos , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Nanotecnología , Receptores Acoplados a Proteínas G , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Internalización del VirusRESUMEN
Cardiovascular (CV) diseases are the major cause of death in industrialized countries. The main function of the CV system is to deliver nutrients and oxygen to all tissues. During most CV pathologies, oxygen and nutrient delivery is decreased or completely halted. Several mechanisms, including increased oxygen transport and delivery, as well as increased blood flow are triggered to compensate for the hypoxic state. If the compensatory mechanisms fail to sufficiently correct the hypoxia, irreversible damage can occur. Thus, hypoxia plays a central role in the pathogenesis and pathophysiology of CV diseases. Hypoxia inducible factors (HIFs) orchestrate the gene transcription for hundreds of proteins involved in erythropoiesis, glucose transport, angiogenesis, glycolytic metabolism, reactive oxygen species (ROS) handling, cell proliferation and survival, among others. The overall regulation of the expression of HIF-dependent genes depends on the severity, duration, and location of hypoxia. In the present review, common CV diseases were selected to illustrate that HIFs, and proteins derived directly or indirectly from their stabilization and activation, are related to the development and perpetuation of hypoxia in these pathologies. We further classify CV diseases into acute and chronic hypoxic states to better understand the temporal relevance of HIFs in the pathogenesis, disease progression and clinical outcomes of these diseases. We conclude that HIFs and their derived factors are fundamental in the genesis and progression of CV diseases. Understanding these mechanisms will lead to more effective treatment strategies leading to reduced morbidity and mortality.
RESUMEN
The adoptive transfer of donor-derived virus-specific T cells (VSTs) is an effective treatment for infections following allogeneic hematopoietic cell transplantation. Acute exercise mobilizes effector lymphocytes and VSTs to the circulation and augments the ex vivo manufacture of VSTs. This study determined if ß2 adrenergic receptor (AR) signaling precipitated the VST response to acute exercise. Healthy participants (n = 12) completed 30 min of steady-state cycling exercise after ingesting a placebo, a ß1 + 2 AR antagonist (nadolol) or a ß1 AR antagonist (bisoprolol). Circulating VSTs to cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV) antigens were enumerated before and after exercise, and peripheral blood mononuclear cells were cultured with viral peptides for 8 days to expand multi-VSTs. Compared with placebo, nadolol blunted the exercise-induced mobilization of CMV-VSTs (Δ VSTs/100,000 CD3+ T cells = 93 ± 104 vs. 22 ± 91 for placebo and nadolol, respectively; p = 0.036), while bisoprolol did not, despite both drugs evoking similar reductions in exercising heart rate and blood pressure. Circulating AdV and EBV VSTs (VSTs/mL blood) only increased after exercise with placebo. Although not significant, nadolol partially mitigated exercise-induced increases in multi-VST expansion, particularly in participants that demonstrated an exercise-induced increase in VST expansion. We conclude that exercise-induced enhancements in VST mobilization and expansion are at least partially ß2 AR mediated, thus highlighting a role for the ß2 AR in targeted therapy for the augmentation of VST immune cell therapeutics in the allogeneic adoptive transfer setting. Moreover, long-term regular exercise may provide additional viral protection in the host through frequent ß2 AR-dependent mobilization and redistribution of VSTs cumulated with each bout of exercise.
Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Antivirales/farmacología , Tratamiento Basado en Trasplante de Células y Tejidos , Ejercicio Físico , Linfocitos T/inmunología , Virus/inmunología , Adulto , Presión Sanguínea/efectos de los fármacos , Catecolaminas/sangre , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Hidrocortisona/sangre , Ácido Láctico/sangre , Masculino , Péptidos/farmacología , Fenotipo , Receptores Adrenérgicos beta/metabolismo , Especificidad de la Especie , Linfocitos T/efectos de los fármacos , Adulto JovenRESUMEN
Benzodiazepines and SSRIs are considered as standard treatment options for anxiety and depression, hallmarks of Post-Traumatic Stress Disorder (PTSD), although their use is often limited by adverse effects. While promising evidence emerged with ß-adrenergic receptor (ß-AR) antagonists (or 'ß-blockers') and PTSD relief, efficacy issues dampened the excitement. However, we believe it is premature to completely eliminate a beneficial role of ß-blockers. Our previous work has suggested that social defeat (SD) results in anxiety-like and depression-like behaviors in rats. Here, using the SD paradigm, we examined the effect of several ß-adrenergic receptor antagonists (propranolol, nadolol, bisoprolol) on these behaviors in rats. Following acclimatization, Sprague-Dawley rats received no treatment (for control groups) or treated with ; propranolol (50 mg/kg/day in water), or nadolol (18 mg/kg/day in rats' chow), or bisoprolol (15 mg/kg/day in water). The treatment lasted for 36 days, following which rats were subjected to SD/control exposures (1 week). Later, anxiety-like and depression-like behaviors, social interaction and learning-memory function tests were conducted. SD rats exhibited anxiety- and depression-like behavior as well as learning-memory impairment. Propranolol and nadolol protected SD rats from exhibiting anxiety-or depression-like behaviors. Bisoprolol treatment did not mitigate SD-induced behavioral impairments in rats. Nadolol, propranolol or bisoprolol have no effect in attenuating SD-induced memory function tests. These results suggest that certain 'ß-blockers' have the potential to mitigate the negative psychological effects of traumatic events.
Asunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Nadolol/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Propranolol/uso terapéutico , Derrota Social , Estrés Psicológico/tratamiento farmacológico , Antagonistas Adrenérgicos beta/farmacología , Animales , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Nadolol/farmacología , Fármacos Neuroprotectores/farmacología , Propranolol/farmacología , Distribución Aleatoria , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Interacción Social/efectos de los fármacos , Estrés Psicológico/psicologíaRESUMEN
Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, ß-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, ß2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/ß, PCG 1α/ß, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Receptores Adrenérgicos beta/metabolismo , Adipogénesis , Adrenérgicos , Reactores Biológicos , Diferenciación Celular/fisiología , Proliferación Celular , Conductividad Eléctrica , Epinefrina/farmacología , Humanos , Hipoxia , Cinética , Miocitos Cardíacos/citología , Transducción de Señal , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido , Factores de Transcripción/metabolismoRESUMEN
LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
Asunto(s)
Receptores Adrenérgicos/metabolismo , Animales , HumanosRESUMEN
ß-arrestins are multifunctional proteins that modulate heptahelical 7 transmembrane receptors, also known as G protein-coupled receptors (GPCRs), a superfamily of receptors that regulate most physiological processes. ß-arrestin modulation of GPCR function includes termination of G protein-dependent signaling, initiation of ß-arrestin-dependent signaling, receptor trafficking to degradative or recycling pathways, receptor transactivation, transcriptional regulation, and localization of second messenger regulators. The pleiotropic influence ß-arrestins exert on these receptors regulates a breadth of physiological functions, and additionally, ß-arrestins are involved in the pathophysiology of numerous and wide-ranging diseases, making them prime therapeutic targets. In this review, we briefly describe the mechanisms by which ß-arrestins regulate GPCR signaling, including the functional cellular mechanisms modulated by ß-arrestins and relate this to observed pathophysiological responses associated with ß-arrestins. We focus on the role for ß-arrestins in transducing cell signaling; a pathway that is complementary to the classical G protein-coupling pathway. The existence of these GPCR dual signaling pathways offers an immense therapeutic opportunity through selective targeting of one signaling pathway over the other. Finally, we will consider several mechanisms by which the potential of dual signaling pathway regulation can be harnessed and the implications for improved disease treatments.
RESUMEN
TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic ß-adrenergic receptor (ß-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic ß-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the ß-AR subtypes involved, by administering a preferential ß1-AR antagonist (bisoprolol) and a non-preferential ß1 + ß2-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40-60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a ß1 + ß2-AR (nadolol), but not a ß1-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic ß-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to ß2-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight ß-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics.
Asunto(s)
Ejercicio Físico/fisiología , Efecto Injerto vs Tumor/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Adrenérgicos/metabolismo , Linfocitos T/inmunología , Agonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/administración & dosificación , Adulto , Bisoprolol/administración & dosificación , Línea Celular , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Humanos , Activación de Linfocitos , Masculino , Nadolol/administración & dosificación , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Trasplante Homólogo , Adulto JovenRESUMEN
Acute exercise preferentially mobilizes cytotoxic T-cells, NK-cells and non-classical monocytes to the bloodstream under the influence of hemodynamic forces and/or ß2-adrenergic receptor (ß2-AR) signaling. However, the relative contribution of these mechanisms to the redeployment of the most exercise-responsive cell types is largely unknown. We determined the lymphocyte and monocyte subtypes mobilized to blood during exercise via ß2-AR signaling whilst controlling for ß1-AR mediated reductions in hemodynamic forces. In a randomized, double blind, complete cross-over design, 14 healthy cyclists exercised for 30-minutes at +10% of blood lactate threshold after ingesting: (1) a placebo, (2) a ß1-preferential antagonist (10â¯mg bisoprolol), or (2) a non-preferential ß1â¯+â¯ß2-antagonist (80â¯mg nadolol) across three trials separated by >7-days. Bisoprolol was administered to reduce hemodynamic forces (heart rate and blood pressure) during exercise to levels comparable with nadolol but without blocking ß2-ARs. The mobilization of total NK-cells, terminally differentiated (CD57+) NK-cells, central memory, effector memory and CD45RA+ effector memory CD8+ T-cells; non-classical monocytes; and γδ T-cells were significantly blunted or abrogated under nadolol compared to both bisoprolol and placebo, indicating that the exercise-induced mobilization of these cell types to the blood is largely influenced by ß2-AR signaling. Nadolol failed to inhibit the mobilization of classical monocytes, CD4+ T-cells (and their subsets) or naïve CD8+ T-cells, indicating that these cell types are mobilized with exercise independently of the ß2-AR. We conclude that the preferential mobilization of NK-cells, non-classical monocytes and differentiated subsets of CD8+ T-cells with exercise is largely dependent on catecholamine signaling through the ß2-AR. These findings provide mechanistic insights by which distinct lymphocyte and monocyte subtypes are preferentially mobilized to protect the host from anticipated injury or infection in response to an acute stress response.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ejercicio Físico/fisiología , Células Asesinas Naturales/inmunología , Monocitos/inmunología , Receptores Adrenérgicos beta 2/inmunología , Adulto , Bisoprolol/farmacología , Linfocitos T CD8-positivos/metabolismo , Catecolaminas/inmunología , Catecolaminas/metabolismo , Estudios Cruzados , Método Doble Ciego , Femenino , Voluntarios Sanos , Humanos , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Masculino , Monocitos/metabolismo , Nadolol/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Transducción de SeñalRESUMEN
Acute dynamic exercise mobilizes CD34+ hematopoietic stem cells (HSCs) to the bloodstream, potentially serving as an economical adjuvant to boost the collection of HSCs from stem cell transplant donors. The mechanisms responsible for HSC mobilization with exercise are unknown but are likely due to hemodynamic perturbations, endogenous granulocyte-colony stimulating factor (G-CSF), and/or ß2-adrenergic receptor (ß2-AR) signaling. We characterized the temporal response of HSC mobilization and plasma G-CSF following exercise, and determined the impact of in vivo ß-AR blockade on the exercise-induced mobilization of HSCs. Healthy runners (nâ¯=â¯15) completed, in balanced order, two single bouts of steady state treadmill running exercise at moderate (lasting 90-min) or vigorous (lasting 30-min) intensity. A separate cohort of healthy cyclists (nâ¯=â¯12) completed three 30-min cycling ergometer trials at vigorous intensity after ingesting: (i) 10â¯mg bisoprolol (ß1-AR antagonist); (ii) 80â¯mg nadolol (ß1â¯+â¯ß2-AR antagonist); or (iii) placebo, in balanced order with a double-blind design. Blood samples collected before, during (runners only), immediately after, and at several points during exercise recovery were used to determine circulating G-CSF levels (runners only) and enumerate CD34+ HSCs by flow cytometry (runners and cyclists). Steady state vigorous but not moderate intensity exercise mobilized HSCs, increasing the total blood CD34+ count by â¼4.15⯱â¯1.62â¯Δcells/µl (+202⯱â¯92%) compared to resting conditions. Plasma G-CSF increased in response to moderate but not vigorous exercise. Relative to placebo, nadolol and bisoprolol lowered exercising heart rate and blood pressure to comparable levels. The number of CD34+ HSCs increased with exercise after the placebo and bisoprolol trials, but not the nadolol trial, suggesting ß2-AR signaling mediated the mobilization of CD34+ cells [Placebo: 2.10⯱â¯1.16 (207⯱â¯69.2%), Bisoprolol 1.66⯱â¯0.79 (+163⯱â¯29%), Nadolol: 0.68⯱â¯0.54 (+143⯱â¯36%)â¯Δcells/µL]. We conclude that the mobilization of CD34+ HSCs with exercise is not dependent on circulating G-CSF and is likely due to the combined actions of ß2-AR signaling and hemodynamic shear stress.
Asunto(s)
Ejercicio Físico/fisiología , Células Madre Hematopoyéticas/fisiología , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Adulto , Antígenos CD34/metabolismo , Bisoprolol , Método Doble Ciego , Femenino , Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Nadolol , Células Madre de Sangre Periférica , Receptores Adrenérgicos beta 2/fisiología , Transducción de SeñalRESUMEN
The mostly widely used bronchodilators in asthma therapy are ß2-adrenoreceptor (ß2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that ß2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that ß2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of ß2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that ß2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent ß2AR ligand shows the receptors are highly expressed in airway epithelium. In ß2AR-/- mice, transgenic expression of ß2ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of ß-arrestin-2 (ßarr-2-/-) attenuates the asthma phenotype as in ß2AR-/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by ß2AR signaling. Together, these results suggest ß2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the ß2AR involves ßarr-2. These results identify ß2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.
Asunto(s)
Asma/etiología , Eosinófilos/patología , Células Epiteliales/metabolismo , Pulmón/patología , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Asma/patología , Bronquios/citología , Modelos Animales de Enfermedad , Epinefrina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-13/toxicidad , Pulmón/citología , Metaplasia , Ratones Endogámicos C57BL , Ratones Transgénicos , Neumonía/inducido químicamente , Neumonía/metabolismo , Receptores Adrenérgicos beta 2/genética , Transducción de SeñalRESUMEN
BACKGROUND: Our previous studies suggested certain ß-adrenoceptor blockers (ß-blockers) attenuate the asthma phenotype in ovalbumin driven murine models of asthma. However, the ovalbumin model has been criticized for lack of clinical relevance. METHODS: We tested the non-selective ß-blockers, carvedilol and nadolol, in house dust mite (HDM) driven murine asthma models where drugs were administered both pre- and post-development of the asthma phenotype. We measured inflammation, mucous metaplasia, and airway hyper-responsiveness (AHR). We also measured the effects of the ß-blockers on extracellular-signal regulated kinase (ERK 1/2) phosphorylation in lung homogenates. RESULTS: We show that nadolol, but not carvedilol, attenuated inflammation and mucous metaplasia, and had a moderate effect attenuating AHR. Following HDM exposure, ERK1/2 phosphorylation was elevated, but the level of phosphorylation was unaffected by ß-blockers, suggesting ERK1/2 phosphorylation becomes dissociated from the asthma phenotype. CONCLUSION: Our findings in HDM models administering drugs both pre- and post-development of the asthma phenotype are consistent with previous results using ovalbumin models and show differential effects for nadolol and carvedilol on the asthma phenotype. Lastly, our data suggest that ERK1/2 phosphorylation may be involved in development of the asthma phenotype, but may have a limited role in maintaining the phenotype.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Asma/tratamiento farmacológico , Carbazoles/farmacología , Nadolol/farmacología , Propanolaminas/farmacología , Animales , Asma/inmunología , Asma/patología , Carvedilol , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ovalbúmina/inmunología , Fenotipo , Fosforilación , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/tratamiento farmacológicoRESUMEN
Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/efectos adversos , Asma/tratamiento farmacológico , Feniletanolamina N-Metiltransferasa/deficiencia , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Aminopiridinas/administración & dosificación , Aminopiridinas/farmacología , Animales , Asma/complicaciones , Asma/patología , Asma/fisiopatología , Benzamidas/administración & dosificación , Benzamidas/farmacología , Hiperreactividad Bronquial/complicaciones , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/patología , Hiperreactividad Bronquial/fisiopatología , Ciclopropanos/administración & dosificación , Ciclopropanos/farmacología , Quimioterapia Combinada , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Fumarato de Formoterol/administración & dosificación , Fumarato de Formoterol/farmacología , Inflamación/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Noqueados , Moco/metabolismo , Fenotipo , Feniletanolamina N-Metiltransferasa/metabolismo , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Inhibidores de Fosfodiesterasa 4/farmacologíaRESUMEN
Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (ß2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related ß2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related ß2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/efectos adversos , Antiasmáticos/efectos adversos , Aspergilosis Broncopulmonar Alérgica/tratamiento farmacológico , Asma/inducido químicamente , Peptidomiméticos/farmacología , Factor de Transcripción STAT6/antagonistas & inhibidores , Albuterol/uso terapéutico , Animales , Arrestinas/deficiencia , Arrestinas/genética , Aspergilosis Broncopulmonar Alérgica/genética , Aspergilosis Broncopulmonar Alérgica/metabolismo , Aspergilosis Broncopulmonar Alérgica/patología , Aspergillus niger/fisiología , Asma/tratamiento farmacológico , Asma/genética , Asma/metabolismo , Broncoconstricción/efectos de los fármacos , Carbazoles/efectos adversos , Carvedilol , Modelos Animales de Enfermedad , Femenino , Fumarato de Formoterol/efectos adversos , Expresión Génica , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Propanolaminas/efectos adversos , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Factor de Transcripción STAT6/agonistas , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Xinafoato de Salmeterol/efectos adversos , beta-ArrestinasRESUMEN
Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes ß2-adrenoceptor (ß2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether ß2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential ß2AR antagonist, but not by CGP-20712A, a preferential ß1AR antagonist. Constitutive ß2AR activity was not sufficient for IL-13 induced mucin production and ß-agonist-induced signaling is required. A clinically important long-acting ß-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that ß2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that ß2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of ß2ARs on epithelial cells.