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Meningococcus utilizes β-arrestin selective activation of endothelial cell β 2 adrenergic receptor (β 2 AR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that β 2 AR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on β 2 AR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host.

An increasing number of studies indicate that adrenergic signalling plays a fundamental role in chronic stress-induced tumour progression and metastasis. However, its function in gastric cancer (GC) and its potential mechanisms remain unknown. The expression levels of β-adrenergic receptor (ADRB) in GC cell lines were examined by using real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of β2 adrenergic receptor (ADRB2) activation and blockade were investigated in vitro in GC cells by using proliferation, migration, invasion, cell cycle and apoptosis assays. Chronic restraint stress (CRS) increased the plasma levels of catecholamines and cortisol and also induced progression and metastasis of GC in vivo. Furthermore, immunohistochemical staining and a TUNEL assay were employed to observe the regulation of cell viability in vivo. The expression levels of ADRB2 in 100 human GC samples were measured by RT-PCR and immunohistochemistry. The stress hormones epinephrine and norepinephrine significantly accelerated GC cell proliferation, invasion and viability in culture, as well as tumour growth in vivo. These effects were reversed by the ADRB antagonists propranolol and ICI118,551 (an ADRB2-specific antagonist). Moreover, the selective ADRB1 antagonist atenolol had almost no effect on tumour cell proliferation and invasion in vitro and in vivo. ADRB2 antagonists suppressed proliferation, invasion and metastasis by inhibiting the ERK1/2-JNK-MAPK pathway and transcription factors, such as NF-κB, AP-1, CREB and STAT3. Analysis of xenograft models using GC cells revealed that ADRB2 antagonists significantly inhibited tumour growth and metastasis, and chronic stress antagonized these inhibitory effects. In addition, chronic stress increased the expression of VEGF, MMP-2, MMP-7 and MMP-9 in transplanted tumour tissue, and catecholamine hormones enhanced the expression of metastasis-related proteins. The expression of ADRB2 was upregulated in tumour tissues and positively correlated with tumour size, histological grade, lymph node metastasis and clinical stage in human GC samples. Stress hormone-induced activation of the ADRB2 signalling pathway plays a crucial role in GC progression and metastasis. These findings indicate that ADRB2 signalling regulates GC progression and suggest β2 blockade as a novel strategy to complement existing therapies for GC.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease resulting in severe respiratory derangements. As such, DMD patients are at a high risk of nocturnal hypoventilation, thereby requiring nocturnal ventilation (NV). To this end, NV is an important clinical milestone in the management of DMD. Emerging evidence suggests that ß 2 adrenergic receptors (ADRB2) may play a role in determining respiratory function, whereby more functional ADRB2 genotype variants (e.g., Gly16) are associated with improved pulmonary function and respiratory muscle strength. These findings suggest that the more functional ADRB2 genotype may help to preserve respiratory function in patients with DMD. The purpose of this study was to identify the influence of ADRB2 genotype on the risk of NV use in DMD. Data from the CINRG Duchenne Natural History Study including 175 DMD patients (3-25 yrs) were analyzed focusing on ADRB2 genotype variants. Time-to-event analyses were used to examine differences in the age at prescription of full-time NV use between genotypes. There were no differences between genotype groups in age, height, weight, corticosteroid use, proportion of ambulatory patients, or age at loss of ambulation. DMD patients expressing the Gly16 polymorphism had a significantly (P < 0.05) lower mean age at NV prescription compared with those patients expressing the Arg16 polymorphism (21.80 ± 0.59 yrs. vs 25.91 ± 1.31 yrs., respectively). In addition, a covariate-adjusted Cox model revealed that the Gly16 variant group possessed a 6.52-fold higher risk of full-time NV use at any given age compared with the Arg16 polymorphism group. These data suggest that genetic variations in the ADRB2 gene may influence the age at which DMD patients are first prescribed NV, whereby patients with the Gly16 polymorphism are more likely to require NV assistance at an earlier age than their Arg16 counterparts.

Purpose: Phosphodiesterase (PDE) terminates the signaling pathway of myometrial relaxation by degradating cAMP to the inactive 5'-AMP. The PDE4 family is one of the most predominant PDE families that display high affinity to cAMP. The objective of this study was to evaluate the effects of PDE4 gene polymorphisms on tocolytic effects and adverse drug events (ADEs) of ritodrine therapy in patients with preterm labor., Methods: A total of 170 preterm labor patients were included in this study. To elucidate the effects of genetic polymorphisms on the inter-individual variability of ritodrine efficacy and ADEs, 8 single nucleotide polymorphisms (SNPs) were genotyped: PDE4D (rs1544791, rs983280, rs1504982, rs10940648, rs829259) and PDE4B2 (rs598961, rs2180335, and rs17128809). Additionally, rs1042719 of the ADRB2 gene was included for multivariate analysis. The primary endpoint of this prospective study was the time to delivery (hr). The secondary endpoint was ritodrine-induced ADEs., Results: The mutant-type homozygote carriers of PDE4B2 rs598961 polymorphism showed shorter median time to delivery than those with other genotypes (adjusted hazard ratio 1.6, 95% confidence interval 1.0 to 2.4, P = 0.035). On the other hand, patients with wild-type homozygotes of PDE4B2 rs17128809 showed 2.6~2.9 times higher ADEs compared to those with other genotypes. Among demographic characteristics, gestational age at start of drug therapy and modified Bishop score were significant factors for time to delivery, whereas height, weight, and BSA were significant factors for ritodrine-induced ADEs after adjusting other factors., Conclusions: This pharmacogenomic study suggested that PDE4 genetic polymorphisms impact individual susceptibility to β2-adrenergic receptor targeted therapy in patients with preterm labor.

Purpose: Antitumor immunity plays an important role in the progression of breast cancer. β 2 -adrenergic receptor (β 2 AR) was found to regulate the antitumor immune response and breast cancer progression in preclinical studies. To understand the clinical role of β 2 AR in cancer progression, we investigated the clinicopathological and prognostic significance of β 2 AR expression in invasive breast cancer., Methods: β 2 AR levels in breast tumors were evaluated by immunohistochemistry in a well-characterized patient cohort with long-term follow-up (n = 278). We evaluated the relationship of β 2 AR expression to patient survival and clinicopathological factors, including immune biomarkers such as tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression. Breast cancer-specific survival was compared between high- and low-β 2 AR expression groups., Results: Although β 2 AR was not related to clinicopathological factors across the whole cohort, high β 2 AR was significantly related to PD-L1 negativity in estrogen receptor (ER)-negative patients. Tumors with high β 2 AR tended to have low TIL grade, and high β 2 AR was an independent prognostic factor for reduced survival in ER-negative patients., Conclusions: β 2 AR is an independent poor prognostic factor in ER-negative breast cancer. The findings suggest that tumor β 2 AR regulates immune checkpoint activity, which may have therapeutic implications for patients with ER-negative breast cancer.

The work was aimed at studying the relationship between the efficiency of bisoprolol and the polymorphism of β1- and β2-adrenergic receptors (β-AR) genes in patients with heart failure. The two-year study included 251 patients with heart failure (with myocardial infarction on the background of coronary heart disease). During hospitalization, a standardized examination and prescription of therapy was carried out, including β-adrenergic blocking agent (β1-AB) - bisoprolol. Afterward, 61 (24.4%) patients stopped taking β1-AB (bisoprolol) as a result of intolerance or violation of compliance; 190 patients took bisoprolol for 2 years. The frequency of rehospitalization (RH) due to decompensation of heart failure (HF) (or intravenous injection of loop diuretics), mortality, and the development of a composite endpoint (CE) for 2 years was taken into account. The control group consisted of 55 healthy individuals. Genotyping was performed using 3 polymorphisms (Gly389Arg of the β1-АR gene, Ser49Gly of the β1-АR gene, Gln27Glu of the β2-АR gene) using the polymerase chain reaction. Genetic and epidemiological analysis was carried out using the SNPStats program. The use of bisoprolol with HF reduces the risk of re-hospitalization (odds ratio (OR)=0.519 (0.278-0.967); p=0.037) and CE (OR=0.494 (0.271-0.900); p=0.030) for 2 years of treatment. Treatment of patients with bisoprolol in a dose of >5 mg leads to a decrease in the risk of CE with G/A polymorphism Ser49Gly (c.145A> G) of the β1-AR gene (OR=0.18 (0.04-0.84), with p=0.014). The use of this drug at this dose also leads to a decrease in the frequency of RH and CE with the homozygous genotype C (C/C) of the Gln27Glu polymorphism (c.79C>G) of the β2-AR gene (OR=0.09 (0.02-0.46), at p=0.018 and OR=0.14 (0.04-0.58), at p=0.006, respectively).

The β 2 -adrenergic receptor (β 2 AR) is a membrane-bound G-protein-coupled receptor and an important drug target for asthma. Clinical studies report that the population variant Gln27Glu is associated with a differential response to common asthma drugs, such as albuterol, isoproterenol and terbutaline. Interestingly, the 27th amino acid is positioned on the N-terminal region that is the most flexible and consequently the least studied part of the receptor. In this study, we probe the molecular origin of the differential drug binding by performing structural modeling and simulations of the wild-type (Gln) and variant (Glu) receptors followed by ensemble docking with the ligands, albuterol, isoproterenol and terbutaline. In line with clinical studies, the ligands were observed to interact preferentially with the Glu variant. Our results indicate that the Glu residue at the 27th position perturbs the network of electrostatic interactions that connects the N-terminal region to the binding site in the wild-type receptor. As a result, the Glu variant is observed to bind better to the three ligands tested in this study. Our study provides a structural basis to explain the variable drug response associated with the 27th position polymorphism in the β 2 AR and is a starting step to identify genotype-specific therapeutics.

Background: Individual susceptibility to exacerbations in chronic obstructive pulmonary disease (COPD) is likely influenced by genetic factors; however, most such variance is unexplained. We hypothesised that β 2 -adrenergic receptor genotypes, Gly16Arg (rs1042713, c.46G>A) and Gln27Glu (rs1042714, c.79C>G) influence risk of severe exacerbations in COPD., Methods: Among 96 762 individuals in the Copenhagen General Population Study, we identified 5262 with COPD (forced expiratory volume in one second divided by forced vital capacity, FEV 1 /FVC, below 0.7, FEV 1 less than 80% of predicted value, age above 40 years and no asthma) who had genotyping performed. Severe exacerbations were defined as acute admissions due to COPD during 5 years of follow-up (mean 3.4 years). 923 individuals with COPD diagnosed similarly in the Copenhagen City Heart Study (CCHS) were used for replication analyses., Results: We recorded 461 severe exacerbations in 5262 subjects. The HRs for severe exacerbations were 1.62 (95% CI 1.30 to 2.03, p=0.00002) for 16Gly/Arg heterozygotes and 1.41 (1.04 to 1.91, p=0.03) for 16Arg homozygotes, compared with 16Gly homozygotes. HRs were 1.35 (1.03 to 1.76, p=0.03) for 27Gln/Glu heterozygotes and 1.49 (1.12 to 1.98, p=0.006) for 27Gln homozygotes, compared with 27Glu homozygotes. Similar trends were observed in the CCHS. Among 27Gln homozygotes only, HRs were 5.20 (1.81 to 14.9, p=0.002) for 16Gly/Arg heterozygotes and 4.03 (1.40 to 11.6, p=0.01) for 16Arg homozygotes, compared with 16Gly homozygotes., Conclusion: Common β 2 -adrenergic receptor genotypes influence risk of severe exacerbations in COPD, potentially mainly by genetic influence of the 16Arg allele in rs1042713., Competing Interests: Competing interests: TSI has received a fee for speaking from AstraZeneca. JV has received honoraria from Chiesi, GlaxoSmithKline, Almirall, AstraZeneca, Boehringer-Ingelheim, and Novartis for consulting and for presenting at meetings and symposia, none of it related to the topic of this study. LR and JLM: none to declare. PL has received honoraria from AstraZeneca, Boehringer Ingelheim, Chiesi, Novartis, Teva, and GlaxoSmithKline for presenting at meetings and symposia, none of it related to the topic of this study. BGN: none to declare., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy., Methods: Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR)., Results: The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%., Conclusion: A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.

β 2 -adrenergic receptor (β 2 -AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of β-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 μg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 μg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified β 2 -AR would have great application potential in detection of β-agonist residues.

Noradrenaline (NE), the main neurotransmitter released by sympathetic nerve terminals, is known to modulate the immune response. However, the role of the sympathetic nervous system (SNS) on the development of autoimmune diseases is still unclear. Here, we report that the SNS limits the generation of pathogenic T cells and disease development in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). β2-Adrenergic receptor (Adrb2) signaling limits T cell autoimmunity in EAE through a mechanism mediated by the suppression of IL-2, IFN-γ, and GM-CSF production via inducible cAMP early repressor (ICER). Accordingly, the lack of Adrb2 signaling in immune cells is sufficient to abrogate the suppressive effects of SNS activity, resulting in increased pathogenic T cell responses and EAE development. Collectively, these results uncover a suppressive role for the SNS in CNS autoimmunity while they identify potential targets for therapeutic intervention., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Evolutionary information contained in the amino acid sequences of proteins specifies the biological function and fold, but exactly what information contained in the protein sequence drives both of these processes? Considerable progress has been made to answer this fundamental question, but it remains challenging to explore the potential space of cooperative interactions between amino acids. Statistical analysis plays a significant role in studying such interactions and its use has expanded in recent years to studies ranging from coevolution-guided rational protein design to protein folding in silico., Results: Here we describe a computational tool named Sibe for use in studies of protein sequence, folding, and design using evolutionary coupling between amino acids as a driving factor. In this study, Sibe is used to identify positionally conserved couplings between pairwise amino acids and aid rational protein design. In this process, pairwise couplings are filtered according to the relative entropy computed from the positional conservations and grouped into several 'blocks', which could contribute to driving protein folding and design. A human β 2 -adrenergic receptor (β 2 AR) was used to demonstrate that those 'blocks' contribute the rational design for specifying functional residues. Sibe also provides folding modules based on both the positionally conserved couplings and well-established statistical potentials for simulating protein folding in silico and predicting tertiary structure. Our results show that statistically inferences of basic evolutionary principles, such as conservations and coupled-mutations, can be used to rapidly design a diverse set of proteins and study protein folding., Conclusions: The developed software Sibe provides a computational tool for systematical analysis from protein primary to its tertiary structure using the evolutionary couplings as a driving factor. Sibe, written in C++, accounts for compatibility with the 'big data' era in biological science, and it primarily focuses on protein sequence analysis, but it is also applicable to extend to other modeling and predictions of experimental measurements.

Pickering, C, Suraci, B, Semenova, EA, Boulygina, EA, Kostryukova, ES, Kulemin, NA, Borisov, OV, Khabibova, SA, Larin, AK, Pavlenko, AV, Lyubaeva, EV, Popov, DV, Lysenko, EA, Vepkhvadze, TF, Lednev, EM, Leońska-Duniec, A, Pająk, B, Chycki, J, Moska, W, Lulińska-Kuklik, E, Dornowski, M, Maszczyk, A, Bradley, B, Kana-ah, A, Cięszczyk, P, Generozov, EV, and Ahmetov, II. A genome-wide association study of sprint performance in elite youth football players. J Strength Cond Res 33(9): 2344-2351, 2019-Sprint speed is an important component of football performance, with teams often placing a high value on sprint and acceleration ability. The aim of this study was to undertake the first genome-wide association study to identify genetic variants associated with sprint test performance in elite youth football players and to further validate the obtained results in additional studies. Using micro-array data (600 K-1.14 M single nucleotide polymorphisms [SNPs]) of 1,206 subjects, we identified 12 SNPs with suggestive significance after passing replication criteria. The polymorphism rs55743914 located in the PTPRK gene was found as the most significant for 5-m sprint test (p = 7.7 × 10). Seven of the discovered SNPs were also associated with sprint test performance in a cohort of 126 Polish women, and 4 were associated with power athlete status in a cohort of 399 elite Russian athletes. Six SNPs were associated with muscle fiber type in a cohort of 96 Russian subjects. We also examined genotype distributions and possible associations for 16 SNPs previously linked with sprint performance. Four SNPs (AGT rs699, HSD17B14 rs7247312, IGF2 rs680, and IL6 rs1800795) were associated with sprint test performance in this cohort. In addition, the G alleles of 2 SNPs in ADRB2 (rs1042713 & rs1042714) were significantly over-represented in these players compared with British and European controls. These results suggest that there is a genetic influence on sprint test performance in footballers, and identifies some of the genetic variants that help explain this influence.

Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting β2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, β2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1α and multiple electron transport chain proteins. Thus, our results support β2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

We created a translational model of chronic heart failure in rats that developed in 3 months after reproducing experimental anterior transmural myocardial infarction. The model simulated the basic clinicodiagnostic criteria of this disease: impaired contractility and dilatation of heart ventricles, signs of venous congestion, elevated plasma content of biochemical markers, and abnormal overexpression of AT1aR and β-adrenoceptors.

In adult mammalian hearts, atrioventricular rings (AVRs) surround the atrial orifices of atrioventricular valves and are hotbed of ectopic activity in patients with focal atrial tachycardia. Experimental data offering mechanistic insights into initiation and maintenance of ectopic foci is lacking. We aimed to characterise AVRs in structurally normal rat hearts, identify arrhythmia predisposition and investigate mechanisms underlying arrhythmogenicity. Extracellular potential mapping and intracellular action potential recording techniques were used for electrophysiology, qPCR for gene and, Western blot and immunohistochemistry for protein expression. Conditions favouring ectopic foci were assessed by simulations. In right atrial preparations, sinus node (SN) was dominant and AVRs displayed 1:1 impulse conduction. Detaching SN unmasked ectopic pacemaking in AVRs and pacemaker action potentials were SN-like. Blocking pacemaker current I f , and disrupting intracellular Ca 2+ release, prolonged spontaneous cycle length in AVRs, indicating a role for SN-like pacemaker mechanisms. AVRs labelled positive for HCN4, and SERCA2a was comparable to SN. Pacemaking was potentiated by isoproterenol and abolished with carbachol and AVRs had abundant sympathetic nerve endings. β 2 -adrenergic and M 2 -muscarinic receptor mRNA and β 2 -receptor protein were comparable to SN. In computer simulations of a sick SN, ectopic foci in AVR were unmasked, causing transient suppression of SN pacemaking.

Background: Thyrotoxicosis increases bone turnover, resulting in net bone loss. Sympathetic nervous system (SNS) activation, via β2-adrenoceptor (β2-AR) signaling, also has osteopenic effects. Because thyroid hormones (TH) interact with the SNS to regulate several physiological processes, we hypothesized that this interaction also occurs to regulate bone mass. Previous studies support this hypothesis, as α2-AR knockout (KO) mice are less susceptible to thyrotoxicosis-induced osteopenia. Here, we evaluated whether TH-SNS interactions in bone involve β2-AR signaling. Methods: Thyrotoxicosis was induced in 120-day-old female and male mice with β2-AR gene inactivation ( β2-AR -/- ) by daily treatment with supraphysiological doses of triiodothyronine (T3) for 12 weeks. The impact of thyrotoxicosis on femoral bone microarchitecture, remodeling, fracture risk, and gene expression of the receptor activator of nuclear factor-kappa-B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) pathway was evaluated. In addition, the effect of the β2-AR-specific agonist clenbuterol (CL) on cAMP accumulation was determined in osteoblastic (MC3T3-E1) cells treated with T3 and/or 17β-estradiol (E2). Results: Thyrotoxicosis negatively affected trabecular bone microarchitecture in wild-type (WT) females, but this effect was milder or nonexistent in β2-AR -/- animals, whereas the opposite was seen in males. T3 treatment increased the femoral RANKL/OPG mRNA ratio and the endosteal perimeter and medullary area of the diaphysis in WT females and males, but not in β2-AR -/- mice, suggesting that T3 promotes endosteal resorption in cortical bone, in a mechanism that involves β2-AR signaling. T3 treatment increased endocortical mineral apposition rate only in WT females but not in β2-AR -/- mice, suggesting that TH also induce bone formation in a β2-AR signaling-dependent mechanism. T3 treatment decreased femoral resistance to fracture only in WT females, but not in KO mice. E2 and CL similarly increased cAMP accumulation in MC3T3-E1 cells; whereas T3 alone had no effect, but it completely blocked E2-stimulated cAMP accumulation, suggesting that some T3 effects on bone may involve E2/cAMP signaling in osteoblasts. Conclusions: These findings sustain the hypothesis that T3 interacts with the SNS to regulate bone morphophysiology in a β2-AR signaling-dependent mechanism. The data also reveal sex as an important modifier of skeletal manifestations of thyrotoxicosis, as well as a modifier of the TH-SNS interactions to control bone microarchitecture, remodeling, and resistance to fracture.

Allergic rhinitis (AR) is a common hypersensitive disease that troubles patients a lot. Nasal epithelial cells (NECs), as the outmost protection of inhalation, play an important role in AR allergic response. Adrenoceptor beta 2 (ADRB2) is an important gene in inflammatory response, which has become the hot spot for AR development and treatment in recent years. MiR-15a-5p has been proved to be involved in AR immune response as the upstream regulator of ADRB2. Human primary NECs were isolated and stimulated by IL-13. qRT-PCR assay was used to detect the RNA level of target genes. ELISA and Western blotting were applied to detect target protein levels. Luciferase reporter assay and biotin pull-down assay were performed to test molecules interaction. ADRB2 was highly expressed in nasal mucosa of AR patients and was positively correlated with IL-13 stimulation, and knockdown of ADRB2 inhibited IL-13-induced expression of GM-CSF, eotaxin, and MUC5AC in NECs. ADRB2 was directly targeted by miR-15a-5p, and miR-15a-5p inhibited IL-13-induced expression of GM-CSF, eotaxin, and MUC5AC in NECs. ADRB2 mediated the effect of miR-15a-5p on the regulation of nasal epithelial immune responses. ADRB2 is negatively regulated by miR-15a-5p, which inhibits IL-13-induced nasal epithelial inflammatory responses.

Chronic heart failure is the terminal stage of various cardiovascular diseases. Despite the availability of several classes of drugs, there is still an unmet need for effective treatment. Based on bench work during the past two decades, we have proposed that enhancement of β 2 -adrenergic receptor signaling in combination with the presently preferred β 1 -adrenergic receptor blockade would be a promising strategy. Chinese herbal medicines have been shown to be effective in the treatment of heart failure, although the mechanisms largely remain unknown. In the present study, we screened an herbal medicine compound/extract library for β -adrenergic receptor ligands to determine the target of certain effective botanical remedies and seek a leading compound(s) for chronic heart failure treatment. Using a high-throughput screening assay, we identified higenamine, which has a long history in chronic heart failure treatment in traditional Chinese medicine, to be a potent β -adrenergic receptor agonist. Further experiments using specific inhibitors showed that higenamine activated both β 1 -adrenergic receptor and β 2 -adrenergic receptor. Inhibition of its action by pertussis toxin (a G i inhibitor) indicated that it is a β 2 -adrenergic receptor G s /G i dual agonist. Contractility experiments demonstrated a positive inotropic effect of higenamine. In conclusion, we found an herbal compound, higenamine, to be a dual agonist for β 1 / β 2 -adrenergic receptors with no preference in stimulating the G s and G i pathways in β 2 -adrenergic receptor signaling. Our results elucidated not only the target of higenamine to explain its pharmacological effect in treating chronic heart failure, but also the mechanisms of its cardiac toxicity., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Drugs targeting the orthosteric, primary binding site of G protein-coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β 2 -adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor's inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β 2 - over the β 1 -adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The evidence from post-mortem biochemical studies conducted on cortisol and catecholamines suggest that analysis of the adrenal gland could provide useful information about its role in human pathophysiology and the stress response. Authors designed an immunohistochemical study on the expression of the adrenal β2-adrenergic receptor (β2-AR), a receptor with high-affinity for catecholamines, with the aim to show which zones it is expressed in and how its expression differs in relation to the cause of death. The immunohistochemical study was performed on adrenal glands obtained from 48 forensic autopsies of subjects that died as a result of different pathogenic mechanisms using a mouse monoclonal β2-AR antibody. The results show that immunoreactivity for β2-AR was observed in all adrenal zones. Furthermore, immunoreactivity for β2-AR has shown variation in the localization and intensity of different patterns in relation to the original cause of death. To the best of our knowledge, this is the first study that demonstrates β2-AR expression in the human cortex and provides suggestions on the possible involvement of β2-AR in human cortex hormonal stimulation. In conclusion, the authors provide a possible explanation for the observed differences in expression in relation to the cause of death.

Background: An interindividual variability in response to short-acting bronchodilator drugs (short-acting inhaled β2-agonists, SABA) exists and this is linked in part to genetic factors. The aim of this study was to verify the influence of single nucleotide polymorphisms (SNPs) of a previously studied gene ( ADRB2 ) and of new candidate genes ( THRB and ARG1 ) on the acute response to SABA in children with asthma. Methods: One hundred asthmatic children (mean age 9.6 ± 3.0 years, 77 boys) underwent allergological and lung function evaluations. Spirometry was performed before and after bronchodilation test (BD test). The ADRB2 region containing the Arg16Gly (rs1042713) and Gln27Glu (rs1042714) variants were amplified by polymerase chain reaction, whereas ARG1 rs2781659 (A>G) and THRB rs892940 (G>A) SNPs were genotyped by high-resolution melting (HRM) analysis. Results: Seventy-seven percent of children developed asthma in the first 6 years of life. Allergic sensitization was observed in 92% (total immunoglobulin G: 529.8 ± 477. kU/L). All patients exhibited respiratory allergy: 43% has multiple respiratory, 22% to single respiratory, and 27% multiple respiratory and food allergies. Fifty four percent children showed positive BD response (forced expiratory volume in 1 second [FEV1] > 12%). Presence of Arg/Gly or Gly/Gly genotypes in position 16 of ADRB2 was significantly associated to a worse BD response (post-BD FEV1: 108.68% ± 15.62% in Arg/Arg vs. 101.86% ± 14.03% in Arg/Gly or Gly/Gly patients, p  = 0.02). No significant association was found between spirometric parameters before and after BD for the other three examined SNPs. Conclusion: The influence of genetic variability on responsiveness to drugs could become a key parameter to optimize a tailored therapy for young patients with asthma, especially if drug-resistance occurs.

Aims: Women with overactive bladder (OAB) have a higher frequency of a single-nucleotide polymorphism (SNP) at codon 64 of the β-3 adrenergic receptor gene (ADRB3). Since the SNP results in an amino acid substitution that could theoretically alter receptor protein function, we hypothesized that those with the SNP would display greater OAB symptom severity. Therefore we aimed to compare OAB severity between women with this SNP and women with the wild type genotype., Methods: A retrospective cohort study was performed in women with bothersome OAB from two academic institutions. Banked blood samples were tested for the codon 64 SNP. Women were divided into two groups based on genotype: wild-type (WT) and heterozygous (HZ). We compared mean OAB Symptom Severity questionnaire (OAB-q) scores between groups using t tests. Linear regression was performed to control for potential confounders., Results: Of the 303 women with OAB, 254 (83.8%) had the WT genotype, and 49 (16.2%) the HZ genotype. There were no homozygous women for the rare allele. The majority were Caucasian (86%) and non-Hispanic (97%). There were no significant differences in mean OAB-q symptom severity scores (WT 21.2 ± 7 vs HZ 22.0 ± 6.6; P = 0.49) and quality of life scores (WT 39.6 ± 15.5 vs HZ 39.1 ± 16.6; P = 0.83) between groups. These remained nonsignificant in a linear regression model., Conclusions: In a predominantly non-Hispanic, Caucasian population of women with bothersome OAB, symptom severity was not related to ADRB3 codon 64 SNP genotype., (© 2019 Wiley Periodicals, Inc.)

BACKGROUND The ADRB2 gene encodes the ß2-adrenergic receptor (ß2-AR). This study aimed to determine the association between the C79G polymorphism of the ADRB2 gene and its association with pediatric asthma using a meta-analysis of the published data. MATERIAL AND METHODS Review of publications up to May 2018 was from the PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), and WanFang databases. The odds ratio (ORs) with 95% confidence interval (CI) were used in evaluating the strength of the reported association between the C79G polymorphism of the ADRB2 gene and pediatric asthma. RESULTS There were 18 controlled studies that included 2,982 pediatric cases of asthma and 2,651 controls. Expression of the C79G polymorphism of the ADRB2 gene was significantly associated with risk of pediatric asthma associated with the C or G allele with comparison of the co-dominant model (GG vs. CC: OR, 0.69; 95% CI, 0.55-0.88) and the recessive model (GG vs. CC+CG: OR, 0.65; 95% CI, 0.53-0.81). Subgroup analysis by ethnicity showed a significantly reduced risk of pediatric asthma in Asian patients for comparison of the co-dominant model (GG vs. CC: OR, 0.59; 95% CI, 0.45-0.78), the recessive model (GG vs. CC+CG: OR, 0.58; 95% CI, 0.45-0.76), and the allelic model (G vs. C: OR, 0.89; 95% CI, 0.79-0.99). CONCLUSIONS The C79G polymorphism of the ADRB2 gene encoding ß2-AR was associated with a reduced risk for the development of pediatric asthma, particularly in the Asian population.

The new technologies for data analysis, such as decision tree learning, may help to predict the risk of developing diseases. The aim of the present work was to develop a pilot decision tree learning to predict overweight/obesity based on the combination of six single nucleotide polymorphisms (SNP) located in feeding-associated genes. Genotype study was performed in 151 healthy individuals, who were anonymized and randomly selected from the TALAVERA study. The decision tree analysis was performed using the R package rpart. The learning process was stopped when 15 or less observation was found in a node. The participant group consisted of 78 men and 73 women, who 100 individuals showed body mass index (BMI) ≥ 25 kg/m 2 and 51 BMI < 25 kg/m 2 . Chi-square analysis revealed that individuals with BMI ≥ 25 kg/m 2 showed higher frequency of the allelic variation Ala67Ala in AgRP rs5030980 with respect to those with BMI <25 kg/m 2 . However, the variant Thr67Ala in AgRP rs5030980 was the most frequently found in individuals with BMI <25 kg/m 2 . There were no statistical differences in the other analyzed SNPs. Decision tree learning revealed that carriers of the allelic variants AgRP (rs5030980) Ala67Ala, ADRB2 (rs1042714) Gln27Glu or Glu27Glu, INSIG2 (rs7566605) 73 + 9802 with CC or GG genotypes and PPARG (rs1801282) with the allelic variants of Ala12Ala or Pro12Pro, will most likely develop overweight/obesity (BMI ≥ 25 kg/m 2 ). Moreover, the decision tree learning indicated that age and gender may change the developed three decision learning associated with overweight/obesity development. The present work should be considered as a pilot demonstrative study to reinforce the broad field of application of new data analysis technologies, such as decision tree learning, as useful tools for diseases prediction. This technology may achieve a potential applicability in the design of early strategies to prevent overweight/obesity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Previous studies have indicated that β2-adrenergic receptor (ADRB2) genetic polymorphism is related to the risk of asthma, but the results still have some controversy and uncertainty. To this end, the meta-analysis was performed, including all studies that can be used to assess the correlation between ADRB2 polymorphism and asthma susceptibility., Materials and Methods: The related papers on ADRB2 polymorphisms and asthma were systematically reviewed in databases of PubMed, EMBASE, Cochrane Library, and WanFang, Odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) were measured. The sensitivity analysis and publication bias were evaluated to investigate the correlation., Results: This meta-analysis included 57 papers in total involving 11,157 cases and 12,281 controls. Results illustrated that the C79G variant genotypes owned a reduced effect on asthma susceptibility (G vs. C: OR=0.94, p=0.037). In the age stratification analysis, C79G polymorphism owned a reduced effect on asthma risk for children (GG vs. CC: OR=0.69, p=0.002; GG vs. CC+CG: OR=0.65, p<0.001). Furthermore, in the ethnic stratification analysis, the C79G variant genotypes also owned a reduced effect on asthma in Asians (GG vs. CC: OR=0.80, p=0.027; GG vs. CC+CG: OR=0.81, p=0.02). Besides, for A46G polymorphism, the ethnic stratification analysis demonstrated that the A46G variant owned an increased effect on asthma susceptibility among Caucasians (G vs. A: OR=1.15, p=0.043). For C491T polymorphism, a considerable reduced effect was found between C491T and asthma susceptibility for children (CT vs. CC: OR=0.70, p=0.03). In the ethnic stratification analysis, the effect was also considerable in the Caucasian subjects., Conclusions: The present meta-analysis demonstrated that C79G and C491T polymorphism may be a defensive factor for asthma, while A46G polymorphism may be a risk factor for asthma among the Caucasian population.

MicroRNAs (miRNAs) are non‑coding RNAs of ~22 nucleotides in length, which serve an important role in numerous diseases. Asthma is a chronic airway inflammatory disease, which is the most common chronic disease among children. The role of miRNA (miR)‑16 in asthma is unclear. The objective of the present study was to examine the underlying molecular mechanism of the involvement of miR‑16 in asthma. A total of 72 volunteers diagnosed with asthma consented to participate in the study, of whom 52 participants were identified to be sensitive to salmeterol and 20 participants were identified to be resistant to salmeterol. Receiver operating characteristic (ROC) curve analysis was performed to compare the expression levels of serum miR‑16 between the sensitive and resistant groups, and to confirm the association between the expression level of serum miR‑16 and forced expiratory volume in 1 sec (FEV1). In silico analysis, a luciferase assay, reverse transcription‑quantitative polymerase chain reaction analysis and western blotting were performed to elucidate the molecular mechanism underlying the role of miR‑16 in asthma. ROC results demonstrated that the serum miR‑16 level may function as a biomarker to predict the response to salmeterol therapy, and the miR‑16 expression level displayed a significant negative correlation with FEV1. According to the in silico analysis, adrenoreceptor β‑2 (ADRB2) was a direct target of miR‑16, and it was further confirmed by luciferase assay that 25 nM miR‑16 mimic had an inhibitory effect on the luciferase activity of the wild‑type ADRB2 3' untranslated region (UTR); the inhibitory effect on the luciferase activity of the wild‑type ADRB2 3'UTR was stronger with 50 nM miR‑16 mimic, and strongest with 75 nM miR‑16 mimic, whereas the luciferase activity of the mutant ADRB2 3'UTR in cells was similar following treatment with 0, 25, 50 or 75 nM miR‑16 mimic. miR‑16 reduced the mRNA and protein expression levels of ADRB2 in a dose‑dependent manner. These results identified that miR‑16 may be used as a predictive biomarker of therapeutic response in asthma.

Rationale: βARs (β-adrenergic receptors) are prototypical GPCRs (G protein-coupled receptors) that play a pivotal role in sympathetic regulation. In heart cells, β 1 AR signaling mediates a global response, including both l-type Ca 2+ channels in the sarcolemma/T tubules and RyRs (ryanodine receptors) in the SR (sarcoplasmic reticulum). In contrast, β 2 AR mediates local signaling with little effect on the function of SR proteins., Objective: To investigate the signaling relationship between β 1 ARs and β 2 ARs., Method and Results: Using whole-cell patch-clamp analyses combined with confocal Ca 2+ imaging, we found that the activation of compartmentalized β 2 AR signaling was able to convert the β 1 AR signaling from global to local mode, preventing β 1 ARs from phosphorylating RyRs that were only nanometers away from sarcolemma/T tubules. This offside compartmentalization was eliminated by selective inhibition of β 2 AR, GRK2 (GPCR kinase-2), βarr1 (β-arrestin-1), and phosphodiesterase-4. A knockin rat model harboring mutations of the last 3 serine residues of the β 1 AR C terminus, a component of the putative βarr1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by β 2 AR activation., Conclusions: β 2 AR stimulation compartmentalizes β 1 AR signaling into nanoscale local domains in a phosphodiesterase-4-dependent manner by targeting the C terminus of β 1 ARs. This finding reveals a fundamental negative feed-forward mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient β 1 AR response of sympathetic excitation.

Background: Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC., Methods: The changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on β2-adrenoceptor (β2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and β2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that β2AR colocalized with the recycling endosome marker Rab11, which indicated β2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth., Results: The microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited β2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and β2AR expression. Interestingly, EHD1 interacted with β2AR and played a novel and critical role in β2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or β2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and β2AR expression in patient specimens., Conclusion: Collectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via β2AR signaling and highlight a potential target for antiangiogenic therapy.

Previous studies demonstrated that depression is more prevalent in women with polycystic ovary syndrome (PCOS). In this study, we aimed to determine whether amitriptyline (AMT), an antidepressant drug, plays a role in preventing PCOS. The results showed that AMT modified ovarian morphology improved the ovarian functions and estrus cycle in estradiol valerate (EV)-induced polycystic ovary (PCO). AMT restored the levels of estradiol (E 2 ), testosterone (T) and progesterone (P 4 ) to normal, and elevated the level of luteinizing hormone (LH) in EV-induced PCO. No significant changes in follicle stimulating hormone (FSH) levels were observed in rats with EV or AMT treatment. The restoration of norepinephrine (NE) level was detected in rats with EV-induced PCO. AMT also altered the expression levels of steroidogenesis genes and beta2-adrenoceptor (beta2-AR) in EV-induced PCO. Our data revealed that AMT improves the ovarian morphology and modifies ovarian expression of beta2-AR and steroidogenesis genes in rats with EV-induced rat PCO. Our data provide support for the hypothesis that AMT is considered as a candidate drug for preventing and treating PCOS along with depression.

Docosahexaenoic acid (DHA), an important ω-3 fatty acid, is abundantly present in the central nervous system and is important in every step of brain development. Much of this knowledge has been based on studies of the role of DHA in the function of the neurons, and reports on its effect on the glial cells are few and far between. We have previously reported that DHA facilitates astrocyte differentiation in primary culture. We have further explored the signaling mechanism associated with this event. It was observed that a sustained activation of the extracellular signal-regulated kinase (ERK) appeared to be critical for DHA-induced differentiation of the cultured astrocytes. Prior exposure to different endocytic inhibitors blocked both ERK activation and differentiation of the astrocytes during DHA treatment suggesting that the observed induction of ERK-2 was purely endosomal. Unlike the β 1 -adrenergic receptor (β 1 -AR) antagonist, atenolol, pre-treatment of the cells with the β 2 -adrenergic receptor (β 2 -AR) antagonist, ICI-118,551 inhibited the DHA-induced differentiation process, indicating a downstream involvement of β 2 -AR in the differentiation process. qRT-PCR and western blot analysis demonstrated a significant induction in the mRNA and protein expression of β 2 -AR at 18-24 h of DHA treatment, suggesting that the induction of β 2 -AR may be due to transcriptional upregulation. Moreover, DHA caused activation of PKA at 6 h, followed by activation of downstream cAMP response element-binding protein, a known transcription factor for β 2 -AR. Altogether, the observations suggest that DHA upregulates β 2 -AR in astrocytes, which undergo endocytosis and signals for sustained endosomal ERK activation to drive the differentiation process.

Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair, however leukocytes lacking β2-adrenergic receptor (β2AR) expression have marked impairments in these processes. β-blockade is a common strategy for the treatment of many cardiovascular etiologies, therefore the objective of our study was to assess the impact of prior β-blocker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and βAR isoform-dependent manner, chronic β-blocker infusion increased splenic vascular cell adhesion molecule-1 (VCAM-1) expression and leukocyte accumulation (monocytes/macrophages, mast cells and neutrophils) and decreased chemokine receptor 2 (CCR2) expression, migration of bone marrow cells (BMC) and peripheral blood leukocytes (PBL), as well as infiltration into the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness was significantly reduced in the PBL of patients receiving β-blocker therapy compared to β-blocker-naïve patients. These results highlight the ability of chronic β-blocker treatment to alter baseline leukocyte characteristics that decrease their responsiveness to acute injury and suggest that prior β-blockade may act to reduce the severity of innate immune responses.

β 2 Adrenergic receptors (β 2 ARs) are G protein-coupled receptors (GPCRs) that are expressed in major insulin target tissues. β 2 ARs play an important role in the regulation of lipid metabolism during aging; however, little is known about the significance of β 2 ARs in the pathogenesis of hepatic fat accumulation in high-fat diet (HFD) mice. This study aims to examine the role of β 2 AR in the development of nonalcoholic fatty liver disease (NAFLD) induced by HFD and the underlying mechanisms. Surprisingly, we found that genetic deletion of β 2 AR significantly increased the liver weight of mice fed a HFD for 20 weeks compared to that of wild-type (WT) mice. Moreover, genetic deletion of β 2 AR could aggravate HFD-induced liver lipid accumulation and liver injury in mice. Mechanistically, we demonstrated that β 2 AR deletion significantly activated PPARγ/CD36 signaling via inactivation of the cAMP response element-binding (CREB) protein to facilitate hepatocellular lipid deposition in HFD mice. Together, our results identify β 2 AR as a plausible therapeutic target for preventing or treating NAFLD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We tested the interactions with four different G protein-coupled receptors (GPCRs) of arrestin-3 mutants with substitutions in the four loops, three of which contact the receptor in the structure of the arrestin-1-rhodopsin complex. Point mutations in the loop at the distal tip of the N-domain (Glu157Ala), in the C-loop (Phe255Ala), back loop (Lys313Ala), and one of the mutations in the finger loop (Gly65Pro) had mild variable effects on receptor binding. In contrast, the deletion of Gly65 at the beginning of the finger loop reduced the binding to all GPCRs tested, with the binding to dopamine D2 receptor being affected most dramatically. Thus, the presence of a glycine at the beginning of the finger loop appears to be critical for the arrestin-receptor interaction., Competing Interests: The authors have declared that no competing interests exist.

G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α 1B -adrenoceptor (α 1B -AR) and β 2 -AR activation. Disrupting this bond by mutagenesis stabilized the α 1B -AR and the β 2 -AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP 1 and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α 1B -AR and β 2 -AR inactive-state mutants. However, the inactive-state β 2 -ARs exhibited decreased agonist affinity, whereas the inactive-state α 1B -ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α 1B -AR and β 2 -AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α 1B -AR mutants but little altered for the β 2 -AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aim: The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion., Methods and Results: By using immunoassaying and western blot analyses, we found that β1 and β2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of β2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a β-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased β1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-β2-AdR levels while decreasing β2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a., Conclusion: Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced β2-AdR and increased β1-AdR levels.

Aims: β-Adrenergic receptors (ADRBs) play a pivotal role in cardiovascular disease. Recently, genetic polymorphisms of ADRB1 and ADRB2 have been suggested to be associated with cardiovascular events and all-cause mortality in coronary artery disease (CAD) patients, but the results of relevant studies are inconsistent and controversial. Therefore, we performed a meta-analysis to investigate the association between ADRB1 and ADRB2 polymorphisms with cardiovascular events and all-cause mortality in CAD patients., Materials and Methods: The PubMed, Ovid, EMBASE, Cochrane, and CINAHL databases were searched for eligible studies published before April 2018. A total of 5495 patients from eight studies were included in our meta-analysis., Results: We found that CAD patients harboring the ADRB2 rs1042714 Glu27 allele exhibited a positive association with cardiovascular events (risk ratio [RR] = 1.31, 95% confidence interval [CI]: 1.08-1.58, p = 0.006), but not with all-cause mortality (RR = 0.97, 95% CI: 0.70-1.35, p = 0.859), compared with patients who were Gln27 homozygotes. No other significant associations were observed between ADRB1 (rs1801252, rs1801253), ADRB2 (rs1042713, rs1800888) polymorphisms and cardiovascular events or all-cause mortality in CAD patients., Conclusion: This study suggests that the identified ADRB2 polymorphism could influence the outcomes of CAD patients, showing important clinical value.

Background: Norepinephrine (NE)-mediated vasoconstriction plays an important role in pulmonary hypertension associated with left heart disease (PH-LHD). However, the role of NE-mediated endothelial cell dysfunction in the pathogenesis of PH-LHD remains to be elucidated., Methods and Results: An enzyme-linked immunosorbent assay showed that the NE concentration in the plasma of patients with PH-LHD was higher and the nitrate-nitrite concentration was lower than those in the control group. NE treatment decreased phospho-Ser633-eNOS and β 2 -adrenergic receptor (β 2 -AR) levels in the membrane of human pulmonary artery endothelial cells (HPAECs) analysed by western blot analysis. Consistently, fluorescence microscopy and flow cytometry showed that nitric oxide (NO) production was also decreased in HPAECs. Coimmunoprecipitation confirmed a direct interaction between β 2 -AR and endothelial NO synthase (eNOS). Overexpression of β 2 -AR attenuated the decline in phospho-Ser633-eNOS and NO production. Additionally, the expression of phospho-Ser633-eNOS and β 2 -AR was decreased in human pulmonary artery endothelium. Finally, our results indicate that NE stimulated HPAEC proliferation, which was blocked by protein kinase A inhibitor or protein kinase B (PKB-AKT) inhibitor., Conclusions: These data provide a novel mechanism for NE-decreased endothelium-derived NO and NE-induced HPAEC proliferation that leads to PH-LHD, suggesting a potential therapeutic target for PH-LHD., (© 2018 Wiley Periodicals, Inc.)

Significant adverse impact of various forms of psychological stress on susceptibility to infection, altered wound healing, increased prevalence and severity of hypersensitivity diseases, and even increased mortality in cancer patients has been well described. Yet these observations are limited by often unpredictable individual responses to various stressful situations. These associations are further clouded by natural variability among diverse forms of and responses to chronic life stressors and associated comorbid conditions. This is particularly true for inflammatory diseases where gene/external environmental interactions are well-described. What is much less understood is gene-internal environmental (i.e., psychological) interactions that commonly affect disease activity and possible susceptibility. We have used selected single nucleotide polymorphisms of stress hormone and regulatory cytokine receptors to categorize both baseline and stress-associated immune parameters for the a priori classification of individuals with the most stress susceptible immune systems to identify those most responsive to a stress reduction/management-based intervention., Competing Interests: Potential Conflicts of Interest: None disclosed.

The region encoding the N-terminal of human β2-adrenergic receptor gene ( ADRB2 ) shows several polymorphisms. To this end, we studied change in susceptibility and/or response to therapy in 175 asthmatic children and adolescents by the two most common variants of the ADRB2 gene namely, rs1042713 (Gly16Arg) and rs1042714 (Gln27Glu). Although, the variants did not correlate with risk of development nor with the severity of the asthma, Gly16/Glu27 haplotype in homozygous individuals conferred protection against development of asthma and was associated with a lower frequency of dyspnea and sputum production. In contrast, the Arg16/Gln27 haplotype was associated with a better response to treatment. These findings show that the risk of development of asthma or response to treatment can be, respectively, deciphered by the detection of both rs1042713 and rs1042714 variants in ADRB2 gene.

Background/aim: The β2-adrenergic receptor (β2AR) is highly expressed in various human cancers and has been linked to tumor growth and metastases. Although β2AR is considered a novel therapeutic target of human neoplasms, the clinicopathological significance of β2AR expression in patients with pulmonary pleomorphic carcinoma (PPC) remains unclear. The aim of this study was to clarify the prognostic impact of β2AR in PPC., Patients and Methods: One hundred and five Japanese patients with surgically resected PPC were included in the study. The expression levels of β2AR were assessed by immunohistochemistry in specimens from the resected tumors, and their association with patient survival, as well as with tumor characteristics was investigated., Results: β2AR was highly expressed in 63% of all patients, irrespective of adenocarcinoma components present. The β2AR expression was significantly associated with lymph node metastasis, lymphatic permeation and tumor cell proliferation in PPC patients with early-stage disease (stage I or II). A high β2AR expression was identified as a significant predictor of worse prognosis for PPC patients during early stages of the disease. Multivariate analysis confirmed that β2AR expression was an independent factor for predicting the overall survival of PPC patients., Conclusion: β2AR can serve as a significant predictor of tumor aggressiveness and poor survival for PPC patients, especially those with early-stage disease., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Primary and secondary lymphoid organs are heavily innervated by the autonomic nervous system. Norepinephrine, the primary neurotransmitter secreted by post-ganglionic sympathetic neurons, binds to and activates β-adrenergic receptors expressed on the surface of immune cells and regulates the functions of these cells. While it is known that both activated and memory CD8 + T-cells primarily express the β2-adrenergic receptor (β2-AR) and that signaling through this receptor can inhibit CD8 + T-cell effector function, the mechanism(s) underlying this suppression is not understood. Under normal activation conditions, T-cells increase glucose uptake and undergo metabolic reprogramming. In this study, we show that treatment of murine CD8 + T-cells with the pan β-AR agonist isoproterenol (ISO) was associated with a reduced expression of glucose transporter 1 following activation, as well as decreased glucose uptake and glycolysis compared to CD8 + T-cells activated in the absence of ISO. The effect of ISO was specifically dependent upon β2-AR, since it was not seen in adrb2 -/- CD8 + T-cells and was blocked by the β-AR antagonist propranolol. In addition, we found that mitochondrial function in CD8 + T-cells was also impaired by β2-AR signaling. This study demonstrates that one mechanism by which β2-AR signaling can inhibit CD8 + T-cell activation is by suppressing the required metabolic reprogramming events which accompany activation of these immune cells and thus reveals a new mechanism by which adrenergic stress can suppress the effector activity of immune cells.

The β2‑adrenergic receptor (β2‑AR, encoded by the ADRB2 gene) is a member of the G‑protein‑coupled receptor superfamily that can be stimulated by catecholamines. Studies in vivo and in vitro have confirmed that β‑blockers (β‑AR antagonists) exert antitumor effects on various tumors. Furthermore, ADRB2 single‑nucleotide polymorphisms (SNPs) have been identified to alter the expression and conformation of β2‑AR, which may alter the β‑blocker drug response. The aim of the present study was to investigate the effect of β‑blockers on triple‑negative breast cancer cells and determine whether ADRB2 SNPs affect the response to β‑blocker drugs. Propranolol and ICI 118,551 significantly inhibited the viability of MDA‑MB‑231 cells, arrested cell cycle progression at G0/G1 and S phase and induced cell apoptosis. Western blot analysis indicated that the phosphorylation levels of extracellular‑signal‑regulated kinase (ERK)1/2 and the expression levels of cyclo‑oxygenase 2 (COX‑2) were significantly decreased following β‑blocker treatment. Four haplotypes, which comprised ADRB2 SNPs rs1042713 and rs1042714, were transfected into 293 cells. After 24 and 48 h of transfection, ADRB2 mRNA expression was significantly decreased in mutant groups compared with the wild‑type group. The ADRB2 SNPs exerted no effect on cell viability, but did affect the drug response of ICI 118,551. Furthermore, ADRB2 SNPs also affected the regulatory function of ICI 118,551 on the ERK/COX‑2 signaling pathway. Collectively, propranolol and ICI 118,551 inhibited the viability of MDA‑MB‑231 cells by downregulating the ERK/COX‑2 signaling pathway and inducing apoptosis. The results of the present study indicated that SNPs rs1042713 and rs1042714 of ADRB2 affected the response to ICI 118,551, and the underlying molecular mechanism was elucidated.

Background/aim: Identifying the role of the sympathetic nervous system (SNS) in tumor progression is among the most important challenges in cancer research. This study aimed to investigate the role of the SNS and β-adrenoreceptor in gastric cancer progression., Materials and Methods: The density of SNS was quantified by immunohistochemical staining for tyrosine hydroxylase in 115 surgically-resected gastric cancer specimens. Immunostaining for β1- and β2-adrenoreceptor was also performed to examine the β-adrenoreceptor expression status in gastric cancer. Then the association of protein expression status with histological grade, pathological tumor stage (pT), and pathological node stage of gastric cancer was investigated., Results: The SNS density of pT4 tumors was significantly lower than that of pT1-3 tumors. The SNS density was positively correlated with β1-adrenoreceptor expression status. In addition, lower β1-adrenoreceptor expression was significantly associated with increased lymph node metastasis. Reduced β2-adrenoreceptor staining proportion was significantly associated with worse histological grade. Furthermore, the proportion of β2-adrenoreceptor staining was significantly lower in tumors with diffuse-type histology, than those with intestinal-type histology., Conclusion: A lower SNS density and β-adrenoreceptor expression was associated with an aggressive oncogenic behavior including worse histological grade, advanced pT, and increased lymph node metastasis. SNS and β-adrenergic pathway are involved in the negative regulation of gastric cancer progression., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Using comprehensive analysis of heart rate (HRV) and blood pressure (BPV) short-term variability we estimated the time course of changes of autonomic nervous system remodeling in two stages of doxorubicin-induced cardiomyopathy (DCM). We also investigated the level of gene expression of cardiac β-1 (β-1AR) and β-2 (β-2AR) adrenoceptors. Experiments were performed in adult male Wistar rats equipped with indwelling catheters for BP recording and blood withdrawal. A 15 mg/kg total cumulative dose of doxorubicin was injected i.p. to rats to induce DCM or saline for control (n=18). Rats were assessed for general toxicity, cardiovascular hemodynamic and echocardiography before treatment (n=6), 35 days (DOX35; n=6) and 70 days (DOX70; n=6) post-treatment. HRV was evaluated by spectral analysis, Poincaré plots, sample and approximate entropy. Expression of β-1AR and β-2AR mRNA was evaluated by RT-qPCR. Doxorubicin-treated rats exhibited poor general condition and lower survival than saline-treated rats. In DOX35 rats, there were no echocardiography signs of decompensation, no increase in serum cardiac troponins, but there was an increase of HRV and decrease of HR complexity. In these rats typical microscopic signs of cardiotoxicity were seen along with over-expression of β-1AR mRNA. 70 days post-treatment echocardiography revealed signs of decompensation and serum cardiac troponin T was increased. At this stage BPV decreased. In conclusion, HRV increase matches transient over-expression of cardiac β-1AR mRNA in compensate stage of DCM while decompensate stage of DCM is characterized by a decrease of BPV and no changes in β-1AR and β-2AR gene expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Depressive symptoms in parents and in youths have been found to relate to disease comorbidity processes in children, including greater disease-related impairment and poorer clinical outcomes. The current study sought to assess whether coming from a family characterized by more depressive symptoms on average would potentiate the effects of changes in youths' own negative mood on the expression of two receptor genes relevant to asthma that are the primary targets of asthma medication, such that the combination of low child negative mood in the context of greater parental depressive symptoms would relate to the lowest levels of gene expression. One-hundred-twenty youths with diagnosed asthma and their parents participated every 6 months for 2 years. Parents reported on their depressive symptoms, children reported negative mood symptoms, and youths completed blood draws from which expression of Glucocorticoid Receptor (GR) and Beta2 Adrenergic Receptor (β 2 -AR) genes was extracted. Multilevel linear modeling revealed significant interactions between average levels of parental depressive symptoms and changes in youths' negative mood symptoms predicting gene expression, such that youths expressed significantly less GR and β 2 -AR during times when they experienced more negative mood symptoms, but this was only true if they came from families with higher levels of average parental depressive symptoms. The current study identifies novel and biologically-proximal molecular signaling patterns that connect depressive symptoms to pediatric asthma while also highlighting the important role of family environment for biological processes that may operate within depression comorbidity.

Background and Objective: Asthma is a complex and heterogeneous disease. We found gene-gene interactions between IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 in asthmatic Chinese Han children. This 4-locus set constituted an optimal statistical interaction model. We examined associations between the 4-gene model (IL13, IL4, FCER1B, and ADRB2) and the Asthma Predictive Index (API) and atopy in Chinese Han children., Methods: Four single-nucleotide polymorphisms in the 4 genes were genotyped in 385 preschool children with wheezing symptoms using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The t test and x2 tests were used for the analysis., Results: Significant correlations were found between the 4-locus gene model and a stringent and loose API (both P<.0001). Additionally, a high-risk asthma genotype was a risk factor for a positive API (stringent API, OR=4.08; loose API, OR=2.36). We also found a statistically significant association between the 4-locus gene model and atopy (P<.01, OR=2.09)., Conclusions: Our results indicated that the 4-locus gene model consisting of L13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 was associated with the API and atopy. These findings provide evidence that this gene model can be used to determine a high risk of developing asthma and atopy in Chinese Han children.

Individual differences in regulation of fear and extinction memory play significant roles in the aetiology development of post-traumatic stress disorder (PTSD). Previous animal based studies showed that the activity of β-adrenergic receptors (β-ARs) are involved in memory modulation. However in humans it is not clear that whether genetic variability in β-ARs contributes to individual differences of fear and extinction memory. In the current study, we investigated the role of a common single-nucleotide polymorphism of β 2 -adrenergic receptor (ADRB2) gene in fear memory acquisition, fear memory extinction, extinction recall and fear generalization in human participants. Ninety-one male participants were exposed to a Pavlovian fear conditioning and their fear responses were assessed by the skin conductance response. Participants were genotyped for a polymorphism (rs2400207) located within the promoter region of the human ADRB2. Differences between genotypes were observed in the extinction memory recall test but not in fear acquisition, extinction learning and fear generalization. Particularly, A-allele carriers of rs2400707 displayed successful retention of extinction memory and prevented the return of fear during recall test. The results revealed the involvement of human noradrenergic system in the retention of extinction memory and genetic variability in this system may underlie individual differences in PTSD. Furthermore, rs2400207 polymorphism of ADRB2 gene may play a key role in the treatment efficacy of PTSD and can be a basis for future studies investigating a personalized medicine for fear memory related disorders., (Copyright © 2018. Published by Elsevier Inc.)