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Background: β-arrestin2 and β2-adrenergic receptor (β2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated β2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of β-arrestin2. Methods: Immunofluorescence and Western blot were used to detect the expression of β-arrestin2 and β2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used β-arrestin2 -/- mice to demonstrate the regulatory role of β-arrestin2 in DEN mice. The subsets of T cells were quantified by flow cytometry. MTT and wound healing assay were applied to detect the proliferation and migration of cells. Co-immunoprecipitation assay was used to detect the link of β-arrestin2 and β2-AR in HSCs. Effect of β-arrestin2 overexpression on β2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA. Results: The expression of β2-AR was significantly increased, while β-arrestin2 was decreased in HSCs of HCC tissues. And β-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased β2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. β-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro , selective activation of β2-AR in HSCs promoted the proliferation and migration of HCC cells. β-arrestin2 overexpression enhanced co-immunoprecipitation of β-arrestin2 and β2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs. Conclusion: Our study demonstrated that β2-AR activation in HSCs induces the proliferation and migration of HCC cells may be through Akt signaling, and this effect appears to be regulated by β-arrestin2., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

β-Adrenergic receptors (βARs) regulate normal and pathophysiological heart function through their impact on contractility. βARs are also regulators of immune function where they play a unique role depending on the disease condition and immune cell type. Emerging evidence suggests an important role for the β 2 AR subtype in regulating remodeling in the pathological heart; however, the importance of these responses has never been examined. In heart failure, catecholamines are elevated, leading to chronic βAR activation and contributing to the detrimental effects in the heart. We hypothesized that immune cell β 2 AR plays a critical role in the development of heart failure in response to chronic catecholamine elevations through their regulation of immune cell infiltration. To test this, chimeric mice were generated by performing bone marrow transplant (BMT) experiments using wild-type (WT) or β 2 AR knockout (KO) donors. WT and β 2 ARKO BMT mice were chronically administered the βAR agonist isoproterenol. Immune cell recruitment to the heart was examined by histology and flow cytometry. Numerous changes in immune cell recruitment were observed with isoproterenol administration in WT BMT mice including proinflammatory myeloid populations and lymphocytes with macrophages made up the majority of immune cells in the heart and which were absent in β 2 ARKO BMT animal. β 2 ARKO BMT mice had decreased cardiomyocyte death, hypertrophy, and interstitial fibrosis following isoproterenol treatment, culminating in improved function. These findings demonstrate an important role for immune cell β 2 AR expression in the heart's response to chronically elevated catecholamines. NEW & NOTEWORTHY Immune cell β 2 -adrenergic receptors (β 2 ARs) are important for proinflammatory macrophage infiltration to the heart in a chronic isoproterenol administration model of heart failure. Mice lacking immune cell β 2 AR have decreased immune cell infiltration to their heart, primarily proinflammatory macrophage populations. This decrease culminated to decreased cardiac injury with lessened cardiomyocyte death, decreased interstitial fibrosis and hypertrophy, and improved function demonstrating that β 2 AR regulation of immune responses plays an important role in the heart's response to persistent βAR stimulation.

Asthma is heterogeneous with respect to clinical presentation, underlying disease mechanisms and response to existing drugs making tailored therapy desirable. Pharmacogenetics, the study of the influence of genetic polymorphisms on drug efficacy and/or adverse effects, is relatively advanced in asthma with replicated genetic associations identified in the main drug classes. In the present issue of Clinical Science, Lipworth and co-workers report a proof-of-concept study and demonstrate that, in asthmatic children carrying the β2-adrenergic receptor gene Arg16 polymorphism, a combination of corticosteroid plus leukotriene receptor antagonist provides superior asthma control (e.g. quality of life scores) compared with corticosteroid plus a long-acting β2-adrenergic receptor agonist as add-on therapy. The basis of these observations is well founded, as it has been demonstrated previously that the Arg16 polymorphism may confer an increased risk of exacerbation following prolonged β2-adrenergic receptor agonist use. These results suggest Gly16Arg genotyping in Caucasian asthma patients may have a role in the clinical management of asthma by influencing the decision of which add-on therapy to prescribe; however, larger studies are required to provide definitive conclusions regarding the clinical utility of this approach. [ABSTRACT FROM AUTHOR]

The high-resolution crystal structure of an engineered human β2-adrenergic receptor has recently been resolved, suggesting a molecular mechanism by which cholesterol may mediate receptor dimerization. Here, we present a critical examination of new structural and functional insights derived from unprecedented preliminary homology modeling of cannabinoid receptors, obtained using the crystal structure of β2-adrenergic receptor as a template. The structural comparison between the two cannabinoid receptor subtypes and the β2-adrenergic receptor may be of particular interest, by providing important clues for the elucidation of the structural determinants involved in cholesterol binding. In addition, the implications of G protein coupled receptor dimerization, as well as the role of cholesterol in this process, are briefly discussed. [ABSTRACT FROM AUTHOR]

Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a β2-adrenergic receptor agonist, nylidrin, was identified as an antiviral compound against influenza A virus. The molecule was effective against multiple isolates of subtype H1N1, but had limited activity against subtype H3N2, depending on the strain. By examining the antiviral activity of its chemical analogues, we found that ifenprodil and clenbuterol also had reliable inhibitory effects against A/H1N1 strains. Field-based pharmacophore modeling with comparisons of active and inactive compounds revealed the importance of positive and negative electrostatic patterns of phenyl aminoethanol derivatives. Time-of-addition experiments and visualization of the intracellular localization of nucleoprotein NP demonstrated that an early step of the virus life cycle was suppressed by nylidrin. Ultimately, we discovered that nylidrin targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational change of HA at acidic pH. In a mouse model, preincubation of a mouse-adapted influenza A virus (H1N1) with nylidrin completely blocked intranasal viral infection. The present study suggests that nylidrin could provide a core chemical skeleton for the development of a direct-acting inhibitor of influenza A virus entry. [ABSTRACT FROM AUTHOR]

β2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of β2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2−/−), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2+/+) and Adrb2−/− mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2+/+ and Adrb2−/− mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2+/+ UAC mice; the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression; the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA. • Adrb2-expressing MSCs in subchondral bone play important roles in TMJ-OA progression. • Conditional deletion of Adrb2 in nestin+ MSCs attenuated TMJ-OA. • Adrb2-expressing MSCs may serve as novel therapeutic targets for TMJ-OA. [ABSTRACT FROM AUTHOR]

Several case-control studies have explored the possible association between polymorphism in the β2 adrenoreceptor gene (β2AR), hypertension, and obesity—the focus being in particular on the Arg16Gly and Gln27Glu substitutions, which appear to modify the extracellular part of the β2AR with possible functional modification. However, controversial results have been obtained.The analysis refers to 993 middle-age men characterized for Arg16Gly and Gln27Glu polymorphism of the β2AR. In this general population sample there were 563 overweight, 160 obese, and 405 hypertensive individuals, of whom 171 were receiving antihypertensive therapy.The genotype frequencies for codon 16 were: GlyGly = 38%; ArGly = 45%; ArgArg = 17%. The frequencies for codon 27 were: GlnGln = 50%; GlnGlu = 39%; GluGlu = 11%. Codon 16 and codon 27 polymorphisms were in linkage disequilibrium. No differences were detected in body mass index and blood pressure across different genotypes. Likewise, no association was detected between either of the two polymorphisms and being overweight (codon 27: χ2 = 0.1, codon 16: χ2 = 1.4), obesity (codon 27: χ2 = 0.1, codon 16: χ2 = 1.7) and hypertension (codon 27: χ2 = 2.7, codon 16: χ2 = 1.9). The odds ratio (with 95% confidence intervals) for overweight, obesity, and hypertension were not different between genotypes. Likewise, no difference in the anthropometric indices of fat distribution, fasting blood glucose, serum insulin, triglycerides, uric acid, and HOMA index could be detected between groups.In summary, in this large unselected sample of adult white men, genetic variation in the β2AR was not associated with blood pressure or with overweight, obesity, and fat distribution. [Copyright &y& Elsevier]

Pseudoexfoliation syndrome (PEXS) and glaucoma (PEXG) are assumed to be caused by a generalized elastosis leading to the accumulation of PEX material in ocular as well as in extraocular tissues. The exact pathophysiology of PEXS is still elusive. PEXG, the most common type of secondary open-angle glaucoma (OAG), is characterized by large peaks of intraocular pressure (IOP) with a progressive loss of the visual field. Agonistic autoantibodies (agAAbs) against the β 2 -adrenergic receptor (AR) have been shown to be present in sera of patients with primary and secondary OAG and ocular hypertension and are seemingly linked to IOP. In the present study, we investigated the autoantibodies directed against the β 2 -AR in sera of patients with PEXS and PEXG. We recruited 15, 10, and 15 patients with PEXG, PEXS, and primary OAG, respectively. Ten healthy individuals served as controls. All patients underwent standard ophthalmological examination with Octopus G1 perimetry. agAAbs prepared from serum samples were analyzed in a rat cardiomyocyte-based bioassay for the presence of agAAbs. We identified the interacting loop of the β 2 -AR and the immunoglobulin G (IgG) subclasses using synthetic peptides corresponding to the extracellular loops of the receptors and enzyme-linked immunosorbent assay, respectively. None of the controls were β 2 -agAAb-positive (0.2 ± 0.5 U). No β 2 -agAAbs (0.2 ± 0.4 U), but inhibitory β 2 -AAbs were observed in 80% of the patients that partially blocked the drug-induced β 2 -adrenergic stimulation; 5.8 ± 1.7 U vs. 11.1 ± 0.9 U for clenbuterol in the absence and the presence of sera from patients with PEXS, respectively. Epitope analyses identified the third extracellular loop of the β 2 -AR as the target of the inhibitory β 2 -AAbs, being of IgG3 subtype in PEXS patients. In contrast, patients with PEXG showed β 2 -agAAbs (5.6 ± 0.9 U), but no inhibitory ones. The β 2 -agAAbs levels of patients with PEXG and primary OAG patients (3.9 ± 2.8 U; p > 0.05) were at a similar level. In two cases of PEXG, the β 2 -agAAbs exert synergistic effects with clenbuterol. The activity increased from 11.5 ± 0.3 (clenbuterol only) to 16.3 ± 0.9 U. As autoimmune mechanisms were reportedly involved in the pathogenesis of glaucoma, agonistic and inhibitory β 2 -AAbs seem to be a part of this multifactorial interplay., Competing Interests: RK as the patent EP1832600A1. MH has the patent EP1832600A1. GW was employed by Berlin Cures GmbH, Berlin, Germany and holds the patent P1832600A1. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hohberger, Schlötzer-Schrehard, Mardin, Lämmer, Munoz, Kunze, Herrmann and Wallukat.)

Activation of the sympathoadrenal system is associated with sleep apnea-related symptoms and metabolic dysfunction induced by chronic intermittent hypoxia (IH). IH can induce hormonal imbalances and growth retardation of the craniofacial bones. However, the relationship between IH and β2-adrenergic receptor signaling in the context of skeletal growth regulation is unclear. This study aimed to investigate the role of β2-adrenergic receptors in IH-induced mandibular growth retardation and bone metabolic alterations. Male 7-week-old Sprague-Dawley rats were subjected to IH for 3 weeks. IH conditions were established using original customized hypoxic chambers; IH was induced at a rate of 20 cycles per hour (oxygen levels changed from 4 to 21% in one cycle) for 8 h per day during the 12 h "lights on" period. The rats received intraperitoneal administration of a β2-adrenergic antagonist (butoxamine) or saline. To exclude dietary effects on general growth, the normoxic rats with saline, normoxic rats with butoxamine, and IH rats with butoxamine were subjected to food restriction to match the body weight gains between IH and other three groups. Body weight, heart rate, blood pressure, and plasma concentrations of leptin, serotonin, and growth hormone were measured. Bone growth and metabolism were evaluated using radiography, microcomputed tomography, and immunohistochemical staining. Plasma leptin levels were significantly increased, whereas that of serotonin and growth hormone were significantly decreased following IH exposure. Leptin levels recovered following butoxamine administration. Butoxamine rescued IH-induced mandibular growth retardation, with alterations in bone mineral density at the condylar head of the mandible. Immunohistochemical analysis revealed significantly lower expression levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the condylar head of IH-exposed rats. Conversely, recovery of RANKL expression was observed in IH-exposed rats administered with butoxamine. Collectively, our findings suggest that the activation of β2-adrenergic receptors and leptin signaling during growth may be involved in IH-induced skeletal growth retardation of the mandible, which may be mediated by concomitant changes in RANKL expression at the growing condyle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hong, Hosomichi, Maeda, Ishida, Usumi-Fujita, Yoshida and Ono.)

Beta-2-adrenergic receptor is a member of the G protein-coupled receptor superfamily, which is highly expressed in most malignancies. There is increasing evidence showing that beta-2-adrenergic receptors are associated with carcinogenesis, proliferation, immune regulation, invasion, angiogenesis, clinical prognosis and treatment resistance in malignancies. Polymorphisms of the ADRB2 gene have been confirmed to be associated with transcriptional activity, mRNA translation, and beta-2-adrenergic receptor expression and sensitivity. This review discusses clinically relevant examples of single nucleotide polymorphisms of ADRB2 in malignancies and the effects of these polymorphisms on cancer susceptibility, prognosis and treatment response of cancer patients.

Background: Failure of the proliferation and infiltration of tumor-specific T cells in tumor site has been considered as one of important reasons induce the inefficiencies of immune checkpoint therapies in advanced cancers. Therefore, we aimed to demonstrate how combinatorial sympathetic and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade affects the tumor growth of melanoma-bearing mice and potential mechanisms., Methods: Tumor growth was measured and the infiltrating immune cell populations were observed with flow cytometry in B16-F10 melanoma-bearing mice treated with combined sympathetic and immune checkpoint blockade, using anti-CTLA-4 antibodies. The expression of adrenergic receptors was investigated in human peripheral blood mononuclear cells and their subpopulations, and the proliferation of T cell subsets was detected when stimulated by norepinephrine and its antagonists., Results: B16-F10 tumor growth was associated with infiltrating CD8 + T cells. Combinatorial sympathetic and CTLA-4 blockade inhibited tumor growth and enhanced CD8 + infiltration. Meanwhile, all β1, β2 and β3 adrenergic receptors were found to be expressed in human peripheral blood mononuclear cells, activated T cells, monocytes, and monocyte-induced dendritic cells. β2-adrenergic receptors were expressed in most CD4 + T cells with increased expression in activated CD8 + T cells. Moreover, norepinephrine was able to prevent CD4 + T cell proliferation and β2-adrenergic receptor antagonists could reverse the inhibition of CD4 + , but not CD8 + cell proliferation., Conclusions: We conclude that the combination of sympathetic and CTLA-4 inhibitors is more effective for inhibiting melanoma progression than a single treatment and might enhance the infiltration of T cells in the tumor site, offering a novel therapeutic approach for immune checkpoint targeting., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr-20-2738). Dr. MJ reports personal fees from Astrazeneca Japan, outside the submitted work. The other authors have no conflicts of interest to declare., (2021 Translational Cancer Research. All rights reserved.)

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. β 2 -Adrenergic receptor (β 2 AR) is a novel and key target for EPC homing. Here, we proposed that attenuated β 2 AR signaling contributes to EPCs dysfunction, whereas enhanced β 2 AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. β 2 AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of β 2 AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The β 2 AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against β 2 AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of β 2 AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired β 2 AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of β 2 AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of β 2 AR in endothelium repair still requires further investigation. NEW & NOTEWORTHY Impaired β 2 -adrenergic receptor (β 2 AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. β 2 AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating β 2 AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that β 2 AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial-mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium-derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors. Recent lines of evidence have suggested that EMT confers cancer cells with tumor-initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a β2-adrenergic receptor (β2-AR) agonist as a low-molecular-weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non-selective β-adrenergic receptor (β-AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non-selective β-AR antagonist, propranolol, and the CRISPR/Cas9 system-mediated deletion of the β2-AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by β2-AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting β2-AR signals to be a promising cancer therapeutic target for treatment of OSCC., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Prolonged hypersecretion of catecholamine induced by chronic stress may correlate with malignant progression of cancer. β2-adrenergic receptor (β2-AR) overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins, such as Her2. In the present study, we demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR-199a-5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ-secretase, leading to shedding of Her2 extracellular domain (ECD) by ADAM10 and subsequent intramembranous cleavage of Her2 intracellular domain (ICD) by presenilin-dependent γ-secretase, nuclear translocation of Her2 ICD, and enhanced transcription of tumor metastasis-associated gene COX-2. Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, nuclear localization of Her2 was significantly correlated with overexpression of β2-AR in human breast cancer tissues, indicating that catecholamine-induced β2-AR activation plays decisive roles in tumor metastasis. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by β2-AR-mediated signaling controls a novel Her2-mediated signaling transduction., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Considerable evidence suggests that the sympathetic nervous system, mainly via adrenergic signaling, contributes to prostate cancer (PCa) progression. However, the underlying molecular mechanisms remain unknown., Methods: The expression level of β 2 -adrenergic receptor (β 2 -AR) in tissue microarray was evaluated by immunohistochemistry. The effects of isoproterenol (ISO) or Sonic Hedgehog (Shh) signaling inhibitor on tumor growth were analyzed in proliferation and colony formation assays. The apoptosis of cells was analyzed by flow cytometry. Small hairpin RNA-based knockdown of β 2 -AR or Gli1 was validated by Western blot analysis and real-time PCR. Effects of β 2 -AR on prostate carcinogenesis in vivo were observed in a mouse xenograft model. The expression levels of the indicated proteins in xenograft tissues were evaluated by immunohistochemistry. Expression levels of Shh signaling components and downstream proteins were assessed by immunoblotting., Results: We determined that β 2 -AR was expressed at significantly higher levels in carcinoma than in normal prostate tissues. β 2 -AR signaling also played an essential role in sustaining PCa cell proliferation in vivo and in vitro. We also found that inhibition of Shh signaling or knockdown of Gli1 expression significantly restrained ISO-induced cell proliferation in vitro. ISO alleviated the apoptosis induced by suppressing or knocking down of Gli1. The β 2 -AR agonist ISO upregulated the transcription and protein expression of target genes of Shh signaling, including c-Myc, Cyclin D1, and VEGFA. Conversely, knocking down β 2 -AR markedly suppressed the expression of Shh components in vivo and in vitro. In Gli1 knockdown cells, ISO failed to increase the expression of target genes of Shh signaling., Conclusions: In this study, we uncovered an important role of β 2 -AR signaling in regulating the Shh pathway activity in PCa tumorigenesis and provide further insight into the mechanism of the involvement of the Hh signaling pathway. Furthermore, given the efficacy of β 2 -adrenergic modulation on PCa, our study might also add evidence for potential therapeutic options of β-blockers for PCa., (© 2020 The Authors. The Prostate published by Wiley Periodicals LLC.)

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the β2-adrenergic receptor (β2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective β2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a β2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the β2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of β2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

The β 2 -adrenergic receptor (β 2 AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein G s and stimulates cAMP formation. Functional studies have shown that the β 2 AR also couples to inhibitory G protein G i , activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001 , re15 (2001)]. A crystal structure of the β 2 AR-G s complex revealed the interaction interface of β 2 AR-G s and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the β 2 AR signaling through G s and its preferential coupling to G s over G i is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β 2 AR in response to the full agonist BI-167107 and G s and G i1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β 2 AR are qualitatively the same for G s and G i1 , we detected distinct differences between the β 2 AR-G s and the β 2 AR-G i1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of G s These differences between the β 2 AR-G s and β 2 AR-G i1 complexes in ICL2 may be key determinants for G protein coupling selectivity., Competing Interests: Competing interest statement: B.K.K. is co-founder of and consultant for ConfometRx, Inc., (Copyright © 2020 the Author(s). Published by PNAS.)

Purpose: The β2-adrenergic receptor (β2AR) is highly expressed in various human cancers. The prognostic significance of its expression in patients with colorectal cancer (CRC) remains unclear. The aim of this study was to assess the prognostic role of β2AR expression in patients with surgically resected CRC., Methods: One hundred and forty-seven patients with surgically resected CRC were examined using immunohistochemistry. The expression of β2AR was assessed in the specimens of resected primary tumors., Results: β2AR was expressed in 52.3% of the patients' tumors. β2AR expression was significantly associated with T factor, N factor, and tumor cell proliferation (Ki-67 labeling index). Univariate analysis demonstrated that T factor, N factor, tumor stage, lymphatic permeation, vascular invasion, perineural invasion, β2AR expression, and Ki-67 labeling index were significant prognostic factors for worse disease-free survival (DFS); all but T factor were also significant predictors for worse overall survival (OS). Multivariate analysis confirmed that expression of β2AR was a significant prognostic marker for predicting worse DFS and OS., Conclusion: β2AR expression was identified as a significant independent prognostic factor in patients with surgically resected CRC.

The β 2 -adrenergic receptor (β 2 AR) is relevant for surfactant formation in alveolar type 2 cells and reduction of intracellular calcium concentration in bronchial muscle cells and thus for secretolytic and bronchospasmolytic effects. Herbal medicinal products that affect the β 2 AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the β 2 -adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of β 2 AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of β 2 AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express β 2 AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the β 2 AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged β 2 AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged β 2 AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß 2 AR internalization either. Furthermore, SNAP-tagged β 2 AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing β 2 AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing β 2 AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the β 2 -adrenergic signal transduction., (© 2020 The Author(s).)

G-protein-coupled receptors (GPCRs) mediate a wide range of human physiological functions by transducing extracellular ligand binding events into intracellular responses. GPCRs can activate parallel, independent signaling pathways mediated by G proteins or β-arrestins. Whereas "balanced" agonists activate both pathways equally, "biased" agonists dominantly activate one pathway, which is of interest for designing GPCR-targeting drugs because it may mitigate undesirable side effects. Previous studies demonstrated that β-arrestin activation is associated with transmembrane helix VII (TM VII) of GPCRs. Here, single-molecule fluorescence spectroscopy with the β 2 -adrenergic receptor (β 2 AR) in the ligand-free state showed that TM VII spontaneously fluctuates between one inactive and one active-like conformation. The presence of the β-arrestin-biased agonist isoetharine prolongs the dwell time of TM VII in the active-like conformation compared with the balanced agonist formoterol, suggesting that ligands can induce signaling bias by modulating the kinetics of receptor conformational exchange. • TM VII of β 2 AR naturally fluctuates between inactive and active-like conformations • Agonists prolong the dwell time of the active-like conformation of TM VII • A β-arrestin-biased agonist is more stabilizing than a balanced agonist TM helix VII of β 2 AR mediates cellular signaling via the β-arrestin pathway. Using an in vitro single-molecule fluorescence system, Lamichhane et al. present evidence that a β-arrestin-biased agonist induces signaling bias by controlling the kinetics of exchange between inactive and active-like conformations of TM VII. [ABSTRACT FROM AUTHOR]

Recent studies have shown that β2-Adrenoreceptor is a regulator of the a-synuclein gene driving risk of Parkinson's disease. The β2-AR agonist (R)-salbutamol, eutomer of rac-salbutamol, may hold therapeutic potential for Parkinson's disease (PD) following nasal administration. In this study, we use desorption electrospray ionization mass spectrometry (DESI-MS) to analyze spatial distribution of (R)-salbutamol in rat brain following nasal and intravenous administration. Here, we report that (R)-salbutamol efficiently deliver to the brain and had more drug dosage exposure in rat's brain through nasal route administration than that of intravenous route administration. In conclusion, administering (R)-salbutamol through nasal route of administration may hold advantages in improving spatial distribution and increased exposure of drug in brain. [ABSTRACT FROM AUTHOR]

In addition to local spinal cord dysfunction, spinal cord injury (SCI) can result in decreased skeletal muscle mitochondrial activity and muscle atrophy. Treatment with the FDA-approved β 2 -adrenergic receptor (ADRB2) agonist formoterol has been shown to induce mitochondrial biogenesis (MB) in both the spinal cord and skeletal muscle and, therefore, has the potential to address comprehensive mitochondrial and organ dysfunction following SCI. Female C57BL/6 mice were subjected to moderate contusion SCI (80 Kdyn) followed by daily administration of vehicle or formoterol beginning 8 h after injury, a clinically relevant time-point characterized by a 50% decrease in mtDNA content in the injury site. As measured by the Basso Mouse Scale, formoterol treatment improved locomotor recovery in SCI mice compared to vehicle treatment by 7 DPI, with continued recovery observed through 21 DPI (3.5 v. 2). SCI resulted in 15% body weight loss in all mice by 3 DPI. Mice treated with formoterol returned to pre-surgery weight by 13 DPI, while no weight gain occurred in vehicle-treated SCI mice. Remarkably, formoterol-treated mice exhibited a 30% increase in skeletal muscle mass compared to those treated with vehicle 21 DPI (0.93 v. 0.72% BW), corresponding with increased MB and decreased skeletal muscle atrophy. These effects were not observed in ADRB2 knockout mice subjected to SCI, indicating that formoterol is acting via the ADRB2 receptor. Furthermore, knockout mice exhibited decreased basal spinal cord and skeletal muscle PGC-1α expression, suggesting that ADRB2 may play a role in mitochondrial homeostasis under physiological conditions. These data provide evidence for systemic ADRB2-mediated MB as a therapeutic avenue for the treatment of SCI. • Daily formoterol treatment beginning 8 h post-SCI induces mitochondrial biogenesis. • Formoterol treatment improves locomotion and decreases lesion volume. • Formoterol decreases skeletal muscle atrophy and increases muscle mass post-SCI. • β 2 -adrenergic receptor KO blocks formoterol-induced recovery post-SCI. [ABSTRACT FROM AUTHOR]

Since the beginning of the last century, substantial evidence has suggested that various aspects of the immune system are influenced by the activity of the nervous system. However, the cellular and molecular basis for the neural control of immune responses has emerged only in the past decade. Recent studies have shown that adrenergic nerves control trafficking of immune cells through cell-type-specific mechanisms. Activation of the β 2 -adrenergic receptor expressed on lymphocytes enhances signals mediated by a particular set of chemokine receptors, and consequently inhibits their exit from lymph nodes. This mechanism is involved in the diurnal variation of adaptive immune responses and the progression of inflammatory diseases. In the present review, we focus on the role of adrenergic nerves in the control of lymphocyte trafficking and adaptive immune responses in physiological and pathological conditions. • Inputs from adrenergic nerves inhibit lymphocyte egress from LNs through β 2 AR. • β 2 AR-mediated signals enhance the responsiveness of specific chemokine receptors. • Adrenergic inputs contribute to the diurnal variation of lymphocyte trafficking. • Activation of β 2 AR suppresses T cell-mediated inflammation. [ABSTRACT FROM AUTHOR]

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. • Sympathetic nervous system (SNS) limits CNS autoimmune inflammation • Adrb2 signaling in immune cells mediates the SNS effects on EAE development • Adrb2-mediated SNS suppressive effects involve ICER-driven inhibition of CD4+ T cells Araujo et al. show that neurotransmitters released by the sympathetic nervous system regulate the generation of adaptive immune responses mitigating autoimmune inflammation within the CNS. [ABSTRACT FROM AUTHOR]

There are report regarding therapeutic effects for Allium cepa L. (A. cepa) in Iranian traditional medicine and the plant has showed anti-inflammatory, anti-allergic, anti-hyperglycemic, antioxidant, anti-cancer, anti-hypertension, anti-hypercholesterolemia and anti-asthmatic activities in previous studies. In this study, the contribution of β2 adrenergic, muscarinic and histamine (H1) receptors, calcium and potassium channels, and cyclooxygenase pathway in the relaxant effect of A. cepa extract on tracheal smooth muscle (TSM) was assessed. TSM was contracted by KCl (60 mM) or methacholine (10 μM) for 5 min and cumulative concentrations of A. cepa extract (2, 4, 8, 16, 32 and 64 mg/ml) were added to organ bath every 5 min. Theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control, and saline (1 ml) as negative control were also examined in non-incubated tissues. The relaxant effect of A. cepa extract was examined on non-incubated and incubated TSM with propranolol, chlorpheniramine, diltiazem, atropine, glibenclamide and indomethacin. A. cepa showed concentration-dependent relaxant effects on non-incubated TSM contracted by KCl (60 mM) or methacholine (10 μM), (P < 0.01 to p < 0.001). There was no significant difference in the relaxant effects of A. cepa between non-incubated and incubated tissues with glibenclamide, atropine, chlorpheniramine and indomethacin. The plant extract showed significant lower relaxant effects in incubated TSM with propranolol and diltiazem compared to non-incubated tissues. EC50 values of A. cepa extract in incubated TSM with propranolol and diltiazem were significantly lower than non-incubated tissues (p < 0.001 and p < 0.05, respectively). The relaxant effects of different concentrations of the extract of A. cepa were not significantly different with those of theophylline. The concentrations of A. cepa extract and theophylline were significant correlated with their relaxant effects (p < 0.05 to p < 0.001). In incubated TSM with propranolol and diltiazem, concentration ratio minus one (CR-1) values was positive (2.65 ± 0.63 and 1.28 ± 0.43 respectively). The A. cepa extract showed relatively potent relaxant effect on TSM which was comparable to the effect of theophylline. The results showed that β2-adrenergic stimulatory and/or calcium channel blockade are the possible mechanisms for the relaxant effects of the plant. Image 1 [ABSTRACT FROM AUTHOR]

Loss of bronchoprotection (LOBP) with a regularly used long-acting β 2 -adrenergic receptor agonist (LABA) is well documented. LOBP has been attributed to β 2 -adrenergic receptor (B2AR) downregulation, a process requiring farnesylation, which is inhibited by alendronate. We sought to determine whether alendronate can reduce LABA-associated LOBP in inhaled corticosteroid (ICS)–treated patients. We conducted a randomized, double-blind, placebo-controlled, parallel-design, proof-of-concept trial. Seventy-eight participants with persistent asthma receiving 250 μg of fluticasone twice daily for 2 weeks were randomized to receive alendronate or placebo while initiating salmeterol for 8 weeks. Salmeterol-protected methacholine challenges (SPMChs) and PBMC B2AR numbers (radioligand binding assay) and signaling (cyclic AMP ELISA) were assessed before randomization and after 8 weeks of ICS plus LABA treatment. LOBP was defined as a more than 1 doubling dose reduction in SPMCh PC 20 value. The mean doubling dose reduction in SPMCh PC 20 value was 0.50 and 0.27 with alendronate and placebo, respectively (P =.62). Thirty-eight percent of participants receiving alendronate and 33% receiving placebo had LOBP (P =.81). The after/before ICS plus LABA treatment ratio of B2AR number was 1.0 for alendronate (P =.86) and 0.8 for placebo (P =.15; P =.31 for difference between treatments). The B2AR signaling ratio was 0.89 for alendronate (P =.43) and 1.02 for placebo (P =.84; P =.44 for difference). Changes in lung function and B2AR number and signaling were similar between those who did and did not experience LOBP. This study did not find evidence that alendronate reduces LABA-associated LOBP, which relates to the occurrence of LOBP in only one third of participants. LOBP appears to be less common than presumed in concomitant ICS plus LABA–treated asthmatic patients. B2AR downregulation measured in PBMCs does not appear to reflect LOBP. [ABSTRACT FROM AUTHOR]

The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the β2-adrenergic receptor (β2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with β2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38. [ABSTRACT FROM AUTHOR]