β2AR Activation Research Articles

Identification of a β-arrestin-biased negative allosteric modulator for the β2-adrenergic receptor

Catecholamine-stimulated β2-adrenergic receptor (β2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β2AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias β2AR signaling toward the Gs pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the β2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit β-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of β-arrestin recruitment to the β2AR while having no effect on β2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the β2AR and protects against the functional desensitization of β-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the β2AR with minimal effects on the β1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the β2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the β2AR.

β2-adrenergic signaling promotes higher-affinity B cells and antibodies

Stress-induced β2-adrenergic receptor (β2AR) activation in B cells increases IgG secretion; however, the impact of this activation on antibody affinity and the underlying mechanisms remains unclear. In the current study, we demonstrate that stress in mice following ovalbumin (OVA) or SARS-CoV-2 RBD immunization significantly increases both serum and surface-expressed IgG binding to the immunogen, while concurrently reducing surface IgG expression and B cell clonal expansion. These effects were abolished by pharmacological β2AR blocking or when the experiments were conducted in β2AR -/- mice. In the second part of our study, we used single B cell sorting to characterize the monoclonal antibodies (mAbs) generated following β2AR activation in cultured RBD-stimulated B cells from convalescent SARS-CoV-2 donors. Ex vivo β2AR activation increased the affinities of the produced anti-RBD mAbs by 100-fold compared to mAbs produced by the same donor control cultures. Consistent with the mouse experiments, β2AR activation reduced both surface IgG levels and the frequency of expanded clones. mRNA sequencing revealed a β2AR-dependent upregulation of the PI3K pathway and B cell receptor (BCR) signaling through AKT phosphorylation, as well as an increased B cell motility. Overall, our study demonstrates that stress-mediated β2AR activation drives changes in B cells associated with BCR activation and higher affinity antibodies.

Norepinephrine as the Intrinsic Contributor to Contact Lens-Induced Pseudomonas aeruginosa Keratitis.

Contact lens wear (CLW) is one of the leading risk factors for Pseudomonas aeruginosa keratitis (PAK). However, the intrinsic factors that contribute to the high susceptibility to keratitis during CLW remain to be elucidated. CLW over an extended period can elevate corneal norepinephrine (NE) concentration. In this study, we investigated the role of NE in promoting PAK. We constructed an injury-induced PAK model and a CLW-induced PAK model to confirm the impact of NE during corneal infection. Pharmacological blockage of NE and gene knockdown mouse were used to investigate the downstream effector of NE. RNA sequencing was performed to explore the cellular alterations during NE treatment. Non-parametric Mann-Whitney U test or Kruskal-Wallis test were used to ascertain the significance (P < 0.05). Supplementation of NE led to PAK even without artificial corneal injury during CLW. The effect was mediated by the β2-adrenergic receptor (β2-AR) in the corneal epithelium. The β2-AR blockage by the NE antagonist ICI118,551 (ICI) or by deleting of its encoding gene Adrb2 significantly alleviated infection during CLW. Conversely, β2-AR activation compromised the integrity of the epithelium and significantly increased the cortical plaque marker ezrin. Transcriptome analysis identified that the protective effect of ICI on the keratitis was mediated by dual-specificity phosphatases. Suramin, a Dusp5 antagonist, abrogated the protective effect of ICI. These data reveal a new mechanism by which NE acts as an intrinsic factor that promotes CLW-induced PAK and provide novel therapeutic targets for treating keratitis by targeting NE-β2-AR.

The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of β2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the β2 adrenergic receptor (β2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting β2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under β2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under β-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Adrenergic receptor signaling regulates the CD40-receptor mediated anti-tumor immunity.

Anti-CD40 agonistic antibody (αCD40), an activator of dendritic cells (DC) can enhance antigen presentation and activate cytotoxic T-cells against poorly immunogenic tumors. However, cancer immunotherapy trials also suggest that αCD40 is only moderately effective in patients, falling short of achieving clinical success. Identifying factors that decrease αCD40 immune-stimulating effects can aid the translation of this agent to clinical reality. Here, we reveal that β-adrenergic signaling on DCs directly interferes with αCD40 efficacy in immunologically cold head and neck tumor model. We discovered that β-2 adrenergic receptor (β2AR) activation rewires CD40 signaling in DCs by directly inhibiting the phosphorylation of IκBα and indirectly by upregulating levels of phosphorylated-cAMP response element-binding protein (pCREB). Importantly, the addition of propranolol, a pan β-Blocker reprograms the CD40 pathways, inducing superior tumor regressions, increased infiltration of cytotoxic T-cells, and a reduced burden of regulatory T-cells in tumors compared to monotherapy. Thus, our study highlights an important mechanistic link between stress-induced β2AR signaling and reduced αCD40 efficacy in cold tumors, providing a new combinatorial approach to improve clinical outcomes in patients.

Activation of β2-adrenergic Receptor Alleviates Collagen-induced Arthritis by Ameliorating Th17/Treg Imbalance.

Recent research in our laboratory shows that CD4+ T cells express the β2 adrenergic receptor (β2-AR), and the sympathetic neurotransmitter norepinephrine regulates the function of T cells via β2-AR signaling. However, the immunoregulatory effect of β2-AR and its related mechanisms on rheumatoid arthritis is unknown. To explore the effects of β2-AR in collagen-induced arthritis (CIA) on the imbalance of T helper (Th) 17/ regulatory T (Treg) cells. In DBA1/J mice, collagen type II was injected intradermally at the tail base to prepare the CIA model. The specific β2-AR agonist, terbutaline (TBL), was administered intraperitoneally beginning on day 31 and continuing until day 47 after primary vaccination, twice a day. Magnetic beads were used to sort CD3+ T cells subsets from spleen tissues. In vivo, β2-AR agonist TBL alleviated arthritis symptoms in the CIA mice including histopathology of the ankle joints, four limbs' arthritis score, the thickness of ankle joints, and rear paws. After TBL treatment, in the ankle joints, the levels of proinflammatory factors (IL-17/22) notably decreased and the levels of immunosuppressive factors (IL-10/TGF-β) significantly increased. In vitro, ROR-γt protein expression, Th17 cell number, mRNA expression and the releasing of IL-17/22 from CD3+ T cells reduced following TBL administration. Moreover, TBL enhanced the anti-inflammatory responses of Treg cells. These results suggest that β2-AR activation exerts anti-inflammatory effects through the amelioration of Th17/Treg imbalance in the CIA disease.

Insights into β3-adrenoceptor agonism through comprehensive in silico investigation

Research onβ3-AR, the new member of the adrenoceptor family, is in its infancy and few β3-AR agonists have been approved for marketing to date. Meanwhile, β3-AR exhibited obvious species differences in pharmacological properties, such as between human and animals, however, the 3D structure of human β3-AR has not been published, which makes it difficult to understand the interaction between human β3-AR and its agonists. Herein, binding patterns of β3-AR agonists are explored starting from the Alphafold predicted structural model, and the obtained model was optimized by using molecular dynamics simulations. Moreover, the human β3-AR and its agonists were subjected to molecular docking, dynamics simulations, binding free energy calculations and pharmacophore modeling to elucidate the characteristics of human β3-AR activity pockets and agonist conformational relationships, including a hydrophobic group, a positively charged group as well as two hydrogen-bonded donors, which provide comprehensive insights into the interactions between human β3-AR and its agonists.

Activation of β2-Adrenergic Receptors in Microglia Alleviates Neuropathic Hypersensitivity in Mice.

Drugs enhancing the availability of noradrenaline are gaining prominence in the therapy of chronic neuropathic pain. However, underlying mechanisms are not well understood, and research has thus far focused on α2-adrenergic receptors and neuronal excitability. Adrenergic receptors are also expressed on glial cells, but their roles toward antinociception are not well deciphered. This study addresses the contribution of β2-adrenergic receptors (β2-ARs) to the therapeutic modulation of neuropathic pain in mice. We report that selective activation of β2-ARs with Formoterol inhibits pro-inflammatory signaling in microglia ex vivo and nerve injury-induced structural remodeling and functional activation of microglia in vivo. Systemic delivery of Formoterol inhibits behaviors related to neuropathic pain, such as mechanical hypersensitivity, cold allodynia as well as the aversive component of pain, and reverses chronically established neuropathic pain. Using conditional gene targeting for microglia-specific deletion of β2-ARs, we demonstrate that the anti-allodynic effects of Formoterol are primarily mediated by microglia. Although Formoterol also reduces astrogliosis at late stages of neuropathic pain, these functions are unrelated to β2-AR signaling in microglia. Our results underline the value of developing microglial β2-AR agonists for relief from neuropathic pain and clarify mechanistic underpinnings.

Beta 2 Adrenergic Receptor Selective Antagonist Enhances Mechanically Stimulated Bone Anabolism in Aged Mice.

The anabolic response of aged bone to skeletal loading is typically poor. Efforts to improve mechanotransduction in aged bone have met with limited success. This study investigated whether the bone response to direct skeletal loading is improved by reducing sympathetic suppression of osteoblastic bone formation via β2AR. To test this possibility, we treated aged wild-type C57BL/6 mice with a selective β2AR antagonist, butaxamine (Butax), before each of nine bouts of cantilever bending of the right tibia. Midshaft periosteal bone formation was assessed by dynamic histomorphometry of loaded and contralateral tibias. Butax treatment did not alter osteoblast activity of contralateral tibias. Loading alone induced a modest but significant osteogenic response. However, when loading was combined with Butax pretreatment, the anabolic response was significantly elevated compared with loading preceded by saline injection. Subsequent studies in osteoblastic cultures revealed complex negative interactions between adrenergic and mechanically induced intracellular signaling. Activation of β2AR by treatment with the β1, β2-agonist isoproterenol (ISO) before fluid flow exposure diminished mechanically stimulated ERK1/2 phosphorylation in primary bone cell outgrowth cultures and AKT phosphorylation in MC3T3-E1 pre-osteoblast cultures. Expression of mechanosensitive Fos and Ptgs2 genes was enhanced with ISO treatment and reduced with flow in both MC3T3-E1 and primary cultures. Finally, co-treatment of MC3T3-E1 cells with Butax reversed these ISO effects, confirming a critical role for β2AR in these responses. In combination, these results demonstrate that selective inhibition of β2AR is sufficient to enhance the anabolic response of the aged skeleton to loading, potentially via direct effects upon osteoblasts. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Sympathetic nerve infiltration promotes stomach adenocarcinoma progression via norepinephrine/β2-adrenoceptor/YKL-40 signaling pathway

ObjectiveThis study aimed to address the status, role, and mechanism of sympathetic nerve infiltration in the progression of stomach adenocarcinoma (STAD). MethodsSympathetic nerve and its neurotransmitter NE, β-ARs, and associated signaling molecules in the STAD tissues and the adjacent tissues from 46 STAD patients were examined using immunostaining, HPLC, and western blotting. The effects and mechanisms of β2-AR activation on the proliferation, migration and invasion of AGS and SGC-7901 gastric cancer (GC) cell lines were examined using CCK-8, transwell, and western blotting assays. Correlations between genes and STAD survival were analyzed using bioinformatics. ResultsStriking sympathetic nerve infiltration, elevations of NGF, TrkA, GAP43, TH, S100, NE, β2-AR, YKL-40, syndecan-1, MMP9, CD206, and CD31 were observed in the STAD tissues compared to the adjacent tissues. Activation of β2-AR in the two GC cell lines significantly amplified the expressions of NGF, YKL-40, MMP9, syndecan-1, p-STAT3 and p-ERK, and increased GC cell proliferation, migration and invasion. Bioinformatic analyses revealed positive correlations of NGF, β2-AR, syndecan-1, and macrophage infiltration, respectively, with low survival of STAD, of β2-AR respectively with STAT3, ERK1/2 (MAPK1/3), YKL-40, MMP9, and syndecan-1, and of YKL-40 with MMP9. ConclusionSympathetic nerves significantly infiltrated into human STAD tissues as a result of high NGF and TrkA expressions; elevated NE led to overactivation of β2-AR-STAT3/ERK-YKL-40 signaling pathway, and finally caused cancer cell growth and invasion, M2 macrophage infiltration, angiogenesis, matrix degradation and STAD metastasis and progression.

Abstract 13223: Calcium/Calmodulin-Dependent Protein Kinase II Decreases KCNH2-Related Current During Sustained Adrenergic Receptor Stimulation

Introduction: KCNH2 codes for a potassium channel that mediates the primary cardiac repolarizing current, I Kr . Calcium/calmodulin-dependent protein kinase II (CaMKII) modulates several ion channels, is enhanced during sustained β-adrenergic receptor (βAR) stimulation, and is proarrhythmic. Furthermore, states of enhanced βAR stimulation/CaMKII activity, such as heart failure, alter microRNA (miR) expression profiles that suppress protein expression. Hypothesis: CaMKII decreases KCNH2-related current during sustained βAR stimulation through miR regulation. Methods: Putative miR binding sites (n=327) on KCNH2 3′-UTR were identified with bioinformatic tools. Binding sites were then constructed into a pIS0 plasmid, amplified, and transfected into hiPSC-cardiomyocytes. Cells were subjected to sustained βAR stimulation (Isoproterenol: 100 nM, 1 μM), constitutively active δCaMKII (Thr287Asp), or control conditions. Next-generation sequencing was used to identify altered miR binding sites expression and endogenous miR alterations. Whole-cell, voltage-clamp experiments were performed in HEK293 cells expressing KCNH2 and δCaMKII T287D . Cells were treated with isoproterenol, and miR mimics were used for validation of the bioassay. Results: Nine and eight predicted miR binding sites were downregulated in the CaMKII group (Δlog fold: -0.287 to -0.59, p&lt;0.05) and sustained βAR group (Δlog fold: -0.29 to -0.72, p&lt;0.05), respectively. Thirty miRs were predicted to bind to these sites on KCNH2 3′-UTR, of which seven were selected for validation based on a Δlog fold &lt; -0.6 in one group (n=4) or &lt; -0.5 in both treatment groups (n=3). Mimics of all seven miRs reduced KCNH2 protein expression, and three decreased KCNH2 currents compared to control (p&lt;0.05). Sustained βAR stimulation decreased peak activation current in cells expressing both KCNH2 and δCaMKII T287D by 25% (17±7 pA/pF, p = 0.03) and tail current density by 27% (21±9 pA/pF, p = 0.03), but had no effect on currents in cells expressing only KCNH2. Conclusions: CaMKII hyperactivity alters miR expression profiles and decreases KCNH2 function during sustained βAR activation. The effect of the identified miRs on arrhythmia susceptibility with enhanced CaMKII activity warrants further study.

RF21 | PSAT78 The beta3-Adrenergic Receptor as a Target for the Prevention of Glucocorticoid-induced Adipose Tissue Dysfunction in Rodents

Abstract Glucocorticoids (GC) are an integral part of anti-inflammatory therapy. However, their therapeutic effectiveness is limited by their deleterious adverse effects such as the development of obesity, diabetes, and cardiometabolic disease. GC-induced obesity arises from excessive fat accumulation in white adipose tissue (WAT) as well as suppressed thermogenic function of brown adipose tissue (BAT). The sympathetic nervous system (SNS) plays a key role in the regulation of both WAT as well as BAT biology. As part of this project, we investigate the interaction between SNS and GC signaling in the adipose organ. To that end, we modulated the SNS activity in adipose tissues of mice by altering the housing temperature of corticosterone-treated mice and respective controls to either 29°C, 22°C, or 13°C. Following the four-week treatment course, GC-treated mice housed at 29°C and 22°C gained more weight and accumulated more visceral and subcutaneous white fat than their temperature-matched controls. Interestingly, maintaining mice in the 13°C environment protected animals from GC-induced weight gain and obesity. Additionally, cold adaptation effectively prevented the development of GC-driven hyperglycemia, hyperinsulinemia and hyperleptinemia, all of which were readily observed at 29°C and 22°C. Furthermore, the sympathetic innervation as well as the thermogenic capacity of BAT were preserved in cold-acclimated mice in spite of corticosterone treatment, whereas mice housed at higher temperatures showed marked suppression of the thermogenic function in BAT following GC exposure. To further test whether sympathetic activation is responsible for the prevention of GC-induced dysmetabolism by prolonged adaption to cold, we investigated the effect of a pharmacological β3-AR agonist, CL316,243 during GC excess. Similar to cold exposure, selective activation of the β3-AR not only averted the development of GC-induced fat accumulation and hyperinsulinemia, but also preserved the thermogenic capacity in BAT despite exposure to supra-physiological levels of corticosterone. Taken together, our data indicate that both cold-acclimation as well as β3-AR activation counteract GC-induced obesity, adipose tissues dysfunction, and related metabolic comorbidities. Thus, targeting the β3-AR may represent a potential therapeutic approach to alleviate GC-induced metabolic abnormalities. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m.

RF36 | PSUN79 Prolonged Stimulation of β-Adrenergic Receptors in Human Brown and White Adipocytes Increases and then Decreases Metabolic Activity

Abstract Background Obesity results from excess adipose triglyceride storage and leads to multiple life-threatening complications that may be amenable to novel pharmacological therapies. Both human white and brown adipocytes express β1-, β2-, and β3-adrenergic receptors (β-AR's), and stimulation of each activates converging signaling pathways that regulate essential metabolic and cellular functions. Clinical data indicate that acute and chronic stimulation of the β3-AR with the selective agonist mirabegron increases lipolysis and thermogenesis, thereby making β3-AR activation a promising target to improve obesity-related metabolic disease. Purpose In this study, we evaluated in vitro the short-term and prolonged activation of β3-ARs via mirabegron and compared the effects to the activation of β1- and β2-AR. Methods Functional cellular assays such as lipolysis and cellular respiration, were measured in immortalized human white (hWA) and brown (hBA) adipocytes derived from the superficial and deep neck depots, respectively. Cells were treated for 6 h (short-term) and 24 h (prolonged) with mirabegron (β3-AR agonist), dobutamine (β1-AR agonist), terbutaline (β2-AR agonist) and isoproterenol (non-selective β-AR agonist). One-way ANOVA was used for statistical analysis. Findings In hWA, only short-term activation of the β2-AR induced lipolysis (1.8-fold increase, P=0.0002), while in hBA, both the β2-AR (1.5-fold increase, P=0.04) and β3-AR (1.6 fold increase, P=0.002) increased lipolysis with 6h treatment, as compared to the vehicle. By 24h, lipolysis was blunted in both cell types, likely due to agonist-induced desensitization and downregulation. Distinct from the lipolytic effects ofβ-AR activation, the basal oxygen consumption rate (OCR) was not significantly different among hBA and hWA treated acutely and chronically with each of the three selective β-ARs and positive control isoproterenol. However, in hBA only, prolonged activation of all three β-AR's lowered maximal mitochondrial respiration (isoproterenol: 25% decrease, P=0.0004; dobutamine: 25% decrease, P=0.0003; terbutaline: 26% decrease, P=0.0004; mirabegron: 38% decrease, P&amp;lt;0.0001, respectively). No significant changes in OCR parameters were detected in hWA. Conclusion In hBA, acute stimulation of either the β2-AR or β3-AR induces lipolysis. Prolonged exposure to β-agonists comparably lowers maximal cellular respiration, suggesting that all β-AR's are subject to agonist-induced desensitization. Moreover, continuous activation of lipolysis may deplete the intracellular fatty acid pool necessary for mitochondrial oxidative respiration. These responses will need to be addressed before novel adrenergic activators can be utilized to reduce triglyceride stores and treat obesity-related metabolic disease. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:00 p.m. - 1:05 p.m.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of β-Adrenoceptors.

Beta-adrenoceptor (β-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting β-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of β-ARs. M2R-dependent contractions were apparent at low frequency (2Hz)electric field stimulation (EFS) and short (10s) stimulus intervals.The β1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 sand was more effective at inhibiting contractions evoked by EFS at 2than 20 Hz.Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the β3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of β1-ARs and that genetic ablation of M2Rs decreased the efficacy of β-AR agonists on cholinergic contractions.

Dimerization of β2-adrenergic receptor is responsible for the constitutive activity subjected to inverse agonism

Dimerization of beta 2-adrenergic receptor (β2-AR) has been observed across various physiologies. However, the function of dimeric β2-AR is still elusive. Here, we revealed that dimerization of β2-AR is responsible for the constitutive activity of β2-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient β2-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gαs preferentially interacts with dimeric β2-AR, but not monomeric β2-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of β2-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric β2-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of β2-AR. Our model not only shows the function of dimeric β2-AR but also provides a molecular insight into the mechanism of the inverse agonism of β2-AR.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

Abstract 2191: Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction

Abstract Bone metastasis is a major cause of morbidity and mortality for breast cancer patients. Bone is a unique metastatic microenvironment because of complex interactions among numerous distinct cell types such as osteo-blasts, -clasts, -cytes and marrow immune cells in physiological and pathological conditions. Imbalanced bone homeostasis by diverse factors such as the sympathetic nerve system (SNS) activation, potentially contribute to the progression of bone metastasis, yet precise mechanisms remain unclear. We investigated the effects of beta 2 adrenergic receptor (β2AR) activation in osteoblasts induced by prolonged SNS stimulation on anti-tumoral immunity in bone metastasis. We treated female Balb/C with chronic immobilization stress (CIS; 2 h. daily) and found that CIS increased syngeneic intra-tibial 4T1 bone metastasis growth as well as the number of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) counterbalancing anti-tumoral cytotoxic T cells in bone microenvironment. A β2AR-specific inhibitor (ICI-118551) or MDSC depletion using anti-Gr1 antibodies reversed CIS-induced bone metastatic tumor growth. More importantly, 2-week CIS pre-treatment increased EDU+ MDSC proliferation in the bone marrow, contributing to subsequent bone metastatic tumor growth and micro-metastatic seeding in intra-tibial and intra-cardiac tumor injection models, respectively. When CIS was administered after the establishment of bone metastatic tumors, the pro-tumorigenic effects were marginal. In osteoblast-specific β2AR knockout mice (Adrb2flox/flox; murine 2.3kb Col1-CreC57BL6 mice), CIS did not increase MDSC proliferation compared with littermate floxed controls, suggesting that β2AR in osteoblasts upregulates MDSC in bone microenvironment. RNA-sequencing transcriptome analysis of saline- or clenbuterol (a selective β2AR agonist)-treated murine osteoblasts showed that interleukin-6 (IL6) was the most significantly increased cytokine. Indeed, CIS or clenbuterol treatment increased IL-6 expression in vivo (immunohistochemistry) and in vitro (cultured osteoblasts), respectively. MDSC isolated from the bone marrow of CIS-treated mice had significantly increased STAT3 phosphorylation by flow cytometry and the expression of immuno-suppressive functional molecules such as arginase 1 (Arg1) and indolamine 2,3-dioxygenase 1 (Ido1) compared to those from control mice. Our data collectively demonstrated that SNS-dependent β2AR in osteoblasts expands and activates MDSC in the pre-metastatic bone microenvironment via the IL6-STAT3-Arg1/IDO1 pathway, creating a fertile soil for bone metastasis progression. Our data provided the first direct evidence for anti-tumoral immunity and pro-tumorigenic effects of autonomous nerve system dysfunction specific to the metastatic bone microenvironment. Citation Format: Eun Jung Lee, Da Hyeon Yun, Seungpil Jung, Serk In Park. Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2191.

Colchicine stimulates browning via antagonism of GABA receptor B and agonism of β3-adrenergic receptor in 3T3-L1 white adipocytes

Colchicine has been used for therapeutic purposes and has attracted considerable attention because of its association with tubulin and the inhibition of small tubular polymerization. Although several studies have examined the possible preventive role of colchicine in metabolic diseases, its role in adipocytes is largely unknown. This study examined the novel functional role of colchicine in adipocytes demonstrating that colchicine stimulates browning in cultured white adipocytes. Colchicine stimulates browning by increasing the brown- and beige fat-specific markers in 3T3-L1 white adipocytes. Interestingly, colchicine decreased the expression of the main lipolytic proteins (ATGL, p-HSL) while it activated Ces3, suggesting a possibility for supplying essential fatty acids for inducing thermogenesis. Molecular docking analysis showed that colchicine has a strong affinity against GABA-BR and β3-AR, and its binding activity with GABA-BR (−26.52 kJ/mol) was stronger than β3-AR (−20.71 kJ/mol). Mechanistic studies were conducted by treating the cells separately with agonists and antagonists of GABA-BR and β3-AR to understand the molecular mechanism underlying the browning effect of colchicine. The results showed that colchicine stimulates browning via the antagonism of GABA-BR and the agonism of β3-AR in 3T3-L1 white adipocytes. The colchicine-mediated activation of β3-AR stimulated the PKA/p38 MAPK signaling pathway, where consequently ATF2 acted as a positive regulator, but AFT4 was a negative regulator for the induction of browning.

In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function

Objective &amp; HypothesisActivation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.MethodsWe employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.ResultsDatabase screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.ConclusionThese findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.

Repeated vagus nerve stimulation produces anxiolytic effects via upregulation of AMPAR function in centrolateral amygdala of male rats

Repeated vagus nerve stimulation (rVNS) exerts anxiolytic effect by activation of noradrenergic pathway. Centrolateral amygdala (CeL), a lateral subdivision of central amygdala, receives noradrenergic inputs, and its neuronal activity is positively correlated to anxiolytic effect of benzodiazepines. The activation of β-adrenergic receptors (β-ARs) could enhance glutamatergic transmission in CeL. However, it is unclear whether the neurobiological mechanism of noradrenergic system in CeL mediates the anxiolytic effect induced by rVNS. Here, we find that rVNS treatment produces an anxiolytic effect in male rats by increasing the neuronal activity of CeL. Electrophysiology recording reveals that rVNS treatment enhances the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR)-mediated excitatory neurotransmission in CeL, which is mimicked by β-ARs agonist isoproterenol or blocked by β-ARs antagonist propranolol. Moreover, chemogenetic inhibition of CeL neurons or pharmacological inhibition of β-ARs in CeL intercepts both enhanced glutamatergic neurotransmission and the anxiolytic effects by rVNS treatment. These results suggest that the amplified AMPAR trafficking in CeL via activation of β-ARs is critical for the anxiolytic effects induced by rVNS treatment.