Adrenergic Receptor Signaling Research Articles

130 Burn Injury Reduces Bone Marrow Mesenchymal Stem Cells and Sensitizes Their Adrenergic Receptor Subgroups in a Murine Model

Abstract Introduction Previous burn and traumatic injury studies have established that adrenergic signaling is increased after burn injury and may lead to an impairment of hematopoietic cell development in the bone marrow (BM). Nonetheless, mesenchymal stem cells (MSCs), which have gained momentum in regenerative medicine also play a predominant role in the BM niche. Understanding the propensity of the adrenergic receptor (AR) response by MSCs can be utilized for devising targeted therapies. However, the traditional plastic adherence procedure using ex vivo culture of BM cells for several weeks may skew the actual characteristics of MSCs. Our current study focused on isolating MSCs from freshly obtained BM in a murine scald burn model with a goal to characterize the expression pattern of native AR subgroups present on BM MSCs as compared to sham mice. Methods Eight, two-month-old adult female mice were subjected to a 15% total body 3rd degree burn or sham burn. The mice were sacrificed 7 days later. Femurs were removed and total bone marrow cells were flushed out. Multi parametric flow cytometry was used to gate for cells negative for hematopoietic cell markers (CD45, CD11B) and positive for MSC markers (CD105, CD106, SSEA, Ly6A) and AR subgroups (α1, α2, β1, β2, β3). We measured the number of BM MSCs, quantified the subtypes of ARs present on MSCs, and compared the ratio of AR antibody binding per total MSC population. Results Overall the frequency of MSCs per million total BM cells decreased by 48% post-burn injury with165,300 ± 194 in sham versus 110,000 ± 30 in burn displayed as bar graph in Panel A. Over 90% of MSCs consistently express β2 AR and only 10% express α2 AR subgroup in both scald and sham burn. Presence of other subgroups ranged from 50% to 80% of MSCs as seen in histograms to the right of dotted line in Panel B. Our AR propensity score based on AR mean fluorescence intensity adjusted to total number of MSCs present was increased by 2.8-fold for α1, 2.5-fold for β1, 1.6-fold for β3, and 1.3-fold for β2 AR subgroups (Panel C). These findings indicate burn injury not only decreases the frequency of BM MSCs but also increases the affinity of certain AR subgroups present on MSCs. Since BM MSCs are the major source of cytokines, chemokines and growth factors; detailed studies on AR mediated signaling in BM MSCs is warranted. Conclusions Polarization of AR signaling in BM MSCs by burn-induced catecholamines may have broader implications for comorbidities such as bone resorption and muscle wasting observed in human patients post burn trauma.

Berries and Their Polyphenols as a Potential Therapy for Coronary Microvascular Dysfunction: A Mini-Review.

Ischemia with no obstructive coronary artery disease (INOCA) is a common diagnosis with a higher prevalence in women compared to men. Despite the absence of obstructive coronary artery disease and no structural heart disease, INOCA is associated with major adverse cardiovascular outcomes as well a significant contributor to angina and related disability. A major feature of INOCA is coronary microvascular dysfunction (CMD), which can be detected by non-invasive imaging and invasive coronary physiology assessments in humans. CMD is associated with epicardial endothelial-dependent and -independent dysfunction, diffuse atherosclerosis, and left-ventricular hypertrophy, all of which lead to insufficient blood flow to the myocardium. Inflammatory and oxidative stress signaling, upregulation of the renin-angiotensin-aldosterone system and adrenergic receptor signaling are major drivers of CMD. Treatment of CMD centers around addressing cardiovascular risk factors; however, there are limited treatment options for those who do not respond to traditional anti-anginal therapies. In this review, we highlight the ability of berry-derived polyphenols to modulate those pathways. The evidence supports the need for future clinical trials to investigate the effectiveness of berries and their polyphenols in the treatment of CMD in INOCA patients.

Chronic Adrenergic Stress Contributes to Metabolic Dysfunction and an Exhausted Phenotype in T Cells in the Tumor Microenvironment.

Metabolic dysfunction and exhaustion in tumor-infiltrating T cells have been linked to ineffectual antitumor immunity and the failure of immune checkpoint inhibitor therapy. We report here that chronic stress plays a previously unrecognized role in regulating the state of T cells in the tumor microenvironment (TME). Using two mouse tumor models, we found that blocking chronic adrenergic stress signaling using the pan β-blocker propranolol or by using mice lacking the β2-adrenergic receptor (β2-AR) results in reduced tumor growth rates with significantly fewer tumor-infiltrating T cells that express markers of exhaustion, with a concomitant increase in progenitor exhausted T cells. We also report that blocking β-AR signaling in mice increases glycolysis and oxidative phosphorylation in tumor-infiltrating lymphocytes (TIL), which associated with increased expression of the costimulatory molecule CD28 and increased antitumor effector functions, including increased cytokine production. Using T cells from Nur77-GFP reporter mice to monitor T-cell activation, we observed that stress-induced β-AR signaling suppresses T-cell receptor (TCR) signaling. Together, these data suggest that chronic stress-induced adrenergic receptor signaling serves as a "checkpoint" of immune responses and contributes to immunosuppression in the TME by promoting T-cell metabolic dysfunction and exhaustion. These results also support the possibility that chronic stress, which unfortunately is increased in many patients with cancer following their diagnoses, could be exerting a major negative influence on the outcome of therapies that depend upon the status of TILs and support the use of strategies to reduce stress or β-AR signaling in combination with immunotherapy.

Abstract PS17-40: Fatty acid metabolism at the connection of histone methylation and mammary cell plasticity

Abstract INTRODUCTION:Metabolic switching has been linked with cancer progression. We initially reported increased expression of lipids metabolism genes in the contralateral breasts of women with unilateral estrogen receptor negative (ER-) breast cancer. We then demonstrated chromatin remodeling in normal mammary epithelial cells following exposure to lipids. Metabolites from intermediate metabolism are substrates that generate chromatin modifications, connecting metabolism to epigenetics. Key to this crosstalk is the fact that the kinetic and thermodynamic properties of chromatin modification reactions are in line with the dynamic range of the physiological concentrations of the corresponding intermediates in metabolism. Since substrates for histone methylation and acetylation reactions often have cellular concentrations that are commensurate with enzyme Km values, and are sensitive and responsive to changes in metabolism, we now present evidence regarding changes in metabolic flux and cellular plasticity connecting lipid metabolism to ER- cancer progression. Methods:MCF-10A cells were grown in the presence of medium chain fatty acid- octanoic acid (5mM) for 24 hours. RNA sequencing was performed on the treated and control cells using Illumina Next Seq 500 for 75bp single-read sequencing. Differential expression and gene set enrichment analyses were utilized to identify significantly enriched biological processes and molecular functions upon fatty acid treatment. Differential metabolic flux was determined using flux balance analysis. For proteomic analysis of post-translational histone modifications, histones were acid extracted from nuclei and LC-MS based mass spectrometry was used to quantitate the modifications and data is represented as the relative abundance in %. Results:Transcriptome data from RNA sequencing, incorporated into the human metabolic network reconstruction, revealed a significant increase in methylation flux in octanoate treated MCF10-A cells. The treated cells showed an increase in the flux in fatty acid oxidation and one-carbon metabolism reactions. The proteomic histone modification profile showed an increase in histone methylation specifically at H3K9, H3K4, H3K27 and H3K36, which corroborates with the GSEA analysis showing a significant correlation of H3K27 methylation (NES = 2.47, q-value = 0.05), a marker of constitutive heterochromatin, in the octanoate treated phenotype. Further, RNA-Seq of octanoate treated MCF-10A cells revealed prominent upregulation of cell-fate commitment and cell differentiation pathways, specifically neural differentiation, and adenylate cyclase-activating adrenergic receptor signaling. Conclusion:Lipids are a source of acetyl-CoA which serves as a donor for histone acetylation; our data shows that resultant increased flux through metabolic reactions produces s-adenosine methionine (SAM), an important methylating agent. These post-translational modifications have profound effects on chromatin structure, effecting changes that range from the expression of a single gene to a complete conversion of phenotype. We observe both an alteration of the molecular phenotype, as well as adenylate cyclase-activating adrenergic receptor signaling, which may be a clue to the development of ER- disease as GWAS have revealed that the adenylate cyclase-activating pathway is enriched in susceptibility to ER-negative disease. Citation Format: Shivangi Yadav, Ranya KA Virk, Carolina H Chung, Sriram Chandrasekaran, Vadim Backman, Seema Ahsan Khan, Susan Elizabeth Clare. Fatty acid metabolism at the connection of histone methylation and mammary cell plasticity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-40.

Divergence of neuroimmune circuits activated by afferent and efferent vagal nerve stimulation in the regulation of inflammation.

It has previously been shown that afferent and efferent vagal nerve stimulation potently inhibits lipopolysaccharide (LPS)-induced inflammation Our data show inhibition of inflammation by efferent but not afferent vagal nerve stimulation requires T-cell derived acetylcholine We show that afferent and efferent neuroimmune circuits require β2 -adrenergic receptor signalling ABSTRACT: Chronic inflammation due to inappropriate immune cell activation can have significant effects on a variety of organ systems, reducing lifespan and quality of life. As such, highly targeted control of immune cell activation is a major therapeutic goal. Vagus nerve stimulation (VNS) has emerged as a therapeutic modality that exploits neuroimmune communication to reduce immune cell activation and consequently inflammation. Although vagal efferent fibres were originally identified as the primary driver of anti-inflammatory actions, the vagus nerve in most species of animals predominantly comprises afferent fibres. Stimulation of vagal afferent fibres can also reduce inflammation; it is, however, uncertain how these two neuroimmune circuits diverge. Here we show that afferent VNS induces a mechanism distinct from efferent VNS, ameliorating lipopolysaccharide (LPS)-induced inflammation independently of T-cell derived acetylcholine (ACh) which is required by efferent VNS. Using a β2 -adrenergic receptor antagonist (β2 -AR), we find that immune regulation induced by intact, afferent, or efferent VNS occurs in a β2- AR-dependent manner. Together, our findings indicate that intact VNS activates at least two distinct neuroimmune circuits each with unique mechanisms of action. Selective targeting of either the vagal efferent or afferent fibres may provide more personalized, robust and effective control over inappropriate immune responses.

Abstract 12717: Enhance Negative Modulation of Beta 3 -Adrenergic Stimulation on Cardiomyocyte Functional Performance, [Ca 2+ ] i Regulation and β-Adrenergic Reserve in Rats Expressing Human Angiotensinogen Gene

Background: Transgenic rats expressing the human angiotensinogen gene [TGR (hAogen) 1623] (hAogen) exhibit sustained hypertension and systolic dysfunction associated with altered cardiac renin-angiotensin system and β- adrenergic receptor (AR) signal transduction systems. We have shown previously, in contrast to β 1 - and β 2 -ARs, β 3 -ARs are linked to G i proteins, and stimulation of β 3 -ARs inhibits cardiac contraction and relaxation. Hypertension is associated with increased cardiac expression of G i proteins. This may alter β 3 -AR stimulation and play an important role in the cardiac functional impairment of this humanized model of hypertension. However, the alteration and functional effect of β 3 -AR activation in this model are unknown. We tested the hypothesis that high cardiac tissue Ang II content in hAogen may cause an up-regulation of cardiac β 3 -AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the cardiac dysfunction. Methods: We compared LV myocyte β 3 -AR expression and myocyte functional and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β- and β 3 -AR stimulation in myocytes obtained from 8 Sprague Dawley (SD) control and 8 hAogen male adult rats. Results: Versus SD, in hAGT rats, basal myocyte contraction (dL/dt max , 107.1 vs 138.8 μm/s), relaxation (dR/dt max , 94.0 vs 103.9 μm/s) and [Ca 2+ ] iT (0.15 vs 0.21) were depressed. A non-selective β-AR agonist, isoproterenol (ISO) (10 -8 M) produced significantly smaller increases in dL/dt max , (42% vs. 61%), dR / dtmax (35% vs. 51%), and [Ca 2+ ] iT (20% vs. 30%). Moreover, versus SD, in hAogen rats, LV myocyte β 3 -AR protein level increased by 32% (0.29 vs 0.22) with resulted significantly altered myocyte functional response to β 3 -AR stimulation. Compared with the changes in SD myocytes, in hAogen myocytes, BRL (10 -8 M) produced a significantly greater decreases in dL/dt max , (27% vs 12%), dR/dt max (28% vs 11%), and [Ca 2+ ] iT (17% vs 10%). Conclusions: TGR (hAogen) 1623, this humanized model of hypertension is associated with an up-regulation of LV myocyte β 3 -AR, which enhances β 3 -AR-caused inhibition of myocyte contractile, relaxation and [Ca 2+ ] iT and exacerbates β-AR desensitization, thereby directly contributing to the cardiac dysfunction.

Activation of β2 adrenergic receptor signaling modulates inflammation: a target limiting the progression of kidney diseases.

Beta 2 adrenergic receptor (β2-AR)-agonists, widely used as bronchodilators, have demonstrated wide-spectrum anti-inflammatory properties in both immune and non-immune cells in various tissues. Their anti-inflammatory properties are mediated primarily, but not exclusively, via activation of the canonical β2-AR signaling pathway (β2-AR/cAMP/PKA). As non-canonical β2-AR signaling also occurs, several inconsistent findings on the anti-inflammatory effect of β2-agonists are notably present. Increasing amounts of evidence have unveiled the alternative mechanisms of the β2-AR agonists in protecting the tissues against injuries, i.e., by augmenting mitochondria biogenesis and SIRT1 activity, and by attenuating fibrotic signaling. This review mainly covers the basic mechanisms of the anti-inflammatory effects of β2-AR activation along with its limitations. Specifically, we summarized the role of β2-AR signaling in regulating kidney function and in mediating the progression of acute and chronic kidney diseases. Given their versatile protective effects, β2-agonists can be a promising avenue in the treatment of kidney diseases.

Alpha adrenergic receptor signaling in the hypothalamic paraventricular nucleus is diminished by the chronic intermittent hypoxia model of sleep apnea

Chronic intermittent hypoxia (CIH) is a model for obstructive sleep apnea. The paraventricular nucleus (PVN) of the hypothalamus has been suggested to contribute to CIH-induced exaggerated cardiorespiratory reflexes, sympathoexcitation and hypertension. This may occur, in part, via activation of the dense catecholaminergic projections to the PVN that originate in the brainstem. However, the contribution of norepinephrine (NE) and activation of its alpha-adrenergic receptors (α-ARs) in the PVN after CIH exposure is unknown. We hypothesized CIH would increase the contribution of catecholaminergic input. To test this notion, we determined the expression of α-AR subtypes, catecholamine terminal density, and synaptic properties of PVN parvocellular neurons in response to α-AR activation in male Sprague-Dawley normoxic (Norm) and CIH exposed rats. CIH decreased mRNA for α1d and α2b AR. Dopamine-β-hydroxylase (DβH) terminals in the PVN were similar between groups. NE and the α1-AR agonist phenylephrine (PE) increased sEPSC frequency after Norm but not CIH. Block of α1-ARs with prazosin alone did not alter sEPSCs after either Norm or CIH but did prevent agonist augmentation of sEPSC frequency following normoxia. These responses to NE were mimicked by PE during action potential block suggesting presynaptic terminal alterations in CIH. Altogether, these results demonstrate that α1-AR activation participates in neuronal responses in Norm, but are attenuated after CIH. These results may provide insight into the cardiovascular, respiratory and autonomic nervous systems alterations in obstructive sleep apnea.

Patients with pheochromocytoma exhibit low aldosterone renin ratio-preliminary reports

BackgroundPlasma renin activity (PRA) is generally increased in patients with pheochromocytoma (PCC) due to low circulating plasma volume and activation of β-1 adrenergic receptor signaling. However, there has been no study on the aldosterone renin ratio (ARR) in patients with PCC. To elucidate the issue, this study aimed to determine the PRA, plasma aldosterone concentration (PAC), and ARR in patients with PCC and compare them with those in patients with subclinical Cushing’s syndrome (SCS) and non-functioning adrenal adenoma (NFA).MethodsIn this retrospective single-center, cross-sectional study, 67 consecutive patients with adrenal tumors (PCC (n = 18), SCS (n = 18), and NFA (n = 31)) diagnosed at Kobe University Hospital between 2008 and 2014 were enrolled.ResultsPRA was significantly higher in patients with PCC than in those with SCS and NFA (2.1 (1.3 ~ 2.8) vs. 0.7 (0.5 ~ 1.8) and 0.9 (0.6 ~ 1.4) ng/mL/h; p = 0.018 and p = 0.025). Although PACs were comparable among the three groups, ARR was significantly lower in patients with PCC than in those with SCS and NFA (70.5 (45.5 ~ 79.5) vs. 156.0 (92.9 ~ 194.5) and 114.9 (90.1 ~ 153.4); p = 0.001 and p < 0.001). Receiver operating characteristic curve analysis demonstrated that, in differentiating PCC from NFA, PRA > 1.55 ng/mL/h showed a sensitivity of 70.0% and specificity of 80.6%. Interestingly, ARR < 95.4 showed a sensitivity of 83.3% and specificity of 86.7%, which were higher than those in PRA.ConclusionsARR decreased in patients with PCC, which was a more sensitive marker than PRA. Further study is necessary to understand the usefulness of this convenient marker in the detection of PCC.Trial registrationThis study was not registered because of the retrospective analysis.

β2 -adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog-Gli1 signaling activation.

BackgroundConsiderable evidence suggests that the sympathetic nervous system, mainly via adrenergic signaling, contributes to prostate cancer (PCa) progression. However, the underlying molecular mechanisms remain unknown.MethodsThe expression level of β2‐adrenergic receptor (β2‐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 β2‐AR or Gli1 was validated by Western blot analysis and real‐time PCR. Effects of β2‐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.ResultsWe determined that β2‐AR was expressed at significantly higher levels in carcinoma than in normal prostate tissues. β2‐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 β2‐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 β2‐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.ConclusionsIn this study, we uncovered an important role of β2‐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 β2‐adrenergic modulation on PCa, our study might also add evidence for potential therapeutic options of β‐blockers for PCa.

Downregulation of USP33 inhibits Slit/Robo signaling pathway and is associated with poor patient survival of glioma.

Gliomas are the most common malignant tumors in the central nervous system originating from brain glial cells. Although characterized as highly invasive and highly malignant, few molecular targeting therapies have been developed. Ubiquitin Specific Protease 33 (USP33), a gene encoding a deubiquinating enzyme important in a variety of processes, including Slit-dependent cell migration and beta-2 adrenergic receptor signaling, participates in the development of several malignant tumors, however, its role in the development of glioma has not been evaluated. Real-time quantitative PCR was performed to examine the expression of USP33 in glioma tissues and cell lines. Immunohistochemistry was performed to determine USP33 expressions in glioma tissue microarray. Transwell assay was performed to analyze the effect of USP33 on glioma cell line migration. The Kaplan-Meier method and log rank test were applied to evaluate the prognostic value of USP33 expression. Univariate and multivariate Cox proportional hazard regression models were used to identify the independent prognostic factors associated with overall survival (OS) or disease-free survival. The present study demonstrated that USP33 expression was significantly downregulated in glioma tissues. Lower expression of USP33 was associated with a poorer patient disease-free survival and overall survival. In vitro studies revealed that overexpression of USP33 significantly inhibited the migration ability of glioma cells. Mechanistically, USP33 inhibits glioma cell migration by regulating the function of Slit/Robo signaling pathway. Downregulation of USP33 is associated with poor patient survival of glioma. USP33 inhibits glioma cell migration by Slit/Robo signaling pathway. This mechanism may be applied for development of targeting therapy especially for the high-grade glioma.

Adrenergic receptors and cardiovascular diseases

<p indent=0mm>The sympatheticoadreno-medullary system plays a critical role in regulating cardiovascular activity, and adrenergic receptors are the gateway to this role. Adrenergic receptors are representative receptors of the G protein-coupled receptor family and mediate important physiological signaling. The abnormality of the adrenergic receptor signaling system is the main mechanism leading to many cardiovascular diseases, so it is also the main target of clinical drug. In this review, we summarize the discovery of adrenergic receptors as well as their research progress, and review the relationship between the adrenergic receptor signal transduction and cardiovascular diseases. It provides a theoretical basis for the further study of adrenergic receptors and the treatment of cardiovascular diseases.

How does stellate ganglion block alleviate immunologically-linked disorders?

BackgroundThe stellate ganglion is an autonomic nervous ganglion, formed by the fusion of the inferior cervical sympathetic ganglion and the first thoracic sympathetic ganglion, which is present in about 80% of people. It is anterior to the neck of the first rib and contains neurons that supply sympathetic innervation to the head and neck. Injection of local anesthetics near the stellate ganglion (stellate ganglion block; SGB) has been used for multiple clinical indications including sympathetic-mediated pain and vascular insufficiency syndromes of the upper extremity. In addition, reports on SGB having significant impact on conditions linked to immune dysfunction have been published for a century, but the mechanisms of SGB action have been poorly understood. HypothesisSGB hinders the sympathetic innervation of the immune organs, thus modulating the immune system activity and leading to the alleviation of the disease. EvidenceAll primary (thymus and bone marrow) and secondary immune organs (spleen, lymph nodes, mucosa-associated lymphoid tissue) receive a substantial sympathetic innervation, with norepinephrine (NE), as the main neurotransmitter. Complementarily, T and B lymphocytes express β2-adrenergic receptors, while innate immune cells express both α- and β-adrenergic receptors. The consequences of adrenergic receptor signaling can be summarized as immuno-modulatory. Activation of adrenergic receptors leads to decreased levels of pro-inflammatory cytokines (e.g. IL-1β, IL-6, TNF-α) and increased levels of anti-inflammatory cytokines, like IL-10 or TGF-β. Cellular changes include increase in the number of regulatory T cells and shift of the Th1/Th2 balance towards the Th2 response. Since the changes in immune response are global, the explanation has to include generalization of the SGB effect. A likely explanation includes centripetal neuronal pathways between the stellate ganglion and deep brain regions such as insula, amygdala, and hippocampus. Those, in turn, have reciprocal innervation with locus ceruleus, a brain-stem structure involved in the control of the autonomous nervous system. ConclusionVarious pathologic conditions have been shown to be SGB responsive, where the symptoms have been reduced or eliminated. Many of those clinical improvements have been mirrored by measurable immunologic changes. A plausible explanation, consistent with the evidence available so far, is that SGB exerts its effects by regulating the immune system, through a central, reflex-like pathway. Our hypothesis provides a theoretical framework for understanding the effects of SGB and could, thus lead to wider usage of the technique in immune-linked disorders such as ulcerative colitis.

Stimulation of the β2 adrenergic receptor rapidly increases phosphorylation of the Na+‐Clcotransporter (NCC)

Adrenergic signaling is proposed to modulate activity of the thiazide‐sensitive Na+‐Clcotransporter (NCC) by direct effects on the renal distal convoluted tubule (DCT). However, there are conflicting data on whether, α1, β1 or β2‐adrenoceptors mediate these effects by activation of the with‐no‐lysine kinases (WNKs) and STE20/SPS1‐related proline alanine‐rich kinase (SPAK) kinase pathway or by other signaling pathways. Here, we determined using RT‐PCR that the β2‐adrenergic receptor was abundantly expressed in mpkDCT cells (a model of the DCT) and in DCT cells isolated from parvalbumin EGFP mice by fluorescence‐activated cell sorting. In mice administered salbutamol (β2 adrenergic receptor agonist used to treat asthma) for 30 min, phosphorylated NCC levels increased, indicating increased activity. However, mice developed hypokalemia, a condition that also increases NCC phosphorylation. To investigate direct effects of β2 adrenergic signaling on DCT cells independent of K+, the effects of salbutamol on mpkDCT cells and in kidney slices/tubule suspensions were examined. Salbutamol gradually increased NCC phosphorylation in the pharmacologically relevant concentration range of 0.01–10 μM, an effect observed after 15 min and maintained at 60 min. Changes in NCC phosphorylation were associated with increased cAMP levels in tubule suspensions, kidney slices and mpkDCT cells. A role for the NCC modulatory PKA/I‐1/PP1 pathway was suggested by attenuated effects of salbutamol on phosphorylated NCC during PKA/PP1 pathway inhibition or in slices from I‐1 KO mice. Inhibition of the WNK‐SPAK pathway with a WNK inhibitor also attenuated, but did not prevent, the salbutamol‐induced phosphorylation of NCC. In conclusion, β2 adrenergic receptor signaling modulates NCC phosphorylation in vivo and ex vivo via PKA and WNK‐dependent pathways. We speculate that this response may be a compensatory mechanism to limit electrogenic renal K+ secretion during β2 adrenergic receptor stimulation of muscle. Furthermore, frequent use of salbutamol in e.g. asthmatic patients may lead to renal NaCl retention and influence blood pressure.Support or Funding InformationThe Novo Nordisk Foundation, The Leducq Foundation and the Danish Medical Research Council.

Novel gut microbiota-derived metabolite promotes platelet thrombosis via adrenergic receptor signalling

A novel metabolite derived from gut microbiota has been shown to promote platelet hyper-responsiveness and modulate platelet function via adrenergic receptors, contributing to increased thrombotic risk.

β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P&lt; 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P&lt;0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

HAND2 TRANSCRIPTION FACTOR ENHANCES NMJ ORGANIZATION AND FUNCTION IN OLD MICE

Over time, declining muscle force and power leads to mobility disability and impaired quality of life. In aging rodents and humans, a denervation and reinnervation process is strongly implicated in sarcopenia: the progressive decline of skeletal muscle mass, composition, and function. We propose that the concomitant decline in expression of Hand2, a key transcription factor (TF) for sympathetic neuron maintenance, induces motor pre- and postsynaptic neuromuscular junction (NMJ) instability and disorganization. To counter the deleterious effect of sympathetic denervation, we developed a novel viral vector (AAV9-Hand2-eGFP, Hand2) carrying Hand2 expression exclusively to sympathetic neurons. Male and female, 16-month-old mice, were examined for signs of muscle denervation and sarcopenia 6 months after IV injection with either Hand2 or control empty virus (AAV9-eGFP, EV). We found that Hand2 increased preterminal synaptic vesicle release, neurofilament phosphorylation (Neurite length: Hand2: 3732±496 µm, EV: 2674±165 µm; P <0.01), NMJ pre/postterminal co-localization, hindlimb muscle mass (EDL: 25%, soleus: 14%, tibialis anterior: 17% and gastrocnemius: 25%; n = 6-8 muscles per treatment group; P < 0.01), myofiber cross-sectional area, and protein kinase-A RIIα/RIα ratio (EV, RIIα:1.05±0.03, RIα:0.93±0.04, ratio: 1.13; Hand2, RIIα:1.81±0.03, RIα:0.94±0.03, ratio: 1.94; P<0.001) which contributes to stability of the NMJ. We also examined Hand2 gene methylation, and RNA-sequencing, muscle metabolomics, and whole body and muscle function with aging in EV and Hand2 injected mice. Our data indicate that expression of Hand2 significantly enhances skeletal muscle adrenergic receptor signaling through the canonical pathway, and prevents in NMJ transmission, and muscle mass and function decline with aging.

Abstract 012: Regulation of Cardiomyocyte Fibrosis by a Negative Feedback Loop Between Aldosterone-Induced Osteopontin Up-Regulation and beta 2 -Adrenergic Receptor Signaling

Cardiac β 2 -adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β 2 AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3`, 5`-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory and pro-fibrotic cytokine, including in the heart. We report here that the cardiotoxic hormone aldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyoblasts. This is prevented by β 2 AR-activated GPCR-kinase (GRK)-5. GRK5 directly phosphorylates and inhibits the MR in cardiomyocytes. Additionally, CRISPR-mediated OPN deletion enhances β 2 AR-dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1. OPN deletion also enables the β 2 AR to completely abrogate transforming growth factor (TGF)-β-dependent fibrosis in H9c2 cardiomyocytes. Mechanistically, OPN interacts with G α s subunits in H9c2 cardiomyocytes to facilitate recruitment of GRK2, the major GRK phosphorylating and desensitizing the cardiac β 2 AR. This, in turn, augments the GRK2-dependent functional desensitization of the β 2 AR, thereby opposing this receptor`s anti-fibrotic cAMP/Epac1 signaling. In conclusion, we have uncovered a direct inhibitory effect of OPN in cardiac β 2 AR`s anti-fibrotic signaling via facilitation of GRK2-mediated receptor desensitization. Thus, OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Synergistic activation of thermogenic adipocytes by a combination of PPARγ activation, SMAD3 inhibition and adrenergic receptor activation ameliorates metabolic abnormalities in rodents.

To treat obesity and related diseases, considerable effort has gone into developing strategies to convert white adipocytes into thermogenic brown-like adipocytes ('browning'). The purpose of this study was to identify the most efficient signal control for browning. To identify the most efficient signal control for browning, we examined rat stromal vascular fraction cells. In addition, physiological changes consequent to signal control were examined in vivo using lean and diet-induced obese (DIO) C57BL/6J mice. Combined treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone, the SMAD3 inhibitor SIS3 and the adrenergic receptor agonist noradrenaline (norepinephrine) synergistically induced Ucp1, Fgf21 and Cited1 expression, triggering brown adipogenesis. Synergistic induction of Ucp1 by the three agents was negatively regulated by forkhead box O (FOXO)3 via the inhibition of PPARγ-dependent gene transcription. Moreover, the administration of rosiglitazone, SIS3 and the selective β3 adrenergic receptor agonist CL316,243 to DIO mice reduced the amount of body-fat deposits (body weight from day 0 to 14, 12.3% reduction), concomitant with morphological changes in white adipose tissue, an increase in mitochondrial biosynthesis and a marked induction of uncoupling protein 1 (UCP1). Furthermore, administration of the three agents significantly increased serum adiponectin levels (mean 65.56μg/ml with the three agents vs 20.79μg/ml in control mice, p< 0.05) and improved glucose and lipid tolerance. These results suggest that the combined regulation of PPARγ, SMAD and the adrenergic receptor signalling pathway synergistically induces brown adipogenesis and may serve as an effective strategy to treat obesity and related diseases, including type 2 diabetes.

Abstract 3440: Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells

Abstract Breast cancer is the world’s leading cause of cancer mortality among women. Despite advances in both diagnosis and treatment a still large number of women die because of metastatic breast cancer. Research demonstrated a strong link between chronic stress and weakened immune system leaving the body prone to disease like cancer. Indeed, psychological stress has been shown to enhance tumor growth and progression. In addition, stress hormones, such as norepinephrine and epinephrine, are implicated in the growth, invasiveness and metastasis of cancer. Therefore, targeting the beta-adrenergic receptors in cancer chemoprevention may offer new promises for the design of therapeutic strategies. For that reason, we determine the cellular processes involved in beta-2 adrenergic receptor (ADRB2) mediated effects on human breast cancer cells in cultures. We particularly focused on the impact of the inhibition of ADRB2 on cell proliferation, migration and invasion of the triple negative breast cancer cell line MDA-MB-231. In order to determine the effects of loss of expression of the gene, we suppressed ADRB2 by knock down, using CRISPR technology. We also used pharmacologically suppression of ADBR by employing a general beta receptor blocker; propranolol. Cell proliferation, migration and invasion assays were performed to define a degree of aggressiveness to the cells phenotype after the knock down or pharmacological suppression of ADRB2 in comparison to control cells. Also, Western Blot analysis on characteristics proteins of the epithelial-mesenchymal transition (EMT) was used to further characterize the phenotype. PCR Array expression studies were done to analyze modifications of genes expression profile and signaling cascades in the ADRB2 knocked down cells. We found that the knocking down by CRISPR of the ADRB2 receptors or, pharmacologically suppressing ADRB2 reduces proliferation, migration and colony formation of MDA-MB-231 cells. Additionally, ADRB2 knock down increased expression of epithelial proteins (beta-catenin and E-cadherin) while decreased mesenchymal markers (vimentin and N-cadherin). The mRNA expression study using a PCR Array targeting cancer stem cell genes shows that the inhibition of ADRB2 significantly modifies signaling involved in cell movement, invasion, migration and proliferation such as Wnt/beta-catenin signaling, ILK signaling and Notch signaling. Within these signaling pathways, the expression of several genes such as ILK, MAP2K6, GSK3B and JUN seem to be critical as they are under-expressed. These data provide preliminary understanding of the possible connection between the beta-2 adrenergic receptor system and signaling pathways use by the tumor cells to grow and acquire a more aggressive phenotype. Also, some genes are identified as new targets that may be valuable for developing effective treatment of advanced breast cancer. (Supported by NIH grant R01 CA20863201) Citation Format: Benedicte Rousseau, Ananya Tirupathur, Sengottuvelan Murugan, Dipak K. Sarkar. Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3440.