β2-AR Expression Research Articles

Functions of beta2-adrenergic receptor in human periodontal ligament cells.

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity.

Obesity results from excess energy intake over expenditure and is characterized by chronic low-grade inflammation involving circulating monocytes (Mo) and group 2 innate lymphoid cells (ILC2s) imbalance. We analyzed circulating Mo subsets and ILC2s percentages and β2-adrenergic receptor (β2AR) expression in lean and obese subjects, and the possible effect of hypocaloric restriction on these innate immune cells. In 139 individuals aged 45 to 57 years, classified in 74 lean individuals (>18.9kg/m2 BMI <24.9kg/m2) and 65 with obesity (n = 65), we collected fasting blood samples to detect Mo subsets, ILC2s number, and β2AR expression by flow cytometry. Lipids, insulin, leptin, and acylated-ghrelin concentrations were quantified. Resting energy expenditure (REE) was estimated by indirect calorimetry. These measurements were repeated in obese subjects after 7-weeks of hypocaloric restriction. Non-classical monocytes (NCM) and β2AR expression on intermediate Mo (IM) were increased in obese individuals (p<0.001, in both cases), whereas the percent of ILC2s was decreased (p<0.0001). Stepwise regression analysis showed significantly negative associations of ILC2s with caloric intake, β2AR expression on IM with REE, but a positive relationship between NCM and HOMA-IR. Caloric restriction allowed a significant diminution of NCM and the β2AR expression on IM, as well as, an increase in the percent of classical Mo (CM), and ILC2s. ΔREE was related to ΔCD16+/CD16- ratio. These findings show that in obesity occur changes in NCM, ILC2s and β2AR expression, which contribute to the low-grade inflammation linked to obesity and might revert with caloric restriction.

Abstract P3-01-02: B2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells

Abstract Chronic stress is thought to suppress immunity; however, details about how chronic stress affects tumor growth and anti-tumor immune responses are still missing or poorly understood. Catecholamines (epinephrine, norepinephrine) released during sympathetic nerve stimulation can activate adrenergic receptors present on nearly every cell type including myeloid derived suppressor cells (MDSCs). MDSCs are increasingly recognized as critical players in tumor immunology because, in addition to their fundamental roles in suppressing effector NK cells, CD8+ T cells and CD4+ T cells, they also play a direct role in promoting tumor growth, differentiation and metastasis. Here, we demonstrate that the chronic adrenergic stress (as manipulated by a) housing temperature (which affects norepinephrine (NE) production, b) use of b-AR agonists or antagonists, or c) using β2-AR knockout mice regulates the accumulation and the pro-tumor phenotype of MDSCs. This same result was seen using two different murine models of breast cancer (4T1 and AT-3) and through the use of β2-AR knockout mice, we found that these effects are dependent on β2-adrenergic receptor (β2-AR) signaling. Using an agonist of β-AR signaling, we found that the activation of β2-AR signaling increases the expression of immunosuppressive molecules such as arginase-I and PDL-1 in MDSCs and enhances their immune-regulatory functions. β2-AR expression in MDSCs was induced by tumor promoting cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF). The survival of MDSCs is increased upon the activation of β2-AR on MDSCs in a Fas-FasL dependent manner. The regulation by β-AR signaling on MDSCs is dependent upon phosphorylation of STAT3 as a pivotal transcription factor in the function and expansion of MDSCs while MDSCs lacking β2-AR had poorer survival in both spleen and tumor. In murine tumor models, we found that the tumor promoting effects of β2-AR activation can be prevented by simple treatment of mice with β-blockers or by peripheral sympathectomy using 6-hydroxydopamine (6-OHDA). Furthermore, treatment with β-AR agonist increases the generation and immunosuppressive functions of MDSCs derived from human PBMCs in vitro. These data reveal a major, physiologically relevant role for nervous activity and adrenergic stress in MDSC-tumor biology and underscore the clinical potential for blockade of a previously unrecognized β2-AR-MDSC axis to improve immunotherapy. Keywords: β2 adrenergic receptor, MDSC, Chronic Stress, Tumor, STAT3 Citation Format: Hemn Mohammadpour, Cameron R. MacDonald, Guanxi Qiao, Minhui Chen, Bowen Dong, Bonnie L. Hylander, Philip L. McCarthy, Scott I. Abrams, Elizabeth A. Repasky. B2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-02.

Tyrosine hydroxylase and β2-adrenergic receptor expression in leukocytes of spontaneously hypertensive rats: putative peripheral markers of central sympathetic activity.

The sympathetic nervous system (SNS) plays a fundamental role in the pathophysiology of cardiovascular diseases, including primary arterial hypertension. In this study, we aimed to investigate whether the expression of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and the β2-adrenergic receptor (β2-AR) in immune cells from peripheral blood, reflect central SNS activity in spontaneously hypertensive rats (SHR). TH expression in the lower brainstem and adrenal glands and β2-AR expression in the lower brainstem were analyzed by western blot analyses. In the leukocytes, TH and β2-AR expression was evaluated by flow cytometry before and after chronic treatment with the centrally-acting sympathoinhibitory drug clonidine. Western blot analyses showed increased TH and β2-AR expression in the lower brainstem and increased TH in adrenal glands from SHR compared to normotensive Wistar Kyoto rats (WKY). Lower brainstem from SHR treated with clonidine presented reduced TH and β2-AR levels, and adrenal glands had decreased TH expression compared to SHR treated with vehicle. Flow cytometry showed that the percentage of leukocytes that express β2-AR is higher in SHR than in WKY. However, the percentage of leukocytes that expressed TH was higher in WKY than in SHR. Moreover, chronic treatment with clonidine normalized the levels of TH and β2-AR in leukocytes from SHR to similar levels of those of WKY. Our study demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment.

Zinc-α2-glycoprotein promotes browning of white adipose tissue in cold-exposed male mice

The promotion of white adipose tissue (WAT) browning has emerged as a promising therapeutic target to increase energy expenditure and decrease weight gain. Zinc-α2-glycoprotein (ZAG) is a newly identified adipokine that regulates lipid metabolism. It shows high expression in brown adipose tissue (BAT), but whether ZAG plays a key role in the browning of white adipose tissue is still largely unclear. In the present study, we explored the relationship between ZAG and the browning of WAT in cold-exposed ZAG knockout (KO) mice and 3T3-L1 adipocytes with overexpressed ZAG. The results showed that cold stress induced marked accumulation of ZAG in wild type (WT) mice. Additionally, ZAG deficiency inhibited the loss of body weight and adipose tissue weight in cold stressed mice. ZAG KO mice were resistant to cold-induced expression of browning markers and energy metabolism in WAT. Furthermore, replenishment ZAG plasmid improved the reduction in cold-induced browning of WAT in ZAG KO mice. In vitro, ZAG overexpression promoted browning and mitochondrial biogenesis and increased the expression of β3-AR and P–P38 in 3T3-L1 adipocytes. These findings demonstrate that ZAG can promote the browning of white adipose tissue and can serve as a potential therapeutic target for treating metabolic diseases such as obesity.

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.

β2 Adrenergic Regulation of the Phagocytic and Microbicide Capacity of Macrophages from Obese and Lean Mice: Effects of Exercise.

Macrophages are crucial in the inflammation associated with obesity. Exercise is the main non-pharmacological strategy against obesity, not only for improving metabolic impairment, but also because of its anti-inflammatory effects, particularly those mediated by β2 adrenergic receptors (β2-AR). Nevertheless, these anti-inflammatory effects could immunocompromise the innate response against pathogen challenge. Thus, the objective of this work was to evaluate the effect of obesity, and of exercise in this condition, on the β2 adrenergic regulation of the innate function of macrophages. High fat diet-induced obese C57BL/6J mice were used to evaluate the effects of acute and regular exercise on the phagocytic and microbicide capacities of peritoneal macrophages. Selective β2-AR agonist terbutaline (1 µM) decreased the phagocytic and microbicide activities of macrophages from control lean and obese sedentary animals. While acute exercise did not modify the inhibitory capacity of terbutaline, regular exercise abolished this inhibitory effect. These effects cannot be explained only by changes in the surface expression of β2-AR. In conclusion, (1) obesity does not alter the β2-AR-mediated decrease of the innate response of macrophages and (2) regular exercise can revert the inhibitory effect of terbutaline on the phagocytic activity of macrophages, although obesity seems to hinder this immunophysiological adaptation.

Efficacy of Quercetin as a potent sensitizer of β2-AR in combating the impairment of fluid clearance in lungs of rats under hypoxia

BackgroundHypoxia reportedly increases free radical generation in the body, causing oxidative stress and inhibiting β2-AR signaling. The present study correlates the prophylactic potential of quercetin and salbutamol in ameliorating fluid clearing capacity of lungs by re-sensitizing β2-AR signaling under hypoxia. MethodsMale SD rats supplemented orally with quercetin (50 mg/Kg BW), and salbutamol (2 mg/Kg BW) were exposed to hypobaric hypoxia at 7620 m for 6 h. Western blotting and ELISA quantitated NFĸB and related genes and GPCR pathway proteins. The binding affinities of drugs with receptor were determined by SPR spectroscopy and further confirmed insilico. ResultsQuercetin and salbutamol pre-treatment significantly up-regulated the expressions of β2-AR, GPR-1, GPR-10, GCSα, cAMP content, and down-regulated GRK-2, β-arrestin, ROS, NFκB (p < 0.001), thus, enhancing alveolar fluid clearance (AFC). SPR and insilico findings revealed a higher binding affinity of β2-AR with quercetin over salbutamol. ConclusionResults indicated quercetin to be a better prophylactic that augmented AFC in rats exposed to hypoxia by attenuating inflammation and stimulating β2-AR.

Blockade of β-Adrenergic Receptors Improves CD8+ T-cell Priming and Cancer Vaccine Efficacy.

β-Adrenergic receptor (β-AR) signaling exerts protumoral effects by acting directly on tumor cells and angiogenesis. In addition, β-AR expression on immune cells affects their ability to mount antitumor immune responses. However, how β-AR signaling impinges antitumor immune responses is still unclear. Using a mouse model of vaccine-based immunotherapy, we showed that propranolol, a nonselective β-blocker, strongly improved the efficacy of an antitumor STxBE7 vaccine by enhancing the frequency of CD8+ T lymphocytes infiltrating the tumor (TIL). However, propranolol had no effect on the reactivity of CD8+ TILs, a result further strengthened by ex vivo experiments showing that these cells were insensitive to adrenaline- or noradrenaline-induced AR signaling. In contrast, naïve CD8+ T-cell activation was strongly inhibited by β-AR signaling, and the beneficial effect of propranolol mainly occurred during CD8+ T-cell priming in the tumor-draining lymph node. We also demonstrated that the differential sensitivity of naïve CD8+ T cells and CD8+ TILs to β-AR signaling was linked to a strong downregulation of β2-AR expression related to their activation status, since in vitro-activated CD8+ T cells behaved similarly to CD8+ TILs. These results revealed that β-AR signaling suppresses the initial priming phase of antitumor CD8+ T-cell responses, providing a rationale to use clinically available β-blockers in patients to improve cancer immunotherapies.

Toxic effects of combination of Aconiti Lateralis Radix Praeparata with Trichosanthis Fructus on inflammatory response and myocardial fibrosis in pressure overload rats based on β2-AR/PKA signal

To study the effects of combination of Aconiti Lateralis Radix Praeparata( Fuzi) with Trichosanthis Fructus( Gualou) on cardiac function,electrocardiogram,inflammatory response and myocardial fibrosis in pressure overload( PO) rats,and further explore the mechanism based on β2-AR/PKA signaling. PO rat model was established by constricting the abdominal aorta. Twelve weeks after the operation,these rats were randomly divided into model goup( PO),low dose Fuzi group( FL,5. 4 g·kg-1·d-1),Gualou group( GL,5. 4 g·kg-1·d-1),Fuzi and Gualou combination group( FG,5. 4 g·kg-1·d-1+5. 4 g·kg-1·d-1) and high dose Fuzi group( FH,10. 8 g·kg-1·d-1). At the same time,sham operation group was set. After intervention for 6 weeks,carotid blood pressure,cardiac function,electrocardiogram and heart mass index were measured. HE staining was used to observe the inflammatory response in the rat heart and kidney. Masson staining was used to determine the myocardial fibrosis. Western blot was used to detect the protein expression of β2-AR and PKA. As compared with sham operation group,the blood pressure and heart mass index were obviously increased in PO model group,but there was no significant difference in various treatment groups in the above indexes. As compared with PO model group,FH treatment significantly increased the ejection fraction( EF) and GL treatment effectively enhanced the cardiac output( CO),but other treatment groups had no significant effect on these parameters. Moreover,FG treatment can synergistically attenuate QT and QTc internal prolongation,but it also aggravated inflammatory response in the heart and kidney tissues and promoted myocardial fibrosis as compared to FZ or GL alone treatment,with toxic effects equivalent to FH treatment group. Following FG and FH treatment,simultaneously,β2-AR and PKA protein levels were significantly elevated,indicating that the increasing toxicity of FG could be associated with activation of β2-AR/PKA signaling. These results suggested that combination of FZ and GL could synergistically enhance toxicity of FZ in special pathological states such as pressure overload,and caution should be taken in clinical application.

α1D-adrenergic receptor expression in human endometrial stromal cells contributes to catecholestradiol-induced cell survival: implications for endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance generation of CCEs in women with endometriosis. During gestation, CCEs bind adrenergic receptors (ARs) to mediate uterine endothelial cell proliferation. Our recent data demonstrated that compared to the basalis layer of the endometrium, the functionalis layer displayed increased β2-AR expression in women with endometriosis. Moreover, binding of CCEs to β-ARs enhanced human endometrial stromal cell (HESC) viability in culture, suggesting a modulatory role for CCE-β-AR binding in retrograde menstruation that contributes to endometriosis development. It is unknown if, in addition to β-ARs, α-ARs also have a role in this process. We, therefore, tested the hypothesis that ectopic endometrial tissue expresses α1D-AR and that 2-OHE2 and 4-OHE2 will potentiate HESC viability via α-ARs. Immunohistochemistry was performed on paired eutopic/ectopic endometriotic tissue and XTT analysis was conducted on cultured CCE-treated HESCs derived from endometrial biopsies. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained using a1D-AR antibody and evaluated semi-quantitatively by HSCORE. Confluent HESCs derived from endometrial biopsies at time of surgery for benign reasons were cultured in 96-well plates (5x103 cells/well) and treated with vehicle (control) or with 10-8 M E2 or 2-OHE2 or 4-OHE2 ± nonspecific α-AR inhibitor (phentolamine) for 48h. XTT assays measured cell viability. Experiments (n=4) were performed in duplicate and results were analyzed by One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed overall weak to moderate α1D-AR staining in endometrial epithelial cells and weak staining in endometrial stromal cells with no significant difference between eutopic and ectopic endometrial tissues in either phase. Compared to the basalis layer, both stromal and epithelial cells in the functionalis layer of eutopic endometrium displayed stronger α1D-AR staining. In vitro XTT analyses revealed phentolamine partially inhibited (P<0.05 and P<0.001, respectively) 2-OHE2 and 4-OHE2-enhanced HESC survival versus control (P<0.05 and P<0.05, respectively). This inhibitory effect of phentolamine was specific to CCEs since E2-enhanced HESC survival was not inhibited by phentolamine. Immunostaining with α1D-AR showed similar expression patterns as β2-AR in that there was increased staining to the functionalis layer of the endometrium; albeit, α1D-AR staining was overall weaker in both ectopic and eutopic tissue. Our in vivo and in vitro results indicate that induction of HESC viability by CCE-α-AR binding may contribute to the propagation of endometriosis. Future studies into different α-AR isoform expressions in endometriotic lesions are warranted.

Propranolol inhibits catecholestrogen-induced human endometrial stromal cell survival mediated by p38 MAPK signaling: potential therapy for endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Studies indicate that local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance local generation of CCEs in women with endometriosis. CCEs have low binding affinity to estrogen receptors whereas CCEs display high binding affinity to β2-adrenergic receptor (AR), which induce uterine endothelial cell proliferation during gestation. Our recent data demonstrated β2-AR expression in eutopic and ectopic endometrial tissue. In addition, binding of CCEs to β-AR enhances human endometrial stromal cell (HESC) viability, suggesting contribution of CCEs to the pathogenesis of endometriosis. Thus, we tested the hypothesis that the mechanism of CCE-enhanced HESC viability involves alterations in either proliferation or apoptosis mediated by β-AR-induced common intracellular signaling pathways, i.e. AKT, MAPK and/or NFκB. BrdU, Apoptotic Cell Detection ELISA, q-PCR, Western blot and XTT analyses were performed on cultured HESCs derived from endometrial biopsies. Cultured HESCs treated with 10-8 M E2 or 2-OHE2 or 4-OHE2 were measured by BrdU for proliferation and ELISA for apoptosis (n=3 with quadruplicate). Total RNA from cultured HESCs was isolated and pro-apoptotic, anti-apoptotic, and proliferation markers were evaluated by q-PCR (n=5 with duplicate). Total and phosphorylated AKT, p38 and ERK1/2 MAPKs, and NFκB levels were detected in lysates of cultured HESCs (n=3 with triplicate) treated for 10 min with vehicle (control) or 10-8 M E2 or 2-OHE2 or 4-OHE2 ± 2x10-5 M non-specific β-antagonist (propranolol). Subsequently, XTT assays were conducted with p38 MAPK inhibitor to assess the role of p38 MAPK on CCE-induced HESC viability (n=4 with triplicate). Results were analyzed by One-way ANOVA and post hoc Tukey test. An increased HESC proliferation index by E2 and 4-OHE2 (P<0.05 and P<0.05, respectively) and decreased apoptosis were detected in HESCs treated with 2-OHE2 and 4-OHE2 vs. control (P<0.01 and P<0.01, respectively). Analysis of apoptotic markers by q-PCR revealed a significant decrease in Bax mRNA expression in response to 2-OHE2 treatment vs. control (P<0.01). Among the several intracellular signaling cascades analyzed, only phosphorylation levels of p38 MAPK were increased by either treatment with 2-OHE2 or 4-OHE2 (P<0.05 and P<0.05 vs. control, respectively), but not with E2. β-AR antagonism with propranolol mitigated this increased phosphorylation in p38 MAPK levels (P<0.05 and P<0.01, respectively) and inhibition of p38 MAPK by SB203580 blocked CCE-induced HESC survival (P<0.05 and P<0.001, respectively). These data indicate that induction of endometrial stromal cell viability by CCE-β-AR interactions results from an imbalance in both proliferation and anti-apoptotic mechanisms and is specifically mediated by the activation of p38 MAPK signaling. These data also suggest that inhibition of β2-ARs with propranolol may be a novel treatment option in endometriosis.

β2‑adrenergic receptor signaling promotes neuroblastoma cell proliferation by activating autophagy.

Accumulating evidence suggests the pivotal role of the sympathetic nervous system in the initiation and aggressive progression of tumors, whereas the role of β-adrenergic receptor (β-AR) signaling in neuroblastoma (NB) and the underlying regulatory mechanisms have not yet been well elucidated. In the present study, it was demonstrated that the expression of both β1-AR and β2-AR was significantly increased in clinical samples of NB compared with those of ganglioneuroma (GN) and ganglioneuroblastoma (GNB), and that β2-AR is the key β-adrenergic receptor responsible for NB cell growth. Further investigation showed that the expression levels of the autophagy markers LC3-II, beclin-1 and unc-51-like autophagy kinase 1 (ULK1) were also elevated in NB, compared to the cases of GN and GNB. Moreover, β2-AR expression was found to be positively associated with autophagy markers in the clinical NB specimens. Cellular functional assays demonstrated that β2-AR activation promoted NB cell growth and activated the autophagy pathway. Pharmacological inhibition of autophagy with 3-methyladenine abolished β2-AR-induced NB cell growth. Mechanistically, β2-AR signaling triggers autophagy through CREB-mediated ULK1 upregulation. In conclusion, the present study uncovered a novel regulatory mechanism of β2-AR-activated autophagy in NB cell growth and provides a novel potential therapeutic approach for treating NB by targeting autophagy and the β2-AR pathway.

Activation of β2-Adrenoceptor Attenuates Sepsis-Induced Hippocampus-Dependent Cognitive Impairments by Reversing Neuroinflammation and Synaptic Abnormalities

Sepsis-associated encephalopathy induces cognitive dysfunction via mechanisms that commonly involve neuroinflammation and synaptic plasticity impairment of the hippocampus. The β2-adrenoceptor (β2-AR) is a G-protein coupled receptor that regulates immune response and synaptic plasticity, whereas its dysfunction has been implicated in various neurodegenerative diseases. Thus, we hypothesized abnormal β2-AR signaling is involved in sepsis-induced cognitive impairment. In the present study, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to mimic the clinical human sepsis-associated encephalopathy. The levels of hippocampal β2-AR, proinflammatory cytokines tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), IL-6, cAMP-response element binding protein (CREB), brain derived neurotrophic factor (BDNF), post-synaptic density protein 95 (PSD95), and NMDA receptor 2 B subtypes (GluN2B) were determined at 6, 12, 24 h and 7 and 16 days after CLP. For the interventional study, mice were treated with β2-AR agonist clenbuterol in two ways: early treatment (immediately following CLP) and delayed treatment (on the 8th day following CLP). Neurobehavioral performances were assessed by open field and fear conditioning tests. Here, we found that hippocampal β2-AR expression was significantly decreased starting from 12 h and persisted until 16 days following CLP. Besides, sepsis mice also exhibited increasing neuroinflammation, down-regulated CREB/BDNF, decreasing PSD95 and GluN2B expression, and displayed hippocampus-dependent cognitive impairments. Notably, early clenbuterol treatment alleviated sepsis-induced cognitive deficits by polarizing microglia toward an anti-inflammatory phenotype, reducing proinflammatory cytokines including IL-1β, TNF-α, and up-regulating CREB/BDNF, PSD95, and GluN2B. Intriguingly, delayed clenbuterol treatment also improved cognitive impairments by normalization of hippocampal CREB/BDNF, PSD95, and GluN2B. In summary, our results support the beneficial effects of both early and delayed clenbuterol treatment, which suggests that activation of β2-AR has a translational value in sepsis-associated organ dysfunction including cognitive impairments.

β2-Adrenergic receptor expression is associated with biomarkers of tumor immunity and predicts poor prognosis in estrogen receptor-negative breast cancer.

Antitumor immunity plays an important role in the progression of breast cancer. β2-adrenergic receptor (β2AR) was found to regulate the antitumor immune response and breast cancer progression in preclinical studies. To understand the clinical role of β2AR in cancer progression, we investigated the clinicopathological and prognostic significance of β2AR expression in invasive breast cancer. β2AR levels in breast tumors were evaluated by immunohistochemistry in a well-characterized patient cohort with long-term follow-up (n = 278). We evaluated the relationship of β2AR expression to patient survival and clinicopathological factors, including immune biomarkers such as tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression. Breast cancer-specific survival was compared between high- and low-β2AR expression groups. Although β2AR was not related to clinicopathological factors across the whole cohort, high β2AR was significantly related to PD-L1 negativity in estrogen receptor (ER)-negative patients. Tumors with high β2AR tended to have low TIL grade, and high β2AR was an independent prognostic factor for reduced survival in ER-negative patients. β2AR is an independent poor prognostic factor in ER-negative breast cancer. The findings suggest that tumor β2AR regulates immune checkpoint activity, which may have therapeutic implications for patients with ER-negative breast cancer.

G protein-coupled receptor kinase 2 regulating β2-adrenergic receptor signaling in M2-polarized macrophages contributes to hepatocellular carcinoma progression.

Background: β2-adrenoceptors (β2-ARs) are expressed on the surface of immune cells, including tumor-associated macrophages (TAMs). Previous studies have demonstrated that the expression of β2-ARs in hepatocellular carcinoma (HCC) is significantly increased in vitro. However, the role of β2-AR in M2-polarized macrophages remains unclear. G protein-coupled receptor kinase 2 (GRK2) can regulate G protein-coupled receptor (GPCR). Previous studies showed that down-regulation of GRK2 in HCC contributes the HCC progression, but it still remains unclear whether the regulation of β2-AR in M2-polarized macrophages by GRK2 can promote HCC.Purpose: The present study was designed to investigate the role of activated β2-AR in M2-polarized macrophages in the HCC progression and GRK2 regulatory effect, as well as the underlying mechanisms involved.Results: The results demonstrated that the M2-polarized macrophages were increased with HCC progression. In vitro, the activation of β2-AR by terbutaline in M2-polarized macrophages elevated the proliferative, migratory and invasive attributes of HCC cells. Furthermore, GRK2 down-regulation in β2-AR activated M2-polarized macrophages activated the downstream cyclic adenosine monophosphate (cAMP)/protein kinase A/cAMP-response element binding protein and cAMP/interleukin-6/signal transducer and the activator of transcription 3 signaling pathways, contributing to the secretion of tumor-associated cytokines, and thus resulting in the promotion of malignant biological behavior in HCC cells.Conclusion: These findings suggest that the regulation of β2-AR occurs through the silencing of GRK2 in M2-polarized macrophages, which is conducive to HCC development, through its engagement in the activation of downstream signaling.

Post-Mortem Immunohistochemical Evidence of β2-Adrenergic Receptor Expression in the Adrenal Gland.

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

Malnutrition screening in patients with pancreatic disorders

Cancer patients often experience multiple emotional distresses, particularly the fear of death. However, there are rare studies to assess the direct effect of the fear of death on disease progression.Xenograft pancreatic cancer animal models were established in nude mice. Fear stress was induced to tumor bearing mice by closely housing with a cat and depressive behaviors were measured using open field test, forced swimming test, and sucrose consumption test. Plasma adrenaline concentration was measured using ELISA.Fear stress induced depression-like behaviors in tumor bearing mice which were accompanied with increases in tumor growth, plasma adrenaline levels as well as the protein expression of alpha 2 adrenergic receptor (α2 AR) and beta 2 adrenergic receptor (β2-AR) in tumor tissues. The β-adrenergic antagonist propranolol (Pro) treatment blocked the effect of stress on tumor growth in pancreatic cancer xenograft animal model, but had no effects on the levels of plasma adrenaline level, and α2 AR and β2-AR expression in tumor tissues. Moreover, fear stress increased Frizzled-1, Wnt1, vimentin, but decreased E-cadherin protein expression in tumor tissues, while Pro reversed the effects of fear stress on the expression of these proteins.Fear of death impacted the growth of PDAC tumor though activation of epithelial-mesenchymal transition. Treating pancreatic cancer patients with β-adrenergic antagonist implicates an effective strategy to treat cancer including PDAC.

Effect of electroacupuncture on myocardial infarct size and expression of inflammatory cytokines and sympathetic-active substances in the myocardial ischemic tissue of rats

To investigate the effect of electroacupuncture (EA) on the expression of interleukin-8 (IL-8), interleukin-10 (IL-10), tyrosine hydroxylase (TH), β3-adrenergic receptor (β3AR), and endothelial nitric oxide synthase (eNOS) in myocardial tissue in ischemic myocardial injury rats, so as to reveal its underlying mechanisms in myocardial protection via anti-inflammation and sympathetic nerve remodeling. A total of 48 male Sprague-Dawley rats were randomly divided into sham-operation (sham, n＝9), sham ＋EA (n＝9), model (n＝15) and EA (n＝15) groups. The myocardial ischemia (MI) model was established by ligation of the left anterior descending branch of the left coronary artery. EA (2 Hz/15 Hz，1.5－2 mA) was applied to bilateral "Neiguan" (PC6) for 30 min, once daily for 4 days. The myocardial infarct size was detected by 2, 3, 5 triphenyltetrazolium chloride (TTC) staining, myocardial histopathological changes and inflammatory infiltration were assessed by H．E. staining, and the expression of IL-8, IL-10, TH, β3AR, and eNOS in the myocardium was determined by using Western blot. Compared with the sham group, a marked myocardial infarction was found in the left ventricle tissue, accompanied with disordered arrangement of myocardial fibers and higher degree of inflammatory cell infiltration, and increased expression of IL-8, TH, β3AR and eNOS in the myocardium in the model group (P<0.01), but without significant change in the expression of IL-10 (P>0.05). After EA intervention and in comparison with the model group, the myocardial infarct size was significantly reduced (P<0.01), the severity of inflammatory cell infiltration and disordered arrangement of myocardial fibers were relieved, and the expression of IL-10 and eNOS proteins were significantly up-regulated (P<0.05), and the markedly up-regulated expression of IL-8, TH, and β3AR were significantly suppressed in the EA group (P<0.01)．. EA intervention can reduce the myocardial infarct size (protective effect) in MI rats possibly by reducing inflammatory reaction and sympathetic nerve remodeling.

Expression of Cutaneous Beta-2 Adrenoceptors Is Similar in Patients with Complex Regional Pain Syndrome and Pain-Free Controls.

Studies in rodents suggest that cutaneous beta-2 adrenoceptors (β2-ARs) mediate inflammation and pain after tissue injury and that inflammation and peripheral nerve injury trigger increases in neuronal β2-AR expression. Hence, the aim of this study was to investigate the expression of β2-ARs on keratinocytes and dermal nerves in patients with complex regional pain syndrome (CRPS). Fifty-eight patients with CRPS were recruited for this study. In addition, skin biopsies were obtained from 13 pain-free women and three pain-free men of similar age and sex distribution as the patients. Quantitative sensory tests for assessing sensitivity to pressure, pinprick, light touch, heat, and cold were administered, and skin biopsies were obtained from the affected and contralateral limbs. Skin biopsies were also obtained from a similar site on the dorsal hand or foot of pain-free controls. Immunohistochemistry and confocal microscopy were used to identify β2-ARs on keratinocytes, dermal nerves, and blood vessels in the skin samples. The distribution of β2-ARs in keratinocytes and nerves was similar in the affected and contralateral limbs of patients and was similar for target cells in patients and controls. However, elevated β2-AR expression in reticular nerve bundles was associated with heightened sensitivity to heat pain. These findings do not support a major role of cutaneous β2-ARs in CRPS. However, activation of neuronal β2-ARs may contribute to thermal hyperalgesia in a subgroup of patients. Whether activation of β2-ARs on keratinocytes mediates inflammation early in the course of CRPS requires further investigation.