Asthma patients with comorbid obesity exhibit increased disease severity, in part, due to airway remodeling, which is also observed in mouse models of asthma and obesity. A mediator of remodeling ...that is increased in obesity is leptin. We hypothesized that in a mouse model of allergic airways disease, mice receiving exogenous leptin would display increased airway inflammation and fibrosis.
Five-week-old male and female C57BL/6J mice were challenged with intranasal house dust mite (HDM) allergen or saline 5 days per week for 6 weeks (n = 6-9 per sex, per group). Following each HDM exposure, mice received subcutaneous recombinant human leptin or saline. At 48 h after the final HDM challenge, lung mechanics were evaluated and the mice were sacrificed. Bronchoalveolar lavage was performed and differential cell counts were determined. Lung tissue was stained with Masson's trichrome, periodic acid-Schiff, and hematoxylin and eosin stains. Mouse lung fibroblasts were cultured, and whole lung mRNA was isolated.
Leptin did not affect mouse body weight, but HDM+leptin increased baseline blood glucose. In mixed-sex groups, leptin increased mouse lung fibroblast invasiveness and increased lung Col1a1 mRNA expression. Total lung resistance and tissue damping were increased with HDM+leptin treatment, but not leptin or HDM alone. Female mice exhibited enhanced airway responsiveness to methacholine with HDM+leptin treatment, while leptin alone decreased total respiratory system resistance in male mice.
In HDM-induced allergic airways disease, administration of exogenous leptin to mice enhanced lung resistance and increased markers of fibrosis, with differing effects between males and females.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Proteinase-Activated rreceptor-2 (PAR ₂), a G-protein–coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these ...opposing actions, both inhibitors and activators of PAR ₂ have been proposed for treating asthma. PAR ₂ can signal through two independent pathways: a β-arrestin–dependent one that promotes leukocyte migration, and a G-protein/Ca ²⁺ one that is required for prostaglandin E ₂ (PGE ₂) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR ₂ activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR ₂-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2 ⁻/⁻ mice. In contrast, PAR ₂-mediated PGE ₂ production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR ₂ “protective” effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2 ⁻/⁻ mice. Using the forced oscillation technique to measure airway resistance reveals that PAR ₂ activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR ₂-enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR ₂ may be β-arrestin independent.
Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote bronchodilation in asthma, but have limited ...efficacy. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of GPCR allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to, and downstream signaling of, guinea pig β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2-agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated β2-agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2-agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.
In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual ...signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Morphine is a potent analgesic, yet, like most opioid narcotics, it exerts unwanted side effects such as constipation and respiratory suppression, thereby limiting its clinical utility. ...Pharmacological approaches taken to preserve the analgesic properties, while eliminating the unwanted side effects, have met with very limited success. Here, we provide evidence that altering mu opioid receptor regulation may provide a novel approach to discriminate morphine's beneficial and deleterious effects in vivo. We have previously reported that mice lacking the G protein-coupled receptor regulatory protein, beta-arrestin 2, display profoundly altered morphine responses. beta-Arrestin 2 knockout mice have enhanced and prolonged morphine analgesia with very little morphine tolerance. In this report, we examine whether the side effects of morphine treatment are also augmented in this animal model. Surprisingly, the genetic disruption of opioid receptor regulation, while enhancing and prolonging analgesia, dramatically attenuates the respiratory suppression and acute constipation caused by morphine.
Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, ...CD4
T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4
T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4
Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4
Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.
ß-arrestins are multifunctional proteins that modulate heptahelical 7 transmembrane receptors, also known as G protein-coupled receptors (GPCRs), a superfamily of receptors that regulate most ...physiological processes. ß-arrestin modulation of GPCR function includes termination of G protein-dependent signaling, initiation of ß-arrestin-dependent signaling, receptor trafficking to degradative or recycling pathways, receptor transactivation, transcriptional regulation, and localization of second messenger regulators. The pleiotropic influence ß-arrestins exert on these receptors regulates a breadth of physiological functions, and additionally, ß-arrestins are involved in the pathophysiology of numerous and wide-ranging diseases, making them prime therapeutic targets. In this review, we briefly describe the mechanisms by which ß-arrestins regulate GPCR signaling, including the functional cellular mechanisms modulated by ß-arrestins and relate this to observed pathophysiological responses associated with ß-arrestins. We focus on the role for ß-arrestins in transducing cell signaling; a pathway that is complementary to the classical G protein-coupling pathway. The existence of these GPCR dual signaling pathways offers an immense therapeutic opportunity through selective targeting of one signaling pathway over the other. Finally, we will consider several mechanisms by which the potential of dual signaling pathway regulation can be harnessed and the implications for improved disease treatments.
Spatial and temporal control of gene expression using cre/loxP technology is a major methodological advance for biomedical research. The ability to alter gene expression after an in vivo disease ...model has been established and allows researchers the opportunity to examine the impact of that gene on the perpetuation of a disease, a mechanistic insight that is arguably more therapeutically relevant than developmental mechanisms.We used the cre/LoxP technology in mice to show that β-arrestin-2, a gene previously shown to be required for the development of the asthma phenotype, is also required for the perpetuation of, at least, the airway hyperresponsiveness characteristic of that phenotype. Here we describe stepwise methods for the activation of the cre-loxP technology and induction of murine allergic inflammatory airway disease. We comment on the unanticipated problems encountered, which we speculate were a result of interactions between the allergen and β-arrestin-2 gene (Arrb2) regulation and the effect of tamoxifen on the asthma phenotype. The issues encountered here may be generally applicable to cre/loxP utilization in inflammatory disease models, especially if the gene of interest is associated with the inflammatory cascade.
ABSTRACT
Arrestins have been shown to regulate numerous G protein‐coupled receptors (GPCRs) in studies employing receptor/arrestin overexpression in artificial cell systems. Which arrestin isoforms ...regulate which GPCRs in primary cell types is poorly understood. We sought to determine the effect of β‐arrestin‐1 or β‐arrestin‐2 inhibition or gene ablation on signaling and function of multiple GPCRs endogenously expressed in airway smooth muscle (ASM). In vitro second messenger (calcium, cAMP generation), ex vivo (ASM tension generation in suspended airway), and in vivo (invasive airway resistance) analyses were performed on human ASM cells and murine airways/whole animal subject to β‐arrestin‐1 or ‐2 knockdown or knockout (KO). In both human and murine model systems, knockdown or KO of β‐arrestin‐2 relative to control missense small interfering RNA or wild‐type mice selectively increased (40‐60%) β2‐adrenoceptor signaling and function. β‐arrestin‐1 knockdown or KO had no effect on signaling and function of β2‐adrenoceptor or numerous procontractile GPCRs, but selectively inhibited M3 muscarinic acetylcholine receptor signaling (∼50%) and function (∼25% ex vivo, >50% in vivo) without affecting EC50 values. Arrestin subtypes differentially regulate ASM GPCRs and β‐arrestin‐1 inhibition represents a novel approach to managing bronchospasm in obstructive lung diseases.—Pera, T., Hegde, A., Deshpande, D. A., Morgan, S. J., Tiegs, B. C., Theriot, B. S., Choi, Y. H., Walker, J. K. L., Penn, R. B. Specificity of arrestin subtypes in regulating airway smooth muscle G protein‐coupled receptor signaling and function. FASEB J. 29, 4227‐4235 (2015). www.fasebj.org
Highlights • β2 AR or PAR2 activation is both protective and pro-inflammatory in asthma. • β2 AR and PAR2 G protein-dependent signaling is protective in asthma. • β2 AR and PAR2 β-arrestin-dependent ...signaling is pro-inflammatory in asthma. • Biased activation of PAR2 and β2 AR G protein signaling may improve asthma treatment. • Biased inhibition of PAR2 and β2 AR β-arrestin signaling may improve asthma treatment.
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