Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Rhinovirus C15 Attenuates Relaxation and cAMP Production in Human Airways and Smooth Muscle.

Rhinoviruses (RVs) evoke as many as 85% of acute asthma exacerbations in children and 50% in adults and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Using human precision-cut lung slices (hPCLSs), primary human air-liquid interface-differentiated airway epithelial cells (HAECs), and human airway smooth muscle (HASM) as preclinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin (Fsk), was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAECs decreased cellular relaxation in response to isoproterenol and prostaglandin E2, but not Fsk. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not Fsk, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV-inactivated RV-C15 showed markedly attenuated airway relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15-mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of ß2-adrenergic receptor function.

Starving a Cell Promotes Airway Smooth Muscle Relaxation: Inhibition of Glycolysis Attenuates Excitation-Contraction Coupling.

Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation-contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5-240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist-induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation-contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.

Mucosal-associated invariant T cells restrict reactive oxidative damage and preserve meningeal barrier integrity and cognitive function.

Increasing evidence indicates close interaction between immune cells and the brain, revising the traditional view of the immune privilege of the brain. However, the specific mechanisms by which immune cells promote normal neural function are not entirely understood. Mucosal-associated invariant T cells (MAIT cells) are a unique type of innate-like T cell with molecular and functional properties that remain to be better characterized. In the present study, we report that MAIT cells are present in the meninges and express high levels of antioxidant molecules. MAIT cell deficiency in mice results in the accumulation of reactive oxidative species in the meninges, leading to reduced expression of junctional protein and meningeal barrier leakage. The presence of MAIT cells restricts neuroinflammation in the brain and preserves learning and memory. Together, our work reveals a new functional role for MAIT cells in the meninges and suggests that meningeal immune cells can help maintain normal neural function by preserving meningeal barrier homeostasis and integrity.

Epinephrine evokes shortening of human airway smooth muscle cells following ß2 adrenergic receptor desensitization.

Epinephrine (EPI), an endogenous catecholamine involved in the body's fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiological functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating ß2-adrenergic receptors (ß2ARs). Conventionally, the selective ß2AR agonism of EPI was thought to be, in part, due to a predominance of ß2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous "switch-like" activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of ß2ARs and/or under experimental conditions that induce ß2AR tachyphylaxis. EPI-induced procontractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or ß2-agonist insensitivity in patients with difficult-to-control, disease subtypes.

Obesity elicits a unique metabolomic signature in human airway smooth muscle cells.

Obesity can aggravate asthma by enhancing airway hyperresponsiveness (AHR) and attenuating response to treatment. However, the precise mechanisms linking obesity and asthma remain unknown. Human airway smooth muscle (HASM) cells exhibit amplified excitation-contraction (EC) coupling and force generation in obesity. Therefore, we posit that airway smooth muscle (ASM) cells obtained from obese donors manifest a metabolomic phenotype distinct from that of nonobese donor cells and that a differential metabolic phenotype, at least in part, drives enhanced ASM cell EC coupling. HASM cells derived from age-, sex-, and race-matched nonobese [body mass index (BMI) ≤ 24.9 kg·m-2] and obese (BMI ≥ 29.9 kg·m-2) lung donors were subjected to unbiased metabolomic screening. The unbiased metabolomic screening identified differentially altered metabolites linked to glycolysis and citric acid cycle in obese donor-derived cells compared with nonobese donor cells. The Seahorse assay measured the bioenergetic profile based on glycolysis, mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates in HASM cells. Glycolytic rate and capacity were elevated in obese donor-derived HASM cells, whereas mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates were comparable between obese and nonobese groups. PFKFB3 mRNA and protein expression levels were also elevated in obese donor-derived HASM cells. Furthermore, pharmacological inhibition of PFKFB3 attenuated agonist-induced myosin light chain (MLC) phosphorylation in HASM cells derived from obese and nonobese donors. Our findings identify elevated glycolysis as a signature metabolic phenotype of obesity and inhibition of glycolysis attenuates MLC phosphorylation in HASM cells. These findings identify novel therapeutic targets to mitigate AHR in obesity-associated asthma.

CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids.

BACKGROUND: CCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function. METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures). RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM. CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma.

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells.

BACKGROUND: Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation-contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. METHODS: In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to ß2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett's test for multiple group comparisons or Student's t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. RESULTS: Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in ß2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. CONCLUSIONS: Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

Dexamethasone rescues TGF-ß1-mediated ß2-adrenergic receptor dysfunction and attenuates phosphodiesterase 4D expression in human airway smooth muscle cells.

Glucocorticoids (GCs) and ß2-adrenergic receptor (ß2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- ß1 (TGF-ß1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes ß2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-ß1-effects on ß2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-ß1 effects on cAMP levels induced by isoproterenol (ISO). TGF-ß1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the ß2AR receptor. Previously, we demonstrated that TGF-ß1 treatment increased ß2AR phosphorylation to induce hyporesponsiveness to a ß2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of ß2AR function, are not altered with TGF-ß1 stimulation. Interestingly, DEX also attenuated TGF-ß1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-ß1 levels.

Budesonide enhances agonist-induced bronchodilation in human small airways by increasing cAMP production in airway smooth muscle.

The nongenomic mechanisms by which glucocorticoids modulate ß2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on ß2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and ß2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.

The Receptor for Hyaluronan-Mediated Motility (CD168) promotes inflammation and fibrosis after acute lung injury.

Acute lung injury results in early inflammation and respiratory distress, and later fibrosis. The glycosaminoglycan hyaluronan (HA) and the Receptor for Hyaluronan-Mediated Motility (RHAMM, CD168) have been implicated in the response to acute lung injury. We hypothesized that, compared to wild type (WT) mice, RHAMM knockout (KO) mice would be protected from, whereas mice with macrophage-specific transgenic overexpression of RHAMM (TG) would have worse inflammation, respiratory distress and fibrosis after intratracheal (IT) bleomycin. Compared to WT mice, 10 days after IT bleomycin, RHAMM KO mice had less weight loss, less increase in respiratory rate, and fewer CD45+ cells in the lung. At day 28, compared to injured WT animals, injured RHAMM KO mice had lower M1 macrophage content, as well as decreased fibrosis as determined by trichrome staining, Ashcroft scores and lung HPO content. Four lines of transgenic mice with selective overexpression of RHAMM in macrophages were generated using the Scavenger Receptor A promoter driving a myc-tagged full length RHAMM cDNA. Baseline expression of RHAMM and CD44 was the same in WT and TG mice. By flow cytometry, TG bone marrow-derived macrophages (BMDM) had increased cell surface RHAMM and myc, but equal CD44 expression. TG BMDM also had 2-fold increases in both chemotaxis to HA and proliferation in fetal bovine serum. In TG mice, increased inflammation after thioglycollate-induced peritonitis was restricted to macrophages and not neutrophils. For lung injury studies, non-transgenic mice given bleomycin had respiratory distress with increased respiratory rates from day 7 to 21. However, TG mice had higher respiratory rates from 4 days after bleomycin and continued to increase respiratory rates up to day 21. At 21 days after IT bleomycin, TG mice had increased lung macrophage accumulation. Lavage HA concentrations were 6-fold higher in injured WT mice, but 30-fold higher in injured TG mice. At 21 days after IT bleomycin, WT mice had developed fibrosis, but TG mice showed exaggerated fibrosis with increased Ashcroft scores and HPO content. We conclude that RHAMM is a critical component of the inflammatory response, respiratory distress and fibrosis after acute lung injury. We speculate that RHAMM is a potential therapeutic target to limit the consequences of acute lung injury.

Obesity increases airway smooth muscle responses to contractile agonists.

The asthma-obesity syndrome represents a major public health concern that disproportionately contributes to asthma severity and induces insensitivity to therapy. To date, no study has shown an intrinsic difference between human airway smooth muscle (HASM) cells derived from nonobese subjects and those derived from obese subjects. The objective of this study was to address whether there is a greater response to agonist-induced calcium mobilization, phosphorylation of myosin light chain (MLC), and greater shortening in HASM cells derived from obese subjects. HASM cells derived from nonobese and obese subjects were age and sex matched. Phosphorylation of MLC was measured after having been stimulated by carbachol. Carbachol- or histamine-induced mobilization of calcium and cell shortening were assessed in HASM cells derived from nonobese and obese donors. Agonist-induced MLC phosphorylation, mobilization of calcium, and cell shortening were greater in obese compared with non-obese-derived HASM cells. The MLC response was comparable in HASM cells derived from obese nonasthma and nonobese fatal asthma subjects. HASM cells derived from obese female subjects were more responsive to carbachol than HASM cells derived from obese male subjects. Insulin pretreatment had little effect on these responses. Our results show an increase in agonist-induced calcium mobilization associated with an increase in MLC phosphorylation and an increase in ASM cell shortening in favor of agonist-induced hyperresponsiveness in HASM cells derived from obese subjects. Our studies suggest that obesity induces a retained phenotype of hyperresponsiveness in cultured human airway smooth muscle cells.

Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination.

Platelet endothelial cell adhesion molecule­1 (PECAM­1) is expressed on the vascular endothelium and has been implicated in the late progression of metastatic tumors. The activity of PECAM­1 appears to be mediated by modulation of the tumor microenvironment (TME) and promotion of tumor cell proliferation, rather than through the stimulation of tumor angiogenesis. The present study aimed to extend those initial findings by indicating that the presence of functional PECAM­1 on the endothelium promotes a proliferative tumor cell phenotype in vivo, as well as in tumor cell (B16­F10 melanoma and 4T1 breast cancer cell lines) co­culture assays with mouse endothelial cells (ECs) or a surrogate EC line (REN­MP). The pro­proliferative effects were mediated by soluble endothelial­derived factors that were dependent on PECAM­1 homophilic ligand interactions, but which were independent of PECAM­1­dependent signaling. Further analysis of the conditioned media obtained from tumor/EC and tumor/REN­MP co­cultures identified TIMP metallopeptidase inhibitor­1 (TIMP­1) as a PECAM­1­regulated factor, the targeting of which in the tumor cell/REN­MP system inhibited tumor cell proliferation. In addition, TIMP­1 expression was decreased in metastatic tumors from the lungs of PECAM­1­null mice, thus providing evidence of the in vivo significance of co­culture studies. Taken together, these studies indicated that endothelial PECAM­1, through PECAM­1­dependent homophilic binding interactions, may induce release of TIMP­1 from the endothelium into the TME, thus leading to increased tumor cell proliferation.

TGF-ß1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3.

Transforming growth factor ß1 (TGF-ß1), a cytokine whose levels are elevated in the airways of patients with asthma, perpetuates airway inflammation and modulates airway structural cell remodeling. However, the role of TGF-ß1 in excessive airway narrowing in asthma, or airway hyperresponsiveness (AHR), remains unclear. In this study, we set out to investigate the direct effects of TGF-ß1 on human airway smooth muscle (HASM) cell shortening and hyperresponsiveness. The dynamics of AHR and single-cell excitation-contraction coupling were measured in human precision-cut lung slices and in isolated HASM cells using supravital microscopy and magnetic twisting cytometry, respectively. In human precision-cut lung slices, overnight treatment with TGF-ß1 significantly augmented basal and carbachol-induced bronchoconstriction. In isolated HASM cells, TGF-ß1 increased basal and methacholine-induced cytoskeletal stiffness in a dose- and time-dependent manner. TGF-ß1-induced single-cell contraction was corroborated by concomitant increases in myosin light chain and myosin phosphatase target subunit 1 phosphorylation levels, which were attenuated by small interfering RNA-mediated knockdown of Smad3 and pharmacological inhibition of Rho kinase. Strikingly, these physiological effects of TGF-ß1 occurred through a RhoA-independent mechanism, with little effect on HASM cell [Ca2+]i levels. Together, our data suggest that TGF-ß1 enhances HASM excitation-contraction coupling pathways to induce HASM cell shortening and hyperresponsiveness. These findings reveal a potential link between airway injury-repair responses and bronchial hyperreactivity in asthma, and define TGF-ß1 signaling as a potential target to reduce AHR in asthma.

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

BACKGROUND AND PURPOSE: PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. EXPERIMENTAL APPROACH: Gα12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction. KEY RESULTS: Knockdown of M3 receptors or Gα12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα12 coimmunoprecipitated with M3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS. CONCLUSIONS AND IMPLICATIONS: Gα12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression.

Airway smooth muscle is a major target tissue for glucocorticoid (GC)-based asthma therapies, however, molecular mechanisms through which the GC receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the nuclear factor-κB (NF-κB) inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-κB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-κB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-κB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied small interfering RNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-κB contributes to cytokine repression in HASM.