Crosstalk between CD4+ T Cells and Airway Smooth Muscle in Pediatric Obesity-related Asthma.

Rationale: Pediatric obesity-related asthma is a nonatopic asthma phenotype with high disease burden and few effective therapies. RhoGTPase upregulation in peripheral blood T helper (Th) cells is associated with the phenotype, but the mechanisms that underlie this association are not known. Objectives: To investigate the mechanisms by which upregulation of CDC42 (Cell Division Cycle 42), a RhoGTPase, in Th cells is associated with airway smooth muscle (ASM) biology. Methods: Chemotaxis of obese asthma and healthy-weight asthma Th cells, and their adhesion to obese and healthy-weight nonasthmatic ASM, was investigated. Transcriptomics and proteomics were used to determine the differential effect of obese and healthy-weight asthma Th cell adhesion to obese or healthy-weight ASM biology. Measurements and Main Results: Chemotaxis of obese asthma Th cells with CDC42 upregulation was resistant to CDC42 inhibition. Obese asthma Th cells were more adherent to obese ASM compared with healthy-weight asthma Th cells to healthy-weight ASM. Compared with coculture with healthy-weight ASM, obese asthma Th cell coculture with obese ASM was positively enriched for genes and proteins involved in actin cytoskeleton organization, transmembrane receptor protein kinase signaling, and cell mitosis, and negatively enriched for extracellular matrix organization. Targeted gene evaluation revealed upregulation of IFNG, TNF (tumor necrosis factor), and Cluster of Differentiation 247 (CD247) among Th cell genes, and of Ak strain transforming (AKT), Ras homolog family member A (RHOA), and CD38, with downregulation of PRKCA (Protein kinase C-alpha), among smooth muscle genes. Conclusions: Obese asthma Th cells have uninhibited chemotaxis and are more adherent to obese ASM, which is associated with upregulation of genes and proteins associated with smooth muscle proliferation and reciprocal nonatopic Th cell activation.

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.

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.

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.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

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.

House Dust Mite-Induced Allergic Airway Disease Is Independent of IgE and FcεRIα.

IgE contributes to disease exacerbations but not to baseline airway hyperresponsiveness (AHR) in human asthma. In rodent allergic airway disease (AAD), mast cell and IgE dependence for the induction of AHR has only been observed when mice are immunized with a relatively weak allergen without adjuvant. To evaluate the role of IgE in murine AAD that is induced by a potent allergen, we inoculated BALB/c and FVB/N background wild-type and IgE- or FcεRIα-deficient mice intratracheally with large or limiting doses of house dust mite extract (HDM) and evaluated AHR, pulmonary eosinophilia, goblet cell metaplasia, serum IgE, and lung mastocytosis. We found that neither IgE nor FcεRIα contributed to AAD, even in mice inoculated with the lowest dose of HDM, which readily induced detectable disease, but did not increase serum IgE or pulmonary mast cell levels. In contrast, high doses of HDM strikingly increased serum IgE and pulmonary mast cells, although both AHR and airway mast cell degranulation were equally elevated in wild-type and IgE-deficient mice. Surprisingly, allergen challenge of mice with severe AAD and pulmonary mastocytosis failed to acutely increase airway resistance, lung Newtonian resistance, or hysteresis. Overall, this study shows that, although mice may not reliably model acute asthma exacerbations, mechanisms that are IgE and FcεRIα independent are responsible for AHR and airway inflammation when low doses of a potent allergen are inhaled repetitively.

Airway responsiveness in CD38-deficient mice in allergic airway disease: studies with bone marrow chimeras.

CD38 is a cell-surface protein involved in calcium signaling and contractility of airway smooth muscle. It has a role in normal airway responsiveness and in airway hyperresponsiveness (AHR) developed following airway exposure to IL-13 and TNF-α but appears not to be critical to airway inflammation in response to the cytokines. CD38 is also involved in T cell-mediated immune response to protein antigens. In this study, we assessed the contribution of CD38 to AHR and inflammation to two distinct allergens, ovalbumin and the epidemiologically relevant environmental fungus Alternaria. We also generated bone marrow chimeras to assess whether Cd38(+/+) inflammatory cells would restore AHR in the CD38-deficient (Cd38(-/-)) hosts following ovalbumin challenge. Results show that wild-type (WT) mice develop greater AHR to inhaled methacholine than Cd38(-/-) mice following challenge with either allergen, with comparable airway inflammation. Reciprocal bone marrow transfers did not change the native airway phenotypic differences between WT and Cd38(-/-) mice, indicating that the lower airway reactivity of Cd38(-/-) mice stems from Cd38(-/-) lung parenchymal cells. Following bone marrow transfer from either source and ovalbumin challenge, the phenotype of Cd38(-/-) hosts was partially reversed, whereas the airway phenotype of the WT hosts was preserved. Airway inflammation was similar in Cd38(-/-) and WT chimeras. These results indicate that loss of CD38 on hematopoietic cells is not sufficient to prevent AHR and that the magnitude of airway inflammation is not the predominant underlying determinant of AHR in mice.

MicroRNA-708 regulates CD38 expression through signaling pathways JNK MAP kinase and PTEN/AKT in human airway smooth muscle cells.

BACKGROUND: The cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38-/- mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3' Untranslated Region (3'UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells. METHODS: Growth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3'UTR to alter gene expression. RESULTS: Using target prediction algorithms, we identified several miRNAs with potential CD38 3'UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3'UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling. CONCLUSIONS: In human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3'UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.

Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases.

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110ß-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, ß, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110ß, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.

miR-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells.

CD38, a membrane protein expressed in airway smooth muscle (ASM) cells, plays a role in cellular Ca(2+) dynamics and ASM contractility. In human ASM (HASM) cells, TNF-α induces CD38 expression through activation of MAPKs, NF-κB, and AP-1, and its expression is differentially elevated in cells from asthmatic patients compared with cells from nonasthmatic subjects. The CD38 3'-untranslated region (UTR) has targets for miR-140-3p. We hypothesized that miR-140-3p regulates CD38 expression in HASM cells by altering CD38 mRNA stability. Basal and TNF-α-induced expression of miR-140-3p was determined in nonasthmatic ASM (NAASM) and asthmatic ASM (AASM) cells. NAASM and AASM cells were transfected with control, miR-140-3p mimic, or miR-140-3p antagomirs, and CD38 expression and CD38 mRNA stability were determined. Luciferase reporter assays were used to determine miR-140-3p binding to the CD38 3'-UTR. Activation of p38, ERK, and JNK MAPKs, NF-κB, and AP-1 was determined in miR-140-3p mimic-transfected NAASM. TNF-α attenuated miR-140-3p expression in NAASM and AASM cells, but at a greater magnitude in AASM cells. CD38 mRNA expression was attenuated by miR-140-3p mimic at comparable magnitude in NAASM and AASM cells. Mutated miR-140-3p target on the CD38 3'-UTR reversed the inhibition of luciferase activity by miR-140-3p mimic. CD38 mRNA stability was unaltered by miR-140-3p mimic in NAASM or AASM cells following arrest of transcription. TNF-α-induced activation of p38 MAPK and NF-κB was attenuated by miR-140-3p mimic. The findings indicate that miR-140-3p modulates CD38 expression in HASM cells through direct binding to the CD38 3'-UTR and indirect mechanisms involving activation of p38 MAPK and NF-κB. Furthermore, indirect mechanisms appear to play a major role in the regulation of CD38 expression.

Differential induction of CD38 expression by TNF-{alpha} in asthmatic airway smooth muscle cells.

The ADP-ribosyl cyclase activity of CD38, a membrane protein expressed in human airway smooth muscle (ASM) cells, generates cyclic ADP-ribose (cADPR), a Ca²(+)-mobilizing agent. cADPR-mediated Ca²(+) responses to agonists are augmented in human ASM cells by TNF-α. CD38-deficient mice fail to develop airway hyperresponsiveness following intranasal TNF-α or IL-13 challenge, suggesting a role in asthma. The role of CD38 in human asthma remains unknown. We hypothesized that CD38 expression will be elevated in ASM cells from asthmatic donors (ASMA cells). CD38 mRNA and ADP-ribosyl cyclase activity were measured in cells maintained in growth-arrested conditions and exposed to vehicle or TNF-α (10-40 ng/ml). TNF-α-induced induction of CD38 expression was greater in ASMA than in ASM cells from nonasthmatic donors (ASMNA). In four of the six donors, basal and TNF-α-induced ERK and p38 MAPK activation were higher in ASMA than ASMNA cells. JNK MAPK activation was lower in ASMA than ASMNA cells. Nuclear NF-κB (p50 subunit) and phosphorylated c-Jun were comparable in cells from both groups, although nuclear c-Fos (part of the AP-1 complex) levels were lower in ASMA than ASMNA cells. NF-κB or AP-1 binding to their consensus sequences was comparable in ASMNA and ASMA cells, as are the decay kinetics of CD38 mRNA. The findings suggest that the differential induction of CD38 by TNF-α in ASMA cells is due to increased transcriptional regulation involving ERK and p38 MAPK activation and is independent of changes in NF-κB or AP-1 activation. The findings suggest a potential role for CD38 in the pathophysiology of asthma.

Role of CD38/cADPR signaling in obstructive pulmonary diseases.

The worldwide socioeconomical burden associated with chronic respiratory diseases is substantial. Enzymes involved in the metabolism of nicotinamide adenine dinucleotide (NAD) are increasingly being implicated in chronic airway diseases. One such enzyme, CD38, utilizes NAD to produce several metabolites, including cyclic ADP ribose (cADPR), which is involved in calcium signaling in airway smooth muscle (ASM). Upregulation of CD38 in ASM caused by exposure to cytokines or allergens leads to enhanced calcium mobilization by agonists and the development of airway hyperresponsiveness (AHR) to contractile agonists. Glucocorticoids and microRNAs can suppress CD38 expression in ASM, whereas cADPR antagonists such as 8Br-cADPR can directly antagonize intracellular calcium mobilization. Bronchodilators act via CD38-independent mechanisms. CD38-dependent mechanisms could be developed for chronic airway diseases therapy.

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.

Altered CD38/Cyclic ADP-Ribose Signaling Contributes to the Asthmatic Phenotype.

CD38 is a transmembrane glycoprotein expressed in airway smooth muscle cells. The enzymatic activity of CD38 generates cyclic ADP-ribose from ß-NAD. Cyclic ADP-ribose mobilizes intracellular calcium during activation of airway smooth muscle cells by G-protein-coupled receptors through activation of ryanodine receptor channels in the sarcoplasmic reticulum. Inflammatory cytokines that are implicated in asthma upregulate CD38 expression and increase the calcium responses to contractile agonists in airway smooth muscle cells. The augmented intracellular calcium responses following cytokine exposure of airway smooth muscle cells are inhibited by an antagonist of cyclic ADP-ribose. Airway smooth muscle cells from CD38 knockout mice exhibit attenuated intracellular calcium responses to agonists, and these mice have reduced airway response to inhaled methacholine. CD38 also contributes to airway hyperresponsiveness as shown in mouse models of allergen or cytokine-induced inflammatory airway disease. In airway smooth muscle cells obtained from asthmatics, the cytokine-induced CD38 expression is significantly enhanced compared to expression in cells from nonasthmatics. This differential induction of CD38 expression in asthmatic airway smooth muscle cells stems from increased activation of MAP kinases and transcription through NF-κB, and altered post-transcriptional regulation through microRNAs. We propose that increased capacity for CD38 signaling in airway smooth muscle in asthma contributes to airway hyperresponsiveness.

Calcium signaling in airway smooth muscle.

Contractility of airway smooth muscle requires elevation of intracellular calcium concentration. Under resting conditions, airway smooth muscle cells maintain a relatively low intracellular calcium concentration, and activation of the surface receptors by contractile agonists results in an elevation of intracellular calcium, culminating in contraction of the cell. The pattern of elevation of intracellular calcium brought about by agonists is a dynamic process and involves the coordinated activities of ion channels located in the plasma membrane and the sarcoplasmic reticulum. Among the signaling molecules involved in this dynamic calcium regulation in airway smooth muscle cells are inositol 1,4,5-trisphosphate and cyclic ADP-ribose, which mobilize calcium from the sarcoplasmic reticulum by acting via the inositol 1,4,5-trisphosphate and ryanodine receptors, respectively. In addition, calcium influx from the extracellular space is critical for the repletion of the intracellular calcium stores during activation of the cells by agonists. Calcium influx can occur via voltage- and receptor-gated channels in the plasma membrane, as well as by influx that is triggered by depletion of the intracellular stores (i.e., store-operated calcium entry mechanism). Transient receptor potential proteins appear to mediate the calcium influx via receptor- and store-operated channels. Recent studies have shown that proinflammatory cytokines regulate the expression and activity of the pathways involved in intracellular calcium regulation, thereby contributing to airway smooth muscle cell hyperresponsiveness. In this review, we will discuss the specific roles of cyclic ADP-ribose/ryanodine receptor channels and transient receptor potential channels in the regulation of intracellular calcium in airway smooth muscle cells.

Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1.

The enzymatic activity of CD38, ADP-ribosyl cyclase, synthesizes the calcium mobilizing molecule cyclic ADP-ribose from beta-NAD. In human airway smooth muscle (HASM) cells, CD38 expression is augmented by the inflammatory cytokine, TNF-alpha, causing increased intracellular calcium response to agonists. The transcriptional and posttranscriptional regulation of CD38 expression involves signaling through MAPKs and requires activation of NF-kappaB and activator protein-1 (AP-1). The cytokine-augmented CD38 expression is decreased by anti-inflammatory glucocorticoids due to inhibition of NF-kappaB activation and other mechanisms. In this study, we investigated glucocorticoid regulation of CD38 expression in HASM cells through the MKP-1. In HASM cells, dexamethasone and TNF-alpha induced MKP-1 expression (both mRNA and protein) rapidly. Dexamethasone decreased TNF-alpha-induced phosphorylation of the major MAPKs, i.e., ERK, p38, and JNK, and decreased the activation of NF-kappaB and AP-1. Dexamethasone also decreased CD38 expression induced by TNF-alpha, and part of this effect was attributable to decreased transcript stability. In cells transfected with MKP-1-specific small interfering RNAs (siRNAs), there was significant attenuation of MKP-1 expression and partial, but nonsignificant, reversal of dexamethasone inhibition of CD38 expression. These results indicate that regulation of CD38 expression in HASM cells by glucocorticoids involves decreased signaling through MAPKs and activation of transcription factors. The glucocorticoid effects on decreased CD38 expression and function result from regulation through transcription and transcript stability.

Role of CD38 in TNF-alpha-induced airway hyperresponsiveness.

CD38 is involved in normal airway function, IL-13-induced airway hyperresponsiveness (AHR), and is also regulated by tumor necrosis factor (TNF)-alpha in airway smooth muscle (ASM) cells. This study aimed to determine whether TNF-alpha-induced CD38 upregulation in ASM cells contributes to AHR, a hallmark of asthma. We hypothesized that AHR would be attenuated in TNF-alpha-exposed CD38-deficient (CD38KO) mice compared with wild-type (WT) controls. Mice (n = 6-8/group) were intranasally challenged with vehicle control or TNF-alpha (50 ng) once and every other day during 1 or 4 wk. Lung inflammation and AHR, measured by changes in lung resistance after inhaled methacholine, were assessed 24 h following the last challenge. Tracheal rings were incubated with TNF-alpha (50 ng/ml) to assess contractile changes in the ASM. While a single TNF-alpha challenge caused no airway inflammation, both multiple-challenge protocols induced equally significant inflammation in CD38KO and WT mice. A single intranasal TNF-alpha challenge induced AHR in the WT but not in the CD38KO mice, whereas both mice developed AHR after 1 wk of challenges. The AHR was suppressed by extending the challenges for 4 wk in both mice, although to a larger magnitude in the WT than in the CD38KO mice. TNF-alpha increased ASM contractile properties in tracheal rings from WT but not from CD38KO mice. In conclusion, CD38 contributes to TNF-alpha-induced AHR after a brief airway exposure to the cytokine, likely by mediating changes in ASM contractile responses, and is associated with greater AHR remission following chronic airway exposure to TNF-alpha. The mechanisms involved in this remission remain to be determined.