Molecular mechanism of bitter taste receptor agonist-mediated relaxation of airway smooth muscle.

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled ß2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

In silico identification of a ß2-adrenoceptor allosteric site that selectively augments canonical ß2AR-Gs signaling and function.

Activation of ß2-adrenoceptors (ß2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and ß2AR agonists (ß-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of ß-agonists is limited by agonist-induced ß2AR desensitization and noncanonical ß2AR signaling involving ß-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on ß2AR that could modulate the activity of ß-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated ß2AR intermediate conformations and identified a putative allosteric binding site. Subsequent database screening using SILCS identified drug-like molecules with the potential to bind to the site. Experimental assays in HEK293 cells (expressing recombinant wild-type human ß2AR) and human ASM cells (expressing endogenous ß2AR) identified positive and negative allosteric modulators (PAMs and NAMs) of ß2AR as assessed by regulation of ß-agonist-stimulation of cyclic AMP generation. PAMs/NAMs had no effect on ß-agonist-induced recruitment of ß-arrestin to ß2AR- or ß-agonist-induced loss of cell surface expression in HEK293 cells expressing ß2AR. Mutagenesis analysis of ß2AR confirmed the SILCS identified site based on mutants of amino acids R131, Y219, and F282. Finally, functional studies revealed augmentation of ß-agonist-induced relaxation of contracted human ASM cells and bronchodilation of contracted airways. These findings identify a allosteric binding site on the ß2AR, whose activation selectively augments ß-agonist-induced Gs signaling, and increases relaxation of ASM cells, the principal therapeutic effect of ß-agonists.

A-Kinase-Anchoring-Protein Subtypes Differentially Regulate GPCR Signaling and Function in Human Airway Smooth Muscle.

A-kinase-anchoring proteins (AKAPs) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent protein kinase A (PKA) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled G protein-coupled receptors (GPCRs). The beta-2-adrenoceptor (ß2AR), as well as the Gs-coupled EP2 and EP4 receptor subtypes of the E-prostanoid (EP) receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes of asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin, in differentially regulating PKA substrates downstream of the ß2AR, EP2 receptor (EP2R) and EP4 receptor (EP4R). Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates vasodilator-stimulated phosphoprotein (VASP) and heat shock protein 20 (HSP20). Ezrin knockdown, as well as combined Ezrin + Gravin knockdown significantly reduced the induction of phospho-VASP and phospho-HSP20 by ß2AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by ß2AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM, and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma.

Diacylglycerol kinase is a keystone regulator of signaling relevant to the pathophysiology of asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Targeted Inhibition of Select Extracellular Signal-regulated Kinases 1 and 2 Functions Mitigates Pathological Features of Asthma in Mice.

ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 µg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.

Diacylglycerol kinase zeta deficiency attenuates papain-induced type 2 airway inflammation.

Allergic airway diseases are caused by inappropriate immune responses directed against inhaled environmental antigens. We previously reported that the inhibition of diacylglycerol (DAG) kinaseζ (DGKζ),an enzyme that terminates DAG-mediated signaling,protects against T cell-mediated allergic airway inflammation by blocking Th2 cell differentiation.In this study, we tested whether DGKζ deficiency also affects allergic airway disease mediated by type 2 innate lymphoid cells (ILC2)s. DGKζ-deficient mice displayed diminished ILC2 function and reduced papain-induced airway inflammation compared to wildtype mice. Unexpectedly, however, mice with hematopoietic cell-specific deletion ofDGKζ displayed intact airway inflammation upon papain challenge. Rather, bone marrow chimera studies revealed thatDGKζ deficiency in the non-hematopoietic compartment was responsible for the reduction in papain-induced airway inflammation. These data suggest that DGK might represent a novel therapeutic target not only for T cell-dependent but also ILC2-dependent allergic airway inflammation by affecting non-hematopoietic cells.

Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

BACKGROUND: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). METHODS: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. RESULTS: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. CONCLUSIONS: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

Proteomics-Based Approach for Detailing the Allergenic Profile of Cannabis Chemotypes.

Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30-37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3.

PD 102807 Induces M3 mAChR-Dependent GRK-/Arrestin-Biased Signaling in Airway Smooth Muscle Cells.

G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated ß-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by ß-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor ß (TGF-ß)-mediated functions, PD 102807 inhibited TGF-ß-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.

Autocrine regulation of airway smooth muscle contraction by diacylglycerol kinase.

Diacylglycerol kinase (DGK), a lipid kinase, catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid, thereby terminating DAG-mediated signaling by Gq-coupled receptors that regulate contraction of airway smooth muscle (ASM). A previous study from our laboratory demonstrated that DGK inhibition or genetic ablation leads to reduced ASM contraction and provides protection for allergen-induced airway hyperresponsiveness. However, the mechanism by which DGK regulates contractile signaling in ASM is not well established. Herein, we investigated the role of prorelaxant cAMP-protein kinase A (PKA) signaling in DGK-mediated regulation of ASM contraction. Pretreatment of human ASM cells with DGK inhibitor I activated PKA as demonstrated by the phosphorylation of PKA substrates, VASP, Hsp20, and CREB, which was abrogated when PKA was inhibited pharmacologically or molecularly using overexpression of the PKA inhibitor peptide, PKI. Furthermore, inhibition of DGK resulted in induction of cyclooxygenase (COX) and generation of prostaglandin E2 (PGE2 ) with concomitant activation of Gs-cAMP-PKA signaling in ASM cells in an autocrine/paracrine fashion. Inhibition of protein kinase C (PKC) or extracellular-signal-regulated kinase (ERK) attenuated DGK-mediated production of PGE2 and activation of cAMP-PKA signaling in human ASM cells, suggesting that inhibition of DGK activates the COX-PGE2 pathway in a PKC-ERK-dependent manner. Finally, DGK inhibition-mediated attenuation of contractile agonist-induced phosphorylation of myosin light chain 20 (MLC-20), a marker of ASM contraction, involves COX-mediated cAMP production and PKA activation in ASM cells. Collectively these findings establish a novel mechanism by which DGK regulates ASM contraction and further advances DGK as a potential therapeutic target to provide effective bronchoprotection in asthma.

Dysregulated retinoic acid signaling in airway smooth muscle cells in asthma.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

Regulation of Airway Smooth Muscle Cell Proliferation by Diacylglycerol Kinase: Relevance to Airway Remodeling in Asthma.

Airway remodeling in asthma involves the hyperproliferation of airway smooth muscle (ASM) cells. However, the molecular signals that regulate ASM growth are not completely understood. Gq-coupled G protein-coupled receptor and receptor tyrosine kinase signaling regulate ASM cell proliferation via activation of phospholipase C, generation of inositol triphosphate (IP3) and diacylglycerol (DAG). Diacylglycerol kinase (DGK) converts DAG into phosphatidic acid (PA) and terminates DAG signaling while promoting PA-mediated signaling and function. Herein, we hypothesized that PA is a pro-mitogenic second messenger in ASM, and DGK inhibition reduces the conversion of DAG into PA resulting in inhibition of ASM cell proliferation. We assessed the effect of pharmacological inhibition of DGK on pro-mitogenic signaling and proliferation in primary human ASM cells. Pretreatment with DGK inhibitor I (DGKI) significantly inhibited platelet-derived growth factor-stimulated ASM cell proliferation. Anti-mitogenic effect of DGKI was associated with decreased mTOR signaling and expression of cyclin D1. Exogenous PA promoted pro-mitogenic signaling and rescued DGKI-induced attenuation of ASM cell proliferation. Finally, house dust mite (HDM) challenge in wild type mice promoted airway remodeling features, which were attenuated in DGKζ-/- mice. We propose that DGK serves as a potential drug target for mitigating airway remodeling in asthma.

Nur77 Attenuates Inflammasome Activation by Inhibiting Caspase-1 Expression in Pulmonary Vascular Endothelial Cells.

Inflammasomes are intracellular multiprotein complexes that help trigger and maintain the inflammatory response as part of the innate immune system. Recently, it has been increasingly recognized that aberrant inflammasome activation is critically involved in endothelial dysfunction in a variety of human diseases, such as atherosclerosis, acute lung injury (ALI), and type 2 diabetes. The molecular mechanisms underlying endothelial inflammasome activation, however, have not been completely elucidated. In the present study, we identified orphan nuclear receptor Nur77 as a novel regulator in controlling inflammasome activation in vascular endothelial cells (ECs). We demonstrated that LPS-induced inflammasome activation was significantly inhibited by ectopic overexpression of Nur77, predominantly through transcriptional suppression of caspase-1 expression in vascular ECs. Consistent with this observation, we found that LPS-induced inflammasome activation was significantly augmented in lung ECs isolated from Nur77-knockout mice. Mechanistically, we showed that Nur77-induced inhibition of caspase-1 expression was due to an inhibition of IRF1 (IFN regulatory factor 1) expression and its subsequent binding to the caspase-1 promoter. Importantly, in a mouse model of LPS-induced ALI, Nur77 knockout led to a marked activation of caspase-1 in the lung, increased alveolar and circulating IL-1ß levels, and exacerbated ALI, all of which were substantially inhibited by administration of caspase-1 inhibitor. Together, our results support the presence of an important role for Nur77 in controlling inflammasome activation in vascular ECs and suggest that Nur77 could be a novel therapeutic target for the treatment of human diseases associated with aberrant inflammasome activation, such as ALI and atherosclerosis.

Diacylglycerol Kinase Inhibition Reduces Airway Contraction by Negative Feedback Regulation of Gq-Signaling.

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors causes airway hyperresponsiveness in asthma. Activation of Gq-coupled G protein-coupled receptors leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKζ and α isoform knockout mice are protected from the development of allergen-induced airway hyperresponsiveness. Here we aimed to determine the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζ, whereas pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Furthermore, we used precision-cut human lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pretreatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and MLC phosphatase in ASM cells. Furthermore, DGK inhibition decreased Gq agonist-induced calcium elevation and generation of IP3 and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio, resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.

A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation.

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

OGR1-dependent regulation of the allergen-induced asthma phenotype.

The proton-sensing receptor, ovarian cancer G protein-coupled receptor (OGR1), has been shown to be expressed in airway smooth muscle (ASM) cells and is capable of promoting ASM contraction in response to decreased extracellular pH. OGR1 knockout (OGR1KO) mice are reported to be resistant to the asthma features induced by inhaled allergen. We recently described certain benzodiazepines as OGR1 activators capable of mediating both procontractile and prorelaxant signaling in ASM cells. Here we assess the effect of treatment with the benzodiazepines lorazepam or sulazepam on the asthma phenotype in wild-type (WT) and OGR1KO mice subjected to inhaled house dust mite (HDM; Dermatophagoides pteronyssius) challenge for 3 wk. In contrast to previously published reports, both WT and OGR1KO mice developed significant allergen-induced lung inflammation and airway hyperresponsiveness (AHR). In WT mice, treatment with sulazepam (a Gs-biased OGR1 agonist), but not lorazepam (a balanced OGR1 agonist), prevented allergen-induced AHR, although neither drug inhibited lung inflammation. The protection from development of AHR conferred by sulazepam was absent in OGR1KO mice. Treatment of WT mice with sulazepam also resulted in significant inhibition of HDM-induced collagen accumulation in the lung tissue. These findings suggest that OGR1 expression is not a requirement for development of the allergen-induced asthma phenotype, but OGR1 can be targeted by the Gs-biased OGR1 agonist sulazepam (but not the balanced agonist lorazepam) to protect from allergen-induced AHR, possibly mediated via suppression of chronic bronchoconstriction and airway remodeling in the absence of effects on airway inflammation.

Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2.

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.