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.

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms.

Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.

CD38 in the pathogenesis of allergic airway disease: Potential therapeutic targets.

CD38 is an ectoenzyme that catalyzes the conversion of ß-nicotinamide adenine dinucleotide (ß-NAD) to cyclic adenosine diphosphoribose (cADPR) and adenosine diphosphoribose (ADPR) and NADP to nicotinic acid adenine dinucleotide phosphate (NAADP) and adenosine diphosphoribose-2'-phosphate (ADPR-P). The metabolites of NAD and NADP have roles in calcium signaling in different cell types including airway smooth muscle (ASM) cells. In ASM cells, inflammatory cytokines augment CD38 expression and to a greater magnitude in cells from asthmatics, indicating a greater capacity for the generation of cADPR and ADPR in ASM from asthmatics. CD38 deficient mice develop attenuated airway responsiveness to inhaled methacholine following allergen sensitization and challenge compared to wild-type mice indicating its potential role in asthma. Regulation of CD38 expression in ASM cells is achieved by mitogen activated protein kinases, specific isoforms of PI3 kinases, the transcription factors NF-κB and AP-1, and post-transcriptionally by microRNAs. This review will focus on the role of CD38 in intracellular calcium regulation in ASM, contribution to airway inflammation and airway hyperresponsiveness in mouse models of allergic airway inflammation, the transcriptional and post-transcriptional mechanisms of regulation of expression, and outline approaches to inhibit its expression and activity.

MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

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.

CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.

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.

Potential role of the CD38/cADPR signaling pathway as an underlying mechanism of the effects of medetomidine on insulin and glucose homeostasis.

OBJECTIVE: To investigate the CD38/cADPR signaling pathway as possible underlying mechanism of the effects of medetomidine on insulin and glucose homeostasis. ANIMALS: Thirty-two C57BL/6 mice of both sexes. METHODS: Wild-type (WT) and CD38-knockout (CD38(-/-) ) mice received medetomidine (50 µg kg(-1) ) or a similar volume of 0.9% NaCl (control) by intraperitoneal (IP) injection (each group n = 8). The mice were euthanized 45 minutes later with sodium pentobarbital IP and blood was sampled via cardiac puncture. Insulin and glucose concentrations were measured by radioimmunoassay and by the oxygen rate method, respectively. Data were analyzed with anova and Bonferroni post hoc (5% significance) and are shown as mean ± SD. RESULTS: Plasma insulin and glucose concentrations were similar between WT and CD38(-/-) mice under control conditions. As compared to controls, medetomidine administration produced a statistically significant decrease in plasma insulin concentrations in the WT mice whereas the decrease in the CD38(-/-) mice was not statistically significant. Correspondingly, medetomidine caused a significantly greater increase in plasma glucose concentrations in the WT than in the CD38(-/-) mice. CONCLUSION: The CD38/cADPR signaling pathway may be one underlying mechanism of the glucose and insulin effects of the alpha-2 adrenergic receptor agonist medetomidine and likely other drugs of its class.

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.

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.

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.

CD38/cyclic ADP-ribose regulates astrocyte calcium signaling: implications for neuroinflammation and HIV-1-associated dementia.

CD38 is a 45-kD ectoenzyme involved in the synthesis of potent calcium (Ca(2+))-mobilizing agents, cyclic adenosine diphosphate-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP+). In HIV-1-infected patients, increased CD38 expression on CD8+ T cells is linked to immune system activation and progression of HIV-1 infection. However, the role of CD38 upregulation in astrocyte function and HIV-1-associated dementia (HAD-now called HAND: HIV-1-associated neurocognitive disorder) neuropathogenesis is unclear. To these ends, we used interleukin (IL)-1beta and HIV-1gp120 to activate primary human astrocytes and measured CD38 expression using real-time polymerase chain reaction and CD38 function by ADP-ribosyl cyclase activity. We also determined cADPR-mediated changes in single-cell intracellular Ca(2+) transients in activated astrocytes in presence or absence of ethylene glycol tetraacetic acid. CD38 levels were downregulated using CD38 small-interfering RNA (siRNA) and intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured. We previously reported a approximately 20-fold rise in CD38 messenger RNA levels in IL-1beta-activated astrocytes. We extend this observation and report that HIV-1gp120 potentiated CD38 expression in a dose-dependent manner and also increased CD38 enzyme activity in control and IL-1beta-activated astrocytes. We demonstrate higher cADPR levels in IL-1beta-activated astrocytes with a corresponding rise in [Ca(2+)](i) upon cADPR application and its non-hydrolysable analog, 3-deaza-cADPR. In activated astrocytes, pre-treatment with the cADPR-specific antagonist 8-Br-cADPR and CD38 siRNA transfection returned elevated [Ca(2+)](i) to baseline, thus confirming a CD38-cADPR specific response. These data are important for unraveling the mechanisms underlying the role of astrocyte-CD38 in HAD and have broader implications in other inflammatory diseases involving astrocyte activation and CD38 dysregulation.

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.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-alpha and dexamethasone.

BACKGROUND: CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-alpha). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-alpha. Regulation of CD38 expression in HASM cells involves the transcription factor NF-kappaB, and glucocorticoids inhibit this expression through NF-kappaB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene. METHODS: We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-kappaB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-kappaB and some of the AP-1 sites, or the promoter with mutations of the NF-kappaB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies. RESULTS: TNF-alpha induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-kappaB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-alpha. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB site and some of the six AP-1 sites was increased by TNF-alpha, and to some of the putative cd38 GREs by dexamethasone. CONCLUSION: The EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-kappaB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

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.

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.

CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge.

The transmembrane glycoprotein CD38 in airway smooth muscle is the source of cyclic-ADP ribose, an intracellular calcium-releasing molecule, and is subject to regulatory effects of cytokines such as interleukin (IL)-13, a cytokine implicated in asthma. We investigated the role of CD38 in airway hyperresponsiveness using a mouse model of IL-13-induced airway disease. Wild-type (WT) and CD38-deficient (CD38KO) mice were intranasally challenged with 5 microg of IL-13 three times on alternate days under isoflurane anesthesia. Lung resistance (R(L)) in response to inhaled methacholine was measured 24 h after the last challenge in pentobarbital-anesthetized, tracheostomized, and mechanically ventilated mice. Bronchoalveolar cytokines, bronchoalveolar and parenchymal inflammation, and smooth muscle contractility and relaxation using tracheal segments were also evaluated. Changes in methacholine-induced R(L) were significantly greater in the WT than in the CD38KO mice following intranasal IL-13 challenges. Airway reactivity after IL-13 exposure, as measured by the slope of the methacholine dose-response curve, was significantly higher in the WT than in the CD38KO mice. The rate of isometric force generation in tracheal segments (e.g., smooth muscle reactivity) was greater in the WT than in the CD38KO mice following incubation with IL-13. IL-13 treatment reduced isoproterenol-induced relaxations to similar magnitudes in tracheal segments obtained from WT and CD38KO mice. Both WT and CD38KO mice developed significant bronchoalveolar and parenchymal inflammation after IL-13 challenges compared with naïve controls. The results indicate that CD38 contributes to airway hyperresponsiveness in lungs exposed to IL-13 at least partly by increasing airway smooth muscle reactivity to contractile agonists.

Transcriptional regulation of CD38 expression by tumor necrosis factor-alpha in human airway smooth muscle cells: role of NF-kappaB and sensitivity to glucocorticoids.

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-alpha. In HASM cells, TNF-alpha augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-alpha increased NF-kappaB expression and its activation, and dexamethasone partially reversed these effects. TNF-alpha increased the expression of IkappaBalpha, and dexamethasone increased it further. An inhibitor of NF-kappaB activation or transfection of cells with IkappaB mutants decreased TNF-alpha-induced CD38 expression. The results indicate that TNF-alpha-induced CD38 expression involves NF-kappaB expression and its activation and dexamethasone inhibits CD38 expression through NF-kappaB-dependent and -independent mechanisms.

Regulation of CD 38 expression and function by steroid hormones in myometrium.

CD 38, a 45-kDa transmembrane glycoprotein, is expressed ubiquitously in many cell types, including the myometrial smooth muscle cells. CD 38 is a bifunctional protein, and has both ADP-ribosyl cyclase (cyclase) and cyclic ADP-ribose (cADPR)-hydrolase (hydrolase) activities. The cyclase converts beta-NAD to cADPR, a calcium mobilizing second messenger involved in fertilization, insulin secretion, and muscle contraction. CD 38 expression in smooth muscle is regulated by cytokines, by the steroid hormones estrogen and progesterone, and during gestation in the rat. Estrogen increases CD 38 expression, which is associated with increased cyclase, but not hydrolase, activity, indicating a differential post-translational regulation. Progesterone attenuates estrogen-induced effects on CD 38 expression and activities. This will have implications for increased calcium mobilization and contractility of the myometrium during parturition.