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

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CD38 and Airway hyperresponsiveness: Studies on human airway smooth muscle cells and mouse models Guedes, Alonso GP1; Deshpande, Deepak A2; Dileepan, Mythili3; Walseth, Timothy F4; Panettieri, Jr, Reynold A5; Subramanian6, Subbaya; Kannan, Mathur S3. Department of Veterinary and Biomedical Sciences3, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108; Department of Medicine2, Thomas Jefferson University, Philadelphia, PA 19107; Department of Pharmacology4 and Surgery6, University of Minnesota Medical School, Minneapolis, MN 55455; Department of Medicine5, University of Pennsylvania, Philadelphia, PA 19104; Department of Surgical & Radiological Sciences1, University of California, Davis, CA 95616. *Address for correspondence: Mathur S Kannan, Ph.D. Department of Veterinary and Biomedical Sciences College of Veterinary Medicine University of Minnesota 1971 Commonwealth Avenue St. Paul, MN 55108, USA Tel: 612-624-3757 Fax: 612-625-5203 E mail: [email protected]


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Abstract: Asthma is an inflammatory disease in which altered calcium regulation, contractility and airway smooth muscle (ASM) proliferation contribute to airway hyperresponsiveness 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 activation of MAP kinases and the transcription factors, NF-ƙB and AP-1 and post-transcriptionally regulated by miR-1403p 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 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 to wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyperresponsiveness to inhaled methacholine in the Cd38 deficient mice. These studies provide

evidence

for

CD38

involvement

in

the

development

of

airway

hyperresponsiveness, a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and/or activity are warranted.

Key Words: Inflammatory cytokines; microRNAs; bone marrow chimeras; cyclic ADP-ribose


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CD38-cyclic ADP ribose (cADPR) signaling regulates a variety of cellular/organ functions, and is known to play an important role in diseases such as chronic lymphocytic leukemia (Malavasi et al. 2011). Studies from our laboratory established that: 1. CD38 is expressed in ASM cells (White et al. 2000); 2. CD38/cADPR-mediated calcium release contributes to calcium homeostasis in ASM cells (Deshpande et al. 2003); 3. ASM contraction and bronchoconstriction are in part mediated by the activation of CD38/cADPR pathway. CD38 is also expressed on the surface of immune cells, and is known to regulate immune responses (Cockayne et al. 1998). Keeping in mind the physiological role of CD38 in the regulation of ASM function, a key modulator of airway tone, and immune cells, we hypothesized that CD38/cADPR signaling pathway regulates airway inflammation and hyperresponsiveness (AHR). This review summarizes the experimental data delineating the pathological roles of CD38 in obstructive airway disease, asthma. CD38: enzyme responsible for cADPR synthesis and degradation Cyclic ADP-ribose is a calcium releasing second messenger derived from nicotinamide adenine dinucleotide (NAD). Results from extensive biochemical studies clearly demonstrated that cADPR is synthesized by the action of ADP-ribosyl cyclase and degraded by cADPR hydrolase (Lee et al. 1999). The observation by Clapper et.al, that addition of NAD to sea urchin egg microsomes results in calcium release from the microsomes led to the discovery of cADPR (Clapper et al. 1987). There were a few key observations made during this seminal study: 1) addition of NAD to egg microsomes results in calcium release, 2) calcium release by NAD is independent of IP3, and 3) the kinetics of calcium release revealed a lag of 1-2 minutes after the addition of NAD


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suggesting involvement of an enzymatic step that converted NAD to a metabolite that was responsible for calcium mobilization. Initial biochemical and biophysical studies and subsequent X-ray crystallographic studies identified the cyclical nature of cADPR, a calcium releasing metabolite of NADcADPR. ADP-ribosyl cyclase was identified as the enzyme responsible for the conversion of NAD to cADPR (Lee and Aarhus 1991). Originally, the NAD metabolizing enzyme was purified from Aplysia ovotestis but was named NADase as methods to determine intermediate products of NAD metabolism were not available at the time (Hellmich and Strumwasser 1991). This ADP-ribosyl cyclase purified from Aplysia ovotestis is a soluble protein of approximately 30 kDa molecular weight (Lee and Aarhus 1991). Initial studies using various extracts obtained from mammalian tissues revealed that the ADP-ribosyl cyclase activity is present in many tissues (Adebanjo et al. 2000; Rusinko and Lee 1989). Subsequently the sequence comparison of the Aplysia cyclase (States et al. 1992) and biochemical analysis revealed that CD38, a membrane bound lymphocyte antigen, possesses ADPribosyl cyclase activity (Lee 2006) and is considered the mammalian homolog of the ADP-ribosyl cyclase (Lee 2006). Interestingly, CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Figure 1). It is ~ 45 kDa in size and found associated with the cell membrane. Subsequent studies demonstrated that ADP-ribosyl cyclase and cADPR hydrolase activities are also associated with other membrane bound proteins such as bone marrow stromal cell surface antigen (BST)-1 or CD157, in mammals (Yamamoto-Katayama et al. 2001). BST-1 was identified to be homologous to CD38. Studies using human, murine and porcine ASM confirmed the expression of CD38 and the enzyme activities associated with CD38 in ASM (Deshpande et al.


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2005a). CD38 is the primary source for cADPR production in ASM although non-CD38 ADP-ribosyl activities have also been described in other cell types (Ceni et al. 2006). siRNA-mediated knockdown or genetic ablation of CD38 results in diminished levels of cADPR (Kang et al. 2005) and increase in CD38 expression results in increased levels of cADPR in ASM (Deshpande et al. 2003) demonstrating a role of CD38 in mediating cADPR production. In ASM cells obtained from Cd38 knockout (KO) mice, a low level of cADPR is detectable in ASM suggesting potential source of non-CD38 ADP-ribosyl cyclases in ASM (Deshpande et al. 2005b). Interestingly, agonist stimulation seems to favor cADPR synthesis in ASM cells. Future studies are needed to establish time kinetics of cADPR synthesis and degradation in ASM cells. In addition to the activities that enable CD38 to produce (ADP-ribosyl cyclase) and degrade cADPR (cADPR hydrolase), this enzyme has been shown to have the ability to produce two other metabolites that are involved in the regulation of calcium homeostasis. CD38 can hydrolyze NAD to ADP-ribose (ADPR) (Zocchi et al. 1993) and synthesize nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP and nicotinic acid via a base-exchange reaction (Aarhus et al. 1995). ADPR and NAADP have been shown to play important roles in calcium signaling. ADPR regulates calcium influx via TRPM2 channels (Perraud et al. 2001), while NAADP regulates calcium release from acidic endolysosomal stores through (Calcraft et al. 2009).

regulation of two pore channels

NAADP was shown to contribute to acetylcholine-induced

contraction of guinea pig trachea and oxytocin-induced contraction of rat uterine smooth muscle (Aley et al. 2010; Aley et al. 2013).

Thus, CD38 produces two second


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messengers, cADPR and NAADP, that are able to regulate calcium release and contractility in ASM. cADPR and regulation of ASM calcium homeostasis and contraction Calcium homeostasis in ASM is regulated by a complex interplay of second messenger molecules, ion channels, signaling regulatory molecules and calcium stores. This includes calcium influx-efflux mechanisms and release/re-uptake processes. NAD metabolites, cADPR and NAADP have emerged as calcium releasing second messengers, and presumably mediating calcium release through ryanodine receptor (RyR) channels (Albrieux et al. 1998; Lee 2011). We and other airway biology investigators have clearly demonstrated the contribution of cADPR-mediated calcium release in ASM using both pharmacological and genetic approaches. The conclusion stems from a series of experiments carried out using freshly isolated porcine ASM cells, cultured primary human ASM cells and murine ASM cells obtained from wild type (WT) and Cd38 KO mice. The following observations support this interpretation: 1. Preincubation of ASM cells with 8-bromo-cADPR, a cell permeable cADPR antagonist, inhibits agonist-induced intracellular calcium ([Ca2+]i) responses in human ASM cells (Prakash et al. 1998; White et al. 2003); 2. addition of

extracellular cADPR to

porcine/bovine ASM cells causes concentration-dependent increase in [Ca2+]i (Franco et al. 2001); 3. up-regulation of CD38 by overexpression or cytokine treatment results in enhanced calcium responses to G protein-coupled receptor (GPCR) agonists (Prakash et al. 1998); 4. down-regulation of CD38 expression by anti-sense CD38 expression attenuates agonist-induced calcium responses in ASM cells (Kang et al. 2005); 5. calcium responses to acetylcholine or endothelin are significantly diminished in ASM


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cells obtained from CD38 KO mice compared to WT mice (Deshpande et al. 2005b); 6. addition of cADPR to equine tracheal smooth muscle cells results in increased amplitude and frequency of spontaneous transient inward currents (STICs) suggesting spontaneous release of calcium by cADPR in ASM cells (Wang et al. 2004); 7. siRNA mediated downregulation of CD38 expression attenuates cytokine-induced increase in store-operated calcium entry in ASM cells (Sieck et al. 2008). cADPR-mediated calcium release is believed to involve activation of RyR channels

on

sarcoplasmic

reticulum

(SR).

Pharmacological

and

biochemical

approaches employed in ASM cells confirmed the role of RyRs in effecting calcium release by cADPR (Deshpande et al. 2005a). Several lines of evidences exist in this regard: 1. Calcium release by cADPR is not sensitive to pretreatment with IP3R antagonist, and 2. inhibitors of RyR channels, ryanodine, ruthenium red, procaine and Mg2+ inhibit cADPR-mediated calcium release. However, RyRs do not possess binding pockets for cADPR although high affinity binding of 32P-cADPR to permeabilized smooth muscle cells and tissue lysates provide the experimental evidence for cADPR-SR interaction. The interaction of cADPR and RyR may involve additional accessory proteins such as calmodulin and FK506 binding protein (Wang et al. 2004). These results indicate that cADPR mediates calcium release via RyR either directly or indirectly via accessory proteins. Calcium elevation in ASM has a causal relationship with contraction. The role of CD38/cADPR in mediating contraction has been investigated in smooth muscle of intestine, seminiferous tubules, blood vessels, trachea and uterus. In longitudinal smooth muscles of intestine, cADPR elicited contractions in a concentration-dependent


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manner (Kuemmerle and Makhlouf 1995; Kuemmerle et al. 1998). This was the first study to demonstrate the contractile response induced by cADPR. Similarly, application of cADPR or 3-deaza cADPR, a non-hydrolyzable cADPR analog, increased acetylcholine-induced contractions of smooth muscle strips isolated from bovine trachea, and pre-incubation of the ASM strips with 8-amino cADPR, a cADPR antagonist, attenuated this response (Franco et al. 2001). Murine tracheal ring contractility ex vivo in WT and Cd38 KO mice is not significantly different under naïve conditions. However, the in vivo airway responsiveness to different doses of inhaled methacholine, as determined by changes in lung resistance and dynamic compliance, are significantly lower in Cd38 KO mice compared with WT controls (Deshpande et al. 2005b; Guedes et al. 2008; Guedes et al. 2006). These evidences clearly demonstrate the functional role of CD38/cADPR in the regulation of ASM contractility and in vivo lung function (Figure 1). In summary, studies in ASM have clearly demonstrated the role of cADPRmediated calcium release in the regulation of calcium homeostasis. Further, CD38 is the primary (if not the only) cADPR generating, membrane bound bi-functional enzyme in ASM. It is imperative to expect that any change in the expression or activity of CD38, the enzyme responsible of cADPR metabolism could result in augmented calcium release in effector cells namely, ASM cells (Figure 1). cADPR and airway hyperresponsiveness Asthma is an inflammatory disease in which pro-inflammatory cytokines have a role in inducing abnormalities of ASM function and in the development of AHR. Inflammatory cytokines alter Ca2+ signaling and contractility of ASM, which results in


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nonspecific hyperreactivity to agonists (Deshpande et al. 2004; Deshpande et al. 2003; Deshpande et al. 2005a). We explored if CD38/cADPR-mediated calcium release in ASM is altered under inflammatory conditions and contributes to AHR. Altered expression and function of CD38 have been demonstrated in pathological conditions such as diabetes (Okamoto et al. 1997), hypoxia-induced vascoconstriction (Wilson et al. 2001) and hematopoietic malignancies (Malavasi et al. 2011). Several lines of evidences suggest the role of CD38/cADPR in AHR: 1. Pretreatment of ASM cells with different concentrations of inflammatory (TNF-α, IL-1β, IFN-γ) or TH2 (IL-13) cytokines results in increased CD38 expression and cADPR production (Deshpande et al. 2004; Deshpande et al. 2003); 2. Augmented calcium responses to different GPCR agonists (indicating AHR) can be attenuated by a cell permeable cADPR antagonist (White et al. 2003); 3. The augmentation of CD38 expression and therefore the capacity for CD38/cADPR signaling is significantly greater following TNF-α exposure in ASM cells derived from asthmatics than in cells from non-asthmatics (Jude et al. 2010); 4. siRNAmediated knockdown of CD38 expression inhibits TNF-α-induced augmented storeoperated calcium entry (another indicator of AHR) in ASM (Sieck et al. 2008); 5. Ex vivo treatment of tracheal rings with IL-13 results in an increased contractility to carbachol exposure in wild-type mice and not in Cd38 KO mice (Guedes et al. 2008); 6. Glucocorticoids (drug of choice in asthma treatment) inhibit cytokine-induced CD38 expression (Kang et al. 2008) and cADPR-mediated calcium release (unpublished observation) in human ASM cells.. These studies collectively demonstrate a causal relationship among CD38 expression, cADPR production, and agonist-induced calcium elevation and ASM contraction, which are altered under inflammatory conditions.


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CD38 gene organization and regulation of expression Regulating expression of CD38 is a potential drug target to mitigate inflammation-induced AHR. In this context, understanding the molecular mechanisms involved in CD38 expression is a first logical and critical step. Studies from our laboratory have shed light on transcriptional and post-transcriptional regulation of CD38 expression in human ASM cells. Sequence analysis of a 3 kb putative CD38 promoter fragment (GenBank Accession # DQ091293) cloned from a human erythropoietic cell line (K562 cells) in our laboratory revealed binding sites for NF-ƙB, AP-1, and glucocorticoid receptor (Tirumurugaan et al. 2008)(Figure 2). There is one NF-ƙB, six AP-1 and four glucocorticoid

response

element

(GRE)

binding

motifs

in

the

CD38

gene.

Electrophoretic mobility shift assay (EMSA) confirmed direct binding of NF-ƙB and AP-1 to putative binding sites on CD38promoter. The AP1–4 site (residing between -2798 to 2789 bp) that shows very strong binding in EMSA studies was found to be functionally important, since mutation of this site profoundly affected TNF-α-induced activation of CD38 expression. The CD38 gene (>80 kb long) comprising of 8 exons is located on chromosome 4 in human and chromosome 5 in the mouse genome (Ferrero et al. 2000). Binding sites for several transcription-activating factors in the CD38 gene have been identified (Ferrero et al. 2000). Previous studies have shown the absence of a canonical TATA or CAAT box sequences in the CD38 promoter region, suggesting that transcription can be initiated at multiple sites (Ferrero et al. 2000). However, TATA-less promoters with


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transcription start sites such as an initiator (Inr) sequence or binding sites for the PU.1 transcription factor have been described in myeloid and B cells. The G/C rich region upstream of ATG may also support the initiation of transcription. Other additional consensus binding sites for T cell transcription factor (TCF-1α), Ig gene box enhancer motifs (µE1, µE5 and κE2), nuclear factor-IL-6 and IFN-responsive factor-1 have been identified

in

the

CD38

5’UTR

region.

Interestingly,

the

DR5

repeat

(TGACCCgaaagTGCCCC) sequence was identified within intron 1, which has a role in retinoic acid induction of CD38 expression in HL-60 cells (Mehta and Cheema 1999). A CpG island of ~900 bp length was identified in the CD38 gene spanning exon 1 and the 5' end of intron 1 with a binding sequence for Sp1, a transcription factor that regulates the constitutive expression of CD38 (Sun et al. 2006). Furthermore, a glucocorticoid response element and an estrogen binding motif have also been described in the promoter region of CD38 (Tirumurugaan et al. 2008). Taken together, it is likely that the transcriptional regulation of CD38 expression by cytokines, glucocorticoids, and hormones may have a physiological and pathophysiological role in the regulation of lung functions and pulmonary diseases. Inflammatory cytokines such as TNF-α, IL-1β and IFN-γ play an important role in diseases such as asthma (Berry et al. 2007; Lappalainen et al. 2005; ten Hacken et al. 1998). TNF-α has been shown to increase the expression of a variety of genes resulting in functional changes in ASM cells (Alrashdan et al. 2012). Studies from our laboratory and others have demonstrated that TNF-α upregulates CD38 expression in ASM. TNFα induced change in CD38 expression in human ASM cells involves NF-ƙB and AP-1 activation, and signaling through the p38 and JNK MAP kinases (Tirumurugaan et al.


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2007). Sequence analysis of the cloned human CD38 promoter also reveals 4 putative binding sites for the transcription factor c/EBPβ, three of which are within a region upstream of the NF-ƙB site. TNF-α via TNFR1 receptor induces CD38 expression in ASM cells in an IFNβ-dependent manner (Tliba et al. 2004). Inflammatory cytokines activate multiple signaling pathways ultimately resulting in transcriptional activation of various genes. Studies described above clearly demonstrate that cytokines via upstream signal activation regulate CD38 expression by multiple transcription factors. This knowledge provides one framework for developing therapeutic strategies aimed at containing CD38 expression induced by inflammatory mediators and hormones. CD38 expression: findings from asthmatic ASM cells ASM obtained from asthmatics is different from smooth muscle obtained from healthy subjects in terms of expression and activity of different signaling molecules and contractile apparatus. Studies of gene expression in airway biopsies from mild allergic asthmatics and non-asthmatics reveal increased expression of fast myosin heavy chain isoform, transgelin, and MLCK in tissues from asthmatics, reflecting faster velocity of actin filament propulsion and AHR (Leguillette et al. 2009). Increased phospho-ERK1/2 and p38 MAPKs, with augmented expression of ERK1/2-inducible proteins sprouty-2 and JunB were noted in the airways of asthmatics (Liu et al. 2008). JunB is a transcription factor that is part of the AP-1 complex and involved in ERK1/2-mediated transcriptional regulation in the airways (Reddy and Mossman 2002). Increased expression

of

CD40

and

OX40

ligand,

cell-surface

molecules

involved

in

immunomodulatory functions of ASM cells (Krimmer et al. 2009) have been reported in


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biopsy specimens. Reports support increased rate of cell proliferation resistant to glucocorticoids, capacity to develop force, and secretion of chemokines in ASM cells obtained from asthmatics compared to cells from healthy subjects (Damera et al. 2009; Ma et al. 2002; Prefontaine et al. 2009). These reports indicate a definite asthmatic phenotype of asthmatic ASM cells characterized by differential expression of genes involved in contractility, MAP kinase signaling, cell to cell and cell to matrix interactions, transcription factors and chemokines involved in the recruitment of inflammatory cells into the airways during allergic airway disease. A systematic study delineating the expression of CD38 in ASM obtained from asthmatics and non-asthmatics is lacking. However, in asthmatic ASM cells maintained in culture we did not find any change in the constitutive expression of CD38 compared to ASM cells obtained from healthy donors. Interestingly, a comparison of CD38 expression induced by TNF-α between ASM cells obtained from asthmatics and nonasthmatics revealed that asthmatic ASM cells are capable of significantly greater level of expression than non-asthmatic ASM cells (Jude et al. 2010). There was greater sensitivity to TNF-α of CD38 expression in asthmatic ASM cells than in non-asthmatic ASM cells. This differential induction of CD38 expression by inflammatory cytokine appears not to be associated with altered activation of the transcription factors NF-ƙB and AP-1 as well as with altered transcript stability. Furthermore, the differential induction of CD38 expression was associated with decreased JNK MAP kinase activation and increased activation of ERK and p38 MAP kinases. These observations indicated the combined role of transcription factors, increased rate of transcription and translational regulation in effecting differential induction of CD38 in asthmatic cells. In


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this context, we investigated post-transcriptional mechanisms in the regulation of CD38 expression in ASM cells derived from asthmatic and non-asthmatic donor lungs. CD38 expression: regulation by post-transcriptional mechanisms MicroRNAs (miRNAs) are a group of small non-coding RNAs that posttranscriptionally regulate gene expression (Bartel 2004; Ha and Kim 2014; He and Hannon 2004). The biogenesis of miRNAs is a complex process. RNA polymerase II (RNA Pol II), or less frequently Pol III, generates a primary miRNA transcript (primiRNA), which is then exported to the cytoplasm by exportin-5. Here, it is further processed to form precursor miRNAs (pre-miRNA). Dicer, an RNAse III enzyme along with its co-factors cleaves the terminal loop from the pre-miRNA to generate miRNA duplex. One of the two strands form mature miRNA (18-23 nucleotides) that generally becomes the guide strand that is incorporated into a ribonucleotide complex to form miRISC (miRNA induced silencing complex). Based on the complementarities between the guide miRNA and the target mRNA, miRNA can either cleave target RNAs with the help of Ago2, or induce a translational suppression. miRNA mediated gene regulation is crucial in the biological system as they can regulate hundreds of different mRNAs that play essential roles in various cellular functions. Therefore, any alteration of miRNA expression can result in various human disease conditions (Subramanian, 2012, sarcomas??), including asthma. Recent reports have demonstrated that expression of several miRNAs is downregulated by inflammatory cytokines in human ASM cells (Chiba et al. 2009; Jude et al. 2012a; Kuhn et al. 2010). There is evidence for control of ASM contractility and relaxation (Chiba et al. 2009; Wang et al. 2011), ASM phenotype (Hu et al. 2014; Kuhn


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et al. 2010), and ASM proliferation (Hu et al. 2014) by specific miRNAs. A recent report showed that miR-10a regulates ASM cell proliferation by a mechanism that involves suppression of expression of the catalytic subunit of PI3 kinase, leading to decreased AKT phosphorylation (Hu et al. 2014). It is worth noting that the PI3 kinase/AKT signaling contributes to the hyperproliferative phenotype of asthmatic ASM cells (Burgess et al. 2008).

Furthermore any role for CD38/cADPR signaling

in the

regulation of ASM growth and proliferation has not been investigated. Based on pleitropic role of this signaling pathway, we predict that CD38 promotes ASM growth. Further, it is imperative to speculate the importance of miRNA in the regulation of expression and activity of numerous intracellular signaling molecules, including CD38, in ASM thereby contributing to AHR. We used multiple web-based target prediction algorithms to determine potential miRNA targets in the CD38 3’Untranslated Region (3’UTR) (Dileepan et al. 2014). We found that miRNAs miR-1272, miR-548, miR-208a, miR-1298, miR-708 and miR-140-3p were predicted to bind to the CD38 3’UTR with high context score. Initial exploratory studies revealed the following: 1. Human ASM cells express miR-708 and miR-140-3p at a relatively higher level compared to the expression of other miRNAs, 2. expression of miR-708 and miR-140-3p is regulated by treatment with TNF-α and 3. asthmatic and non-asthmatic ASM cells demonstrate different sensitivity to cytokine-induced expression of miR-708 and miR-140-3p (Dileepan et al. 2014; Jude et al. 2012a). Expression of miR-140-3p was found to be lower and reduced further to a greater extent by TNF-α in ASM cells from asthmatics compared to levels in cells from non-asthmatics. On the contrary, miR-708 expression was found to be reduced by TNF-α in non-


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asthmatic ASM cells compared to expression in vehicle-treated cells, while it caused an increase in miR-708 expression in asthmatic ASM cells (Dileepan et al. 2014). We hypothesized that dysregulation of expression of these two miRNAs is involved in the augmented cytokine-induced CD38 expression in human ASM cells. The mechanisms by which these two miRNAs regulate the expression of CD38 were also determined in these studies. miRNAs can inhibit gene expression by binding to the target gene at the 3’UTR or indirectly by inhibiting other signaling pathways (Dileepan et al. 2014; Jude et al. 2012a). We carried out CD38 3’UTR-luciferase reporter assays to determine whether there is evidence for control of CD38 expression by direct binding. Our results revealed that regulation of CD38 expression by miR-708 and miR-140-3p results from binding to 3’-UTR of CD38. However, this binding did not have any effect on CD38 transcript stability, suggesting that translational repression may be an underlying mechanism of regulation. We also evaluated the effects of these two miRNAs on the signaling mechanisms and transcription factors involved in the regulation of CD38 expression in human ASM cells. Prior investigations in the laboratory have demonstrated that CD38 expression is regulated by p38 and JNK MAP kinases at the transcriptional level, whereas ERK and p38 MAP kinase regulation involved post-transcriptional mechanisms (Tirumurugaan et al. 2007). Furthermore, specific isoforms of the PI3 kinases (Jude et al. 2012b) and the transcription factors NF-ƙB and AP-1 are critical in this regulation (Tirumurugaan et al. 2007). The evidence for this came from use of isoform-specific pharmacological inhibitors of the catalytic subunit of PI3 kinase, transfection of cells with dominant negative constructs for the transcription factors, and deletion of the binding


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sites for the transcription factors (Tirumurugaan et al. 2007). Transfection of human ASM cells with miR-140-3p mimic oligonucleotides revealed down-regulation of p38 MAP kinase and NF-κB activation (Jude et al. 2012a). On the other hand, transfection of cells with miR-708 mimic oligonucleotides caused down-regulation of JNK MAP kinase activation, with no significant change in the expression of total JNK MAP kinase (Dileepan et al. 2014). In addition, miR-708 transfection of the cells caused upregulation of phosphatase and tensin homolog (PTEN), which regulates PI3 kinase signaling through activation of AKT (AKT) and a dual-specificity phosphatase, also known as MAP kinase phosphatase-1 (DUSP-1 or MKP-1) that regulates MAP kinase signaling (Figure 2; (Dileepan et al. 2014). These findings indicated that miR-140-3p regulation of CD38 expression stems from altered p38 MAP kinase and NF-ƙB activation, while regulation by miR-708 stems from altered PI3 kinase/AKT signaling and JNK MAP kinase activation. The combined effect on PTEN and DUSP-1 induction by miR-708 should have a profound effect on two signaling pathways involved in the inflammation and cell proliferation. Airway remodeling is another key component of asthma and future studies should focus on assessing the role of CD38 in the regulation of ASM growth. These new findings support the concept that the capacity for CD38 signaling in the asthmatic airways is significantly higher than in the airways of healthy subjects. These findings have a significant impact in terms of the role of CD38 in asthma and understanding the regulation of CD38 expression and function in the asthmatic airways may help identify novel therapeutic targets. The miRNAs that were investigated in the studies target different signaling mechanisms involved in the regulation of CD38


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expression and may provide another therapeutic strategy in inflammatory airway diseases such as asthma and COPD. Airway inflammation and hyperresponsivess Airway hyperresponsiveness to contractile agonists is a key feature of asthma, a chronic inflammatory disease of the airways that results from a complex interplay between genetic and environmental factors (Chen et al. 2003; Factor 2003; Fredberg 2004; O'Byrne and Inman 2003). In asthma, inhaled allergens are taken up by airway dendritic cells, which subsequently migrate to bronchial lymph nodes where they mature into immunostimulatory cells capable of efficiently presenting antigen to T-helper (CD4+) cell precursors. The cytokine balance in the lymph node determines the fate of these precursor cells. If interleukin (IL)-12 predominates, then precursor cells become type 1 T-helper cells (Th1) that produce interferon (IFN)-γ, IL-2, tumor necrosis factor (TNF)-α, and lymphotoxin, collectively referred to as Th1 cytokines. Conversely, if IL-4 predominates in the node, CD4+ precursor cells become type 2 T-helper cells (Th2), which secrete the Th2 cytokines IL-4, IL-5, IL-9, and IL-13. These Th2 cytokines, among other mediators, recruit eosinophils to the airways. Binding and antigen-dependent cross-linking of immunoglobulin E (IgE)-bound high affinity receptors (Fc€RI) on eosinophils and resident mast cells results in the activation and degranulation with local release of mediators that cause airway injury, increased mucous production, and the development of AHR (Cohn et al. 2004; Factor 2003; Komai et al. 2003; Robinson 2004; Williams 2004). Once a Th2 type response has occurred, Th2 memory cells alone appear to determine the development of AHR and pulmonary eosinophilia to antigen challenge in a manner that is refractory to inhibition by Th1 cells or endogenous


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inhibitory mechanisms (Aronica et al. 2004). However, the effects of Th1 responses on Th2 response-induced lung injury seem to be ambiguous since the Th1 cytokine IFN-γ was shown to be concomitantly inhibitory and stimulatory on the effects of the Th2 cytokine IL-13 in a mouse model of lung inflammation (Ford et al. 2001). Furthermore, airway exposure to TNF-α, a Th1 cytokine, can cause substantial AHR and inflammation (Guedes et al. 2008; Thomas and Heywood 2002; Thomas et al. 1995). Inflammatory cells (i.e., lymphocytes, mast cells, eosinophils) and inflammatory mediators (i.e., cytokines, eicosanoids) interact with ASM, inducing important anatomic and functional changes (Damera and Panettieri 2011; Tliba et al. 2003; Tliba and Panettieri 2008; Webb et al. 2000). After repeated allergen exposure, the ASM may continue to proliferate even after resolution of the inflammatory process (Leung et al. 2004). Therefore, while airway wall swelling and airway plugging by excessive secretion are important amplifying factors, the major end-effector of acute airway narrowing in asthma is the ASM (FitzGerald and Macklem 1996; Fredberg 2004). Collectively these studies suggest that pathogenesis of asthma involves a 2-step process: 1. Inflammation and 2. Cellular changes leading to functional abnormalities (e.g. AHR). Interestingly, CD38 is expressed on immune cells as well as resident airway cells including ASM cells. Predictably CD38 plays a pivotal role both in the onset of allergen-induced inflammatory response and AHR (Figure 1). Studies on integrative models using CD38 KO mice have provided insights into multiple roles of CD38/cADPR signaling in the pathogenesis of asthma (Table 1). CD38 in airway inflammation and AHR: findings from mouse models of asthma


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Naïve Cd38 KO mice exhibit very low cADPR levels in the lungs, attenuated [Ca2+]i responses to spasmogens such as endothelin-1 and acetylcholine in ASM, and decreased airway responsiveness to inhaled methacholine compared to WT counterparts (Deshpande et al. 2005b; Guedes et al. 2008; Guedes et al. 2006). Cytokine-induced changes in CD38/cADPR signaling have also been investigated using mouse models of airway inflammation and AHR (Guedes et al. 2008; Guedes et al. 2006). Intranasal challenge with the Th2 cytokine IL-13 or with the pro-inflammatory cytokine TNF-α caused significantly lower AHR to inhaled methacholine in the Cd38 KO mice compared to WT controls, but a robust and comparable airway inflammation was elicited in both groups of mice. Isometric force generation of tracheal segments incubated with IL-13 or TNF-α is also significantly lower in the Cd38 KO mice compared to WT controls (Guedes et al. 2008; Guedes et al. 2006). Together with the demonstrated role of CD38/cADPR signaling in calcium homeostasis in ASM cells under naïve conditions and after cytokine treatment, these studies suggest the contribution of this signaling system to ASM contractility under normal and inflammatory conditions. The CD38/cADPR/Ca2+ signaling system also plays a role in the regulation of humoral immune responses in vivo by directing dendritic cells chemotaxis, antigen presentation and adequate T cell priming as shown in studies using Cd38 KO mice (Cockayne et al. 1998; Partida-Sanchez et al. 2004a; Partida-Sanchez et al. 2004b). Furthermore, CD38 ligation with agonist monoclonal antibodies (mAb) or its natural ligand CD31 in peripheral blood T cells and monocytes induces the expression and secretion of a mixed Th1- (IFN-γ, IL-2) and Th2-type (IL-6, IL-10) cytokine profile


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(Ausiello et al. 1996; Deaglio et al. 1998; Lande et al. 2002). Since airway inflammation is a hallmark of asthma, the above observations suggest that the CD38/cADPR signaling specifically in immune cells may have a role in the pathophysiology of allergic airway diseases. There is strong evidence from independent studies using mouse models that CD38 is involved in airway inflammation and AHR (Gally et al. 2009; Guedes et al. 2008; Guedes et al. 2006). However, whether or not the involvement of CD38 in asthma pathogenesis is primarily due to its role in inflammatory or resident lung cells has not been fully resolved. A recent study using mouse model of ovalbumin-induced airway inflammation and AHR showed that Cd38 KO mice did not develop inflammation or eosinophilia in the lungs, had consistently lower levels of IL-4, IL-5 and IL-13 in culture supernatants of peribronchial lymph nodes, and had reduced AHR compared to controls (Table 1). Reciprocal bone marrow transfer experiments indicated that CD38 acts both as modulator of the immune response and plays an equally important role as an intrinsic pulmonary component (Gally et al. 2009). However, intranasal challenge with an extract of the environmental fungus Alternaria alternata, an epidemiologically relevant allergen, caused pronounced and equivalent increases in eosinophil numbers and IL-13 concentrations in bronchoalveolar lavage fluid in Cd38 KO and WT mice, but AHR was significantly lower in the CD38 KO mice (Guedes et al. 2006). Transferring CD38 KO bone marrow into WT mice or vice-versa does not alter the typical responses seen in intact mice challenged with IL-13. In other words, WT bone marrow transferred into CD38 KO recipients does not restore AHR to the WT levels. Similarly, transfer of Cd38 KO bone marrow into WT mice does not diminish their AHR to the levels seen in the


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Cd38 KO mice. Furthermore, airway inflammation in WT and Cd38 KO mice following reciprocal bone marrow transfer and IL-13 challenge is similar. Hence, reciprocal bone marrow transfer does not alter their typical inflammatory and methacholine responses (unpublished results). These results argue in favor of a hypothesis that CD38 expression in lung structural cells is the primary determinant of AHR in the fungal- and cytokine-induced asthma in murine models. It would be interesting to determine structural changes (e.g. ASM mass) in the Cd38 KO v.s. WT lungs after allergen challenge or cytokine treatment. Conclusions and future directions Studies from human ASM cells (asthmatic and non-asthmatic) and murine models of asthma clearly demonstrated the role of CD38/cADPR-mediated signaling in the regulation of airway function, inflammation and hyperresponsiveness. Can CD38 serve as a potential anti-asthma target? Basic and pre-clinical studies strongly support this notion. Regulation of CD38 expression or activity could mitigate severity of allergeninduced airway inflammatory diseases. Challenge lies in the development of pharmacological tools to inhibit the enzyme (ADP-ribosyl cyclase) activity associated with CD38. Advanced medicinal chemistry approaches and computer assisted drug design tools may assist in developing inhibitors of ADP-ribosyl cyclase. Delivering drugs to inhibit CD38 function in ASM or immune cells could be achieved by the use of nanotechnology. Recent studies have demonstrated the use of nanoparticle technology to augment the drug delivery effectively. The concept of one-bead-one-compound combinatorial chemistry to discover ligands against several different cell surface integrins has gained momentum in recent years. One of these ligands, LLP2A (a high-


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affinity peptidomimetic antagonist against activated α4β1 integrin), was found to bind strongly to human airway fibroblasts (HAF) leaving other cell types intact. This approach could be used for targeted delivery of novel drugs to specific resident airway cells. miRNAs seem additional attractive target, however, off-target effects of miRNA pose a major challenge. Future studies using new bioinformatics tools may offer much required solution by establishing specificity for miRNAs in the regulation of CD38 expression.


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Acknowledgments: Authors wish to thank the contribution of Drs. Jude, Kang, Tirumurugaan and White whose work has been cited in this review. The investigations from the laboratory cited in this review have been supported by grants from the National Institutes of Health, Academic Health Center, University of Minnesota and the Comparative Medicine Research Program, College of Veterinary Medicine, University of Minnesota.


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References Aarhus, R., Graeff, R.M., Dickey, D.M., Walseth, T.F., and Lee, H.C. 1995. ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP. The Journal of biological chemistry 270(51): 30327-30333. Adebanjo, O.A., Koval, A., Moonga, B.S., Wu, X.B., Yao, S., Bevis, P.J., et al. 2000. Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases. Biochem Biophys Res Commun 273(3): 884-889. doi: 10.1006/bbrc.2000.3041. Albrieux, M., Lee, H.C., and Villaz, M. 1998. Calcium signaling by cyclic ADP-ribose, NAADP, and inositol trisphosphate are involved in distinct functions in ascidian oocytes. J Biol Chem 273(23): 14566-14574. Aley, P.K., Noh, H.J., Gao, X., Tica, A.A., Brailoiu, E., and Churchill, G.C. 2010. A functional role for nicotinic acid adenine dinucleotide phosphate in oxytocin-mediated contraction of uterine smooth muscle from rat. The Journal of pharmacology and experimental therapeutics 333(3): 726-735. doi: 10.1124/jpet.110.165837. Aley, P.K., Singh, N., Brailoiu, G.C., Brailoiu, E., and Churchill, G.C. 2013. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger in muscarinic receptor-induced contraction of guinea pig trachea. The Journal of biological chemistry 288(16): 10986-10993. doi: 10.1074/jbc.M113.458620. Alrashdan, Y.A., Alkhouri, H., Chen, E., Lalor, D.J., Poniris, M., Henness, S., et al. 2012. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement. Am J Physiol Lung Cell Mol Physiol 302(10): L1118-1127. doi: 10.1152/ajplung.00232.2011.


Page 26 of 43

Aronica, M.A., McCarthy, S., Swaidani, S., Mitchell, D., Goral, M., Sheller, J.R.,et al. 2004. Recall helper T cell response: T helper 1 cell-resistant allergic susceptibility without biasing uncommitted CD4 T cells. Am J Respir Crit Care Med 169(5): 587-595. doi: 10.1164/rccm.200301-100OC. Ausiello, C.M., la Sala, A., Ramoni, C., Urbani, F., Funaro, A., and Malavasi, F. 1996. Secretion of IFN-gamma, IL-6, granulocyte-macrophage colony-stimulating factor and IL-10 cytokines after activation of human purified T lymphocytes upon CD38 ligation. Cell Immunol 173(2): 192-197. doi: 10.1006/cimm.1996.0267. Bartel, D.P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2): 281-297. Berry, M., Brightling, C., Pavord, I., and Wardlaw, A. 2007. TNF-alpha in asthma. Curr Opin Pharmacol 7(3): 279-282. doi: 10.1016/j.coph.2007.03.001. Burgess, J.K., Lee, J.H., Ge, Q., Ramsay, E.E., Poniris, M.H., Parmentier, J., et al. 2008. Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma. J Cell Physiol 216(3): 673-679. doi: 10.1002/jcp.21450. Calcraft, P.J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., et al. 2009. NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459(7246): 596-600. doi: 10.1038/nature08030. Ceni, C., Pochon, N., Villaz, M., Muller-Steffner, H., Schuber, F., Baratier, J., et al. 2006. The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain. Biochem J 395(2): 417-426. doi: 10.1042/BJ20051321.


Page 27 of 43

Chen, H., Tliba, O., Van Besien, C.R., Panettieri, R.A., Jr., and Amrani, Y. 2003. TNF[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl. J Appl Physiol (1985) 95(2): 864-872; discussion 863. doi: 10.1152/japplphysiol.00140.2003. Chiba, Y., Tanabe, M., Goto, K., Sakai, H., and Misawa, M. 2009. Down-regulation of miR-133a contributes to up-regulation of Rhoa in bronchial smooth muscle cells. Am J Respir Crit Care Med 180(8): 713-719. doi: 10.1164/rccm.200903-0325OC. Clapper, D.L., Walseth, T.F., Dargie, P.J., and Lee, H.C. 1987. Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J Biol Chem 262(20): 9561-9568. Cockayne, D.A., Muchamuel, T., Grimaldi, J.C., Muller-Steffner, H., Randall, T.D., Lund, F.E., et al. 1998. Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses. Blood 92(4): 13241333. Cohn, L., Elias, J.A., and Chupp, G.L. 2004. Asthma: mechanisms of disease persistence

and

progression.

Annu

Rev

Immunol

22:

789-815.

doi:

10.1146/annurev.immunol.22.012703.104716. Damera, G., Fogle, H.W., Lim, P., Goncharova, E.A., Zhao, H., Banerjee, A., et al. 2009. Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1. Br J Pharmacol 158(6): 1429-1441. doi: 10.1111/j.1476-5381.2009.00428.x.


Page 28 of 43

Damera, G., and Panettieri, R.A., Jr. 2011. Does airway smooth muscle express an inflammatory phenotype in asthma? Br J Pharmacol 163(1): 68-80. doi: 10.1111/j.14765381.2010.01165.x. Deaglio, S., Morra, M., Mallone, R., Ausiello, C.M., Prager, E., Garbarino, G., et al. 1998. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J Immunol 160(1): 395-402. Deshpande, D.A., Dogan, S., Walseth, T.F., Miller, S.M., Amrani, Y., Panettieri, R.A., et al. 2004. Modulation of calcium signaling by interleukin-13 in human airway smooth muscle: role of CD38/cyclic adenosine diphosphate ribose pathway. Am J Respir Cell Mol Biol 31(1): 36-42. doi: 10.1165/rcmb.2003-0313OC. Deshpande, D.A., Walseth, T.F., Panettieri, R.A., and Kannan, M.S. 2003. CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyperresponsiveness. FASEB J 17(3): 452-454. doi: 10.1096/fj.02-0450fje. Deshpande, D.A., White, T.A., Dogan, S., Walseth, T.F., Panettieri, R.A., and Kannan, M.S. 2005a. CD38/cyclic ADP-ribose signaling: role in the regulation of calcium homeostasis in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 288(5): L773-788. doi: 10.1152/ajplung.00217.2004. Deshpande, D.A., White, T.A., Guedes, A.G., Milla, C., Walseth, T.F., Lund, F.E., et al. 2005b. Altered airway responsiveness in CD38-deficient mice. Am J Respir Cell Mol Biol 32(2): 149-156. doi: 10.1165/rcmb.2004-0243OC. Dileepan, M., Jude, J.A., Rao, S.P., Walseth, T.F., Panettieri, R.A., Subramanian, S., et al. 2014. MicroRNA-708 regulates CD38 expression through signaling pathways JNK


Page 29 of 43

MAP kinase and PTEN/AKT in human airway smooth muscle cells. Respir Res 15: 107. doi: 10.1186/s12931-014-0107-0. Factor, P. 2003. Gene therapy for asthma. Mol Ther 7(2): 148-152. Ferrero, E., Saccucci, F., and Malavasi, F. 2000. The making of a leukocyte receptor: origin, genes and regulation of human CD38 and related molecules. Chem Immunol 75: 1-19. FitzGerald, J.M., and Macklem, P. 1996. Fatal asthma. Annu Rev Med 47: 161-168. doi: 10.1146/annurev.med.47.1.161. Ford, J.G., Rennick, D., Donaldson, D.D., Venkayya, R., McArthur, C., Hansell, E., et al. 2001. Il-13 and IFN-gamma: interactions in lung inflammation. J Immunol 167(3): 17691777. Franco, L., Bruzzone, S., Song, P., Guida, L., Zocchi, E., Walseth, T.F., et al. 2001. Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 280(1): L98-L106. Fredberg, J.J. 2004. Bronchospasm and its biophysical basis in airway smooth muscle. Respir Res 5: 2. doi: 10.1186/1465-9921-5-2. Gally, F., Hartney, J.M., Janssen, W.J., and Perraud, A.L. 2009. CD38 plays a dual role in allergen-induced airway hyperresponsiveness. Am J Respir Cell Mol Biol 40(4): 433442. doi: 10.1165/rcmb.2007-0392OC. Guedes, A.G., Jude, J.A., Paulin, J., Kita, H., Lund, F.E., and Kannan, M.S. 2008. Role of CD38 in TNF-alpha-induced airway hyperresponsiveness. Am J Physiol Lung Cell Mol Physiol 294(2): L290-299. doi: 10.1152/ajplung.00367.2007.


Page 30 of 43

Guedes, A.G., Paulin, J., Rivero-Nava, L., Kita, H., Lund, F.E., and Kannan, M.S. 2006. CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge.

Am

J

Physiol Lung Cell

Mol Physiol

291(6):

L1286-1293.

doi:

10.1152/ajplung.00187.2006. Ha, M., and Kim, V.N. 2014. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15(8): 509-524. doi: 10.1038/nrm3838. He, L., and Hannon, G.J. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7): 522-531. doi: 10.1038/nrg1379. Hellmich, M.R., and Strumwasser, F. 1991. Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme. Cell Regul 2(3): 193202. Hu, R., Pan, W., Fedulov, A.V., Jester, W., Jones, M.R., Weiss, S.T.,et al. 2014. MicroRNA-10a controls airway smooth muscle cell proliferation via direct targeting of the PI3 kinase pathway. FASEB J 28(5): 2347-2357. doi: 10.1096/fj.13-247247. Jude, J.A., Dileepan, M., Subramanian, S., Solway, J., Panettieri, R.A., Jr., Walseth, T.F., et al. 2012a. miR-140-3p regulation of TNF-alpha-induced CD38 expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303(5): L460468. doi: 10.1152/ajplung.00041.2012. Jude, J.A., Solway, J., Panettieri, R.A., Jr., Walseth, T.F., and Kannan, M.S. 2010. Differential induction of CD38 expression by TNF-{alpha} in asthmatic airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 299(6): L879-890. doi: 10.1152/ajplung.00021.2010.


Page 31 of 43

Jude, J.A., Tirumurugaan, K.G., Kang, B.N., Panettieri, R.A., Walseth, T.F., and Kannan, M.S. 2012b. Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases. Am J Respir Cell Mol Biol 47(4): 427435. doi: 10.1165/rcmb.2012-0025OC. Kang, B.N., Deshpande, D.A., Tirumurugaan, K.G., Panettieri, R.A., Walseth, T.F., and Kannan, M.S. 2005. Adenoviral mediated anti-sense CD38 attenuates TNF-alphainduced changes in calcium homeostasis of human airway smooth muscle cells. Can J Physiol Pharmacol 83(8-9): 799-804. doi: 10.1139/y05-081. Kang, B.N., Jude, J.A., Panettieri, R.A., Jr., Walseth, T.F., and Kannan, M.S. 2008. Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1. Am J Physiol Lung Cell Mol Physiol 295(1): L186193. doi: 10.1152/ajplung.00352.2007. Komai, M., Tanaka, H., Masuda, T., Nagao, K., Ishizaki, M., Sawada, M., et al. 2003. Role of Th2 responses in the development of allergen-induced airway remodelling in a murine

model

of

allergic

asthma.

Br

J

Pharmacol

138(5):

912-920.

doi:

10.1038/sj.bjp.0705105. Krimmer, D.I., Loseli, M., Hughes, J.M., Oliver, B.G., Moir, L.M., Hunt, N.H., et al. 2009. CD40 and OX40 ligand are differentially regulated on asthmatic airway smooth muscle. Allergy 64(7): 1074-1082. doi: 10.1111/j.1398-9995.2009.01959.x. Kuemmerle, J.F., and Makhlouf, G.M. 1995. Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca(2+)-induced Ca2+ release in intestinal longitudinal muscle. J Biol Chem 270(43): 25488-25494.


Page 32 of 43

Kuemmerle, J.F., Murthy, K.S., and Makhlouf, G.M. 1998. Longitudinal smooth muscle of the mammalian intestine. A model for Ca2+ signaling by cADPR. Cell Biochem Biophys 28(1): 31-44. doi: 10.1007/BF02738308. Kuhn, A.R., Schlauch, K., Lao, R., Halayko, A.J., Gerthoffer, W.T., and Singer, C.A. 2010. MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype. Am J Respir Cell Mol Biol 42(4): 506513. doi: 10.1165/rcmb.2009-0123OC. Lande, R., Urbani, F., Di Carlo, B., Sconocchia, G., Deaglio, S., Funaro, A., et al. 2002. CD38 ligation plays a direct role in the induction of IL-1beta, IL-6, and IL-10 secretion in resting human monocytes. Cell Immunol 220(1): 30-38. Lappalainen, U., Whitsett, J.A., Wert, S.E., Tichelaar, J.W., and Bry, K. 2005. Interleukin-1beta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung. Am J Respir Cell Mol Biol 32(4): 311-318. doi: 10.1165/rcmb.2004-0309OC. Lee, H.C. 2006. Structure and enzymatic functions of human CD38. Mol Med 12(11-12): 317-323. doi: 10.2119/2006–00086.Lee. Lee, H.C. 2011. Cyclic ADP-ribose and NAADP: fraternal twin messengers for calcium signaling. Sci China Life Sci 54(8): 699-711. doi: 10.1007/s11427-011-4197-3. Lee, H.C., and Aarhus, R. 1991. ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite. Cell Regul 2(3): 203-209. Lee, H.C., Munshi, C., and Graeff, R. 1999. Structures and activities of cyclic ADPribose, NAADP and their metabolic enzymes. Mol Cell Biochem 193(1-2): 89-98.


Page 33 of 43

Leguillette, R., Laviolette, M., Bergeron, C., Zitouni, N., Kogut, P., Solway, J., et al. 2009. Myosin, transgelin, and myosin light chain kinase: expression and function in asthma. Am J Respir Crit Care Med 179(3): 194-204. doi: 10.1164/rccm.2006091367OC. Leung, S.Y., Eynott, P., Noble, A., Nath, P., and Chung, K.F. 2004. Resolution of allergic airways inflammation but persistence of airway smooth muscle proliferation after repeated allergen exposures. Clin Exp Allergy 34(2): 213-220. Liu, W., Liang, Q., Balzar, S., Wenzel, S., Gorska, M., and Alam, R. 2008. Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic airways. J Allergy Clin Immunol 121(4): 893-902 e892. doi: 10.1016/j.jaci.2008.02.004. Ma, X., Cheng, Z., Kong, H., Wang, Y., Unruh, H., Stephens, N.L., et al. 2002. Changes in biophysical and biochemical properties of single bronchial smooth muscle cells from asthmatic subjects. Am J Physiol Lung Cell Mol Physiol 283(6): L1181-1189. doi: 10.1152/ajplung.00389.2001. Malavasi, F., Deaglio, S., Damle, R., Cutrona, G., Ferrarini, M., and Chiorazzi, N. 2011. CD38 and chronic lymphocytic leukemia: a decade later. Blood 118(13): 3470-3478. doi: 10.1182/blood-2011-06-275610. Mehta, K., and Cheema, S. 1999. Retinoid-mediated signaling pathways in CD38 antigen expression in myeloid leukemia cells. Leuk Lymphoma 32(5-6): 441-449. doi: 10.3109/10428199909058401. O'Byrne, P.M., and Inman, M.D. 2003. Airway hyperresponsiveness. Chest 123(3 Suppl): 411S-416S.


Page 34 of 43

Okamoto, H., Takasawa, S., and Nata, K. 1997. The CD38-cyclic ADP-ribose signalling system in insulin secretion: molecular basis and clinical implications. Diabetologia 40(12): 1485-1491. doi: 10.1007/s001250050854. Partida-Sanchez, S., Goodrich, S., Kusser, K., Oppenheimer, N., Randall, T.D., and Lund, F.E. 2004a. Regulation of dendritic cell trafficking by the ADP-ribosyl cyclase CD38: impact on the development of humoral immunity. Immunity 20(3): 279-291. Partida-Sanchez, S., Iribarren, P., Moreno-Garcia, M.E., Gao, J.L., Murphy, P.M., Oppenheimer, N., et al. 2004b. Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose. J Immunol 172(3): 1896-1906. Perraud, A.L., Fleig, A., Dunn, C.A., Bagley, L.A., Launay, P., Schmitz, C., et al. 2001. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature 411(6837): 595-599. doi: 10.1038/35079100 [doi] 35079100 [pii]. Prakash, Y.S., Kannan, M.S., Walseth, T.F., and Sieck, G.C. 1998. Role of cyclic ADPribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle. Am J Physiol 274(6 Pt 1): C1653-1660. Prefontaine, D., Lajoie-Kadoch, S., Foley, S., Audusseau, S., Olivenstein, R., Halayko, A.J., et al. 2009. Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells. J Immunol 183(8): 5094-5103. doi: 10.4049/jimmunol.0802387.


Page 35 of 43

Reddy, S.P., and Mossman, B.T. 2002. Role and regulation of activator protein-1 in toxicant-induced responses of the lung. Am J Physiol Lung Cell Mol Physiol 283(6): L1161-1178. doi: 10.1152/ajplung.00140.2002. Robinson, D.S. 2004. The role of the mast cell in asthma: induction of airway hyperresponsiveness by interaction with smooth muscle? J Allergy Clin Immunol 114(1): 58-65. doi: 10.1016/j.jaci.2004.03.034. Rusinko, N., and Lee, H.C. 1989. Widespread occurrence in animal tissues of an enzyme catalyzing the conversion of NAD+ into a cyclic metabolite with intracellular Ca2+-mobilizing activity. The Journal of biological chemistry 264(20): 11725-11731. Sieck, G.C., White, T.A., Thompson, M.A., Pabelick, C.M., Wylam, M.E., and Prakash, Y.S. 2008. Regulation of store-operated Ca2+ entry by CD38 in human airway smooth muscle.

Am

J

Physiol

Lung

Cell

Mol

Physiol

294(2):

L378-385.

doi:

10.1152/ajplung.00394.2007. States, D.J., Walseth, T.F., and Lee, H.C. 1992. Similarities in amino acid sequences of Aplysia ADP-ribosyl cyclase and human lymphocyte antigen CD38. Trends in biochemical sciences 17(12): 495. Sun, L., Iqbal, J., Zaidi, S., Zhu, L.L., Zhang, X., Peng, Y., et al. 2006. Structure and functional regulation of the CD38 promoter. Biochem Biophys Res Commun 341(3): 804-809. doi: 10.1016/j.bbrc.2006.01.033. ten Hacken, N.H., Oosterhoff, Y., Kauffman, H.F., Guevarra, L., Satoh, T., Tollerud, D.J., et al. 1998. Elevated serum interferon-gamma in atopic asthma correlates with increased airways responsiveness and circadian peak expiratory flow variation. Eur Respir J 11(2): 312-316.


Page 36 of 43

Thomas, P.S., and Heywood, G. 2002. Effects of inhaled tumour necrosis factor alpha in subjects with mild asthma. Thorax 57(9): 774-778. Thomas, P.S., Yates, D.H., and Barnes, P.J. 1995. Tumor necrosis factor-alpha increases airway responsiveness and sputum neutrophilia in normal human subjects. Am J Respir Crit Care Med 152(1): 76-80. Tirumurugaan, K.G., Jude, J.A., Kang, B.N., Panettieri, R.A., Walseth, T.F., and Kannan, M.S. 2007. TNF-alpha induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-kappaB and AP-1. Am J Physiol Lung Cell Mol Physiol 292(6): L1385-1395. doi: 10.1152/ajplung.00472.2006. Tirumurugaan, K.G., Kang, B.N., Panettieri, R.A., Foster, D.N., Walseth, T.F., and Kannan, M.S. 2008. Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-alpha and dexamethasone. Respir Res 9: 26. doi: 10.1186/1465-9921-926. Tliba, O., Deshpande, D., Chen, H., Van Besien, C., Kannan, M., Panettieri, R.A., Jr., et al. 2003. IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle. Br J Pharmacol 140(7): 1159-1162. doi: 10.1038/sj.bjp.0705558. Tliba, O., and Panettieri, R.A., Jr. 2008. Regulation of inflammation by airway smooth muscle. Curr Allergy Asthma Rep 8(3): 262-268. Tliba, O., Panettieri, R.A., Jr., Tliba, S., Walseth, T.F., and Amrani, Y. 2004. Tumor necrosis factor-alpha differentially regulates the expression of proinflammatory genes in human airway smooth muscle cells by activation of interferon-beta-dependent CD38 pathway. Mol Pharmacol 66(2): 322-329. doi: 10.1124/mol.104.001040.


Page 37 of 43

Wang, W.C., Juan, A.H., Panebra, A., and Liggett, S.B. 2011. MicroRNA let-7 establishes expression of beta2-adrenergic receptors and dynamically down-regulates agonist-promoted down-regulation. Proc Natl Acad Sci U S A 108(15): 6246-6251. doi: 10.1073/pnas.1101439108. Wang, Y.X., Zheng, Y.M., Mei, Q.B., Wang, Q.S., Collier, M.L., Fleischer, S., et al. 2004. FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells. Am J Physiol Cell Physiol 286(3): C538-546. doi: 10.1152/ajpcell.00106.2003. Webb, D.C., McKenzie, A.N., Koskinen, A.M., Yang, M., Mattes, J., and Foster, P.S. 2000. Integrated signals between IL-13, IL-4, and IL-5 regulate airways hyperreactivity. J Immunol 165(1): 108-113. White, T.A., Johnson, S., Walseth, T.F., Lee, H.C., Graeff, R.M., Munshi, C.B., et al. 2000. Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle. Biochim Biophys Acta 1498(1): 64-71. White, T.A., Kannan, M.S., and Walseth, T.F. 2003. Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle. FASEB J 17(3): 482-484. doi: 10.1096/fj.02-0622fje. Williams, T.J. 2004. The eosinophil enigma. J Clin Invest 113(4): 507-509. doi: 10.1172/JCI21073. Wilson, H.L., Dipp, M., Thomas, J.M., Lad, C., Galione, A., and Evans, A.M. 2001. Adpribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction. J Biol Chem 276(14): 11180-11188. doi: 10.1074/jbc.M004849200.


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Yamamoto-Katayama, S., Sato, A., Ariyoshi, M., Suyama, M., Ishihara, K., Hirano, T., et al. 2001. Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity. Biochem J 357(Pt 2): 385-392. Zocchi, E., Franco, L., Guida, L., Benatti, U., Bargellesi, A., Malavasi, F., et al. 1993. A single protein immunologically identified as CD38 displays NAD+ glycohydrolase, ADPribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes.

Biochem

S0006291X83724162 [pii].

Biophys

Res

Commun

196(3):

1459-1465.

doi:


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Figure Legends: Figure 1: Synthesis and degradation of cADPR by CD38. β-NAD is converted into cADPR by ADP-ribosyl cyclase and hydrolyzed by cADPR hydrolase, both enzyme activities are associated with a single transmembrane protein, CD38 in mammals. CD38 expressed on immune cells, contributes to humoral and cell-mediated immunity whereas CD38 on ASM cells by releasing cADPR regulates calcium homeostasis, contraction and bronchoconstriction. Important to note that increase in expression of CD38 (dotted arrows) results in an altered calcium homeostasis, hyper-contractility of ASM, and AHR.

Figure 2: Organization of CD38 gene and potential regulatory sites. Genomic analysis of CD38 gene reveals the presence of several transcription factor binding sites upstream of the ATG. Inflammatory cytokines such as TNF-α through the MAP kinase pathway activate transcription factors and regulate CD38 expression. Full length CD38 mRNA has binding sites for several microRNAs, suggesting potential posttranscriptional regulation of CD38 expression (Dileepan et al. 2014). Transfection of ASM cells with miR-140-3p causes decreased p38 MAP kinase and NF-ƙB activation and CD38 expression. Transfection of ASM cells with miR-708-5p causes decreased JNK MAP kinase activation, increased expression of the MAP kinase phosphatase MKP-1 and PTEN that regulates PI3K/AKT signaling and CD38 expression. Solid lines represent increased expression/activation of proteins; dotted lines represent inhibition of expression/activation.


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Table: WT Naïve mice Inflammation Airway responsiveness

0 +

Cd38KO

Reference Deshpande et al. 2005

0 -

IL-13 challenge Inflammation Airway responsiveness

Guedes et al. 2008 ++++ ++++

++++ ++

TNF-α α challenge Inflammation Airway responsiveness

0 ++++

0 ++

Guedes et al. 2006

Fungal Antigen Challenge Inflammation Airway responsiveness

Guedes et al. 2006 ++++ ++++

++++ +

Ovalbumin challenge Inflammation Airway responsiveness

+++ ++

++ +

Gally et al. 2009

0 = unchanged response compared to naïve - = decreased response compared to naïve ++++ = maximal response compared to naïve


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Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/16/14 ly. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official

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Cytokines MAP kinases

TNF-a, IL-1b, INF-g

p38

Transcription factors

ERK

JNK

MKP-1

PTEN PI3K/pAKT

NF-kB, AP-1 miR-708-5p miR-140-3p

3kb

2kb

1kb

+1 ATG

Promoter region (kbp) AP-1

NF-kB

GRE

Coding region c/EBP

9

CD38

22

Post-transcriptional regulators

3’ UTR (bp)

Smooth muscle CaMKIIδ promotes allergen-induced airway hyperresponsiveness and inflammation.

Commentaries on Viewpoint: Airway smooth muscle and airway hyperresponsiveness in human asthma: have we chased the wrong horse?

Airway smooth muscle stretch and airway hyperresponsiveness in asthma: have we chased the wrong horse?

Airway smooth muscle and asthma.

Receptor-activated calcium influx in human airway smooth muscle cells.

Control of human airway smooth muscle.

NHLBI Workshop Summary. Models of airway hyperresponsiveness.

Receptors on target cells. Receptors on airway smooth muscle.

Asthma and airway hyperresponsiveness.

Airway epithelial cells regulate membrane potential, neurotransmission and muscle tone of the dog airway smooth muscle.

Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling.

Interaction between bronchoconstrictor stimuli on human airway smooth muscle.

Cigarette Smoke and Estrogen Signaling in Human Airway Smooth Muscle.

Adrenoceptors in airway smooth muscle.

Nelumbo nucifera leaves extracts inhibit mouse airway smooth muscle contraction.

Inhaled toxicants and airway hyperresponsiveness.

Airway blood flow modifies allergic airway smooth muscle contraction.

Eosinophil supernatant causes hyperresponsiveness of airway smooth muscle in guinea pig trachea.

Epithelium-generated neuropeptide Y induces smooth muscle contraction to promote airway hyperresponsiveness.

Bronchial smooth muscle mechanics of a canine model of allergic airway hyperresponsiveness.

Effects of cigarette smoke extract on human airway smooth muscle cells in COPD.

Exposure of airway smooth muscle cells to cigarette smoke extract.

TGF-β1 feedback circuit regulates airway remodeling in airway smooth muscle cells.

IgE induces proliferation in human airway smooth muscle cells: role of MAPK and STAT3 pathways.