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Modulation of Calcium Signaling by Interleukin-13 in Human Airway Smooth Muscle

Role of CD38/Cyclic Adenosine Diphosphate Ribose Pathway

Departments of Veterinary PathoBiology and Pharmacology, University of Minnesota, St. Paul; Department of Physiology, Mayo Clinic, Rochester, Minnesota; and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Address correspondence to: Mathur S. Kannan, Professor of Pharmacology, Department of Veterinary PathoBiology, College of Veterinary Medicine, 205 Veterinary Science, 1971 Commonwealth Avenue, St. Paul, MN 55108. E-mail: kanna001{at}umn.edu

	Abstract

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CD38/cyclic adenosine diphosphate ribose (cADPR) signaling plays an important role in the regulation of intracellular calcium responses to agonists in a variety of cells, including airway smooth muscle (ASM) cells. The present study was aimed at determining the effect of interleukin (IL)-13, a cytokine implicated in the pathogenesis of asthma, on CD38/cADPR signaling and to ascertain the contribution of CD38/cADPR signaling to IL-13–induced airway hyperresponsiveness. Human ASM cells maintained in culture were exposed to 50 ng/ml IL-13 for 22 h and levels of CD38 expression and intracellular calcium responses to agonists were measured. Treatment of human ASM cells with IL-13 resulted in increased CD38 expression as determined by real-time polymerase chain reaction, Western blot analysis, and indirect immunofluorescence. Increased CD38 expression was reflected as increased ADP-ribosyl cyclase activity in the ASM cell membranes. The net intracellular calcium responses to bradykinin, thrombin, and histamine were significantly (P 0.05) higher in cells treated with IL-13 compared with controls. Furthermore, 8-bromo-cADPR, a cADPR antagonist, attenuated IL-13–induced augmented intracellular calcium responses to agonists in human ASM cells. These findings indicate that the CD38/cADPR–dependent pathway has a major role in IL-13–induced modulation of calcium signaling in human ASM.

Abbreviations: 8-bromo-cyclic adenosine diphosphate ribose, 8br-cADPR • airway hyperresponsiveness, AHR • airway smooth muscle, ASM • bovine serum albumin, BSA • cyclic adenosine diphosphate ribose, cADPR • number of cycles required to achieve threshold fluorescence, C_t • cyclic guanosine diphosphoribose, cGDPR • human airway smooth muscle, HASM • Hanks' balanced salt solution, HBSS • interleukin, IL • nicotinamide adenine dinucleotide, NAD • reverse transcriptase–polymerase chain reaction, RT-PCR • T helper, Th

	Introduction

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Chronic inflammatory diseases such as asthma are characterized by severe airway dysfunction and smooth muscle lining the airways is a target of inflammatory changes. Several studies have demonstrated that cytokines induce functional changes in the airway smooth muscle (ASM) characterized by altered responses to contractile and relaxant agents, leading to severe bronchoconstriction (1, 2). Recent studies have provided evidence that the inflammatory response in asthma is predominantly mediated by the release of T helper (Th) 2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 (3). Subsequent studies have confirmed that IL-13 mediates inflammatory responses in asthmatic airways (4, 5). IL-13 inhalation mimics the effects of allergen-induced airway inflammation in mice (4, 5). Allergen-induced pathological changes in lungs can be reversed by blocking IL-13 in a mouse model of asthma (4, 5). While IL-13 null mice do not exhibit airway hyperresponsiveness (AHR), transgenic mice overexpressing IL-13 in lungs exhibit asthmatic features such as AHR and eosinophil infiltration (6, 7). Furthermore, analysis of bronchoalveolar lavage (8) and biopsy samples (9) from individuals with asthma has demonstrated increased levels of IL-13. These findings demonstrate that the Th2 cytokine IL-13 plays a key role in the pathogenesis of asthma.

Understanding the pathogenesis of IL-13–induced changes in the airways has been the focus of recent investigations. These include characterization of IL-13 receptors and the signaling mechanisms associated with the effects of IL-13 on the target cells. ASM expresses receptors for IL-13 (10) and is an effector cell type that regulates bronchomotor tone (11). Direct interaction of IL-13 with ASM cells is thought to be responsible for the development of AHR. Furthermore, studies have demonstrated that the responsiveness of ASM to contractile and relaxing agents changes upon exposure to IL-13 (1, 2, 11, 12). However, the precise mechanisms of these changes have not been identified.

Calcium is a common second messenger for mediating contraction and contractile agonists elevate intracellular calcium concentration in ASM cells to initiate contraction. Alterations in the agonist–induced calcium changes in the ASM may account for the altered contractile response of airways, a feature of asthma. Previous studies related to inflammatory cytokine effects on ASM cells have demonstrated that altered calcium homeostasis may lead to exaggerated contractile responses (1, 13, 14). However, such mechanisms have not been described for IL-13–induced changes in ASM cells.

Calcium concentration in ASM cells is regulated by multiple mechanisms (13, 15, 16). Studies from our laboratory have demonstrated that cyclic adenosine diphosphate ribose (cADPR), a ß-nicotinamide adenine dinucleotide (NAD) metabolite, contributes to agonist–induced elevation of intracellular calcium concentration ([Ca²⁺]_i) in airway and coronary artery smooth muscle cells (17, 18). cADPR mobilizes Ca²⁺ through ryanodine receptors and potentially contributes to calcium-induced calcium release mechanism (16, 17). Furthermore, we and others have demonstrated that CD38, a bifunctional membrane-bound protein expressed on ASM cells, is responsible for the synthesis and degradation of cADPR (19). CD38 is constitutively expressed in a variety of cells and its expression is influenced by factors such as hormones and cytokines (20, 21). Changes in the expression of CD38 or cADPR–mediated calcium release may account for the altered calcium homeostasis as demonstrated in pancreatic ß cells in response to glucose (22, 23). IL-13 has been shown to bring about physiologic changes in ASM cells, leading to hyperreactivity to contractile agents (11). Whether IL-13 effects are mediated through CD38/cADPR signaling is not established. In this study we determined the effect of IL-13 on intracellular calcium responses to contractile agonists in ASM cells and the contribution of CD38/cADPR–mediated calcium signaling to this process.

	Materials and Methods

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Materials
Routinely used reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Molecular biology reagents were obtained from Invitrogen (Grand Island, NY). Mouse anti-human CD38, goat polyclonal anti-rat CD38 antibody, donkey anti-goat immunoglobulin (Ig) G, and horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cy3-conjugated anti-mouse IgG was obtained from Chemicon (Temecula, CA). Gradient gels and the protein assay kit were purchased from Bio-Rad Laboratories (Hercules, CA). Protease inhibitors and IL-13 were obtained from Calbiochem (La Jolla, CA). RNeasy mini kit was obtained from Qiagen (Valencia, CA). The SYBR Green Master mix and Taq-man RT reagents were purchased from Applied Biosystems (Foster City, CA). Fura-2/AM was purchased from Molecular Probes (Eugene, OR). 8-Br-cADPR was synthesized as described previously (24).

Cell Culture
Human airway smooth muscle (HASM) cells maintained in culture were used in this study. HASM cells were isolated and propagated as described earlier (25). Briefly, trachealis muscle isolated from a segment of trachea was enzymatically dissociated using 640 U/ml of collagenase, 1 mg/ml of soybean trypsin inhibitor, and 10 U/ml of elastase at 37°C for 90 min. Subsequently, the cell suspension was filtered through 105-µm Nytex mesh (Millipore, Billerica, MA). The cells were washed with equal volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and were plated at a density of 1.0 x 10⁴ cells/cm². The cells were cultured in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and amphotericin B. The cells were maintained in Dulbecco's minimum essential medium with no serum and supplemented with 5.7 µg/ml insulin and 5 µg/ml transferrin (arresting medium) for 72–96 h before the experiments. All the experiments were performed using the cells between second and fifth passages. All the experiments were performed with a minimum of three different cell preparations obtained from different donors.

HASM cells arrested at G_o of cell cycle were exposed to 50 ng/ml of IL-13 for 22 h before the studies. HASM cells exposed to 0.05% bovine serum albumin (BSA) were used as control.

Reverse Transcriptase–Polymerase Chain Reaction
CD38 mRNA expression was determined by reverse transcriptase–polymerase chain reaction (RT-PCR) followed by PCR as described (25, 26). Total cellular RNA was isolated from control and IL-13–treated HASM cells using RNeasy mini kit (Qiagen) as per the manufacturer's protocol. Equal amounts of RNA from control and IL-13–treated HASM cells were used for RT. The RT reaction was performed at 25°C for 10 min and subsequently at 48°C for 2 h using Taq-man RT reagents. The first strand cDNA synthesized by RT was denatured at 94°C for 5 min and used subsequently for amplification by PCR.

Human CD38 (GenBank accession no. gi1911098)–specific primers were used to amplify CD38 cDNA by PCR. The following primers were used: forward 5'-ATG TTC ACC CTGGAG GACACG CTG CT-3' (positions 527–552); reverse 5'-CTC AGG ATT TTT CAC ACA CTG AAG -3' (positions 955–980). ß-Actin primers that produced an 250-bp product were used as internal controls. Water with no template was used as a negative control. The PCR was performed under the following conditions: 94°C for 4 min denaturing, 30 cycles of 94°C for 45 s, 45°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were separated on agarose gels and stained with ethidium bromide. CD38 and ß-actin expressions were determined by densitometric analysis of respective bands on the gel. The results were expressed as the ratio of intensity of CD38 to that of ß-actin in samples from control and IL-13–treated HASM cells.

Quantitative Real-Time PCR
CD38 expression was determined by quantitative real-time PCR using SYBR Green PCR master mix as described previously (25, 26). cDNA obtained by reverse transcription from total RNA from control and IL-13–treated HASM was amplified using SYBR green master mix and CD38 or ß-actin specific primers. The reactions were performed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) under the following conditions: 50°C for 2 min, 95°C for 5 min, 40 cycles at 95°C for 45 s, 45°C for 45 s, and 72°C for 45 s, and one cycle of 72°C for 10 min. The fluorescence was determined three times during each cycle and Sequence Detection System version 1.7 software (Applied Biosystems) was used to analyze the data. The fluorescence obtained in the reaction was normalized using the passive reference dye included in the master mix. Results of the real-time PCR were expressed as C_t (cycle at threshold), and the level of expression of CD38 was indicated by the number of cycles required to achieve the threshold level of amplification. The C_t value from control HASM was compared with that of IL-13–treated HASM cells.

HASM Lysate and Membrane Preparation
The HASM cells grown to confluence were scraped in Hanks' balanced salt solution (HBSS) (pH 7.4). The cell suspension was centrifuged at 3,000 rpm for 5 min, and the pellet resuspended in 20 mM Tris HCl, pH 7.2, containing 0.25 M sucrose and protease inhibitors (homogenization buffer). Cells were homogenized by sonication for 10 s three times and the homogenate was stored at –80°C before use.

The cell lysate was centrifuged at 10,000 rpm for 15 min. The pellet was resuspended in Tris HCl buffer and referred to as P10. The supernatant was subsequently centrifuged at 100,000 x g for 1 h and the pellet was resuspended in Tris HCl buffer (referred to as microsomes). The supernatant obtained after high-spin centrifugation was also used for biochemical analysis. The protein content in the whole cell lysate, P10, microsome, and supernatant was determined using the Bio-Rad protein assay kit, with BSA as the standard.

Western Blot Analysis
CD38 protein in HASM cells was detected by Western blot analysis, as described earlier (25, 26). Cell lysates from control and IL-13–treated HASM (100 µg protein) were applied onto 4–15% gradient polyacrylamide gels and proteins were separated by electrophoresis. After transferring the proteins on the gel to polyvinylidene difluoride membrane, the proteins were probed with polyclonal goat anti-rat CD38 antibody for 1 h. Subsequently the membrane was washed and incubated with horseradish peroxidase–conjugated donkey anti-goat IgG for 1 h. The blots were developed using chemiluminescence substrate before exposure to X-ray film. The molecular weight of CD38 was determined by comparing with the standard molecular weight markers. The densities of CD38 bands on the membrane were determined to compare the expression of CD38 protein in control and IL-13–treated HASM cells.

Indirect Immunofluorescence and Confocal Microscopic Examination of CD38 Expression on HASM Cells
HASM cells were grown on glass coverslips and fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 10 min. After washing the cells three times with PBS, the cells were exposed to 10% normal goat serum for 20 min. Subsequently the cells were incubated with mouse anti-human CD38 antibody in 1.5% normal goat serum overnight at 4°C. The cells were washed with PBS five times and incubated with Cy3–conjugated goat anti-mouse IgG for 1 h. The cells were washed with PBS five times and mounted onto a glass slide using SlowFade (Molecular Probes) mounting medium. The stained cells were visualized using confocal microscopy. Cells stained with no primary antibody were used as negative control.

Immunostained cells were examined with a laser scanning confocal microscope (model LSM 510; Carl Zeiss, Inc., Thornwood, NY) equipped with an argon–krypton laser. A 40x-water immersion objective (numerical aperture 1.2) was used with additional electronic zoom, when necessary. Cells were imaged using the 543-nm laser line with a 560-nm long-pass emission filter appropriate for CY3. Typically, 12–18 confocal optical sections (512 x 512 pixels) were sequentially collected at 0.6–1.0-µm intervals through each cell. The images were merged together into a single "stacked" image using Zeiss-LSM software. Typically, images from 3–4 fields were collected from each slide.

Measurement of ADP-Ribosyl Cyclase Activity
Nicotinamide guanine dinucleotide, an analog of NAD, was used as the substrate to determine the ADP-ribosyl cyclase activity (19, 25, 26). The reactions were performed in 20 mM Tris HCl buffer, pH 7.2, at 37°C by incubating HASM cell lysate (equal amounts of cellular proteins from control and cytokine-treated HASM), P10, microsomes, or supernatant with 200 µM nicotinamide guanine dinucleotide. Production of cyclic guanosine diphosphoribose (cGDPR) was measured fluorometrically at an excitation wavelength of 305 nm and an emission wavelength of 410 nm. The fluorescence intensity was converted to nanomoles of cGDPR using a cGDPR standard curve and activity expressed as nmol mg^–1 min^–1.

Measurement of cADPR Hydrolase Activity
cADPR hydrolase activity in HASM cells was determined using ³²P-cADPR as the substrate and the conversion of ³²P-cADPR to ³²P-ADPR was measured using a method described in previous publications (19, 26).

Measurement of cADPR Levels
HASM cells were grown to confluence in 175-mm² flasks and treated with vehicle or IL-13. The cells were washed with HBSS (pH 7.4) twice and cells were scraped in ice-cold 0.5-M perchloric acid (PCA) solution. Subsequently the levels of cADPR in the control and IL-13–treated HASM cells were determined using a cycling assay as described previously (27).

Measurement of Intracellular Calcium Concentration
Cell permeant, ratiometric calcium indicator dye fura-2/AM was used to determine the intracellular calcium concentration in HASM cells. HASM cells were grown on glass coverslips and exposed to BSA (control) or 50 ng/ml of IL-13 for 22 h. Cells were washed and maintained in HBSS containing 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 11 mM glucose, 2.5 mM CaCl₂, and 1.2 mM MgCl₂ (pH 7.4) and incubated with 5 µM fura-2/AM for 30 min at 37°C and 5% CO₂. An open slide chamber was used to mount the coverslip, which was subsequently placed on the stage of a Nikon Diaphot inverted microscope (Nikon Instruments Inc., Melville, NY). The cells were perfused with HBSS and the basal intracellular calcium concentration ([Ca²⁺]_i) was determined using real-time digital video fluorescence imaging (Metafluor; Universal Imaging Corporation, Downing, PA) as described previously (25, 28).

Fura-2–loaded cells were alternately excited at 340 and 380 nm using a Lambda DG-4 high-speed filter changer (Sutter Instrument Co., Novato, CA). Fluorescence emissions were collected separately for each wavelength using a 510-nm barrier filter. A Photometric Cool Snap 12-bit digital camera (Roper Scientific, Trenton, NJ) was used to collect images at the rate of one image for every 0.75 s, and ratios of fluorescence intensities at 340 nm and 380 nm were determined at each time point. 4-Br-A23187 was used to determine the maximum while ethyleneglycol-bis-(ß-aminoethyl ether)-N-N'-tetraacetic acid was used to determine the minimum calcium concentration in HASM cells, and these images were used to obtain calcium calibration curve, as described previously (25, 28).

Agonist–Induced Intracellular Ca²⁺ Responses
Bradykinin (BK), thrombin (TH), and histamine–induced changes in [Ca²⁺]_i were measured in control and IL-13–treated HASM cells. All experiments were performed at room temperature (25°C). The basal [Ca²⁺]_i was determined by perfusing the fura-2–loaded HASM cells with HBSS. Subsequently the cells were perfused with HBSS containing 1 nM BK, 1 IU/ml TH, or 50 µM histamine for at least 2–3 min. The net intracellular Ca²⁺ responses to agonists were calculated by subtracting basal [Ca²⁺]_i from the peak [Ca²⁺]_i.

Effect of 8Bromo-cADPR on Intracellular Ca²⁺ Response to Agonists
A cell-permeant cADPR antagonist, 8 bromo-cADPR (8Br-cADPR) was used to determine the role of cADPR in the agonist–induced intracellular Ca²⁺ release in control and IL-13–treated HASM cells. Control and IL-13–treated HASM cells were incubated with either HBSS or 100 µM 8Br-cADPR for 15 min. The concentration and the duration were determined based on our previous studies (25, 28). The intracellular Ca²⁺ responses to BK, TH, and histamine were measured as described above. The net [Ca²⁺]_i was determined for each agonist in the presence or absence of 8Br-cADPR. The intracellular Ca²⁺ responses to agonists in control and IL-13–treated cells in the presence of 8Br-cADPR were compared with that of respective controls.

Statistical Analysis
HASM cells isolated from at least three different donors were used to repeat all the experiments. CD38 expression in control and IL-13–treated HASM cells were analyzed by Student's t test. The net [Ca²⁺]_i responses to different agonists in control and IL-13–treated HASM cells were compared using one-way analysis of variance with Bonferroni's test for multiple comparison. All the statistical analyses were done using GraphPad PRISM statistical software (Verbatim Co., Charlotte, NC). Two means were considered significantly different when P value was less than 0.05.

	Results

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IL-13 Increases CD38 Expression, ADP-Ribosyl Cyclase Activity, and cADPR Levels in Human ASM Cells
IL-13 alters the transcriptional activity of a variety of genes in ASM cells (29). To determine the IL-13–induced changes in CD38/cADPR signaling, we measured CD38 expression, ADP-ribosyl cyclase activity, and cADPR levels in HASM cells. CD38 mRNA expression was determined by RT-PCR and real-time PCR. In cells treated with 50 ng/ml of IL-13, CD38 mRNA expression was significantly higher than in controls. Densitometric analysis of the RT-PCR product separated by agarose gel electrophoresis revealed an 2.5-fold increased expression of CD38 mRNA in cells exposed to IL-13 as compared with controls (Figure 1A).

View larger version (43K): [in this window] [in a new window]   Figure 1. IL-13 upregulates CD38 expression in HASM cells. (A) The figure shown is a representative PCR analysis of CD38 expression in control and IL-13–treated HASM cells. Expression of ß-actin was used as internal control. C: Control; L: 100-bp DNA ladder. Note an increased expression of CD38 by IL-13. (B) The figure shown is a representative Western blot analysis for CD38 expression in control and IL-13–treated (two different preparations) HASM cells. Note an increased expression of CD38 in IL-13–treated HASM cells compared with control.

CD38 protein expression was also determined by confocal microscopy using indirect immunofluorescence staining of HASM cells. Representative confocal images are shown in Figure 2. IL-13 treatment resulted in more intense staining in HASM cells compared with control cells, reflecting increased CD38 protein expression.

View larger version (57K): [in this window] [in a new window]   Figure 2. HASM cells treated with IL-13 (50 ng/ml) show higher CD38 expression. CD38 expression on the surface of HASM cells was determined by indirect immunofluorescence staining and visualized using laser confocal microscopy. Anti-human CD38 antibody was used as a primary antibody and cells treated with no primary antibody were used as negative control. Shown are the representative confocal images of control (upper panels), IL-13–treated (middle panels), and negative control (lower panels) HASM cells (three different preparations) stained for CD38. Note more intense immunostaining in cells treated with IL-13 compared with controls.

View larger version (17K): [in this window] [in a new window]   Figure 3. IL-13–treated HASM have higher ADP-ribosyl cyclase activity. Whole cell lysate, P10, microsomes, or supernatant fractions from control and IL-13–treated HASM cells were incubated with NGD, and the formation of cGDPR was monitored fluorimetrically. The data represent the specific ADP-ribosyl cyclase activity (determined using a cGDPR standard curve) in (A) whole cell lysate and (B) microsomal fraction obtained from control and IL-13–treated HASM cells. cADPR hydrolase activity in the fractions was determined using 32P-cADPR as the substrate. The data represents specific cADPR hydrolase activity in HASM microsomal fraction (C).

cADPR levels were measured in control and IL-13–treated HASM cells. The basal cADPR levels were 889.8 ± 316.3 and 1,280 ± 275.7 fmol/mg in the control and IL-13–treated HASM cells (n = 5), respectively, demonstrating a 58% increase in the basal cADPR levels upon IL-13 treatment (P 0.05).

Agonists Elicit Augmented Intracellular Calcium Responses in HASM Cells Treated with IL-13
Exposure of HASM cells to 50 ng/ml of IL-13 had no significant effects on basal intracellular Ca²⁺ (data not shown). Stimulation of HASM cells with BK, TH, or histamine resulted in elevation of [Ca²⁺]_i. Figure 4 demonstrates the representative traces of calcium responses from control and IL-13–treated HASM cells. The net intracellular Ca²⁺ responses to BK, TH, or histamine were significantly (P 0.05) higher in IL-13–treated HASM cells compared with controls (Figure 5). In control cells, the net intracellular Ca²⁺ response to 1 nM BK, 1 IU/ml TH, and 50 µM histamine were 661 ± 23, 1,031 ± 36.98, 614 ± 25 nM, respectively. In IL-13–treated cells, the net intracellular Ca²⁺ responses to 1 nM BK, 1 IU/ml TH, and 50 µM histamine were 1,108 ± 48, 1,443 ± 109, and 973 ± 59 nM, respectively. These values were significantly (P 0.05) higher than values in control cells.

View larger version (30K): [in this window] [in a new window]   Figure 4. Bradykinin, thrombin, and histamine elevate intracellular calcium in HASM cells. Representative traces of calcium responses to bradykinin (A, B), thrombin (C, D), and histamine (E, F) in control (A, C, E) and IL-13–treated (B, D, F) HASM cells obtained using digital videofluorescence imaging. The vertical lines indicate the time of stimulation of cells with the agonist.

View larger version (18K): [in this window] [in a new window]   Figure 5. Exposure of HASM cells to IL-13 results in augmented intracellular calcium responses compared with controls and 8Br-cADPR attenuates these responses. Net intracellular response to bradykinin, thrombin, and histamine were determined by subtracting basal [Ca2+]i from the peak [Ca2+]i. Control and IL-13–treated HASM cells were preincubated with 100 µM 8Br-cADPR for 15 min and the net intracellular Ca2+ responses to bradykinin, thrombin, and histamine were determined. *Denotes statistically significant (P 0.05) difference between the responses in the presence or absence of 8Br-cADPR and **denotes statistical significance between control and IL-13–treated cells. Note a significant augmentation of intracellular Ca2+ response in IL-13–treated HASM cells and attenuation by 8Br-cADPR.

8Br-cADPR Attenuates Agonist-Induced Intracellular Calcium Responses in Control and IL-13–Treated HASM Cells
8Br-cADPR, a cell-permeant cADPR antagonist, was used to determine the contribution of cADPR to agonist-induced intracellular Ca²⁺ responses in control and IL-13–treated HASM cells. [Ca²⁺]_i in response to agonists was determined in cells preincubated for 15 min with 100 µM 8Br-cADPR or HBSS. The net Ca²⁺ responses to BK were 661.2 ± 23 and 512.5 ± 21 nM in control cells in the absence and presence of 8Br-cADPR, respectively (Figure 5). In IL-13–treated HASM cells, the BK–induced intracellular Ca²⁺ responses were 1,108 ± 48 and 649 ± 24 nM in the absence and presence of 8Br-cADPR, respectively. The net Ca²⁺ responses to BK in the presence of 8Br-cADPR were significantly (P 0.05) lower than in its absence in both control and IL-13–treated cells (Figure 5).

Intracellular Ca²⁺ responses to TH and histamine were also significantly (P 0.05) attenuated by 8Br-cADPR in control and IL-13–treated HASM cells (Figure 5). One International Unit per milliliter TH elicited a net Ca²⁺ response of 654 ± 20 nM in the presence of 8Br-cADPR in control cells, whereas the response was 1,031 ± 37 nM in its absence. In cells treated with IL-13, the net Ca²⁺ response was significantly higher (1,443 ± 109 nM) than controls and 8Br-cADPR attenuated this response (978 ± 47 nM) (P 0.05). The net intracellular Ca²⁺ responses to histamine (50 µM) were 614 ± 25 and 506 ± 35 nM in control and 973 ± 59 and 742 ± 32 nM in IL-13–treated HASM cells in the absence and presence of 8Br-cADPR, respectively. In the presence of 8Br-cADPR, the net intracellular Ca²⁺ responses to all three agonists in IL-13–treated HASM cells were similar to responses in control cells in the absence of 8Br-cADPR, reflecting a significant contribution of cADPR-mediated Ca²⁺ release in the augmented responses to the agonists (Figure 5).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The study was aimed at determining the contribution of the CD38/cADPR signaling to IL-13–induced changes in calcium homeostasis in ASM cells. Exposure of HASM cells to IL-13 resulted in upregulation of CD38 expression and function as shown by increased ADP-ribosyl cyclase activity and basal cADPR concentrations. Contractile agonists such as BK, histamine, and TH elicited augmented intracellular Ca²⁺ responses in IL-13–treated human ASM cells compared with controls. Furthermore, a cADPR antagonist attenuated agonist-induced intracellular Ca²⁺ response in IL-13–treated HASM cells. The findings indicate that CD38/cADPR signaling contributes to the modulation of calcium responsiveness to contractile agonists by IL-13 in ASM cells.

Recent studies have demonstrated that allergen-induced airway diseases are predominantly mediated through the Th2 cytokines. In this context, two important cytokines, IL-4 and IL-13, have been identified as central players in the pathogenesis of asthma. Differentiation of lymphocytes into a Th2 phenotype is mediated by IL-4, but it does not appear to have an obligatory role in the development of allergen-induced AHR (5). However, the events that lead to the development of AHR are mediated by IL-13 (3, 30). This is supported by several lines of evidence provided in studies involving murine models of asthma (4, 7, 10, 31).

Understanding the mechanisms by which IL-13 promotes AHR has been the focus of recent investigations. Transcriptional activation is an important mechanism by which IL-13 induces mucus secretion, eosinophilia, cell proliferation, and AHR (31, 32). High throughput microarray analysis of ASM cells has revealed that IL-13 alters transcriptional activity of a variety of genes (29). In this study, we demonstrate that exposure of HASM cells to IL-13 results in increased levels and activity of CD38, an important ectoenzyme that regulates calcium signaling via the production of cADPR. CD38 expression is not only regulated by hormones such as estrogen, corticosteroids, vitamin D3, and chemical agents such as retinoic acid, but also by cytokines such as IL-1ß, tumor necrosis factor and interferon (20, 21). Alterations in the expression of CD38 are known to lead to certain pathologic conditions (23, 33). However, the molecular mechanism associated with alterations in the CD38 expression is not known. 5' upstream region of CD38 gene contains GC-rich region and binding sites for transcription factors such as nuclear factor IL-6, interferon responsive factor-1 PEA-3, CP2, and PuF (20, 21, 34). Other studies have demonstrated that IL-13 exerts its effects in the target cells through the IL-13R/IL-4R-STAT6 signaling pathway (11, 31, 32, 35). Other transcription factors such as IRS-2 are also altered by IL-13, although STAT-6–dependent pathway has been shown to be critical in the development of AHR (10, 35, 36). Whether CD38 expression is primarily dependent on STAT-6 phosphorylation or secondary to the primary response genes needs to be elucidated. Furthermore, recent studies have described non-CD38 source of ADP-ribosyl cyclase for cADPR production. The role of such sources of cADPR in intracellular calcium regulation in smooth muscle is not known. Therefore, we cannot rule out the contribution of non-CD38 sources of ADP-ribosyl cyclase to IL-13–induced changes in ADP-ribosyl cyclase activity in ASM cells.

A study by Venkayyaand colleagues (12) demonstrated that the cells lining the airways are the direct target of Th2 cytokines in the development of AHR. Smooth muscle being the principal contractile component of the airways, changes in the contractile properties of ASM cells would directly contribute to AHR. In this regard, overexpression of IL-13 in the lung and exogenous administration of IL-13 are known to result in altered responsiveness to contractile and relaxant agents (4, 5, 7). Exposure of tracheal rings to IL-13 also revealed similar effects (37). The mechanism of IL-13–induced AHR has been attributed to its effects on ASM lining the airways. In support of this hypothesis, IL-13 receptor components (IL-13R1, IL-13RII, and IL-4) are known to be expressed on ASM cells (11), indicating a potential for direct effects. Similarly, Laporte and colleagues (11) demonstrated that IL-13 alters the responsiveness of isolated HASM cells to contractile and relaxant agents in an extracellular signaling regulated kinase–mitogen activated protein kinase–dependent mechanism. The results of these investigations provide evidence for ASM as a primary target in the development of IL-13–induced AHR.

Contractility of airways being calcium-dependent, we determined the effect of IL-13 on agonist-induced intracellular Ca²⁺ responses. Exposure of HASM cells to IL-13 resulted in augmented intracellular Ca²⁺ responses to multiple agonists. Previous investigations have shown that inflammatory cytokines such as tumor necrosis factor and IL-1ß alter calcium homeostasis in ASM cells (13, 14, 25). The present study demonstrates that IL-13 modulates calcium homeostasis in ASM cells and provides evidence for calcium signaling in ASM as a potential mechanism in the development of IL-13–induced AHR.

The role of CD38/cADPR signaling has been investigated in smooth muscle cell calcium homeostasis and contractility. We have demonstrated that cADPR, a ß-NAD metabolite, contributes to agonist–induced elevation of intracellular Ca²⁺ in ASM cells (17, 18, 25, 28). Studies using smooth muscle cells lining seminiferous tubules revealed the contribution of cADPR–mediated Ca²⁺ release to agonist-induced contraction (38). Furthermore, CD38/cADPR signaling contributes to inflammatory cytokine–induced ASM cell hyperresponsiveness (25). A similar role of CD38/cADPR-mediated Ca²⁺ release has been demonstrated in hypoxia-induced vasoconstriction (39–41). Increased CD38 expression, ADP-ribosyl cyclase activity, and the augmented intracellular Ca²⁺ responses to agonists and their sensitivity to the cADPR antagonist collectively provide evidence for a role of CD38/cADPR signaling in IL-13–induced AHR. Whether sensitivity of cADPR-mediated calcium release through ryanodine receptor channels is altered during IL-13 treatment remains to be determined.

Altered expression of other molecules involved in signal transduction is known to lead to increased responsiveness of ASM cells. In support of this, genes encoding mitogen activated protein kinases, phosphlipase A2, and diacylglycerol kinase have been identified to be upregulated by IL-13 (29). These and our present findings indicate that IL-13, by altering the expression of second messenger and/or transduction molecules, modulates calcium homeostasis in ASM cells, which may lead to AHR. In summary, we found that IL-13, by upregulating CD38 expression, enhances agonist–evoked intracellular Ca²⁺ responses in ASM cells, which may contribute to changes in the responsiveness of airways to contractile agonists.

	Acknowledgments

 
The help of Andrew Eszterhas in preparing the cells for this study is highly appreciated. This study was supported through grants from the National Institutes of Health (HL057498 to M.S.K., DA11806 to T.F.W., and HL55301 and HL64063 to R.A.P.), a University of Minnesota Academic Health Center grant to M.S.K. and T.F.W., and an American Lung Association grant RG-062-N to Y.A. Y.A. is a Parker B. Francis Fellow in Pulmonary Research.

Received in original form August 21, 2003

Received in final form January 29, 2004

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