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Am J Physiol Lung Cell Mol Physiol 294: L378-L385, 2008. First published January 4, 2008; doi:10.1152/ajplung.00394.2007
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Regulation of store-operated Ca2+ entry by CD38 in human airway smooth muscle

Gary C. Sieck,1,2 Thomas A. White,1 Michael A. Thompson,2 Christina M. Pabelick,1,2 Mark E. Wylam,1,3 and Y. S. Prakash1,2

Departments of 1Physiology and Biomedical Engineering, 2Anesthesiology, and 3Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 21 September 2007 ; accepted in final form 28 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ectoenzyme CD38 catalyzes synthesis and degradation of cyclic ADP ribose in airway smooth muscle (ASM). The proinflammatory cytokine TNF{alpha}, which enhances agonist-induced intracellular Ca2+ ([Ca2+]i) responses, has been previously shown to increases CD38 expression. In the present study, we tested the hypothesis that the effects of TNF{alpha} on CD38 expression vs. changes in [Ca2+]i regulation in ASM cells are linked. Using isolated human ASM cells, CD38 expression was either increased (transfection) or knocked down [small interfering RNA (siRNA)], and [Ca2+]i responses to sarcoplasmic reticulum depletion [i.e., store-operated Ca2+ entry (SOCE)] were evaluated in the presence vs. absence of TNF{alpha}. Results confirmed that TNF{alpha} significantly increased CD38 expression and ADP-ribosyl cyclase activity, an effect inhibited by CD38 siRNA, but unaltered by CD38 overexpression. CD38 suppression blunted, whereas overexpression enhanced, ACh-induced [Ca2+]i responses. TNF{alpha}-induced enhancement of [Ca2+]i response to agonist was blunted by CD38 suppression, but enhanced by CD38 overexpression. Finally, TNF{alpha}-induced increase in SOCE was blunted by CD38 siRNA and potentiated by CD38 overexpression. Overall, these results indicate a critical role for CD38 in TNF{alpha}-induced enhancement of [Ca2+]i in human ASM cells, and potentially to TNF{alpha} augmentation of airway responsiveness.

bronchial smooth muscle; tumor necrosis factor-{alpha}; sarcoplasmic reticulum; ADP ribosyl cyclase; cyclic ADP ribose; small interfering RNA

IN AIRWAY SMOOTH MUSCLE (ASM), changes in intracellular Ca2+ concentration ([Ca2+]i) underlie a number of cellular responses including contraction and relaxation. Increased [Ca2+]i in response to agonist stimulation (e.g., ACh, histamine) can occur via Ca2+ release from sarcoplasmic reticulum (SR) (11, 15, 20, 26, 38, 39, 46) or by plasma membrane Ca2+ influx.

TNF{alpha}, a potent proinflammatory cytokine found in bronchoalveolar lavage fluid (12) and sputum (50) from asthmatics, has been implicated as a mediator in the pathophysiology of asthma (45, 51, 52) and chronic obstructive pulmonary disease (8, 16). Indeed, TNF{alpha} has been shown to enhance ASM contractility (13, 43, 47). Other studies have shown that TNF{alpha} augments agonist-induced [Ca2+]i responses (1, 4). The mechanisms underlying TNF{alpha}-induced enhancement of [Ca2+]i are being actively investigated. TNF{alpha} increases both peak [Ca2+]i (thought to represent SR Ca2+ release) as well as the subsequent, steady-state plateau phase of [Ca2+]i responses (3) (thought to represent Ca2+ influx). In ASM, SR Ca2+ release occurs through both inositol trisphosphate (IP3) receptor channels (11, 20, 46) as well as via ryanodine receptor (RyR) channels (15, 26), the latter resulting from increased levels of the novel second messenger cyclic ADP ribose (cADPR) (27, 39). cADPR, in turn, is synthesized and degraded by the bifunctional ectoenzyme CD38 via ADP ribosyl cyclase and cADPR hydrolase activities, respectively (22). Indeed, the CD38/cADPR pathway has been implicated in [Ca2+]i regulation in ASM (39, 57) and intestinal (30, 31), uterine (7, 53), and vascular (17, 25) smooth muscles. Recent studies suggest that the CD38/cADPR signaling pathway contributes to TNF{alpha}-induced augmentation of [Ca2+]i responses in ASM (14). Such an effect is thought to result from an increase in cADPR-induced Ca2+ mobilization from the SR. However, this does not explain the effect of TNF{alpha} on both peak and plateau responses. While it is likely that SR Ca2+ release is augmented by TNF{alpha}, whether Ca2+ influx is also increased remains to be determined.

Ca2+ influx in ASM can occur through voltage-gated (59), receptor-operated (33), and/or store-operated channels (6). In the last case, store-operated Ca2+ entry (SOCE) is triggered by depletion of SR Ca2+ stores (6, 41, 42, 49, 55, 56). We and others have shown that different transient receptor potential channel (TRPC) isoforms are expressed in ASM (6, 58). In a recent study using human ASM, we further demonstrated that TNF{alpha} treatment increases SOCE (58). TNF{alpha} treatment also increases CD38 expression. Studies in other cell types suggest that CD38 itself can also regulate SOCE (10, 21). Whether altered CD38 expression contributes to TNF{alpha}-induced changes in Ca2+ influx is not known. In the present study, we hypothesized that the effects of TNF{alpha} treatment on CD38 expression and SOCE in human ASM cells are linked. Accordingly, we examined the effects of CD38 overexpression vs. knockdown of CD38 expression [via specific small interfering RNA (siRNA)] on TNF{alpha}-induced enhancement of SOCE.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human ASM cells. Human bronchi were obtained from discarded surgical specimens in accordance with procedures reviewed and approved (as well as deemed exempt from Human Subjects classification under 45CFR 46) by the Mayo Clinic Institutional Review Board. The techniques for isolation of ASM cells from bronchi have been recently described (26, 58). Briefly, third to sixth generation bronchi were immersed in ice-cold Hanks' balanced salt solution [in mM: 2.25 Ca, 0.8 Mg, 12 glucose, pH 7.4 (HBSS)] aerated with 100% O2. Under a dissecting microscope, epithelium was removed, and the remaining tissue was finely minced in ice-cold Ca2+-free HBSS ("0" Ca2+ HBSS). The HBSS was removed, and the tissue was incubated for 1 h at 37°C in Earl's balanced salt solution (EBSS) containing 30 mg/ml BSA, 20 U/ml papain, and 0.005% DNase (Worthington Biochemical, Lakewood, NJ). After 1 h, 1 mg/ml of type IV collagenase and 0.4 U/ml elastase were added for an additional 45 min at 37°C. The tissue was gently triturated, centrifuged, and redispersed several times in EBSS with final resuspension in DMEM/F-12 medium containing antibiotics/antimycotic and 10% fetal bovine serum (DMEM complete). The final resuspension was seeded into culture flasks and maintained in incubators (21% O2, 5% CO2 at 37°C). Cells were passaged upon reaching 70–90% confluence and were used for experiments between passages 1 and 5. Presence of ASM cells was confirmed by immunohistochemistry and RT-PCR for smooth muscle {alpha}-actin.

CD38 overexpression. Full-length CD38 was originally generated from myometrial smooth muscle cells using RT-PCR and primers designed from a published sequence (accession no. D84276) that includes the endogenous translation start and stop codons (forward primer sequence: 5'-ACCCCGCCTGGAGCCCTATG-3', reverse primer sequence: 5'-GCTAAAACAACCACAGCGACTGG-3'). Agar-purified PCR product (to eliminate nonspecific product) was incubated with 5 units of Taq polymerase and 1 pmol dATP at 72°C for 15 min to create a 3'(A) overhang, cloned into the pcDNA3.1/V5-HIS-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into bacteria. Correct insertion of CD38 DNA was confirmed by sequencing. A green fluorescent protein (GFP)-tagged CD38 DNA construct was constructed using standard procedures. ASM cells at 70–90% confluence were transfected with GFP-CD38 (for [Ca2+]i imaging studies), CD38 (for biochemical studies), or Lipofectamine 2000 (Invitrogen) alone according to the manufacturer's protocol. Cells were incubated with the transfection mix in DMEM-F12 media without serum for 24 h and analyzed for CD38 expression. Functionality and overexpression of in situ-generated CD38 protein was confirmed either by fluorescence imaging of GFP-CD38 [epifluorescence attachment to Nikon imaging system (see below), excitation 488 nm, emission 510 nm] or by treating transfected cells with 300 µM nicotinamide guanine dinucleotide (NGD) and measuring conversion to the fluorescent product cGDPR as detailed below for measurement of ADP ribosyl cyclase activity of CD38. For SOCE studies, DMEM/F-12 media containing 10% FBS was added 4 h posttransfection and maintained for 24 h. This medium was replaced with serum-free DMEM/F-12 media for an additional 48 h to drive cells to quiescence before SOCE measurements.

CD38 knockdown via siRNA. At time of passage, ASM cells were plated (8-well borosilicate coverglass chambers for Ca2+ studies and 6-well chambers for biochemical measurements) and maintained in DMEM with 10% FBS (no antibiotics or antimycotics). Cells were grown to 50–60% confluence and transfected with 150 nM stealth siRNA directed toward CD38 (CD38si, Invitrogen) or with a negative control siRNA with similar guanosine and cytosine (GC) content as CD38si (siCon, Invitrogen). Stealth CD38si is a 25-bp duplex corresponding to nucleotides 329–353 of the reported human CD38 mRNA sequence. The sense sequence of CD38si is 5'-UUU GGC AGU CUA CAU GUC UCA UCU C-3'. Lipofectamine (Invitrogen) was used as the transfection reagent at a concentration of 2 µl/ml in the final transfection volume. Cells were transfected with siRNA Lipofectamine complexes diluted in DMEM/F-12 without serum or antibiotics. Transfection efficiency was determined to be 81 ± 2% using an FITC-tagged siRNA and counting the proportion of fluorescing cells at 24 h (n = 6 trials). Six hours after initiation of transfection, DMEM containing 10% FBS was added to the transfection medium. After an additional 48 h, the medium was replaced with serum-free medium to elicit an ACh-responsive phenotype (19, 32). For treatment with TNF{alpha} (20 ng/ml), the cytokine was introduced followed by 24 h of serum deprivation and continued for an additional 24 h. Nonspecific effects of CD38si were evaluated using Western blots for TRPC3 using previously described techniques (58).

Real-time semiquantitative RT-PCR for CD38. To determine that CD38 mRNA was indeed increased by TNF{alpha} treatment and CD38 overexpression, but decreased by CD38si knockdown, real-time PCR using LightCycler (Roche Applied Science, Indianapolis, IN) and SYBR Green (Molecular Probes, Eugene, OR) technologies were employed. The technique has been previously described for TRPC isoforms (58). Total RNA from human ASM cells was isolated using an RNeasy Mini kit (Qiagen, Valencia, CA) per the manufacturer's protocol. RNA was quantified via spectrophotometry, and equimolar amounts from different cell groups were used for RT-PCR. First-strand cDNA synthesis was carried out using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). A LightCycler human-glucose-6-phosphate dehydrogenase housekeeping (hG6PDH) gene set (Roche Applied Science) was also used to normalize expression levels of CD38 mRNA to expression levels of hG6PDH in all samples examined. Primers for CD38 as well as for smooth muscle {alpha}-actin and GAPDH were designed against known mRNA sequence obtained from GenBank and were as follows: CD38 forward (CTC TGT CTT GGC GTC AGT ATC CTG), reverse (AGC AAG GTA GCC TAG CAG CGT GTC); smooth muscle {alpha}-actin forward (GAT CAC GGC CCT AGC ACC), reverse (AGG CCC GGC TTC ATC GTA); and GAPDH forward (AAC AGC GAC ACC CAC TCC TC), reverse (GGA GGG GAG ATT CAG TGT GGT). Products from some of the real-time PCR reactions were separated in 1% agarose gels containing ethidium bromide and visualized using UV luminescence. A DNA ladder was run in parallel to approximate product sizes. Bands corresponding to the predicted size of each product were excised from the gels, and the products were extracted using a QIAquick gel extraction kit (Qiagen) and subsequently sequenced by Mayo Clinic's core facilities to verify authenticity of all products. CD38 mRNA levels were also examined in RNA extracts from cells transfected with CD38si or siCon with or without TNF{alpha} to confirm the effectiveness of the siRNA transfections at reducing CD38 mRNA.

Measurement of ADP ribosyl cyclase activity. The ADP ribosyl cyclase activity of CD38 was measured in cell lysates using a fluorescence assay based on the fact that ADP ribosyl cyclase can cyclize the NAD analog, NGD, to produce the non-hydrolyzable fluorescent product, cGDPR (18). ASM cells from different groups (controls, CD38si and CD38 overexpression with or without TNF{alpha} exposure) were processed with lysis buffer, and 400 µM NGD was added. NGD in acellular medium served as background. Fluorescence was measured using a Hitachi F-2000 spectrofluorometer (excitation 305 nm, emission 410 nm). Changes in fluorescence above background were considered resulting from conversion of NGD to cGDPR.

[Ca2+]i imaging. The techniques for [Ca2+]i measurements in ASM using fluorescent indicators have been previously described (6, 58). Briefly, cells plated in eight-well borosilicate coverglass chambers were incubated for 45 min in 5 µM fura-2 AM (Molecular Probes), washed, and perfused with HBSS. Fura-2 emissions (excitation 340 vs. 380 nm; emission 510 nm recorded every 750 ms) were recorded using a video fluorescence imaging system (Metafluor; Molecular Devices, Sunnyvale, CA and Micromax 12-bit camera system; Princeton Instruments, Trenton, NJ) mounted on an inverted Nikon Diaphot microscope and a x20 objective lens. After background and shading corrections, [Ca2+]i was calculated from the 340/380 ratios based on an in vitro calibration as previously described (57).

SOCE. The techniques for SOCE measurements in ASM cells have also been previously described (6, 58). Fura-2-loaded ASM cells were initially perfused with HBSS, and baseline [Ca2+]i levels were recorded. Extracellular Ca2+ was then removed by replacing HBSS with 0 Ca2+ (nominally Ca2+-free) HBSS. In the absence of extracellular Ca2+, SR Ca2+ was depleted using 10 µM cyclopiazonic acid (CPA; inhibitor of SR Ca2+ ATPase). In the continued presence of CPA, extracellular Ca2+ was rapidly reintroduced to trigger SOCE.

Materials. DMEM, antibiotic/antimycotic mixture, and fura-2 were obtained from Invitrogen. All fine chemicals were purchased from Sigma-Aldrich (St. Louis, MO). CPA was purchased from Calbiochem (La Jolla, CA).

Statistical analysis. Differences between cell groups were analyzed for statistical significance using one- or two-way ANOVA as appropriate (control vs. TNF{alpha}, nontransfected vs. transfected, etc.). Multiple comparison tests (Tukey or Scheffé) were employed as appropriate. Experiments were performed using human ASM cells obtained from six different patients. A value of P < 0.05 was considered significant. All results are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of TNF{alpha} treatment on CD38 expression. Treatment of human ASM cells with TNF{alpha} resulted in a marked increase in CD38 mRNA expression compared with control cells not exposed to TNF{alpha} (Fig. 1; P < 0.05). It should be noted that basal levels of CD38 mRNA expression in control cells were extremely low, barely within the limits of detection using the assay we used.


Figure 1
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  		Fig. 1. Effects of CD38 small interfering RNA (siRNA; CD38si) on CD38 mRNA expression in human airway smooth muscle (ASM) cells. Treatment of (nontransfected) ASM cells with 20 ng/ml TNF{alpha} leads to significant increases in CD38 mRNA expression. However, transfection with CD38si blunts TNF{alpha}-induced increase in CD38 mRNA, with minimal effect in control cells (no TNF{alpha} stimulation). Nonspecific effects of CD38si on other proteins involved in store-operated Ca2+ entry (SOCE) were ruled out by lack of effect on transient receptor potential channel (TRPC)3 (Western analysis). Values are means ± SE. *Significant effect of TNF{alpha}, #significant effect of CD38si (P < 0.05).

 
The enhancing effect of TNF{alpha} treatment on CD38 mRNA expression was blunted by ~65% by prior transfection of the cells CD38si (Fig. 1; P < 0.05). In control cells, CD38si alone had no detectable effect on basal levels of CD38 mRNA expression (which were low to begin with). In contrast to CD38si, transfecting cells with siCon (nonspecific siRNA) had no effect on either basal CD38 mRNA expression or the elevation of CD38 mRNA expression induced by TNF{alpha} (Fig. 1). In cells exposed to CD38si, there was no significant change in TRPC3 protein expression (Fig. 1).

Effect of TNF{alpha} on ADP ribosyl cyclase activity. Compared with control ASM cells, TNF{alpha} treatment significantly increased ADP ribosyl cyclase activity (Fig. 2; P < 0.05). Transfection of ASM cells with CD38si significantly blunted TNF{alpha}-induced increase in ADP ribosyl cyclase activity (Fig. 2; P < 0.05) but had no detectable effect on cyclase activities in the absence of TNF{alpha} stimulation. Transfecting cells with siCon had no effect on basal ADP ribosyl cyclase activity or on TNF{alpha}-induced increase in ADP ribosyl cyclase activity (Fig. 2).


Figure 2
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  		Fig. 2. Effects of CD38si on ADP ribosyl cyclase activity. Treatment of human ASM cells with TNF{alpha} significantly increased ADP ribosyl cyclase activity (indirect measure of CD38 protein content; see METHODS for details) compared with control. The TNF{alpha} induced increase in ADP ribosyl cyclase activity was significantly attenuated by CD38si, with minimal effect in CD38si-transfected cells not exposed to TNF{alpha}. Values are means ± SE. *Significant effect of TNF{alpha}, #significant effect of CD38si (P < 0.05).

 
Effect of TNF{alpha} treatment on SOCE. Compared with control ASM cells, TNF{alpha} treatment resulted in significant enhancement of SOCE (Fig. 3; P < 0.05). Transfection of ASM cells with CD38si resulted in significant attenuation of TNF{alpha}-induced increase in SOCE (Fig. 3; P < 0.05). However, CD38si transfection alone (i.e., without TNF{alpha} stimulation) had no effect on SOCE (Fig. 3). Similarly, transfecting cells with siCon had no effect on either basal SOCE or TNF{alpha}-induced increase in SOCE (Fig. 3).


Figure 3
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  		Fig. 3. Effects of TNF{alpha} and CD38si on SOCE in human ASM cells. In all cell groups, depletion of sarcoplasmic reticulum Ca2+ in zero extracellular Ca2+ followed by rapid reintroduction of Ca2+ resulted in activation of SOCE (6, 58). Compared with controls, TNF{alpha} significantly enhanced SOCE. In contrast, CD38si significantly blunted TNF{alpha}-elicited increase in SOCE. Values are means ± SE. *Significant effect of TNF{alpha}, #significant effect of CD38si (P < 0.05).

 
Effect of CD38 overexpression. Transfecting human ASM cells with a vector containing the intact CD38 gene tagged to GFP resulted in a preponderance of ASM cells being fluorescent for GFP (excitation 488, emission 510 nm; Fig. 4). Furthermore, in these cells, there was a significant increase in CD38 mRNA expression compared with control cells (Fig. 4; P < 0.05). In addition, CD38 overexpression also significantly increased ADP ribosyl cyclase activity compared with controls (Fig. 5; P < 0.05). In CD38 overexpressing cells, TNF{alpha} treatment resulted in further enhancement of ADP ribosyl cyclase activity compared with cells exposed to TNF{alpha} alone. Finally, in CD38 overexpressing cells, TNF{alpha} treatment resulted in a significant augmentation of SOCE compared with both nontransfected controls as well as with TNF{alpha} exposure of nontransfected cells (Fig. 6; P < 0.05). CD38 overexpression by itself also increased SOCE (i.e., without TNF{alpha} stimulation) compared with controls.


Figure 4
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  		Fig. 4. CD38 overexpression in human ASM cells. Transfection with a green fluorescent protein (GFP)-tagged CD38 vector resulted in uptake of the vector in a majority of cells (B) compared with controls (A). Furthermore, vector transfection significantly increased CD38 expression as determined by RT-PCR (C). A DNA ladder was run in the left lane; the expected product size for the CD38 PCR product was 399 bp. In these cells, exposure to TNF{alpha} significantly enhanced CD38 expression as determined by RT-PCR (D). Values are means ± SE (bottom). *Significant effect of TNF{alpha} (P < 0.05).

 

Figure 5
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  		Fig. 5. Effect of CD38 overexpression on ADP ribosyl cyclase activity. Transfection with the CD38 vector significantly increased ADP ribosyl cyclase activity compared with control cells, indicating increased CD38 enzyme activity. Values are means ± SE. *Significant effect of TNF{alpha}, #significant effect of CD38 vector (P < 0.05).

 

Figure 6
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  		Fig. 6. Effect of CD38 overexpression on SOCE. In ASM cells transfected with CD38 vector, SOCE was significantly increased compared with controls. Furthermore, CD38 overexpression enhanced TNF{alpha}-induced increase in SOCE. Values are means ± SE. *Significant effect of TNF{alpha}, #significant effect of CD38 vector (P < 0.05).

 
Role of cADPR. To determine whether the effects of TNF{alpha} and/or CD38 on SOCE were mediated via the second messenger cADPR, additional experiments were performed in ASM cells where the cell-permeant cADPR antagonist 8-Br-cADPR (10 µM) was introduced into the extracellular medium following CPA in zero Ca2+ for at least 5 min (thus inhibiting cADPR action before triggering of SOCE). In control ASM cells (i.e., no CD38si, CD38 overexpression, or TNF{alpha}), 8-Br-cADPR had a less than 10% effect on SOCE (compared with cells not exposed to 8-Br-cADPR). In comparison, 8-Br-cADPR produced an ~30% reduction in TNF{alpha}-induced enhancement of SOCE (Fig. 7, P < 0.05). With CD38si (where SOCE was already decreased), 8-Br-cADPR had no significant effect with or without TNF{alpha} exposure. With CD38 overexpression (but without TNF{alpha} exposure), 8-Br-cADPR decreased SOCE by ~30% (P < 0.05; compared with CD38 overexpression alone). In comparison, 8-Br-cADPR blunted TNF{alpha}-induced enhancement of SOCE in CD38 overexpressing cells by ~40% (Fig. 7; P < 0.05).


Figure 7
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  		Fig. 7. Effect of cyclic ADP ribose (cADPR) antagonism on SOCE. In different groups, ASM cells were exposed to the cell-permeant cADPR antagonist 8-Br-cADPR (10 µM) before triggering of SOCE (see METHODS for details). In control ASM cells with normal levels of CD38, as well as CD38si cells with or without TNF{alpha} exposure, 8-Br-cADPR had minimal effect on SOCE. In comparison, 8-Br-cADPR significantly blunted TNF{alpha}-induced enhancement of SOCE in nontransfected as well as transfected cells. *Significant effect of 8-Br-cADPR (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrate a role for CD38 in the process of SOCE in human ASM cells. Treatment of ASM cells with the cytokine TNF{alpha} caused a significant increase in CD38 expression and ADP ribosyl cyclase activity. Furthermore, in the presence of TNF{alpha}, SOCE was significantly increased. siRNA knockdown of CD38 expression blunted TNF{alpha}-elicited increase in CD38 expression as well as SOCE. Conversely, CD38 overexpression increased CD38 expression by itself as well as the effect of TNF{alpha} on CD38, with potentiation of TNF{alpha} effects on SOCE. These data suggest that CD38 regulates SOCE in normal ASM cells, as well as with inflammation. Furthermore, our results using the cADPR antagonist 8-Br-cADPR suggest that the effects of CD38 on SOCE may be only partially mediated via cADPR, especially with TNF{alpha} exposure.

There is now considerable evidence for store-operated or capacitative Ca2+ entry in different smooth muscle types. The presence of store-operated Ca2+ influx pathways has been demonstrated previously in guinea pig, rat, human, and porcine airway smooth muscle (6, 34, 35, 48, 58). Previous studies from our group have established that SOCE exists in human ASM (6, 58). Furthermore, we demonstrated that 1) it is SR Ca2+ depletion that matters for triggering SOCE, regardless of whether the Ca2+ release occurs via IP3 receptor or RyR channels (6); 2) ASM expresses different TRPC channels that are thought to mediate SOCE; and 3) siRNA suppression of TRPC3 expression blunts SOCE, whereas suppression of other isoforms do not necessarily lead to altered SOCE (58). Thus the intracellular machinery for SOCE exists in human ASM. However, SOCE regulation in human ASM, especially under conditions of altered [Ca2+]i homeostasis as occurs with airway inflammation, is still being investigated. Sweeney et al. (48) demonstrated a role for SOCE in bronchial contraction and proliferation, proposing that SOCE may play an important role in airway constriction and remodeling with inflammation. We found that treatment of ASM with TNF{alpha} significantly increases SOCE (58), a finding we confirmed in the present study. In another study, we (5) reported that SOCE in ASM is modulated by cyclic nucleotides. Thus, there is increasing evidence that changes in SOCE may contribute to altered [Ca2+]i in ASM inflammation. The present study indicates that CD38, a key [Ca2+]i regulatory molecule already shown to be involved in ASM inflammation (14), also regulates SOCE, and may mediate the effect of inflammation on SOCE.

CD38 is a multifunctional ectoenzyme with ADP ribosyl cyclase, cADPR hydrolase, and NAD glycohydrolase activities (60). cADPR has been shown to modulate Ca2+ release from the SR of ASM through activation of RyR channels (39). Previous studies have shown that TNF{alpha} treatment of human ASM cells enhances contractile agonist-elicited [Ca2+]i responses (1, 4). While TNF{alpha} can potentially activate a number of intracellular mechanisms involved in ASM [Ca2+]i regulation (2, 3), recent studies indicate that the CD38/cADPR signaling pathway is involved in TNF{alpha}-induced increased responsiveness (14). For example, 8-bromo-cADPR, a cADPR antagonist, has been shown to reduce TNF{alpha}-augmented [Ca2+]i responses to agonists including acetylcholine, bradykinin, and thrombin (14). More recently, Kang et al. (24) confirmed a role for CD38 in TNF{alpha}-induced changes in Ca2+ responses using an adenoviral mediated antisense CD38 approach. In each of these studies, the presumption has been that TNF{alpha} is acting through the CD38/cADPR pathway to enhance Ca2+ release from the SR Ca2+ stores either directly or via enhanced calcium-induced calcium release (the "classic" view of cADPR action). Whether CD38 modulates other [Ca2+]i regulatory pathways remains to be determined. The current study suggests that CD38 indeed modulates Ca2+ influx. These data are consistent with studies in other tissues showing CD38 modulation of influx via different channels, although not necessarily SOCE. Indeed, only one other study in myometrial smooth muscle has demonstrated CD38 modulation of SOCE (54).

The mechanisms by which CD38 modulates SOCE were not specifically examined and are currently unknown. The original premise of our study was that CD38 directly modulates SOCE and was supported by the siRNA vs. overexpression studies. The lack of effect of CD38si (or overexpression) on TRPC3 channel expression suggests that as with airway inflammation, effects on channel expression may not be the mechanism. However, this does not rule out CD38 regulation of TRPC3 channels per se. This may occur within caveolae expressing CD38 and TRPC3, via direct complexing of CD38 and TRPC3, or through modulation of the recently recognized trigger for SOCE, stromal interaction molecule (STIM1) (34, 36, 40). Our additional studies showing partial blockade of SOCE (or of TNF{alpha} and CD38 overexpression effects) by 8-Br-cADPR suggest that at least some of the CD38 effects may be mediated via cADPR (which is produced by CD38 activation following agonist exposure). How cADPR may affect SOCE remains to be determined. Some studies on capacitative Ca2+ entry have proposed the idea of a diffusible Ca2+ influx factor that triggers SOCE (35). While STIM1 may be that protein, other proteins such as cADPR may play a role. These scenarios of CD38/cADPR mechanisms remain to be explored.

Previous studies have demonstrated the pivotal role of TNF{alpha} in airway inflammation and hyperreactivity (12, 28, 52). Treatment of severe asthmatics with soluble TNF{alpha} receptor IgG1 Fc fusion proteins appears to be effective (23). However, the specific targets in the lung affected by TNF{alpha} and the mechanisms of altered airway hyperreactivity are still under investigation. In this regard, studies have shown that exposure of ASM cells to TNF{alpha} leads to potentiation of [Ca2+]i responses to various contractile agonists including ACh and bradykinin (3, 4). Such responses may be mediated via effects on SR Ca2+ release as well as Ca2+ influx. We previously found that in ASM cells treated with TNF{alpha}, basal [Ca2+]i levels were increased compared with controls (58), an effect reversed by removal of extracellular Ca2+. While TNF{alpha} does affect Ca2+ influx, the specific channels typically present in ASM that may be involved remain to be determined. The present study suggests that SOCE is one such channel.

Studies in human ASM have already shown that CD38 expression increases with TNF{alpha} treatment (14, 58), again a finding confirmed in the present study. An interesting observation has been the magnitude of TNF{alpha}-induced increase in CD38 mRNA expression (~60-fold) compared with the relatively small increase in TRPC isoform expression for the same concentration and time period of cytokine exposure (~3-fold). Given that TNF{alpha} enhances SOCE, these comparisons (albeit qualitative) suggest that TNF{alpha} effects on SOCE are more likely to be mediated via its effects on SOCE regulation rather than effects on expression of channels mediating SOCE. Indeed, in the present study, we found that CD38 knockdown via siRNA substantially blunts TNF{alpha}-induced enhancement of SOCE. Conversely, overexpression of CD38 potentiates TNF{alpha} effects on Ca2+ influx via SOCE. The consistent effects of CD38 knockdown vs. overexpression on ADP ribosyl cyclase activities confirm the efficacy and specificity of the transfection techniques. Therefore, the present study supports the idea that CD38 not only modulates SOCE but also mediates the effect of TNF{alpha} on Ca2+ influx. In this regard, an interesting observation in this study was that enhancement of SOCE by CD38 overexpression alone (i.e., without TNF{alpha} stimulation) was not as dramatic as that in the presence of TNF{alpha}. This would suggest that CD38 may not be the only regulator of SOCE in the presence of TNF{alpha}. Certainly, TNF{alpha} is known to activate several intracellular signaling cascades that may, in turn, affect TRPC channels (2, 3). The roles of these cascades in TNF{alpha} effects on SOCE remain to be determined. An alternative mechanism may yet involve ADPR, an end product of the CD38/cADPR pathway. Recent evidence suggests that ADPR is involved in the gating of TRPM2 (37, 44), a Ca2+-permeable cation channel member of the TRP family, activated by ADPR and cADPR (9, 29, 37, 44). In ongoing experiments, we have found that human ASM expresses TRPM2, suggesting that the CD38/cADPR pathway may activate this channel. However, there is currently no direct evidence for TRPM2 in SOCE of ASM. Future studies should examine this aspect of SOCE regulation.

In conclusion, based on the present study as well as previous reports (58), we propose a model in which TNF{alpha} induces an increase in CD38 and TRPC3 expression. Agonist stimulation results in enhanced cADPR and ADPR production, leading to enhanced SR Ca2+ release and activation of SOCE, and thus an overall increase in [Ca2+]i.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Institutes of Health (NIH) Grants HL-74309 (G. C. Sieck) and 1-UL1-RR-024150-01* (Mayo Clinic Clinical Research awards to C. M. Pabelick and Y. S. Prakash from the National Center for Research Resources, a component of NIH, and the NIH Roadmap for Medical Research). This work is additionally supported by a Mayo Clinic Early Career Development Award to Y. S. Prakash.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical assistance of Dr. Sandra Soares.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology & Biomedical Engineering, 4-184 W Jos SMH, Mayo Clinic College of Medicine, Rochester, MN 55905 (e-mail: sieck.gary{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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