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Published as doi: 10.1096/fj.05-4585fje.
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(The FASEB Journal. 2006;20:1000-1002.)
© 2006 FASEB

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

Bit-Na Kang*, K. G. Tirumurugaan*, Deepak A. Deshpande*, Yassine Amrani§, Reynold A. Panettieri§, Timothy F. Walseth{dagger} and Mathur S. Kannan*,{ddagger},1

* Department of Veterinary and Biomedical Sciences,

{dagger} Pharmacology and

{ddagger} Pediatrics, University of Minnesota, St. Paul, Minnesota; and

§ Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

1Correspondence: Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Ave., St. Paul, MN 55108, USA. E mail: kanna001{at}umn.edu

ABSTRACT

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-{alpha}. In HASM cells, TNF-{alpha} augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-{alpha} increased NF-{kappa}B expression and its activation, and dexamethasone partially reversed these effects. TNF-{alpha} increased the expression of I{kappa}B{alpha}, and dexamethasone increased it further. An inhibitor of NF-{kappa}B activation or transfection of cells with I{kappa}B mutants decreased TNF-{alpha}-induced CD38 expression. The results indicate that TNF-{alpha}-induced CD38 expression involves NF-{kappa}B expression and its activation and dexamethasone inhibits CD38 expression through NF-{kappa}B-dependent and -independent mechanisms.— Kang, B.-N., Tirumurugaan, K. G., Deshpande, D. A., Amrani, Y., Panettieri, R. A., Walseth, T. F., Kannan, M. S. Transcriptional regulation of CD38 expression by tumor necrosis factor-{alpha} in human airway smooth muscle cells: role of NF-{kappa}B and sensitivity to glucocorticoids.


Key Words: cytokines • transcription factors • smooth muscle • airway • glucocorticoid

CD38 IS A ~45-KDA transmembrane glycoprotein expressed ubiquitously in many different cell types (1 2 3) . CD38 catalyzes the conversion of ß-NAD to cADPR, a calcium-mobilizing agent (4) . This conversion is catalyzed by the enzyme ADP-ribosyl cyclase that is an integral part of CD38. CD38 also catalyzes the hydrolysis of cADPR to ADPR, and this is catalyzed by the enzyme cADPR hydrolase. Thus, CD38 is widely considered as a bifunctional protein. In smooth muscle cells, there is now evidence to support the role of cADPR generated through CD38 in intracellular calcium mobilization during stimulation by contractile agonists (5 , 6) .

Although CD38 is constitutively expressed in smooth muscle cells and other cells, its expression has been shown to be highly regulated. In this regard, cytokines such as tumor necrosis factor-alpha (TNF-{alpha}), interleukin (IL)-1ß, IFN-{gamma}, and IL-13, estrogen, retinoic acid, and vitamin D3 have been shown to increase its expression in different cell types (5 6 7 8 9 10) . Our earlier findings demonstrated that the inflammatory cytokines TNF-{alpha}, IL-1ß, and IFN-{gamma} and the Th-2 cytokine IL-13 augment CD38 expression in airway smooth muscle cells (ASM; refs 6 , 8 ). This resulted in increased intracellular calcium responses to agonists. The augmented calcium responses were sensitive to an antagonist of cADPR, indicating a contribution of the CD38/cADPR signaling to cytokine-induced hyperresponsiveness. These results also indicated that CD38 may have a role in ASM hyperresponsiveness in inflammatory diseases such as asthma.

Inflammatory mediators such as TNF-{alpha} play a critical role in the pathogenesis of airway hyperresponsiveness (11) . Studies have shown that TNF-{alpha} acts directly on ASM cells, resulting in structural and functional changes (12) . Furthermore, activation of transcription factors such as NF-{kappa}B has been found to be important in mediating the effects of TNF-{alpha} on ASM (13) and other cell types (14 , 15) . However, the mechanisms by which TNF-{alpha} augments CD38 expression in ASM are not understood. Therefore, an objective of the present study was to determine the role of NF-{kappa}B in TNF-{alpha}-mediated CD38 expression in ASM cells.

Glucocorticoids are widely used in the prevention and management of asthma. The mechanisms of action of glucocorticoids are complex and involve the participation of a variety of transcription factors, including NF-{kappa}B (16 17 18 19 20) . With respect to the cd38, earlier studies (21) have described a glucocorticoid response element, thus providing a basis for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression in ASM cells stimulated with TNF-{alpha}.

MATERIALS AND METHODS

Tris base, glucose, HEPES, TNF-{alpha}, dexamethasone, and other chemicals were purchased from Sigma Chemical (St. Louis, MO). Hanks’ balanced salt solution (HBSS) and Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco-BRL (Grand Island, NY). RNeasy mini kit was obtained from Qiagen (Valencia, CA). The SYBR Green Master mix was purchased from Stratagene (Cedar Creek, TX). Superscript III reverse transcriptase, TaqDNA polymerase, and the 100-base pair (bp) DNA ladder were purchased from Invitrogen (Carlsbad, CA). Gel shift assay kit was purchased from Promega (Madison, WI). Antibodies against the p65 (sc-109) and p50 (sc-1191) subunits of NF-{kappa}B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Basic Nucleofector kit for primary smooth muscle was purchased from Amaxa Inc. (Gaithersburg, MD).

Cell culture
Human airway smooth muscle (HASM) cells maintained in culture were used in this study. HASM cells were isolated from trachealis muscle and propagated as described earlier (22) . The cells were plated at a density of 1.0 x 104 cells/cm2 and were cultured in DMEM supplemented with 10% FBS, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.25 µg/ml of amphotericin B. For studies described below, HASM cells were growth-arrested at Go of cell cycle by maintaining for at least 48 h in arresting medium containing no serum, but in the presence of transferrin and insulin, as described previously (23) .

Reverse transcription-polymerase chain reaction
CD38, p50 subunit of NF-{kappa}B, and I{kappa}B{alpha} mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Total cellular RNA was isolated from control, TNF-{alpha} treated cells, and cells treated with TNF-{alpha} in the presence of various concentrations of dexamethasone using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. RNA was quantified using a spectrophotometer, and equal amounts from the different samples were used for the RT-PCR. The RT reaction was carried out using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), as per manufacturer’s instructions.

Primers used for the studies
ß-Actin primers that produced a ~250 bp product were used as an internal control and nontemplate control was also used. The PCR was performed under the following conditions: 94°C for 3 min denaturing, 30 cycles of 94°C for 30 s, 50–58°C (50°C for I{kappa}B{alpha}, 55°C for CD38 and 58°C for p50) for 30 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.

Quantitative real-time PCR
Quantitative real-time PCR was performed using the SYBR green PCR master mix (Stratagene, Cedar Creek, TX). cDNA obtained by reverse transcription of total RNA from the different samples was amplified using the SYBR green master mix and CD38, p50, I{kappa}B{alpha}, or ß-actin specific primers (Table 1 ). The reactions were performed in the Stratagene Mx3000p sequence detection system under the following conditions: 94°C for 3 min, 45 cycles at 94°C for 30 s, 50°C for I{kappa}B{alpha}, 55°C for CD38 and 58°C for p50 for 30 s, and 72°C for 45 s. All samples were run in duplicates, and the readings were normalized using nontemplate control and passive reference dye included in the SYBR Green Master mix. Normalized fluorescence was plotted against the cycle number (amplification plot), and the threshold suggested by the software was used to calculate Ct (cycle at threshold). Real-time PCR results were expressed as fold change (2–{Delta}{Delta}CT) in expression in the cells after subtraction of internal ß-actin control and expressed relative to the levels in untreated control cells.


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  		Table 1. Specific primers

Measurement of ADP-ribosyl cyclase activity
The CD38 protein content in the HASM cell lysates was assessed by measuring the ADP-ribosyl cyclase activity. The ADP-ribosyl cyclase activity was determined by a competitive binding assay employing 3-deaza-NAD as the substrate. HASM cells were sonicated,and the homogenate was incubated with 3-deaza-NAD (final concentration of 65.3 µM) for 2 h at room temperature. The reaction was stopped by adding an equal volume of 100 mM HCl. The amount of 3-deaza-cADPR formed in this step was determined using a competitive binding assay using sea urchin egg homogenates as a source of cADPR binding protein and [32P]3-deaza-cADPR as the radioligand. The binding reaction was incubated at 4°C for 15 min, and the amount of [32P]3-deaza-cADPR bound was determined using 96-well GF/C filtration plates (Millipore, Bedford, MA). After each well was washed with 15% polyethylene glycol to remove any free radioligand, the filter plate was exposed to a phosphorimage screen for 16 to 24 h and the results were read using a Packard Cyclone phosphorimager. A standard curve was developed employing the displacement of [32P]3-deaza-cADPR with known concentrations of unlabeled 3-deaza cADPR. The specific activities of the ADP-ribosyl cyclase were expressed as fmoles of 3-deaza-cADPR/mg protein/min.

Nuclear protein extraction
Nuclear extracts were prepared by following a modified procedure based on previously described protocol (24) . After being harvested from the culture dishes, the cells were washed with ice-cold PBS before resuspending in ice-cold lysis buffer (10 mM Tris-HCl, pH 8, 60 mM KCl, 1 mM EDTA, 0.5 x Nonidet P-40, 1 mM DTT, 1 mM PMSF, and 2 µg/ml leupeptin and aprotinin). Nuclei were separated from the cytoplasm by centrifugation at 200 g for 5 min at 4°C. The resulting pellets were resuspended in nuclear extraction buffer (20 mM Tris-HCl, pH 8, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol) for 40 min on ice. The suspensions were centrifuged at 20,000 g for 30 min at 4°C, and the supernatants harvested as nuclear extracts and stored at –80°C.

Electrophoretic mobility shift assay
Protein concentration in the nuclear extract was quantitated using the Brad Ford protein assay (Bio-Rad, Hercules, CA). The double-stranded oligonucleotide containing the consensus binding sites for NF-{kappa}B (-5'-AGTTGAGGGGACTTTCCCAGGC-3') was labeled with [{gamma}-32P]ATP (3.00 Ci/mmol at 10 mCi/ml) by T4 Polynucleotide Kinase (Promega, Madison, WI). Nuclear extracts (2 µg) were incubated for 30 min at room temperature with 0.4 pmol of double-stranded 32P-labeled oligonucleotide containing the consensus binding sites for NF-{kappa}B in a total volume of 10 µl in a buffer containing 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 0.25 mg/ml poly (dI-dC). After 30 min at room temperature, samples were separated on a nonreducing 4% polyacyrlamide gel using TBE buffer (0.5 M, containing 107.8 g Tris base, 55 g boric acid, and 7.44 g disodium EDTA · 2H2O in 1 l of water). The gels were dried and autoradiographed with intensifying screens at –70°C. To confirm specificity of the EMSA, competition assays were performed with a 100-fold excess of unlabeled NF-{kappa}B probe or the activating protein (AP)-2 probe as a nonspecific competitor. For the gel supershift assay, 1 µl of antibodies specific for the p65 (sc-109) or p50 (sc-1191) subunits of NF-{kappa}B was incubated with the nuclear extracts 30 min before the addition of the oligonucleotide probe.

Western blot detection of the NF-{kappa}B subunits
For Western blot detection of the NF-{kappa}B subunits, nuclear extracts with equal amount of protein were fractionated on 4–15% gradient gels and the proteins were transferred electrophoretically onto a polyvinylidene difluoride (PVDF) membrane. For immunostaining, the membranes were blocked in PBS containing 1% milk diluent (KPL, Gaithersburg, MD) and 0.05% Tween-20 for 30 min. Membranes were incubated first with goat anti-human p50 (SC1191) polyclonal antibody (pAb) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by donkey anti-goat IgG-conjugated with horseradish peroxidase for 1 h. The membrane was stripped and reprobed with goat anti-human p65 pAb (sc-109), followed by donkey anti-goat IgG-conjugated with horseradish peroxidase for 1 h. The signals were visualized on X-ray film after the application of chemiluminescence SuperSignal West Dura Extended Duration Substrate (Pierce Chemical, Rockford, IL).

Electroporation of I{kappa}B{alpha} mutants
TNF-{alpha}-induced CD38 expression was determined by transient transfection of HASM cells with I{kappa}B{alpha} mutants using the Amaxa nucleofection technology (Amaxa, Gaithersburg, MD) and the Basic Smooth Muscle Kit (VPI-1004). A transfection efficiency of ~70% with a cell viability of >80% was standardized with the program D-33 and 2 µg of pmaxGFP Vector. Deletion mutants of inhibitor {kappa}B (I{kappa}B)-{alpha} encoding amino acids 37–317 (I{kappa}Ba{Delta}normal) and 1–242 (I{kappa}B{alpha}{Delta}C) and a dominant negative I{kappa}B kinase (IKK)-ß with Ser-177 and Ser-181 replaced by alanine (IKK-AA) in pCMV4 were used for the studies (kindly provided by Dr. Marc Hershenson, University of Michigan, Ann Arbor, MI). After electroporation, the cells were maintained in growth medium overnight, growth arrested for 8 h and then exposed to 20 ng/ml TNF-{alpha} for additional 24 h. In each experiment, the electroporated samples were split into two different wells and one sample was used as control and another for treatment. Each experiment was repeated at least three times.

Role of NF-{kappa}B in TNF-{alpha}-induced CD38 expression and function
First, HASM cells growth-arrested for 48 h were exposed to an inhibitor of NF-{kappa}B (SN-50 at 18 µM final concentration) or the control peptide (SN-50M, 18 µM final concentration). The concentration of the inhibitory peptide was chosen based on published reports (25) . One hr following addition of the peptides, the cells were exposed to either the vehicle or 50 ng/ml of TNF-{alpha} for an additional 22 h. Nuclear extracts were prepared for detection of NF-{kappa}B activation by Western blot analysis as well as by EMSA. CD38 expression and ADP-ribosyl cyclase activity were determined as described above. Second, TNF-{alpha}-induced CD38 expression was determined by RT-PCR, as described above, in cells transfected with I{kappa}B{alpha} mutants.

Effect of dexamethasone pretreatment on TNF-{alpha}-induced CD38 expression and function in HASM cells: Role of NF-kB activation
HASM cells growth arrested at G0 of cell cycle were treated with different concentrations of dexamethasone for 1 h, followed by TNF-{alpha} (50 ng/ml) or vehicle (PBS containing 0.1% BSA) for 22 h. At the end of the incubation period, the expressions of CD38, NF-{kappa}B, and I{kappa}B{alpha} were measured by RT-PCR, followed by quantitative real-time PCR. ADP-ribosyl cyclase activity was measured by competitive binding assay and NF-{kappa}B activation was determined by Western blot analysis as described above. In other experiments, cells were treated with TNF-{alpha} (10 ng/ml) for 1 h in the presence or absence of different concentrations of dexamethasone. NF-{kappa}B activation was measured in the nuclear extracts using EMSA, as described above.

Effect of dexamethasone on CD38 expression after exposure to TNF-{alpha}
In the first set of experiments, HASM cells growth-arrested for 48 h were exposed to TNF-{alpha} for 22 h and the medium was replaced with fresh arresting medium with or without 1 µM dexamethasone. After an additional 24 and 48 h of incubation, the cells were processed for determination of CD38 expression by RT-PCR and ADP-ribosyl cyclase activity using the binding assay as described above. In the second set of experiments, HASM cells growth-arrested for 48 h were exposed to TNF-{alpha} for 22 h and the medium was replaced with fresh medium containing TNF-{alpha} or medium alone either in the absence or presence of 1 µM dexamethasone for an additional 24 and 48 h period. The cells were harvested for determination of CD38 expression and ADP-ribosyl cyclase activity.

Statistical analysis
HASM cells isolated from at least three different donors were used in the experiments. The experiments involving EMSA and transfections of the I{kappa}B{alpha} mutant constructs were repeated three times. The quantitative PCR results and ADP-ribosyl cyclase activities in the various samples were compared by one-way ANOVA with Bonferroni’s test for multiple comparisons. Statistical analyses were done using the GraphPad PRISM statistical software. Two means were considered significantly different when P value was <0.05.

RESULTS

Role of NF-{kappa}B in TNF-{alpha}-induced CD38 expression
To determine the role of NF-{kappa}B in TNF-{alpha}-induced augmented CD38 expression, HASM cells were pretreated with SN-50 (18 µM), an inhibitor of NF-{kappa}B translocation to the nucleus, or the control peptide SN-50M (18 µM). The cells were subsequently exposed to 50 ng/ml TNF-{alpha} for 30 min or 22 h. Nuclear extracts prepared from the cells were fractionated on 4–15% SDS gradient gels, and the separated proteins were transferred to PVDF membranes for Western blot detection of the p50 or the p65 subunit of NF-{kappa}B. In HASM cells maintained in arresting medium, there were no detectable NF-{kappa}B subunits in the nuclear extract. Exposure to TNF-{alpha} for 30 min (data not shown) or 22 h resulted in NF-{kappa}B activation, which was significantly (P<0.05) inhibited by pretreatment with SN-50 (Fig. 1 A). In the presence of SN-50, the TNF-{alpha}-induced augmented CD38 expression was significantly (P<0.05) inhibited (Fig. 1B ). In the presence of the control peptide, TNF-{alpha}-induced NF-{kappa}B activation and CD38 expression were reduced, although the reduced expression levels did not reach statistical significance (Fig. 1) .


Figure 1
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  		Figure 1. A) Representative Western blot image showing the presence of p50 subunit of NF-{kappa}B in the nuclear extracts prepared from HASM cells. C: control cells; TNF-{alpha}: cells treated with 50 ng/ml TNF-{alpha} for 22 h; SN50: cells pretreated with 18 µM SN50 followed by 50 ng/ml TNF-{alpha} for 22 h; and SN50M: cells pretreated with 18 µM SN50M followed by 50 ng/ml TNF-{alpha} for 22 h. Note decreased amount of p50 in nuclear extracts prepared from SN50-treated cells indicating decreased NF-{kappa}B activation. Nuclear actin is shown as an internal control; n = 3. B) Results of quantitative real-time PCR shown as fold change in CD38 mRNA expression in HASM cells. Note significant (P<0.05) attenuation of CD38 expression in cells pretreated with SN50; n = 3. C) Results of transient transfection using the various I{kappa}B mutants on TNF-{alpha}-induced CD38 expression in HASM cells. Lanes 1 and 2: untreated and TNF-{alpha}-treated cells after electroporation; lanes 3 and 4: cells transfected with the vector alone; lanes 5 and 6: cells transfected with the N-terminal deletion mutant; lanes 7 and 8: cells transfected with the C-terminal deletion mutant; and lanes 9 and 10: cells transfected with the IKK dominant negative mutant. Note attenuation of TNF-{alpha}-induced CD38 expression in the presence of N-terminal deletion and IKK dominant negative mutants and increased expression in the presence of the C-terminal deletion mutant. Representative of 3 experiments.

NF-{kappa}B activation was also confirmed by EMSAs using nuclear extracts from cells and consensus NF-{kappa}B binding sequence. NF-{kappa}B binding was increased after treatment with TNF-{alpha} for 1 h (Fig. 2 C). In the presence of SN-50 or the control peptide SN-50M, there was significant inhibition of NF-{kappa}B binding after treatment with TNF-{alpha}. Supershift analysis using anti-p65 or anti-p50 antibodies confirmed the specificity of the NF-{kappa}B complexes (Fig. 2C ).


Figure 2
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  		Figure 2. A) Dexamethasone inhibits TNF-{alpha}-induced CD38 mRNA expression in HASM cells. Results of quantitative real-time PCR shown as fold change in CD38 mRNA expression in control cells and in cells treated with 50 ng/ml TNF-{alpha} for 22 h alone, or TNF-{alpha} in the presence of 0.01, 0.1, and 1 µM dexamethasone. C + D refers to cells exposed to 1 µM dexamethasone. Different letters denote significant (P<0.05) differences between treatments in this and other figures. Note significant inhibition of TNF-{alpha}-induced CD38 expression in the presence of 0.1 and 1 µM dexamethasone; n = 5. Values are mean ± SE in this and other figures. B) ADP-ribosyl cyclase activity in HASM cells. ADP-ribosyl cyclase activity was measured in cell lysates with a competitive binding assay using sea urchin egg microsomes (see Materials and Methods). Note significant (P<0.05) enhancement of ADP-ribosyl cyclase activity in lysate obtained from TNF-{alpha}-stimulated cells. Dexamethasone pretreatment resulted in inhibition of ADP-ribosyl cyclase activity only at a concentration of 1 µM; n = 5. C) Nuclear extracts were prepared from HASM cells and assayed for NF-{kappa}B binding to 32P end-labeled NF-{kappa}B oligonucleotide probe by EMSA. Effect of different concentrations of dexamethasone pretreatment (1 h) on TNF-{alpha} (10 ng/ml)-induced NF-{kappa}B activation was also measured. Specificity of NF-{kappa}B binding was determined using 100-fold excess of unlabeled probe as well as a nonspecific competitor (activating protein-2). Specificity of NF-{kappa}B complex is demonstrated by gel supershift using p65 or the p50 antibody. Note TNF-{alpha}-induced NF-{kappa}B activation is decreased by 1 µM dexamethasone as well as by the NF-{kappa}B inhibitory peptides (SN-50 and SN-50M). Representative of 3 assays.

To further confirm the role of NF-{kappa}B in TNF-{alpha}-induced CD38 expression, HASM cells transfected with the various I{kappa}B{alpha} mutants were used to decrease NF-{kappa}B activation. In cells transiently transfected with I{kappa}B{alpha}{Delta}normal or the IKK-AA constructs, TNF-{alpha}-induced CD38 expression was reduced as compared to levels in cells transfected with the I{kappa}B{alpha}{Delta}C construct (Fig. 1C ).

Dexamethasone decreases TNF-{alpha}-induced CD38 expression and function and NF-{kappa}B activation in human ASM cells
We measured CD38 expression and ADP-ribosyl cyclase activity in TNF-{alpha}-treated HASM cells in the presence of different concentrations of dexamethasone. CD38 mRNA expression was determined by RT-PCR followed by quantitative real-time PCR. In HASM cells treated with TNF-{alpha} for 22 h, there was a significant increase (P<0.05) in CD38 expression compared to controls. There was a significant (P<0.05) attenuation of TNF-{alpha}-induced augmented CD38 expression at 0.1 and 1 µM but not at 0.01 µM of dexamethasone. Figure 2A shows the effect of different concentrations of dexamethasone on TNF-{alpha}-induced CD38 expression. Dexamethasone had no effect on the constitutive expression of CD38.

To measure CD38 protein content, ADP-ribosyl cyclase activity was determined in whole cell lysates from HASM cells. ADP-ribosyl cyclase activity was ~50% higher (P<0.05) in the whole cell lysate fraction obtained from HASM cells treated with TNF-{alpha} than in controls (Fig. 2B ). The TNF-{alpha}-induced increased ADP-ribosyl cyclase activity was lower in cells pretreated with 1 µM dexamethasone but not in cells treated with lower concentrations of dexamethasone.

TNF-{alpha}-induced NF-{kappa}B activation was also determined in the presence of different concentrations of dexamethasone by EMSA (see Fig. 2C ). There was a decrease in NF-{kappa}B activation in the presence of 1 µM but not in the presence of the lower concentrations of dexamethasone.

We also determined NF-{kappa}B activation and p50 mRNA expression in cells after exposure to TNF-{alpha} both in the absence and presence of dexamethasone. TNF-{alpha} treatment of cells for 22 h caused an increase in p50 mRNA compared to the untreated controls (Fig. 3 A). Pretreatment of the cells with dexamethasone resulted in inhibition of p50 expression only at 1 µM concentration. We measured NF-{kappa}B activation by detection of the p50 and p65 subunits in the nuclear extracts in cells treated with TNF-{alpha} in the presence of dexamethasone. As shown in Fig. 3B , dexamethasone treatment resulted in attenuation of TNF-{alpha}-induced NF-{kappa}B activation.


Figure 3
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  		Figure 3. A) Results of quantitative real-time PCR shown as fold change in expression of p50 mRNA in HASM cells. C, cells treated with TNF-{alpha} (T), cells treated with 0.01, 0.1, or 1.0 µM dexamethasone, and control cells treated with 1 µM dexamethasone. Note significant (P<0.05) attenuation of expression only in the presence of 1 µM dexamethasone. B) Representative Western blot image showing the presence of p50 and p65 subunits of NF-{kappa}B in nuclear extracts prepared from HASM cells. C: control; T: treated with 50 ng/ml TNF-{alpha} for 22 h; 1D+T: pretreated with 1 µM dexamethasone followed by 50 ng/ml TNF-{alpha} for 22 h; 1D: treated with 1 µM dexamethasone alone. Note inhibition of TNF-{alpha}-induced NF-{kappa}B activation in the presence of dexamethasone; n = 3. C) I{kappa}B{alpha} expression in HASM cells. Top) Results of quantitative real-time PCR shown as fold change in expression of I{kappa}B{alpha} mRNA in HASM cells. Labels below bars correspond to treatment conditions described in A. Note increased I{kappa}B{alpha} expression in the presence of TNF-{alpha} and greater expression in the presence of TNF-{alpha} and dexamethasone; n = 3.

The expression of I{kappa}B{alpha} after exposure to TNF-{alpha} was also measured in cells pretreated with different concentrations of dexamethasone. I{kappa}B{alpha} expression was significantly (P<0.05) greater in cells treated with TNF-{alpha} than in controls. In cells treated with dexamethasone in the presence of TNF-{alpha}, the I{kappa}B{alpha} expression was greater than in cells treated with TNF-{alpha} alone (Fig. 3C ).

Effect of dexamethasone on CD38 expression after exposure to TNF-{alpha}
In HASM cells exposed to TNF-{alpha} for 48 or 72 h, CD38 expression was significantly (P<0.05) higher than in cells exposed to TNF-{alpha} for 22 h (Fig. 4 A). After an initial treatment with TNF-{alpha} for 22 h, withdrawal of TNF-{alpha} resulted in a significant decline in CD38 expression at 24 and 48 h. In the presence of 1 µM dexamethasone, the CD38 expression was significantly lower at 24 h, but not at 48 h, after removal of TNF-{alpha}. The ADP-ribosyl cyclase activity in the lysates obtained from the cells after exposure to TNF-{alpha} for 48 or 72 h was ~500% higher than in lysates obtained from cells exposed to TNF-{alpha} for 22 h. The ADP-ribosyl cyclase activity determined at 24 and 48 h after removal of TNF-{alpha} was significantly attenuated compared to activity in samples before removal of TNF-{alpha}, although statistical significance was reflected only at 24 h (Fig. 4B ). In the presence of dexamethasone, the ADP-ribosyl cyclase activity in the cells was further attenuated at 24 and 48 h after removal of TNF-{alpha} than in cells not treated with dexamethasone. Dexamethasone had no effect on ADP-ribosyl cyclase activity in control cells, i.e., not treated with TNF-{alpha}.


Figure 4
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  		Figure 4. A) Results of quantitative real-time PCR shown as fold change in CD38 mRNA expression in control cells (1) ; cells treated with 50 ng/ml TNF-{alpha} for 48 h (2) ; cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium for 24 h (3) ; cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium containing 1 µM dexamethasone for 24 h (4) ; cells treated with 50 ng/ml TNF-{alpha} for 72 h (5) ; cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium for 48 h (6) ; cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium containing 1 µM dexamethasone for 48 h (7) ; and control cells treated with 1 µM dexamethasone for 48 h (8) . Note significant (P<0.05) attenuation of CD38 expression on removal of TNF-{alpha}. Dexamethasone inhibits CD38 expression in cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium for 24 h; n = 4. B) ADP-ribosyl cyclase activity in HASM cells. Numbers below bars correspond to treatment conditions described in A. Note exposure of cells to TNF-{alpha} for 48 (2) or 72 h (5) results in significantly greater ADP-ribosyl cyclase activity than in controls (1) or in cells exposed to TNF-{alpha} for 22 h (see Fig. 1B ). Dexamethasone inhibits ADP-ribosyl cyclase activity in cells treated with 50 ng/ml TNF-{alpha} for 24 h and returned to arresting medium for 48 h; n = 6.

DISCUSSION

In this study, we have demonstrated that TNF-{alpha}-induced augmented CD38 expression in HASM cells requires NF-{kappa}B activation. This conclusion is supported by the following findings: pretreatment of the cells with an inhibitor or transient transfection of cells with I{kappa}B mutants to decrease NF-{kappa}B activation attenuated CD38 expression. In addition, TNF-{alpha}-induced CD38 expression was attenuated by dexamethasone and this inhibition was concentration dependent. However, dexamethasone inhibited NF-{kappa}B activation only at the highest concentration used. In cells initially exposed to TNF-{alpha} for 22 h, there was a decline in CD38 expression and ADP-ribosyl cyclase activity over time on withdrawal of TNF-{alpha}. This decline was greater in the presence of dexamethasone than in its absence. These results suggest a slow turnover of CD38 in HASM cells. Dexamethasone inhibited TNF-{alpha}-induced expression of mRNA for the p50 subunit of NF-{kappa}B. TNF-{alpha} increased the expression of I{kappa}B{alpha}, and in the presence of dexamethasone there was a greater increase in its expression. The results indicate that in HASM, CD38 expression is regulated by TNF-{alpha} through NF-{kappa}B. The inhibitory effects of dexamethasone on TNF-{alpha}-induced CD38 expression are consistent with inhibition of expression of NF-{kappa}B and its activation, the latter through increased I{kappa}B{alpha} expression. However, NF-{kappa}B-independent effects of dexamethasone in the regulation of CD38 expression cannot be ruled out.

Inflammatory cytokines such as TNF-{alpha}, IL-1ß, and IFN-{gamma} play an important role in diseases such as asthma (11 , 26 , 27) . Previous investigations have demonstrated that the levels of inflammatory cytokines are elevated in the bronchoalveolar lavage fluid obtained from asthmatic subjects (11 , 27) . TNF-{alpha} has been shown to increase the expression of a variety of genes resulting in functional changes in the resident cells such as ASM cells (13 , 28) . The effects of TNF-{alpha} involve the transcription factor NF-{kappa}B (15) . However, other transcription factors such as AP-1 are also involved in the effects of TNF-{alpha} (29 , 30) . Recent investigations (6 , 8 , 31) from our laboratory have shown that the inflammatory cytokines increase the expression of CD38 in HASM. In the present study, we demonstrate that TNF-{alpha} causes NF-{kappa}B expression and its activation. Inhibition of NF-{kappa}B activation results in attenuation of CD38 expression. However, there is significant CD38 expression in response to TNF-{alpha} in the presence of an inhibitor of NF-{kappa}B activation. Transcription factors such as AP-1 are also shown to be involved in mediating the effects of TNF-{alpha}. Analysis of the promoter region of CD38 reveals the presence of binding sites for different transcription factors, although NF-{kappa}B binding motifs have not been described previously (21) . These studies indicate that NF-{kappa}B effects on CD38 expression may be secondary to the primary response genes. Previous studies from our laboratory have demonstrated that TNF-{alpha}-induced CD38 expression in ASM cells involves TNFR1-associated signaling pathways and the autocrine action of IFNß that is generated in response to TNF-{alpha} (32) . Thus, the induction of CD38 expression by TNF-{alpha} may involve regulation by multiple transcription factors such as IFN regulatory factor-1, NF-{kappa}B, and others.

Glucocorticoids are used extensively as anti-inflammatory therapy in asthma. The mechanisms of action of glucocorticoids are complex and have been studied extensively (33) . The genomic effects of glucocorticoids in regulating the expression of a multitude of genes involve the glucocorticoid receptor (19) . The nuclear translocation of the glucocorticoid-receptor complex and its binding to specific DNA motifs results in both transactivation and repression of a variety of genes (17 18 19 , 34) . In this context, analysis of the promoter region of cd38 reveals the presence of binding sites for different transcription factors (21) and response elements for steroid hormones, cytokines, retinoic acid, and other agents (9 , 21) . In this context, cloning and sequence analysis of the 5'-untranslated region of CD38 from our laboratory reveal a putative NF-{kappa}B binding site, four glucocorticoid response elements (GREs) and binding sites for other transcription factors (Acc. No. DQ091293). Presence of the GREs provides a basis for transcriptional regulation of CD38 expression. The glucocorticoid-receptor complex is known to interfere with NF-{kappa}B binding to DNA (16 , 20) , thereby decreasing the expression of genes that are regulated by this transcription factor (17 , 18) . In the present study, we demonstrate inhibition of NF-{kappa}B activation by dexamethasone in cells exposed to TNF-{alpha}. The effects of dexamathasone are consistent with regulation of NF-{kappa}B expression, since it inhibits TNF-{alpha}-induced NF-{kappa}B expression. In addition, dexamethasone increases I{kappa}B{alpha} expression, which should result in decreased NF-{kappa}B activation due to increased nuclear export of the NF-{kappa}B-I{kappa}B{alpha} complex (35 , 36) . These effects of dexamethasone should result in decreased NF-{kappa}B binding to DNA. This mechanism of regulation of NF-{kappa}B activation has been described in other cell systems (18 , 37) . An interesting observation of the present study is that TNF-{alpha} increased the expression of I{kappa}B{alpha} while at the same time increasing the nuclear translocation of NF-{kappa}B. A previous report in alveolar epithelial cells has shown that TNF-{alpha} can induce the expression of not only genes involved in inflammation but also the genes that silence the NF-{kappa}B pathway (38) . The presence of the NF-{kappa}B response element in the promoter region of I{kappa}B{alpha} gene would favor increased I{kappa}B{alpha} expression during activation by TNF-{alpha}, thus modulating the expression of NF-{kappa}B-dependent transcription (14 , 16) . In this study, dexamethasone inhibited CD38 expression and increased I{kappa}B{alpha} expression at a concentration where it had minimal, if any, effect on NF-{kappa}B expression or its activation. These findings are consistent with both a NF-{kappa}B-dependent and -independent mechanisms in the regulation of CD38 expression. The NF-{kappa}B-independent mechanisms may involve transcriptional regulation through GRE and inhibition of NF-{kappa}B activation by direct protein-protein interaction, referred to as transcriptional crosstalk (17 , 18 , 34) . In this context, evidence for transcriptional regulation via crosstalk with other transcription factors in the actions of glucocorticoids has been presented (34) . These and other studies reveal direct interactions between the glucocorticoid receptor and transcription factors (39) , such as NF-{kappa}B (16 , 40 , 41) , activator protein-1 (34 , 42 43 44 45) , and signal transducers and activators of transcription (46 , 47) . Further studies are required to clearly delineate the role of other transcription factors and the mechanisms by which glucocorticoids regulate CD38 expression and its function in ASM cells.

In conclusion, we have shown that TNF-{alpha}-induced CD38 expression in HASM cells requires NF-{kappa}B activation and is attenuated by dexamethasone. The effects of glucocorticoid on TNF-{alpha}-induced CD38 expression involve decreased NF-{kappa}B expression and increased I{kappa}B{alpha} expression, resulting in inhibition of NF-{kappa}B activation. However, other mechanisms of regulation cannot be ruled out. Figure 5 shows a model that incorporates the principal findings reported in this study. The CD38/cyclic ADP-ribose signaling contributes to normal (N) airway function (5) and to cytokine-mediated ASM hyperresponsiveness (6 , 8 , 31) . Therefore, elucidation of how this signaling pathway is regulated in inflammatory airway diseases such as asthma is important. Future investigations must address whether regulation of CD38 expression in HASM muscle cells by NF-{kappa}B results from binding to motifs within the CD38 gene (as shown in Fig. 5B ) or secondarily through regulation of NF-{kappa}B-dependent genes (as shown in Fig. 5A ).


Figure 5
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  		Figure 5. A model showing the regulation of CD38 expression in HASM by the inflammatory cytokine TNF-{alpha} and glucocorticoids. Activation of TNF receptor 1 (TNFR1) by TNF causes receptor trimerization, recruitment of an adaptor protein complex and a downstream signaling cascade, resulting in I{kappa}B phosphorylation, dissociation of the p50/p65 subunits of NF-{kappa}B and their nuclear translocation to activate transcription of NF-{kappa}B-dependent genes. Two possible modes of transcriptional regulation are shown. A) Binding of the p50/p65 subunits of NF-{kappa}B to response elements activates transcription of NF-{kappa}B-dependent genes (including I{kappa}B{alpha}); B) Binding resulting in transactivation of cd38. Glucocorticoid (GC) binding to its receptor (GR) causes dissociation of GR/heat shock protein complex, nuclear translocation of GR homodimer, and binding to glucocorticoid response element (GRE). This results in transactivation (e.g., I{kappa}B{alpha}) or transrepression (e.g., NF-{kappa}B and cd38) (as shown in A and B). Model also shows direct protein-protein interaction involving the GCR (GC bound to its receptor)/NF-{kappa}B, which may result in mutual repression.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (HL057498 to M. S. Kannan, DA11806 to T. F. Walseth, and HL55301 to R. A. Panettieri). Current address for D. A. Deshpande: Department of Medicine, Wake Forest University Medical School, Winston-Salem, NC. The authors thank Drs. Michael Murtaugh, Mitchell Abrahamsen and Douglas Foster for helpful discussions. We thank Dr. Herhsenson for providing the I{kappa}B mutants used in the study.

Received for publication July 20, 2005. Accepted for publication December 2, 2005.

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