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CD38 Expression Is Insensitive to Steroid Action in Cells Treated with Tumor Necrosis Factor- and Interferon- by a Mechanism Involving the Up-Regulation of the Glucocorticoid Receptor Isoform

Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania (O.T., Y.A.); and Molecular Endocrinology Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (J.A.C.)

Received October 6, 2005; accepted November 16, 2005

	Abstract

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Evidence shows that the CD38 molecule, recently involved in the two main features of asthma, bronchial hyper-responsiveness and airway inflammation, could represent a new potential therapeutic target for asthma. In this study, we investigated whether glucocorticoid (GC), the most effective treatment for lung diseases, can affect CD38 expression in human airway smooth muscle (ASM) cells treated with different pro-inflammatory cytokines, such as tumor necrosis factor- (TNF) and interferons (IFNs). We found that CD38 expression induced by TNF alone was completely abrogated by fluticasone (100 nM), dexamethasone (1 µM), or budesonide (100 nM). In contrast, the synergistic induction of CD38 by the combination of TNF with IFN or IFN, but not with IL-1 or IL-13, was completely insensitive to the GC inhibitory effects. We also found that TNF and IFN impaired GC responsiveness by inhibiting steroid induced both 1) GR-DNA binding activity and 2) GC-responsive element-(GRE)-dependent gene transcription. Although levels of the GC receptor (GR) isoform remained unchanged, expression of GR, the dominant-negative GR isoform, was synergistically increased by TNF and IFN with a GR/GR ratio of 1 to 3. More importantly, fluticasone failed to induce GRE-dependent gene transcription and to suppress TNF-induced CD38 expression in ASM cells transfected with constitutively active GR. We conclude that, upon pro-inflammatory cytokine stimulation, CD38 expression becomes insensitive to GC action by a mechanism involving the up-regulation of GR isoform, thus providing a novel in vitro cellular model to dissect GC resistance in primary cells.

Glucocorticoids (GCs) are the treatment of choice for chronic inflammatory diseases such as asthma (Lemanske and Busse, 2003). Most of their anti-inflammatory effects are mediated via glucocorticoid receptor isoform (GR), which suppresses expression of inflammatory genes through mechanisms known as transactivation or transrepression (Leung and Bloom, 2003). Transactivation results from a direct binding of activated GR to DNA sequences called GC-response elements (GRE) present on inducible anti-inflammatory genes. Transrepression is mediated via the direct interaction between activated GR and different transcription factors, such as nuclear factor B and activator protein 1, thus repressing transcription factor-inducible pro-inflammatory genes (Pujols et al., 2004). It is noteworthy that in addition to GR, and as a result of alternative splicing mechanisms, another GR isoform, GR, has been described previously (Hollenberg et al., 1985). Although the role of GR is not well understood, a previous report in immune and transformed cells demonstrated its dominant-negative effects on GR-dependent transcriptional activities (Leung et al., 1997).

Increasing evidence suggests that ASM cells play a central role in the pathogenesis of asthma (Parris et al., 1999; Amrani and Panettieri, 2003; Hunter et al., 2003). In fact, when exposed to inflammatory conditions, ASM can become a source of different chemokines/cytokines that are capable of orchestrating and/or perpetuating airway inflammation (Howarth et al., 2004). Although the GC effects on human ASM cells have been investigated, their modulatory effects on gene expression are quite complex, and the signaling mechanisms underlying their suppressive effects have been poorly characterized. First, the effects of GC seem to be highly gene-specific. For example, reports showed that dexamethasone can effectively inhibit cytokine-induced IL-6, RANTES (Ammit et al., 2002), eotaxin (Pang and Knox, 2001), or cyclooxygenase-2 expression (Vlahos and Stewart, 1999), whereas it has no effect on cytokine-induced ICAM-1 expression (Amrani et al., 1999). Second, GC suppressive effect seems to be time-dependent, because dexamethasone partially abrogates cytokine-mediated ICAM-1 expression at early time points but has no effect at later time points (Amrani et al., 1999). Third, unexpectedly, instead of acting as potent inhibitors, GC synergistically enhanced cytokine-induced fractalkine expression and secretion (Sukkar et al., 2004). Finally, GC inhibitory action is also stimuli-specific. Indeed, whereas dexamethasone significantly inhibits granulocyte macrophage-colony stimulating factor secretion induced by IL-1, it partially inhibits the secretion induced by thrombin. Together, these observations demonstrate that ASM is a unique and complex model in which GC actions are differentially modulated by cytokines.

In the present report, we demonstrate that CD38 expression induced by cytokine combination becomes refractory to the suppressive action of steroid. We found that the specific combination of TNF with IFNs, but not with IL-1 or IL-13, reduces ASM cell responsiveness to different classes of GC, including fluticasone, dexamethasone, and budesonide. This steroid insensitivity seems to be the result of increased levels of GR that impair GR transcriptional activity.

	Materials and Methods

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ASM Cell Culture and Treatment. Human tracheal tissue culture was obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. The culture of human ASM cells was performed as described elsewhere (Panettieri et al., 1989).

Reverse Transcription-Polymerase Chain Reaction Analysis. Total RNA was extracted from human ASM cells using RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. In preliminary experiments, we determined, for each primer pair, the melting temperature and number of amplification cycles necessary to yield the appropriate hybridization signal. The PCR of CD38, GR, GR, and -actin was performed using previously published primers (Orii et al., 2002; Tliba et al., 2004). Each of 35 cycles of the PCR was programmed to carry out denaturation at 94°C for 30 s, primers annealing at 55°C for 45 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. The semiquantitative PCR approach was performed in parallel by investigating the -actin mRNA level. The intensity of the area density was analyzed using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD), and the final PCR product was expressed as a ratio of area density of CD38, GR, or GR to -actin.

Real-Time PCR. Real-time PCR analysis was performed on a Smart Cycler (Cepheid, Sunnyvale, CA) by using a SYBR-Green kit according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). PCR was performed using the same GR, GR, and -actin primers in a total volume of 25 µl in the presence of SYBR Green PCR Master Mix. For GR and GR PCR amplification, the program consists of 60 s for the initial denaturation period at 95°C, and 45 cycles of 95°C for 1 s, 60°C for 5 s, and 72°C for 15 s. For -actin, the program consists of 60 s for the initial denaturation period at 95°C, 40 cycles of 95°C for 1 s, 55°C for 5 s, and 72°C for 18 s. The results were calculated using a quantitation approach termed the comparative cycle threshold (C_t) method as described elsewhere (Ponchel et al., 2003).

GR-DNA Binding Activity. Nuclear extraction was performed as described previously (Tliba et al., 2003). Ten micrograms of nuclear extracts were tested for GR-DNA binding activity by using TransAM GR kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA). The results (optical densities measured at 450 nm) were expressed as a percentage of increase over untreated cells.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis. Immunoblot analyses for GR and GR were performed as described previously (Tliba et al., 2003) using specific anti-GR and anti-GR antibodies (Affinity BioReagents, Golden, CO). To ensure equal loading, the membranes were stripped and reprobed with anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The area densities for each GR isoform and the total actin were calculated using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD), and the data were expressed as a ratio of area density of GR or GR to actin.

Transfection of ASM Cells. Because most standard transfection methods yield poor transfection efficiencies for ASM cells (<10%), here we have optimized a high-efficiency transfection technique. This technique is an extension of electroporation, using Nucleofector kit for primary smooth muscle (Amaxa Biosystems, Cologne, Germany), in which plasmid DNA is transfected directly into the cell nucleus. Transfection was performed according to the manufacturer's instructions, and the program used was D-33. Sixteen to eighteen hours after transfection, the media were changed with serum-free media for the next 24 h. Transfection efficiency of GFP-tagged plasmids was monitored by flow cytometry as described below. This method, using green fluorescence protein (GFP)-pmax control vector (Amaxa Biosystems), enabled us to reach a transfection efficiency of 70%.

Recombinant Plasmids. To assess the dominant negative activity of GR, ASM cells were transfected with constitutively active (CA) GR GFP-tagged (Oakley et al., 1996) or with pCMV-GFP empty vector (Clontech, Mountain View, CA). To monitor fluticasone transactivating activity, cells were cotransfected with GRE-secreted alkaline phosphatase (SEAP) reporter vector and with pSV--galactosidase vector used to normalize transfection efficiencies (Promega, Madison, WI).

SEAP and -Galactosidase Assays. The activities of SEAP and -galactosidase were evaluated using Great EscAPe SEAP detection kit (Clontech) and -galactosidase detection kit (Promega, Madison, WI), respectively, according to their manufacturer's instructions (Tliba et al., 2003; An et al., 2005).

Fluorescence-Activated Cell Sorting Analysis. Flow cytometry was performed as described previously (Tliba et al., 2002). Antibody used for CD38 expression was purchased from Santa Cruz Biotechnology. Phycoerythrin-conjugated secondary antibody was bought from Jackson ImmunoResearch Laboratories (West Grove, PA). GR-GFP transfection efficiency was monitored by a shift to green fluorescence (FL-1), and CD38 expression level was monitored by a shift to red fluorescence (FL-2). Fluorescence-activated cell sorting analysis was performed with EPICS XL flow cytometer (Beckman Coulter, Inc., Fullerton, CA) using the Cellquest software.

Materials and Reagents. Tissue culture reagents and primers were obtained from Invitrogen (Carlsbad, CA). Human recombinant (r) TNF, rIL-1, and rIFN were provided by Roche Diagnostics. Human rIFN and rIL-13 were purchased from R&D Systems (Minneapolis, MN). Fluticasone propionate (FP) was generously supplied by GlaxoSmithKline, Inc. (King of Prussia, PA). Dexamethasone and budesonide were obtained from Sigma (St. Louis, MO).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Dose-dependent effect of fluticasone (FP) on TNF-induced CD38 expression in ASM cells. Cells were pretreated with indicated concentrations of FP for 2 h and then stimulated with TNF (10 ng/ml) for 24 h. A, representative agarose gel showing the CD38 PCR products stained with ethidium bromide. Cells stimulated as indicated were lysed, total mRNA was isolated, and RT-PCR was performed as described in the Materials and Methods using specific primers for CD38 and -actin genes. B, CD38 protein expression was assessed by flow cytometry as described in the Materials and Methods and the results are expressed as the -fold increase in mean fluorescence intensity over basal (untreated cells) ± S.E.M. of three separate experiments. *, P < 0.05 compared with untreated cells; #, P < 0.05 compared with cells treated with TNF alone; NS, not significant compared with cells treated with TNF alone. C, representative flow cytometry histograms of GC effect on CD38 expression.

	Results

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Fluticasone Inhibits in a Dose-Dependent Manner TNF-Induced CD38 Expression. We first investigated the effect of GC on TNF-induced CD38 expression, a gene potentially involved in modulating airway inflammation via the transcriptional regulation of different inflammatory mediators (Tliba et al., 2004). For this purpose, human ASM cell cultures were pretreated for 2 h with increasing concentrations of fluticasone (1–100 nM) before TNF treatment, and CD38 expression was measured by RT-PCR. As shown in Fig. 1A, fluticasone at 100 nM completely inhibited TNF-induced CD38 mRNA expression (P < 0.05). In addition, flow cytometry analyses revealed that fluticasone also inhibited, in a concentration-dependent manner, TNF-induced CD38 expression (P < 0.05) with complete inhibition achieved at 100 nM (CD38 level decreased from 5.2 ± 0.26- to 1.42 ± 0.13-fold increase over basal in cells treated with TNF in the absence or presence of fluticasone, respectively) (Fig. 1, B and C). In a parallel experiment, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay (Amrani et al., 1996), we found that fluticasone did not affect ASM cell viability (data not shown). Together, these data suggest that GC suppresses TNF-induced CD38 expression by involving transcriptional mechanisms.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effect of TNF and IFNs combination on CD38 sensitivity to FP. Human ASM cells were treated with TNF (10 ng/ml) and IFN (500 IU/ml) (A) or TNF (10 ng/ml) and IFN (500 IU/ml) (B) for 24 h in the presence or absence of FP (100 nM) added 2 h before. CD38 expression was assessed by flow cytometry as described under Materials and Methods. The results are expressed as the -fold increase in mean fluorescence intensity over basal (untreated cells) ± S.E.M. of three separate experiments. *, P < 0.05 compared with untreated cells; **, P < 0.01 compared with untreated cells; # and , P < 0.05 compared with cells treated with TNF or IFNs alone, respectively; NS, not significant compared with cells treated with TNF and IFNs combination.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effect of TNF and IL-1 or TNF and IL-13 combination on CD38 sensitivity to FP. Human ASM cells were treated with TNF (10 ng/ml) and IL-1 (10 ng/ml) (A) or TNF (10 ng/ml) and IL-13 (50 ng/ml) (B) for 24 h in the presence or absence of FP (100 nM) added 2 h before. CD38 expression was assessed by flow cytometry as described under Materials and Methods. The results are expressed as the -fold increase in mean fluorescence intensity over basal (untreated cells) ± S.E.M. of three separate experiments. *, P < 0.05 compared with untreated cells; # and , P < 0.05 compared with cells treated with cytokine alone or in combination, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effect of TNF and IFN on CD38 sensitivity to dexamethasone and budesonide. Human ASM cells were treated with TNF (10 ng/ml) and IFN (500 IU/ml) for 24 h in the presence or absence of dexamethasone (DEX) (1 µM) or budesonide (BUD) (100 nM) added 2 h before. CD38 expression was assessed by flow cytometry as described under Materials and Methods. The results are expressed as the -fold increases in mean fluorescence intensity over basal (untreated cells) ± S.E.M. of three separate experiments. *, P < 0.05 compared with untreated cells; **, P < 0.01 compared with untreated cells; #, P < 0.05 compared with cells treated with TNF alone; NS, not significant compared with cells treated with TNF and IFN combination.

View larger version (11K): [in this window] [in a new window]   Fig. 5. TNF and IFN combination impairs GC activities. A, ASM cells were treated with fluticasone (100 nM) for 24 h in the presence or absence of wild-type oligonucleotide, mutated oligonucleotide, or TNF and IFN combination. Nuclear extracts were then tested for GR-DNA binding activity using the TransAM GR kit as described in Materials and Methods. *, P < 0.05 compared with untreated cells; #, P < 0.05 compared with cells treated with fluticasone alone. NS, not significant compared with cells treated with fluticasone alone. B, cells were transfected with 4 µg of SEAP reporter construct containing GRE motifs before fluticasone and cytokines were added. Cells were then lysed, and the SEAP activity was performed as described in Materials and Methods. *, P < 0.05 compared with untreated cells; #, P < 0.05 compared with cells treated with fluticasone alone.

TNF and IFN Enhance Selectively the Expression of GR Isoform. Evidence suggests that changing the GR/GR cellular ratio in different inflammatory conditions, where GR predominates, may promote GC resistance (Pujols et al., 2004). Therefore, we next investigated whether cytokine combination could alter GR/GR ratio in human ASM cells. Using RT-PCR analyses, we found that the steady state of GR mRNA levels, which is constitutively expressed in untreated cells, was not affected by TNF or IFN either alone or in combination (Fig. 6A). Similar results were obtained by real-time PCR (data not shown). Even though TNF or IFN alone have only a slight stimulatory effect on GR levels, their combination significantly increased by 470% the steady state of GR mRNA levels (Fig. 7A), a result that was also confirmed by real-time PCR (Fig. 7B). Using immunoblot analyses, we found that although the constitutive expression of GR protein was not affected by TNF or IFNs ( or ), either alone or in combination (Fig. 6B), GR protein expression was significantly increased by TNF and IFNs combination (Fig. 7, C and D). Semiquantitation analyses of GR isoform expression showed that GR/GR ratio was 8:1 in untreated cells, 1:1 in TNF-, IFN-, or IFN-treated cells, and 1:2 and 1:3 in TNF/IFN- and TNF/IFN-treated cells, respectively. Together, these data demonstrate that in cells exposed to cytokine combination, GR becomes the predominant GR isoform.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Effect of TNF and IFN combination on GR expression. ASM cells were stimulated with TNF (10 ng/ml) and IFN (500 IU/ml) either alone or in combination for 24 h. A, total mRNA (2 µg) was subjected to RT-PCR with -actin and GR primers. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. B, in separate experiments, total cell lysates were prepared and assayed for GR by immunoblot analysis.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of TNF and IFN combination on GR expression. ASM cells were stimulated with TNF (10 ng/ml) and IFN (500 IU/ml) either alone or in combination for 24 h. A, total mRNA (2 µg) was subjected to RT-PCR with -actin and GR primers. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. B, real-time PCR analysis of GR gene. GR mRNA levels were normalized to the corresponding levels of -actin mRNA using the comparative cycle threshold (Ct) method as described under Materials and Methods. The results are expressed as a percentage of untreated control values. *, P < 0.05 compared with untreated cells; **, P < 0.01 compared with untreated cells. C, total cell lysates were prepared and assayed for GR by immunoblot analysis. D, scanning densitometry of three representative immunoblots with each condition normalized over the area density of the corresponding actin content. The results are expressed as the -fold increase over basal. *, P < 0.05 compared with untreated cells; **, P < 0.01 compared with untreated cells.

GR Overexpression Prevents GC-Mediated Transactivation and Reduces the Sensitivity of CD38 to Fluticasone. To further support the association between the reduced action of GC (Figs. 2, 4, and 5) and the enhancement of GR expression (Fig. 7) in cells treated with cytokine combination, we next examined the effect of GR overexpression on GC activities. To determine whether GR interferes with GC-induced transactivation activity, ASM cells were cotransfected with 2 µg of CA-GR or pCMV empty vector, and 2 µg of SEAP-reporter construct containing GRE motifs. As shown in Fig. 8A, fluticasone-induced GRE-dependent reporter activity was completely abrogated in GR-transfected but not in pCMV-transfected cells. To test whether GR interferes with GC actions on CD38 expression, ASM cells were transfected with 4 µg of GFP-tagged CA-GR then treated with TNF for 24 h in the presence or absence of fluticasone, and CD38 expression in GR-transfected cells was assessed by two-color flow cytometry. As shown in Fig. 8B, TNF significantly enhanced CD38 expression in GR-transfected cells (35.1 ± 2.2-fold increase over basal), a response that was completely insensitive to fluticasone action (filled bars). However, fluticasone was still effective in inhibiting TNF-induced CD38 expression in cells transfected with control vector (pCMV-GFP) (open bars). Together, these data suggest that GR up-regulation is associated with a significant reduction of GC activities in ASM cells.

View larger version (10K): [in this window] [in a new window]   Fig. 8. Effect of GR over-expression on fluticasone activities. A, cells were cotransfected with a 2 µg of pCMV empty vector or CA-GR construct and 2 µg of SEAP reporter plasmid containing GRE motifs before FP (100 nM) was added for 24 h. SEAP activity was measured as described in the Materials and Methods. The results are expressed as a percentage of control values from untreated cells. Data are representative of three separate experiments. *, P < 0.05 compared with untreated cells; #, P < 0.05 compared with cells treated with FP alone; NS, not significant compared with cells treated with fluticasone alone. B, flow cytometry analysis of CD38 expression in GR-transfected cells. Cells were transfected with 4 µg of pCMV-empty vector or CA-GR construct before FP (100 nM) and TNF (10 ng/ml) were added for 24 h. Cells were then harvested by trypsinization and fixed, and two-color flow cytometry was performed to assess CD38 expression on GR-positive cells as described under Materials and Methods. The percentage of double-positive cells [i.e., pCMV-GFP/CD38 (open bars) or GR-GFP/CD38 (filled bars)] was then calculated and presented in bar graph. *, P < 0.05 compared with untreated cells; #, P < 0.05 compared with cells treated with TNF alone; NS, not significant compared with cells treated with TNF alone.

	Discussion

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Our data show the TNF and IFNs render ASM cells insensitive to GC as well as reduce GC-induced transactivation activities. This is an important finding, in that numerous studies suggest a strong association between GC insensitivity and a variety of inflammatory diseases, such as asthma, nasal polyps, and inflammatory bowel disease (Adcock et al., 1995; Honda et al., 2000; Farrell and Kelleher, 2003). These observations prompted investigators to examine whether inflammatory mediators could be responsible for the altered GC cellular responses. Impairment of GC activity can be reproduced in vitro in immune cells exposed to IL-2 and IL-4 that reduced the inhibitory effect of methylprednisolone on mitogen-induced T cell proliferation (Leung and Bloom, 2003). Other studies in human peripheral blood mononuclear cells showed that the combination of IL-1, IL-6, and IFN also decreased the suppressive action of dexamethasone on mitogen-induced proliferation (Almawi et al., 1991). In T cells, monocytes, and neutrophils, GC action is as well impaired when exposed to a single cytokine such as IL-2, IL-7, IL-13, IL-15, or IL-8 (Spahn et al., 1996; Pipitone et al., 2001; Strickland et al., 2001; Goleva et al., 2002). Although all in vitro studies describing GC insensitivity have been performed in either immune or transformed cells, very little is known about the modulation of GC signaling in structural cells that are relevant for lung diseases. We now show that the specific combination of TNF with IFNs reduces ASM cell responsiveness to different classes of GC, including fluticasone, dexamethasone, and budesonide (Figs. 2 and 4). This TNF- and IFN-dependent GC insensitivity was not observed when TNF was combined with other cytokines, such as IL-1 or IL-13 (Fig. 3), although these latter cytokines significantly reduced GC cellular response in other cell types (Spahn et al., 1996; Pariante et al., 1999; Webster et al., 2001). Our findings could explain, at least in part, the lack of dexamethasone to suppress fractalkine secretion in ASM cells treated similarly with both TNF and IFN (Sukkar et al., 2004). These data suggest that TNF and IFNs is a new cytokine combination inducing GC insensitivity in human ASM cells, thus providing a novel in vitro model to dissect GC insensitivity in structural cells. The mechanisms underlying TNF/IFNs suppressive effects on GC signaling and function are unknown, but our study demonstrates the potential role of GR.

The fact that the inhibitory effect of cytokine combination on GC actions could be mimicked in cells transfected with CA-GR (Fig. 8) strongly suggests that GR represents one molecular mechanism responsible for the impaired GC activity. We found that the combination of TNF and IFNs significantly increased GR isoform at both mRNA and protein levels but had a weak effect on GR expression. In peripheral blood mononuclear cells, where the levels of GR were significantly increased by IL-7 or IL-18, no changes in GR levels were observed (Orii et al., 2002). In neutrophils, however, IL-8 enhanced GR expression, whereas the GR expression was decreased (Strickland et al., 2001). Moreover, Torrego et al. (2004) showed that IL-2 and IL-4 combination had no effect on GR expression but enhanced significantly GR expression contrasting with the selective induction of GR by the same cytokine combination observed by Leung et al. (1997). Together, these observations suggest that GR isoforms are differentially regulated in a complex stimuli- and cell-specific manner. The important question that remains unanswered is the nature of the specific mechanisms that regulate the selective induction of GR. Because GR expression is a result of alternative splicing (Hollenberg et al., 1985), it is plausible that the selective GR induction by TNF and IFNs occurs because of the possible activation of specific alternative splicing factors. Indeed, Xu et al. (2003) showed that increased levels of the serine-arginine–rich protein SRp30 is required for the alternative splicing of GR pre-mRNA to GR isoform in neutrophils. In our study, cytokine combination failed to increase SRp30 content in ASM cells (O. Tliba and Y. Amrani, unpublished observations). Further investigations are needed to determine the alternative splicing factors that are involved in the differential induction of GR isoforms in ASM cells.

So far, the inhibitory role of GR on GR activity remains controversial. Oakley et al. (1999) found that the transient transfection of increasing amounts of GR-expressing vectors into immune and transformed cells inhibits GC actions by blocking the transactivating ability of GR. In contrast, Hecht et al. (1997) and de Lange et al. (1999) were unable to confirm the inhibitory role of GR when overexpressed into COS-1 cells. These controversial results may be explained by the use of different experimental conditions, such as the transfection methods, the promoter used to overexpress GR, and the relative amounts of transfected plasmids, which could lead to insufficient GR expression. Indeed, a high expression level of GR isoform is necessary to significantly inhibit GC-mediated gene expression in COS-1 cells (Oakley et al., 1999). This hypothesis is supported in our study by the fact that in cells exposed to cytokine alone (TNF or IFN), where GR expression was slightly enhanced, reaching a level comparable with that of GR (Figs. 6 and 7), fluticasone was still able to suppress cytokine-induced CD38 expression (Fig. 1). In contrast, we showed that in cells resistant to fluticasone action [i.e., treated with both TNF/IFNs (Fig. 2)], GR level was significantly predominant over that of GR (ratio of GR to GR was 1:3). The dominant-negative properties of GR were difficult to demonstrate in primary cells because most standard transfection methods yield poor transfection efficiencies (<10%). To overcome the low transfection efficiencies, we have optimized a high-efficiency transfection technique. This technique is an extension of electroporation, using a Nucleofector kit for primary smooth muscle (Amaxa Biosystems). Using this method, we showed that the GC ability to block TNF-induced CD38 expression as well as to induce GRE-dependent gene transcription was completely impaired in ASM cells transfected with CA-GR (Fig. 8). To our knowledge, this is the first study in primary cells showing that proasthmatic cytokines alter steroid function via the induction of GR.

In summary, we found that the combination of TNF and IFNs renders CD38 expression refractory to the suppressive action of steroid by a mechanism involving the GR-dependent alteration of GR signaling and function. The importance of IFNs in promoting GC insensitivity in airway resident cells could explain, at least in part, the increased GC requirements in patients with severe asthma experiencing viral infections that produce high levels of IFNs in the airways (Johnston, 1999; Yamada et al., 2000). Our study opens a new area of investigation to determine the molecular mechanisms by which cytokine-induced GR expression impairs GC signaling in structural cells.

	Acknowledgements

 
We thank Mary McNichol for assistance in the preparation of the manuscript.

	Footnotes

 
This work was supported by grant HL64063 from the National Institutes of Health (to Y.A.), by the Francis Families Foundation (to Y.A.), and by GlaxoSmithKline. Y.A. is a Parker B. Francis Fellow in Pulmonary Research.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.019679.

ABBREVIATIONS: cADPr, cyclic ADP ribose; TNF, tumor necrosis factor-; IFN, interferon; IL, interleukin; RANTES, regulated upon activation normal T-cell expressed and secreted; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; ICAM, intercellular adhesion molecule; ASM, airway smooth muscle; PCR, polymerase chain reaction; CA, constitutively active; SEAP, secreted alkaline phosphatase; r, recombinant; COX-2, cyclooxygenase 2; GFP, green fluorescent protein; FP, fluticasone propionate; RT, reverse transcription; CMV, cytomegalovirus.

Address correspondence to: Dr. Omar Tliba, Pulmonary, Allergy and Critical Care Division, University of Pennsylvania Medical Center, 421 Curie Boulevard, BRB II/III, Philadelphia, PA 19104-6160. E-mail: omartlib{at}mail.med.upenn.edu

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