Dexamethasone suppresses iNOS gene expression by upregulating I-kappa Balpha and inhibiting NF-kappa B

Michael E. De Vera, Bradley S. Taylor, Qi Wang, Richard A. Shapiro, Timothy R. Billiar, and David A. Geller

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression is dependent on nuclear factor-kappa B (NF-kappa B) activation and is suppressed by glucocorticoids (GC). In this study we examined the molecular mechanisms of GC inhibition of iNOS expression in rat hepatocytes. Combinations of tumor necrosis factor-alpha , interleukin-1beta , and interferon-gamma (cytokine mixture CM) induced high levels of iNOS mRNA and NO synthesis. The synthetic GC dexamethasone markedly repressed iNOS mRNA and protein expression, and nuclear run-on assays showed that this inhibition was occurring at the level of transcription. In addition, transfection studies showed that CM-stimulated activity of a 1.6-kb murine iNOS promoter fragment linked upstream of luciferase was suppressed by dexamethasone. Electromobility shift assays demonstrated that CM induced the appearance of an NF-kappa B complex composed of p50 and p65 subunits; the addition of dexamethasone markedly decreased this band shift. I-kappa Balpha expression was decreased by CM and upregulated in the presence of dexamethasone. Subsequently, nuclear p65 levels were decreased by dexamethasone compared with CM-treated cells. Thus GC repress NF-kappa B DNA-binding activity in rat hepatocytes in part through the upregulation of its inhibitor I-kappa Balpha . These data indicate that one mechanism by which GC block iNOS expression is through the inhibition of NF-kappa B activation resulting in decreased iNOS transcription.

inducible nitric oxide synthase; nuclear factor-kappa B; tumor necrosis-alpha ; interleukin-1beta ; interferon-gamma ; glucocorticoids; gene regulation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

NITRIC OXIDE (NO) synthesized by the inducible form of NO synthase (iNOS) plays a role in numerous inflammatory and immune processes, such as sepsis, hemorrhagic shock, transplant rejection, and autoimmune diseases (5, 20). Although some epithelial cells have been shown to express iNOS constitutively (1), most cells express iNOS mRNA and protein only after exposure to specific stimulating agents. In these cells cytokines, viruses, or lipopolysaccharide (LPS) stimulate iNOS expression in a widespread fashion, leading to the production of large amounts of NO. NO exerts cytoprotective and beneficial actions (e.g., antitumor, antimicrobial, and anti-atherogenic effects); however, overproduction of NO can have cytotoxic or other detrimental effects (e.g., septic shock, apoptosis, or direct cellular injury). iNOS expression therefore must be tightly regulated to achieve the maximal benefit of NO while avoiding toxicity.

Previously, we reported that tumor necrosis factor-alpha (TNF-alpha ), interleukin-1beta (IL-1beta ), interferon-gamma (IFN-gamma ), and LPS can act synergistically to induce iNOS gene expression in rodent and human hepatocytes, whereas glucocorticoids (GC) inhibit iNOS expression (9, 10). This increase in cytokine-induced hepatic iNOS mRNA levels is due in part to the transcriptional activation of the iNOS gene (7, 8). The cis-regulatory elements required for the activation of the murine iNOS promoter have been identified and shown to reside within 1 kb of the transcriptional start site of the iNOS gene (18, 31). In contrast, the proximal 1 kb of the 5'-flanking region of the human iNOS gene is not sufficient to activate the iNOS promoter. We recently demonstrated that cytokine-responsive elements upstream of -3.8 kb are required for activation of the human iNOS promoter (7). Notwithstanding these intrinsic and important differences between the human and murine iNOS promoters, many studies have now shown that cytokine activation of the iNOS gene is dependent on the transcription factor nuclear factor-kappa B (NF-kappa B) (11, 30). NF-kappa B in its inactive state resides in the cytoplasm bound by its inhibitor, I-kappa Balpha . Activation of NF-kappa B by a wide variety of inflammatory stimuli leads to degradation of I-kappa Balpha either through a ubiquitin-proteosome pathway or proteosome-independent pathway (12). This allows NF-kappa B to translocate to the nucleus and bind to the promoter of its target genes. Numerous inflammatory and immunomodulatory genes including cytokines, cell adhesion molecules, and major histocompatibility complex proteins are known to be regulated by NF-kappa B.

Despite extensive clinical use of GC as immunosuppressive agents, we have only begun to understand the molecular mechanisms by which these drugs exert their anti-inflammatory actions. It is known that GC bind to steroid hormone receptors, leading to activated GC receptors that then bind to either GC response elements (GRE) or negative GRE, resulting in transcriptional activation or repression of genes, respectively. The majority of genes that are downregulated by steroids, however, lack negative GRE in their promoters. Furthermore, GC have been shown to inhibit the transcription factor AP-1 through protein-protein interactions (13), and yet only a few of the genes affected by GC contain functional binding sites for AP-1 in their promoters. Recently, several researchers have demonstrated that GC are able to suppress NF-kappa B through cross-coupling mechanisms (26) or through the induction of I-kappa Balpha (2, 25); it now appears that NF-kappa B inhibition may be the predominant mechanism by which proinflammatory genes are suppressed by GC. However, the precise mechanism by which GC downregulate iNOS expression is unknown. In this study, we explored the molecular mechanisms underlying GC downregulation of iNOS gene expression in rat hepatocytes. We demonstrate that GC upregulate I-kappa Balpha expression and decrease nuclear p65 translocation, thus inhibiting iNOS gene transcription and promoter activation by decreasing NF-kappa B DNA binding activity.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Reagents. Murine recombinant TNF-alpha was purchased from Genzyme (Cambridge, MA), human recombinant IL-1beta was generously provided by Craig Reynolds (National Cancer Institute), and murine recombinant IFN-gamma was obtained from GIBCO BRL (Grand Island, NY). Dexamethasone and pyrrolidine dithiocarbamate (PDTC) were from Sigma (St. Louis, MO), and T4 polynucleotide kinase and lipofectin were purchased from United States Biochemical (Cleveland, OH) and GIBCO BRL, respectively.

Cell culture. Rat hepatocytes were isolated from male Sprague-Dawley rats weighing 200-250 g (Harlan Sprague Dawley, Madison, WI), using a modified in situ collagenase (type IV, Sigma) perfusion technique as previously described (10). Hepatocytes (5 × 106) were plated onto 100-mm gelatin-coated cell culture dishes (Corning, Corning, NY) and maintained at 37°C, 95% air-5% CO2 in William's E (GIBCO) supplemented with L-arginine (0.50 mM), insulin (10-6 M), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (15 mM), L-glutamine, penicillin, streptomycin, and 10% low endotoxin calf serum (Hyclone Laboratories, Logan, UT). After an 18-h incubation hepatocytes were treated with a cytokine mixture (CM) of TNF-alpha (500 U/ml), IL-1beta (200 U/ml), and IFN-gamma (100 U/ml) or different combinations of these cytokines in the presence or absence of dexamethasone (10-9 to 10-5 M).

Nitrite plus nitrate assay. Culture supernatants were collected 24 h after cytokine treatment and assayed for nitrite plus nitrate, the stable end-products of NO oxidation, using an automated procedure based on the Griess reaction (9).

Northern blot analysis. The iNOS probe used was a 2.3-kb BamH I fragment from the human hepatocyte iNOS cDNA (11). The I-kappa Balpha cDNA probe was generously provided by Gary Nabel (Univ. of Michigan). RNA extraction, Northern blot analysis, and autoradiography were performed as described previously (10). After being probed for iNOS or I-kappa Balpha , membranes were stripped with boiling 5 mM EDTA and 0.1% sodium dodecyl sulfate (SDS) and rehybridized with a probe for 18S ribosomal RNA. Relative mRNA levels were quantitated by PhosphorImager scanning with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

SDS-polyacrylamide gel electrophoresis and Western blotting. Cytosolic and nuclear proteins were prepared as described (6, 14) and quantitated with bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL). Western blot analysis was carried out by separating proteins (75 µg) with 8% SDS-polyacrylamide gel electrophoresis and electrophoretically transferring the products to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) (14). Nonspecific binding to the membrane was blocked by 5% nonfat dry milk in phosphate-buffered saline-Tween overnight at 4°C. Blots were washed in phosphate-buffered saline-Tween and then incubated for 1 h with either monoclonal mouse anti-iNOS antibody (1:5,000 dilution, Transduction Laboratories, Lexington, KY), polyclonal rabbit anti-I-kappa Balpha antibody (1:2,000 dilution; Rockland, Gilbertsville, PA), and polyclonal rabbit anti-p65 antibody (1:1,000 dilution; Rockland). The secondary antibody used was a peroxidase-conjugated goat anti-mouse immunoglobulin G in a 1:1,000 dilution (Schleicher & Schuell). Membranes were developed with the enhanced chemiluminescence-detection system (DuPont-NEN, Boston, MA) and exposed to film.

Nuclear run-on assay. Hepatocytes were stimulated with CM with or without dexamethasone for 4 h. Nuclei were purified as previously reported (7, 8). Linearized plasmid containing full-length human iNOS, glyceraldehyde-3-phosphate dehydrogenase, and glutaminase cDNA were immobilized and crosslinked on a GeneScreen membrane (9). Hybridizations were carried out as described, and results were quantitated with the PhosphorImager (7, 8).

Promoter analysis. A deletional construct containing 1.6 kb of the 5'-flanking region of the murine iNOS gene ligated upstream of the reporter gene luciferase (gift of Charles Lowenstein, Johns Hopkins Univ.) was transiently transfected into hepatocytes using liposomes as described (6). Serum-free transfections were carried out in six-well plates (Corning) using 1 µg of DNA and 10 µg of lipofectin for 6 h. After an overnight recovery hepatocytes were treated with cytokines for 6 h in the presence or absence of dexamethasone (10-6 M) or the NF-kappa B inhibitor PDTC (100 µM), after which luciferase activity was measured using a commercially available assay kit (Promega, Madison, WI) (6).

Electromobility shift assays. Hepatocytes were treated with cytokines in the presence or absence of dexamethasone (10-6 M) or PDTC (100 µM) for 1 h. Preparation of nuclear extracts and electromobility shift assays were carried out essentially as described (6). The oligonucleotide used was derived from the murine iNOS promoter (positions -90 to -71) (18) and contained a functional NF-kappa B element (italized): CAACTGGGGACTCTCCCTTTG. A mutant NF-kappa B oligonucleotide was also used as a mutant competitor: AAGCTGGGTCCACTCCCTTTG. Nuclear extracts (10 µg) were incubated with ~0.5 ng (~40,000 counts/min) of 32P-end-labeled oligonucleotide (T4 polynucleotide kinase) for 30 min at room temperature. In other experiments nuclear extracts were incubated with excess unlabeled NF-kappa B oligomers or antibodies against the different subunits of NF-kappa B (Santa Cruz Biotechnology and kind gift of Nancy Rice, National Cancer Institute) for 15 min before the addition of labeled probe. DNA-protein complexes were electrophoretically resolved on a 5% nondenaturing polyacrylamide gel, which was then dried and subjected to autoradiography.

Statistical analysis. The significance of differences was determined by analysis of variance (ANOVA) using the Statview statistics program (Abacus Concepts, Berkeley, CA). Statistical significance was established at P < 0.01.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Dexamethasone inhibits cytokine-induced iNOS mRNA and protein synthesis. To investigate the effects of GC on iNOS mRNA and NO production in rat hepatocytes, combinations of TNF-alpha , IFN-gamma , and IL-1beta were added to cultures in the presence or absence of the synthetic GC dexamethasone. Consistent with our previous results (8, 10) combinations of these cytokines (CM) or IL-1beta alone induced iNOS mRNA expression (Fig. 1A, top) and NO production as measured by nitrite and nitrate release (Fig. 1B). The addition of dexamethasone significantly inhibited iNOS mRNA levels (Fig. 1A, bottom) and NO production (~50-75% maximum inhibition) in all groups. As shown in Fig. 2, the inhibitory effect on CM-induced NO synthesis was concentration dependent, with an ~60% decrease in nitrite and nitrate release at the highest concentration of dexamethasone (10-5 M). Western blot analysis showed expression of iNOS protein with CM stimulation (Fig. 3), and similar to the decrease in mRNA expression, protein levels were also lower in the presence of dexamethasone. These data therefore indicate that cytokine-induced NO synthesis in hepatocytes is inhibited by GC at the mRNA and protein levels.


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Fig. 1.   Dexamethasone (Dex) inhibits inducible nitric oxide synthase (iNOS) gene expression and NO synthesis. Cultured rat hepatocytes (HC) were treated with tumor necrosis factor-alpha (TNF-alpha , 500 U/ml), interferon-gamma (IFN-gamma , 100 U/ml), interleukin-1beta (IL-1beta , 200 U/ml), or the combination of all 3 cytokines (CM, cytokine mixture) with or without dexamethasone (10-6 M) for 6 h. A: Northern blot analysis for iNOS mRNA. Equal loading of RNA in each lane was confirmed by 18S ribosomal RNA probing (not shown). B: 24-h nitrite plus nitrate release (stippled bars, cytokines alone; hatched bars, cytokines plus dexamethasone). Values are expressed as nmol/million hepatocytes and represent means ± SE (n = 4/group). * P < 0.01 vs. control. dagger  P < 0.01 vs. cytokines (by ANOVA).


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Fig. 2.   Dose-dependent suppression of NO production by dexamethasone. Rat hepatocytes were stimulated with CM (TNF-alpha , IL-1beta , and IFN-gamma ) and dexamethasone (10-5 M to 10-9 M). Culture supernatants were collected 24 h later and nitrite plus nitrate release was quantitated (n = 4/group). * P < 0.01 vs. control (Ctl). dagger  P < 0.01 vs. CM (by ANOVA).


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Fig. 3.   Western blot analysis of iNOS protein. Cultured rat hepatocytes were exposed to CM (TNF-alpha , IL-1beta , and IFN-gamma ) in the presence or absence of dexamethasone (10-6 M). Cellular protein was collected 18 h after stimulation and SDS-polyacrylamide gel electrophoresis and Western immunoblotting were performed as described in the text. Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells served as positive control. These studies are representative of 2 similiar Western blots.

Dexamethasone suppresses iNOS gene transcription and promoter activation. To examine whether the suppressive effects of GC occurred at the level of transcription, nuclear run-on assays were performed. Shown in Fig. 4 is a representative nuclear run-on experiment looking at the effects of CM with or without dexamethasone on iNOS gene transcription. An average of several experiments indicates that CM stimulation increases iNOS transcription 8- to 10-fold in rat hepatocytes, and the addition of dexamethasone results in a 50% inhibition of iNOS transcription.


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Fig. 4.   Dexamethasone transcriptionally inhibits iNOS gene expression. Hepatocytes were stimulated with CM (TNF-alpha , IL-1beta , and IFN-gamma ) with or without dexamethasone (10-6 M) for 4 h. Nuclei were isolated, and in vitro transcription was performed after which 32P-labeled nuclear RNA was hybridized to a GeneScreen membrane containing 5 µg of immobilized cDNA probes of iNOS. Membranes were quantitated using PhosphorImager scanning. A representative blot is shown. These experiments were performed twice.

We next analyzed the effects of GC on cytokine activation of the iNOS promoter. Several groups have carried out reporter gene analyses of the murine iNOS promoter, and NF-kappa B binding sites within the proximal 1 kb of the 5'-flanking region have been found to mediate LPS- and cytokine-induced iNOS gene expression in macrophages and vascular smooth muscle cells (29, 30). Transfection of hepatocytes with a 1.6-kb murine iNOS promoter-luciferase construct followed 6 h later by CM stimulation resulted in a sixfold increase in luciferase activity over baseline (Fig. 5). The addition of dexamethasone (10-6 M) suppressed CM-induced iNOS promoter activity by 40%, whereas dexamethasone alone had no measurable effects. Treatment with the NF-kappa B inhibitor PDTC completely abrogated CM-stimulated luciferase expression, suggesting that NF-kappa B is required for cytokine activation of the iNOS promoter in rat hepatocytes. Collectively, these data show that GC inhibit iNOS gene transcription by suppressing iNOS promoter activation.


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Fig. 5.   Effect of dexamethasone on iNOS promoter activation. After transient transfection of a 1.6-kb iNOS promoter-luciferase construct, rat hepatocytes were treated with CM (TNF-alpha , IL-1beta , and IFN-gamma ) with or without dexamethasone (10-6 M) or the NF-kappa B inhibitor pyrrolidine dithiocarbamate (PDTC, 100 µM). Luciferase activity was measured after 6 h of cytokine stimulation and normalized to protein content. Values represent multiples of induction of luciferase activity ± SE of cytokine-stimulated cells over control cells. Three experiments were performed in duplicate.

NF-kappa B DNA binding activity is suppressed by dexamethasone. Several studies in other cell types have shown that GC inhibit NF-kappa B activation (2, 25, 26). To investigate the effects of GC on the DNA-binding activity of NF-kappa B in hepatocytes, electromobility shift assays were carried out. A single NF-kappa B complex appeared on CM stimulation of hepatocytes (Fig. 6A). Both dexamethasone and PDTC partially prevented the appearance of this shifted band, indicating that the repressor effect of dexamethasone on cytokine-induced iNOS gene transcription is mediated in part by downregulating NF-kappa B activity. The specificity of the binding was confirmed by cold competition with wild-type and mutant probes (Fig. 6B). Immunodepletion supershift studies with antibodies revealed that the complex was composed of p50 and p65 subunits. Antibodies against C-rel had no effect (data not shown).


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Fig. 6.   Dexamethasone inhibits NF-kappa B binding activity. A: rat hepatocytes were stimulated with CM (TNF-alpha , IL-1beta , and IFN-gamma ), and the effects of dexamethasone (10-6 M) or PDTC (100 µM) on NF-kappa B binding were analyzed. Nuclear extracts were collected 1 h after stimulation and incubated with 32P-end-labeled probes containing a binding site for NF-kappa B. Resulting complexes were resolved by electromobility shift assay. B: specificity was determined by cold competition, wild-type, and mutant oligonucleotides. In addition, supershift experiments using antiserum against the p50 and p65 subunits of NF-kappa B were performed. Three independent gel shifts were performed.

I-kappa Balpha expression is upregulated by cytokines and dexamethasone. Dexamethasone has been shown to upregulate the synthesis of I-kappa Balpha in monocytes and lymphocytes (2, 25) but not in primary endothelial cells or A549/8 cells (3, 15). To analyze the effects of GC on I-kappa Balpha expression in rat hepatocytes, we performed Northern blot analysis for I-kappa Balpha mRNA. Control cells showed basal expression of I-kappa Balpha mRNA, and this was decreased by the addition of CM. When dexamethasone was added alone or with CM there was a two- to threefold upregulation in I-kappa Balpha mRNA (Fig. 7). Consistent with the mRNA findings Western blot analysis revealed basal I-kappa Balpha protein levels that were also decreased by CM and preserved by dexamethasone (Fig. 8). Thus GC positively affect I-kappa Balpha mRNA and protein expression in rat hepatocytes.


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Fig. 7.   Effect of dexamethasone on I-kappa Balpha expression. Shown is a Northern blot analysis (representative of 3 studies) of I-kappa Balpha mRNA collected from rat hepatocytes treated with CM (TNF-alpha , IL-1beta , and IFN-gamma ) for 1 h in the presence or absence of dexamethasone (10-6 M). Equal loading of RNA in each lane was confirmed by 18S ribosomal RNA probing.


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Fig. 8.   Effect of dexamethasone on I-kappa Balpha protein levels. Western blot analysis of I-kappa Balpha protein. Cultured rat hepatocytes were exposed to CM (TNF-alpha , IL-1beta , and IFN-gamma ) in the presence or absence of dexamethasone (10-6 M). Cellular proteins were collected 3 h after stimulation, and SDS-polyacrylamide gel electrophoresis and Western immunoblotting were performed as described in the text. These studies are representative of 3.

Nuclear p65 levels are decreased by dexamethasone. To discern whether the increase in I-kappa Balpha expression resulted in a decrease in p65 nuclear translocation cytosolic and nuclear proteins levels were assayed by Western blot analysis. These studies revealed that nuclear p65 levels were increased in CM-treated hepatocytes and were decreased when dexamethasone was added to CM (Fig. 9), suggesting that p65 is sequestered by the newly formed I-kappa Balpha in the cytoplasm, thereby preventing nuclear translocation of NF-kappa B. Western blot analysis for cytosolic p65 protein showed basal expression in control hepatocytes; both CM alone or CM and dexamethasone increased cytosolic p65 protein levels (data not shown). The lack of a reciprocal change in cytosolic p65 for the CM and dexamethasone group may be due to the already increased level of cytosolic p65 expression and the fact that the cytosolic content reflects the steady state between synthesis and degradation.


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Fig. 9.   Effect of CM and dexamethasone on nuclear p65 protein levels. Cultured rat hepatocytes were exposed to CM (TNF-alpha , IL-1beta , and IFN-gamma ) in the presence or absence of dexamethasone (10-6 M and 10-8 M). Nuclear proteins were collected 3 h after stimulation, followed by Western immunoblotting as described in the text. The autoradiograph shown is representative of 2 similar experiments.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

GC inhibit the expression of many cytokine and immunomodulatory genes, including iNOS (10, 15, 16, 23). In this study we characterized some of the mechanisms involved in GC inhibition of iNOS gene expression in rat hepatocytes. The synthetic GC dexamethasone markedly suppressed cytokine-stimulated iNOS expression and NO synthesis in rat hepatocytes (Figs. 1 and 2). Because iNOS mRNA levels were reduced, it was not surprising that cytokine-induced iNOS protein levels were also decreased by dexamethasone (Fig. 3). Nuclear run-on analysis showed that dexamethasone-mediated iNOS repression in rat hepatocytes occurred at the level of transcription (Fig. 4). The transcriptional suppression by GC was confirmed by reporter gene analysis of a 1.6-kb murine iNOS promoter fragment (Fig. 5). The ~40% suppression of luciferase activity that we measured was less than the 60-75% decrease in mRNA levels, suggesting that dexamethasone may have posttranscriptional effects on iNOS expression in hepatocytes. Although we did not explore the possibility of posttranslational effects, others have shown that GC prolong iNOS mRNA half-life in other cell types (16, 23), and we are currently investigating the possibility that dexamethasone enhances iNOS mRNA stability. A recent study in rat mesangial cells showed that steroids reduced iNOS translational rates and also increased the degradation of iNOS protein (16). Simmons et al. (28) demonstrated an additional regulatory mechanism whereby dexamethasone decreases NO production by limiting L-arginine and tetrahydrobiopterin (BH4) availability in cardiac microvascular endothelial cells.

The transcription factor NF-kappa B is required for LPS and cytokine induction of the murine iNOS promoter (11, 30), and we have also reported that iNOS gene expression is dependent on NF-kappa B in rat hepatocytes (6). The addition of the antioxidant PDTC resulted in an almost complete abrogation of CM-induced luciferase activity, suggesting that NF-kappa B is required for iNOS promoter activation in rat hepatocytes (Fig. 5). Electromobility shift assays demonstrated that NF-kappa B DNA binding activity was suppressed by dexamethasone (Fig. 6). Two major mechanisms appear to be at work in GC repression of NF-kappa B. The first involves the upregulation of I-kappa Balpha expression by dexamethasone (Figs. 7 and 8), resulting in a reduction of the amount of NF-kappa B that translocates to the nucleus (Fig. 9). This is in agreement with the findings of Scheinman et al. (25) and Auphan et al. (2) who reported that dexamethasone increased I-kappa Balpha expression in HeLa and Jurkat cells, respectively. The second mechanism involves cross-coupling and physical association between the activated GC receptor and NF-kappa B, leading to the inhibition of DNA transactivation (24, 26). The relative importance of each mechanism appears to be cell-type dependent. For instance, I-kappa Balpha levels in endothelial cells and lung epithelial cells do not seem to be influenced significantly by dexamethasone (3, 15) compared with monocytes and lymphocytes (2, 25). Our studies show that I-kappa Balpha mRNA expression was upregulated by dexamethasone (Fig. 7) and that I-kappa Balpha protein levels are stabilized (Fig. 8). This suggests that the decrease in the DNA-binding activity of NF-kappa B in rat hepatocytes is due in part to the prevention of NF-kappa B translocation to the nucleus. The fact that nuclear p65 levels were diminished in our study confirms that newly formed I-kappa Balpha decreases the nuclear translocation of p65, resulting in a reduction in NF-kappa B-DNA binding activity.

In summary, dexamethasone inhibits iNOS gene transcription in rat hepatocytes by increasing I-kappa Balpha expression and decreasing NF-kappa B activity. This documents the importance of NF-kappa B in the regulation of iNOS gene expression and suggests that targeting NF-kappa B activation may be a more specific way of modulating NO production.

    ACKNOWLEDGEMENTS

We thank Debra Williams for excellent technical assistance, Charles J. Lowenstein for providing the murine macrophage iNOS promoter, and Gary Nabel for the I-kappa Balpha probe.

    FOOTNOTES

M. E. de Vera and B. S. Taylor contributed equally to the work in this study.

This work was presented in part at the 30th Annual Meeting of the Association for Academic Surgery, Chicago, IL, November 14, 1996.

This study was supported by National Institute of General Medical Sciences Grants GM-52021, GM-44100, and GM-37753

Address for reprint requests: D. A. Geller, Dept. of Surgery, 497 Scaife Hall, Pittsburgh, PA 15261.

Received 11 March 1997; accepted in final form 22 August 1997.

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Abstract
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Methods
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Discussion
References

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AJP Gastroint Liver Physiol 273(6):G1290-G1296
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