A Dual Mechanism Mediates Repression of NF-{kappa}B Activity by Glucocorticoids

S. Wissink, E. C. van Heerde, B. van der Burg and P. T. van der Saag

Hubrecht Laboratory Netherlands Institute for Developmental Biology 3584 CT Utrecht, The Netherlands


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Repression of nuclear factor (NF)-{kappa}B-dependent gene expression is one of the key characteristics by which glucocorticoids exert their antiinflammatory and immunosuppressive effects. In vitro studies have shown protein-protein interactions between NF-{kappa}B and the glucocorticoid receptor, possibly explaining their mutual repression of transcriptional activity. Furthermore, glucocorticoid-induced transcription of I{kappa}B{alpha} was presented as a mechanism in mediation of immunosuppression by glucocorticoids. At present, the relative contribution of each mechanism has not been investigated. We show that dexamethasone induced I{kappa}B{alpha} gene transcription in human pulmonary epithelial A549 cells. However, this enhanced I{kappa}B{alpha} synthesis did not cause repression of NF-{kappa}B DNA-binding activity. In addition, dexamethasone was still able to inhibit the expression of NF-{kappa}B target genes (cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of protein synthesis. Furthermore, we show that the antihormone RU486 did not induce I{kappa}B{alpha} expression. However, RU486 was still able to induce, albeit less efficiently, both glucocorticoid- and progesterone receptor-mediated repression of endogenous NF-{kappa}B target gene expression in A549 cells and the breast cancer cell line T47D, respectively. Taken together, these results indicate that induced I{kappa}B{alpha} expression accounts for only part of the repression of NF-{kappa}B activity by glucocorticoids and progestins. In addition, protein-protein interactions between NF-{kappa}B and the glucocorticoid or progesterone receptor, resulting in repression of NF-{kappa}B activity, seem also to be involved. We therefore conclude that NF-{kappa}B activity is repressed via a dual mechanism involving both protein-protein interactions and induction of I{kappa}B{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Glucocorticoids are widely used as immunosuppressive and antiinflammatory agents. They have been shown to inhibit the expression of cytokines, adhesion molecules, and enzymes involved in the inflammatory process (1). Glucocorticoids mediate these effects through an intracellular receptor, the glucocorticoid receptor (GR), a member of the steroid/thyroid hormone receptor superfamily. Upon hormone binding, the cytoplasmic GR can enter the nucleus, dimerize, and bind to specific DNA sequences, the glucocorticoid response elements (GREs), and activate transcription of target genes (2). However, the antiinflammatory and immunosuppressive effects of glucocorticoids are achieved by inhibition rather than by activation of target gene expression. Many negatively regulated genes involved in the inflammatory response do not contain GREs in their promoter and therefore must be negatively regulated by a different mechanism, e.g. through transcriptional interference between GR and other transcription factors, such as AP-1 and nuclear factor (NF)-{kappa}B (3, 4, 5).

The NF-{kappa}B/Rel family of transcription factors regulates the expression of many genes involved in immune and inflammatory responses. NF-{kappa}B was originally identified as a heterodimer of NF-{kappa}B1 and RelA (6), but a variety of other {kappa}B/Rel homo- and heterodimers have now been described. NF-{kappa}B is present in an inactive state in the cytoplasm, sequestered by an inhibitor protein, designated I{kappa}B. After stimulation of the cells, I{kappa}B becomes phosphorylated, ubiquitinated, and subsequently degraded (7). As a result, NF-{kappa}B is free to translocate to the nucleus and activate transcription of target genes. In the nucleus, NF-{kappa}B can also induce the synthesis of I{kappa}B{alpha}, which terminates the NF-{kappa}B response, explaining its transient nature (8).

Glucocorticoids were shown to be potent inhibitors of NF-{kappa}B activation. In addition, the NF-{kappa}B subunit, RelA, has been shown to physically interact with GR in vitro (9, 10, 11, 12) as well as with other steroid receptors, such as the estrogen receptor (ER; Ref.13), the progesterone receptor (PR; Ref.14), and the androgen receptor (AR; Ref.15). Since it has been demonstrated that NF-{kappa}B was also able to repress ligand-dependent activation of steroid receptor-regulated promoters in vitro, a mutually inactive complex formed either by direct protein-protein interaction of the receptor and RelA or via a third partner has been proposed (9, 10, 11, 12, 13, 14, 15).

A second independent mechanism through which glucocorticoids could repress NF-{kappa}B activity has been described (16, 17). Glucocorticoids were shown to induce transcription of the I{kappa}B{alpha} gene in HeLa cells, monocytic cells, and T-lymphocytes. This induction resulted in increased synthesis of I{kappa}B{alpha} protein, which is able to interact with activated NF-{kappa}B, thereby terminating the NF-{kappa}B response. However, Brostjan et al. (18) reported that glucocorticoid-mediated repression of NF-{kappa}B activity did not involve induction of I{kappa}B{alpha} synthesis in endothelial cells.

The physiological relevance of both these mechanisms has not been clearly established, and it remains unclear which pathway represents the major mechanism. Therefore, we investigated the contribution of each mechanism to the antiinflammatory and immunosuppressive activity of glucocorticoids. Here we show that dexamethasone (Dex) induces expression of I{kappa}B{alpha} in human pulmonary epithelial A549 cells. Furthermore, we show that Dex is able to inhibit the expression of two endogenous NF-{kappa}B target genes, cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) partially independent of newly synthesized I{kappa}B{alpha}. On the basis of these results, we conclude that glucocorticoids repress NF-{kappa}B activity in A549 cells via a dual mechanism, which involves both protein-protein interaction and induction of I{kappa}B{alpha}.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Glucocorticoids Induce I{kappa}B{alpha} in A549 Cells
Glucocorticoids have been described to induce I{kappa}B{alpha} synthesis in monocytic and lymphocytic cells (16, 17), but not in endothelial cells (18). To determine whether glucocorticoids increased I{kappa}B{alpha} mRNA in human pulmonary epithelial A549 cells, Northern blotting analysis was performed on mRNA derived from these cells treated with Dex for increasing periods of time. As shown in Fig. 1Go, Dex induced an increase in I{kappa}B{alpha} mRNA in these cells, which peaked by 2–8 h (3- to 4-fold) and remained elevated to 24 h (2-fold).



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Figure 1. Dex-Induced I{kappa}B{alpha} Gene Expression in A549 Cells

A549 cells were treated for increasing periods of time with 1 µM Dex. Total RNA was isolated and Northern blotting analysis was performed. Upper panel shows blot sequentially hybridized with a probe containing I{kappa}B{alpha} and GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the transcripts of I{kappa}B{alpha} and GAPDH are indicated. Lower panel shows PhosphoImager (Molecular Dynamics, Sunnyvale, CA) quantification of I{kappa}B{alpha} hybridization signal. Fold induction indicates hybridization signal for cells treated with Dex over untreated cells. Error bars indicate SE.

 
Repression of NF-{kappa}B-Regulated Genes Is Not Only Mediated by I{kappa}B{alpha} Induction
To investigate the mechanism(s) involved in repression of NF-{kappa}B activity by glucocorticoids, we determined the repression by Dex of NF-{kappa}B-regulated genes in the absence of I{kappa}B{alpha} protein synthesis. Treatment of A549 cells with interleukin (IL)-1ß resulted in a 5-fold increase in I{kappa}B{alpha} mRNA expression, and a 3-fold induction was observed after treatment with Dex. The combination of IL-1ß and Dex showed a similar induction as IL-1ß treatment alone (Fig. 2AGo, lanes 1–4). Dex-mediated I{kappa}B{alpha} induction and I{kappa}B{alpha} resynthesis after IL-1ß-induced degradation can be observed for I{kappa}B{alpha} protein (Fig. 2BGo, lanes 1–4), indicating that IL-1ß and Dex can induce both I{kappa}B{alpha} transcription and protein synthesis in A549 cells. Cycloheximide (CHX), an inhibitor of protein synthesis, also induced I{kappa}B{alpha} mRNA expression (5-fold; Fig. 2AGo, lane 5), as has been observed for other NF-{kappa}B target genes, e.g. ICAM-1 (19). CHX together with IL-1ß superinduced I{kappa}B{alpha} mRNA (29-fold; Fig. 2AGo, lane 6), whereas CHX in combination with Dex resulted in a weaker induction (9-fold; Fig. 2AGo, lane 7). No resynthesis of I{kappa}B{alpha} protein could be observed in the presence of CHX (Fig. 2BGo, lanes 6 and 8).



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Figure 2. Repression of IL-1ß-Induced COX-2 and ICAM-1 Expression by Dex in the Absence of Protein Synthesis

A549 cells were treated with IL-1ß (I; 100 U/ml) and Dex (D; 1 µM) in the absence or presence of CHX (10 µg/ml) for 6 h. A, Total RNA was isolated and Northern blotting analysis was performed. Left panel shows blot sequentially hybridized with a probe containing I{kappa}B{alpha} and GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the transcripts of I{kappa}B{alpha} and GAPDH are indicated. Right panel shows quantification of I{kappa}B{alpha} hybridization signal. Fold induction indicates hybridization signal for cells treated with IL-1ß, Dex, and/or CHX over untreated cells. B, Whole cell extracts were prepared and fractionated on a 12.5% SDS-PAGE, and Western blotting analysis was performed. Blots were immunostained with a polyclonal antibody to I{kappa}B{alpha}. I{kappa}B{alpha} was visualized after incubation with a peroxidase-conjugated second antibody and ECL. C, Total RNA was isolated and Northern blotting analysis was performed. Left panel shows blots sequentially hybridized with a probe containing COX-2, ICAM-1, or GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the transcripts of COX-2, ICAM-1, and GAPDH are indicated. Right panel shows PhosphoImager quantification of COX-2 (upper panel) and ICAM-1 (lower panel) hybridization signal. The relative induction, indicating hybridization signal of cells treated with IL-1ß over untreated cells, in the absence (black bars) and presence (white bars) of CHX is set at 100%. Error bars indicate SE.

 
To study whether protein synthesis was necessary for the repressive effect of Dex on endogenous NF-{kappa}B target gene expression, COX-2 (20) and ICAM-1 (21) mRNA expression was investigated. As shown in Fig. 2CGo, IL-1ß induced the expression of both COX-2 and ICAM-1, which could be strongly repressed by Dex (lanes 2 and 4). Interestingly, in the absence of protein synthesis and I{kappa}B{alpha} protein induction, Dex was still able to repress IL-1ß-induced COX-2 and ICAM-1 expression (lanes 6 and 8), although the repression was less strong than in the absence of CHX (Fig. 2CGo, right panel). This suggests that induction of I{kappa}B{alpha} plays a role, but is obviously not the only mechanism mediating the repressive effect of Dex.

NF-{kappa}B DNA-Binding Activity Is Not Inhibited by Dex
It has been shown that Dex-induced I{kappa}B{alpha} was able to inhibit NF-{kappa}B activity by preventing nuclear translocation and DNA binding (16, 17). To determine whether Dex-induced I{kappa}B{alpha} could block NF-{kappa}B DNA-binding activity in A549 cells, the cells were pretreated with Dex for 15 h and subsequently treated with IL-1ß for 1 h. NF-{kappa}B binding to the radiolabeled probe containing the human immunodeficiency virus (HIV) long terminal repeat (LTR) was observed with nuclear extracts from cells treated with IL-1ß (Fig. 3Go, lane 3). Pretreatment with Dex did not result in inhibition of binding (lane 5). The same results were obtained after pretreatment with Dex for 5 h (data not shown). The observed binding activity was specific because it could be competed with a 100-fold excess of unlabeled {kappa}B probe but not with a mutant {kappa}B probe (lanes 6 and 7). The {kappa}B-binding activity was composed mostly of NF-{kappa}B1 and RelA heterodimer (NF-{kappa}B) as determined by supershift analysis (lanes 8 and 9). The faster migrating complexes appeared to contain NF-{kappa}B1 protein in other combinations.



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Figure 3. No Effect of Dex on DNA-Binding Activity of NF-{kappa}B

A549 cells were pretreated with Dex (1 µM) for 15 h and stimulated with Il-1ß (100 U/ml) for 1 h. Subsequently, nuclear extracts were analyzed by electrophoretic mobility shift assay with 32P-labeled probe containing the {kappa}B site from the HIV LTR. Specificity of binding was demonstrated by competition with 100-fold molar excess of unlabeled probe containing the {kappa}B site from the HIV LTR (lane 6) or a mutant {kappa}B site (lane 7). {kappa}B-Binding complexes were characterized by supershift analysis using antisera to NF-{kappa}B1 (lane 8) and RelA (lane 9).

 
These results show that, in A549 cells, Dex-induced I{kappa}B{alpha} is not able to prevent nuclear translocation or DNA binding of NF-{kappa}B, suggesting a minor contribution of I{kappa}B{alpha} in the mechanism of repression.

Antihormones Repress NF-{kappa}B Activity without Induction of I{kappa}B{alpha}
We recently showed that the antiglucocorticoid/antiprogestin RU486 was able to induce PR-mediated repression of RelA activity (14). To examine the repression of NF-{kappa}B target genes by RU486-occupied GR, we transiently transfected COS-1 cells with a reporter construct containing four NF-{kappa}B sites from the ICAM-1 promoter. Cotransfection with expression vector encoding RelA (20 ng) resulted in an induction of luciferase activity, which could be repressed by cotransfection of an expression vector for GR (200 ng) and treatment of the cells with RU486 (Fig. 4AGo). The repressive activity of GR was only slightly reduced with an RU486-occupied receptor (~65% repression) as compared with a receptor occupied with the agonist Dex (~85% repression).



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Figure 4. Effects of the Antiglucocorticoid RU486 on GR-RelA Interaction

A, COS-1 cells were transiently transfected with 0.4 µg 4xNF-kB(IC)tkluc reporter, 20 ng RelA, and 200 ng GR expression constructs and treated with Dex or RU486 for 24 h. The concentration of Dex or RU486 used was 1 µM (black bars) or 0.1 µM (hatched bars). Depicted is the induction of luciferase activity evoked by RelA over cells transfected with empty expression vector. B, COS-1 cells were transiently transfected with 0.4 µg 2xGREtkluc reporter and 20 ng GR expression construct and treated with Dex or RU486 for 24 h. The concentration of Dex or RU486 used was 1 µM (black bars) or 0.1 µM (hatched bars). Fold induction indicates reporter activity in cells treated with Dex or RU486 over untreated cells. Error bars indicate SE.

 
In addition to being antagonistic, RU486 has also partial agonistic activity with respect to PR- and GR-mediated transcription (22). To investigate the partial agonistic activity of RU486, COS-1 cells were transfected with a reporter construct containing two GREs. As shown in Fig. 4BGo, cotransfection of an expression vector encoding GR resulted in a hormone-dependent induction of luciferase activity after treatment of the cells with Dex (65-fold) and very low induction upon RU486 treatment (3-fold). This indicates that repression by RU486 is not correlated with transcriptional activation mediated by RU486.

In A549 cells, both Dex and RU486 were able to repress IL-1ß-induced COX-2 mRNA expression, although the anti-hormone was less efficient (Fig. 5AGo, lanes 1–4). As expected, the antagonist RU486 was unable to induce I{kappa}B{alpha} mRNA (Fig. 5AGo, lane 6) or I{kappa}B{alpha} protein (Fig. 5CGo, lanes 4 and 6) in these cells, indicating that I{kappa}B{alpha} synthesis is not necessary for repression of NF-{kappa}B activity by RU486.



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Figure 5. Repression of Il-1ß-Induced NF-{kappa}B Target Gene Expression by RU486

A, A549 cells were treated with Il-1ß (I; 100 U/ml) and Dex (D; 1 µM) or RU486 (R; 1 µM) for 24 h. Total RNA was isolated and Northern blotting analysis was performed. Left panel shows blot sequentially hybridized with a probe containing COX-2 or I{kappa}B{alpha} and GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the transcripts of COX-2, I{kappa}B{alpha}, and GAPDH are indicated. Right panel shows quantification of COX-2 (black bars) and I{kappa}B{alpha} (white bars) hybridization signal. Fold induction indicates hybridization signal for cells treated with IL-1ß, Dex, and/or RU486 over untreated cells. B, T47D cells were treated with Il-1ß (I; 100 U/ml) and Org2058 (O; 10 nM) or RU486 (R; 1 µM) for 24 h. Total RNA was isolated and Northern blotting analysis was performed. Left panel shows blot sequentially hybridized with a probe containing ICAM-1 or I{kappa}B{alpha} and GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the transcripts of ICAM-1, I{kappa}B{alpha}, and GAPDH are indicated. Right panel shows PhosphoImager quantification of ICAM-1 (black bars) and I{kappa}B{alpha} (white bars) hybridization signal. Fold induction indicates hybridization signal for cells treated with IL-1ß, Org2058, and/or RU486 over untreated cells. Error bars indicate SE. C, A549 cells were treated as in panel A. Whole cell extracts were prepared and fractionated on a 12.5% SDS-PAGE, and Western blotting analysis was performed. Blots were immunostained with a polyclonal antibody to I{kappa}B{alpha}. I{kappa}B{alpha} was visualized after incubation with a peroxidase-conjugated second antibody and ECL. D, T47D cells were treated as in panel B, and Western blotting was performed as in panel C.

 
Previously, we described mutual repression between progesterone receptor (PR) and RelA in the breast tumor cell line T47D (14). To investigate whether RU486 could also repress NF-{kappa}B target genes in these cells containing endogenous PR, the same experiment was performed in T47D cells. Both the progestagen Org2058 and the progesterone antagonist RU486 were able to repress IL-1ß-induced ICAM-1 expression (Fig. 5BGo, lanes 3 and 4). Whereas Org2058 induced I{kappa}B{alpha} mRNA expression, RU486 was unable to induce I{kappa}B{alpha} mRNA in these cells (Fig. 5BGo, lanes 5 and 6), although a small increase in I{kappa}B{alpha} protein could be observed (Fig. 5DGo, lanes 4 and 6). The fact that RU486-occupied receptors are able to repress NF-{kappa}B target gene expression in the absence of induced I{kappa}B{alpha} expression indicates that the repression of endogenous NF-{kappa}B target genes by GR and PR is at least partially mediated by an I{kappa}B{alpha}-independent mechanism.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
NF-{kappa}B plays a pivotal role in the regulation of a variety of genes involved in immune and inflammatory responses. Therefore, inhibition of NF-{kappa}B activity can account for many of the immunosuppressive and antiinflammatory activities of glucocorticoids. In the present study, we show that glucocorticoids can control immune response and inflammation by repressing NF-{kappa}B activity via a dual mechanism.

First, Dex was shown to induce I{kappa}B{alpha} mRNA expression in A549 cells, which has also been reported for HeLa cells, monocytic cells, and T lymphocytes (16, 17). The fact that this induction occurs in the presence of CHX suggests that glucocorticoids activate I{kappa}B{alpha} gene transcription directly. For these cells it has been shown that Dex-induced I{kappa}B{alpha} was able to inhibit NF-{kappa}B activity by preventing nuclear translocation and DNA binding of NF-{kappa}B (16, 17). However, in A549 cells we observed no inhibition of NF-{kappa}B DNA-binding activity by Dex, suggesting that in this case the Dex-induced I{kappa}B{alpha} was not able to efficiently sequester NF-{kappa}B in the cytoplasm and to prevent DNA binding. Similar results have been described for endothelial cells (18). Nevertheless, repression of NF-{kappa}B activity by protein-protein interaction can occur via tethering of GR to NF-{kappa}B in its DNA-bound form, without affecting DNA binding.

Second, we showed that in the absence of I{kappa}B{alpha} protein synthesis, Dex was still able to repress IL-1ß-induced expression of the NF-{kappa}B target genes, COX-2 and ICAM-1. The repressive activity of Dex in the presence of CHX was less strong than in the absence of CHX, providing evidence for more than one mechanism involved in Dex-mediated repression of NF-{kappa}B activity. In contrast to this observation, Auphan et al. (16) and Scheinman et al. (17) showed that in the presence of CHX, inhibition of NF-{kappa}B DNA binding activity could no longer be observed, suggesting a requirement of I{kappa}B{alpha} for repression of NF-{kappa}B activity. However, we showed that in A549 cells, I{kappa}B{alpha} was unable to prevent NF-{kappa}B DNA binding, suggesting that inhibition of NF-{kappa}B DNA binding is not essential for repression of NF-{kappa}B target genes in these cells.

As we showed previously for the repression of RelA activity by PR (14), we found that the antiprogestin/antiglucocorticoid RU486 was also able to induce GR-mediated repression of RelA activity. In addition, RU486 could repress IL-1ß-induced expression of COX-2 in A549 cells, albeit less efficiently than the agonist, Dex. Furthermore, RU486 was able to induce PR-mediated repression of the IL-1ß-induced expression of the NF-{kappa}B target gene, ICAM-1, in T47D cells. In contrast to the agonists, Dex and Org2058, RU486 was not able to induce I{kappa}B{alpha} synthesis in both cell lines. Taken together, these findings demonstrate that in addition to Dex- and Org2058-induced I{kappa}B{alpha} synthesis, a second mechanism must be involved in the repression of NF-{kappa}B activity by both glucocorticoids and progestins. Furthermore, Dex-mediated repression of NF-{kappa}B activity has been shown to be independent of I{kappa}B{alpha} synthesis in endothelial cells (18) and in rat kidney epithelial cells (23), which again suggests that the induction of I{kappa}B{alpha} is not a universal mechanism explaining NF-{kappa}B repression by glucocorticoids in all cell types. In addition to the induction of IkB{alpha} synthesis, glucocorticoids have been shown to repress transcription of target genes by transcriptional interference, a mechanism likely to involve protein-protein interactions between GR and NF-{kappa}B (9, 10, 11, 12). In this way, GR can interfere with the transcriptional activity of NF-{kappa}B by 1) forming a complex with NF-{kappa}B and inhibiting its DNA-binding activity or by 2) forming a complex with NF-{kappa}B in its DNA-bound form without affecting DNA binding, or by 3) contacting a cofactor that is bound to NF-{kappa}B and thereby inhibiting the transactivation potential of NF-{kappa}B. Further experiments will have to be carried out to determine which of the mechanisms is involved. GR has been found to associate in vitro with NF-{kappa}B either in a manner leading to inhibition of DNA binding (9, 10) or without affecting DNA binding (18, 21). However, previous reports regarding a decreased AP-1 DNA-binding activity in the presence of GR in vitro (24) could not be confirmed by in vivo footprinting studies (25). Therefore in vivo footprinting analysis could be used to study NF-{kappa}B binding to DNA in the presence of GR. Similar to GR, other steroid receptors, such as ER (13), PR (14), and AR (15), have also been shown to physically interact with NF-{kappa}B in vitro and inhibit its transcriptional activity, suggesting an important role for protein-protein interactions in repression of NF-{kappa}B activity by steroids.

In contrast to the previously described mechanism, which indicates that inhibition of NF-{kappa}B activity does not rely on interaction between GR and NF-{kappa}B but is predominantly based on induction of I{kappa}B{alpha} expression, we provide evidence that both mechanisms, resulting in repression of NF-{kappa}B activity, contribute to the antiinflammatory action of glucocorticoids. The involvement of both these mechanisms emphasizes the importance of multiple levels of regulation of NF-{kappa}B activity by glucocorticoids in modulation of the antiinflammatory response. This sustains the possibility of developing ligands that specifically activate the repression function of GR and that may therefore be more efficient in the treatment of inflammatory diseases without undesirable side effects.


    MATERIALS and METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Special Reagents and Antibodies
Dexamethasone and cycloheximide were obtained from Sigma Chemical Co. (St. Louis, MO). The progestin Org2058 was provided by Organon International (Oss, The Netherlands). IL-1ß was obtained from NCI Biological Resources Branch (Frederick, MD). RU486 was obtained form Roussel-Uclaf (Romainville, France). Polyclonal antibody against I{kappa}B{alpha} was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The polyclonal antibody against the N terminus of RelA was from Santa Cruz Biotechnology (Santa Cruz, CA). Antiserum 2 against NF-{kappa}B1 was a kind gift of Dr. A. Israël (Paris, France).

Cell Culture
Human pulmonary epithelial A549 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD). Cells were cultured in DMEM from Life Technologies Inc. (Gaithersburg, MD), buffered with bicarbonate, and supplemented with 7.5% FCS from Integro (Linz, Austria). Monkey COS-1 cells (ATCC) and human breast tumor T47D cells, originally provided by Dr. R. L. Sutherland (Sydney, Australia), were cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium (DF; Life Technologies Inc.), buffered with bicarbonate, and supplemented with 7.5% FCS. Dextran-coated charcoal-FCS was prepared by treatment of FCS with dextran-coated charcoal to remove steroids, as described previously (26).

Plasmids and Transient Transfections
The luciferase reporter plasmid (4xNF-{kappa}B(IC)tkluc) containing four NF-{kappa}B sites from the ICAM-1 promoter was constructed by ligating two copies of the annealed oligonucleotides (5'-AGCTTATGGAAATTCCGAGATCATGGAAATTCCGAC-3') and (5'-AGCTGTCGGAATTT-CCATGATCTCGGAATTTCCATA-3'), containing two NF-{kappa}B sites from the ICAM-1 promoter and HindIII linkers, into the HindIII site of ptkluc. The reporter plasmid 2xGREtkluc has been described elsewhere (27). The CMV4 expression vectors containing full-length cDNAs encoding human RelA and GR have been described previously (11). For transient transfections, COS-1 cells were cultured in 24-well plates and transfected using calcium-phosphate coprecipitation with 0.4 µg luciferase reporter, 0.6 µg PDMlacZ, and the indicated amount of expression plasmids. pBluescript was added to obtain a total amount of 2 µg DNA/well. After 16 h, the medium was refreshed and hormone was added. Cells were harvested 24 h later and assayed for luciferase activity using the Luclite luciferase reporter gene assay kit (Packard Instruments, Meriden, CT) according to the manufacturer’s protocol and the Topcount liquid scintillation counter (Packard Instruments). Values were corrected for transfection efficiency by measuring ß-galactosidase activity (28).

Northern Blotting Analysis
A549 cells were cultured in 10-cm dishes, treated as indicated, and harvested. T47D cells were cultured in DF+, supplemented with 5% dextran-coated charcoal-FCS, and treated as A549 cells. Total RNA was isolated using the acid-phenol method of Chomczynski and Sacchi (29). Twenty micrograms of RNA were fractionated on a 0.8% agarose gel and transferred to Hybond C-extra membranes by capillary transfer using 10xstandard sodium citrate (SSC). The blots were baked under vacuum for 2 h at 80 C. The blots were hybridized to cDNA probes overnight at 42 C in hybridization buffer. Subsequently, blots were washed with 2xSSC/0.1%SDS, 1xSSC/0.1%SDS, 0.2xSSC/0.1%SDS, and 0.1xSSC/0.1%SDS when necessary. cDNA probes were labeled with [{alpha}32P]dCTP by random priming according to the manufacturer’s protocol (Amersham Pharmacia Biotech., Rainham, Essex, UK). As probes for Northern blotting, a 1-kb HindIII fragment of the I{kappa}B{alpha} cDNA, a 1.8-kb XbaI fragment of the ICAM-1 cDNA, a 1-kb EcoRI/XhoI fragment of the murine COX-2 cDNA, a kind gift from Dr. H. Herschman, and a 1.4-kb PstI fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used.

Western Blotting Analysis
For isolation of whole cell extracts A549 cells were cultured in 5-cm dishes, treated as described, and harvested in buffer containing 50 mM Tris (pH 7.4), 50 mM NaCl, 0.5% nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin at 4 C. Subsequently, cells were centrifuged for 15 min at 4 C, and protein concentration of the supernatant was determined by the Bio-Rad (Richmond, CA) protein assay according to the manufacturers protocol. Twenty five micrograms of extract were separated on SDS-PAGE gels and transferred to Immobilon (Milipore, MA). For the polyclonal antibody against I{kappa}B{alpha} (Upstate Biotechnology Inc.), all incubations were carried out according to the manufacturer’s protocol. Immunoreactive bands were visualized with enhanced chemiluminescence (ECL) (Amersham).

Electrophoretic mobility shift assay (EMSA)
A549 cells were cultured in 10-cm dishes and pretreated with Dex (1 µM) for 15 h and with IL-1ß (100 U/ml) for 1 h. Cells were harvested and nuclear extracts were prepared according to Lee et al. (30). A double-stranded oligonucleotide containing the {kappa}B site from the HIV LTR (5'-agcttcagaGGGGACTTTCCgagagg-3') was labeled with [32P]dCTP using the Klenow fragment of DNA polymerase I. Labeled probe was separated from unincorporated nucleotides by gel filtration on Sephadex G-50 spin columns and eluted overnight from 5% polyacrylamide gels in 0.5 M CH3COONH4/1 mM EDTA at 37 C. Nuclear extracts of A549 cells (10 µg per assay) were incubated with 10.000 cpm of probe (0.1 to 0.5 ng) and 1 µg poly(dI-dC), respectively, for 30 min at room temperature in a total reaction mixture of 20 µl containing 20 mM HEPES, pH 7.5, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 µg/µl BSA. Samples were loaded on a 5% polyacrylamide (29:1) gel, containing 0.25 x TBE as running buffer, and the gel was run at room temperature at 150 V for 2–2.5 h. Excess unlabeled competitor oligonucleotide, containing the HIV {kappa}B site or a mutant {kappa}B site (5'-AGCTTGTAAATTGTGGAGC-3') or antisera to NF-{kappa}B1 and RelA, was added to the reaction mixture before addition of labeled probe. After electrophoresis, gels were dried and autoradiographed for 1 day at -80 C.


    ACKNOWLEDGMENTS
 
We thank J. Heinen and F. Vervoordeldonk for photographic reproductions.

Note added in Proof. Recently two papers have appeared reporting findings similar to those reported here: Heck et al. (1997) EMBO J 16:4698–4707; de Bosscher et al. (1997) Proc Natl Acad Sci USA 94:13504–13509.


    FOOTNOTES
 
Address requests for reprints to: Dr. P. T. van der Saag, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

This research was supported by grants from the Netherlands Asthma Foundation (92.96), the European Community (BIOMED. 2, PL 95–1358), and Boehringer Ingelheim GmbH.

Received for publication June 2, 1997. Revision received November 11, 1997. Accepted for publication December 18, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 

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