Dexamethasone regulates AP-1 to repress TNF-{alpha} induced MCP-1 production in human glomerular endothelial cells

Su-Kil Park1, Won Seok Yang1, Nam Jung Han1, Sang Koo Lee1, Hanjong Ahn2, In Kyu Lee4, Joong Yeol Park1, Ki-Up Lee1 and Jae Dam Lee3

1Department of Internal Medicine, 2Department of Urology and 3Department of Biochemistry, Asan Medical Center, College of Medicine, University of Ulsan, Seoul and 4Department of Internal Medicine, Keimyung University School of Medicine, Taegue, Korea

Correspondence and offprint requests to: Su-Kil Park, MD, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea. Email: skpark{at}www.amc.seoul.kr



   Abstract
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Background. Glomerular endothelial cells play a role in the pathogenesis of glomerulonephritis by producing chemotactic factors. We investigated the role of NF-{kappa}B and AP-1 in tumour necrosis factor {alpha} (TNF-{alpha}) induced monocyte chemoattractant protein 1 (MCP-1) production in cultured human glomerular endothelial cells (HGEC). We also examined whether or not these processes could be modified by glucocorticoid.

Methods. MCP-1 protein and mRNA levels were measured by ELISA and northern blot. NF-{kappa}B and AP-1 binding activity were assessed by electrophoretic mobility shift assay. Cytosolic I{kappa}B{alpha} and nuclear p65 protein were evaluated by western blot. For specific inhibition of NF-{kappa}B or AP-1, we used a decoy oligodeoxynucleotide.

Results. TNF-{alpha} (10 ng/ml) increased MCP-1 mRNA expression in HGEC and also the release of MCP-1 protein into culture media. These effects could be partially inhibited by dexamethasone (10 nM). TNF-{alpha} induced MCP-1 production appeared to be NF-{kappa}B and AP-1 interdependent, based on the following results. (i) TNF-{alpha} increased NF-{kappa}B and AP-1 binding activity. (ii) Both NF-{kappa}B decoy oligodeoxynucleotide and AP-1 decoy oligodeoxynucleotide partially suppressed TNF-{alpha} induced MCP-1 mRNA expression. On the other hand, dexamethasone decreased TNF-{alpha} induced DNA-binding activity of AP-1 without an effect on the DNA-binding activity of NF-{kappa}B, cytosolic I{kappa}B{alpha} degradation or p65 nuclear translocation.

Conclusions. These data demonstrate that while TNF-{alpha} induced MCP-1 production is mediated by the cooperative action of NF-{kappa}B and AP-1 in HGEC, dexamethasone represses TNF-{alpha} induced MCP-1 production via suppression of AP-1 binding activity.

Keywords: AP-1; decoy oligonucleotide; dexamethasone; endothelial cells; MCP-1; NF-{kappa}B



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Acute or chronic inflammatory renal disease including glomerulonephritis develops by the process of leukocyte trafficking from the peripheral circulation into tissue spaces, as in other inflammation. Leukocytes are recruited along the chemotactic gradient to the site of inflammation. Glomerular endothelial cells produce and release monocyte chemoattractant protein-1 (MCP-1), a chemotactic peptide for monocytes, in response to proinflammatory stimuli such as TNF-{alpha}, IL-1ß or lipopolysaccharides [1,2]. Glucocorticoid, a therapeutic agent for a certain form of glomerulonephritis, was shown to decrease the production of MCP-1 [3] in human fibrosarcoma cell lines, but there was also a report that dexamethasone had no effect on TNF-{alpha} induced expression of MCP-1 in bovine glomerular endothelial cells [1].

In the transcription of a gene, gene-specific transcription factors bind to specific recognition sites within the regulatory region of the gene and regulate the initiation of transcription [4]. For the induction of MCP-1, transcription factor NF-{kappa}B has been reported to be important [5], but several other studies suggest that both transcription factors NF-{kappa}B and AP-1 are required for MCP-1 gene induction [6,7].

Glucocorticoid is known to mediate repression of NF-{kappa}B [8], though not all of the studies agree with this result through experiments using other cell types [912], and there are also several studies showing that glucocorticoid could suppress inflammation by inhibition of AP-1 activity [13,14].

So far, little of the intracellular signalling process in MCP-1 production and the effect of glucocorticoid on this process in glomerular endothelial cells have been evaluated. In the present study, we investigate whether NF-{kappa}B and/or AP-1 mediate TNF-{alpha} induced production of MCP-1 in cultured human glomerular endothelial cells (HGEC), and whether it can be modified by glucocorticoid.



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Human glomerular endothelial cell cultures
We isolated HGEC from a normal portion of nephrectomized tissues from patients with renal cell carcinoma, as described previously [15]. In brief, after passing the minced renal cortex through 250 µm sieves, the glomerular suspension was filtered sequentially through 200 and 150 µm sieves. Then the suspension was passed through a 25 gauge needle five times to rupture the Bowman's capsules. After resuspension in Waymouth MB 752/1 supplemented with 20% FCS, fetuin (100 mg/ml; Gibco Laboratories), glutamine (2 mM; Gibco Laboratories) and antibiotics (basal culture medium), the pellet was incubated for 30 min at 37°C in medium containing 6 mg/ml collagenase type III (Worthington Biomedical, NJ), and filtered sequentially through 100 and 50 µm sieves. Glomerular segments retained by the 50 µm filter were collected and plated onto two fibronectin-coated surfaces (60 mm dish) in basal culture medium containing heparin (100 mg/ml; Sigma Chemical, St Louis, MO) and endothelial cell growth factor (200 mg/ml; Sigma Chemical). After 3–4 weeks, the cells were transferred to a T 25 flask coated with fibronectin and cultured in the presence of puromycin (10 mg/ml; Sigma) for 24 h to prevent epithelial cell contamination. The experiments were performed on cells between passages 4 and 8.

Endothelial cells were identified by immunohistochemical staining with rabbit antihuman factor VIII antibody (Dako A0082; Dako, Glostrup, Denmark) and their ability to take up fluorescently labelled (1,1-dioctadecyl-1-1-3,3,3,3,-tetramethyl-indocarbocyanine perchlorate)-acetylated low-density lipoprotein (DiI-Ac-LDL) (Biomedical Technologies, Stoughton, MA). More than 95–99% of cells in homogenous monolayers endocytosed DiI-Ac-LDL in a perinuclear pattern, which also exhibited diffuse perinuclear staining of factor VIII antigen.

MCP-1 sandwich ELISA
We quantified MCP-1 using sandwich ELISA according to the manufacturer's instructions. Murine anti-MCP-1 monoclonal antibody precoated 96 well microtitre plates (R&D Systems, Abingdon, UK) were used. After 2 h of incubation with samples at 37°C, the plates were washed with 400 ml of washing buffer. Then, 200 ml of MCP-1 conjugate (polyclonal antibody against MCP-1 conjugates to horseradish peroxidase) was added and incubated for 1 h at room temperature. After washing, 200 ml of substrate solution (1:1 of hydrogen peroxide and tetramethylbenzidine) was incubated for 20 min. We stopped the reaction by adding 50 ml of 2N sulfuric acid and measured optical densities at 450 nm. Each experiment was done in duplicate. The results were expressed as a percentage of control (control = 100%).

Northern blot analysis of MCP-1 mRNA
Endothelial cells grown to confluence in 100 mm fibronectin-coated dishes (Corning) were treated with TNF-{alpha} (10 ng/ml) in serum-free media. At the indicated time points, we isolated total cellular RNA using a Tri reagent kit® (Molecular Research Center Inc., Cincinnati, OH). The amount of RNA was measured by absorbance at 260 nm and purity of RNA was assessed by absorbance ratio at 260/280 nm. For the northern blot assay, RNA (10 mg/lane) was electrophoresed through 1% agarose, 2.2 mmol/l formaldehyde-denaturing gel with MOPS buffer, followed by capillary transfer to nylon membranes. The prehybridized filters were hybridized with 32P-labelled cDNA probes and 100 mg/ml salmon sperm DNA at 65°C overnight. cDNA probes were 32P-labelled using random primers (MegaprimeTM DNA labelling system; Amersham International, UK). The same filters were rehybridized with a cDNA specific for glycealdehyde-3-phosphate-dehydrogenase to correct for variation in RNA loading and transfer efficiency.

Preparation of decoy oligodeoxynucleotide (ODN)
The sequences of the phosphorothioate double-stranded ODN against the NF-{kappa}B and AP-1 binding site and the mismatched ODN used in this study were prepared as previously reported [16,17]: NF-{kappa}B decoy ODN, 5'-AGTTGAGGGGACTTTCCCAGGC-3', mismatched NF-{kappa}B decoy ODN, 5'-AGTTGAGGCGACTTTCCCAGGC-3'; AP-1 decoy ODN, 5'-AGCTTGTGAGTCAGAAGCT-3', mismatched AP-1 decoy ODN, 5'-AGCTTGAATCTCAGAAGCT-3'. The double-stranded ODNs were prepared from complementary single-stranded phosphorothiolate-bonded oligonucleotides. The ODNs were annealed for 2 h, while the temperature descended from 80 to 25°C.

Transfection of ODNs into HGEC
Cells were grown in a 100 mm dish for northern blot analysis to 80% confluence. DNA was precomplexed with the PLUS reagents (Life Technologies, Rockville, MD) at room temperature for 15 min. The pre-complexed DNA was combined with diluted LipofectAMINE reagent (Life Technologies), mixed and incubated for 15 min at room temperature. While complexes were forming, we replaced the medium with serum-free transfection medium. Next we added the DNA-PLUS LipofectAMINE reagent complex to each dish containing fresh medium on cells, and then incubated at 37°C at 5% CO2 for 6 h. After incubation, complete medium with serum for normal growth was added. After a 2 day transfection period, we assayed cell extracts for northern blot analysis.

Western blot analysis
HGEC were maintained in 100 mm tissue culture dishes and stimulated at confluency with TNF-{alpha} (R&D Systems) and dexamethasone (Sigma) as indicated after 24 h culture with 2% FCS. Dexamethasone was pretreated for 12 h. After being washed with PBS, the cell layer was incubated on ice for 5 min into 100–150 ml of lysis buffer containing 50 mM KCl, 25 mM HEPES (pH 7.8), 0.5% Igepal CA-630, 1 mM PMSF, 2 mM leupeptin, 1 x Aprotinin and 100 mM DTT. Cytosolic fractions were separated using a pipette and nuclear fractions were lysated again with lysis buffer (500 mM KCl, 25 mM HEPES pH 7.8), 10% glycerol, 0.1 M PMSF, 2 mM leupeptin, 1000 x Aprotinin and 1 M DTT. After repeated freezing and thawing with liquid nitrogen and water at 37°C, the nuclear lysates were incubated at 4°C for 20 min on a rocking platform. After centrifugation at 20 000 g for 20 min, the amount of protein was measured using the Bradford method. We separated 10–20 mg of protein extracts by 12% SDS–polyacrylamide gel electrophoresis, transferred the extracts to an Immobilon-P transfer membrane (Millipore, Bedford, MA) by electroblotting and probed with the rabbit IgG antibody directed against a C-terminal epitope of human p65 and I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were visualized using horseradish peroxidase conjugated anti-rabbit IgG (Santa Cruz Biotechnology) and the Enhanced Chemiluminescence assay (Amersham International) according to the manufacturers’ instructions.

Electrophoretic mobility shift assay (EMSA)
As a baseline study, HGEC were either left untreated or incubated with TNF-{alpha} (10 ng/ml) for the time indicated. To determine the effect of dexamethasone after culture with 2% FCS for 24 h, the cells were preincubated with dexamethasone (10 nM) for 12 h, then stimulated with TNF-{alpha} (10 ng/ml) for the time indicated. NF-{kappa}B and AP-1 consensus oligonucleotides (5'-AGTTGAGGGGACTTTCAGGA-3' and 5'-CGCTTGATGAGTCAGCCGGAA-3', respectively) (Promega, Madison, WI) was end-labelled with [{gamma}-32P]ATP (50 mCi at 3000 Ci/mmol) (New England Nuclear, Boston, MA) and T4 polynucleotide kinase. Binding reactions in 25 ml contained 10 mg nuclear extract protein, binding buffer (50 mM HEPES, pH 7.9, 300 mM KCl, 5 mM EDTA, 35% glycerol, 1 mg poly dI-dC, 10 mM DTT, 1 mg probe), and 1.75 pmol 32P-labelled DNA. Reactions were incubated at room temperature for 15 min and analyzed by electrophoresis on a 6% non-denaturing polyacrylamide gel at 100–150 V for 2 h using high ionic strength conditions. After electrophoresis, gels were dried and DNA–protein complexes localized by autoradiography for 18 h. For the competitive assay, 20-fold excess unlabelled consensus NF-{kappa}B or AP-1 oligonucleotide and anti-p65, anti-c-jun or anti-c-fos antibody (Santa Cruz Biotechnology) were applied in an EMSA assay.

Statistical analysis
Data are presented as mean±SE with n representing the number of different experiments. Comparisons of the values between groups were performed by ANOVA and Scheffe test. P<0.05 was considered statistically significant.



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TNF-{alpha} induced MCP-1 production: inhibition by dexamethasone
TNF-{alpha} (10 ng/ml) was applied to stimulate HGEC with or without pretreatment with dexamethasone (10 nM) for 1 h. After 6 h incubation, the supernatants were collected and assessed for MCP-1 production. As depicted in Figure 1, TNF-{alpha} (10 ng/ml) increased the release of MCP-1 into culture supernatants as compared to control (266±24%, mean±SE, P<0.01, n = 10, each n is in duplicate). Pretreatment with dexamethasone (10 nM) partially, but significantly, inhibited TNF-{alpha} induced MCP-1 production (190±31% vs 266±24%, mean±SE, P<0.05, n = 10).



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Fig. 1. TNF-{alpha} induced MCP-1 production: inhibition by dexamethasone. TNF-{alpha} (10 ng/ml) was applied to stimulate HGEC with or without pretreatment (1 h) with dexamethasone (10 nM). After 6 h incubation, the supernatants were assessed for MCP-1 production using the sandwich ELISA method. *TNF-{alpha} (10 ng/ml) increased the release of MCP-1 as compared to control (266±24%, mean±SE, P<0.01, n = 10, each n is in duplicate). #Pretreatment (1 h) with dexamethasone (10 nM) partially, but significantly, inhibited TNF-{alpha} induced MCP-1 production (190±31% vs 266±24%, mean±SE, P<0.05, n = 10).

 
TNF-{alpha} induced MCP-1 mRNA expression: downregulated by dexamethasone
Treatment of HGEC with TNF-{alpha} (10 ng/ml) caused a significant increase in MCP-1 mRNA, which was evident 2 h after stimulation as shown in Figure 2A. After 8 h, the expression of MCP-1 mRNA in cells stimulated with TNF-{alpha} (10 ng/ml) persistently increased (n = 3). In keeping with the results of ELISA, pretreatment with dexamethasone partially inhibited TNF-{alpha} induced elevation of MCP-1 mRNA levels as shown in Figure 2B.



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Fig. 2. TNF-{alpha} induced MCP-1 mRNA expression: downregulated by dexamethasone. Treatment of HGEC with TNF-{alpha} (10 ng/ml) caused a significant increase in MCP-1 mRNA as shown in (A). Pretreatment (1 h) with dexamethasone partially inhibited TNF-{alpha} induced elevation of MCP-1 mRNA level (B). The results shown are representative of four independent experiments. *P<0.05 compared to control. #P<0.05 compared to TNF-{alpha}.

 
NF-{kappa}B and AP-1 in TNF-{alpha} induced MCP-1 mRNA expression
In EMSA, the DNA-binding activity of NF-{kappa}B began to increase 15–30 min after stimulation with TNF-{alpha} (10 ng/ml), and a 20-fold excess of unlabelled consensus NF-{kappa}B oligonucleotides or anti-p65 antibody abolished the DNA binding of NF-{kappa}B (Figure 3A). Similarly, the DNA-binding activity of AP-1 also began to increase 15–30 min after TNF-{alpha} (10 ng/ml) stimulation while in the presence of a 20-fold excess of unlabelled consensus AP-1 oligonucleotides, anti-c-fos or anti-c-jun antibody, the DNA-binding activity of AP-1 disappeared (Figure 3B).



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Fig. 3. NF-{kappa}B and AP-1 in TNF-{alpha} stimulated HGEC. After stimulation with TNF-{alpha} (10 ng/ml) in HGEC, the DNA-binding activity of NF-{kappa}B began to increase after 15–30 min, while in the presence of excess unlabelled consensus NF-{kappa}B oligonucleotide or anti-p65 antibody, the DNA-binding activity of NF-{kappa}B disappeared (A). Similarly, the DNA-binding activity of AP-1 also increased 15–30 min after TNF-{alpha} (10 ng/ml) stimulation (B), and unlabelled consensus AP-1 oligonucleotide or anti-c-fos and anti-c-jun antibody abolished the DNA-binding activity of AP-1. The results shown are representative of four independent experiments using four different preparations of nuclear extracts.

 
To assess whether activation of NF-{kappa}B or AP-1 was implicated in TNF-{alpha} induced MCP-1 gene transcription, the cells were stimulated for 6 h with TNF-{alpha} (10 ng/ml) after transfection of NF-{kappa}B decoy ODN, an inhibitor of NF-{kappa}B, or AP-1 decoy ODN, an inhibitor of AP-1. NF-{kappa}B decoy ODN partially inhibited TNF-{alpha} induced MCP-1 mRNA expression (Figure 4A). AP-1 decoy ODN also downregulated TNF-{alpha} induced MCP-1 mRNA expression (Figure 4B). Therefore, our experiments demonstrated that both NF-{kappa}B and AP-1 had a cooperative activity for TNF-{alpha} induced MCP-1 production in HGEC.



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Fig. 4. Implication of NF-{kappa}B and AP-1 in TNF-{alpha} induced MCP-1 mRNA production. HGEC were stimulated with TNF-{alpha} (10 ng/ml) for 6 h after transfection of NF-{kappa}B decoy ODN, an inhibitor of NF-{kappa}B, or AP-1 decoy ODN, an inhibitor of AP-1 to assess the role of NF-{kappa}B and AP-1 in TNF-{alpha} induced MCP-1 gene transcription. NF-{kappa}B decoy ODN partially inhibited TNF-{alpha} induced MCP-1 mRNA level (A), and AP-1 decoy ODN also partially downregulated TNF-{alpha} induced MCP-1 mRNA level (B). However, mismatched control of NF-{kappa}B and AP-1 had no effect on TNF-{alpha} induced MCP-1 mRNA level. These results are representative of four independent experiments. *P<0.05 compared to control. #P<0.05 compared to TNF-{alpha}.

 
TNF-{alpha} induced p65 nuclear translocation or cytosolic I{kappa}B{alpha} degradation, or DNA-binding activity of NF-{kappa}B: not affected by dexamethasone
To evaluate the dynamics of I{kappa}B{alpha} and p65 protein, the cells were treated with TNF-{alpha} (10 ng/ml) for the times shown in Figure 5, and cytosolic or nuclear extracts from cells were analyzed by western blotting. In HGEC treated with TNF-{alpha}, cytoplasmic I{kappa}B{alpha} disappeared almost completely within 15 min of stimulation and reappeared in the cytoplasm after 30 min (Figure 5A). On the other hand, p65 gradually appeared in the nuclear proteins after 15–30 min of TNF-{alpha} exposure (Figure 5B). To evaluate the effect of dexamethasone, HGEC were treated with TNF-{alpha} (10 ng/ml) for the indicated time (Figure 6) after pre-incubation with dexamethasone (10 nM) for 12 h. This experiment showed that dexamethasone had no influence on cytosolic degradation of I{kappa}B{alpha}, and nuclear translocation of p65 in TNF-{alpha} stimulated HGEC. In EMSA, TNF-{alpha} induced DNA-binding activity of NF-{kappa}B was not affected by pretreatment with dexamethasone (10 nM) (Figure 7).



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Fig. 5. TNF-{alpha} induced p65 nuclear translocation or cytosolic I{kappa}B{alpha} degradation. In HGEC, after stimulation of TNF-{alpha} (10 ng/ml), cytosolic I{kappa}B{alpha} disappeared almost completely within 15 min and reappeared in the cytoplasm after 30 min (A). On the other hand, p65 gradually appeared in the nuclear proteins after 15–30 min of TNF-{alpha} exposure in the western blot (B). The results shown are representative of four independent experiments.

 


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Fig. 6. The effect of dexamethasone on the TNF-{alpha} induced p65 nuclear translocation or cytosolic I{kappa}B{alpha} degradation. To evaluate the effect of dexamethasone, HGEC were treated with TNF-{alpha} (10 ng/ml) for the indicated times after preincubation with dexamethasone (10 nM) for 12 h. This experiment shows dexamethasone has no effect on cytosolic degradation of I{kappa}B{alpha}, and nuclear translocation of p65 (A and B). The results shown are representative of four independent experiments.

 


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Fig. 7. The effect of dexamethasone on the TNF-{alpha} induced DNA-binding activity of NF-{kappa}B. HGEC were treated with TNF-{alpha} (10 ng/ml) for the indicated time after preincubation with dexamethasone (10 nM) for 12 h. In EMSA, TNF-{alpha} induced DNA-binding activity of NF-{kappa}B was not affected by pretreatment with dexamethasone. These results were consistent in eight independent experiments using eight different preparations of nuclear extracts.

 
TNF-{alpha} induced DNA-binding activity of AP-1: downregulated by dexamethasone
To assess the effect of dexamethasone on activation of AP-1, the DNA-binding activity of AP-1 was analyzed by EMSA. As shown in Figure 8, pretreatment with dexamethasone (10 nM) downregulated TNF-{alpha} induced DNA-binding activity of AP-1.



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Fig. 8. The effect of dexamethasone on the TNF-{alpha} induced DNA-binding activity of AP-1. To assess the effect of dexamethasone on activation of AP-1, the DNA-binding activity of AP-1 was analyzed by EMSA. Pretreatment with dexamethasone for 12 h downregulated TNF-{alpha} (10 ng/ml) induced the DNA-binding activity of AP-1. The results shown are representative of three independent experiments.

 


   Discussion
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The infiltration of monocytes/macrophages into the glomerulus plays a major role in glomerular injury in proliferative glomerulonephritis [16]. A murine model of immune-complex glomerulonephritis, studied by Anders et al. [17], showed increased expression of glomerular MCP-1 and RANTES in the early phase with subsequent glomerular macrophage influx and mesangial proliferation, which then resulted in proteinuria. In a rat model of anti-glomerular basement membrane glomerulonephritis, Panzer et al. [18] demonstrated the dominant role of MCP-1 in the infiltration of mononuclear cells into the glomerulus. Induction of glomerulonephritis resulted in a significant increase in glomerular MCP-1 expression with monocyte/macrophage recruitment into glomeruli, whereas treatment with an anti-MCP-1 antibody selectively reduced glomerular monocyte/macrophage recruitment. Renal glomeruli are composed of glomerular capillary and supporting mesangial cells. The present study shows that cultured HGEC produce MCP-1 in response to TNF-{alpha}, which is in agreement with the previous study of bovine glomerular endothelial cells. Thus, besides mesangial cells, glomerular endothelial cells are also thought to participate in the pathogenesis of glomerulonephritis by releasing MCP-1.

NF-{kappa}B ubiquitously serves as a critical regulator of the inducible expression of many genes [8]. A major form of NF-{kappa}B is composed of a dimer of p50 and p65 subunits, and is retained in the cytoplasm by an I{kappa}B family. The activation of NF-{kappa}B involves the targeted degradation of its cytoplasmic inhibitor, I{kappa}B{alpha}, and the translocation of NF-{kappa}B to the nucleus. In the present study, TNF-{alpha} increased the DNA-binding activity of NF-{kappa}B, while NF-{kappa}B decoy ODN partially inhibited TNF-{alpha} induced elevation of the MCP-1 mRNA level, which indicated that the production of MCP-1 was dependent on NF-{kappa}B.

The transcription factor complex AP-1 has been identified as a target of mitogen-activated protein kinase signalling pathways [19], and AP-1 complexes are composed of members of the Jun and Fos families. In the present study, the DNA-binding activity of AP-1 also increased 15–30 min after TNF-{alpha} stimulation along with an increase in MCP-1 mRNA, and transfection of an AP-1 decoy ODN into the cells downregulated the TNF-{alpha} induced MCP-1 mRNA level. Therefore, TNF-{alpha} induced MCP-1 production was interdependent on NF-{kappa}B and AP-1. These findings are in agreement with the previous study of human umbilical vein endothelial cells, which showed two cis-acting elements within the human MCP-1 5' flanking region; an NF-{kappa}B binding site located 90 bp upstream of the transcriptional start site and an AP-1 consensus binding site located 68 bp upstream of the transcriptional start site were essential for maximal cytokine induction [7].

In contrast to the study by Kakizaki et al. [1], dexamethasone suppressed the TNF-{alpha} induced expression of MCP-1 at the protein and gene levels in HGEC. There is evidence that the immunosuppressive and anti-inflammatory actions of glucocorticoid hormones are mediated by their transrepression of AP-1 and NF-{kappa}B [20]. Generally, glucocorticoid is known to have a dual effect in inhibiting the activity of NF-{kappa}B [8,21]. The first mechanism is glucocorticoid-induced transcription of I{kappa}B{alpha} [8,22] and the second mechanism is protein–protein interactions between NF-{kappa}B and the glucocorticoid receptor [21]. In experiments using lymphocytes, HeLa cells, or vascular smooth muscle cells, glucocorticoid affected the transcription rate or mRNA stability of I{kappa}B{alpha} and prevented nuclear translocation of NF-{kappa}B [22]. However, in other cell types, not all the studies demonstrated the same findings, suggesting cell type-specific differences. Auwardt et al. [12] reported that dexamethasone was unable to prevent nuclear translocation or DNA binding of NF-{kappa}B to the nucleus in rat mesangial cells in spite of partial inhibition of mRNA of MCP-1. Moreover, in transfection studies using an NF-{kappa}B reporter construct, the increase in luciferase activity seen after stimulation with IL-1ß was not significantly reduced by dexamethasone in rat mesangial cells. Similarily, studies of synoviocytes and brain cells showed that the anti-inflammatory effect of glucocorticoid was independent of NF-{kappa}B activation [9,10]. In our experiments, dexamethasone had no effect on I{kappa}B{alpha} degradation, or nuclear translocation of p65, or DNA-binding activity of NF-{kappa}B.

Dexamethasone is known to repress AP-1 by the inhibition of Jun-N-terminal kinase [13], by the glucocorticoid-inducible leucine zipper [14] or by binding to AP-1 after forming the glucocorticoid-receptor complex [23]. However, so far little is known about whether the inhibition of AP-1 could suppress the MCP-1 production at the protein and gene levels. In an experiment using retinoic acids, Lucio-Cazana et al. [24] reported that retinoic acids selectively inhibited the constitutive expression of MCP-1 via intervention in the AP-1 pathway. In the present study, dexamethasone suppressed TNF-{alpha} induced MCP-1 production and AP-1 binding activity. As the AP-1 decoy suppressed TNF-{alpha} induced mRNA production of MCP-1, these results might suggest that dexamethasone suppressed MCP-1 production via inhibition of the AP-1 pathway.

In conclusion, our study showed that in HGEC, TNF-{alpha} induced MCP-1 gene transcription required both NF-{kappa}B and AP-1, and dexamethasone downregulated TNF-{alpha} induced MCP-1 production. Inhibition of the AP-1 pathway, not of the NF-{kappa}B pathway, might be the downregulatory mechanism of dexamethasone in TNF-{alpha} induced MCP-1 production in HGEC.



   Acknowledgments
 
This study was supported by a grant (2203-070) from the Asan Institute for Life Sciences, Seoul, Korea.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 26. 3.03
Accepted in revised form: 24. 9.03