Ceramide-induced and Age-associated Increase in Macrophage COX-2 Expression Is Mediated through Up-regulation of NF-kappa B Activity*

Dayong WuDagger , Melissa MarkoDagger , Kate ClaycombeDagger §, K. Eric Paulson, and Simin Nikbin MeydaniDagger ||**

From the Dagger  Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, the  Department of Biochemistry, and the || Department of Pathology, Sackler Graduate School of Biomedical Sciences, Tufts University, Boston, Massachusetts 02111

Received for publication, July 25, 2002, and in revised form, December 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (Mphi ) prostaglandin E2 (PGE2) production is because of ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription that leads to increased COX-2 expression and enzyme activity. To determine the mechanism of the age-related and ceramide-dependent increase in COX-2 transcription, we investigated the role of various transcription factors involved in COX-2 gene expression. The results showed that LPS-initiated activations of both consensus and COX-2-specific NF-kappa B, but not AP-1 and CREB, were significantly higher in Mphi from old mice than those from young mice. We further showed that the higher NF-kappa B activation in old Mphi was because of greater Ikappa B degradation in the cytoplasm and p65 translocation to the nucleus. An Ikappa B phosphorylation inhibitor, Bay 11-7082, inhibited NF-kappa B activation, as well as PGE2 production, COX activity, COX-2 protein, and mRNA expression in both young and old Mphi . Similar results were obtained by blocking NF-kappa B binding activity using a NF-kappa B decoy. Furthermore, NF-kappa B inhibition resulted in significantly greater reduction in PGE2 production and COX activity in old compared with young Mphi . Addition of ceramide to the young Mphi , in the presence or absence of LPS, increased NF-kappa B activation in parallel with PGE2 production. Bay 11-7082 or NF-kappa B decoy prevented this ceramide-induced increase in NF-kappa B binding activity and PGE2 production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-kappa B activation, which is, in turn, because of a greater Ikappa B degradation in old Mphi .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well documented that T cell-mediated immune function declines in old animals and elderly humans compared with their young counterparts (1, 2). The age-associated dysregulation in macrophages (Mphi )1 contributes to the impaired T cell function with aging. We, as well as others, have demonstrated that immune cells, including Mphi , from old animals and humans produced more PGE2 than those from their young counterparts (3-7). We further showed that the increased PGE2 production by Mphi contributes to the decline in T cell-mediated function with aging (8).

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid (AA) to PG endoperoxide (PGH2), which is further converted to different PGs and thromboxane. COX is hence a key factor in PG synthesis. Two isoforms of COX have been identified: a constitutive form, COX-1 (9, 10), and the inducible counterpart, COX-2 (11, 12). We have demonstrated that the age-associated increase in Mphi PGE2 production is because of higher COX activity in Mphi from old mice compared with those from young mice. This increased COX activity is, in turn, a result of increased expression of COX-2 protein and mRNA (13). In a recent study, we further demonstrated that the age-related increase in COX-2 mRNA was because of a higher level of ceramide in old Mphi compared with those of young, which induced up-regulation of COX-2 transcription (14). In addition, we showed that the effect of ceramide was not mediated through components of the mitogen-activated protein kinase pathway, c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, or p38 (14).

To determine the mechanism of the age-related and ceramide-induced increase in COX-2 transcription, we investigated the role of various nuclear transcription factors that are involved in COX-2 gene expression. The binding sites for several nuclear transcription factors, such as nuclear factor kappa B (NF-kappa B), nuclear factor interleukin-6, and cAMP-responsive element (CRE), have been identified on the promoter region of the COX-2 gene (15-17). A number of studies have suggested that NF-kappa B activation and binding to its cognate site on the COX-2 promoter region are required to induce COX-2 expression (16, 18, 19). Dysregulation of NF-kappa B activation has been indicated in certain inflammatory diseases (20-22), in which COX-2-catalyzed prostaglandin production may play an important role. It is thus feasible that the age-associated increase in COX-2 expression may be mediated through a corresponding change in the regulation of NF-kappa B with aging. However, to date, age-related changes in Mphi NF-kappa B activity and the role of NF-kappa B in age-related up-regulation of COX-2 have not been demonstrated.

Along with NF-kappa B, another redox-sensitive transcription factor, activator protein-1 (AP-1), has been shown to be involved in COX-2 transcriptional regulation (23, 24). Although an independent AP-1 binding site has not been recognized on the COX-2 promoter, it was reported that the binding site for AP-1 in the COX-2 promoter is a CRE binding site (15, 24, 25). A number of studies suggest that binding of nuclear proteins, such as CRE-binding protein (CREB) and c-Jun, to CRE, an element of COX-2 promoter, induces COX-2 transcription (26, 27). Accordingly, we examined the roles of NF-kappa B, AP-1, and CREB in the age-associated up-regulation of Mphi COX-2 transcription.

Our recent study showed that the intracellular concentration of ceramide was higher in LPS-stimulated Mphi of old mice compared with those of young mice and that this increased level of ceramide mediates the age-associated up-regulation of COX-2 transcription (14). Whereas the effect of ceramide on regulation of transcription factors has not been well defined, previous studies showed that ceramide induced AP-1 (28) and NFkappa B activation (29). We hypothesize that up-regulated COX-2 transcription with aging is because of altered activation of transcription factors involved in COX-2 expression by ceramide. We demonstrate here that of the three transcription factors studied, only NF-kappa B contributes to the age-associated up-regulation of COX-2. The age-associated increase in NF-kappa B activation is because of enhanced Ikappa B degradation in the cytoplasm, resulting in increased nuclear translocation of activated NF-kappa B. Ceramide induces increased COX-2 activation and the consequent PGE2 production through up-regulating NF-kappa B activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals-- Specific pathogen-free male young (4-6 months) and old (22-24 months) C57BL/6NIA mice were obtained from National Institute on Aging colonies at Harlan Teklad (Madison, WI). Mice exhibiting skin lesions, visible tumors, or splenomegaly were excluded from the study. Mice were housed individually in microisolator cages at a constant temperature (23 °C) with a 12-h light-dark cycle and were fed autoclaved mouse chow Harlan 7012 (Harlan Teklad) and water ad libitum. All conditions and handling of the animals was approved by the Animal Care and Use Committee of the Jean Mayer Human Nutrition Research Center on Aging, Tufts University, and were in accordance with guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Peritoneal Macrophage Isolation-- Mice were injected per peritoneum with 3 ml of 2.98% thioglycollate to elicit Mphi . Three days later, the mice were euthanized via CO2 asphyxiation. Peritoneal exudate cells were obtained by peritoneal lavage with cold Ca2+- and Mg2+-free Hanks' balanced salt solution (Sigma). Cells were centrifuged and resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10 mM HEPES (Sigma), 2 mM glutamine (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen), and 2% fetal bovine serum. Peritoneal exudates were enriched for Mphi by their adherence to tissue culture-treated plastic dishes or plates for 2 h at 37 °C in 5% CO2. Nonadherent cells were removed by vigorous washing and the remaining cells were at least 90% macrophages as assessed by the expression of cell surface markers Mac-1 and F4/80. Cells were monitored for their general condition and viability throughout the study as assessed by morphology, adherence, and trypan blue exclusion. No cytotoxicity was observed in treated cells compared with untreated controls. We previously conducted a number of studies using resident macrophages (8, 13, 30, 31). Because of a large number of Mphi that were necessary for various experiments and the limited number of resident Mphi obtainable from each mouse, we used thioglycollate-elicited Mphi . Prior to use of thioglycollate in our experiments, we compared the magnitude and pattern of responsiveness between resident and thioglycollate-elicited Mphi . Although there are differences between the two types of cells in their ability to response to certain stimulation agents, the relative response pattern, the age-related difference, as well as the response to in vitro intervention are the same between these two cell types. Particularly and of relevance to this study, thioglycollate-elicited Mphi showed the age-associated difference in COX-2 expression similar to that observed with resident Mphi (8, 13, 30).

Preparation of Cytosolic and Nuclear Extracts-- Mphi (2 × 107) in a 10-cm dish were incubated overnight in serum-free RPMI 1640 medium. The cells were washed and then stimulated by lipopolysaccharide (LPS, Escherichia coli serotype 0111:B4, Sigma) at 5 µg/ml for various lengths of time. This concentration of LPS was used because our testing experiments indicated it to be optimal for production of PGE2 and nitric oxide. A parallel experiment was conducted using IL-1beta (R & D Systems, Minneapolis, MN) at 50 ng/ml as stimulator. For the NF-kappa B inhibition study, the cells were preincubated with an inhibitor of Ikappa B-alpha phosphorylation, Bay 11-7082 (Biomol Research Laboratories, Plymouth Meeting, PA) for 30 min, or a NF-kappa B decoy (see "NF-kappa B Decoy Approach" below) for 24 h before LPS stimulation. To increase intracellular ceramide levels, cell-permeable C2-ceramide (30 µM) (Matreya, Pleasant Gap, PA) was added to the cell cultures with or without the presence of LPS and the cells were incubated for different times. The concentration of 30 µM was chosen based on our previous study in which different doses of ceramide were used and shown to induce an efficient COX-2 expression and PGE2 production at this level (14). At the end of the stimulation period, the cells were washed with cold PBS and then collected with a cell scraper. The cells were resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 2 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, and 0.5% Nonidet P-40) and incubated on ice for 10 min. After the cell lysates were centrifuged at 15,000 × g for 1 min, the supernatants were collected as a cytosolic fraction and stored at -70 °C. The remaining pellets were resuspended in a high salt buffer (50 mM HEPES, pH 7.9, 300 mM NaCl, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, and 10% glycerol) and incubated in rotation at 4 °C for 30 min. The nuclear lysate was centrifuged at 15,000 × g at 4 °C for 30 min. The supernatant was collected as a nuclear fraction and stored at -70 °C.

Electrophoretic Mobility Shift Assay (EMSA)-- For the NF-kappa B binding activity assay, both consensus and COX-2 promoter-specific sequences were used. A double-stranded oligonucleotide containing an NF-kappa B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGG C-3') was purchased from Promega (Madison, WI). A COX-2-specific NF-kappa B binding oligonucleotide (distal, -408/-388, 5'-GAGGTGAGGGGATTCCCTTAG-3') and its complementary sequence were synthesized by the Tufts University Core Facility laboratory and were annealed before labeling. For AP-1 and CREB binding assays, the double-stranded consensus oligonucleotides (AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3' and CREB, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') were purchased from Promega. All the oligonucleotides were end labeled using [gamma -32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences) and T4 polynucleotide kinase (Promega). 32P-Labeled probes were purified using MicroSpinTM G-25 columns (Amersham Biosciences). For each reaction, nuclear extracts (2 µg of protein) were incubated with labeled oligonucleotide in the presence of binding buffer (Promega) at room temperature for 20 min. To ensure specificity of probe binding, a 50-fold excess of unlabeled (cold) and mutant oligonucleotides were added to the nuclear samples and incubated for 10 min before the labeled oligonucleotide was added. In supershift assays, antibodies specific for the p65 or p50 subunit of NF-kappa B (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the binding reaction and incubated for 30 min at room temperature before the labeled oligonucleotide was added. Protein-DNA complexes were resolved at 350 V for 1 h in 4% polyacrylamide gels and visualized using Kodak x-ray film. Bands were quantified by ChemiImager (Alpha Innotech Corp., San Leandro, CA).

Western Blot-- The cytosolic and nuclear samples were prepared as described under "Preparation of Cytosolic and Nuclear Extracts" and were used for Ikappa B-alpha and p65 detection, respectively. For COX-2 and inducible nitric-oxide synthase (iNOS) detection, Mphi were preincubated with or without Bay 11-7082 for 30 min and then stimulated by LPS (5 µg/ml) for 16 h. Total cellular lysates were collected and 25 µg of protein from each sample was electrophoresed in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk in TBS containing 0.1% Tween 20 overnight, the membranes were incubated with the respective antibodies (all from Santa Cruz Biotechnology) for 1 h. The membranes were rinsed and then incubated with the corresponding secondary antibodies conjugated with alkaline phosphatase (Tropix, Inc., Bedford, MA) for 1 h. After being rinsed, the membrane was incubated in a Chemiluminescent Detection System (Tropix) for 4 min and then exposed to film. The equal loading across the samples was first estimated by staining the membranes with Ponceau S (Sigma) and further confirmed by reprobing the stripped membranes with beta -actin antibody (Sigma). All bands were quantified by ChemiImager (Alpha Innotech). The bands of interest molecules were normalized with beta -actin bands and presented as relative density ratio.

PGE2 Production and COX Enzyme Activity-- Peritoneal Mphi (1 × 106 cells/well) were plated to 24-well plates and isolated by adherence as described above. The cells were preincubated with or without Bay 11-7082 for 30 min before being stimulated by 5 µg/ml LPS (Sigma), 30 µM ceramide (Matreya), or both for 12 to 24 h. After the supernatants were removed and stored at -70 °C for analysis of accumulated production of PGE2, the cells were layered with 1 ml of medium containing 30 µM AA and incubated at 37 °C for 10 min for determination of COX activity as described by Fu et al. (32). Total cellular COX activity can be measured by adding excess exogenous AA to Mphi , because the intracellular enzyme pool is saturated with the substrate and is functioning at maximal velocity. After 10 min, 2.1 mM aspirin was added to inactivate the COX enzyme activity. Supernatants were immediately removed and stored at -70 °C. Cells were then incubated with 1 M NaOH for 5 min, at which time the supernatant was removed and stored at -20 °C for protein analysis by the bicinchoninic acid protein assay kit (Pierce). PGE2 was measured by radioimmunoassay (RIA) as previously described (4).

COX-2 mRNA Reverse Transcriptase-PCR-- Mphi were preincubated with or without Bay 11-7082 for 30 min and then stimulated by LPS (5 µg/ml) for 4 h. Total RNA was isolated using the Totally RNA Isolation kit (Ambion, Austin, TX). Two µg of total RNA was reverse-transcribed to first-strand cDNA using random hexamer, and amplified by PCR using the Superscript amplification kit (Invitrogen). The PCR conditions for COX-2 mRNA were 1 cycle for 2 min at 94 °C, followed by 30 cycles of 1 min at 94 °C and 5 min at 55 °C. Mouse exon 8 sense primer (5'-ACTCACTCAGTTTGTTGAGTCATTC-3') and exon 10 antisense primer (5'-GTAATTGGGATGTCATGATTAGTTT-3') were used to generate 583-bp PCR products. To normalize the COX-2 mRNA reverse transcriptase-PCR results, 18 S rRNA primers and the competitors at a ratio of 4:6 (Ambion) were used to generate 18 S rRNA PCR products from the same cDNA samples used in COX-2 mRNA PCR assays. Our PCR conditions for both murine COX-2 mRNA and 18 S rRNA were tested to be within the linear range of PCR product formation (14). The PCR products were resolved by electrophoresis in an ethidium bromide-stained 1.2% agarose gel and the bands were visualized by ethidium bromide staining and quantified using ChemiImager (Alpha Innotech).

NF-kappa B Decoy Approach-- The COX-2-specific NF-kappa B binding oligonucleotide (distal, 5'-GAGGTGAGGGGATTCCCTTAG-3') and its complementary sequence (5'-CTAAGGGAATCCCCTCACCTC-3'), and their mutated counterparts (5'-GAGGTGAGGGCCTTCCCTTAG-3' and 5'-CTAAGGGAAGGCCCTCACCTC-3') were custom synthesized by Qiagen Operon (Alameda, CA). The underlined letters denote phosphorothioated bases and the bold letters mark mutations. The complementary oligonucleotides were annealed to double strands by heating at 90 °C for 5 min and then cooling down to room temperature within 3 h. To be efficiently delivered to the cells, the double-stranded oligonucleotides were mixed with LipofectAMINE reagent (Invitrogen) and incubated at room temperature for 40 min. The complex was then added to the cell cultures at 0.5 to 10 nM for oligonucleotides and 5 µg/ml for LipofectAMINE reagent. The cells were incubated in antibiotics and serum-free RPMI 1640 medium for 24 h, after which the cells were washed and then stimulated for varied times depending on the purpose.

Statistical Analysis-- Data were analyzed using SYSTAT statistical software (SYSTAT 10, 2000; Evanston, IL). Paired Student's t test was used to determine the effect of incubation time and inhibitor. The difference between two age groups was assessed using nonpaired Student's t test. Results are expressed as mean ± S.E. Significance was set at p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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NF-kappa B Binding Activity in Murine Mphi Increases with Aging-- We previously showed that the age-associated increase in PGE2 production is because of the ceramide-induced up-regulation of COX-2 transcription with aging (14). Of the transcription factors found in the promoter region of the COX-2 gene, NF-kappa B is the most intensively studied and several investigations have linked its activity to the activation of the COX-2 gene (16, 18). Although the age-related up-regulation of NF-kappa B activation has been shown in rat gastric mucosa (33), kidney (34), liver, heart (35), and brain (36), its binding activity in Mphi , a major source of PGE2, has not been compared between young and old animals. In this study, the peritoneal Mphi from young and old mice were stimulated with LPS for different times as indicated in Fig. 1. NF-kappa B binding activity in nuclear fractions was assessed by EMSA. Fig. 1A shows the results obtained using an NF-kappa B consensus oligonucleotide as the binding motif. Without stimulation, there was no detectable binding activity in either young or old Mphi . At every time point following LPS stimulation, the NF-kappa B binding activity was higher in old Mphi compared with that of young Mphi . To assure the specificity of the binding, a 50-fold concentrated unlabeled (cold) NF-kappa B consensus oligonucleotide was added to compete with the 32P-labeled NF-kappa B oligonucleotide. The binding was competed out in both young and old Mphi at peak time (2 h). When a mutated NF-kappa B oligonucleotide was added, however, the binding was not affected, further indicating the sequence specificity. To determine the composition of the NF-kappa B proteins in the binding complex, we used antibody supershift in EMSA. As seen in Fig. 1A, the binding complex was shifted by antibodies to p65 and p50 units. Next, we used a COX-2-specific NF-kappa B oligonucleotide in EMSA. Similar to the experiments in which the consensus oligonucleotide was used, a consistently higher binding activity was observed in old compared with young Mphi (Fig. 1B). The specificity was examined using the samples from old Mphi at peak stimulation time (2 h) and 50-fold cold oligonucleotide was shown to completely compete out the band. The supershift assay yielded similar results to those observed in the assay using the consensus oligonucleotide.


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Fig. 1.   NF-kappa B binding activity in murine Mphi increases with aging. Peritoneal Mphi from young and old mice were stimulated with LPS (5 µg/ml) for 0, 15 min, 30 min, 1, 2, or 4 h prior to the preparation of nuclear extracts as described under "Experimental Procedures." Nuclear extracts (2 µg) were incubated with 32P-labeled consensus (A) or COX-2-specific NF-kappa B oligonucleotide (B) and subjected to EMSA. Cold competition was conducted in the presence of 50-fold excess unlabeled corresponding NF-kappa B oligonucleotide for 10 min before the 32P-labeled oligonucleotide was added. In supershift analysis, antibody specific for p65 or p50 were added to the binding reaction and incubated for 30 min before the 32P-labeled oligonucleotide was added. The gel figures are representative samples of six independent experiments. The bar figures are the mean ± S.E., n = 6. Different from young at *, p < 0.05, and #, p < 0.1.

AP-1 and CREB Binding Activity in Mphi Does Not Change with Aging-- After establishing an age-related difference in NF-kappa B binding activity, we examined whether two other transcription factors, AP-1 and CREB, which have been shown to be involved in COX-2 regulation, are affected by the aging process. Culture conditions and LPS doses were the same as those used in the NF-kappa B gel shift assay. Both AP-1 and CREB were activated by LPS treatment, but the induction was less potent than that seen in NF-kappa B so that the autoradiography required 5-8-fold longer exposure times than that with the NF-kappa B probe. The binding activity for both AP-1 and CREB peaked at around 1 h poststimulation and no significant difference was detected between young and old mice in either AP-1 or CREB activation (data not shown).

Old Mphi Have Higher Ikappa B Degradation and p65 Translocation Than Young Mphi -- To determine the mechanism of the age-related increase in NF-kappa B activation, we examined the two key steps preceding the NF-kappa B binding activity: Ikappa B degradation in the cytoplasm and NF-kappa B translocation to the nucleus. The results indicated that there was no difference between young and old Mphi in expression of Ikappa B under resting conditions. However, after LPS stimulation, there was greater Ikappa B-alpha degradation in old Mphi than that in young Mphi . The degradation also occurred faster, as shown at 15 min after stimulation, and appeared to recover more slowly when compared with young Mphi (Fig. 2A). We then examined the p65 appearance in nuclear extracts following its translocation from the cytoplasm. As shown in Fig. 2B, p65, which was not detected in the nucleus before stimulation, gradually increased during the time from 15 min to 2 h after LPS stimulation, followed by a decline between 2 and 4 h. These results indicate that the increased NF-kappa B binding activity with age is because of different rates of Ikappa B degradation, and subsequent NF-kappa B translocation, two immediate events prior to the binding of the NFkappa B to its target genes.


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Fig. 2.   Old Mphi have higher Ikappa B degradation and p65 translocation than young Mphi . Peritoneal Mphi from young and old mice were stimulated with LPS (5 µg/ml) for 0, 15 min, 30 min, 1 h, 2 h, or 4 h. The cytosolic and nuclear samples were prepared as described under "Experimental Procedures" and were used for Ikappa B-alpha and p65 detection, respectively. The corresponding samples (25 µg of protein per lane) were used to determine Ikappa B-alpha or p65 protein using Western blot analysis. After Ikappa B-alpha or p65 bands were visualized, the membranes were stripped and reprobed with the antibody to beta -actin to be used as normalization control. The results for Ikappa B-alpha and p65 are shown in A and B, respectively. Each bar represents mean ± S.E. of three separate experiments. * indicates a significant difference at p < 0.05 or less.

An Ikappa B Inhibitor Prevents NF-kappa B Activation in Both Young and Old Mouse Mphi -- As no age-related change was observed in AP-1 and CREB activation, the relationship between NF-kappa B activation and age-related increase in COX-2 expression was further investigated. Because increased NF-kappa B activation in old Mphi was associated with a higher Ikappa B phosphorylation and the consequent degradation, to inhibit NF-kappa B activation, we used Bay 11-7082, an inhibitor of Ikappa B phosphorylation, and therefore NF-kappa B activation (37). This inhibitor has been shown not to have as broad an effect as do other NF-kappa B activation inhibitors such as aspirin, caffeic acid, and N-acetylcysteine. Doses of Bay 11-7082 between 0.5 and 5 µM were used. These doses were chosen based on preliminary experiments in which a range of 0.1 to 100 µM Bay 11-7082 was tested. Below 0.5 µM, Bay 11-7082 did not inhibit NF-kappa B binding and at 5 µM, it almost completely inhibited NF-kappa B binding. We stimulated Mphi from young and old mice with LPS for 2 h and used the COX-specific NF-kappa B binding oligonucleotide to detect the formation of the DNA-protein complex in the nuclear fractions. As shown in Fig. 3A, there were no detectable levels of NF-kappa B binding activity in unstimulated samples. Consistent with the above mentioned experiments, LPS induced higher NF-kappa B activation in old rather than in young Mphi . Preincubation with unlabeled COX-2-specific NF-kappa B binding oligonucleotide (cold competition) or mutant NF-kappa B binding oligonucleotide was conducted to confirm the specificity. NF-kappa B activation was partially inhibited at doses of 0.5-2 µM and almost completely inhibited at 5 µM by Bay 11-7082. 


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Fig. 3.   Ikappa B inhibitor inhibits the NF-kappa B activation in mouse Mphi . Peritoneal Mphi from young and old mice were preincubated with the Ikappa B-alpha phosphorylation inhibitor, Bay 11-7082, for 30 min and then stimulated with LPS (5 µg/ml) for 2 h prior to the preparation of nuclear extracts as described under "Experimental Procedures." Nuclear extracts (2 µg) were incubated with 32P-labeled COX-2-specific NF-kappa B (A), AP-1 (B), or CREB oligonucleotide (C) and subjected to EMSA as described under "Experimental Procedures." Cold competition was conducted in the presence of 50-fold excess unlabeled corresponding oligonucleotides for 10 min before the 32P-labeled oligonucleotide was added. In NF-kappa B EMSA (A), the specificity of the binding was determined only in old Mphi by adding cold or mutated NF-kappa B prior to the 32P-labeled NF-kappa B. The results are representative of three independent experiments.

To demonstrate that the effect of Bay 11-7082 is specific to NFkappa B activation, we also examined its effect on AP-1 and CREB activation. As shown in Fig. 3B, neither AP-1 nor CREB binding activity was affected by Bay 11-7082 under the same condition as that used for determination of NF-kappa B activation. These results indicate that Bay 11-7082 can be used as a tool to determine the role of NF-kappa B activation in age-related up-regulation of COX-2 expression.

Inhibition of NF-kappa B Preferentially Reduces PGE2 Production and COX Activity in Old Mphi -- Of the transcription factors that control COX-2 transcriptional activation, NF-kappa B was the only one that exhibited the age-related increase. Therefore, we next examined whether changing NF-kappa B activation would alter the LPS-stimulated production of PGE2, a representative COX product in Mphi . After Mphi from either young or old mice were preincubated with Ikappa B inhibitor Bay 11-7082 for 30 min, the cells were stimulated by LPS for 24 h and PGE2 production was then determined. As shown in Fig. 4A, PGE2 production was low in unstimulated cells and greatly increased with LPS stimulation. This LPS-induced PGE2 production was 5-fold higher in old compared with young mice. LPS-induced PGE2 production was inhibited by Bay 11-7082 in a dose-dependent manner between 0.5 and 2 µM, whereas at a dose of 5 µM, no further inhibition was observed. Because old Mphi have higher NF-kappa B activation and COX-2 expression than those of young Mphi , we next examined the effect of NF-kappa B inhibition on the ability of young and old Mphi to produce PGE2. Results indicated that when NF-kappa B activation was reduced with Bay 11-7082, PGE2 production in old Mphi was inhibited more significantly (p < 0.05) compared with that of young Mphi (80 versus 58, 27 versus 12, and 39 versus 13% of their control levels were observed in young and old mice in the presence of Bay 11-7082 at 1, 2, and 5 µM, respectively). COX is the rate-limiting enzyme in prostaglandin biosynthesis and we have demonstrated that increased COX activity is the major contributing factor to the age-related increase in PGE2 production (13). We, therefore, examined COX activity in cells treated under the same condition as in the test for PGE2 production. As shown in Fig. 4B, LPS-stimulated Mphi from old mice have significantly higher COX activity than those from young mice. NF-kappa B inhibition by Bay 11-7082 reduced COX activity in a dose-dependent manner. Furthermore, the inhibition of COX activity in old Mphi was more significant (p < 0.05) compared with that of young Mphi (75 versus 48, 36 versus 12, and 3 versus 0.2% of their control levels were observed in young and old mice in the presence of Bay 11-7082 at 1, 2, and 5 µM, respectively).


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Fig. 4.   Inhibition of NF-kappa B activation dose dependently reduces Mphi PGE2 production and Mphi COX activity. The peritoneal Mphi from young and old mice were pretreated with increasing concentrations of Ikappa B phosphorylation inhibitor Bay 11-7082 for 30 min and then stimulated with LPS (5 µg/ml) for 24 h at 37 °C. The supernatant was collected and analyzed for PGE2 production. After the supernatant was collected for PGE2 production, the cells were washed and then further incubated in the medium containing 30 µM AA for 10 min at 37 °C. Aspirin (2.1 mM) was added at the end of incubation to terminate the reaction. The supernatant was collected and analyzed for the PGE2 synthesized utilizing exogenous AA to assess the COX enzyme activity. PGE2 concentrations in the samples were determined using RIA and adjusted for total cell protein. The results for PGE2 production and COX enzyme activity are shown in A and B, respectively. The data are mean ± S.E. of four independent experiments in each of which a duplicate measurement was conducted. The bars bearing different letters within the same case (lower or upper) represent significant difference with p < 0.05. The lowercase and uppercase letters represent young and old mice, respectively.

To rule out the possibility that Bay 11-7082 may directly inactivate the COX enzyme rather than inhibit transcriptional activation of COX-2 through reducing NF-kappa B activation, we also tested its direct effect on COX activity by adding Bay 11-7082 to cultures after LPS stimulation. After 24 h of LPS stimulation, COX-2 would be fully activated. The presence of Bay 11-7082 for 30 min thereafter should not change the levels of COX-2 enzyme, but would be adequate to affect enzyme activity if it did have a direct effect on the enzyme. The results showed that addition of Bay 11-7082 after LPS stimulation did not change COX activity in either young or old Mphi (data not shown), thus a direct effect on COX enzyme activity can be ruled out.

Age-related Increase in PGE2 Production as Well as the Involvement of NF-kappa B Is Not Limited to LPS as Stimulant-- Increased PGE2 production with age is not limited to that stimulated by LPS. Previously we showed that in addition to LPS, calcium ionophore or T cell mitogens also stimulated more PGE2 production in splenocytes of old mice or peripheral blood mononuclear cells of elderly humans compared with their young counterparts (4-6). To further confirm this in the elicited peritoneal Mphi , we conducted a dose-response experiment using IL-1beta , another common stimulant of COX-2. Fig. 5A shows that IL-1beta dose dependently induced PGE2 production in both young and old Mphi , but old Mphi produced significantly more PGE2 than young Mphi in response to IL-1beta stimulation. Next, we stimulated the cells with 50 ng/ml IL-1beta in the presence of Bay 11-7082 and found that IL-1beta -stimulated PGE2 production was also inhibited by Bay 11-7082 in a dose-dependent manner (Fig. 5B). The patterns of response in young and old Mphi were similar to those when LPS was used as a stimulant. Similar results were obtained when COX activity was evaluated (data not shown).


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Fig. 5.   Age-associated difference is also present in IL-1beta -induced PGE2 production, which is also inhibited by blocking NF-kappa B activation. A, the peritoneal Mphi from young and old mice were stimulated by IL-1beta at different concentrations as indicated for 24 h at 37 °C. The supernatant was collected and analyzed for PGE2 production. PGE2 concentrations in the samples were determined using RIA and adjusted for total cell protein. Data are mean ± S.E. of four individual experiments. * indicates a significant difference between young and old mice at p < 0.05, and # at p = 0.1. B, as mentioned above the peritoneal Mphi were pretreated with increasing concentrations of Ikappa B phosphorylation inhibitor Bay 11-7082 for 30 min and then stimulated with IL-1beta (50 ng/ml) for 24 h at 37 °C. The supernatant was collected and analyzed for PGE2 production. The data are mean ± S.E. of four independent experiments. The bars bearing different letters within the same case (lower or upper) represent significant difference with p < 0.05. The lowercase and uppercase letters represent young and old mice, respectively.

Inhibition of NF-kappa B Reduces COX-2 mRNA and Protein Levels-- Because our previous studies (13, 14) showed that the age-related increase in COX activity is because of increased expression of the COX-2 mRNA and protein, we determined COX-2 mRNA and protein levels in cells that were incubated with Bay 11-7082 prior to LPS stimulation. As shown in Fig. 6A, unstimulated Mphi had very low expression of COX-2. However, LPS significantly induced COX-2 expression and the LPS-stimulated COX-2 expression was higher in old compared with young Mphi . Inhibition of NF-kappa B activation dose-dependently reduced the COX-2 mRNA expression in both young and old Mphi . The change in COX-2 protein levels was generally in accordance with the change in COX-2 mRNA levels, although the dose response was not as pronounced as seen in mRNA expression (Fig. 6B). These results demonstrate that there is an age-dependent increase in activation of NF-kappa B, which results in higher transcription of the COX-2 gene, increased COX-2 mRNA, COX-2 protein, and greater PGE2 production.


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Fig. 6.   Inhibition of NF-kappa B dose dependently inhibits expression of COX-2 mRNA and protein, and iNOS protein in young and old mouse Mphi . Peritoneal Mphi from young and old mice were pretreated with increasing concentrations of Ikappa B phosphorylation inhibitor Bay 11-7082 for 30 min and then stimulated with LPS (5 µg/ml) at 37 °C for 4 and 16 h for mRNA and protein assays, respectively. A, the isolated total RNA (2 µg) was used to generate first-strand cDNA and then COX-2 mRNA level was determined using PCR as described under "Experimental Procedures." To normalize COX-2 mRNA, the 18 S rRNA PCR products were generated from the same cDNA samples used in COX-2 mRNA PCR assays. The results are representative of three independent experiments. Total cell lysates (25 µg of protein per lane) were used to determine COX-2 (B) or iNOS (C) protein using Western blot analysis. After COX-2 or iNOS bands were visualized, membranes were stripped and reprobed with the antibody to beta -actin to serve a loading control. Results are representative of four independent experiments for COX-2 and two independent experiments for iNOS.

Nitric oxide and iNOS have been shown to be up-regulated with age (31, 38). The promoter region of the murine iNOS gene has a NF-kappa B binding site (39). Thus, to further prove the link between NF-kappa B activation and its target genes in the context of age-related events, we chose to measure iNOS protein expressed by young and old Mphi under the same condition used for COX-2 determination. As demonstrated in Fig. 6C, LPS-induced iNOS expression was higher in old rather than in young Mphi and was also inhibited by Bay 11-7082 in a dose-dependent manner.

Ceramide Increases LPS-induced Activation of NF-kappa B in Young Mphi -- Our recent work (14) showed that old Mphi produce higher levels of intracellular ceramide, compared with those from young mice after LPS stimulation. Furthermore, we showed that increasing ceramide levels in young Mphi , by adding exogenous ceramide, significantly increased COX-2 expression. This effect was specific to ceramide and did not depend on its downstream metabolite, sphingosine (14). Because no age-related difference in mitogen-activated protein kinase activity (14) or AP-1 and CREB activation was observed, we hypothesized that NF-kappa B mediates ceramide-induced COX-2 up-regulation. In this study, we added exogenous ceramide to the young Mphi and determined its effect on NF-kappa B activation. First, we tested the effect of different concentrations of ceramide on NF-kappa B activation. The results showed that incubating cells in the presence of ceramide for 2 h induced, although not as strongly as LPS, NF-kappa B activation in a dose-dependent manner (Fig. 7A). We then tested the time course of ceramide-induced NF-kappa B activation in the absence or presence of Bay 11-7082. As shown in Fig. 7B, ceramide induced NF-kappa B activation at all time points tested. This ceramide-induced NF-kappa B activation was prevented by addition of the Ikappa B phosphorylation inhibitor Bay 11-7082. Furthermore, we determined the effect of ceramide on LPS-stimulated NF-kappa B activation and demonstrated that LPS induced higher NF-kappa B activation in the cells supplemented with ceramide compared with those treated with vehicle control (Fig. 7C). It should be mentioned that ceramide by itself is a weak inducer of NF-kappa B activation and a much longer exposure time was needed to obtain a comparable band density to that seen with LPS. However, ceramide had an additive effect on LPS-induced NF-kappa B activation.


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Fig. 7.   Ceramide induces by itself, and also enhances LPS-stimulated NF-kappa B binding activity, which is inhibited by blocking NF-kappa B activation. A, peritoneal Mphi from young mice were incubated in the presence of ceramide at different concentrations as indicated for 2 h at 37 °C. B, peritoneal Mphi from young mice were preincubated with or without Bay 11-7082 (5 µM) for 30 min. Ceramide (30 µM) was added to the cells and incubation was continued at 37 °C for the additional times as indicated. C, in a separate experiment, Mphi were stimulated with LPS (5 µg/ml) during the same time course in the presence or absence of ceramide (30 µM). The nuclear extracts were prepared as described under "Experimental Procedures." Nuclear extracts (2 µg) were incubated with 32P-labeled COX-2-specific NF-kappa B oligonucleotide and subjected to an EMSA. The experiments were repeated twice and similar results were obtained.

To confirm that the altered NF-kappa B activation by ceramide or Bay 11-7082 is coupled to the changes in COX-2 activation, we measured PGE2 production under the same condition. As shown in Fig. 8, addition of exogenous ceramide increased PGE2 production and this ceramide-induced increase was prevented by inhibiting NF-kappa B activation. Similar to that seen in the NF-kappa B binding assay, addition of ceramide increased LPS-stimulated PGE2 production, an effect that was also prevented by inhibiting NF-kappa B activation.


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Fig. 8.   Addition of ceramide enhances LPS-induced PGE2 production, which is reduced by inhibition of NF-kappa B activation. The peritoneal Mphi from young mice were preincubated with or without Ikappa B phosphorylation inhibitor Bay 11-7082 (5 µM) for 30 min and then treated with ceramide (30 µM), LPS (5 µg/ml), or ceramide plus LPS for 12 h at 37 °C. The supernatants were collected and analyzed for PGE2 production. PGE2 concentrations in samples were determined using RIA and adjusted for total cell protein. Data are mean ± S.E. of four independent experiments in each of which a duplicate measurement was conducted. The bars bearing different letters represent significant difference at p < 0.05.

NF-kappa B Decoy Blocks NF-kappa B Binding Activity and Inhibits PGE2 Production and COX-2 Expression-- To further confirm the role of NF-kappa B, we repeated some of the above experiments by employing NF-kappa B decoy as an alternative and more specific approach to block NF-kappa B binding to COX-2 promoter. Use of the NF-kappa B decoy has been shown to successfully suppress COX-2 expression (40). The COX-2-specific NF-kappa B decoys have the identical sequences to those used for EMSA but modified on the 3 bases at each end by phosphorothioation to prevent being digested in the cells. The NF-kappa B decoy competes with the COX-2 promoter for binding to the activated NF-kappa B dimers and thus, block COX-2 gene activation. As shown in Fig. 9A, NF-kappa B decoy dose-dependently inhibited LPS-induced NF-kappa B binding activity while its mutated form did not have any effect. The role of NF-kappa B in the additive effect of ceramide on LPS-induced PGE2 production was further confirmed by data shown in Figs. 9 and 10. As seen in Fig. 9, NF-kappa B decoy but not the mutant abrogated the ceramide and LPS-induced NF-kappa B binding activity (Fig. 9B).


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Fig. 9.   NF-kappa B decoy blocks LPS or LPS plus ceramide induced NF-kappa B binding activity. A, NF-kappa B decoy or mutated NF-kappa B decoy at indicated concentrations were prepared as described under "Experimental Procedures." Peritoneal Mphi from young mice were preincubated in the presence of the decoys for 24 h at 37 °C. The culture medium was then replaced with new medium containing LPS (5 µg/ml) and incubated for 2 h. The nuclear extracts were prepared as described under "Experimental Procedures." Nuclear extracts (2 µg) were incubated with 32P-labeled COX-2-specific NF-kappa B oligonucleotide and subjected to an EMSA. B, the cells were preincubated with only one high concentration (10 nM) of NF-kappa B decoy or its mutant. LPS (5 µg/ml) were than added to stimulate the cells in the presence or absence of ceramide (30 µM). The experimental procedures are the same as described above in A.


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Fig. 10.   NF-kappa B decoy inhibits ceramide, LPS, or LPS plus ceramide-induced PGE2 production through suppressing COX-2 expression. A, the peritoneal Mphi from young mice were preincubated with NF-kappa B decoy or its mutant for 24 h. LPS (5 µg/ml), ceramide (30 µM), or both were then added to stimulate the cells for 12 h. The supernatants were collected and analyzed for PGE2 production. PGE2 concentrations in samples were determined using RIA and adjusted for total cell protein. Data are mean ± S.E. of four independent experiments. * indicates a significant difference at p < 0.05. B, the cell treatments were similar to those described above. Total cell lysates (25 µg of protein per lane) were used to determine COX-2 protein levels using Western blot analysis. After COX-2 bands were visualized, membranes were stripped and reprobed with the antibody to beta -actin to serve as a loading control.

Furthermore, we examined whether this blocked NF-kappa B binding would similarly affect PGE2 production and COX-2 expression. Mutated NF-kappa B decoy did not have a significant effect on PGE2 production (data not shown). NF-kappa B decoy, however, significantly inhibited LPS-stimulated PGE2 production both in the presence and absence of ceramide (Fig. 10A). The changes in PGE2 production was associated with similar changes in COX-2 protein expression (Fig. 10B).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously showed that the higher PGE2 production by Mphi from old mice was because of their increased expression of COX-2 mRNA (13). Furthermore, we showed that the increase in COX-2 mRNA was because of transcriptional up-regulation of COX-2 (14). Transcription factors NF-kappa B, AP-1, and CREB have been indicated in regulation of COX-2 activation. In this study, we tested the involvement of these transcription factors in the age-associated and ceramide-induced up-regulation of COX-2 activation. First we compared the activation levels of NF-kappa B, AP-1, and CREB in peritoneal Mphi from young and old mice. We found that in response to LPS stimulation, old Mphi had significantly higher consensus as well as COX-2-specific NF-kappa B binding activity compared with young Mphi . However, neither AP-1 nor CREB showed any significant change with age. The age-associated change in transcription factor activation has not been well studied and in particular, no information was available regarding their changes with age in Mphi . Kim et al. (34) reported that NF-kappa B binding activity in rat kidney increased with age. Helenius et al. (35) showed an age-related increase in the nuclear binding activity of NF-kappa B, but not those of Sp1 and AP-1, in rat liver and heart. Increased NF-kappa B and AP-1 activation with age was also observed in rat gastric mucosa cells (33). Mphi are the major source of inflammatory mediators including PGE2. These Mphi -originated mediators are involved in the pathogenesis of inflammatory, cardiovascular, and neoplastic diseases (41-43). Most of these diseases are more prevalent in the elderly population. Because NF-kappa B has been indicated in regulation of various Mphi -originated mediators, our finding that consensus NF-kappa B activation in Mphi is up-regulated with aging might shed light on the mechanism of age-related changes in other Mphi -originated mediators such as nitric oxide and proinflammatory cytokines.

Binding of activated NF-kappa B to the COX-2 promoter region has been suggested to be necessary for COX-2 transcriptional activation (16, 18, 19). To confirm this and also determine its role in the age-associated increase of COX-2 activation, we inhibited NF-kappa B activation by employing an Ikappa B phosphorylation inhibitor, Bay 11-7082. In agreement with the result reported by others (37), this inhibitor effectively prevented NF-kappa B activation in this study. Furthermore, the NF-kappa B inhibition dose dependently, up to 2 µM, reduced LPS-induced PGE2 production in both young and old Mphi . However, inhibition of NF-kappa B activation resulted in a significantly larger reduction in PGE2 synthesis in old Mphi compared with that in young Mphi . Because old Mphi have higher NF-kappa B activity as well as PGE2 production compared with young Mphi , these results further confirm the involvement of NF-kappa B in the age-related up-regulation of COX-2. Because PGE2 synthesis is determined by both substrate availability and COX activity, we measured COX activity by providing excessive exogenous arachidonic acid so that substrate availability would not be a limiting factor. The results showed dose-dependent inhibition of COX activity by Bay 11-7082. The degree of inhibition was significantly higher in old Mphi compared with that in young Mphi .

To stimulate Mphi for the activation of COX-2 and PGE2 production, we have mainly used LPS. It has been questioned whether the age-related difference in COX-2 expression is a phenomenon specific for LPS, merely reflecting the difference in LPS signal transduction at its receptor level. This is not likely because we previously observed an age-related increase in PGE2 production when different immune cells from both mice and humans and several other stimulating agents were used (4, 6). In fact, Mphi Toll-like receptor 4 expression decreases with aging (57). Thus, the age-related up-regulation of LPS-stimulated COX-2 activation involves a post-receptor signal transduction event, such as NF-kappa B, as suggested by this study. This was further strengthened in this study by the observation that when IL-1beta was used in place of LPS to stimulate Mphi , those from old mice have significantly higher PGE2 production compared with those from young mice. Furthermore, the IL-1beta -induced increase in PGE2 production was abrogated by inhibiting NF-kappa B activation.

If age-associated up-regulation of COX-2 is mediated through increased NF-kappa B activation, it will be predicted that other NF-kappa B target genes may also demonstrate an up-regulation with age and their expression could also be suppressed when NF-kappa B activation is blocked. We chose iNOS as such a candidate to substantiate this speculation. Increased NO production and iNOS expression have been shown in old compared with young murine Mphi (31, 38). The murine iNOS gene has an NF-kappa B binding site in its promoter region and pyrrolidine dithiocarbamate, an NF-kappa B inhibitor, blocks both NF-kappa B activation and NO production in LPS-stimulated Mphi (39). Increased iNOS expression has also been shown in vascular smooth muscle cells from old rats compared with those from young rats and this age-related up-regulation is associated with NF-kappa B activation (44). In this study, we confirmed the previous finding by showing higher iNOS expression in old Mphi compared with young Mphi . More importantly, we further demonstrated that blocking NF-kappa B activation reduced iNOS expression. This observation added further support for the involvement of NF-kappa B as a mechanism underlying the age-associated up-regulation of certain genes and their products.

The prototypical and most abundant form of NF-kappa B complex is the p65 and p50 heterodimer (45). In most resting cells, NF-kappa B is sequestered in the cytoplasm as an inactive precursor in complex with the inhibitory protein Ikappa B. By binding to the p65 component, Ikappa B inhibits transactivation of the p65 and p50 heterodimer, and thus blocking the translocation of the dimer to the nucleus (46, 47). In response to activation signals, Ikappa B is phosphorylated and degraded, allowing NF-kappa B release and further translocation to the nucleus where it regulates gene expression. Thus, the degradation of Ikappa B and translocation of NF-kappa B are closely linked to the activity of NF-kappa B binding to DNA. Although several members of the Ikappa B family have been identified, the best characterized Ikappa B is Ikappa B-alpha . In the current study, we found that upon LPS stimulation, old Mphi had increased degradation of Ikappa B-alpha in the cytoplasm, which was accompanied by an increased appearance of p65 in the nucleus. These results strongly suggest that the increased degradation of Ikappa B-alpha and the subsequent p65 translocation to the nucleus are the main contributors to the increased NF-kappa B binding activity seen in old Mphi compared with that in young Mphi . An age-related decrease in cytoplasmic Ikappa B-alpha and an age-related increase in nuclear p65 was observed in unstimulated rat kidney tissue by Kim et al. (34). However, another study showed that NF-kappa B activation increased with age but Ikappa B-alpha and p65 levels were unaffected in the rat gastric mucosa (33). Ikappa B is phosphorylated by the action of Ikappa B kinase (IKK) (48). The activation of IKK is in turn mediated by phosphorylation through NFkappa B-inducing kinase (49, 50). The cause of increased Ikappa B degradation was not determined in this study and is the subject of our future investigation. However, our recent work (14) suggested that ceramide might be involved in the age-associated increase in Ikappa B degradation. We showed that, following LPS stimulation, Mphi from old mice generated significantly more ceramide than those from young mice. Furthermore, we showed that C2-ceramide significantly increased LPS-induced COX activity and COX-2 expression in young Mphi ; this effect of ceramide was not mediated through mitogen-activated protein kinase. In this study, we showed that ceramide dose dependently induced NF-kappa B activation in young Mphi . This effect of ceramide was blocked by the Ikappa B phosphorylation inhibitor, Bay 11-7082. Addition of ceramide increased LPS-stimulated NF-kappa B activation, which was also inhibited by Bay 11-7082. These changes in NF-kappa B activation caused by ceramide, in the presence or absence of LPS, were mirrored by those in PGE2 production. Bay 11-7082 treatment prevented the ceramide-induced effect on both NF-kappa B activation and PGE2 production in the presence or absence of LPS. In this study, we also used a COX-2-specific NF-kappa B decoy as an alternative approach to block NF-kappa B activation. The decoy competes with the COX-2 promoter for binding to the activated NF-kappa B dimers and as a result, blocks COX-2 gene activation. The results obtained using the NF-kappa B decoy were similar to those obtained with Bay 11-7082. Taken together, because old Mphi have higher levels of ceramide compared with young Mphi and because increasing the level of ceramide in young Mphi enhances NF-kappa B activation, COX-2 expression, and PGE2 production, all of which are abrogated by Bay 11-7082 or an NF-kappa B decoy, these data strongly suggest that the age-associated increase in COX-2 transcription is because of the ceramide-induced up-regulation of NF-kappa B activation.

Determining NF-kappa B activation after reducing ceramide levels in old Mphi would have provided further support for our proposed mechanism, however, the approach is not feasible at the present time. Although knockout mice for acidic sphingomyelinase are available, these animals develop Neimann-Pick disease and die by the age of 10 months, making them unsuitable to address the role of ceramide in NF-kappa B up-regulation in aged mice (typically more than 20 months old). In addition, the neutral, but not the acidic sphingomyelinase has been indicated to be responsible for the age-related increase of ceramide levels in other tissues. However, specific neutral sphingomyelinase inhibitors are not commercially available.

The mechanism for the ceramide-induced increase in NF-kappa B activation has not been determined and will be the subject of our future study. It has been suggested that ceramide may induce NF-kappa B activation through its effect on the zeta  isotype of protein kinase C (PKC-zeta ). PKC-zeta was shown to activate NF-kappa B through phosphorylation of Ikappa B-alpha in NIH-3T3 fibroblasts (51). Furthermore, overexpression of PKC-zeta positively modulates IKKbeta activity, whereas the transfection of a PKC-zeta dominant negative mutant severely impairs the activation of IKKbeta (52). PKC-zeta -deficient mice have impaired NF-kappa B activation (53). It was reported that PKC-zeta can be activated in vitro by ceramide and in vivo by sphingomyelinase, which produces ceramide, in NIH-3T3 fibroblasts (54). Finally, ceramide was shown to induce the translocation of PKC-zeta to both the nucleus and membrane in rat astrocytes (55) and hepatocytes (56).

In summary, our data demonstrated for the first time, that NF-kappa B activation is up-regulated with aging in murine Mphi , and that this age-associated up-regulation of NF-kappa B activation mediates the higher age-associated expression of COX-2. Increased Ikappa B degradation and p65 translocation with aging represent important determinants of the increased NF-kappa B activation observed in old Mphi . Combined with our previous study (14), in which ceramide was shown to mediate the age-associated up-regulation of COX-2 transcription, the current study suggests that increased ceramide levels in old Mphi induces higher NF-kappa B activation, leading to increased COX-2 transcription. These findings will help to further understand the mechanism of the age-associated increase in COX-2 expression and associated diseases.

    ACKNOWLEDGEMENTS

We thank Dr. Sung Nim Han for technical assistance and Stephanie Marco for preparation of the manuscript.

    FOOTNOTES

* This work was supported by NIA National Institutes of Health Grant RO1-AG09140-09, National Institutes of Health Grant ES11518, and United States Department of Agriculture agreement 58-1950-9-001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Food Science and Human Nutrition Department, Michigan State University, East Lansing, MI 48824.

** To whom correspondence should be addressed: Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111. Tel.: 617-556-3129; Fax: 617-556-3224; E-mail: smeydani@hnrc.tufts.edu.

Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M207470200

    ABBREVIATIONS

The abbreviations used are: Mphi , macrophages; LPS, lipopolysaccharide; PGE2, prostaglandin E2; COX, cyclooxygenase; NF-kappa B, nuclear factor kappa B; AP-1, activator protein-1; CREB, cAMP-responsive element-binding protein; EMSA, electrophoretic mobility shift assay; AA, arachidonic acid; IL, interleukin; iNOS, inducible nitric-oxide synthase; RIA, radioimmunoassay; IKK, Ikappa B kinase; CRE, cAMP-response element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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