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© 2008 American Society for Nutrition J. Nutr. 138:17-23, January 2008

Biochemical, Molecular, and Genetic Mechanisms

3,3'-Diindolylmethane Suppresses the Inflammatory Response to Lipopolysaccharide in Murine Macrophages1–3,

Han Jin Cho5, Mi Ra Seon4, Yeo Myeong Lee4, Jaebong Kim6, Jin-Kyung Kim5, Sang Geon Kim7 and Jung Han Yoon Park4,5,*

4 Department of Food Science and Nutrition, 5 Center for Efficacy Assessment and Development of Functional Foods and Drugs, and 6 Department of Biochemistry, Hallym University, Chuncheon 200-702, South Korea; and 7 National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea

* To whom correspondence should be addressed. E-mail: jyoon{at}hallym.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
3,3'-Diindolylmethane (DIM), a major acid-condensation product of indole-3-carbinol, has been shown to have multiple anticancer effects in experimental models. Because recurrent or chronic inflammation has been implicated in the development of a variety of human cancers, this study examined the antiinflammatory effects of DIM and the underlying mechanisms using lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. DIM significantly decreased the release of nitric oxide (NO), prostaglandin (PG)E2, tumor necrosis factor {alpha}, interleukin (IL)-6, and IL-1β by RAW264.7 cells treated with LPS. DIM inhibited LPS-induced increases in protein levels of inducible NO synthase (iNOS), which were accompanied by decreased iNOS mRNA levels and transcriptional activity. The mRNA levels of phospholipase A2 decreased, whereas neither cyclooxygenases-2 protein nor transcript was altered by DIM. In addition, DIM suppressed LPS-induced nuclear factor-{kappa}B (NF-{kappa}B) transcriptional activity, NF-{kappa}B DNA-binding activity, translocation of p65 (RelA) to the nucleus, and degradation of inhibitor of {kappa}B{alpha}. Furthermore, DIM decreased LPS-induced transcriptional activity of activator protein (AP)-1, AP-1 DNA-binding activity, and phosphorylation of stress-activated protein kinase/Jun-N-terminal kinase and c-Jun. We demonstrate that DIM inhibits LPS-induced release of proinflammatory mediators in murine macrophages. Downregulation of NF-{kappa}B and AP-1 signaling may be one of the mechanisms by which DIM inhibits inflammatory responses.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Recurrent or chronic inflammation has been implicated in the development of a variety of human cancers, including those of the esophagus, stomach, colon, bladder, and prostate (1). Accordingly, developing strategies to reduce inflammation could be effective in preventing these diseases. In this regard, the evaluation of the antiinflammatory effects of various bioactive food components has gained widespread attention.

Macrophages are the main proinflammatory cells that respond to invading pathogens by releasing many pro-inflammatory molecules, including short-living free radical nitric oxide (NO)8 and prostaglandins (PG). NO is synthesized from L-arginine by NO synthase (NOS) in numerous types of cells. In mammals, 3 distinct isoforms of NOS have been cloned: endothelial, neuronal, and inducible NOS (iNOS) (2). Among these isoforms, iNOS plays an important role in the regulation of inflammatory responses. iNOS is synthesized in immune cells that have been stimulated by lipopolysaccharide (LPS) or cytokines. Cyclooxygenases (COX) are enzymes that catalyze the conversion of arachidonic acid to PG and have 2 known isoforms. COX-1 is normally expressed in many tissues and organs and plays an important role in maintaining physiologic functions. In contrast, COX-2 is induced by various stimuli, including inflammation, growth factors, and the cytokines produced by tumor cells. It has been shown that COX-2 and iNOS inhibitors suppress carcinogenesis (3,4), indicating that antiinflammatory agents that inhibit COX-2 and iNOS might be used for cancer prevention.

Inflammation is mediated by a range of soluble factors and cellular signaling events (1). Among these, considerable evidence indicates that nuclear factor-{kappa}B (NF-{kappa}B)-dependent gene expression plays an important role in inflammatory responses and increases the transcription of genes encoding cytokines and pro-inflammatory enzymes such as iNOS and COX-2 (1). In addition, activator protein (AP)-1, another transcriptional activator, is also involved in pro-inflammatory responses and is the transcription factor that regulates the genes encoding COX-2 and iNOS (5,6).

Indole-3-carbinol (I3C) is an autolysis product of glucosinolate present in Brassica plants such as turnips, kale, broccoli, cabbage, Brussels sprouts, and cauliflower. It has been shown to be protective against several cancers (7,8). When I3C is exposed to gastric acid, it is converted to many self-condensation products, among which 3,3'-diindolylmethane (DIM) is the major product (9,10). DIM was readily detected in the liver and feces of rodents fed I3C, whereas the original I3C was not detected in these animals (11). In addition, DIM is gradually formed from I3C in cell culture at a neutral pH over extended incubation periods (12). Furthermore, it has been reported that in women who underwent a phase I trial, I3C itself was not detectable in plasma after I3C administration and that the only detectable I3C-derived product was DIM (13). These results suggest that DIM, not I3C, may mediate the observed physiological effects of dietary I3C.

In vivo and in vitro studies indicated that DIM exhibits promising cancer preventive effects (14–20). However, to the best of our knowledge, the effect of DIM on inflammatory responses and the molecular mechanisms of DIM's antiinflammatory effects have not been elucidated. This study examined antiinflammatory effects of DIM and its underlying mechanisms using LPS-stimulated RAW264.7 murine macrophages.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Reagents. The following reagents were purchased from the indicated suppliers: DIM, LKT Laboratories; antibodies against iNOS and COX-2, BD Transduction Laboratories; anti-NF-{kappa}B p65 (C-20) antibody, Santa Cruz Biotechnology; antibodies against inhibitor of {kappa}B (I{kappa}B){alpha}, stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK), p-SAPK/JNK (Thr-183/Tyr-185), c-Jun, p-c-Jun (Ser-63), extracellular signal-regulated kinase (ERK)-1/2, p-ERK-1/2 (Thr-202/Tyr-204), p38 mitogen-activated protein kinase (MAPK), and p-p38 MAPK (Thr-180/Tyr-182), Cell Signaling Technology; pNF-{kappa}B-Luc and pAP-1-Luc containing the firefly luciferase (luc) coding sequence from Photinus pyralis (21), and pCMV-β, Takara Bio. pNF-{kappa}B-Luc contains multiple copies of NF-{kappa}B consensus sequence and pAP-1-Luc contains multiple copies of the AP-1 enhancer.

    Cell culture and cell viability assay. The RAW264.7 cell line was purchased from the American Type Culture Collection and maintained in DMEM containing 10% fetal bovine serum (FBS), 100 kU/L penicillin, and 100 mg/L streptomycin. For all experiments, the cells were subjected to no more than 20 cell passages. To examine the effect of DIM and LPS on cell viability, cells were plated in 24-well plates at 50,000 cells per well with DMEM containing 10% FBS. One day later, the monolayers were serum deprived in DMEM containing 1% FBS for 24 h. After serum deprivation, cells were treated with various concentrations (0–10 µmol/L) of DIM in the absence or presence of 100 µg/L LPS. Viable cell numbers were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (22).

    cDNA microarray. RAW264.7 cells were plated in 100-mm dishes at 1.5 x 106 cells per dish and serum deprived as described above. After serum deprivation, cells were incubated for 18 h in DMEM containing 1% FBS with 0 or 10 µmol/L DIM. Cells were then treated with or without 100 µg/L LPS for another 12 h. Total RNA was isolated using Tri reagent (Sigma) and a cDNA microarray was performed using a mouse whole genome chip (38.5K) in GenomicTree. The RNA pooled from 3 independent experiments was used in each chip. The images from the scanned microarrays were analyzed using Agilent GeneSpringGX 7.3 software and normalized by the intensity dependent (LOWESS) normalization method. The microarrays were performed by comparing the gene expression of each treatment group (10 µmol/L DIM, 100 µg/L LPS, or LPS + DIM) with that of the control group (0 µmol/L DIM) and the fold changes in gene expression by LPS, LPS + DIM, or DIM relative to the control are listed.

    NO, PGE2, and cytokine assays. RAW264.7 cells were treated with DIM and/or LPS as described above. The 24-h conditioned media were collected for NO, PGE2, and cytokine assays. The NO concentrations were measured using the Griess reagent system (Promega) and the PGE2 concentrations were measured using a PGE2 assay kit (R&D Systems). The concentrations of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6, IL-1β, and IL-10 were measured using ELISA kits according to the manufacturer's instructions (eBioscience).

    Western blot analysis. RAW264.7 cells were plated in 100-mm dishes at 1.5 x 106 cells per dish, serum deprived, and treated with various concentrations (0–10 µmol/L) of DIM in the absence or presence of LPS. Cell lysates were prepared as described previously (22). Total cell lysates were resolved on an SDS-PAGE (4–20%) and transferred onto polyvinylidene fluoride membrane (Millipore). The blots were blocked for 1 h in 5% milk in 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, and 0.1% Tween 20 and incubated overnight with their relevant antibodies (1:1000). The blots were then incubated with anti-mouse or anti-rabbit horseradish peroxidase–conjugated antibody. Signals were detected by the enhanced chemiluminescence method using SuperSignal West Dura Extended Duration Substrate (Pierce). The relative abundance of each band to its own β-actin was quantified by densitometric scanning of the exposed film using the Bio-profile Bio-1D application (Vilber-Lourmat). The control levels were set at 100% and the adjusted mean ± SEM (n = 3) of each band is shown above each blot.

    RT-PCR and real-time RT-PCR. Total RNA was isolated using Tri reagent and cDNA synthesized using 3 µg of total RNA with SuperScript II reverse transcriptase (Invitrogen), as described previously (22). Sequences for PCR primer sets, annealing temperatures, and numbers of cycles used for PCR amplification were published by Park et al. (23). The PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The relative abundance of bands corresponding to each specific PCR product to its own β-actin was quantified and the LPS control levels (100 µg/L LPS + 0 µmol/L DIM) were set at 100%. The adjusted mean ± SEM (n = 3) of each band is shown above each blot.

For quantification of TNF{alpha}, IL-6, IL-1β, phospholipase (PL)A2, and glyceraldehyde-3-phosphate dehydrogenase transcripts, real-time PCR was performed using a Rotor-gene 3000 PCR (Corbett Research). The reaction was carried out in 20 µL of reaction mixture that contained 2 µL cDNA, 0.5 µmol/L of both primers, and 10 µL SYBR PCR Master Mix (Qiagen). Sequences for PCR primer sets have been previously published [TNF{alpha}, IL-6, IL-1β, and glyceraldehyde-3-phosphate dehydrogenase in (24) and PLA2 in (25)]. PCR amplification of cDNA was performed at 94°C for 5 min, followed by 45 cycles as follows: 94°C for 20 s, 56°C for 15 s, and 72°C for 20 s. The progress of the PCR amplification was monitored in real-time by fluorescent measurement during each amplification cycle for the quantification of the target DNA molecules. The analysis of PCR results and calculation of the relative concentrations were performed using the Rotor-gene software (ver. 6) and the control levels (0 µmol/L DIM) were set at 1.

    Preparation of nuclear extract. RAW264.7 cells were washed briefly with ice-cold PBS containing 1 mmol/L iodoacetic acid and 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF), and lysed with hypotonic buffer (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2 mmol/L PMSF, 0.5 mmol/L dithiothreitol (DTT), 5 mL/L NP-40, 10 mmol/L iodoacetic acid, 20 mg/L aprotinin, 10 mg/L antipain, 10 mg/L leupeptin, 80 mg/L benzamidine HCl) on ice for 10 min. After centrifugation (2300 x g; 15 min at 4°C), pellets were resuspended with hypotonic buffer (without NP-40). The nuclei were pelleted by centrifugation and resuspended in 150 µL low salt buffer (20 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2 mmol/L PMSF, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 100 mL/L glycerol). Then 50 µL high salt buffer (containing 1.6 mol/L KCl) was added dropwise and the mixture was incubated for 1 h on ice. After centrifugation (25,000 x g; 30 min at 4°C), the supernatant was retained for use in the western blot and DNA-binding assay.

    Electrophoretic mobility shift assay. Double-stranded DNA probes for the consensus sequences of NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') were used for electrophoretic mobility shift assay after end-labeling of each probe with [{gamma}-32P]ATP and T4 kinase. Nuclear extracts (10 µg) were incubated with 30 µL binding buffer (10 mmol/L Tris-HCl, 100 mmol/L NaCl, 1 mmol/L EDTA, 4% glycerol, 1 mmol/L DTT) and 1 µg poly(dIdC) for 30 min at 37°C. For competitive and supershift experiments, nuclear extracts were preincubated with a 20-fold molar excess of unlabeled probe and 1 µg antibody, respectively, for 15 min. Each sample was subjected to 5% nondenaturing gel and the gels were dried and visualized by autoradiography.

    Luc reporter gene assay. The cells were cotransfected with pGL-miNOS-1588 containing murine iNOS gene promoter with the Luc coding region (5), NF-{kappa}B-Luc, or AP-1-Luc reporter plasmid and pCMV-β-galactosidase control vector using Nucleofector-II (Amaxa). The transfected cells were plated in 6-well plates at 2 x 105 cells per well. After 24 h, cells were serum deprived for 24 h, incubated for 18 h with 0, 5, or 10 µmol/L DIM, and then treated with or without LPS for another 6 h. Cell lysates were prepared to measure Luc and β-galactosidase activities using the Luc assay system (Promega). Luciferase activity was normalized to β-galactosidase activity.

    Statistical analyses. Data were expressed as means ± SEM and analyzed by ANOVA. Differences between treatment groups were analyzed by Duncan's multiple range test. Differences were considered significant at P < 0.05.


   Results
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    DIM inhibits LPS-induced NO and PGE2 production by RAW264.7 cells. The cytotoxic effects of DIM were evaluated in the presence and absence of LPS using the MTT assay. Because cell viability was not significantly affected by DIM up to 10 µmol/L (data not shown), we chose a concentration of 5–10 µmol/L DIM for the subsequent experiments.

To examine the effects of DIM on LPS-induced NO and PGE2 production, RAW264.7 cells were treated with various concentrations of DIM in the presence or absence of LPS. LPS markedly increased NO and PGE2 production. DIM inhibited LPS-induced NO production in a dose-dependent manner and 5 µmol/L DIM almost completely blocked LPS-induced PGE2 production. In unstimulated cells, NO and PGE2 production were low and DIM had no effect on either NO or PGE2 production (Table 1).


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  		TABLE 1 Effects of DIM on LPS-induced NO and PGE2 production in RAW264.7 cells12

 
We next assessed the effects of DIM on the expression of iNOS and COX-2, which catalyze the reactions for NO and PGE2 production, respectively. Western blot analysis revealed that DIM dose-dependently inhibited LPS-induced protein expression of iNOS, while having no effect on COX-2 protein levels (Fig. 1A). RT-PCR analysis showed that the steady-state levels of iNOS and COX-2 mRNA correlated with their corresponding protein levels (Fig. 1B). To examine whether the transcriptional activation of iNOS gene is inhibited by DIM, reporter gene assays were performed using RAW264.7 cells transfected with a mammalian cell expression vector pGL-miNOS-1588, which contained the Luc structural gene and –1.59 kb murine iNOS promoter (5). When transfected cells were treated with LPS, Luc activity increased by 200%, an increase that was significantly inhibited by DIM (Fig. 1C). These data indicate that DIM inhibits LPS-induced NO production through downregulation of iNOS expression.


Figure 1
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  		FIGURE 1  Effect of DIM on LPS-induced expression of iNOS and COX-2 in RAW264.7 cells. (A) Cell lysates were subjected to western blotting with their relevant antibodies. (B) Total RNA was isolated and RT-PCR performed. Photographs of chemiluminescent detection of the blots (A) or the ethidium bromide-stained gels (B), which were representative of 3 independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified and the LPS control levels (100 µg/L LPS + 0 µmol/L DIM) were set at 100%. The adjusted mean ± SEM, n = 3, of each band is shown above each blot. (C) Cells were cotransfected with murine iNOS reporter gene construct and pCMV-β-galactosidase control vector and the transfected cells were plated in 6-well plates at 2 x 105 cells per well. After serum deprivation, cells were treated with the indicated concentrations of DIM in the absence or presence of LPS for 6 h. Cell lysates were prepared to measure Luc and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity. Each bar represents the mean ± SEM, n = 3. Means without a common letter differ, P < 0.05.

 
    DIM inhibits LPS-induced activation of NF-{kappa}B and AP-1 signaling. Because activation of NF-{kappa}B plays an important role in the transcriptional activation of iNOS, we examined next the effects of DIM on NF-{kappa}B signaling. LPS decreased I{kappa}B{alpha} and pretreatment of cells with DIM significantly prevented LPS-induced decrease in I{kappa}B{alpha} (Fig. 2A). Western blot analysis using nuclear extracts revealed that LPS-induced translocation of p65 to the nucleus was inhibited by DIM pretreatment (Fig. 2B). To test whether the decreased p65 translocation is functionally linked to decreased DNA-binding capacity, we performed electrophoretic mobility shift assay. DIM pretreatment significantly inhibited LPS-induced NF-{kappa}B DNA-binding activity (Fig. 2C). Supershift analysis indicated that the band induced by LPS contained p50/p65 proteins, because this complex was partially supershifted by anti-p50 and anti-p65 antibody. Reporter gene analysis using a Luc plasmid containing the NF-{kappa}B binding sequence indicated that LPS-stimulated NF-{kappa}B reporter activity was significantly inhibited by DIM pretreatment (Fig. 2D).


Figure 2
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  		FIGURE 2  Effects of DIM on LPS-induced NF-{kappa}B signaling in RAW264.7 cells. Serum-deprived cells were treated with DIM for 18 h in DMEM containing 1% FBS. LPS was then added and incubated for another 20 min. (A) Cell lysates were subjected to western blotting with an anti-I{kappa}B{alpha} antibody. Nuclear extracts were prepared for western blotting with an anti-p65 antibody (B) and electrophoretic mobility shift assay (C). Photographs of chemiluminescent detection of the blots (A,B) or an autoradiography of the dried gels (C), which were representative of 3 independent experiments, are shown. The relative abundance of each band was quantified and the control levels (0 µmol/L DIM) were set at 100%. The adjusted means ± SEM, n = 3 of each band is shown above each blot. (D) Cells were cotransfected with NF-{kappa}B-Luc reporter plasmid and pCMV-β-galactosidase vector and plated in 6-well plates at 2 x 105 cells per well. After serum deprivation, cells were incubated for 18 h with 0, 5, or 10 µmol/L DIM. LPS was then added and the incubation continued for another 6 h. Luciferase activity was normalized to β-galactosidase activity. Each bar represents the mean ± SEM, n = 3. Means without a common letter differ, P < 0.05.

 
For transcriptional activation of any single gene that is involved in the induction of the inflammatory response, NF-{kappa}B often needs help from other sequence-specific transcription factors. An example is the AP-1 family, whose activation is dependent on phosphorylation by MAPKs (26). Three mammalian MAPK pathways have been identified; the ERK, SAPK/JNK, and p38 MAPK pathways. Treatment of RAW264.7 cells with LPS dramatically activated the MAPK family proteins. DIM pretreatment significantly inhibited LPS-induced phosphorylation of SAPK/JNK, whereas the phosphorylation of other MAPK family proteins (p38 or ERK-1/2) was unaffected by DIM pretreatment (Fig. 3A). LPS stimulated phosphorylation of c-Jun, a component of AP-1 and a downstream target of SAPK/JNK (27); this phosphorylation was decreased by pretreatment with DIM. DIM pretreatment also inhibited LPS-induced DNA-binding activity (Fig. 3B) and transcriptional activity of AP-1 (Fig. 3C).


Figure 3
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  		FIGURE 3  Effects of DIM on LPS-induced AP-1 signaling in RAW264.7 cells. Cells were treated with DIM and/or LPS as described in Figure 2. (A) Cell lysates were subjected to western blotting with their relevant antibodies. (B) Nuclear extracts were prepared for electrophoretic mobility shift assay. Photographs of chemiluminescent detection of the blots (A) or an autoradiography of the dried gels (B), which were representative of 3 independent experiments, are shown. The relative abundance of each band was quantified and the control levels (0 µmol/L DIM) set at 100%. The adjusted means ± SEM, n = 3 of each band is shown above each blot. (C) Cells were cotransfected with AP-1-Luc reporter plasmid and pCMV-β-galactosidase control vector and plated in 6-well plates at 2 x 105 cells per well. After serum deprivation, cells were incubated for 18 h with 0, 5, or 10 µmol/L DIM. Cells were untreated or treated with LPS for another 6 h. Luciferase activity was normalized to β-galactosidase activity. Each bar represents the mean ± SEM, n = 3. Means without a common letter differ, P < 0.05.

 
    DIM inhibits LPS-induced TNF{alpha}, IL-6, and IL-1β production. To evaluate changes in gene expression following treatment with LPS and DIM, cDNA microarrays were performed using a mouse whole genome chip (38.5K). A total of 2294 genes were found to be induced at least 2-fold by LPS, from which we chose inflammation and immune-like response related genes. Among these genes, those down-regulated by DIM are listed in Supplemental Table 1. LPS dramatically increased gene expression of pro-inflammatory cytokines such as TNF{alpha}, IL-1β, and IL-6, and chemokine ligands. This increased expression was substantially suppressed by DIM treatment. DIM inhibited LPS-induced gene expression of iNOS, whereas DIM had no effect on COX-2 gene expression. These results are consistent with the results shown in Figure 1 obtained with western blot and RT-PCR analysis showing that DIM decreased protein and mRNA levels of iNOS but not those of COX-2. Expression of PLA2 gene was increased by LPS, an effect mitigated by DIM treatment.

Results of real time RT-PCR analysis revealed that LPS increased the steady-state levels of TNF{alpha}, IL-6, IL-1β, and PLA2 transcripts, which were suppressed by DIM treatment (Table 2) confirming the cDNA microarray results.


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  		TABLE 2 Effects of DIM on LPS-induced mRNA expression of TNF{alpha}, IL-6, IL-1β, and PLA2 in RAW264.7 cells1

 
To examine whether the changes in mRNA levels of cytokines led to alterations of their protein levels, we performed ELISA. LPS-induced TNF{alpha} production was inhibited by DIM treatment in a dose-dependent manner. In addition, DIM treatment significantly inhibited LPS-induced production of IL-6 and IL-1β (Table 3).


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  		TABLE 3 Effects of DIM on LPS-induced TNF{alpha}, IL-6, and IL-1β production in RAW264.7 cells12

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
In this study, we addressed the question of whether DIM modulates LPS-induced production of pro-inflammatory mediators and the signal transduction cascades involved in these processes in RAW264.7 murine macrophages. We observed that DIM inhibited the effects of LPS stimulation. The inhibition resulted in decreased LPS-stimulated production of PGE2, NO, TNF{alpha}, IL-6, and IL-1β; attenuated LPS-stimulated increase in mRNA levels of iNOS, IL-6, IL-1β, TNF{alpha}, and PLA2; reduced LPS-stimulated activation of SAPK/JNK and c-Jun; decreased LPS-stimulated AP-1 DNA binding and transcription activity; and reduced LPS-stimulated nuclear translocation of p65, NF-{kappa}B DNA binding, and transcription activity. To the best of our knowledge, these findings are the first to show that DIM inhibits inflammatory responses in macrophages through the inhibition of the JNK/AP-1 and NF-{kappa}B signaling pathways. Future animal studies are required to determine whether DIM inhibits inflammatory processes in vivo.

NO produced by iNOS is known to be an important mediator in inflammation (28). Transcription of iNOS is increased in activated macrophages, resulting in a prolonged high level of NO production (29). In this study, NO production, iNOS protein and mRNA levels, and iNOS transcription activity were increased in LPS-treated cells. All of these effects were suppressed by DIM. These results clearly demonstrated that DIM decreased NO production through downregulation of iNOS gene transcription.

Most surprisingly, we observed that DIM markedly suppressed LPS-induced PGE2 production (Table 1) without any change in COX-2 protein or mRNA levels (Fig. 1A,B). cDNA microarray and real time RT-PCR analysis revealed that LPS increased PLA2 mRNA, which was suppressed by DIM treatment. PLA2 regulates fatty acid release from membrane phospholipids (30) and released arachidonic acid is converted to PG by COX. These results suggest that DIM inhibits arachidonate release by decreasing PLA2 expression, which leads to decreased PGE2 synthesis. It remains to be determined whether DIM directly inhibits the activity of COX-2.

Substantial evidence indicates that the most important signaling pathway involved in the initiation and amplification of inflammatory responses is the one that leads to activation of NF-{kappa}B transcription factors (31,32). NF-{kappa}B is maintained in a latent form in the cytoplasm where it is in complex with I{kappa}B. The interaction of NF-{kappa}B with I{kappa}B masks the nuclear localization signal. Phosphorylation of I{kappa}B by I{kappa}B kinase leads to ubiquitination of the protein and its subsequent degradation by the 26S proteasome (33). NF-{kappa}B is then free to translocate to the nucleus where it binds to DNA leading to the activation of a wide variety of inflammatory response target genes (34,35). NF-{kappa}B transcription factors bind to DNA as hetero- or homodimers that are selectively derived from 5 possible subunits [RelA (p65), c-Rel, RelB, p50, and p52]. The most common heterodimer is RelA (p65)-p50. The roles of NF-{kappa}B in the induction of TNF{alpha}, IL-6, IL-1β, PLA2, and iNOS gene transcription have been well established (36–38). In this study, we have shown that DIM regulates protein levels of the inflammatory-related cytokines and enzymes at the RNA level. In addition, we observed that DIM mitigated the LPS-induced decrease in I{kappa}B{alpha} and increases in nuclear translocation of p65, NF-{kappa}B DNA binding, and NF-{kappa}B reporter activity. These results indicate that inhibition of NF-{kappa}B signaling contributes to the decreased production of TNF{alpha}, IL-6, IL-1β, PLA2, and iNOS in DIM-treated macrophages.

Despite its central role, it is unlikely that the simple activation of NF-{kappa}B is sufficient for transcription activation or induction of any single NF-{kappa}B target gene that is involved in the initiation of inflammatory responses. For most promoters, NF-{kappa}B requires assistance from other sequence-specific transcription factors such as AP-1 (26). AP-1 controls transcription of many genes, including TNF{alpha} (6), IL-6 (39), IL-1β (40), and iNOS (6). Here, we observed that DIM significantly inhibited AP-1 DNA-binding activity and transcription activity. These results indicate that inhibition of AP-1 activity contributes to the decreased production of TNF{alpha}, IL-6, IL-1β, and iNOS in DIM-treated macrophages. Because AP-1 activity is stimulated by a complex network of signaling pathways that involves MAPKs of the ERK, p38, and JNK families (41), this study examined whether DIM inhibits phosphorylation of these 3 MAPKs. DIM did not inhibit phosphorylation of either ERK or p38 MAPK but significantly inhibited LPS-induced phosphorylation of SAPK/JNK. JNK has the double phosphorylation motif Thr-Pro-Tyr, which is phosphorylated by the upstream kinases MKK4 and MKK7 leading to activation of this kinase. JNK is a Ser/Thr protein kinase that phosphorylates c-Jun, a part of the transcription factor AP-1 (42). DIM not only inhibited LPS-induced phosphorylation of JNK, but also phosphorylation of c-Jun, indicating that inhibition of AP-1 activity by DIM involves inhibition of JNK/c-Jun activation.

In addition to the antiinflammatory effect, DIM was reported to induce cell cycle arrest and apoptosis of various cancer cells, inhibit tumor invasion and angiogenesis, modulate estrogen metabolism [reviewed in (43)], and stimulate the immune functions (44). DIM can exert these effects by binding to some signaling molecules on the plasma membranes or inside of the cells. DIM has been reported to bind to the aryl hydrocarbon receptor in the cytoplasm resulting in the induction of relevant gene transcription (45,46), and induce peroxisome proliferator-activated receptor {gamma}-dependent transactivation (47). This suggests that DIM is translocated into the cells where it exerts its biological effects. More studies will be needed to address the question as to how DIM modulates the antiinflammatory effects such as suppression of I{kappa}B{alpha} degradation and SAPK/JNK phosphorylation.

This study used DIM at concentrations of 5–10 µmol/L. However, the biological relevance of these doses in relation to prevention or therapy has not been fully evaluated. Anderton et al. (48) examined concentrations of DIM in tissues following the dosing of mice with 250 mg/kg DIM or an equivalent amount of the enhanced absorption BioResponse DIM. The levels of DIM peaked at ~0.5–1 h in the liver (160 µmol/L) and plasma (6 µmol/L) following administration of DIM. The BioResponse resulted in concentrations ~50% higher than those with DIM. Unfortunately, there is a paucity of information on the DIM concentrations in the human blood. Recently, it was reported that in a phase I trial of women, the level of DIM in plasma increased to 2.46 µmol/L (607 µg/L) after a single oral dose of 1000 mg I3C (13). These results suggest that the concentrations of DIM used in this study can be achieved in vivo. However, to determine whether the DIM concentrations used in the cell culture studies are clinically relevant, more studies will be needed to determine the concentrations of DIM in human serum after the administration of I3C or DIM.

In conclusion, this study demonstrates that DIM inhibits the inflammatory responses of murine macrophages. Downregulation of NF-{kappa}B and AP-1 signaling contributes the antiinflammatory actions of DIM. This study provides some of the molecular basis for using DIM as a potential antiinflammatory agent. DIM deserves further evaluation of its potential as an antiinflammatory agent in experimental animal models.


   FOOTNOTES
 
1 Supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-311-C00176) and a grant (code no. 20070301034039) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. Back

2 Author disclosures: H. J. Cho, M. R. Seon, Y. M. Lee, J. Kim, J. K. Kim, S. G. Kim, J. H. Y. Park, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

8 Abbreviations used: AP, activator protein; COX, cyclooxygenase; DIM, 3,3'-diindolylmethane; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; I3C, Indole-3-carbinol; I{kappa}B{alpha}, inhibitor of {kappa}B{alpha}; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; Luc, luciferase; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor-{kappa}B; NO, nitric oxide; NOS, nitric oxide synthase; PG, prostaglandin; PL, phospholipase; SAPK/JNK, stress-activated protein kinase/Jun-N-terminal kinase; TNF, tumor necrosis factor. Back

Manuscript received 21 August 2007. Initial review completed 18 September 2007. Revision accepted 19 October 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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