Abstract
Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-κB signaling while also activating the c-Jun NH2-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-κB translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-κB plays an inhibitory role. Moreover, NF-κB stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-κB protein, is knocked down. Thus, NF-κB signaling pathways negatively regulate the CYP4F11 gene.
Introduction
The release of several cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1, and IL-6 from activated immune cells in patients with an infection or a disease with an inflammatory component occurs as part of the activation of systemic host defense mechanisms. This defense mechanism and the release of these cytokines usually result in the down-regulation of many isoforms of cytochrome P450 (P450) (Ghezzi et al., 1986; Shedlofsky et al., 1987, 1997; Bertini et al., 1988; Morgan 1989; Wright and Morgan 1990). This is important because of the major roles of the P450 systems in the metabolism of drugs used in the treatment of inflammatory conditions, as well as steroids, lipid-soluble vitamins, prostaglandins, and leukotrienes. Changes in the gene expression of P450s can in turn cause changes in detoxification and metabolic bioactivation in tissues.
TNF-α, one of the cytokines prominent in the inflammatory response, is known to be released during many types of infections and inflammatory diseases (Shedlofsky et al., 1997). In the current study, we used this cytokine to define the effects of its presence on the regulation of CYP4F11, an isoform of the 4F family whose substrate profile includes not only endogenous compounds but also xenobiotics, specifically pharmaceuticals (Kalsotra et al., 2004). TNF-α stimulation causes an activation of two signal transduction pathways, the c-Jun NH2-terminal kinase (JNK) pathway and the nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) pathway (De Smaele et al., 2001; Tang et al., 2001; Deng et al., 2003). Our previous studies have examined the roles the JNK pathway and retinoic acids play in the regulation of CYP4F11 and have found that JNK stimulation causes an increase in CYP4F11 mRNA, whereas retinoids cause down-regulation of CYP4F11 mRNA (Wang et al., 2010). However, we did not examine the effects of NF-κB on CYP4F11 expression during TNF-α stimulation.
To define at a deeper level the regulation of CYP4F11 expression during inflammation, we report in this study that TNF-α and mitogen-activated protein kinase kinase kinase (MEKK) overexpression can down-regulate CYP4F11 expression in an NF-κB-dependent manner. Our results suggest that there is an intricate multipathway regulatory system for control of expression of CYP4F11 during conditions wherein inflammatory modulators are released.
Materials and Methods
Chemicals.
N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354) was obtained from Sigma-Aldrich (St. Louis, MO). TNF-α was a gift from Dr. Jianping Jin (University of Texas-Houston). IL-1 was obtained from Invitrogen (Carlsbad, CA). Polyclonal anti-CYP4F11 antibody was provided by Proteintech Group, Inc. (Chicago, IL). Rel B and p65 antibodies were purchased from Cell Signaling Technology (Danvers, MA).
Cell Culture.
HepG2 cell line was obtained from American Type Culture Collection (Manassas, VA). Cells were grown at 37°C in a humidified incubator with 5% CO2 in minimal essential medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO), l-glutamine, and penicillin/streptomycin antibiotics.
Quantitative Real-Time Polymerase Chain Reaction.
Cells were rinsed with phosphate-buffered saline (PBS), and total RNA was isolated using TRIzol reagent (Invitrogen). DNase I (Invitrogen) was used to alleviate DNA contamination. Aliquots (100 ng) of total RNA were reverse-transcribed by SuperScript II reverse transcriptase (Invitrogen) in triplicate, including a reverse transcription blank to evaluate the presence of contaminating genomic DNA. Amplification was performed using Taq DNA polymerase (Invitrogen) with an ABI Prism 7700 (Applied Biosystems, Foster City, CA) at 95°C for 1 min, followed by 40 cycles at 95°C for 12 s and 60°C for 30 s. CYP4F11 mRNA levels were measured using standard curves generated by plotting threshold cycle versus the log of the amount of purified amplicon for CYP4F11 (custom synthesis by Invitrogen) (200 ag to 2 pg). Abundance of human 18S RNA was used as an internal control. Primers and fluorescent probe sequences for CYP4F11 and 18S RNA are reported in Table 1.
Plasmids.
pGL3-CYP4F11 plasmid mutants were constructed using human genomic DNA as a template for polymerase chain reaction (PCR). The primer pairs for each mutant are also listed in Table 1. The products were all 3′ promoter regions of the CYP4F11 (GenBank accession number NG_0008335). The PCR products were then cloned into HindIII- and NheI-digested linear pGL3-Basic luciferase vector (Promega, Madison, WI) using the In-Fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA).
Transfection and Luciferase Assays.
Cells were transfected using FuGENE HD reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. Cells were seeded onto 24 well plates (5 × 104 cells/well). Twenty-four hours after seeding, cells were transfected at the recommended reagent/DNA ratio of 4.5:1 with 0.5 μg of DNA/well including 0.03 μg of phRL-SV40 (Promega). The total amount of DNA was maintained at a constant concentration by adding empty pcDNA3 vector when appropriate. After 24 h of transfection, TNF-α (10 ng/ml) or IMD-0354 (1 ng/ml) treatment was given for 24 h.
RNA Interference.
Double-stranded, small interfering RNAs (siRNAs) targeting p65 were designed and synthesized by Sigma-Aldrich, 5′-GACAUUGAGGUGUAUUUCA-3′ and 5′-UGAAAUACACCUCAAUGUC-3′, and were reverse-transfected into HepG2 cells using Lipofectamine RNAiMAX (Invitrogen). In short, 24-well plates were seeded (5 × 104 cells/well) in the presence of 6 pmol and 1 μl of Lipofectamine RNAiMAX in 100 μl of Opti-Mem I serum (Invitrogen). Total medium volume was 600 μl for a final RNA interference duplex concentration of 10 nM.
Chromatin Immunoprecipitation Analysis.
Binding of p65 transcription factor to the human CYP4F11 promoter region was determined by chromatin immunoprecipitation (ChIP) assay according to manufacturer's protocol (Active Motif, Carlsbad, CA). HepG2 cells after either 1 or 24 h of treatment with TNF-α (10 ng/ml) were fixed using 1% formaldehyde in modified Eagle's medium for 10 min at 37°C. The cells were then washed 1× in ice-cold PBS and were treated with 125 mM glycine to terminate the cross-linking reaction. The cells were washed again with ice-cold PBS and were collected in PBS containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Active Motif). The cells were lysed and sonicated to shear chromatin. Fifteen micrograms of chromatin was then incubated overnight at 4°C with antibody directed against p65. PCR was used to amplify the 222-base pair (bp) region upstream of the start site of CYP4F11 from the purified DNA-protein immune complexes using the 222-bp CYP4F11 primers described in Table 1. PCR products were run on 2% agarose gel and were visualized after ethidium bromide staining. Controls for the assay were performed using IgG and input template DNA.
Statistical Analysis.
Data are presented as mean ± S.E.M. One-way analysis of variance followed by Dunnett's multiple comparison test was used for the statistical analysis. Statistical differences were considered significant if P < 0.05.
Database Sequence Analysis.
The TRANSFAC database (BIOBASE, Beverly, MA) was searched using AliBaba 2.1 (BIOBASE) for putative NF-κB binding sites on the 5′ flanking sequence (2000 bp from the ATG start codon) of the CYP4F11 gene.
Results
NF-κB Inhibition Increases Expression of Endogenous CYP4F11.
Previously published work reported that CYP4F11 expression is increased upon stimulation with TNF-α in keratinocyte HaCaT cells (Wang et al., 2010). TNF-α activates two pathways: the JNK pathway, which was shown to up-regulate CYP4F11 expression through activator protein 1 (AP-1) binding sites, and the NF-κB signaling pathway. This current study was designed to understand what role the NF-κB transcription factor played in the regulation of CYP4F11. A 24-h treatment with TNF-α (10 ng/ml) caused an up-regulation of CYP4F11 mRNA expression in HepG2 cells measured by real-time quantitative PCR. To understand the role NF-κB may have on the expression of CYP4F11, inhibition of NF-κB translocation to the nucleus from the cytoplasm using the chemical inhibitor IMD-0354 (1 ng/ml) was used. HepG2 cells were cotreated with both the inhibitor and the TNF-α (10 ng/ml) (Fig. 1). There was a significant increase in the expression of CYP4F11 transcripts compared with vehicle control. Realizing that NF-κB played a role in CYP4F11 expression after observing an up-regulation of the gene when translocation of NF-κB to the nucleus was inhibited, the 2000-bp upstream promoter region of the CYP4F11 gene was analyzed in the TRANSFAC database using the program AliBaba 2.1 for predictions of transcription factor binding sites (Fig. 2). The website predicted five different NF-κB binding sites in the promoter region of CYP4F11.
CYP4F11 Promoter Activity is Down-Regulated after HepG2 Activation by TNF-α and MEKK Overexpression.
To determine the TNF-α regulatory region in the CYP4F11 promoter, the 1.7-kilobases (kb) region upstream of CYP4F11 exon-2 transcription initiation site was cloned into pGL3 basic luciferase vector. The basal promoter activity was determined, and activity after treatment with TNF-α and MEKK overexpression in HepG2 cells was assessed. As seen in Fig. 3A, treatment of the transfected cells with MEKK or TNF-α resulted in a 70 to 75% reduction in CYP4F11 promoter activity. TNF-α and MEKK treatment up-regulated control NF-κB luciferase reporter vector activity (Fig. 3B) but had no effect on pGL3 basic luciferase control vectors (Fig. 3C). This suggests that even though TNF-α causes an overall up-regulation of endogenous CYP4F11 transcripts, as seen previously in Fig. 1, the promoter inhibition is accomplished through NF-κB activation. This was seen in both the TNF-α treatment condition and through the overexpression of MEKK, which constitutively activates NF-κB protein, both of which down-regulate CYP4F11 promoter activity.
NF-κB-Responsive Region Is Present in the First 200 bp of CYP4F11 Promoter.
Three deletion mutants were created to determine the functional importance of the NF-κB binding sites predicted by AliBaba 2.1 (Fig. 4A). Each construct deletes a predicted NF-κB binding site. The last 1.5-kb mutant construct deleted three predicted NF-κB binding sites that were within a single 200-bp segment. HepG2 cells were transiently transfected with each of the 5′-deletion constructs with or without MEKK expression plasmid and were treated with or without TNF-α. All of the 5′-deletion constructs of the CYP4F11 promoter except the 1.5-kb construct were down-regulated by TNF-α and MEKK (Fig. 4B). This result suggests that the NF-κB-responsive region should be within the first 200 bp of CYP4F11 promoter. A ChIP assay was conducted on the first 222 bp of CYP4F11 promoter region to determine whether the p65 transcription factor was binding to the promoter region to cause an inhibition of the promoter. Figure 5 shows that the protein does bind to the region, and the relative amount of protein binding to the region is dependent on the length of time that has elapsed after TNF-α activation. There is an increase in NF-κB protein binding to the promoter region of CYP4F11 after 1 h of TNF-α treatment, whereas there is no difference between control and treatment after 24 h. The density difference for the 1-h time point is 83.291/11608.5283 between control and 1-h TNF-α treatment, whereas the difference at the 24-h time point is 5584.8798/5647.9126 between control and TNF-α treatment. Many investigators have reported the time dependencies of activation of NF-κB and JNK (Román et al., 2000; Deng et al., 2003; Papa et al., 2006). Román (2000) showed that the degrees of binding of NF-κB and AP-1 were enhanced by acetaldehyde but were time-dependent, with NF-κB binding first and then AP-1 binding after 4 h. Papa et al. (2006) have also reported that the binding of TNF-α leads to rapid activation of NF-κB that can inhibit some functions of the JNK pathway. This mechanistic control is reported to be prosurvival and time-dependent; the longer TNF-α is present to activate downstream signaling pathways, the more JNK signaling prevails over NF-κB (Deng et al., 2003; Papa et al., 2006).
NF-κB Is Required for CYP4F11 Down-Regulation.
The next goal was to determine the importance of NF-κB in the down-regulation of CYP4F11 in response to TNF-α. We approached this goal by using the inhibitor of nuclear factor-κB kinase complex-α phosphorylation inhibitor IMD-0354. This drug inhibits the translocation of NF-κB to the nucleus, thus preventing any regulatory controls NF-κB might exert on its response genes. HepG2 cells were transiently transfected with each CYP4F11 deletion luciferase promoter construct and then were cotreated with TNF-α (10 ng/ml) and IMD-0354 (1 ng/ml). As shown in Fig. 6, CYP4F11 promoter activity is not down-regulated by TNF-α in the presence of IMD-0354. We did observe an increase in expression of the 1.5-kb CYP4F11 mutant construct in the presence of IMD-0354 without the presence of TNF-α. We saw a trend for increase with the use of IMD-0354 among all the deletion constructs; however, this increase was significant only without the presence of NF-κB regulatory binding sites in the 1.5-kb construct. To confirm these results and rule out any treatment drug interactions, we repeated the experiment in HepG2 cells in the presence of siRNA that knocks down p65 (an NF-κB protein). The data presented in Fig. 7 show that after p65 knockdown and in the presence of TNF-α or overexpression of MEKK conditions, down-regulation of CYP4F11 does not occur. These data indicate that CYP4F11 promoter down-regulation requires p65 or NF-κB, and that the data obtained from the use of IMD-0354 are valid.
Discussion
The ability of cytokines, prostaglandins, and other inflammatory mediators to alter the expression of many different P450s has been the subject of numerous studies (Wright and Morgan, 1990; Iber et al., 2000; Ke et al., 2001; Abdulla et al., 2005). The significance of this work stems from the effects that changes in P450 levels have on the metabolism of many drugs, on the homeostasis of steroid hormones, and on the ability of P450s to detoxify xenobiotics. The effects of inflammation on P450 expression vary, but the predominant effect is that inflammatory cytokines suppress the gene expression of most P450s. In this work, we have presented a novel mode of regulation for CYP4F11 expression during an inflammatory response. We have shown that activation of the NF-κB pathway by TNF-α stimulation or overexpression of MEKK causes down-regulation of CYP4F11 promoter transcripts. However, what makes this finding unique is that the inhibition of endogenous CYP4F11 is not seen in the presence of TNF-α after 24 h. This regulation by TNF-α was reported in a previous study from our laboratory (Wang et al., 2010) and is verified in this study in Fig. 1, which also shows that the presence of TNF-α and an inhibitor of NF-κB cause a greater increase in CYP4F11 transcript quantity than TNF-α alone. This large increase in the number of transcripts shows that although JNK is a strong stimulator of CYP4F11 expression, NF-κB may play an inhibitory role. We believe this is due to the signal transduction properties of TNF-α. It has been shown that the JNK and the NF-κB pathways are both stimulated in the presence of TNF-α; however, they are competing forces (Papa et al., 2006). The up-regulation of CYP4F11 mRNA in the presence of TNF-α is due to the stimulation of the JNK pathway (Wang et al., 2010); however, this is time-dependent and does not reach a level of significance until 24 h after treatment, as observed in our experiments. This was confirmed by ChIP analysis, where there was a large increase in p65-bound CYP4F11 transcripts at 1 h after treatment that was not present at 24 h.
When we then examined a specific promoter region of CYP4F11 that had many NF-κB binding sites, we were able to show that NF-κB activation causes an inhibition of CYP4F11 promoter construct expression. This was consistent with our findings for endogenous CYP4F11 mRNA transcripts, where the deactivation of NF-κB resulted in a release of inhibition and an increase in CYP4F11 mRNA expression was increased. After determining that NF-κB inhibition caused a decrease in promoter activity in our constructs (Fig. 4), we used chemical inhibition (Fig. 6) and protein knockdown (Fig. 7) of NF-κB to determine the effects on the promoter activity. We found that in those cases, there was either control-level expression or overexpression of the CYP4F11 gene. This led us to believe that the down-regulation of CYP4F11 is NF-κB-dependent.
The overexpression of the endogenous CYP4F11 gene seen in Fig. 1, and also seen in the CYP4F11 promoter mutants after inhibition, can be explained partly by the activation of areas in the promoter region that have AP-1 binding sites, which would create higher levels of expression of CYP4F11 through JNK activation. This mechanism was described in an article published by our laboratory (Wang et al., 2010). In short, JNK activation causes activation of AP-1, which regulates expression through the AP-1 binding sites on the promoter region of the gene. In the 1.7-kb CYP4F11 promoter construct, there are five AP-1 binding sites positioned throughout the promoter region, and when TNF-α is present, JNK signaling can still occur and elicit regulation of the CYP4F11 promoter construct. These sites are shown in Fig. 2. Overall, this finding is one piece of a complicated mix of regulatory networks that lead to the regulation of CYP4F11, and with each finding, the pieces of the puzzle fit more coherently. This is pictorially represented in Fig. 8. Overall, we believe that this regulation of CYP4F11 may be an important mechanism of compensation in cells. Most P450s are down-regulated during the inflammatory response, so it is unique that CYP4F11 is up-regulated (Morgan, 2001; Kalsotra et al., 2003; Morgan et al., 2008). For instance, CYP4F3A/B and CYP4F2 have been shown in our laboratory to be down-regulated during an inflammatory response (data not shown); thus, it may be beneficial to up-regulate CYP4F11 as a partial compensatory mechanism for activities of down-regulated isoforms. For instance, CYP4F11 can metabolize leukotriene B4 and arachidonic acid but at a much lower activity than CYP4F2 or CYP4F3A/B (Kalsotra et al., 2004). However, because no direct assessment of this possibility has been made to our knowledge, this must await further study.
Authorship Contributions
Participated in research design: Bell and Strobel.
Conducted experiments: Bell.
Performed data analysis: Bell.
Wrote or contributed to the writing of the manuscript: Bell and Strobel.
Acknowledgments
We acknowledge provision of TNF-α by Dr. Jianping Jin. We also thank the Dr. Jacqueline Hecht laboratory for assistance in experiments.
Footnotes
This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS44174]; the National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases [Grant AR45603]; and the National Institutes of Health National Institute of General Medical Sciences [Grant 1F31-GM081907-01].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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ABBREVIATIONS:
- TNF-α
- tumor necrosis factor-α
- IL
- interleukin
- P450
- cytochrome P450
- AP-1
- activator protein 1
- IMD-0354
- N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- ChIP
- chromatic immunoprecipitation
- bp
- base pair
- kb
- kilobase
- siRNA
- small interfering RNA
- NF-κB
- nuclear factor κB of the light-chain-enhancer in activated B cells
- JNK
- c-Jun NH2-terminal kinase
- MEKK
- mitogen-activated protein kinase kinase kinase.
- Received June 28, 2011.
- Accepted October 19, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics