Background
The NF-κB proteins are sequence-specific transcription factors that play critical roles in the immune system. NF-κB proteins regulate the expression of cytokines, chemokines, growth factors, and inflammatory enzymes in response to activation of T-cell, B-cell, Toll/IL-1R, and TNF-α receptors [
1,
2]. The NF-κB family of proteins is characterized by the presence of a conserved 300 amino acid Rel Homology Domain (RHD) which controls dimerization, DNA binding, and association with the inhibitory IκB proteins [
3]. The five members of the mammalian NF-κB family; RelA (p65), RelB, c-Rel, NF-κB1 (p50) and NF-κB2 (p52) are present in unstimulated cells as homo- or heterodimers bound to inhibitory IκB proteins. This association prevents NF-κB proteins from translocating to the nucleus, thereby maintaining an inactive state [
4]. In response to inflammatory stimuli such as TNF-α, IL-1, or LPS, multiple signaling pathways are activated resulting in the phosphorylation of IκB-α [
5,
6]. Subsequent poly-ubiquitination and proteosomal degradation of IκB-α permits the translocation of NF-κB proteins into the nucleus where transcription is activated [
7,
8]. NF-κB dimers exhibit variable binding affinities for consensus κB binding sites. These proteins also differ in their ability to initiate transcription; RelA, RelB and c-Rel have been shown to have potent trans-activating domains, while NF-κB proteins that lack transactivating domains such as p50 and p52 have been to shown to mediate transcriptional repression [
3]. Activated NF-κB proteins can be inhibited by newly synthesized IκB proteins which cause re-export back to the cytosol [
9].
Extracts of the Creosote bush,
Larrea tridentata, found in deserts of the Southwestern United States and Northern Mexico, have been used for centuries by indigenous peoples to treat inflammatory disorders. Many of the medicinal effects of
L. tridentata have been ascribed to the polyphenolic compound nordihydroguaiaretic acid (NDGA) [
10]. In addition,
L. tridentata also contains polyphenolic compounds with modifications to the backbone structure of NDGA [
11]. A number of these compounds have been examined for their antiviral activity. For example, an analysis of eight methylated forms of NDGA for their ability to inhibit HIV replication revealed that tetra-O-methyl NDGA, also known as terameprocol (TMP), displayed the highest level of activity. Mechanistic studies suggest that TMP mediates this effect by inhibiting HIV Tat-mediated transactivation [
12]. TMP has also been shown to block the replication of herpes simplex virus
in vitro and this effect has been attributed to the drug's ability to block the binding of the transcription factor Sp1 to viral DNA, which is required for virus replication [
13].
Based on these reports, we have recently evaluated the efficacy of TMP as an anti-inflammatory agent. We reasoned that since inflammation is heavily dependent on
de novo transcription, TMP might be a useful therapeutic compound. We found that TMP exerted a range of effects on various inflammatory cytokines, chemokines and lipid mediators both
in vivo and
in vitro following treatment with LPS or infection with H1N1 influenza A virus strain PR/8/34 [
14]. TMP strongly inhibited the production of TNF-α, MCP-1/CCL2, G-CSF, and several prostaglandins, while modestly inhibiting the production of IL-6 and MIP-1α/CCL3. Since the NF-κB RelA protein has been reported to regulate the expression of several of these genes [
15‐
18]; we have focused our current studies on how TMP modulates RelA activation and occupancy at its cognate DNA binding motifs. We report that TMP did not affect the cytoplasmic activation and nuclear localization of RelA in RAW 264.7 cells following treatment with LPS. However, reporter assays revealed strong inhibition of NF-κB-dependent transcription. Chromatin immunoprecipitation (ChIP) assays revealed that TMP abrogated the LPS-induced binding of RelA at the TNF-α, MCP-1/CCL2, and RANTES/CCL5 promoters despite its inability to block NF-κB association with electrophoretic mobility shift assay (EMSA) probes
in vitro. We conclude, therefore, that TMP acts indirectly to inhibit the binding of RelA to the promoters of certain key pro-inflammatory cytokine and chemokine genes. Taken together our data suggest that TMP could be useful for the treatment of inflammatory disorders where NF-κB RelA-dependent transcription plays a pathogenic role.
Methods
RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in Dulbecco's modification of minimal essential medium (DMEM) with 4 mM L-glutamine, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate with 10% FCS. Media and supplements were obtained from Sigma-Aldrich, St. Louis, MO and Cellgro, Manassas, VA. FCS was obtained from Atlanta Biologicals, Atlanta, GA and Cellgro. Constitutive TLR3(293/TLR3-YFP), TLR8(293/TLR8) (InvivoGen, San Diego, CA) and TLR4(293/TLR4-YFP/MD2) (a gift from D. Golenbock) expressing HEK293 cells were grown in DMEM supplemented with 10% FCS, 1% antibiotics, 20 μg/ml gentamicin at 37°C. Stable expression of TLRs was maintained with the addition of 10 μg/ml blasticidin for (293/TLR3) and (293/TLR8) cells, and 400 μg/ml of G418 (Geneticin) for (293/TLR4) cells.
Chemicals and biological reagents
Unless otherwise indicated, reagents were purchased from Sigma-Aldrich. TMP was supplied by Erimos Pharmaceuticals, Raleigh, NC. DMSO was used as the solvent for TMP in all experiments. The maximum DMSO concentration was 0.1% in all assays. This concentration of DMSO was tested in all assays and did not affect the results. LPS from Salmonella Minnesota R595 was purchased from LIST Biological Laboratories, Inc. (Campbell, CA).
Quantitative RT-PCR analysis
Total RNA was extracted using the RNAeasy kit (Qiagen, Valencia, CA) according to the manufacturer's specifications. Residual genomic DNA was eliminated using on-column DNase digestion with the RNase-free DNase set (Qiagen) and resulting extracts were resuspended in nuclease free water. Amount and purity of RNA was determined using a Nanodrop 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA). RNA (1 μg) was denatured and reverse transcription was performed with the Improm ll reverse transcription kit (Promega, Madison, WI) in a reaction mix containing random hexamers as primers (50 ng/μl) for 60 min at 42°C. The iQTM SYBR Green supermix kit (BioRad, Hercules, CA), was used for Real-time PCR analysis. cDNA was amplified using primers specific for murine GAPDH, TNF-α, MCP-1/CCL2, and RANTES/CCL5 genes. Primer combinations are GAPDH [antisense: 5' ATG TCA GAT CCA CAA CGG ATA GAT 3'; sense: 5' ACT CCC TCA AGA TTG TCA GCA AT 3']; TNF-α [antisense: 5' AGA AGA GGC ACT CCC CCA AAA 3'; sense: 5' CCG AAG TTC AGT AGA CAG AAG AGC G 3']; MCP-1/CCL2 [sense: 5' CAC TAT GCA GGT CTC TGT CAC G 3'; antisense: 5' GAT CTC ACT TGG TTC TGG TCC A 3']; RANTES/CCL5: [sense: 5' CCC CAT ATG GCT CGG ACA CCA 3'; antisense: 5' CTA GCT CAT CTC CAA ATA GTT GAT 3']. All primer pairs were purchased from Integrated DNA Technologies (Coralville, IA). PCR was performed in 96 well plates (Eppendorf AG, Hamburg, Germany). Samples were amplified for a total of 50 cycles, followed by a meltcurve analysis to ensure the specificity of reactions. To generate a standard curve, total RNA was isolated from the cells and 300-600 bp fragments of the gene of interest were amplified by RT-PCR using cognate primer sets. PCR fragments were gel purified, quantified, and the copy number was calculated. Serial tenfold dilutions were prepared for use as templates to generate standard curves. All samples were normalized to amplified murine GAPDH. GAPDH control was analyzed per plate of experimental gene to avoid plate-to-plate variation. Final RT-PCR data is expressed as the ratio of copy numbers of experimental gene per 103 or 104 copies of GAPDH for samples performed in duplicates.
Western blot analysis
After treatments, cell monolayers were washed twice with cold phosphate buffered saline (PBS), solubilized in lysis buffer (50 mM Hepes, pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM leupeptin, 0.5% SDS) and collected by scraping. The protein concentration for each sample lysate was determined using the Pierce BCA system (Pierce, Rockford, IL). Equal protein samples (25 μg) were loaded on 12% Tris-Glycine gels and subjected to electrophoresis using the Novex Mini-Cell System (Invitrogen). Following transfer, and blocking, blots were probed with antibodies specific for the phosphorylated serine 32 residue of IκB-α and total IκB-α protein (Cell Signaling; Beverly, MA). Bands were visualized using the SuperSignal Chemiluminescent system (Pierce).
Immunofluorescence
RAW 264.7 cells were seeded onto 8 well chamber slides and stimulated with 1 μg/ml of LPS or co-stimulated with 1 μg/ml of LPS and 25 μM TMP for various amounts of time. To visualize NF-κB subcellular localization at the end of each treatment period, cells were briefly washed with phosphate-buffered saline, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked (2% bovine serum albumin, 5% normal horse serum, and 10 mM glycine in phosphate-buffered saline). The cells were then incubated with a rabbit monoclonal anti-NF-κB (p65) antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), followed by incubation with a goat anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (Southernbiotech, Birmingham, AL). Fluorescence was viewed using a Zeiss Axioskop 2 microscope (Zeiss AG, Oberkochen, DE). Images were captured using a spot camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Cytokine Measurements
MCP-1/CCL2 and TNF-α ELISA kits were purchased from R&D Systems (Minneapolis, MN), Assay Designs (Ann Arbor, MI) or eBioscience (San Diego, CA). RAW 264.7 cells were stimulated with 1 μg/ml of LPS for 24 hrs and supernatants were collected for ELISA assays. In each case, sample values were interpolated from standard curves. Optical density was determined using a PolarStar microplate reader (BMG Labtechnologies, Durham, NC).
Reporter Assays
Reporter assays were performed using a luciferase gene under the control of an NF-κB response element (NF-κB -Luc; Stratagene, Santa Clara, CA). Briefly, the plasmid contains 5 consecutive NF-κB binding motifs designed from a consensus sequence cloned into a PGL3 vector. 100 ng each of NF-κB-Luc and pCMV beta (β-Gal) (Clontech) and 300 ng of pcDNA6 (Invitrogen) were cotransfected into 293/TLR3, 293/TLR4-YFP/MD2 and 293/TLR8 cells using the TransIT-LT1 transfection reagent (Mirus, Madison, WI). pcDNA6 was used to keep the overall DNA concentration at a total of 500 ng which has proven itself suitable for reporter assay in this system. At 24 h post-transfection, cells were either treated for 4 hours with 20 μg/ml poly(I:C) (pIC; Calbiochem, Gibbstown, NJ), 1 μg/ml LPS or 1 μg/ml resiquimod (R-848; Axxora, San Diego, CA) alone, or co-treated with 25 μM TMP. Following treatment, cells were lysed in reporter lysis buffer (Promega, Madison, WI) containing 0.1% Triton X-100 and assayed for Luc and β-Gal activities using a Promega Luc assay system and an ONPG (o-nitrophenyl-β-D-galactopyranoside)-based β-Gal assay. β-Gal activity was used to normalize the Luc data for all experiments. All data are expressed as relative light units/mU of β-Gal activity.
Chromatin immunoprecipitation (ChIP) assays
4.5 × 10
7 RAW 264.7 cells were stimulated with 1 μg/ml LPS or co-treated with 1 μg/ml LPS and 25 μM TMP for 4 hours and chromatin was isolated by methods previously described [
19]. Briefly, after treatments, cells were harvested and nucleoprotein complexes were crosslinked with formaldehyde (1% final) with shaking for 10 min at room temperature, followed by incubation with glycine (125 mM final) for an additional 5 min. Cells were pelleted, washed and resuspended in 500 μl lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 3 mM MgCl
2, and 0.5% NP-40) supplemented with 1 mM PMSF and 1× Protease Inhibitor Cocktail (PIC, Roche). Nuclei were pelleted and resuspended in Micrococcal nuclease buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl
2, 1 mM CaCl
2, 4% NP-40) supplemented with 1 mM PMSF and 1× PIC, and chromatin was sheared with the addition of 10 U MNase for 7 min at 37°C. Digestion was stopped with the addition of EDTA (10 mM final), and the resultant chromatin was stored at -80°C. Shearing was confirmed by electrophoresis and >80% of the DNA was in fragments <400 bp.
Using magnetic capture, Protein A and G-coupled Dynabeads (Invitrogen) (5 μl each/IP) were washed 2× (100 μl/IP) in RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDeoxycholate and sheared salmon sperm DNA (0.5 mg/ml). Beads were conjugated with 1-5 μg antibody for 1 h at 4°C in RIPA buffer supplemented with 1 mM PMSF and 1× PIC. Conjugated antibody:bead complexes were washed 3× in RIPA buffer as described above, and protein-DNA complexes were immunoprecipitated for 2 h at 4°C with rotation in RIPA buffer (100 μl) supplemented with 1 mM PMSF, 1× PIC and chromatin (105 cell equivalents). Following IP, beads were successively washed 4× in RIPA buffer and 2× in TE 8.0, and protein-DNA complexes were eluted in 100 mM NaHCO3 by gentle vortexing for 15 min at room temp. Supernatants were recovered and crosslinks were reversed in NaCl (100 mM final) together with matched input samples by heating at 95°C for 15 min. Proteins were removed using Proteinase K (10 μg/ml final) for 1 h at 45°C and DNA was purified using Qiaquick nucleotide removal columns (Qiagen) according to the manufacturer's instructions.
ChIP Q-PCR and data analysis
For realtime PCR, bound (3 μl) and input samples were amplified in a MyIQ thermal cycler (Bio-RAD) using 1× SensiMix
Plus (Quantace, London, UK) and primers specific for the RelA binding sites at the TNF-α, MCP-1/CCL2 and RANTES/CCL5 promoters. TNF-α: (forward:5'-TCTCAAGCTGCTCTGCCTTC-3'; reverse:5'CACCAGGATTCTGTGGCAAT-3'). RANTES/CCL5:(Forward:5'-TGGAGGGCAGTTAGAGGCAGAG-3';reverse:5'-AGCCAGGGTAGCAGAGGAAGTG-3') and MCP-1/CCL2:(Forward:5'-ATTCTTCCCTCTTTCCCCCCCC-3';reverse:5'-TCCGCTGAGTAAGTGCAGAGCC-3') Cycling parameters for 20 μl reactions were 95°C 10 min, followed by 50 cycles of 95°C, 20 s; 60°C, 30 s; 72°C, 30 s, for all genes listed. Fold enrichment in the bound fractions relative to input was calculated as previously described [
20], and the average enrichment for triplicate amplifications was reported.
Electrophoretic mobility shift assays (EMSA)
RAW 264.7 nuclear extracts and radioactive probes were prepared and EMSA reactions performed as previously described [
21]. Sequences of wildtype and mutant oligonucleotide EMSA probes include: wildtype TNF-α κB3 sense (5'-AACAGGGGGCTTTCC-3') and antisense (5'-AGGAGGGAAAGCCCC-3'), and mutant TNF-α κB3 sense (5'-AACAGGGGGCTGAGCCTC-3') and antisense (5'-GAGGCTCAGCCCCCTGTT-3').
Statistical Analysis
All graphs and statistical analyses were produced using Prism software (GraphPad Software Inc., La Jolla CA).
Discussion
Previously we showed that TMP could inhibit the expression of a number of cytokines and chemokines following stimulation with LPS [
14]. The production of TNF-α, MCP-1/CCL2, and G-CSF were most strongly inhibited and we hypothesized that these effects might stem from effects on NF-κB RelA, which is thought to play a key role in the activation of these genes. The results of reporter and ChIP assays confirmed this hypothesis. We found strong inhibition of NF-κB-dependent transcriptional activation and loading of RelA to the promoters of several genes. Based on these results, a series of experiments was performed in an attempt to understand the molecular mechanism underlying this activity of TMP.
One hypothesis we considered was a direct inhibitory effect of TMP on the interaction between RelA and its cognate sites on the DNA. TMP could be acting on RelA itself, binding to conserved motifs present in the amino terminus RHD thereby preventing RelA from recognizing its DNA binding site. Alternatively, TMP could be interacting with the DNA, preventing RelA from occupying its binding sites. In support of this hypothesis, Chen et al., [
13] have shown that TMP can bind the HSV ICP4 promoter and prevent Sp1 binding. Additionally, compounds with structures similar to TMP; 3'-
O-methyl NDGA [
13,
31] and tetra-O-glycyl-NDGA [
32] have been shown to bind DNA and prevent Sp1 binding. The recent finding that Sp1 can directly bind to certain NF-κB sites on the DNA [
33] also supported this hypothesis and raised the possibility that it is the same activity of TMP that is responsible for both RelA and Sp1 inhibition of binding. However, the results of our EMSA experiments did not support this hypothesis. TMP did not interfere with the abilty of RelA to bind its cognate site when TMP was incubated with cells prior to nuclear extract preparation. Similarly, TMP did not inhibit RelA:DNA binding when it was added
in vitro to the nuclear extracts and DNA. We conclude, therefore, TMP is working indirectly, upstream of DNA binding in the NF-κB pathway to prevent RelA from loading its promoter following LPS stimulation.
We next considered the hypothesis that TMP inhibits the signaling pathway that results in RelA translocation into the nucleus. TLR3/8 transcription was blocked more effectively than was TLR4. Since both TLR3 and 8 are localized to endosomal compartment this difference could suggest an effect of TMP on endocytosis. However, the phosphorylation of IκB-α and nuclear translocation of RelA were not altered following treatment with TMP suggesting that TMP is affecting additional regulatory systems. The results of our experiments also showed that, in the presence of TMP, IκB-α was resynthesized normally after treatment with LPS. Transcription of IκB-α is dependent on RelA [
30] suggesting that the effect of TMP is selective for only certain RelA:promoter interactions. Phosphorylation of RelA is a mechanism that has been shown to confer selectivity for certain promoters. For example, phosphorylation at Ser
276 has been shown to be critical for transcription of IL-8 and GROβ/CXCL2 but not IκB-α [
34]. RelA which is phosphorylated at this site interacts with positive transcription elongation factor b (PTEF-b), which is required for IL-8 and GROβ/CXCL2 transcription but not IκB-α [
34]. Similarly, phosphorylation at Ser
311 has been shown to regulate the interaction of RelA with other transcriptional coactivators such as cyclic AMP-responsive element binding protein/p300 and RNA polymerase II [
35‐
37] while acetylation of RelA is also known to be a molecular switch that regulates its activity [
38]. Clearly future experiments with TMP will need to evaluate its effects on the post-translational modification of RelA.
The range of inhibitory effects seen with TMP with different cytokines and chemokines may arise from the differential requirements of these genes for the various modified forms of RelA as discussed above. Alternatively, the variation might stem from the degree to which NF-κB RelA is required for transcription of each gene. For example, several groups have reported that transcriptional activation of the TNF-α and MCP-1/CCL2 genes is strongly dependent on the trans-activating activities of NF-κB RelA [
17,
39], likely explaining the strong inhibition of these molecules by TMP. Similarly, inhibition of NF-κB RelA binding might explain the strong inhibition of G-CSF production by TMP we noted previously [
14]. NF-κB binding sites have been shown to be present at the G-CSF promoter (CSF box) [
40] and nuclear factors have been shown to associate with these sequences. In contrast, TMP only weakly inhibited production of IL-6, MIP-1α/CCL3, and RANTES/CCL5 [
14]. It is possible that for these genes, although NF-κB sites are present in their promoters, their transcription in RAW 264.7 cells treated with LPS is not predominantly dependent on NF-κB. Transcription of IL-6, for example, can be entirely dependent on NF-IL-6 (C/EBPβ) [
41]. Similarly, while the MIP-1α/CCL3 LPS response element does contain an NF-κB c-rel binding site it also contains four C/EBP family binding sites [
42]. For RANTES/CCL5, although Fessele, et. al. [
43] reported that NF-κB is essential for LPS-induced transcription in mono mac 6 cells [
43] Shin et. al. [
44] observed that NF-κB is not required for its LPS-induced transcription in RAW 264.7 cells [
45] (the cells we used in our investigation). In agreement, our ChIP assays showed complete inhibition of RelA binding to the RANTES/CCL5 promoter, while at the same time levels of RANTES/CCL5 mRNA and protein were not blocked by TMP [
14].
In addition to the effects we noted on NF-κB, in our experiments we also noted an effect of TMP on the steady state levels of MCP-1/CCL2 mRNA (Figure
1D). To our knowledge, post-transcriptional regulation of MCP-1/CCL2 mRNA has not been reported. It is possible, that the effects of TMP may be related to the normal regulation of this mRNA. If levels of TTP-mediated degradation are normally low, they may be masked by the high levels of LPS-induced MCP-1/CCL2 transcription and only revealed when transcription is effectively blocked by TMP. In support of this hypothesis, MCP-1/CCL2 mRNA does contain the TTP AUUUA recognition site in its 3' untranslated region. It is also possible that TMP could be modifying TTP or the 3' untranslated region to enhance rates of degradation. If so, then one might also predict enhanced rates of TNF-α message degradation, which did not occur.
In summary, we have examined the effects of TMP on NF-κB activation, translocation and binding. We report that TMP inhibited NF-κB- dependent transcription and NF-κB RelA binding at the promoters of TNF-α, MCP-1/CCL2, and RANTES/CCL5. Since NF-κB RelA-dependent transcription is critical to numerous inflammatory and pathological responses, TMP might be useful to treat a variety of disorders. The safety of TMP has been established in several clinical trials, and testing for efficacy in inflammation should begin immediately.
Competing interests
Erimos Pharmaceuticals produced TMP but is no longer in existence. None of the authors was paid by Erimos nor did they have stock or shares in the company.
Authors' contributions
AOO performed the experiments and drafted the manuscript. FS and JRW supervised the reporter assays. MLS supervised the ChIP and EMSA experiments. SML, FS, and MLS participated in design and coordination of the experiments, acquisition of funding, and drafting of the manuscript. All authors read and approved the final draft.