Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, 37232-2650; and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37203
Submitted 22 September 2003 ; accepted in final form 11 December 2003
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ABSTRACT |
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sodium butyrate; phosphorylation; acetylation; mitogen-activated protein kinase
In the resting state, transcriptionally inactive chromatin is tightly wrapped around histone proteins and inhibits DNA binding of transcriptional factors. The NH2-terminal histone tails protrude from the nucleosomes, and specific amino acids are subjected to a wide array of posttranslational modifications, including phosphorylation and acetylation (1, 7). Differential posttranslational modifying mechanisms strongly influence histone wrapping and loosen or tighten their grip to DNA, differentially exposing gene promoter elements to various transcription factors. These posttranslational modifications of histones also generate specific docking surfaces for proteins that regulate chromatin unfolding and transcription, such as histone acetyltransferase (HAT)-associated transcriptional coactivators (28, 30). Transcriptional factors that are involved the regulation of COX-2 expression in macrophage, including NF-B, interact with p300, which contains an acetyltransferase enzyme complex. This suggests that histone modification may be involved in COX-2 gene expression (14).
Several enzymes regulate histone modification; the steadystate levels of histone acetylation in vivo are maintained by balance of opposing HAT and histone deacetylase (HDAC) activities, and these enzymes are associated with activation and/or repression of gene expression (28). For histone phosphorylation, diverse stimuli activate MAP kinase cascades and elicit the rapid and transient phosphorylation of serine residues on histone H3. The extent and duration of acetylation and phosphorylation correlate with transcriptional activation of many immediate-early genes, suggesting a fundamental role for histone modification in transcriptional activation (19, 29). For example, ERK-activated ribosomal S6 kinase (Rsk)-2 is directly involved in H3 phosphorylation in vivo (26), and the p38 pathway is responsible for H3 phosphorylation through the mitogen and stress-activated protein kinase (MSK1) (29). Recently, IKK- has been shown to have H3 kinase activity, providing a closer link between activation of NF-
B and H3 phosphorylation (2, 35). These data together indicate that signaling cascades triggered by biological stimuli activate transcription factors and modify histone structure. These processes act in concert to regulate expression of inflammatory genes such as COX-2.
Few studies have investigated how posttranslational modifications of histone H3 influence gene expression in macrophages. Here we demonstrate that H3 modification at the COX-2 promoter plays a key role in regulation of COX-2 gene expression in macrophage. We showed that sodium butyrate (NaBT), a well-known HDAC inhibitor, requires a secondary signal to increase histone acetylation, through the MAP kinase pathway. Our data indicate that histone acetylation and phosphorylation at the promoter site of COX-2 gene have a major effect on COX-2 gene expression by macrophages.
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METHODS |
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Reagents. SB-203580 (Calbiochem) and U-0126 (Calbiochem, San Diego, CA) were dissolved in DMSO, and an equivalent amount of DMSO was added to cells in mock-treated control samples. Escherichia coli endotoxin (LPS; Sigma, St. Louis, MO) and NaBT (Sigma) were added to cell cultures for various lengths of time.
Western blot. Modification-specific antibodies that recognize either phosphorylated H3 (Ser10) or acetylated H3 (Lys14) were from Upstate Biotechnology (Lake Placid, NY). Anti-COX-2 antibody was from Cayman Chemical (Ann Arbor, MI). Antibodies against phospho-p42/44, total p42/44, phospho-p38, and p38 were obtained from Cell Signaling Technology (Beverly, MA). Total cell lysate was prepared, and the total amount of proteins was quantified by the Bradford assay. Equal amounts of protein were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, blocked with 5% milk, and incubated overnight with primary antibodies. After extensive washing, blots were incubated with horseradish peroxidase-conjugated anti-rabbit antibody diluted 1:5,000 and developed using standard ECL+ reagent (Amersham, Arlington Heights, IL). The harvesting and electrophoresis of histone protein were performed as previously described (8).
Reporter assay. A DNA fragment (nucleotides -815 to +123) prepared from the 5'-franking region of the mouse COX-2 gene was obtained from Dr. R. C. Harris (Vanderbilt University, Nashville, TN) and was inserted into the luciferase plasmid pGL3 Basic (Promega, Madison, WI). For the transfection of luciferase plasmid, RAW cells were treated with plasmids by GenePORTER 2 (Gene Therapy Systems, San Diego, CA), as specified by the manufacturer. The transfected cells were further incubated with G418 (Sigma) to select the stably transfected cells. For the treatment, the cells were washed, incubated in serum-free media overnight, and treated with various conditions.
Chromatin immunoprecipitation assay. Cells were grown to 90% confluence. Cells were then cultured in the serum-starved media overnight before specific stimulation. We generally use 12 x 107 cells for each condition. Cells were cross-linked with 1% formaldehyde for 5 min and rinsed three times with ice-cold 1x phosphate-buffered saline. After the harvest by brief centrifuge, cell pellets were resuspended in SDS-lysis buffer (50 mM Tris·HCl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors). Sonication of chromatin was performed four times for 12 s each at one-fifth of the maximum potency followed by centrifugation at 4°C for 10 min. Supernatants were collected and diluted 1:10 with dilution buffer (16.7 mM Tris·HCl, pH 8.1, 1.2 mM EDTA, 167 mM NaCl, 0.01% SDS, and 1.1% Triton X-100) followed by preclearing the extract with 60 µl of salmon sperm-saturated protein A (Zymed, San Francisco, CA) for 2 h at 4°C. Immunoprecipitation was carried out overnight at 4°C with 1 µg of specific antibodies as indicated. After immunoprecipitation, 40 µl of salmon sperm-saturated protein A were added and incubated for 30 min and followed by brief centrifuge. Precipitates were washed twice (5 min each at 4°C) with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 150 mM NaCl), once with high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 500 mM NaCl), and once with LiCl buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris·HCl, pH 8.1). Then precipitates were washed again with Tris-buffer twice for 5 min each. The antigen-antibody complexes were extracted three times with 200 µl of elution buffer (1% SDS and 0.1 M NaHCO3). Elutes were heated at 65°C for at least 4 h to reverse formaldehyde cross-linking. The samples were treated with 10 µg of proteinase K at 45°C for 1 h. The recovered DNA was purified with a DNA clean-up kit (Qiagen), and samples of input DNA were also prepared in the same way. PCR conditions were as follows: 94°C for 240 s; 30
32 cycles at 94°C for 40 s, 54°C for 40 s, and 72°C for 60 s; and final elongation at 72°C for 10 min. PCR for the input was performed with 100 ng of genomic DNA. The PCR products were analyzed on a 1% agarose gel or 8% polyacrylamide gel. Primers for the promoter region of the mouse COX-2 gene were 5'-CTAATTCC ACCAGTACAGATG-3' and 5'-ACTAGGCGAGACTCAGCGAAC-3'. This primer set covers the COX-2 promoter segment from -540 to -265, which contains NF-
B and c-ETS-1 binding sites.
Statistical analysis. For comparison among groups, paired or unpaired t-tests and one-way analysis of variance tests were used (with the assistance of InStat; Graphpad Software, San Diego, CA). P values <0.05 were considered significant.
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RESULTS |
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NaBT augments induction of the COX-2 promoter activity in the LPS-treated RAW cells. To assess whether NaBT affects COX-2 promoter activity, we used RAW cells stably transfected with a luciferase reporter construct containing the mouse COX-2 promoter region. We isolated the promoter region of the COX-2 gene (-815 to +123) from genomic DNA of RAW cells and cloned it upstream of the luciferase gene and developed clones of stably transfected RAW cells through selection with G418. This segment was previously shown to be sufficient for regulation of COX-2 gene expression and contains binding sites for the key transcription factors (36). With these stably transfected RAW cells containing 5'-promoter regions of COX-2, we evaluated the effect of NaBT on promoter activity of the COX-2 gene by treating the cells with various doses of NaBT in the presence and absence of endotoxin. The luciferase activity of whole cell lysates was assayed 8 h after stimulation. Compared with basal activity, endotoxin significantly increased COX-2 promoter activity in a dose-dependent manner (10 ng/ml LPS, 2.0 ± 0.8-fold increase; 100 ng/ml LPS, 3.1 ± 1.0-fold increase; Fig. 2). However, when NaBT (5 mM) was added along with 10 ng/ml of LPS, it resulted in a 10.4 ± 3.9-fold superinduction of COX-2 promoter activity, whereas this concentration of NaBT by itself had no effect on COX-2 promoter activity (1.08 ± 0.11-fold). These data suggest that NaBT augments COX-2 gene expression by endotoxin-treated RAW cells at the transcriptional level.
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Phosphorylation of histone H3 at the COX-2 promoter is increased in a time-dependent manner in response to treatment with NaBT. To examine global changes in H3 modification in vivo, we examined extracts from NaBT-stimulated cells by Western blot using antiacetylated histone H3 and antiphosphorylated histone H3 (Ser10) antibody. RAW cells were stimulated with 5 mM of NaBT for various lengths of time (02 h), and equal amounts of cell lysate protein were immunoblotted with specific antibodies for phosphorylated, acetylated, or total histone H3. NaBT globally induced histone H3 phosphorylation and acetylation in a time-dependent manner with an increase in phosphorylated and acetylated histone H3 detected by 1 and 2 h of treatment (Fig. 3A).
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To determine whether treatment with NaBT alters the acetylation and phosphorylation state of histone H3 that is associated with the COX-2 promoter in RAW cells, we developed a chromatin immunoprecipitation (ChIP) assay that employs antiacetylated histone H3 (Lys14) and antiphosphorylated histone H3 (Ser10) antibodies, respectively. In this experiment, RAW cells were stimulated with NaBT (5 mM) or LPS (100 ng/ml) for various time points (02 h), and then protein was cross-linked to DNA by formaldehyde treatment. Acetylated and phosphorylated histone H3 were immunoprecipitated with specific antibodies, and binding to the COX-2 promoter was detected by PCR from the immunoprecipitant. To quantify the relative amount of acetylated and phosphorylated histone H3 associated with COX-2 promoters, we used the total input DNA and the immunoprecipitated DNA as templates for PCR with primers for the mouse COX-2 gene promoter.
These results show that treatment with NaBT resulted in a large increase in phosphorylation of histone H3 at the COX-2 promoter starting at 15 min, which peaked at 12 h of treatment (Fig. 3B). There was no change in acetylation of histone H3 at the COX-2 promoter by NaBT treatment alone (Fig. 3B), even though there was a global increase of NaBT-induced histone acetylation detected by Western blot (Fig. 3A). These findings suggest that NaBT modified histone H3 in a genespecific manner. Inhibition of HDAC results in alteration of only a limited subset of genes that comprise <2% of total cellular genes (31), and histone phosphorylation is induced in an extremely small fraction of total H3 in response to mitogen (3). In combination with our data, it seems apparent that there are large differences between global cellular levels of acetylated and phosphorylated histone H3 that are measured by Western immunoblot and those that are associated with the COX-2 promoter that can be measured by a specific ChIP assay.
Combined treatment with NaBT and endotoxin results in a synergistic increase in the phosphorylation and acetylation of H3 at the COX-2 gene promoter. To assess whether the synergistic effect on COX-2 gene expression of combined treatment with NaBT and endotoxin is associated with modifications of histone H3 at the COX-2 gene promoter, we examined the states of acetylated and phosphorylated histone H3 using a ChIP assay. We detected a small increase in the acetylation of histone H3 at the COX-2 promoter in response to treatment with endotoxin (Fig. 4A, lane 2). There was a marked increase in acetylation of H3 in the cells treated with combined endotoxin and NaBT compared with the cells treated only with LPS (Fig. 4A, lane 4 compared with lane 2), but NaBT, by itself, had no effect on acetylation of histone H3 (Fig. 4A, lane 3).
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In contrast, endotoxin, by itself, had no effect on phosphorylation of histone H3 at the COX-2 promoter (Fig. 4B, lane 2). However, there was a marked increase in the phosphorylation of H3 at the COX-2 promoter in the cells treated with combined endotoxin and NaBT compared with the cell treated only with endotoxin (Fig. 4B, lane 4 compared with 2).
MAP kinase inhibitors block the accentuation effect of NaBT on H3 acetylation and phosphorylation at the COX-2 promoter. Our data indicated that phosphorylation of histone H3 was related to potentiation effect of NaBT (Fig. 4B). This prompted us to examine whether MAP kinase is involved in the synergistic effect of NaBT on LPS-induced COX-2 gene expression, because the MAP kinase pathway is involved in the H3 kinase cascade (26, 29). The inhibitory effects of U-0126, an MEK1/2 inhibitor, and SB-203580, a p38 inhibitor, on the phosphorylation of MAPK proteins were evaluated by Western blot with antibodies against phosphorylated p42/44 and phosphorylated p38. Compared with combined treatment with endotoxin and NaBT (Fig. 5A, lane 2), the pretreatment of U-0126 and SB-203580 successfully inhibited phosphorylation of p42/44 (lane 3) and p38 (lane 4). We found that blockade of the ERK pathway with U-0126 markedly decreased in H3 acetylation at the COX-2 promoter but resulted in minimal inhibition of the H3 phosphorylation (Fig. 5, B and C). We also examined whether the p38-MAP kinase pathway is involved by employing a relatively specific p38 inhibitor, SB-203580. In contrast to the effect of U-0126, inhibition of p38 MAP kinase diminished both the increase of acetylation and phosphorylation of H3 that were associated with combined treatment of endotoxin and NaBT at the COX-2 promoter (Fig. 5, B and C, lane 4). These data suggest that H3 phosphorylation and acetylation at the promoter site of COX-2 gene are coupled and that the potentiation effect of NaBT on endotoxin-induced H3 modification is dependent on MAP kinase signaling.
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COX-2 promoter activity and the production of immunoreactive COX-2 are dependent on MAP kinase activity. We measured transcriptional activity of the COX-2 promoter by the luciferase reporter system using the RAW cell line stably transfected with COX-2 promoter. There was a slight increase in luciferase activity in response to treatment with 10 ng/ml of LPS, and this was augmented by combined treatment with 10 ng/ml of LPS and 5 mM NaBT. Luciferase activity that was stimulated by this combined treatment was blocked to a variable extent by both SB-203580 and U-0126 (Fig. 6A). A similar trend was seen when we examined the expression of the endogenous COX-2 protein by RAW cells with a Western blot. As shown, treatment with endotoxin resulted in a dose-dependent increase in production of immunoreactive COX-2 (Fig. 6B, lanes 13). Although treatment with NaBT alone did not increase the production of COX-2 (lane 4), there was a marked increase in COX-2 production by combined treatment with endotoxin and NaBT (lane 5), and this increase was completely inhibited by treatment with either SB-203580 or U-0128 (lanes 6 and 7). This indicates that both ERK and p38 are required for efficient transcription of the COX-2 gene in response to endotoxin.
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DISCUSSION |
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Histone modification affects chromosome function by providing a platform for nuclear factors to bind and target their activities to selected regions of the genome (21, 28). Acetylated histones are associated with promoters of actively transcribed genes, whereas silent genes appear to either lack or have only minimal histone acetylation at their promoter regions (4, 22). Our data show that even though there is increased histone H3 phosphorylation at the COX-2 promoter site (Fig. 4B, lane 4), COX-2 gene expression is not induced without acetylation of histone H3, indicating that acetylation of histone H3 lysine is involved in the recruitment of transcriptional factors to the COX-2 promoter. Although treatment with LPS alone can induce histone H3 acetylation at the COX-2 promoter site and is followed by COX-2 gene expression, combined treatment with LPS and NaBT markedly accentuates both histone H3 modifications at the promoter site of COX-2 gene and synergistically increases COX-2 gene expression.
Diverse stimuli that induce phosphorylation of histone H3 include growth factors (EGF, FGF), phorbol esters, okadaic acid, and protein synthesis inhibitors such as anisomycin and cycloheximide (10). Our data show that NaBT induces phosphorylation and acetylation of histone H3 at the COX-2 promoter. This observation is similar to a recent report that NaBT increases susceptibility of histone H3 to nuclear histone kinase due to secondary alterations in chromatin conformation by NaBT, but the exact mechanism of NaBT-induced H3 phosphorylation is unclear (33, 34). Another report indicates that HDAC is closely associated with protein phosphatase 1, which has been known to regulate histone H3 dephosphorylation, and that HDAC inhibitor suppresses this phosphatase activity, suggesting that HDAC and phosphatase act as an enzyme complex (6). This observation raises the possibility that NaBT increases H3 phosphorylation by interaction with HDAC that is associated with phosphatase.
Histone H3 phosphorylation is mediated by various MAP kinase cascade systems, depending on the stimulus (9, 25). Several reports strongly suggest that p38- and ERK-activated MSK1/2 is a major histone H3 kinase activated by both mitogens and stress (25, 29). Other reports also suggest that ERK-activated Rsk-2 kinase is a histone H3 kinase (26). In our experiments, we used relatively specific MAP kinase inhibitors U-0126 to inhibit the activation of MEK1/2, the upstream activator of ERKs, and SB-203580 to inhibit p38 pathway. Both agents block the potentiation effect of NaBT on LPS-induced COX-2 transcriptional and translational activity. This finding indicates that histone H3 modification is MAP kinase dependent at the COX-2 promoter site, which is closely associated with the level of COX-2 gene expression.
NF-B and C/EBP
are well-known key transcriptional factors in the COX-2 gene expression of macrophage, and these factors are directly involved in COX-2 gene expression (5, 13, 17), but MAP kinase pathways are also involved in COX-2 gene expression in a different way. Inhibition of MAP kinase impairs COX-2 induction (27) by repressing the NF-
B pathway and abolishing the induction of C/EBP
/
DNA binding activities at the murine COX-2 promoter site (5, 23, 24, 32). This finding cannot be totally explained by simple inhibition of the MAP kinase pathway activation because LPS-induced phosphorylation of c-Jun is unaffected in these experiments (5). This suggests that MAP kinase pathways are strongly involved in the DNA binding affinity of key transcription factors on COX-2 gene expression. Our data support the conclusion that the MAP kinase pathway modulates COX-2 transcriptional activity by mediating H3 modification at COX-2 promoter in murine macrophage cells. These findings could provide an explanation for the observation that the MAP kinase pathway seems to regulate DNA binding affinities for multiple transcriptional factors at the COX-2 promoter.
The functional interrelationship between two histone modifications, acetylation and phosphorylation, is not understood yet. Several lines of evidence indicate that coupled histone acetylation and phosphorylation may act in concert to induce chromatin remodeling events facilitating gene activation. The rapid mitogen-induced phosphorylation of Ser10 in the NH2-terminal of H3 has been coupled to the activation of the immediate-early genes. Phosphorylation is followed by rapid increase of acetylation of Lys14 by HAT activity (19). Furthermore, MAP kinase-regulated Rsk-2 kinase phosphorylates histone H3 upon mitogenic stimulation and is physically associated with CREB-binding protein that has HAT activity (20). This observation was supported by another in vitro study that showed that HAT displays a 10-fold greater preference for Ser10-phosphorylated H3 peptide as a substrate compared with the unmodified peptide supported this observation (8). These findings seem to indicate that there is a close linkage between acetylation and phosphorylation of histone, but little is known about in vivo coupling of phosphorylation and acetylation. Our data indicate that MAP kinase inhibitors SB-203580 and U-0126 attenuate both acetylation and phosphorylation of histone H3, which suggests that both modifications are important in macrophages.
In summary, our data show that NaBT accentuates LPS-induced COX-2 gene expression through MAP kinase-dependent increase of phosphorylation and acetylation of histone H3 at the COX-2 promoter site.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants P01 HL-66196-01A1 and R01 HL-075557 and by the Department of Veterans Affairs.
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FOOTNOTES |
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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.
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REFERENCES |
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