Published online before print June 7, 2007, doi: 10.1161/ATVBAHA.107.144352
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© 2007 American Heart Association, Inc.
Vascular Biology |
Interferon-
Suppresses Cyclooxygenase-2 Promoter Activity by Inhibiting C-Jun and C/EBPß Binding
From the University of Texas Health Science Center (W.-G.D., A.J.M., K.K.W.) and M.D. Anderson Cancer Center (W.-G.D., A.J.M., K.K.W.), Houston, Tex; and the National Health Research Institutes (K.K.W.), Zhunan, Miaoli, Taiwan. Present address for A.J.M.: Department of Internal Medicine, Medical University of South Carolina, Charleston.
Correspondence to Kenneth K. Wu, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan. E-mail kkgo{at}nhri.org.tw; or University of Texas Health Science Center, 6431 Fannin, MSB 5.016, Houston, TX 77030-1503. E-mail Kenneth.K.Wu@uth.tmc.edu
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Abstract |
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Objective— Cyclooxygenase-2 (COX-2) and interferon
(IFN) are overexpressed in vascular inflammatory and atherosclerotic lesions. We postulated that IFN suppresses COX-2 expression at the transcriptional level.
Methods and Results— The effect of IFN on COX-2 expression was evaluated in several types of human cells stimulated with phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1ß, or tumor necrosis factor (TNF) 
. IFN concentration-dependently inhibited COX-2 proteins and promoter activities induced by PMA or cytokines in human fibroblasts and monocytic and endothelial cells. PMA and cytokines stimulate binding of C-Jun, C-Fos, CCAAT/enhancer binding protein ß (C/EBPß), or NF-
B to their respective regulatory elements on COX-2 promoter. IFN blocked C-Jun and C/EBPß but not C-Fos or p50 NF-B binding as determined by in vitro binding assays and chromatin immunoprecipitation assay. p300 binding to COX-2 promoter was inhibited by IFN in a manner comparable to C-Jun and C/EBPß binding.
Conclusions— IFN suppresses proinflammatory mediator-induced COX-2 transcription by selective inhibition of C-Jun and C/EBPß DNA binding activity and p300 recruitment in human cells. Because IFN is coexpressed with COX-2 in vascular lesions, it may play a role in controlling COX-2–mediated inflammatory changes.
INF- suppressed proinflammatory mediator-induced transcriptional activation in human cells by selective inhibition of C-Jun and C/EBPß binding to their respective response elements on COX-2 promoter. We propose that IFN may represent an important factor for controlling COX-2–mediated vascular inflammatory diseases including the development of atheromatous plaque.
Key Words: interferon • cyclooxygenase-2 • atherosclerosis • plaque instability • inflammation
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Introduction |
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Cyclooxygenase-2 (COX-2) is inducible by diverse proinflammatory and mitogenic factors.1 COX-2 expression stimulated by exogenous factors plays a critical role in multiple pathophysiological processes including inflammation, tissue injury, and tumorigenesis.2–5 Increased COX-2 expression in atherosclerotic lesions is considered to play a role in vascular inflammation. COX-2 transcriptional activation by proinflammatory mediators (PIM) has been extensively characterized.6,7 A core promoter region within 500 bp from the COX-2 transcription start site harbors several regulatory elements, notably cAMP response element, CCAAT/enhancer binding protein (C/EBP) enhancer element, and NF-
B binding sites, that are essential for COX-2 promoter activity in response to inflammatory signals.8–10 Binding of multiple transactivators to their respective cis-acting elements on the core COX-2 promoter results in sustained overexpression of COX-2 which has dire consequences unless it is controlled. COX-2 expression induced by PIM is reported to be abrogated by an array of small molecular weight compounds such as salicylate.11 Less is known about the endogenous control of COX-2 expression. Only a few endogenous factors have been reported to inhibit COX-2 expression stimulated by PIM.12,13
Interferon (IFN) is a pleiotropic cytokine with diverse biological activities including antimicrobial actions, immune modulation, and antiproliferative activities.14 Its actions are mediated through the Jak-STAT-1 transcriptional pathway.15 On IFN stimulation, tyrosine-phosphorylated STAT-1 translocates to the nucleus where it binds -activated sites (GAS) and thereby induces the transcription of a group of GAS-bearing IFN-stimulated genes.15–17 It has been reported that IFN may also stimulate gene expression by a STAT-independent mechanism, which involves the extracellular signal regulated kinase (ERK) pathway.18 Paradoxically, IFN has also been reported to suppress the expression of a small number of genes.19–23 The mechanism by which IFN inhibits the expression of these genes remains to be elucidated. In search for endogenous factors that control COX-2 expression, we postulated that IFN suppresses PIM-induced COX-2 expression. The results reveal that IFN abrogated COX-2 expression induced by phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1ß, and tumor necrosis factor (TNF) at the transcriptional level. Results from in vitro binding assays and chromatin immunoprecipitation (ChIP) assay show that IFN-inhibited binding of C-Jun and C/EBPß and recruitment of p300 to the core COX-2 promoter region.
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Materials and Methods |
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Cell Culture and Treatment
Human foreskin fibroblasts (HFb) were obtained from American Type Culture Collection (ATCC, Manassas, Va) and cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO2 incubator. Human umbilical vein endothelial cells (HUVECs) were cultured from fresh umbilical veins by a method previously described.11 Only cells at passage 2 to 4 were used. Human monocytic cell line, U937, was maintained in RPMI 1640 medium containing 10% FBS. In all experiments, 80% to 90% of confluent cells were washed and incubated in serum-free medium for 24 hours before treatment with or without IFN
for 12 hours followed by PMA (100 nmol/L) for 4 hours or IL-1ß (10 ng/mL) or TNF (30 ng/mL) for 12 hours.
Assessment of COX-2 Promoter Activity
COX-2 promoter activity was determined by transient transfection of a luciferase expression vector pGL3 containing a well established COX-2 promoter region –891 to +9 as previously described.8 This region contains essential regulatory elements for PIM-induced COX-2 promoter activation.24
DNA Binding by Streptavidin-Agarose Pulldown Assay
Transactivator and p300 coactivator binding to a 424-bp COX-2 core promoter as well as to 20- to 24-bp authentic COX-2 C/EBP-, CRE-, and B-specific probes were determined by a streptavidin-agarose pulldown assay as previously described.25 This assay allows for simultaneous quantitative determination of transactivators and coactivators that complex with the DNA probes. Several 5'-biotinylated double-strand COX-2 promoter probes were used in the binding experiments1: a 24-bp sequence (5'-ACCGGCTTACGCAATTTTTTTAAG-3') corresponding to COX-2 promoter sequence –115 to –138 which harbors C/EBP enhancer element and its mutant (5'-ACCGGCgcgatagcTTTTTTTAAG-3')2; a 20-bp sequence containing CRE (–52 to –58) (5'-CAGTCATTTCGTCACATGGG-3') and its mutant (5'-CAGTCATcgaGTCACATGGG-3')3; a 22-bp sequence corresponding to human COX-2 promoter sequence –207 to –228 which harbors a B site (–213 to –222); and4 a 424-bp sequence (–30 to –453) containing all the sites required for PIM-induced promoter activation. To assess the specificity of C/EBPß and C-Jun binding, a 50-fold molar excess of unlabeled WT or mutant sequences were coincubated with biotinylated C/EBP or CRE probes. Furthermore, a nonrelevant 22-bp probe with a sequence of 5'-AGAGTGGTCACTACCCCCTCTG-3' was included as a negative control. The assay was performed according to a procedure previously described.25 In brief, 500 µg nuclear extracts were incubated in a 500 µL mixture containing 5 µg of a 5'-biotinylated probe and 4% streptavidin-conjugated agarose beads (Sigma) at room temperature for 1 hour in a rotating shaker. Beads were pelleted by centrifugation. After washing, proteins in the complex were analyzed by immunoblots using rabbit polyclonal antibodies (1 µg/mL each) specific for the indicated transcription factors. A non-immune rabbit IgG (1 µg/mL) and a rabbit polyclonal IgG (1 µg/mL) directed against von Willebrand factor (vWF) were also used as negative controls.
Western Blot Analysis
Immunoblots for protein analysis were performed by a procedure previously described.12 To determine cellular levels of transactivators and coactivators, HFb cells were lysed with RIPA buffer containing multiple protease and phosphatase inhibitors as previously described.12 Proteins were detected by enhanced chemiluminescence.
Electrophoretic Mobility Shift Assay
24 bp oligonucleotide probes corresponding to C/EBP binding site (–115 to –138) or C-Jun binding site (–42 to –65) on the core COX-2 promoter were synthesized by Sigma. The probes were end-labeled with [32P] ATP using T4 kinase (Promega). Electrophoretic mobility shift assay (EMSA) was performed by incubating 10 µg of nuclear extract with a labeled probe (10 000 cpm; 
10 fmol) in a binding buffer as previously described.26 To assess the specificity, a 50-fold molar excess of unlabeled C/EBP or CRE wild-type or mutant oligonucleotides (their sequences were given above) was added.
ChIP Assay
The ChIP assay was performed as previously described.27 In brief, 80% confluent HFbs were serum-starved for 24 hours and treated with PMA (100 nmol/L) for 4 hours. Chromatin was crosslinked to proteins by 1% formaldehyde, and cell lysates were sonicated. One third of the samples were used as DNA input and the remaining were subjected to IP with candidate rabbit polyclonal antibodies or a nonimmune rabbit IgG. COX-2 promoter region was amplified with two primers: 5' primer, sequence –709 to –690 and 3' primer, –32 to –51 by polymerase chain reaction (PCR) yielding an expected 678-bp product. The amplified DNA product was analyzed by 1% agarose gel electrophoresis.
PGE2 Assay
Amounts of PGE2 in the conditioned media collected from control or PMA or IL-1ß stimulated cells were determined by enzyme-linked immunosorbent assay (Amersham).
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Results |
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IFN
Inhibited COX-2 Protein ExpressionIFN
inhibited PMA-induced COX-2 but not COX-1 protein levels in a concentration-dependent manner. Significant inhibition was noted at 50 U/mL and reached plateau at 500 U/mL (Figure 1A). Densitometric analysis of COX-2 in the Western blots revealed that IFN at 500 U/mL reduced PMA-stimulated COX-2 protein levels by about 70% to 80% (data not shown). IFN at 500 U/mL also suppressed IL-1ß– and TNF-induced COX-2 protein levels to a similar extent (Figure 1B). IFN similarly inhibited PMA- and IL-1ß–induced COX-2 protein expression in HUVECs and U937 (supplemental Figure I, available online at http://atvb.ahajournals.org). In accordance with inhibition of COX-2 protein levels, IFN reduced PGE2 production in HFb (Figure 1C) and HUVECs (data not shown) stimulated with PMA or IL-1ß.
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IFN Abrogated PMA- and IL-1ß–Induced COX-2 Promoter Activity
To determine whether IFN reduced COX-2 expression at the transcriptional level, we evaluated COX-2 promoter activity by transfecting HFb with a core COX-2 promoter fragment (–891 to +9) constructed into a luciferase expression vector. Treatment of transfected HFb with PMA (100 nmol/L) or IL-1ß (10 ng/mL) resulted in an increase in COX-2 promoter activity which was abrogated by pretreatment with IFN (Figure 2A). Similarly, IFN abrogated PMA- or IL-1ß–induced COX-2 promoter activity in HUVECs (Figure 2B).
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IFN Suppressed C-Jun and C/EBPß Binding to COX-2 Promoter Probes
We initially evaluated the effect of IFN on binding of C-Jun/C-Fos, C/EBPß, and NF-B to several short (20 to 24 bp) probes harboring specific regulatory elements. Streptavidin-agarose pulldown assay was used to determine the binding activity. Nuclear extracts were incubated with a biotinylated probe, and the DNA-protein complex was pulled down by streptavidin-conjugated agarose beads. Proteins complexed with the probe were analyzed by Western blots using antibodies against the candidate transactivators. Consistent with previous data, PMA increased binding of C-Jun and C-Fos to the CRE probe, C/EBPß to the C/EBP probe, and p50 to the B probe (Figure 3A). Coincubation of biotinylated CRE or C/EBP probes with a 50-fold molar excess of unlabeled WT probes blocked C-Jun or C/EBPß binding whereas a 50-fold molar excess of mutant probes had no effect (supplemental Figure II). IFN reduced C-Jun and C/EBPß binding but had no effect on C-Fos or p50 NF-B binding (Figure 3A). Neither C-Jun nor C/EBPß protein levels were altered by PMA, IL-1ß, or IFN (supplemental Figure III). Several isoforms of C/EBPß were detected notably the 46-kDa full-length, 41-kDa LAP (liver transcription activating protein), and 16-kDa LIP (liver transcription inhibitory protein) (Figure 3A). IFN reduced full-length and LAP and completely abolished LIP binding to the C/EBP probe (Figure 3A). There was no detectable binding to a control probe (data not shown). We next transfected HFb with a 424-bp COX-2 core promoter probe (–30 to –453) and performed binding assays. We detected basal binding of C/EBPß and C-Jun but not vWF to this core promoter probe (Figure 3B). Binding to the control probe was not detected (Figure 3B). The basal binding activity of C-Jun and C/EBPß was reduced by IFN. PMA increased C-Jun and C/EBPß binding which was abrogated by IFN (Figure 3B). IL-1ß– and TNF-induced C-Jun and C/EBPß binding to the COX-2 promoter probe was also blocked by IFN (Figure 3C). Furthermore, PMA- and IL-1ß–induced C-Jun and C/EBPß binding to COX-2 promoter probe in HUVECs was similarly inhibited by IFN (supplemental Figure IV).
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The effect of IFN on C-Jun and C/EBPß binding to their respective binding sequences was further evaluated by EMSA. IFN at 500 U/mL inhibited C-Jun/probe complex (Figure 4A) and C/EBPß/probe complex (Figure 4B). As previously reported, C-Jun or C/EBPß complex formation was blocked by a 50-fold molar excess of unlabeled WT probes but not a 50-fold molar excess of unlabeled mutant probes (data not shown). Identity of C-Jun and C/EBPß binding was confirmed by supershift assay using specific antibodies for C-Jun and C/EBPß, respectively (Figure 4).
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IFN Inhibited C-Jun and C/EBPß Binding to Chromatin COX-2 Promoter Region
The effect of IFN on C-Jun and C/EBPß binding to COX-2 promoter was evaluated by ChIP assay. The results are in agreement with those obtained from the in vitro binding assays (Figure 5). IFN inhibited basal binding and abrogated PMA-induced binding of C/EBPß and C-Jun to the COX-2 promoter region (Figure 5). It had no effect on C-Fos or p50 binding. The effect of IFN on PMA-induced C-Jun and C/EBPß binding to chromatin COX-2 promoter in HUVECs was evaluated by identical ChIP assays. IFN similarly suppressed PMA-induced C-Jun and C/EBPß in HUVECs (supplemental Figure V).
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IFN Reduced p300 Recruitment to COX-2 Promoter
p300 is a transcription coactivator that integrates the transcriptional signal by interacting with promoter-bound transactivators such as CREB, C/EBPß, C-Jun, and NF-B.28–30 Because IFN inhibited C-Jun and C/EBPß binding, we suspected a consequent reduction in the level of p300 in the complex. The streptavidin-agarose pulldown and ChIP assays are well suited for analysis of multiple proteins in the promoter-protein complex. We analyzed p300 levels in the same HFb and HUVEC samples used in the pulldown assay as shown in Figure 3. p300 binding in HFb was detected at the basal state and its level was increased in cells stimulated by PMA, which was abrogated by IFN (Figure 6A). PMA-induced p300 binding was similarly inhibited by IFN (data not shown). The p300 level in the binding complex was correlated with that of C/EBPß and C-Jun. ChIP analysis reveals that IFN not only abrogated PMA-stimulated p300 binding but also reduced basal p300 binding in HFb (Figure 6B) and HUVECs (data not shown). As previously reported,30 neither the agonist nor IFN influenced p300 protein levels (data not shown).
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Discussion |
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Our results indicate that IFN
suppresses COX-2 transcriptional activation in a number of human cell types stimulated by PMA and cytokines. We demonstrate by in vitro binding assays as well as by ChIP assay that IFN abrogates PIM-induced COX-2 promoter activation through interfering with binding of C-Jun and C/EBPß to COX-2 promoter. Its inhibition of transactivator DNA binding is selective as it has no effect on C-Fos or p50 NF-B binding. DNA-bound C-Jun and C/EBPß recruit p300 coactivators to COX-2 promoter where the coactivators interact with the transcriptional machinery and thereby integrate the signal for promoter activation.29,30 We have recently reported that p300 is the predominant coactivator expressed in human fibroblasts and is essential for COX-2 expression induced by PIM.30 We provide evidence in this study for a reduced p300 recruitment to the COX-2 promoter by IFN. Taken together, our data suggest that IFN inhibits PIM-induced COX-2 expression in human cells by suppressing C/EBPß and C-Jun DNA binding activities, thereby compromising p300 recruitment resulting in a decline in COX-2 promoter activity. The mechanism by which IFN blocks C-Jun and C/EBPß DNA binding remains to be elucidated. PMA and cytokines activate C/EBPß and C-Jun and thereby COX-2 transcription via multiple signaling pathways including ERK1/2, JNK, and RSK1/2.6,7,31 It is possible that IFN suppresses C/EBPß and C-Jun activation by inhibiting a common signaling pathway. Further work is needed to identify this important signaling pathway.
It is unclear why IFN blocks C-Jun but not C-Fos binding to CRE of COX-2 promoter. One possible explanation is that C-Jun and C-Fos form dimers with distinct classes of partner transactivators. It has been shown that C-Jun dimerizes with diverse transactivators notably Jun, Fos, and ATF family proteins.32 C-Fos does not form homodimers but heterodimerizes with Jun and ATF proteins.32 These dimers exhibit dimer-specific DNA binding site preferences.33 For example, C-Jun/C-Fos dimers bind with high affinity to the canonical AP-1 binding site whereas C-Jun/ATF binds to CRE.33 Because IFN inhibits PMA-induced C-Jun but not C-Fos binding, it may be speculated that the key partner of C-Jun under PMA stimulation is not C-Fos but may be ATF whereas the partner for C-Fos may be ATF-4 or other Jun proteins. It is important to note that C-Jun has been implicated as a target of transcriptional inhibition of macrophage scavenger receptor gene by IFN. It was reported that IFN inhibits transcription of macrophage scavenger receptor gene in human monocytic cell lines U937 and THP-1 by blocking C-Jun transactivation activity without a direct effect on C-Jun binding to the promoter.19 It was proposed that suppression of C-Jun transactivation activity is attributed to limited availability of p300 coactivators as a result of IFN-induced STAT-1 overexpression which steals the coactivators.19,21 Paradoxically, IFN stimulates the transcription of a series of genes by activating C-Jun via the ERK pathway in murine embryonic fibroblasts.18 Taken together, these results suggest that C-Jun is pivotal in the transcriptional program of IFN and that IFN exerts transcriptional activation or suppression of genes in a C-Jun-dependent manner. The opposite effects of IFN on C-Jun DNA binding activity and transactivation function are likely attributable to the involvement of different signal transduction pathways, which remain to be delineated.
Several reports have shown that IFN stimulates gene expressions in murine cells. It is well known that IFN primes murine macrophages for their augmented response to myriad stimuli. For example, IFN synergizes with lipopolysaccharide (LPS) and cytokines in stimulating the expression of inducible nitric oxide synthase (iNOS) in RAW/264.7 cells.34–36 It has been shown that IFN and LPS exert the synergistic action by inducing IRF-1 which binds to several cognate sites on iNOS promoter, thereby upregulating gene expression.34–36 On the other hand, results from previous studies do not show that IFN synergizes with LPS in stimulating COX-2 expression in RAW/264.7 cells.37 Nor does it have an apparent effect on inhibiting COX-2 expression.37 IRF-1 does not appear to influence COX-2 promoter activity in RAW/264.7 cells. This is contrary to a report which showed that IFN-induced IRF-1 upregulated COX-2 expression in murine peritoneal macrophages.38 Taken together, these results suggest that IFN has a complex action on gene expressions, depending on animal species as well as on type of genes, cells, and stimuli.
COX-2 promoter activation by PIMs, growth factors, and angiogenic factors is mediated by binding of several transactivators to enhancer elements located within about 500 bp 5'- from the transcription start site. CREB/ATF, C/EBPß, C-Jun/C-Fos, and NF-B have been identified as crucial for COX-2 promoter activation. Previous reports from several laboratories including ours have shown that C/EBPß binding to its enhancer element on COX-2 promoter is required for COX-2 promoter activation not only by PMA but also by IL-1ß, LPS, src oncogene, and growth factors.8–10,39 Similarly, C-Jun binding is required for COX-2 promoter activation by PMA and oncogenic factors.39 Because IFN is capable of repressing C/EBPß and C-Jun binding, it is likely that it may exert a wide spectrum of effect against COX-2 expression induced by diverse agonists.
Excessive COX-2 expression stimulated by potent proinflammatory cytokines and endotoxins plays a key role in inflammatory disorders. COX-2 overexpression has also been implicated in vascular inflammatory lesions including atherosclerotic plaque formation and instability.40 There is strong evidence for accumulation of lymphocytes in atheromatous lesions, and IFN produced by lymphocytes has been implicated in plaque instability.41 As IFN and COX-2 are coexpressed in these lesions, it is likely that IFN also plays a role in suppressing COX-2 expression and attenuating COX-2–meidated plaque instability. However, it should be cautioned that evidence has not been provided that IFN produced at the vascular lesion reaches high enough concentrations to exert the multiple actions of IFN including COX-2 transcriptional inhibition. Nonetheless, IFN may have a potential for treating COX-2–mediated inflammatory and neoplastic diseases.
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Acknowledgments |
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We thank Nathalie Huang for excellent editorial assistance.
Sources of Funding
This work was supported by grants from the National Institutes of Health (HL-50675) and National Health Research Institutes intramural funds.
Disclosures
None.
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Footnotes |
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Original received August 19, 2003; final version accepted May 23, 2007.
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