Tài liệu Báo cáo khoa học: The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes doc

The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes Franck T Grall, Wolf C Prall, Wanjiang Wei, Xuesong Gu, Je-Yoel Cho, Bob K Choy, Luiz F Zerbini, Mehmet S Inan, Steven R Goldring, Ellen M Gravallese, Mary B Goldring, Peter Oettgen and Towia A Libermann New England Baptist Bone and Joint Institute and BIDMC Genomics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, USA Keywords COX-2; ESE-1; Ets; gene expression; LPS Correspondence T A Libermann, New England Baptist Bone & Joint Institute, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Blackfan Circle, Boston, MA 02115, USA Fax: +1 617 975 5299 Tel: +1 617 667 3393 E-mail: tliberma@bidmc.harvard.edu (Received 21 July 2004, revised 19 January 2005, accepted February 2005) doi:10.1111/j.1742-4658.2005.04592.x Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte ⁄ macrophages ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-jB and nuclear factor of activated T cells Neutralization of COX-2 is the goal of many anti-inflammatory drugs As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation Cyclooxygenase (COX) is an enzyme that converts arachidonic acid into the prostaglandin H2 This product is the critical point of the synthetic pathway of numerous members of the prostaglandin family COX exists as two major isoforms derived from two separate genes: COX-1 and COX-2 COX-1 is constitutively expressed, whereas COX-2 expression is inducible Pro-inflammatory substances are some of the major activators of COX-2 Examples include interleukin (IL)-1 [1], tumor necrosis factor (TNF)-a [2], and bacterial lipopolysaccharide (LPS) [3] A third isoform COX-3 has also been reported [4] The mechanisms leading to COX-2 expression involve various combinations of different transcription factors, depending on the cell type and stimulus The members of the C ⁄ EBP family have been identified as Abbreviations Ad, Adenovirus; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; COX, cyclooxygenase; CRE, cAMP responsive element; ESE, epithelium specific Ets factor; Ets, E26 transformation specific; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HRP, horseradish peroxidase; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; NFAT, nuclear factor of activated T cells; NF-jB, nuclear factor-jB; TNF, tumor necrosis factor 1676 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS F T Grall et al important regulators of COX-2 expression in osteoblasts [5], T lymphocytes [6], amnion epithelial cell WISH [7], macrophages [8,9], and chondrocytes [10] The nuclear factors of activated T cells (NFAT) are essential for COX-2 activation in T lymphocytes [6] The role of nuclear factor-jB (NF-jB) appears to be more cell-specific According to Allport et al [7], the mutation of the major NF-jB site of the COX-2 promoter abolishes IL-1-induced COX-2 expression in the human amnion epithelial cell line WISH Crofford et al [11] successfully employed p65 antisense oligonucleotides to inhibit IL-1-mediated induction of the COX-2 promoter in synoviocytes Furthermore, a fragment of the COX-2 promoter starting downstream of the NF-jB sites could not be activated by IL-1 in chondrocytes [10] However, in macrophages, Wadleigh et al [9] mutated the NF-jB site of the murine COX-2 promoter without loss of LPS mediated COX-2 activation The cAMP responsive element (CRE) site overlapping an E-box element is another important site for transcription factors as the mutation of the CRE site within the COX-2 promoter or the expression of a dominant negative mutant of CREB reduces inducibility of the COX-2 promoter [9,12–14] The transcription factors binding this site include CREB and cJun as well as USF-1 for the E-box [12] Some Ets factors have also been suggested to play a role in the regulation of COX-2 such as Ets-1 [14,15], PEA3 [16–18] and Pu.1 [19] The multiple activation modalities observed across the different studies as well as the similarity of the recognition sites of the NFAT and Ets factors led us to investigate the potential involvement of the epithelial specific Ets factor (ESE-1 ⁄ ESX ⁄ ELF3 ⁄ ERT ⁄ JEN) in this process This factor is a likely regulator of COX-2 expression, as we recently discovered that ESE-1 plays an important role in the responses of various cell types to inflammatory mediators [20,21] We reported the induction of ESE-1 in response to IL-1, TNF-a and LPS in vascular and connective tissue cells This induction was mediated through the activation of NF-jB [21] We showed that ESE-1 could activate the expression of another important player in the inflammatory response: inducible nitric oxide synthase (iNOS) [20] Although ESE-1 was initially described as exclusively expressed in epithelial cells in a variety of tissues [22–25], we subsequently observed a broader expression pattern for ESE-1 under inflammatory conditions [21] We now report that ESE-1 can bind to the promoter of COX-2 and that the integrity of the Ets sites is required for LPS-induced COX-2 expression ESE-1 can activate the COX-2 promoter in the monocyte cell FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS ESE-1 activates COX-2 expression line RAW 264.7 as well as chondrocytic cells where it acts synergistically with NF-jB and NFAT Moreover, we show the capacity of a dominant-negative form of ESE-1 to diminish COX-2 promoter induction in response to LPS or IL-1 exposure Results COX-2 induction by pro-inflammatory stimuli correlates with ESE-1 induction Our previous data had indicated that ESE-1 expression is rapidly and transiently induced by pro-inflammatory cytokines in a variety of vascular and connective tissue cell types [20,21] We also demonstrated that the iNOS gene, a target for pro-inflammatory cytokines, is a downstream target for ESE-1 To further our understanding of ESE-1 function during inflammatory processes, we have now explored the involvement of ESE-1 in the regulation of another inflammation-related gene, COX-2, in monocytic cells and chondrocytes We previously demonstrated that LPS stimulation of human monocytic THP1 cells leads to an induction of ESE-1 mRNA expression within h of exposure, reaching a peak at h and leveling off after 24 h [12] To investigate the level of ESE-1 protein following LPS exposure, we performed western blot analysis in murine monocytic RAW 264.7 cells The intensity of the ECL signal was determined using the alphaease fc sofware and divided by the protein concentration of the sample ESE-1 protein was detected h after LPS stimulation and increased levels were observed until 10 h Analysis of COX-2 protein expression in response to LPS in RAW 264.7 cells by western blot revealed that the temporal pattern of COX-2 protein induction upon stimulation by LPS correlated with the expression pattern of ESE-1 (Fig 1) Thus, COX-2 may be a potential target for ESE-1 during inflammation ESE-1 transactivates the human COX-2 promoter To elucidate whether the COX-2 gene could be an ESE-1 target gene in inflammatory processes, we screened the regulatory region of the COX-2 promoter for potential ESE-1 binding sites Inspection of the COX-2 promoter sequence (GeneBank accession number AY229989) revealed the presence of five possible Ets binding sites within the first 200 bp upstream of the transcription initiation site, one of which overlaps with a potential NFAT site (Fig 2A) To determine whether the COX-2 gene may be regulated by ESE-1, we constructed two human 1677 ESE-1 activates COX-2 expression A B F T Grall et al in a dose–response curve Different amounts of ESE-1 and PEA3 expression vector DNA were cotransfected with the COX-2 promoter luciferase construct, maintaining the total amount of transfected DNA constant by adding the parental pCI vector As illustrated in Fig 2D, ESE-1 at all concentrations was more effective than PEA3 in transactivating the COX-2 promoter This result does not appear to be due to a higher production of the ESE-1 protein Western blot analysis of ESE-1 and PEA3 expression after transfection of equal amounts of expression vector into 293ft cells, shows that ESE-1 protein expression is lower than PEA3 (Fig 2E) Indeed we have observed that generally ESE-1 protein expression after transfection is significantly lower than most other Ets factors ESE-1 binds to the human COX-2 promoter Fig Induction of ESE-1 and COX-2 expression in monocytic cells RAW cells were grown in the absence or presence of LPS (100 ngỈmL)1) for 0, 2, 4, 10 or 12 h ESE-1 and COX-2 protein levels were measured by western blotting using ESE-1 and COX-2 specific antibodies (A) ECL signal on a photographic film (B) Levels of ESE-1 and COX-2 shown as integrated intensity divided by the protein load for each lane COX-2 promoter luciferase reporter plasmids, pXP2 ⁄ COX-2–170 and pXP2 ⁄ COX-2–831, starting at )170 and )831, respectively, upstream of the transcription start site, which we transiently transfected into RAW 264.7 cells Cotransfections of these promoter plasmids together with the ESE-1 expression vector, pCI ⁄ ESE-1, enhanced COX-2 promoter activity 10- and 30-fold, when the long or the short promoter constructs, respectively, were used (Fig 2B) Stimulation with LPS resulted in a more than 20-fold induction of the COX-2 promoter (Fig 2B) This ESE-1-mediated transactivation of the COX-2 promoter was not restricted to RAW 264.7 cells, since transfection of pCI ⁄ ESE-1 also stimulated transcription of the )170 bp COX-2 promoter in the human chondrocyte cell line T ⁄ C28a2 (Fig 2C), a cell type shown to express ESE-1 in response to IL-1 [21] As another Ets factor, PEA3, has previously been shown to activate the COX-2 promoter, we compared the relative activities of ESE-1 and PEA3, cloned downstream of the cytomegalovirus (CMV) promoter, 1678 To investigate whether this induction could be due to a direct effect of ESE-1 on the COX-2 promoter, we assessed the ability of ESE-1 to bind to the COX-2 promoter by performing an EMSA We used as probes the five putative Ets binding sites of the COX-2 promoter present within the )170 COX-2 promoter We tested their ability to form a complex with in vitro translated ESE-1 protein As shown in Fig 3A, sites and formed strong protein ⁄ DNA complexes with ESE-1 The specificity of this complex was confirmed in supershift assays using two different anit-ESE-1 Igs (Fig 3) Site 4, which overlaps with the NFAT binding site, did not appear to bind specifically to ESE-1 in this assay The mobility of the ESE-1 complex was consistent with that reported in Rudders et al and was absent when unprogrammed reticulocyte lysate was used To further determine whether ESE-1 binds to the COX-2 promoter in vivo, we performed a chromatin immunoprecipitation experiment in chondrocyte cells which express both ESE-1 and COX-2 in response to IL-1 (Fig 3B) T ⁄ C28a2 chondrocyte cells were transfected with either pcDNA3-1 ⁄ Flag-ESE-1 or pcDNA3-1 ⁄ Flag After cross-linking the proteins bound to DNA with formaldehyde followed by sonication, the cell extracts were immunoprecipitated using the anti-Flag Ig or nonspecific serum As shown in Fig 4B, a precipitate specifically retaining the COX-2 promoter region spanning all five Ets sites shown in Fig ()186 to +56 of the transcription start site) was only obtained in cells transfected with the plasmid containing ESE-1 This experiment most clearly demonstrates that ESE-1 directly binds to the COX-2 promoter in vivo FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS F T Grall et al ESE-1 activates COX-2 expression A B C D E Fig The COX-2 promoter is a target for ESE-1 (A) Sequence of the COX-2 promoter The five putative Ets binding sites present in the )170 COX-2 construct (starting at the asterisk) are highlighted as well as additional NFAT and NF-jB sites within the COX-2 promoter sequence Two extra Ets sites described in Howes et al [16] are underlined (B) Transcriptional activation of the COX-2 promoter by ESE-1 and LPS RAW cells were cotransfected with the pXP2 luciferase construct containing the COX-2 promoter (pXP2 ⁄ COX-2) starting either at )831 or at )170 and the pCI ⁄ ESE-1 expression vector and incubated in the absence or presence of LPS Luciferase activity in the lysates was determined 16 h later, as described Data shown are means of duplicate measurements from one representative transfection The experiment was repeated three times with different plasmid preparations with comparable results Error bars represent the SD for the two replicates (C) Transcriptional activation of the COX-2 promoter by ESE-1 in chondrocytes T ⁄ C28a2 cells were cotransfected with the pXP2 luciferase construct containing the )170 COX-2 promoter (pXP2 ⁄ COX-2) and the pCI ⁄ ESE-1 expression vector Luciferase activity in the lysates was determined 16 h later, as described Data shown are means of duplicate measurements from one representative transfection Error bars represent SD of the two replicates (D) Transcriptional activation of the COX-2 promoter by ESE-1 and PEA3 RAW cells were cotransfected with the COX-2 promoter luciferase construct (pXP2 ⁄ COX-2–170) and different amounts of expression vectors for ESE-1 or PEA3 maintaining constant (700 ng) the total amount of DNA with pCI vector Luciferase activity in the lysates was determined 16 h later, as described Data shown are means of duplicate measurements from one representative transfection The experiment was repeated three times with different plasmid preparations with comparable results Error bars represent SD for the two replicates (E) Protein expression levels of ESE-1 and PEA3 after transfection into 293ft cells Myc-tagged Ets factors were transfected into 293 cells The cells were lysed 16 h later and equal amounts of lysate were loaded on a gel for a western blot analysis using anti-myc Ig Mutation of multiple ESE-1 binding sites drastically reduces activation of the COX-2 promoter by ESE-1 and by LPS To examine whether the Ets sites in the COX-2 promoter are responsive to ESE-1 and to determine whether LPS induction of the COX-2 promoter is mediated via ESE-1, we introduced mutations into individual or multiple Ets sites of the COX-2 promoter Wild type or mutant constructs of pXP2 ⁄ COX-2–170 were cotransfected into RAW 264.7 cells in the absence or presence of pCI ⁄ ESE-1 These experiments indicated FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS that individual mutations of the Ets binding sites 2, and led to more than 50% reduction of ESE-1-mediated COX-2 promoter activation (Fig 4A) Simultaneous mutation of site along with sites 1, or almost completely eliminated inducibility by ESE-1 suggesting that ESE-1 acts on the COX-2 promoter via multiple Ets sites (Fig 4A), but that sites 2, and are crucial for inducibility, since their mutations gave the strongest reductions of activity compared to other isolated mutations This is in line with the findings of Liu et al [18] who showed that the Ets site number was critical for COX-2 induction by NO No inducibility 1679 ESE-1 activates COX-2 expression F T Grall et al A B Fig ESE-1 binds to several Ets sites in the COX-2 promoter (A) Interaction of ESE-1 with Ets binding sites in the COX-2 promoter EMSA using either unprogrammed reticulocyte lysate [23] or in vitro translated ESE-1 (ESE-1) and five labeled oligonucleotide probes containing different COX-2 promoter Ets sites The in vitro translated proteins were alternatively preincubated with antibody (Ab1, east-acres Biological; Ab2, QED; C, negative control, normal rabbit serum) The white arrow indicates the specific ESE-1 DNA–protein complex and the black arrow shows the supershift form with the antibody (B) Binding of ESE-1 to endogenous human COX-2 promoter by ChIP The anti-Flag Ig was used to specifically enrich COX-2 promoter DNA sequences in a ChIP assay Chromatin proteins from IL-1 treated T ⁄ C28a2 cells transfected with either pcDNA3 ⁄ Flag (lanes 1–3) or pcDNA ⁄ Flag-ESE-1 (lanes 4–6) were crosslinked to DNA with formaldehyde, and purified nucleoprotein complexes were immunoprecipitated using either anti-Flag Ig or nonspecific rabbit serum The precipitated DNA fractions were analyzed by PCR for the presence of the COX-2 promoter region encompassing the Ets sites The input and genomic DNA (gDNA) were used as a positive control, and water was used as a negative control for PCR was left when sites 1, 2, and were mutated in combination LPS response was also significantly affected when sites 3, or were mutated individually (Fig 4B) Combined mutation of the Ets sites 1, 2, 3, and 5, leaving the NFAT element in site number intact, led to a drastic inhibition of promoter activation in response to LPS (Fig 4B) This experiment demonstrates that LPS activation of the COX-2 promoter is at least partially mediated via ESE-1 or a related Ets factor The mutation of the C ⁄ EBPb site that inhibited the activity of PEA3 [16] led to only a diminution of the activity of ESE-1 ESE-1 and NFAT act synergistically on the COX-2 promoter As the NFAT factors have been reported as activators of COX-2 [6], we evaluated whether ESE-1 and NFAT could cooperate in the context of the COX-2 promoter The )170 COX-2 promoter luciferase construct was 1680 cotransfected into RAW cells together with pCI ⁄ ESE-1 or a constitutively active form of one member of the NFAT family, NFAT3, cloned into pRK5, or a combination thereof, and either empty pRK5 or pCI, respectively (Fig 5A) ESE-1 enhanced COX-2 promoter activity 10-fold compared to only 2.5-fold activation by NFAT3 Combined expression of ESE-1 and NFAT3 synergistically enhanced COX-2 promoter activity more than 20-fold These results indicate that ESE-1 and NFAT3 most likely act via different sites or different DNA sides at the same sites and may actually cooperate in transactivation of the COX-2 promoter ESE-1 cooperates with NF-jB in the transactivation of the COX-2 promoter In addition to the Ets binding sites the COX-2 promoter contains at least four putative NF-jB binding sites As NF-jB has been demonstrated previously to play a role in regulating COX-2 promoter activity in at least some cell types [7,11] and NF-jB cooperates with ESE-1 in transactivating the iNOS promoter in endoFEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS F T Grall et al ESE-1 activates COX-2 expression A B C Fig ESE-1 and LPS transactivate the COX-2 promoter through multiple Ets binding sites (A) Mutation of multiple Ets binding sites within the COX-2 promoter inhibits induction by ESE-1 RAW cells were cotransfected with the pCI ⁄ ESE-1 expression vector and the COX-2 promoter luciferase constructs containing either wild-type (WT) or multiple mutants of potential binding sites (mut) alone or in combination Luciferase activity in the lysates was determined 16 h later, as described elsewhere [23] Data shown are means of duplicate measurements from one representative transfection The experiment was repeated four times with different plasmid preparations with comparable results Error bars represent SD of the two replicates (B) Mutation of the Ets binding sites reduces LPSinduced transactivation of the COX-2 promoter RAW cells were transfected with the wild-type or Ets mutant COX-2 promoter luciferase constructs and then stimulated with LPS Luciferase activity in the lysates was determined 16 h later Error bars represent the SD of the two replicates thelial cells [20], we evaluated whether ESE-1 and NF-jB cooperate in the context of the COX-2 promoter as well The )831 COX-2 promoter luciferase FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS Fig ESE-1 cooperates with NFAT and NF-jB in transactivating the COX-2 promoter (A) RAW cells were cotransfected with the pCI ⁄ ESE-1 expression vector or the pRK5 ⁄ NFAT3 expression vectors or a combination thereof and the )170 COX-2 promoter luciferase construct Luciferase activity in the lysates was determined 16 h later, as described elsewhere [23] Data shown are means of duplicate measurements from one representative transfection The experiment was repeated twice with different plasmid preparations with comparable results Error bars represent the standard deviation of the two replicates (B and C) RAW cells were cotransfected with the pCI ⁄ ESE-1 expression vector, the NF-jB p50 and p65 expression vectors or a combination thereof and the )831 or )170 COX-2 promoter wild-type (B) or )170 mut ets +2 + +4 + (C) luciferase constructs Luciferase activity in the lysates was determined 16 h later, as described elsewhere [23] Data shown are means of duplicate measurements from one representative transfection The experiment was repeated twice with different plasmid preparations with comparable results Error bars represent the standard deviation of the two replicates construct was cotransfected into RAW cells together with either ESE-1, NF-jB p50 or p65 alone as well as with various combinations thereof (Fig 5B) Whereas 1681 ESE-1 activates COX-2 expression F T Grall et al p50 alone did not significantly enhance COX-2 promoter activity, p65 increased promoter activity by three to fourfold The p50 ⁄ p65 combination enhanced transactivation of the COX-2 promoter to 10-fold, similar to the effect of ESE-1 alone ESE-1 cooperated with both p50 and p65, which enhanced COX-2 promoter activity by 30-fold in cotransfection with ESE-1 This cooperativity was most striking when ESE-1 was cotransfected with the p50 ⁄ p65 heterodimer, which increased transactivation of the COX-2 promoter by 70-fold Mutation of the Ets sites within the COX-2 promoter did not significantly affect NF-jB mediated transactivation, but strongly reduced cooperativity between ESE-1 and NF-jB (Fig 5C) Dominant-negative ESE-1 mutants inhibit LPS and IL-1 mediated induction of COX-2 gene expression To evaluate whether ESE-1 is indeed involved in regulation of inducible COX-2 gene expression we used dominant-negative mutants of ESE-1 as tools to block endogenous COX-2 gene expression We constructed two dominant-negative forms of ESE-1 One of these two constructs, Dominant Negative (DN1), encompasses the carboxy-terminal Ets DNA binding domain of ESE-1 and competes with intact endogenous ESE-1 for binding to target gene promoters The second dominant-negative mutant, Dominant Negative (DN2), encompasses the amino-terminal transactivation domain and Pointed domain fused to a nuclear localization signal and presumably acts as a dominant negative ESE-1 due to its ability to interact with coactivators and other cofactors needed for transactivation of ESE-1, thereby depriving intact ESE-1 of its factors needed for transactivation DN1 and DN2 were cloned into adenovirus vectors and the expression plasmid pCI We tested the effect of dominant-negative ESE-1 on inducible COX-2 gene expression in the human chondrocyte cell line T ⁄ C28a2 Very little COX-2 mRNA expression was detected in unstimulated cells, but a strong induction was observed upon exposure to IL-1 (Fig 6A) Infection with AdE1-DN1 or AdE1-DN2 inhibited IL-1-induced expression of COX-2 mRNA by 50–70% compared to Ad-bGal infection (Fig 6A) As RAW monocytic cells are difficult to infect with adenoviruses and also not transfect with high efficiency, they are not suitable for assessing the effects of the dominant negative ESE-1 on endogenous COX-2 mRNA levels Therefore, we evaluated the effects of the dominant-negative ESE-1 mutants in RAW cells transfected with the pXP2 ⁄ COX-2 promoter luciferase 1682 Fig Dominant-negative mutants of ESE-1 reduce expression of the endogenous COX-2 gene in response to LPS and inhibit the transactivation its promoter (A) T ⁄ C28a2 chondrocyte cells were infected with the adenoviruses Ad ⁄ ESE-1 DN1, DN2 or b-galactosidase (beta-gal) and 16 h later treated with IL-1 (500 pgỈmL)1) The RNA was harvested 28 h later and used for real-time PCR using COX-2 specific primers Data shown are means of duplicate measurements from one representative transfection representing the ratio of the measurements of COX-2 to GAPDH mRNA Error bars represent the SD of the two replicates (B) RAW cells were cotransfected with the pCI expression vector containing the dominant-negative mutant of ESE-1 (ESE-1 DN1) and the COX-2 promoter Luciferase ()170) construct Cells were grown in the absence or presence of LPS (500 ngỈmL)1) for 16 h Luciferase activity in the lysates was determined 16 h after addition of LPS Data shown are means of duplicate measurements from one representative transfection Error bars represent the SD of the two replicates construct ESE-1 DN1 expression completely blocked LPS-mediated induction of the COX-2 promoter, again confirming the involvement of ESE-1 in LPS-mediated activation of the COX-2 promoter (Fig 6B) These data most vividly indicate that ESE-1 may play a critical role in induction of the COX-2 gene by pro-inflammatory stimuli Discussion The regulation of COX-2 expression during inflammation has been the focus of numerous studies The heterogeneity of parameters such as the cell type and the stimulus used has made it difficult to describe precisely FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS F T Grall et al the mechanisms by which the promoter of COX-2 is activated Several transcription factor families have been shown to be involved in this process such as C ⁄ EBP [5,9,10,26], NF-jB [7,10,11], NFAT [6,10] and Ets [16,18,27] We now report the involvement of another Ets transcription factor ESE-1 in the regulation of COX-2 expression in monocytes ⁄ macrophages and chondrocytes ESE-1 (also named ELF3, Jen, ERT, ESX) is an Ets family transcription factor, recently discovered by us and others [22,23,28,29], whose expression under normal physiological conditions is restricted to epithelial cells However, we uncovered an unexpected function for ESE-1 in the vascular system and in connective tissue cells where its expression is induced following exposure to pro-inflammatory stimuli such as IL-1, TNF-a, and LPS [20,21] We show here that LPS-mediated induction of COX-2 gene expression is, at least partially, dependant upon ESE-1 upregulation ESE-1 binds to the promoter of COX-2 on several sites and activates its expression The integrity of these sites is required for full COX-2 promoter activation by LPS, since mutation of two or more sites markedly attenuates the response This is true even when the NFAT site is left intact (identical to Ets site number 4) ESE-1 mediated transactivation of the COX-2 promoter is synergistic with NFAT and NF-jB, since ESE-1 can enhances the activity of COX-2 promoter due to NFAT or NF-jB transactivation more than additive This cooperativity can be due to the previously demonstrated [20] direct binding of ESE-1 to the NF-jB family members p50 and p65 NF-jB itself also upregulates endogenous ESE-1 expression in response to pro-inflammatory stimuli via a high affinity NF-jB binding site within the ESE-1 promoter [20,21] Thus, ESE-1 may be involved in a positive feedback loop during the inflammatory response to enhance the transactivation effect of NF-jB on some of its target genes by cooperating with ESE-1 The mutation of the Ets site decreases the ESE-1 activation of COX-2, even though no binding could be detected in vitro by EMSA This site may be functionally mixed or it is possible that these two factors interact with each other and bind as a complex on this site That would explain the further increase of COX-2 activation when ESE-1 is transfected together with NFAT The involvement of an Ets factor in the regulation of COX-2 was reported by Howe et al [16], who demonstrated that overexpression of another member of the Ets family, PEA3, could activate the COX-2 promoter When we compared the activities of PEA3 to ESE-1, we found that ESE-1 is a more effective transactivator of the COX-2 promoter than PEA3, further FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS ESE-1 activates COX-2 expression supporting the notion that ESE-1 may be relevant for COX-2 regulation However, in contrast to ESE-1, PEA3 does not appear to be regulated by pro-inflammatory stimuli Surprisingly, the effect of PEA3 seems to be mediated via the C ⁄ EBPb site Our data show that mutation of the C ⁄ EBPb site also affects ESE-1 mediated transactivation, but only partially This suggests that ESE-1 may also cooperate with C ⁄ EBPb or another factor binding to this site in this process but that does not account for the entire activation activity as the mutation of Ets sites leads to a more drastic abolition of this induction It has also been shown that the pattern of expression of PEA3 in breast cancer samples correlates with the patterns of expression of HER-2 ⁄ neu and COX-2 [17] suggesting that the levels of COX-2 may results from an HER-2 ⁄ neu stimulation of PEA3 Interestingly, a similar correlation between the expression of HER-2 ⁄ neu and ESE-1 has also been observed [28,30,31] and HER-2 ⁄ neu could itself activate the expression of ESE-1 [31] The role of Ets factors in inflammation and in the regulation of cytokine-responsive genes has not been studied in detail However, several genes, including urokinase-type plasminogen activator, matrix metalloproteinase (MMP)-1, MMP-3, TNF-a, scavenger receptor, intercellular adhesion molecule (ICAM)-1, ICAM-2, and IL-12 have been shown to depend on Ets factors for their inducibility by cytokines such as IL-1 or TNF-a [32–36] Many additional cytokineresponsive genes contain putative Ets binding sites within their regulatory regions, including COX-2, iNOS, and MMP-13 We demonstrate here that endogenous COX-2 gene expression can be inhibited by using dominant negative forms of ESE-1 The inhibitory effect of these dominant negative ESE-1 mutants on COX-2 expression, confirm the importance of ESE-1 or a related factor that would bind to these Ets sites These results give some insight in the potential use of new therapeutic approaches manipulating the activity of ESE-1 or other Ets factors as a tool to reduce inflammation Indeed, several pro-inflammatory agents such as IL-1 [21], and TNF-a [21] can also induce ESE-1, and ESE-1 target genes with regard to inflammation so far identified by us include iNOS, COX-2 and potentially MMP-1 and MMP-13 (X Gu, F Grall, M Joseph, L Zerbini & T Libermann, unpublished data) The kinetic of activation of ESE-1 seems to implicate ESE-1 at later stages of inflammation These results suggest that ESE-1 regulates a subset of the genes whose inhibition could be of significant interest in the management of the inflammatory reaction 1683 ESE-1 activates COX-2 expression In conclusion, this report demonstrates further that ESE-1 is a relevant player in the inflammatory process ESE-1 is upregulated in several cell types in response to pro-inflammatory stimuli through the NF-jB pathway It can activate some important genes such as iNOS and COX-2 Our studies also suggest that, by modulating the activity of ESE-1, we could decrease the inflammatory reaction in response to LPS exposure Experimental procedures Cell culture and patient samples THP-1 (human monocytic) and RAW 264.7 (murine monocytic) cells (ATCC, Manassas, VA, USA) were cultured in DMEM with 10% serum (Hyclone, Logan, UT, USA) with undetectable levels of endotoxin Immortalized human chondrocytes T ⁄ C28a2 were grown and treated with cytokines as described [37,38] Lipopolysaccharide was obtained from Sigma (St Louis, MO, USA) (catalogue number L8274) RT ⁄ PCR analysis Total RNA was harvested using QIAshreder (Qiagen, Valencia, CA, USA) and RNeasyÒ Mini Kits (Qiagen) The cDNAs were generated from lg total RNA using ReadyTo-GoTM You-prime First-Strand Beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) SYBR Green I-based real-time PCR was carried out on the Opticon Monitor (MJ Research, Inc., Waltham, MA, USA) All PCR mixtures contained: PCR buffer (final concentration 10 mm Tris ⁄ HCl pH 9.0, 50 mm KCl, mm MgCl2, 0.1% TritonX-100), 250 lm deoxy-NTP (Roche Pleasanton, CA, USA), 0.5 lm each PCR primer, 0.5 · SYBR Green I, 5% dimethylsulfoxide, U Taq DNA polymerase (Promega, Madison, WI, USA) with lL cDNA in a final volume of 25 lL The samples were loaded into wells of Low Profile 96-well microtiter plates After an initial denaturation step at 95 °C for min, conditions for cycling were 38 cycles of denaturation (95 °C, 30 s), annealing (54 °C, 30 s), and extension (72 °C, min) Then, the fluorescence signal was measured immediately following incubation at 78 °C for s that follows each extension step, thereby eliminating possible primer dimer detection At the end of the PCR cycles, a melting curve was generated to identify the specificity of the PCR product For each run, serial dilutions of human GAPDH plasmids were used as standards for quantitative measurement of the amount of amplified cDNA For normalization of each sample, hGAPDH primers were used to measure the amount of hGAPDH cDNA All samples were run as duplicates and the data were presented as ratios of Cox-2 ⁄ hGAPDH The primers used for real time PCR are as follows For hGAPDH forward, 5¢-CAAAGTTGTCATG 1684 F T Grall et al GATGACC-3¢; reverse, 5¢-CCATGGAGAAGGCTGGG G-3¢, which will amplify 195 bp of human GAPDH For Cox2 forward, 5¢-TTCAAATGAGATTGTGGGAAAA TTGCT-3¢; reverse, 3¢-ATATCATCTCTGCCTGAGTAT CTT-3¢, which will amplify 304 bp of human Cox2 Expression vector and luciferase reporter gene constructs Full-length and dominant negative mutant ESE-1 cDNAs were inserted into the EcoRI site of the pCI (Promega) eukaryotic expression vector downstream of the T7 and CMV promoter as described [39] The dominant negative form of ESE-1, DN1, encodes the ESE-1 peptidic sequence deleted of the amino acid residues 76–198, and DN2 encodes the amino acid residues 1–231 fused in frame to a nuclear localization signal motif repeated three times and a sequence coding for EYFP in the pEYFP-NLS vector from Clontech Full length ESE-1 was fused in-frame with the Flag peptide at the amino terminus in the pcDNA3-1 vector The human COX-2 promoter sequences spanning )831 and )170 to +103, kindly provided by L J Crofford, Division of Rheumatology, University of Michigan [11], were cloned into the pXP2 luciferase vector in the HindIII and XhoI sites (pXP2 ⁄ COX-2) An expression vector for the mouse PEA3 gene downstream of the CMV promoter (pCANMycPEA3) was a gift from L Howe, Strang Cancer Research Laboratory, Rockefellar University EMSA In vitro transcription ⁄ translation was performed in TNT rabbit reticulocyte lysate (Promega) using the pCI ⁄ ESE-1 vector as described [23] EMSAs were performed using lL of in vitro translation product and [32P]-labeled doublestranded oligonucleotide probes [40] Supershift assays were performed by preincubating the in vitro translated protein 20 at room temperature with lL antibody Oligonucleotides used as probes and for competition studies were: (a) COX-2 promoter Ets site #1, 5¢-GCA CGTCCAGGAACTCCTCAGC-3¢; (b) COX-2 promoter Ets site #2, 5¢-GAGAGAACCTTCCTTTTTATAA-3¢; (c) COX-2 promoter Ets site #3, 5¢-CGAAAAGGCGGAAAG AAACAGT-3¢; (d) COX-2 promoter Ets site #4, 5¢-GAGA GGAGGGAAAAATTTGTGG-3¢; 3¢-CTCTCCTCCCTTT TTAAACACC-5¢; (5) COX-2 promoter Ets site #5, 5¢-TCTCATTTCCGTGGGTAAAAA-3¢ Site-directed mutagenesis Mutations in the different COX-2 promoter ETS sites were generated by site-directed mutagenesis with the QuikChange Site-directed Mutagenesis kit (Stratagene, Cedar Creek, TX, USA) and confirmed by sequencing FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS F T Grall et al The following primers were used (the mutated bases are underscored): (a) COX-2 promoter Ets site #1, 5¢-GCT GAGGAGTAGCTGGACGTGCTCCTGAC-3¢; (b) COX-2 promoter Ets site #2, 5¢-CAGTCTTATAAAAACCAA GGTTCTCTCGGTTAGCGACC-3¢; (c) COX-2 promoter Ets site #3, 5¢-GACGAAATGACTGTTTCTTTGAGCC TTTTCGTACCCC-3¢; (d) COX-2 promoter Ets site #4, 5¢-AGGGGAGAGGAGGGTTAAATTTGTGGGGGGTA CGAAAAGGCGG-3¢; (e) COX-2 promoter Ets site #5: 5¢-GGGTTTTTTACCCACGCTAATGAGAAAATCGGAA ACC-3¢ ESE-1 activates COX-2 expression signal intensity was determined with the alphaease software (AlphaInnotech, San Leandro, CA, USA) and then divided for normalization by the protein concentration for each lane 293ft cells (3 · 105; Invitrogen, Carlsbad, CA, USA) were transfected with lg ESE-1 or PEA3 expression plasmid using lipofectamine and lysed 16 h later Cell lysate (225 lg) was loaded onto a SDS ⁄ PAGE gel Anti-myc ⁄ HRP conjugated Ig (Santa-Cruz) was used at : 200 dilution for h to detect myc-tagged ESE-1 and PEA3 Chromatin immunoprecipitation (ChIP) DNA transfection assays Cotransfections were carried out in 6-well plates containing 3–8 · 105 cells per well using 600 ng of reporter gene construct DNA and 200 ng expression vector DNA using LipofectAMINE PLUS (Gibco-BRL) for 16 h as described [23] Transfections were performed independently in duplicate, repeated three to four times with different plasmid preparations and gave similar results Cotransfection of a second plasmid for determination of transfection efficiency was omitted, because potential artifacts with this technique have been reported [41] and many commonly used viral promoters contain binding sites for Ets factors Adenovirus infection Adenoviruses encoding the dominant negative forms of ESE-1 were constructed using the Adeno-X expression system from Clontech TC ⁄ 28a2 cells were infected with adenovirus for h in serum-free medium using a multiplicity of infection of 300 After infection the cells were washed with medium and incubated for 16 h in DMEM containing 10% fetal calf serum in the absence or presence of IL-1 (500 pgỈmL)1) (R & D Systems, Minneapolis, MN, USA) Western blot analysis RAW 264.7 cells were plated at · 105 cells per well 16 h before being exposed to LPS (100 ngỈmL)1) in fresh medium for different periods of time The cells were rinsed with NaCl ⁄ Pi, harvested in 200 lL RIPA lysis buffer containing protease inhibitors (Roche) and frozen and thawed once before being sonicated Forty microliters of lysate were loaded on a 10% polyacrylamide gel containing SDS Proteins were transferred to a poly(vinylidene difluoride) (PVDF) membrane and blocked with 5% milk in NaCl ⁄ Pi ⁄ Tween (0.2%) A polyclonal antibody directed against the aminoterminal half of ESE-1 (East Acres Biologicals, Southbridge, MA, USA) or an anti-COX-2 Ig (Santa-Cruz, Santa Cruz, CA, USA) were used to detect the presence of these proteins A secondary antibody labeled with horseradish peroxidase (HRP) was used for detection by ECL The FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS ChIP was conducted as previously reported [21] Briefly, TC28 ⁄ a2 chondrocytes cells (2 · 107) were plated on 150-mm dishes and transfected with either pcDNA3Flag ⁄ ESE-1 or pcDNA3Flag and after 24 h stimulated with IL-1 (500 pgỈmL)1) for an additional h A 10-min formaldehyde cross-linking step was stopped by adding glycine (0.125 m, at room temperature) After two washes, the cells were resuspended in 0.3 mL lysis buffer, sonicated and then centrifuged at °C Supernatants were collected and 100 lL of chromatin preparation were aliquoted as the input fraction The remainder of the supernatants was diluted : 10 in dilution buffer for immunoclearing with sheared salmon sperm DNA, normal rabbit serum and protein A–Sepharose for h at °C Immunoprecipitation was performed overnight at °C with 80 lL M2 Agarose (anti-Flag Ig at 50% slurry in TE) (Sigma) or with lL rabbit IgG and 80 lL protein A– Sepharose as a negative control Precipitates 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enhancer pi site J Biol Chem 271, 26007–26012 Sokoloff MH, Tso CL, Kaboo R, Taneja S, Pang S, deKernion JB & Belldegrun AS (1996) In vitro modulation of tumor progression-associated properties of hormone refractory prostate carcinoma cell lines by cytokines Cancer 77, 1862–1872 1687 ... examine whether the Ets sites in the COX-2 promoter are responsive to ESE-1 and to determine whether LPS induction of the COX-2 promoter is mediated via ESE-1, we introduced mutations into individual... construct ESE-1 DN1 expression completely blocked LPS- mediated induction of the COX-2 promoter, again confirming the involvement of ESE-1 in LPS- mediated activation of the COX-2 promoter (Fig 6B) These... activate the expression of ESE-1 [31] The role of Ets factors in inflammation and in the regulation of cytokine-responsive genes has not been studied in detail However, several genes, including urokinase-type