Tài liệu Báo cáo khoa học: Liver X receptor agonists inhibit tissue factor expression in macrophages docx

Liver X receptor agonists inhibit tissue factor expression in macrophages Naoki Terasaka 1 , Ayano Hiroshima 1 , Akiko Ariga 1 , Shoko Honzumi 1 , Tadashi Koieyama 1 , Toshimori Inaba 2 and Toshihiko Fujiwara 1 1 Pharmacology and Molecular Biology Research Laboratories, Sankyo Co. Ltd, Tokyo, Japan 2 Pharmacodynamics Research Laboratories, Sankyo Co. Ltd, Tokyo, Japan Tissue factor (TF) is the cell surface glycoprotein that functions as the major cellular initiator of the coagula- tion protease cascades [1–4]. It is a high-affinity recep- tor for serine protease factors VII and VIIa. The resulting TF–factor VIIa complex provides the first catalytic event which is responsible for initiation of the coagulation protease cascades. TF-initiated thrombosis is associated with many diseases, including Gram- negative sepsis, cancer, and atherosclerosis [5–8]. Atherosclerosis is a chronic inflammatory disease as well as a disorder of lipid metabolism [9–11]. As modulators of both lipid metabolism and immune responses, macrophages play a central role in the ath- erogenic process. The accumulation of cholesterol- loaded macrophages in the arterial wall is the hallmark of early atherosclerotic lesions. TF plays an important role in the pathogenesis of thrombus formation at sites of atherosclerotic plaque disruption resulting in acute Keywords atherosclerosis; genes; lipopolysaccharide; liver X receptor; macrophage; tissue factor Correspondence N. Terasaka, Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan Fax: +81 3 5436 8566 Tel: +81 3 3492 3131 E-mail: terasa@shina.sankyo.co.jp (Received 5 November 2004, revised 17 January 2005, accepted 4 February 2005) doi:10.1111/j.1742-4658.2005.04599.x Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in throm- bus formation after plaque rupture. Macrophage TF expression is induced by pro-inflammatory stimuli including lipopolysaccharide (LPS), inter- leukin-1b and tumor necrosis factor-a. Here we demonstrate that activation of liver X receptors (LXRs) LXRa and LXRb suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP-1, NFjB, Egr-1 and Sp1, but no LXR-binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS-induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFjB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimula- ted with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease. Abbreviations ABC, ATP-binding cassette; apoE, apolipoprotein E; COX, cyclo-oxygenase; DMEM, Dulbecco’s modified Eagle’s medium; Egr-1, early growth response-1; IL, interleukin; iNOS, inducible nitric oxide synthase; KLF, Kru ¨ ppel-like factor; LDLR, low-density lipoprotein receptor; LPS, lipopolysaccharide; LPDS, lipoprotein protein-deficient serum; LXR, liver X receptor; MMP, matrix metalloproteinase; NFjB, nuclear factor-jB; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; TBST, Tween 20 Tris buffered saline; TF, tissue factor; TNFa, tumor necrosis factor-a. 1546 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS coronary events [5,12]. Physical disruption of the pla- que promoted by macrophage-derived proteases such as matrix metalloproteinases (MMPs) permits access of blood coagulation proteins to TF in the lipid-rich core [13,14]. Unlike other cofactors of the coagulation protease cascades, which circulate as nonfunctional precursors, TF is a potent initiator that is fully functional when expressed on the cell surface. Therefore, transcriptional regulation of TF would appear to be a crucial step in the control of protease cascades. TF expression and activation is increased by a variety of stimuli, such as lipopolysaccharide (LPS), oxidized low-density lipo- protein, shear stress, tumor necrosis factor-a (TNFa), interleukin (IL)-1b, and CD40 ligand [14,15]. The tran- scriptional regulation of the TF gene varies depending on the cell type and stimulus. Functional analysis of the TF promoter has identified putative AP-1, nuclear factor-jB (NFjB), Sp1 and early growth response (Egr)-1 binding sites [16]. Liver X receptors (LXRs), LXRa and LXRb, are members of the nuclear receptor superfamily and are involved in regulation of cholesterol and lipid metabo- lism [17,18]. LXRs bind to DNA as obligate hetero- dimers with retinoid X receptors (RXRs). Uptake of oxidized low-density lipoprotein by macrophages leads to increased cellular concentration of oxysterols, the natural ligands for LXRs. LXRs directly regulate the expression of ATP-binding cassette transporters ABCA1 and ABCG1, and apolipoprotein E (apoE), which mediate cellular cholesterol efflux in the pres- ence of acceptors such as high-density lipoprotein (HDL). In addition, recent studies suggest that LXRs may also inhibit inflammatory responses [19,20]. LXR agonists can suppress induction of inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2) and MMP-9. In this study, we investigated whether LXR activation inhibits inducible TF expression and activity in macrophages. Results LXR agonists inhibit TF expression induced by inflammatory stimuli in mouse peritoneal macrophages The effect of LXR activation on LPS-induced TF mRNA concentrations in mouse peritoneal macro- phages was determined by real-time quantitative PCR assays. Macrophages were pretreated with 1 lm LXR agonist T0901317 for 18 h and then stimulated with LPS (100 ngÆmL )1 ). In a time course experiment, macrophages stimulated with LPS exhibited a fivefold induction of TF mRNA, which was maximal at 2–6 h and reduced to low levels by 24 h (Fig. 1A). Preincu- bation of macrophages with T0901317 resulted in a reduction in TF mRNA concentrations (Fig. 1A). Agonists for peroxisome proliferator-activated recep- tor (PPAR)a (Wy14643), PPARc (rosiglitazone), PPARoad (GW501516) and farnesoid X receptor (GW4064) had minimal effects on TF expression at a concentration of 1 lm (Fig. 1B). The inhibitory effect of T0901317 on TF expression was dose-dependent over the concentration range 0.01–1 lm (Fig. 1C). LXR agonist GW3965, which has a different chemical structure from T0901317, also inhibited TF expression in a dose-dependent manner (Fig. 1C). We further examined the effect of LXR agonists on induction of TF mRNA by TNFa (20 ngÆmL )1 ) and IL-1b (20 ngÆmL )1 ). Pretreatment with T0901317 also reduced induction of TF mRNA by the stimuli in macrophages (Fig. 1D). LXR agonists inhibit LPS-induced expression of TNFa, but not IL-1b or IL-6, in mouse peritoneal macrophages To investigate whether LXR agonists alter LPS- induced inflammatory cytokine secretion in mouse peritoneal macrophages, we examined the effects of T0901317 and GW3965 on IL-1b, IL-6 and TNFa protein secretion after LPS stimulation. Pretreatment of macrophages for 18 h with T0901317 significantly reduced LPS-induced TNFa protein secretion in a dose-dependent manner (Fig. 2A), whereas neither LPS-induced IL-1b nor IL-6 protein secretion was affected (Fig. 2B,C). LPS-induced TNFa mRNA con- centrations were also reduced by T0901317 (Fig. 2D). LXR agonists inhibit LPS-induced TF expression and activity in human monocytes To determine whether LXR agonists had similar effects on TF expression in human cells, human mono- cytes were used for LPS stimulation experiments. The data show that induction of TF mRNA was greater in human monocytes than in mouse macrophages (Fig. 3A). Pretreatment of human monocytes for 18 h with T0901317 or GW3965 significantly reduced LPS- induced TF activity in a dose-dependent manner in the concentration range 0.01–1 lm (Fig. 3B). The effect on inhibition of TF expression was greater than that observed in mouse macrophages. LPS-induced TF activity in human monocytes was also inhibited by pre- treatment with T0901317 or GW3965 and correlated with TF mRNA concentrations (Fig. 3B). In addition, N. Terasaka etal. Repression of tissue factor in macrophages by LXR FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1547 T0901317 reduced TF protein expression, as revealed by western blot analysis (Fig. 3C). LXR agonists inhibit LPS-induced TF promoter activity The results described above suggest that LXR agonists inhibit transcription of the TF gene. However, sequence analysis of the 5¢-flanking region of the TF gene did not reveal the presence of potential LXR response elements. The TF promoter contains binding sites for AP-1, NFjB, Egr-1 and Sp1 [14]. These bind- ing sites are highly conserved in human, porcine and mouse TF genes. Then, we examined the possibility that LXR agonists antagonize the signaling pathways that induce TF expression. We investigated the effects of LXRs on a luciferase reporter containing the human TF gene promoter ()278 bp to 121 bp) (Fig. 4A). The TF promoter was transiently transfected into RAW 264.7 macrophages along with expression plasmids for LXRa and RXRa [21]. After transfection, cells were treated with LPS and ⁄ or T0901317. TF promoter activity was increased about 30-fold in response to LPS (Fig. 4A). Pretreatment with T0901317 resulted in a significant reduction in luciferase activity induced by LPS when LXRa ⁄ RXRa expression plasmids were cotransfected (Fig. 4A). Inhibition of TF promoter activation induced by LPS was also observed when Fig. 1. LXR agonists inhibit TF expression induced by inflammatory stimuli in mouse peritoneal macrophages. Thioglycolate-elicited peritoneal macrophages were obtained from C57Bl ⁄ 6 J mice. For each condition, data are represented as mean ± SEM (n ¼ 4). (A) Mouse peritoneal macrophages were pretreated with 1% Me 2 SO or 1 lM LXR agonist T0901317 for 18 h and then stimulated with LPS (100 ngÆmL )1 ) for 0.5, 1, 2, 4, 6, 8 and 24 h. TF mRNA concentrations were determined by real-time quantitative PCR assay. (B) Mouse peritoneal macrophages were pretreated with 1% Me 2 SO or 1 lM T0901317 (LXR), Wy14643 (PPARa), rosiglitazone (PPARc), GW501516 (PPARoad) or GW-3965 (farnesoid X receptor) for 18 h and then stimulated with LPS (100 ngÆmL )1 ) for 6 h. TF mRNA concentrations were determined by real-time quantitative PCR assay. (C) Mouse peritoneal macrophages were pretreated with 1% Me 2 SO or the indicated concentrations (lM)of T0901317 or GW-3965 for 18 h and then stimulated with LPS (100 ngÆmL )1 ) for 6 h. TF mRNA concentrations were determined by real-time quantitative PCR assay. (D) Mouse peritoneal macrophages were pretreated with 1% Me 2 SO or the indicated concentrations (lM)of T0901317 for 18 h and then stimulated with TNFa (20 ngÆmL )1 ) or IL-1b (20 ngÆmL )1 ) for 6 h. TF mRNA concentrations were determined by real-time quantitative PCR assay. *P<0.05, as compared with the vehicle control group using Dunnett’s multiple comparison test. Repression of tissue factor in macrophages by LXR N. Terasaka etal. 1548 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS LXRb ⁄ RXRa expression plasmids were cotransfected instead of LXRa ⁄ RXRa (Fig. 4A). The extent of inhi- bition of TF promoter activity by LXRb ⁄ RXRa cotransfection was equivalent to that by LXRa ⁄ RXRa. Next, transient reporter assays were performed using various TF gene promoter constructs (Fig. 4B). Serial deletions revealed that the T0901317-mediated inhibi- tion of luciferase activities induced by LPS was well maintained when the constructs contained the reg- ion )228 ⁄ )188, which contains AP-1-binding sites (Fig. 4B). Deletion of the region )188 ⁄ )181, which contains the NFjB-binding site, decreased T0901317- mediated inhibition of luciferase activities (Fig. 4B). To determine which binding site is important for LXR-dependent repression of TF, transient reporter assays were also performed using pGL3 ⁄ 3 · hTF- dAP1-TK-Luc or pGL3 ⁄ 3 · hTFjB-TK-Luc (Fig. 4C). Luciferase activity of these reporter plasmids was aug- mented by LPS stimulation (Fig. 4C). Activation of pGL3 ⁄ 3 · hTFjB-TK-Luc induced by LPS was sig- nificantly inhibited by T0901317 when LXRa was overexpressed. On the other hand, T0901317 did not affect activation of pGL3 ⁄ 3 · hTFdAP1-TK-Luc by LPS. These results indicate that the ability of LXRs to inhibit the TF promoter requires the NFjB-binding site. To further investigate the mechanism of inhibition of the NFjB pathway by LXR agonists, we examined DNA-binding activity of NFjB. Mouse peritoneal macrophages were preincubated with 1 lm T-0901317 Fig. 2. LXR agonists inhibit LPS-induced expression of TNFa, but not IL-1b or IL-6, in mouse peritoneal macrophages. Thioglyco- late-elicited peritoneal macrophages were obtained from C57Bl ⁄ 6J mice. For each condition, data are represented as mean ± SEM (n ¼ 3). Mouse peritoneal macropha- ges were pretreated with 1% Me 2 SO or the indicated concentrations (l M) of LXR agon- ists T0901317 or GW3965 for 18 h and then stimulated with LPS (100 ngÆmL )1 )for6h. (A) IL-1a, (B) IL-6 and (C) TNFa protein con- centrations in culture medium were deter- mined as described in Experimental procedures. (D) TNFa mRNA concentrations were determined by real-time quantitative PCR assay. *P<0.05, as compared with the vehicle control group using Dunnett’s multiple comparison test. AB C Fig. 3. LXR agonists inhibit LPS-induced TF expression and activity in human mono- cytes. Human monocytes were pretreated with 1% Me 2 SO or the indicated concentra- tions (l M) of T0901317 or GW-3965 for 18 h and then stimulated with LPS (100 ngÆmL )1 ) for 6 h. For each condition, data are repre- sented as mean ± SEM. (n ¼ 3). (A) TF mRNA concentrations were determined by real-time quantitative PCR assay. (B) TF activity was determined using a standard chromogenic assay. (C) TF protein expres- sion was analyzed by western blotting. *P<0.05, as compared with the vehicle control group using Dunnett’s multiple comparison test. N. Terasaka etal. Repression of tissue factor in macrophages by LXR FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1549 or GW3965 and then activated with LPS (100 ngÆmL )1 ). After 6 h of LPS stimulation, nuclear extracts were collected and then DNA-binding activity of NFjB p50 and p65 was assessed. There were no sig- nificant changes exhibited in the protein–DNA com- plexes induced by LPS in the presence of LXR agonists (Fig. 5). LXR agonists inhibit TF expression induced by LPS stimulation To address whether the LXR pathway also functions to modulate TF gene expression in vivo, we first inves- tigated the effect of administration of LXR agonists on induction of TF expression by LPS stimulation in C57Bl ⁄ 6 mice. LPS at 4 mgÆkg )1 induced TF mRNA 3.5-fold in kidney and 2.2-fold in lung 6 h after injec- tion (Fig. 6). Administration of T0901317 at 3 mgÆkg )1 or GW3965 at 30 mgÆkg )1 significantly reduced induc- tion of TF mRNA by LPS, but did not affect baseline expression (Fig. 6). LXR agonists inhibit TF expression in atherosclerotic lesions We previously observed that T0901317 augmented ABCA1 expression in atherosclerotic lesions and resul- ted in the prevention of lesion progression in LDLR – ⁄ – mice [22]. Next, we investigated the effect of T0901317 on expression of TF mRNA within the atherosclerotic lesion in LDLR – ⁄ – mice. T0901317 significantly redu- ced TF mRNA concentrations in the atherosclerotic lesion in a dose-dependent manner (Fig. 7A). On the other hand, ABCA1 mRNA concentrations in the atherosclerotic lesion were increased by T0901317 (Fig. 7B), consistent with previous work [22], evaluated by immunohistochemical analysis. Discussion LXRs are members of the nuclear receptor superfamily and are highly expressed in macrophages. They play a crucial role in the cholesterol efflux pathway through Fig. 4. LXR agonists inhibit LPS-induced TF promoter activity. For each condition, data are represented as mean ± SEM. (n ¼ 4). (A) RAW 264.7 cells were transiently transfected with TF promoter construct (pGL3 ⁄ )278+121hTF-Luc) with or without pCMX, pCMX-LXRa, pCMX- LXRb, pCMX-RXRa and pRL-CMV as described in Experimental procedures. After transfection, RAW 264.7 cells were incubated with 1% Me 2 SO or 1 lM T-0901317 for 12 h in DMEM containing 10% LPDS. Cells were then stimulated with 100 ngÆmL )1 LPS for 18 h. (B) RAW 264.7 cells were transiently transfected with TF promoter constructs (pGL3 ⁄ )228+121hTF-Luc, pGL3 ⁄ )211+121hTF-Luc, pGL3 ⁄ )188+121hTF-Luc and pGL3 ⁄ )181+121hTF-Luc) with or without pCMX-LXRa, pCMX-RXRa and pRL-CMV, respectively, as described in Experimental procedures. (C) RAW 264.7 cells were transiently transfected with promoter reporter constructs (pGL3 ⁄ 3 · hTFdAP1-TK-Luc or pGL3 ⁄ 3 · hTFjB-TK-Luc) with or without pCMX-LXRa, pCMX-RXRa and pRL-CMV, respectively, as described in Experimental procedures. Luciferase activity was normalized to Renilla luciferase activities. *P<0.05, as compared with the vehicle control group using Dunnett’s multiple comparison test. Repression of tissue factor in macrophages by LXR N. Terasaka etal. 1550 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS the regulation of target genes, including ABCA1, ABCG1 and apoE [23–26]. Recently we [22] and Joseph et al. [27] demonstrated that synthetic LXR agonists inhibit development of atherosclerotic lesion area in LDLR – ⁄ – and apoE – ⁄ – mice without major changes in plasma lipid concentrations. In addition, Tangirala et al. [28] reported that LXRs in macro- phages play a protective role in the development of atherosclerosis in a bone marrow transplantation study using LXR-deficient mice. In this study, we focused on the molecular mechanism of the anti-atherosclerotic effect of LXR agonists. We identified TF as a novel LXR target gene in macrophages. We also demonstra- ted that activation of LXRs in macrophages inhibits expression of the TF gene in atherosclerotic lesions in mice. TF is abundantly expressed in macrophages and the lipid-rich core in atherosclerotic plaques [5,29]. In chronic atherosclerosis, macrophages appear to be the major source of TF within the plaque. Macrophages accumulate lipid, becoming foam cells, and finally degenerating into a lipid core. Plaque rupture exposes active TF in the lipid core to circulating blood, trigger- ing thrombosis. As the initiator of coagulation, TF is a potential target for inhibiting the thrombotic compli- cations of atherosclerosis. Another study revealed that deficiency of TFPI, an intrinsic TF inhibitor, reduced atherosclerosis and thrombosis in apoE – ⁄ – mice [30]. In addition, the absence of Egr-1, which is an important regulator of TF expression, ameliorated progression of atherosclerosis in apoE – ⁄ – mice [31]. These observa- tions support the notion that TF has a crucial role in the pathogenesis of atherosclerosis. However, the avail- ability of synthetic TF inhibitors as therapeutic agents has still not been reported. Our current data indicate that LXR agonists would be useful as suppressants of TF as well as activators of reverse cholesterol transport. Macrophages and foam cells secrete a number of inflammatory mediators that increase inflammation in the vessel wall and contribute to additional leukocyte accumulation and smooth muscle cell proliferation. Fig. 5. LXR agonists do not affect NFjB binding to DNA induced by LPS. Thioglycolate-elicited peritoneal macrophages were obtained from C57Bl ⁄ 6J mice. For each condition, data are represented as mean ± SEM. (n ¼ 3). Mouse peritoneal macrophages were pre- treated with 1% Me 2 SO or 1 lM T0901317 or GW-3965 for 18 h and then stimulated with LPS (100 ngÆmL )1 ) for 6 h. Nuclear extracts were prepared from peritoneal macrophages using a com- mercial kit. DNA-binding activity of NFjB p50 (A) and p65 (B) was assessed as described in Experimental procedures. Fig. 6. LXR agonists inhibit TF expression induced by LPS stimulation. LXR agonist T-0901317 at 3 mgÆkg )1 or GW-3965 at 30 mgÆkg )1 was orally administered daily to male C57Bl ⁄ 6J mice for 7 days (n ¼ 5 per group). The day after the last administration of LXR agonists, LPS at 4mgÆkg )1 was intraperitoneally administered to C57Bl ⁄ 6 mice, and then mice were killed 6 h later. TF mRNA concentrations in kidney and lung were determined by real-time quantitative PCR assay. N. Terasaka etal. Repression of tissue factor in macrophages by LXR FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1551 Thus, inflammation plays a central role in the incep- tion and progression of atherosclerosis as well as accu- mulation of lipid. Recently, LXR agonists have been reported to repress expression of inflammatory genes such as iNOS, COX-2 and MMP-9 in macrophages [19,20]. In this study, we also found that LXR agonists inhibit LPS-induced expression of TNFa in macro- phages. Our observation also supports the notion that LXRs play a role in the regulation of the macrophage inflammatory response. Activated NFjB can be detected in atherosclerotic lesions, mainly within macrophages, whereas poor NFjB activation is present in healthy vessels [32]. LXR agonists appear to directly downregulate the enhanced expression of inflammatory genes including TF during the development of atherosclerosis induced by the acti- vation of NFjB. The current data suggest that LXRs allow binding of NFjB to the promoter, but prevent functional activation of NFjB. Several models might explain the inhibitory effect of LXR agonists on func- tional NFjB activity. It is known that transcriptional activation by nuclear receptors involves at least two separate processes: derepression and activation [33]. Ligand binding triggers dissociation of corepressors and recruitment of coactivators. Coactivators bridge transcription factors including not only nuclear recep- tors, but also CERB, STATs, bHLH factors, AP-1, NFjB and the components of basal transcriptional machinery. One possible explanation for the ligand- dependent transcriptional repression is that LXRs com- pete with NFjB for limited amounts of coactivators. Indeed, LXRs and NFjB appear to recruit the same coactivators, such as steroid receptor coactivator-1 (SRC-1) and activating signaling cointegrator-2 [34–39]. Another possible mechanism is that a direct protein– protein interaction between LXR and NFjBor between LXR and another transcription factor such as Foxo1, a winged helix transcription factor, affects coactivator recruitment to NFjB. Delerive et al. [40] demonstrated that PPARa physically interacts with the NFjB p65 subunit. PPARa agonists are also reported to inhibit TF expression induced by LPS in macro- phages [41,42]. Alternatively, a recent report by Dowell et al. [43] revealed that PPARc interacts directly with Foxo1 and inhibits each transcriptional activity in a ligand-dependent manner. Although the mechanism of PPARc-dependent repression remains to be elucidated, PPARc agonists can also inhibit iNOS and COX-2 induction by LPS in macrophages as well as LXR agonists [44]. Further studies focusing on coactivator recruitment are needed to clarify the molecular basis of inhibition of NFjB activity by LXR. TF expression is also downregulated by all-trans- retinoic acid, which is a ligand for a nuclear receptor family known as the retinoic acid receptors (RARs) [45–47]. In RAR activation, unlike LXR, the inhibitory effect appears to be independent of the NFjB or AP-1 pathway. Whereas LXR and PPARa agonists inhibit LPS-induced TNFa, no such inhibition by RAR agon- ists was observed [45]. In addition, whereas LXR or PPARa agonists do not affect basal TF expression, RAR agonists inhibit both basal and LPS-induced TF expression [45]. These data suggest that RAR activation affects other nuclear factors in the transcription complex required for TF expression. The zinc finger transcription factor Egr-1 is also known to be a key player in TF expression [16]. Shindo et al. [48] showed that a RAR agonist reduces platelet-derived growth factor-A Fig. 7. LXR agonists inhibit TF expression in atherosclerotic lesions. Male LDLR – ⁄ – mice were fed an atherogenic diet (1.25% choles- terol, 7.5% cocoa butter and 0.5% sodium cholate; Oriental Yeast, Tokyo, Japan). T-0901317 at doses of 3 or 10 mgÆkg )1 was orally administered to LDLR – ⁄ – mice daily for 8 weeks (n ¼ 9 per group). (A) TF and (B) ABCA1 mRNA concentrations in atherosclerotic lesions were determined by real-time quantitative PCR assay. *P<0.05, as compared with the vehicle control group using Dun- nett’s multiple comparison test. Repression of tissue factor in macrophages by LXR N. Terasaka etal. 1552 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS promoter activity via interaction with the transcription factor Kru ¨ ppel-like factor 5 (KLF5), which is a target gene of Egr-1. Furthermore, both KLF and Egr-1 tran- scription factors appear to bind to the GC-rich binding site of the platelet-derived growth factor-A promoter [49]. Taken together, these findings suggest that suppres- sion of TF expression by RAR is dominantly regulated by the Egr-1 ⁄ KLF pathway, and not the NFjB or AP-1 pathway. These distinct mediations by nuclear receptors provide clues to the elucidation of the cell-type-specific manner of TF expression. Finally, this study demonstrates that LXR agonists antagonize inflammatory stimuli-induced TF expression by inhibiting NFjB activity. Previous studies indicate that LXR agonists strongly induce cholesterol efflux by upregulation of ABCA1, ABCG1, and apoE expression [22,27]. In addition, LXR agonists can suppress iNOS, COX-2, MMP-9 and TNFa expression [19,20]. Taking these observations together, the anti-atherosclerotic effects of LXR agonists appear to be due to enhance- ment of reverse cholesterol transport, anti-inflammation and anti-thrombosis. Various mechanisms of action of LXR agonists can be suggested from the function of LXRs as positive, as well as negative, regulators of tar- get genes. LXR agonists would be useful therapeutic agents for the treatment of cardiovascular diseases. Experimental procedures Reagents LXR agonists T0901317 and GW3965, farnesoid X recep- tor agonist GW4064, peroxisome proliferator-activated receptor-d (PPAR-d) agonist GW501516, and PPARc agon- ist rosiglitazone were synthesized by Sankyo Co., Ltd. PPARa agonist Wy14643 was purchased from Cayman Chemical (Ann Arbor, MI, USA). 9-cis-Retinoic acid was obtained from Sigma Chemical (St. Louis, MO, USA). Agonists were dissolved in Me 2 SO before use in cell culture. LPS from Salmonella typhimurium was purchased from Sigma Chemical. Mouse TNFa and IL-1b were from Bio- source International (Camarillo, CA, USA). Plasmids pCMX expression plasmids for LXRa, LXR b and RXRa have been described previously [21]. The 5¢-flanking region of the human TF gene ()278 ⁄ +121) was prepared by PCR using human genomic DNA (Roche Applied Science, India- napolis, IN, USA) as a template and a forward primer tailed with a KpnI restriction site (5¢-AAGGTACCAACCCACCT AAGCTGCACGT-3¢) and a reverse primer tailed with BglII (5¢-GAAGATCTATGTCTACCAGTTGGCGGCGA-3¢). The PCR product was digested with KpnI and BglII and sub- cloned into the KpnI ⁄ BglII-digested luciferase reporter plasmid pGL3-basic (Promega, Madison, WI, USA), gener- ating pGL3 ⁄ )278+121hTF-Luc. For the construction of pGL3 ⁄ )228+121hTF-Luc, pGL3 ⁄ )211+121hTF-Luc, pGL3 ⁄ )188+121hTF-Luc and pGL3 ⁄ )181+121hTF-Luc, the PCR products amplified by the reverse primer and a for- ward primer tailed with a KpnI restriction site (5¢-AAGG TACCGGTTGAATCACCTGGGGT-3¢)(5¢-AAGGTACC TGAGTCATCCCTTGCAGGGT-3¢)(5¢-AAGGTACCGG AGTTTCCTACCGGGAGGA-3¢) and (5¢-AAGGTACCT ACCGGGAGGAGGCGG-3¢), respectively, was subcloned into the pGL3-basic. For generation of pGL3 ⁄ 3 · hTF- dAP1-TK-Luc and pGL3 ⁄ 3 · hTFjB-TK-Luc, oligonucleo- tides encoding three copies of the downstream AP-1 site (5¢-GGGTGAGTCATCC-3¢) and the NFjB site (5¢-CCC GGAGTTTCCTA-3¢), respectively, on the 5¢-flanking region of TF was subcloned into the HindIII and SalI sites of TK-luc. Cell culture and transfection Peritoneal macrophages were obtained from thioglycolate- injected C57Bl ⁄ 6J mice as described previously [22]. Cells (4 · 10 5 ) were plated on 24-well plates and cultured in Dul- becco’s modified Eagle’s medium (DMEM) supplemented with 10% lipoprotein protein-deficient serum (LPDS) (Sig- ma Chemical). Human monocytes were isolated from lym- phocyte preparations obtained from freshly drawn blood of healthy volunteers. Lymphocyte preparations were diluted with an equal volume of NaCl ⁄ P i . This mixture was under- layered with Ficoll-Paque (Amersham Biosciences, Piscat- away, NJ, USA) and centrifuged at 500 g for 10 min. The lymphocytes were then harvested from the interface. The lymphocytes were washed twice with NaCl ⁄ P i and resus- pended in RPMI 1640 medium containing 10% LPDS (1 · 10 6 cellsÆmL )1 ). Monocytes were isolated from lympho- cytes by adherence (4 h at 37 °C). RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum. For ligand treatment, cells were cultured in DMEM supplemen- ted with 10% LPDS and receptor ligands for 18 h before LPS or cytokine stimulation. Transient transfection of RAW 264.7 cells was performed in triplicate in 48-well plates. Cells (2 · 10 5 ) were transfected for 6 h with reporter plasmid (100 ng per well), receptor plasmids (50 ng per well) and pRL-CMV (50 ng per well) as internal control using OptiMEM medium and LipofectaminePlus reagent (Invitro- gen, Carlsbad, CA, USA). After transfection, cells were incubated in medium containing 10% LPDS and the indica- ted ligands or vehicle for 12 h before stimulation with LPS for another 18 h. Firefly and Renilla luciferase activities were measured in a luminometer, Analyst TM HT (Molecular Devices, Atlanta, GA, USA) using the Dual-Luciferase Reporter Assay System (Promega). N. Terasaka etal. Repression of tissue factor in macrophages by LXR FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1553 Western blot analysis Cell lysates (50 lg per lane) were separated by SDS ⁄ PAGE using a 12% separating gel and transferred to an Immobi- lon TM -P membrane (Millipore, Bedford, MA, USA) for immunoblotting. The blot was blocked overnight at 4 °Cin 0.01% Tween 20 Tris buffered saline (TBST) containing 5% nonfat milk, incubated with goat antibody to human TF (American Diagnostica, Greenwich, CT, USA) for 1 h at room temperature, and washed with TBST. The blot was then incubated with horseradish peroxidase-conjugated sec- ondary antibody, washed in TBST, and proteins were detected by ECL (enhanced chemoluminescence) (Amer- sham Biosciences). Assay of TF activity TF activity was determined using a standard chromogenic assay kit ActichromeÒ TF (American Diagnostica). RNA analysis Total RNA was extracted using RNeasyÒ mini kit (Qiagen, Valencia, CA, USA). Real-time quantitative PCR (TaqMan) assay was performed using an Applied Biosystems 7700 sequence detector as described [22]. The sequences of forward primers, reverse primers and TaqMan probes, respectively, were as follows: mouse TF: 5¢-GCTCTCAGGTGGGATG CAG-3¢,5¢-GGCTCGTCCAGAATGACAAC-3¢,5¢-FAM- CTTGGCCTTCGTGGGTGGATCC-TAMRA-3¢; human TF: 5¢-CCCGTCAATCAAGTCTA CAC-3¢,5¢-GTCTGCT TCACATCCTTCAC-3¢,5¢ -FAM-TACACAACAGACAC AGAGTGTGACCTCACC-TAMRA-3¢; mouse TNFa:5¢- CGGAGTCCGGGCAGGT-3¢,5¢-GCTGGGTAGAGAAT GGATGAACA-3¢,5¢-FAM-ACTTTGGAGTCATTGCTCT GTGAAGGG-TAMRA-3¢; cyclophilin: 5¢-CGATGACGAG CCCTTGG-3¢,5¢-TCTGCTGTCTTTGGAACTTTGTC-3¢, 5¢-FAM-CGCGTCTCCTTTGAGCTGTTTGCA-TAMRA- 3¢. All assays were performed in duplicate, and cycle thresholds of individual genes were normalized to that of cyclophilin. Determination of cytokine concentrations Concentrations of IL-1b, IL-6 and TNFa were determined by the Bio-Plex Cytokine Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Nuclear NFjB p50 and p65 activity Nuclear extracts were prepared from peritoneal macro- phages using a nuclear extraction kit (Transfactor Extraction Kit; BD Biosciences Clontech Laboratories, Palo Alto, CA, USA) following the manufacturer’s instructions. Briefly, DNA-binding activity of NFjB p50 and p65 was assessed in nuclear extracts using an ELISA-based format (BD Mercury NFjB p50 and p65 Transfactor Kits; BD Biosciences Clon- tech Laboratories) following the manufacturer’s instructions. Animals Male C57Bl ⁄ 6J mice were obtained from Charles River Japan. Vehicle [propylene glycol ⁄ Tween 80 (4 : 1, v ⁄ v)] or LXR agonists T-0901317 at 3 mgÆkg )1 or GW-3965 at 30 mgÆkg )1 was orally administered daily to the mice for 7 days (n ¼ 5 per group). The day after the last administra- tion of LXR agonist, saline or LPS at 4 mgÆkg )1 was intra- peritoneally administered to C57Bl ⁄ 6 mice at 9 : 00 a.m. The mice were anaesthetized with ethyl ether and killed at 3 : 00 p.m. (6 h after LPS administration). Blood was obtained from the abdominal vein, and tissues were rapidly removed and snap-frozen in liquid nitrogen for further ana- lysis. Male low-density lipoprotein receptor (LDLR) – ⁄ – mice were obtained from Charles River Japan. LDLR – ⁄ – mice were fed an atherogenic diet (1.25% cholesterol, 7.5% cocoa butter and 0.5% sodium cholate). Vehicle or T-0901317 at doses of 3 or 10 mgÆkg )1 was orally administered daily for 8 weeks (n ¼ 9 per group), and the tissues were obtained as previously described in [22]. For gene expression analysis, atherosclerotic lesions from aortic arch were visualized using a stereo-microscope (Leica MZ12) and harvested using a sterile scalpel to ensure the material collected contained only atherosclerotic lesion and not underlying aortic tissue. All animal care and experimental procedures complied with the Sankyo Animal Care and Use Committee. Statistical analysis Significant difference from the vehicle control was assessed using Dunnett’s multiple comparison test. A difference was considered to be significant when the P value was less than 0.05. Acknowledgements We thank the Medicinal Chemistry Research Laborat- ories at Sankyo for synthesizing ligands, Drs Richard Heyman and Raju Mohan at X-Ceptor Therapeutics for providing LXRs and RXR expression plasmids, and Drs Hiroyuki Koike, Hiroaki Yanagisawa, Teii- chiro Koga and Jun Ohsumi at Sankyo for critical reading and helpful discussion. References 1 Bach RR (1988) Inhibition of coagulation by tissue factor. CRC Crit Rev Biochem 23, 339–368. Repression of tissue factor in macrophages by LXR N. Terasaka etal. 1554 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 2 Harlos K, Martin DM, O’Brien DP, Jones EY, Stuart DI, Polikarpov I, Miller A, Tuddenham EG & Boys CW (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370, 662–665. 3 Rapaport SI & Rao LV (1995) The tissue factor path- way: how it has become a ‘prima ballerina’. Thromb Haemost 74, 7–17. 4 Camerer E, Kolsto AB & Prydz H (1996) Cell biology of tissue factor, the principal initiator of blood coagula- tion. Thromb Res 81, 1–41. 5 Wilcox JN, Smith KM, Schwartz SM & Gordon D (1989) Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 86, 2839–2843. 6 Weinberg JB, Pippen AM & Greenberg CS (1991) Extravascular fibrin formation and dissolution in syno- vial tissue of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum 34, 996–1005. 7 Osterud B & Flaestad T (1983) Increased tissue throm- boplastin activity in monocytes of patients with menin- gococcal infection: related to an unfavorable prognosis. Thromb Haemost 49, 5–7. 8 Dvorak HF (1987) Thrombosis and cancer. Hum Pathol 18, 275–284. 9 Libby P (2002) Inflammation in atherosclerosis. Nature 420, 868–874. 10 Glass CK & Witztum JL (2001) Atherosclerosis: the road ahead. Cell 104, 503–516. 11 Li AC & Glass CK (2002) The macrophage foam cell as a target for therapeutic intervention. Nat Med 8, 1235– 1242. 12 Moreno PR, Bernardi VH, Lopez-Cuellar J, Murcia AM, Palacios IF, Gold HK, Mehran R., Sharma SK, Nemerson Y, Fuster V & Fallon JT (1996) Macro- phages, smooth muscle cells, and tissue factor in unstable angina. Implications for cell-mediated throm- bogenicity in acute coronary syndromes. Circulation 94, 3090–3097. 13 Galis ZS, Sukhova GK, Kranzhofer R., Clark S & Libby P (1995) Macrophage foam cells from experi- mental atheroma constitutively produce matrix-degra- ding proteinases. Proc Natl Acad Sci USA 92, 402–406. 14 Mackman N (1995) Regulation of the tissue factor gene. FASEB J 9, 883–889. 15 Mach F, Schonbeck U, Bonnefoy JY, Pober JS & Libby P (1997) Activation of monocyte ⁄ macrophage functions related to acute atheroma complication by ligation of CD40. Circulation 96, 396–399. 16 Mackman N (1997) Regulation of the tissue factor gene. Thromb Haemost 78, 747–754. 17 Janowski BA, Willy PJ, Devi TR, Falck JR & Mangels- dorf DJ (1996) An oxysterol signalling pathway mediated by the nuclear receptor LXRa. Nature 383, 728–731. 18 Repa JJ & Mangelsdorf DJ (1999) Nuclear receptor reg- ulation of cholesterol and bile acid metabolism. Curr Opin Biotechnol 10, 557–563. 19 Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ & Tontonoz P (2003) Reciprocal regulation of inflamma- tion and lipid metabolism by liver X receptors. Nat Med 9, 213–219. 20 Castrillo A, Joseph SB, Marathe C, Mangelsdorf DJ & Tontonoz P (2003) Liver X receptor-dependent repres- sion of matrix metalloproteinase-9 expression in macro- phages. J Biol Chem 278, 10443–10449. 21 Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ & Tontonoz P (2001) LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci USA 98, 507–512. 22 Terasaka N, Hiroshima A, Koieyama T, Ubukata N, Morikawa Y, Nakai D & Inaba T (2003) T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice. FEBS Lett 536, 6–11. 23 Costet P, Luo Y, Wang N & Tall AR (2000) Sterol- dependent transactivation of the ABC1 promoter by the liver X receptor ⁄ retinoid X receptor. J Biol Chem 275, 28240–28245. 24 Repa JJ, Turley SD, Lobaccaro J, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM & Mangelsdorf DJ (2000) Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR hetero- dimers. Science 289, 1524–1529. 25 Venkateswaran A, Repa JJ, Lobaccaro JM, Bronson A, Mangelsdorf DJ & Edwards PA (2000) Human white ⁄ murine ABC8 mRNA levels are highly induced in lipid- loaded macrophages. J Biol Chem 275, 14700–14707. 26 Wang N, Lan D, Chen W, Matsuura F & Tall AR (2004) ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipo- proteins. Proc Natl Acad Sci USA 26, 9774–9779. 27 Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, Chen M, Noh G, Goodman J, Hagger GN, Tran J, Tippin TK, Wang X, Lusis AJ, Hsueh WA, Law RE, Collins JL, Willson TM & Tontonoz P (2002) Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci USA 99, 7604–7609. 28 Tangirala RK, Bischoff ED, Joseph SB, Wagner BL, Walczak R., Laffitte BA, Daige CL, Thomas D, Hey- man RA, Mangelsdorf DJ, Wang X, Lusis AJ, Tonto- noz P & Schulman IG (2002) Identification of macrophage liver X receptors as inhibitors of athero- sclerosis. Proc Natl Acad Sci USA 99, 11896–11901. 29 Libby P (1995) Molecular bases of the acute coronary syndromes. Circulation 91, 2844–2850. 30 Westrick RJ, Bodary PF, Xu Z, Shen YC, Broze GJ & Eitzman DT (2001) Deficiency of tissue factor pathway N. Terasaka etal. Repression of tissue factor in macrophages by LXR FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1555 [...]... 41 42 43 44 45 46 47 48 49 vascular in ammatory gene response by negative crosstalk with transcription factors NF-kappaB and AP-1 J Biol Chem 274, 32048–32054 Neve BP, Corseaux D, Chinetti G, Zawadzki C, Fruchart JC, Duriez P, Staels B & Jude B (2001) PPARalpha agonists inhibit tissue factor expression in human monocytes and macrophages Circulation 103, 207–212 Marx N, Mackman N, Schonbeck U, Yilmaz... (2001) PPARalpha activators inhibit tissue factor expression and activity in human monocytes Circulation 103, 213–219 Dowell P, Otto TC, Adi S & Lane MD (2003) Convergence of peroxisome proliferator-activated receptor gamma and Foxo1 signaling pathways J Biol Chem 278, 45485–45491 Ricote M, Li AC, Willson TM, Kelly CJ & Glass CK (1999) The peroxisome proliferator-activated receptorgamma is a negative... macrophage activation Nature 391, 79–82 Oeth P, Yao J, Fan ST & Mackman N (1998) Retinoic acid selectively inhibits lipopolysaccharide induction of tissue factor gene expression in human monocytes Blood 8, 2857–2865 Conese M, Montemurro P, Fumarulo R., Giordano D, Riccardi S, Colucci M & Semeraro N (1991) Inhibitory effect of retinoids on the generation of procoagulant activity by blood mononuclear phagocytes...Repression of tissue factor in macrophages by LXR 31 32 33 34 35 36 37 38 39 40 inhibitor promotes atherosclerosis and thrombosis in mice Circulation 103, 3044–3046 Harja E, Bucciarelli LG, Lu Y, Stern DM, Zou YS, Schmidt AM & Yan SF (2004) Early growth response-1 promotes atherogenesis: mice deficient in early growth response-1 and apolipoprotein E display decreased atherosclerosis and vascular in ammation... by liver X receptors in mice Mol Cell Biol 23, 3583–3592 Huuskonen J, Fielding PE & Fielding CJ (2004) Role of p160 coactivator complex in the activation of liver X receptor Arterioscler Thromb Vasc Biol 24, 703–708 Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G & Staels B (2001) Peroxisome proliferatoractivated receptor alpha negatively... Activated transcription factor nuclear factor- kappa B is present in the atherosclerotic lesion J Clin Invest 97, 1715–1722 Rosenfeld MG & Glass CK (2001) Coregulator codes of transcriptional regulation by nuclear receptors J Biol Chem 276, 36865–36868 Na SY, Lee SK, Han SJ, Choi HS, Im SY & Lee JW (1998) Steroid receptor coactivator-1 interacts with the p50 subunit and coactivates nuclear factor kappaBmediated... Kruppel-like zinc-finger transcription ¨ factor KLF5 ⁄ BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling Nat Med 8, 856–863 Aizawa K, Suzuki T, Kada N, Ishihara A, KawaiKowase K, Matsumura T, Sasaki K, Munemasa Y, Manabe I, Kurabayashi M, Collins T & Nagai R (2004) Regulation of platelet-derived growth factor- A chain by Kruppel-like factor 5 J Biol... activity by blood mononuclear phagocytes Thromb Haemost 66, 662–665 Barstad RM, Hamers MJ, Stephens RW & Sakariassen KS (1995) Retinoic acid reduces induction of monocyte tissue factor and tissue factor ⁄ factor VIIa-dependent arterial thrombus formation Blood 86, 212–218 Shindo T, Manabe I, Fukushima Y, Tobe K, Aizawa K, Miyamoto S, Kawai-Kowase K, Moriyama N, Imai Y, Kawakami H, Nishimatsu H, Ishikawa... Endocrinol 14, 915–925 Gan X, Kaplan R., Menke JG, MacNaul K, Chen Y, Sparrow CP, Zhou G, Wright SD & Cai TQ (2001) Dual mechanisms of ABCA1 regulation by geranylgeranyl pyrophosphate J Biol Chem 276, 48702–48708 Kim SW, Park K, Kwak E, Choi E, Lee S, Ham J, Kang H, Kim JM, Hwang SY, Kong YY, Lee K & Lee JW (2003) Activating signal cointegrator 2 required for liver lipid metabolism mediated by liver X receptors... Sheppard KA, Rose DW, Haque ZK, Kurokawa R., McInerney E, Westin S, Thanos D, Rosenfeld MG, Glass CK & Collins T (1999) Transcriptional activation by NF-kappaB requires multiple coactivators Mol Cell Biol 19, 6367–6378 Lee SK, Na SY, Jung SY, Choi JE, Jhun BH, Cheong J, Meltzer PS, Lee YC & Lee JW (2000) Activating protein-1, nuclear factor- kappaB, and serum response factor as novel target molecules of the . and activity in macrophages. Results LXR agonists inhibit TF expression induced by in ammatory stimuli in mouse peritoneal macrophages The effect of LXR activation. 2D). LXR agonists inhibit LPS-induced TF expression and activity in human monocytes To determine whether LXR agonists had similar effects on TF expression in