Tamoxifen inhibits macrophage FABP4 expression ...

3 downloads 0 Views 1MB Size Report
5 Uysal, K. T., Scheja, L., Wiesbrock, S. M., Bonner-Weir, S. and Hotamisligil, .... 41 Dewar, J. A., Horobin, J. M., Preece, P. E., Tavendale, R., Tunstall-Pedoe, ...
Biochem. J. (2013) 454, 467–477 (Printed in Great Britain)

467

doi:10.1042/BJ20130580

*The State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 30071, China, †The College of Life Sciences, Nankai University, Tianjin 30071, China, ‡The Tianjin University of Traditional Chinese Medicine, Tianjin 30071, China, §Weill Medical College of Cornell University, New York, NY 10067, U.S.A., and Collaborative Innovation Center of Biotherapy, Nankai University, Tianjin 30071, China

Macrophage adipocyte fatty acid-binding protein (FABP4) plays an important role in foam cell formation and development of atherosclerosis. Tamoxifen inhibits this disease process. In the present study, we determined whether the anti-atherogenic property of tamoxifen was related to its inhibition of macrophage FABP4 expression. We initially observed that tamoxifen inhibited macrophage/foam cell formation, but the inhibition was attenuated when FABP4 expression was selectively inhibited by siRNA. We then observed that tamoxifen and 4-hydroxytamoxifen inhibited FABP4 protein expression in primary macrophages isolated from both the male and female wild-type mice, suggesting that the inhibition is sex-independent. Tamoxifen and 4-hydroxytamoxifen inhibited macrophage FABP4 protein expression induced either by activation of GR (glucocorticoid receptor) or PPARγ (peroxisome-proliferator-activated receptor γ ). Associated with the decreased protein expression, Fabp4 mRNA expression and promoter activity were also inhibited by tamoxifen and 4-hydroxytamoxifen, indicating transcriptional

regulation. Analysis of promoter activity and EMSA/ChIP assays indicated that tamoxifen and 4-hydroxytamoxifen activated the nGRE (negative glucocorticoid regulatory element), but inhibited the PPRE (PPARγ regulatory element) in the Fabp4 gene. In vivo, administration of tamoxifen to ApoE (apolipoprotein E)-deficient (apoE − / − ) mice on a high-fat diet decreased FABP4 expression in macrophages and adipose tissues as well as circulating FABP4 levels. Tamoxifen also inhibited FABP4 protein expression by human blood monocyte-derived macrophages. Taken together, the results of the present study show that tamoxifen inhibited FABP4 expression through the combined effects of GR and PPARγ signalling pathways. Our findings suggest that the inhibition of macrophage FABP4 expression can be attributed to the antiatherogenic properties of tamoxifen.

INTRODUCTION

isobutylmethylxanthine. The combination eventually induces expression of PPARγ (peroxisome-proliferator-activated receptor γ ), a ligand-activated transcription factor. PPARγ alone or in co-operation with C/EBPα (CCAAT/enhancer-binding protein α) induces expression of adipocyte-related molecules, including FABP4, and maintains the adipocyte phenotype [6]. At the molecular level, regulatory elements responding to activation of GR (glucocorticoid receptor) and PPARγ [pGRE (positive glucocorticoid regulatory element) and PPRE (PPARγ regulatory element)] exist [2]. Therefore both GR and PPARγ synthetic ligands, such as dexamethasone and TZDs (thiazolidinediones), can induce FABP4 expression in different cell types, including macrophages. In addition, our recent study shows a nGRE (negative GRE) in the Fabp4 promoter [7]. Statins, a class of HMG-CoA (3-hydroxy-3-methylglutarylCoA) reductase inhibitors which are used in treatment of hypercholesterolaemia patients, also possess non-cholesterollowering pleiotropic effects. Although statins alone reduce FABP4 expression [8], they synergize dexamethasone-induced macrophage FABP4 expression by inactivating the nGRE [7]. FABP4 is highly expressed in macrophages, suggesting the implication of FABP4 in macrophage/foam cell formation and the development of atherosclerosis. In vitro, overexpressing FABP4 facilitates foam cell formation in human THP-1

FABPs (fatty acid-binding proteins) are a family of proteins with a small molecular mass and a high affinity for fatty acids [1]. FABPs facilitate the intracellular transport of hydrophobic fatty acids which are required to serve as an energy source and metabolic signals in cell growth/survival and inflammatory responses by activating enzymatic or transcriptional networks. On the basis of the expression pattern, FABPs can be classified as liver, intestinal, heart, adipocyte, epidermal, ileal, brain, myelin and testis FABP [2]. The adipocyte FABP (FABP4, A-FABP or aP2) is the bestcharacterized isoform among the entire FABP family. It was originally identified in adipose tissues and mature adipocytes [3]. In addition to fatty acid transport, FABP4 also plays an important role in other biological processes, particularly in many aspects of metabolic syndrome. Deficiency of FABP4 expression protects mice from hyperinsulinaemia and insulin resistance, and improves systemic glucose and lipid metabolism in the context of dietary or genetic obesity [4,5]. Expression of FABP4 can be regulated at both cellular and molecular levels [2]. During adipocyte differentiation, FABP4 is highly induced and regarded as a marker of terminal cell differentiation. Adipocyte differentiation in vitro is triggered by a combination of dexamethasone, insulin and

Key words: adipocyte fatty acid-binding protein (FABP4), glucocorticoid receptor, macrophage, peroxisome-proliferatoractivated receptor γ (PPARγ ), tamoxifen.

Abbreviations used: ApoE, apolipoprotein E; ER, oestrogen receptor; FABP, fatty acid-binding protein; GR, glucocorticoid receptor; HEK, human embryonic kidney; IL, interleukin; LDL, low-density lipoprotein; nGRE, negative glucocorticoid regulatory element; oxLDL, oxidized LDL; pGRE, positive glucocorticoid regulatory element; PPARγ, peroxisome-proliferator-activated receptor γ; PPRE, PPARγ regulatory element; TNFα, tumour necrosis factor α; TZD, thiazolidinedione. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).  c The Authors Journal compilation  c 2013 Biochemical Society

Biochemical Journal

Meixiu JIANG*†1 , Ling ZHANG†1 , Xingzhe MA†, Wenquan HU†, Yuanli CHEN*†, Miao YU†, Qixue WANG†, Xiaoju LI†, Zhinan YIN†, Yan ZHU‡, Xiumei GAO‡, David P. HAJJAR§, Yajun DUAN*†2 and Jihong HAN*†2

www.biochemj.org

Tamoxifen inhibits macrophage FABP4 expression through the combined effects of the GR and PPARγ pathways

468

M. Jiang and others

macrophages [9]. In vivo, genetic deletion of FABP4 expression inhibits high-fat-diet-induced atherosclerosis in mice without significant changes in serum lipids or insulin sensitivity [10–12]. More importantly, lack of FABP4 expression specifically in macrophages by bone marrow transplantation inhibits lesion development, suggesting an important role for macrophage FABP4 in atherosclerosis [11]. Deficiency of FABP4 expression in macrophages results in decreased cholesteryl ester accumulation and secretion of inflammatory cytokines [TNFα (tumour necrosis factor α), MCP-1 (monocyte chemoattractant protein-1) and IL (interleukin)-6], and resistance to PMA-induced inflammatory cytokines (TNFα, IL-1β and IL-6) [11]. Clinically, it has been reported that the FABP4 levels in atherosclerotic lesions are associated with an unstable plaque phenotype and an increased risk for cardiovascular events [13]. A 10-year prospective study demonstrates that circulating FABP4 levels are associated with long-term prognosis in patients with coronary heart disease and may represent an important pathophysiological mediator of atherosclerosis [14]. Tamoxifen and its derivative, 4-hydroxytamoxifen, are selective ER (oestrogen receptor) modulators [15]. Dependent on the target cell types/tissues, tamoxifen functions either as an agonist or antagonist [16]. For instance, in breast tissue, tamoxifen inactivates the ER, thereby inhibiting the growth of breast cancer cells. In endometrium, tamoxifen behaves as an agonist. Clinically, tamoxifen has been used for more than two decades to treat both early and advanced ER-positive breast cancer in women, and as the most common hormone treatment for male breast cancer. Tamoxifen also has a preventive effect on breast cancer in women with a high risk of developing the disease [17]. Tamoxifen has been demonstrated to have multiple pleiotropic biological effects. Both pre-clinical and clinical studies have suggested the cardioprotective properties of tamoxifen. For example, tamoxifen reduces the incidence of fatal myocardial infarction and the intima-media thickness of the common carotid artery in post-menopausal women. It also increases ED-FMD (endothelium-dependent flow-mediated dilation) in men with advanced atherosclerosis [18–20]. In animal models, tamoxifen reduces atherosclerotic lesions induced by high-fat diet in both wild-type and ApoE (apolipoprotein E)-deficient (apoE − / − ) mice and surgically post-menopausal monkeys [21–23]. It is also well known that atherosclerosis is an underlying cause of coronary heart disease. The lipid-laden macrophage/foam cells are a prominent part of atherosclerotic lesions. Thus the formation of foam cells is an initial and critical step in lesion development [24]. Although tamoxifen has been shown to have anti-atherogenic properties, the molecular mechanisms involved which define its role are unknown. Therefore we hypothesize that macrophage FABP4 is a molecular target of tamoxifen. The inhibition of macrophage FABP4 expression can be, at least in part, attributed to the tamoxifen effects on macrophage/foam cell formation and atherosclerosis. In the present study, we attempt to identify the signalling pathways by which tamoxifen can regulate macrophage FABP4 expression. MATERIALS AND METHODS Materials

Fulvestrant (ICI182780) was purchased from Cayman Chemical. Tamoxifen, 4-hydroxytamoxifen, 17β-oestradiol and other chemicals were purchased from Sigma–Aldrich. Goat anti(mouse FABP4), rabbit anti-(mouse GR) and rabbit anti-(mouse PPARγ ) polyclonal antibodies were obtained from Santa Cruz Biotechnology.  c The Authors Journal compilation  c 2013 Biochemical Society

Cell culture and in vivo study

HEK (human embryonic kidney)-293T cells were purchased from the A.T.C.C. and cultured in complete DMEM (Dulbecco’s modified Eagle’s medium) containing 10 % FBS, 50 μg/ml penicillin/ streptomycin and 2 mM glutamine. Cells at 95 % confluence were used for transfection and analysis of promoter activity. The protocol for animal studies was approved by the Ethics Committee of Nankai University and conforms to the Guide for the Care and Use of Laboratory Animals published by the NIH (National Institutes of Health). C57BL/6 wild-type mice and ApoE-deficient mice (apoE − / − ) purchased from the Jackson Laboratory were bred at the Animal Center of Nankai University. Mouse peritoneal macrophages were collected as follows. Mice at approximately 8 weeks old were injected intraperitoneally with 3 ml of 4 % thioglycollate solution and maintained with access to water and normal chow for 5 days. The animals were anaesthetized and killed within a CO2 chamber. The peritoneal macrophages were collected from the mouse abdomen by lavage with PBS. Cells were cultured in complete RPMI 1640 medium containing 10 % FBS, 50 μg/ml penicillin/streptomycin and 2 mM glutamine for 2 h. After the removal of all floating cells, the adhesive cells (macrophages) were cultured in complete RPMI 1640 medium for a further 2 days followed by treatment in serumfree medium. To investigate the effect of tamoxifen on macrophage and adipose tissue FABP4 expression as well as the circulating FABP4 levels in vivo, both male and female apoE − / − mice (∼ 8-week-old) were fed a high-fat diet (0.5 % cholesterol and 21 % fat) or the diet containing tamoxifen (2 mg/100 g of food) for 1 month. The mice were injected intraperitoneally with 4 % thioglycollate solution 5 days before the end of the experiments. After killing, mouse blood, peritoneal macrophages, and both white and brown adipose tissues were individually collected. Blood samples were kept for more than 2 h at room temperature (22 ◦ C). After centrifugation for 20 min at 2000 g (at room temperature), the serum was transferred into a new test tube and kept at − 20 ◦ C until assaying of circulating FABP4 using an ELISA assay kit purchased from USCN Life Science. Total cellular proteins were extracted from the above macrophage or tissue samples for determination of FABP4 protein expression. Human blood monocyte-derived macrophages were obtained as described previously with blood obtained from a healthy donor (Tianjin Blood Bank, China) [25]. Briefly, after removal of red blood cells by incubating the blood with dextran sedimentation mixture, the remained cells were collected by centrifugation (250 g for 5 min at 4 ◦ C) and re-suspended in serum-free RPMI 1640 medium. The suspension was overlaid on the top of a FicollHypaque solution. After gradient centrifugation for 20 min at 1000 g, monocytes in the interface layer were collected and washed twice with serum-free medium followed by culture in complete RPMI 1640 medium (20 % FBS) for 7 days to differentiate into macrophages. Inhibition of macrophage FABP4 expression by siRNA and determination of foam cell formation

The siRNA against FABP4 (FABP4 siRNA) with the target sequence of 5 -AATGTAGTGTTTGATGCAAAT-3 was constructed using the Ambion Silencer siRNA Construction kit (Invitrogen). Peritoneal macrophages isolated from male apoE − / − mice were plated in six-well plates (for the transfection efficiency assay) or on coated 12-mm glass slides in 24-well plates (for Oil Red O staining) and transfected with scrambled siRNA or FABP4 siRNA using LipofectamineTM (Invitrogen) in RPMI 1640

Tamoxifen inhibits macrophage FABP4 expression

medium. After 6 h of transfection, the same volume of medium was added to the cells and the transfection was continued for another 24 h. The transfected cells were then switched into complete medium for another 48 h followed by extraction of total cellular protein or determination of foam cell formation as follows. The transfected cells were treated with 5 μM tamoxifen or 4-hydroxytamoxifen for 12 h followed by incubation with 50 μg/ml oxLDL [oxidized LDL (low-density lipoprotein)] for 2 h. The cells were then fixed in 4 % paraformaldehyde for 30 min, washed twice with PBS for 5 min, and stained with Oil Red O solution (a mixture of three parts 0.5 % Oil Red O in propan-2-ol and two parts water) for 50 min at room temperature followed by washing twice with water. The cells were then re-stained with a haematoxylin solution for 30 s, kept in water for 5 min and photographed. Cells containing lipid droplets (more than ten) were counted as foam cells and at least ten fields of each sample were counted for quantitative analysis [26]. Determination of FABP4 protein expression by Western blot analysis

After treatment, cells were washed twice with ice-cold PBS and lysed with a lysis buffer [20 mM Tris (pH 7.5), 137 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 25 mM 2-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 μg/ml aprotinin/ leupeptin and 100 mM sodium orthovanadate]. The cellular lysate was spun for 10 min at 16 200 g at 4 ◦ C, and the supernatant was transferred into a new test tube. Proteins (40 μg) from each sample were separated by SDS/PAGE (15 % gel) and transferred on to a nylon-enhanced nitrocellulose membrane. The membrane was blocked with a solution of 5 % BSA in PBS for 1 h, and then incubated with a goat anti-(mouse FABP4) polyclonal antibody for 1 h at room temperature followed by washing for 3×10 min with a solution of PBS-T (0.5 % Tween 20/PBS). The membrane was then exposed to HRP (horseradish peroxidase)-conjugated rabbit anti-(goat IgG) and incubated for 1 h at room temperature. After three washes with PBS-T (10 min each), the membrane was incubated for 5 min in a mixture of equal volumes of Western blot chemiluminescence reagents 1 and 2 (Millipore). The membrane was then exposed to film for development. Isolation of total RNA, purification of poly(A + ) RNA and Northern blot analysis of macrophage Fabp4 mRNA expression

After treatment, cells were used to extract total cellular RNA. The poly(A + ) RNA isolated from 100 μg of total RNA was used to conduct Northern blot analysis of Fabp4 mRNA expression as described previously [27]. Preparation of plasmid DNA and determination of FABP4 promoter activity

The GRα expression vector was prepared as described previously [7]. The PPARγ 2 expression vector was prepared as follows. A cDNA encoding mouse Ppargamma2 was generated by reverse transcription with total RNA isolated from the differentiated 3T3L1 cells and oligo(dT)18 primer. A PCR was carried out with the cDNA and the primers 5 -TCTCGAGCTCAATGGGTGAAACTCTGGGAG-3 (forward) and 5 -CCGCGGTACCCTAATACAAGTCCTTGTAGATCTCCT-3 (reverse). After the sequence was confirmed, the PCR product was digested with SacI and KpnI and then subcloned into an expression vector, pEGFP-C2 (pC2-PPARγ ). A mouse Fabp4 promoter (from − 4029 to + 25) was constructed by PCR with mouse genomic DNA and the following primers: 5 -CCGCTCGAGAGACACAAACAGAGCAAATG-3 (forward) and 5 -CCCAAGCTTAGGAGCTGTCTTCCAGGTG-

469

3 (reverse). After the sequence was confirmed, the PCR product was digested with XhoI and HindIII followed by ligation with the pGL4 luciferase reporter vector from Promega (pFABP44K), transformed into Escherichia coli for amplification. The Fabp4 promoter with PPRE1 (from − 3693 to − 3681) deletion (pFABP4-4K-PPRE1del) or PPRE2 (from − 2033 to − 2021) deletion (pFABP4-4K-PPRE2del) was constructed using the Phusion Site-Directed Mutagenesis kit from New England Biolabs with pFABP4-4K DNA and primers with the corresponding PPRE deletion respectively. To disclose the role of the nGRE or pGRE (nGRE is from − 114 to − 99; pGRE is from − 416 to − 411) [7] in tamoxifen-mediated Fabp4 promoter activity, another Fabp4 promoter (from − 1880 to + 25, pFABP4-1.9K) containing no PPRE was constructed by PCR with pFABP4-4K and primer 5 -CCGCTCGAGGAAATTTTCACGATGCATTGC3 (forward) and the same reverse primer as above. The promoter with nGRE deletion was constructed with pFABP4-1.9K and the primers with nGRE deletion. To analyse FABP4 promoter activity, HEK-293T cells at ∼ 95 % confluence in 24-well plates were transfected with the DNA of the FABP4 promoter or pC2-GR or pC2-PPARγ and Renilla (for internal normalization) using LipofectamineTM 2000 (Invitrogen). After 24 h transfection plus treatment, the cells were lysed and the cellular lysate was used to determine the activity of firefly and Renilla luciferases using the Dual-Luciferase Reporter Assay system (Promega). EMSA and ChIP assay of GR or PPARγ DNA-binding activity

After treatment, nuclear protein was extracted from the cells and was used for an EMSA to analyse the binding of nGRE and PPRE1 as described previously [28]. The sequences of the nGRE and PPRE1 probes were GGTCATGAAGgaaaTGATCTGGCC and AAAGTAGGTCAcAGTTTATCTGCAG respectively (the underlined nucleotides are putative nGRE and PPRE in FABP4 in which the uppercase letters are conserved nucleotides, whereas the lowercase letters are variable nucleotides). The ChIP assay was conducted as follows. After treatment, cells were cross-linked by the addition of formaldehyde and then sonicated in a lysis buffer [50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 % Triton X-100, 1 mM EDTA, 0.1 % sodium deoxycholate, 0.1 % SDS and protease inhibitors aprotinin/leupeptin] to fragment DNA into an average size of 500–1000 bp. The input PCR was conducted with DNA extracted from the sonicated chromatin after reversal of the cross-linking. Immunoprecipitation was conducted with the same amount of chromatin from each sample based on the input and anti-PPARγ or anti-GR polyclonal antibody, followed by PCR. The primers for the ChIP assays were: PPRE1, 5 -GGCCTGCTGGTAAGTTCTTC-3 (forward) and 5 -GTAAAGACAGACTGAGAGTG-3 (reverse); nGRE, 5 -GTACTCTAAGTCCAGTGATC-3 (forward) and 5 -GTAAACCTTCGAGGAGGAGC-3 (reverse). Data analysis

Data were generated from three independent experiments. All statistical results were obtained by Student’s t test using GraphPad Prism. P < 0.05 was considered significant. RESULTS Tamoxifen inhibits macrophage FABP4 expression in a sex-independent manner

To determine whether tamoxifen inhibits macrophage/foam cell formation by its effect on macrophage FABP4 expression,  c The Authors Journal compilation  c 2013 Biochemical Society

470

M. Jiang and others

inhibition was concentration-dependent, with a greater effect on cells isolated from male mice (Figures 2A and 2B). We also conducted a time-course study in which the cells were treated with 5 μM tamoxifen or 4-hydroxtamoxifen for different times. The results in Figures 2(C) and 2(D) show that the inhibition occurred quickly (∼ 2 h) and reached a steady state after 6 h of treatment. Similarly, we treated the macrophages with either an ER agonist (17β-oestradiol) or antagonist (fulvestrant or ICI182780). 17βOestradiol reduced macrophage FABP4 protein expression in a sex-independent manner (Figures 2E and 2F). Fulvestrant slightly inhibited FABP4 protein expression in macrophages isolated from female mice (Figure 2G), but slightly increased expression in the cells isolated from male mice at high concentrations (Figure 2H). Taken together, the results in Figure 2 indicate that tamoxifen inhibited macrophage FABP4 expression in a sex-independent manner. Owing to the similar inhibitory effects of tamoxifen on FABP4 expression between peritoneal macrophages isolated from male and female mice, we chose to conduct the rest of our in vitro experiments with cells isolated from male C57BL/6 wild-type mice.

Tamoxifen inhibits GR or PPARγ -induced macrophage FABP4 expression Figure 1

Tamoxifen inhibits macrophage/foam cell formation

(A) Peritoneal macrophages were isolated from male apoE − / − mice. The cells were transfected with scrambled siRNA (NS) or FABP4 siRNA at the indicated concentrations for 3 days. Expression of FABP4 protein was determined by Western blot analysis. (B) After transfection with 20 μM scrambled siRNA or FABP4 siRNA for 3 days, the cells were treated with 5 μM tamoxifen or 4-hydroxytamoxifen (4-OH Tam) for 12 h followed by staining with Oil Red O and haematoxylin (top panel) and foam cells were quantified (bottom panel) as described in the Materials and methods section. *P < 0.05 (n = 10), significantly different from control (oxLDL alone). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

we isolated peritoneal macrophages from male apoE − / − mice. We transfected the cells with FABP4 siRNA at different concentrations for 3 days and then determined FABP4 protein expression. Compared with scrambled siRNA, FABP4 siRNA reduced macrophage FABP4 protein expression in a concentration-dependent manner (Figure 1A). We then determined the effects of tamoxifen and 4-hydroxytamoxifen on macrophage/foam cell formation and investigated whether the inhibitory effect is related to FABP4 expression. After 3 days of transfection with scrambled or FABP4 siRNA, the cells were treated with tamoxifen or 4-hydroxytamoxifen and foam cell formation was assessed. The results shown in Figure 1(B) indicate that tamoxifen or 4-hydroxytamoxifen reduced lipid accumulation in the cells transfected with scrambled siRNA, indicating inhibition of foam cell formation (top panel of Figure 1B). The reduction in macrophage FABP4 expression by siRNA also decreased lipid accumulation by macrophages. Interestingly, compared with scrambled siRNA, inhibition of FABP4 expression by siRNA attenuated the inhibitory effect of tamoxifen on lipid accumulation in macrophages (Figure 1B), indicating that the inhibition of foam cell formation by tamoxifen was related to its effect on FABP4 expression. To test the effect of tamoxifen on macrophage FABP4 expression and whether the effect is sex-related, we isolated peritoneal macrophages from both male and female C57BL/6 wild-type mice and treated the cells with tamoxifen and 4hydroxytamoxifen at different concentrations overnight. The results of Western blot analysis indicate that tamoxifen or 4-hydroxtamoxifen inhibited FABP4 protein expression in macrophages isolated either from male or female mice. The  c The Authors Journal compilation  c 2013 Biochemical Society

Macrophage FABP4 expression can be activated by ligands for the GR, such as dexamethasone, and the activation can be synergized by statins [7]. To test whether tamoxifen is able to antagonize the above induction of FABP4 expression, we treated macrophages with tamoxifen or 4-hydroxytamoxifen at different concentrations in the presence or absence of dexamethasone. Expression of FABP4 protein was induced ∼ 2.5-fold by dexamethasone. However, both tamoxifen and 4hydroxytamoxifen blocked this induction in a concentrationdependent manner (Figure 3A). The co-treatment of macrophages with dexamethasone and pitavastatin further induced macrophage FABP4 protein expression (>3-fold). However, tamoxifen or 4hydroxytamoxifen displayed a similar inhibitory effect on this induction as with dexamethasone alone (Figures 3B and 3C). Therefore the inhibition of FABP4 expression by tamoxifen may be related to its inactivation of the GR, although we observed that tamoxifen or 4-hydroxytamoxifen had little effect on GR expression and dexamethasone-induced GR nuclear translocation (results not shown). To determine whether the inhibition of FABP4 protein expression by tamoxifen was associated with FABP4 mRNA expression, we conducted Northern blot analysis to determine the expression of macrophage Fabp4 mRNA in response to tamoxifen and 4-hydroxytamoxifen. Expression of Fabp4 mRNA in control macrophages is undetectable, but can be significantly induced by dexamethasone or dexamethasone plus pitavastatin [7,27]. Therefore we determined the effect of tamoxifen on macrophage Fabp4 mRNA expression in the presence of dexamethasone or dexamethasone plus pitavastatin. The results of both concentration and time-course studies (Figures 4A and 4B) demonstrate that tamoxifen or 4-hydroxytamoxifen can inhibit the induction of Fabp4 mRNA expression. These findings indicate that tamoxifen may regulate FABP4 transcription. PPARγ , a transcription factor, can be activated by natural ligands such as 15-deoxy12,14 prostaglandin J2, oxLDL and synthetic ligands, such as TZDs. Activation of PPARγ can also induce macrophage FABP4 expression [29–31]. To test whether the inhibition of macrophage FABP4 expression by tamoxifen is related to inactivation of PPARγ , we initially treated cells with tamoxifen or 4-hydroxytamoxifen in the presence of

Tamoxifen inhibits macrophage FABP4 expression

Figure 2

471

Tamoxifen and 4-hydroxytamoxifen inhibit mouse peritoneal macrophage FABP4 protein expression in a sex-independent manner

Peritoneal macrophages were isolated from male and female C57BL/6 mice respectively. After treatment in serum-free medium, the cells were used to extract total cellular proteins. Expression of FABP4 in cellular proteins (40 μg/sample) was determined by Western blot analysis. (A and B) Cells were treated with tamoxifen and 4-hydroxytamoxifen (4-OH tamoxifen) at the indicated concentrations for 16 h. (C and D) Cells were treated with 5 μM tamoxifen (Tam) or 4-hydroxytamoxifen (4-OH Tam) for the indicated times. (E–H) Cells were treated with 17β-oestradiol (E and F) or fulvestrant (G and H) at the indicated concentrations for 16 h. The results of statistical analysis of blots are shown at the bottom of each panel. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mϕ, macrophage.

oxLDL. The results in both concentration and time-course studies indicate that tamoxifen and 4-hydroxytamoxifen blocked the induction of FABP4 protein expression by oxLDL (Figures 5A and 5B). Troglitazone is a synthetic PPARγ ligand. Treatment of macrophages with troglitazone increased FABP4 protein expression (∼ 3-fold). However, the induction was reduced by tamoxifen and 4-hydroxytamoxifen (Figure 5C). In particular, 4hydroxytamoxifen totally blocked the increased FABP4 protein expression. To further identify the mechanism by which tamoxifen inhibits PPARγ -induced FABP4 expression, we determined the effect of tamoxifen or 4-hydroxytamoxifen on PPARγ expression. As seen in Figure 5(D), PPARγ protein expression was reduced by tamoxifen and 4-hydroxytamoxifen.

Inhibition of macrophage FABP4 expression by tamoxifen occurs at the transcriptional level

To further characterize the inhibition of FABP4 expression by tamoxifen, we constructed a Fabp4 promoter (pFABP4-1.9K, from − 1880 to + 25, Figure 6A) which includes both the pGRE (from − 416 to − 411) and nGRE (from − 114 to − 99). The results in Figure 6(B) indicate that both tamoxifen and 4-hydroxytamoxifen inhibited pFABP4-1.9K promoter activity.

Highly expressing GR alone by transfection of cells with a GR expression vector had little effect on pFABP4-1.9K activity, but significantly increased the promoter activity when the highly expressed GR was activated by dexamethasone. However, both tamoxifen and 4-hydroxytamoxifen blocked the induction by GR activation (Figure 6C). Interestingly, when the nGRE (ATGAAGgaaaTGATCT, from − 114 to − 99) was deleted from the pFABP4-1.9K promoter [7], both tamoxifen and 4hydroxytamoxifen had little effect on the promoter activity (Figure 6D), suggesting that tamoxifen and 4-hydroxytamoxifen enhanced the nGRE activity. To test the effect of tamoxifen on PPARγ -induced FABP4 transcription, we constructed another Fabp4 promoter (pFABP44K, from − 4029 to + 25, Figure 6A), which includes two putative PPREs (PPRE1, AGGTCAcAGTTTA, from − 3693 to − 3681; PPRE2, AGAGCAcAGAGCA, from − 2033 to − 2021; the underlined nucleotides are identical with the sequence of the conserved PPRE, AGGTCAnAGGTCA). pFABP4-4K also contains nGRE thus, compared with the effect on pFABP41.9K, tamoxifen and 4-hydroxytamoxifen demonstrated greater inhibition on pFABP4-4K (Figure 6E). Overexpressing PPARγ alone induced pFABP4-4K activity, and activation of PPARγ by troglitazone further increased the promoter activity. However, the activation was blocked by tamoxifen and 4-hydroxytamoxifen  c The Authors Journal compilation  c 2013 Biochemical Society

472

M. Jiang and others

Figure 3 Tamoxifen and 4-hydroxytamoxifen macrophage FABP4 protein expression

inhibit

GR-induced

Peritoneal macrophages isolated from male C57BL/6 mice received the treatment indicated in serum-free medium. FABP4 protein expression was determined by Western blot analysis. (A) Cells were treated with tamoxifen or 4-hydroxytamoxifen at the concentrations indicated in the presence of dexamethasone for 16 h. (B and C) Cells were treated with dexamethasone plus pitavastatin in the presence or absence of tamoxifen or 4-hydroxytamoxifen at the concentrations indicated for 16 h (B) or plus 5 μM tamoxifen/4-hydroxytamoxifen for the times indicated (C). Ctrl, control; Dex, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 4-OH Tam, 4-hydroxytamoxifen; Tam, tamoxifen.

in a concentration-dependent manner (Figure 6F), indicating the inactivation of PPARγ by tamoxifen. To distinguish the role of PPRE1 and PPRE2 in regulation of FABP4 transcription, we constructed the promoter with deletion of PPRE1 or PPRE2, and determined activity of the promoter in response to treatment. As

Figure 5

Figure 4 Tamoxifen and 4-hydroxytamoxifen inhibit macrophage Fabp4 mRNA expression After treatment, peritoneal macrophages isolated from male C57BL/6 mice were used to extract total cellular RNA. Poly(A + ) RNA was isolated from 100 μg of total RNA and used to determine macrophage Fabp4 mRNA expression by Northern blot analysis as described in the Materials and methods section. The same blot was re-hybridized with a 32 P-labelled GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe as an internal control. (A) Macrophages were treated with dexamethasone or dexamethasone plus pitavastatin in the presence or absence of tamoxifen or 4-hydroxytamoxifen at the concentrations indicated for 16 h. (B) Cells were treated with dexamethasone plus pitavastatin or plus 5 μM tamoxifen or 4-hydroxytamoxifen for the times indicated. Ctrl, control; Dex, dexamethasone; 4-OH Tam, 4-hydroxytamoxifen; Pit, pitavastatin.

Tamoxifen and 4-hydroxytamoxifen inhibit PPARγ -induced macrophage FABP4 expression

(A) Mouse peritoneal macrophages isolated from male C57BL/6 mice were treated with tamoxifen or 4-hydroxytamoxifen at the concentrations indicated in the presence of 50 μg/ml oxLDL for 16 h. (B) Macrophages were pre-treated with 50 μg/ml oxLDL for 8 h followed by treatment with 5 μM tamoxifen or 4-hydroxytamoxifen for the times indicated. (C) Peritoneal macrophages were treated with tamoxifen or 4-hydroxytamoxifen at the concentrations indicated in the presence of 2 μM troglitazone for 16 h. (D) Macrophages were treated with tamoxifen and 4-hydroxytamoxifen at the concentrations indicated for 16 h. For all panels, expression of FABP4 or PPARγ protein was determined by Western blot analysis. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 4-OH Tam/4-OH T, 4-hydroxytamoxifen; Tam, tamoxifen.  c The Authors Journal compilation  c 2013 Biochemical Society

Tamoxifen inhibits macrophage FABP4 expression

Figure 6

473

Tamoxifen and 4-hydroxytamoxifen inhibit Fabp4 promoter activity by inactivating GR and PPARγ pathways

(A) Two Fabp4 promoters (pFABP4-1.9K and pFABP4-4K) as indicated were constructed as described in the Materials and methods section. (B–G) HEK-293T cells at ∼ 95 % confluence were transfected with the indicated Fabp4 promoter or promoter plus expression vector and Renilla (as an internal control) for 4 h followed by the treatment indicated overnight. Activity of firefly and Renilla luciferases in the cellular lysate was determined using the Dual-Luciferase Reporter Assay system. Promoter and expression vector used are: (B) pFABP4-1.9K; (C) pFABP4-1.9K, C2-GR; (D) pFABP4-1.9K-nGREdel (nGRE deleted), C2-GR; (E) pFABP4-4K; (F) pFABP4-4K, C2-PPARγ ; and (G) pFABP4-4K, pFABP4-4K-PPRE1del (PPRE1 deleted), pFABP4-4K-PPRE2del (PPRE2 deleted), C2-PPARγ . *P < 0.05 (paired Student’s t test; n = 3), significantly different from promoter alone (B–G) or promoter plus expression vector (C, D, F and G). ¶P < 0.05 (n = 3), significantly different from promoter plus agonist [dexamethasone (C); troglitazone (F and G)] in the corresponding groups. 4-OH Tam, 4-hydroxytamoxifen.

shown in Figure 6(G), the deletion of PPRE1, but not PPRE2, dramatically decreased the inductive effect of PPARγ activation, suggesting that PPRE1 plays a critical role in regulating FABP4 transcription (Figure 6G). Tamoxifen and 4-hydroxytamoxifen partially inhibited the activity of the PPRE1- or the PPRE2-deleted promoter owing to the existence of the nGRE in these promoters (Figure 6G). The effects of tamoxifen on the activity of GR and PPARγ were further investigated by an EMSA to assess the binding of nuclear proteins to the nGRE and PPRE1 in response to tamoxifen treatment. As expected, tamoxifen and 4-hydroxytamoxifen enhanced the binding of the nGRE with nuclear proteins (Figure 7A). In contrast, tamoxifen and 4-hydroxytamoxifen

reduced the affinity of PPRE1 to nuclear proteins (Figure 7B). The binding of the nGRE or PPRE1 with nuclear proteins was partially reduced by an anti-GR antibody (lane 5 compared with the lane 6 in Figure 7A) or by an anti-PPARγ antibody (the first lane compared with the last lane in Figure 7B) indicating specific binding. In addition, the results in ChIP assays indicated a strong interaction between PPRE1 and PPARγ protein, and a weak interaction between nGRE and GR protein (Figure 7C). However, tamoxifen and 4-hydroxytamoxifen reduced the interaction of PPRE1–PPARγ but enhanced the interaction of nGRE–GR (Figure 7C). Taken together, these results further indicate that tamoxifen inhibits macrophage FABP4 expression at the transcriptional level by inactivating GR and PPARγ pathways.  c The Authors Journal compilation  c 2013 Biochemical Society

474

Figure 7

M. Jiang and others

Tamoxifen and 4-hydroxytamoxifen enhance the binding of GR with the nGRE but inhibit the binding of PPARγ with PPRE1

(A and B) Macrophages isolated from male C57BL/6 mice received the treatment indicated overnight. Nuclear proteins were extracted and used to determine the binding of GR with nGRE (A) and PPARγ with PPRE1 (B) by EMSA. The sequences of probes are: ggtcATGAAGgaaaTGATCTggcc (nGRE) and aaagtAGGTCAcAGTTTAtctgcag (PPRE1) (the underlined nucleotides are putative nGRE and PPRE in FABP4 in which the uppercase letters are conserved nucleotides, whereas the lowercase letters are variable nucleotides). (C) After overnight treatment, chromatin was isolated from the cells by sonication and a ChIP assay was conducted (left-hand panel) as described in the Materials and methods section. The ratio of the density of PCR product of PPRE1 or nGRE between IP (immunoprecipitate) and input in the corresponding control sample was defined as 100 % for the statistical analysis (right-hand panel). 4-OH Tam, 4-hydroxytamoxifen; Tam, tamoxifen.

Tamoxifen inhibits FABP4 expression in vivo and human macrophage FABP4 expression

To define the in vivo effect of tamoxifen on FABP4 expression, we fed apoE − / − mice a high-fat diet or a diet containing tamoxifen (2 mg/100 g of food) for 1 month. We then determined changes in FABP4 protein expression in peritoneal macrophages. The results in Figure 8(A) indicate that administration of mice with tamoxifen reduced macrophage FABP4 expression in vivo. In addition to macrophages, FABP4 is also highly expressed in adipocytes. Therefore we collected both white and brown adipose tissue samples from mice after tamoxifen treatment. The results in Figure 8(B) indicate that tamoxifen also inhibited FABP4 expression in adipose tissues in vivo. To determine the effect of tamoxifen on circulating FABP4 levels, we also collected the blood samples after treatment and determined serum FABP4 levels by ELISA. The results in Figure 8(C) indicate that tamoxifen reduced circulating FABP4 levels similarly between female and male mice (by approximately one-third; female, 20.8 compared with 14.1 μg/l; male, 18.4 compared with 12.0 μg/l). The similar inhibitory effects on macrophage or adipose FABP4 expression or circulating FABP4 levels in vivo between male and female mice further confirm that the regulation of FABP4 expression by tamoxifen is sex-independent. Finally, to determine the effects of tamoxifen on human macrophage FABP4 protein expression, we isolated human monocytes from blood and differentiated them into macrophages. We then treated the cells with tamoxifen or 4-hydroxytamoxifen alone or in the presence of troglitazone. Similar to mouse macrophages, we observed that tamoxifen and 4-hydroxytamoxifen inhibited human macrophage FABP4 protein expression in a dose-dependent manner (Figure 9A). Furthermore, tamoxifen decreased PPARγ -induced human macrophage FABP4 protein expression (Figure 9B).  c The Authors Journal compilation  c 2013 Biochemical Society

DISCUSSION

A lower incidence of cardiovascular disease is observed among women than men in the age group 20–39 years owing to the endogenous production of oestrogen in women [32]. However, this gap narrows with advancing age, indicating that cardiovascular disease can contribute to a significant health threat to older post-menopausal women, independent of breast cancer. Tamoxifen, as an adjuvant therapy for breast cancer, has been observed to possess several pleiotropic functions, including cardioprotection [33,34]. At the molecular level, tamoxifen functions as an antioxidant to protect LDL particles from oxidative damage and lipid peroxidation [35,36]. It also inhibits cholesterol biosynthesis by inhibiting 8 isomerase and 24 reductase [37,38]. Tamoxifen alone, and with lovastatin, can enhance LDLR (LDL receptor) expression which contributes to the hypolipidaemic effect of tamoxifen [39]. Clinically, patients receiving tamoxifen treatment have reduced total and LDLcholesterol levels [40,41]. Formation of macrophage/foam cells is the initial and critical step in the development of atherosclerosis. We have previously reported that both tamoxifen and 4-hydroxytamoxifen induce macrophage SR-BI (scavenger receptor BI) expression in a sex-dependent manner, indicating another mechanism by which tamoxifen inhibits macrophage/foam cell formation [42]. However, some of the cardioprotective effects of tamoxifen occur in both males and females, indicating that tamoxifen can also function in a sex-independent manner. Indeed, in the present study we observed that tamoxifen and 17β-oestradiol inhibit FABP4 expression in macrophages isolated from male and female wild-type mice, and fulvestrant has little effect (Figure 2). More importantly, our in vivo study demonstrates a similar inhibitory effect of tamoxifen on FABP4 expression in macrophages and adipose tissues, and circulating FABP4 levels between male

Tamoxifen inhibits macrophage FABP4 expression

Figure 8

475

Tamoxifen inhibits FABP4 expression in vivo

Both male and female apoE − / − mice were fed a high-fat diet or the diet containing tamoxifen (2 mg/100 g of food) for 1 month. At the end of experiment, peritoneal macrophages (A) and both white and brown adipose tissues (B) were collected and total proteins were extracted, followed by determination of FABP4 protein expression by Western blot analysis. (C) Mouse serum samples were prepared and used to determine circulating FABP4 levels by ELISA. *P < 0.05 (n = 5), significantly different from the corresponding control. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 9 Tamoxifen and 4-hydroxytamoxifen inhibit human blood-derived macrophage FABP4 protein expression Human monocytes were isolated from blood and differentiated into macrophages as described in the Materials and methods section. The human macrophages were treated with tamoxifen or 4-hydroxytamoxifen at the concentrations indicated in the absence (A) or presence (B) of 2 μM troglitazone overnight. Expression of human macrophage FABP4 protein was determined by Western blot analysis. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 4-OH Tam, 4-hydroxytamoxifen.

and female mice (Figure 8). These findings confirm that the regulatory effect of tamoxifen on FABP4 expression is independent of sex. Meanwhile, we demonstrate for the first time that the inhibition of macrophage FABP4 expression by tamoxifen occurs through the combined effects of GR and PPARγ , the two classical signalling pathways regulating FABP4 expression (Figures 3 and 5–7). FABP4 expression is highly induced during fibroblast/ adipocyte and monocyte/macrophage differentiation. Activation of PPARγ by ligands or oxLDL can induce FABP4 expression in monocyte/macrophages [29,30]. Tamoxifen blocks the induction of FABP4 expression induced by troglitazone or oxLDL (Figure 5), suggesting an involvement of PPARγ in the inhibition

of macrophage FABP4 expression by tamoxifen. Indeed, tamoxifen and 4-hydroxytamoxifen inhibited PPARγ expression that can result in a decreased interaction between PPARγ and PPRE1 in the FABP4 gene (Figures 5D, 7B and 7C). Two putative PPREs were found in the distal region of the Fabp4 promoter (Figure 6A); deletion of PPRE1, but not PPRE2, profoundly altered the promoter activity in response to treatment (Figure 6G), since PPRE1 has more nucleotides than PPRE2 which are identical with the conserved PPRE sequence (Figure 6A). GR, a typical transcription factor, functions after ligand-induced nuclear translocation. Activation of GR induces transcription of target genes containing the pGRE alone. It can also inhibit transcription of some genes containing the nGRE alone. However, the transcription of genes containing both the pGRE and nGRE, such as FABP4, is determined by activity of these two elements [7,43]. Dexamethasone alone activates GR nuclear translocation and pGRE, thereby inducing macrophage FABP4 expression [27]. The statins alone reduce macrophage FABP4 expression [8]. However, statins synergize dexamethasone-induced FABP4 transcription since they can suppress nGRE activity [7]. In the present study, we observed that tamoxifen has little effect on GR expression as well as dexamethasone-induced nuclear translocation, yet dexamethasone-induced FABP4 expression and promoter activity remain decreased by tamoxifen (Figures 3A, 4A and 6C). Interestingly, the dexamethasone-activated Fabp4 promoter with nGRE deletion is little affected by tamoxifen (Figure 6D), suggesting an important role of the nGRE in the tamoxifen-inhibited macrophage FABP4 expression. In fact, the results of EMSA and ChIP assays suggest that tamoxifen enhances the interaction between GR and nGRE (Figures 7A and 7C). Taken together, we observed that tamoxifen inactivates PPARγ by reducing its expression. Meanwhile, it reduces GR activity by activating the nGRE. Therefore the results of the present study suggest that tamoxifen inhibits macrophage FABP4 expression through the combined effects of GR and PPARγ signalling pathways. Such findings suggest that the inhibition of FABP4 expression can result from the anti-atherosclerotic properties of tamoxifen.  c The Authors Journal compilation  c 2013 Biochemical Society

476

M. Jiang and others

AUTHOR CONTRIBUTION Meixiu Jiang, Xingzhe Ma, Miao Yu and Xiaoju Li conducted biochemical experiments. Ling Zhang, Wenquan Hu, Yuanli Chen and Qixue Wang conducted molecular biology experiments. Zhinan Yin, Yan Zhu and Xiumei Gao conducted in vivo experiments. David P. Hajjar edited the paper before submission. Yajun Duan and Jihong Han designed experiments and prepared the paper.

FUNDING This work was supported by the Ministry of Science and Technology of China [grant number 2010CB945003 (to J.H.)]; the National Science Foundation of China (NSFC) [grant numbers 81272460 (to J.H.) and 81000128 (to Y.D.)]; and the Tianjin Municipal Science and Technology Commission of China [grant number 11JCYBJC10600 (to J.H.)].

REFERENCES 1 Haunerland, N. H. and Spener, F. (2004) Fatty acid-binding proteins: insights from genetic manipulations. Prog. Lipid Res. 43, 328–349 2 Chmurzynska, A. (2006) The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism. J. Appl. Genet. 47, 39–48 3 Spiegelman, B. M., Frank, M. and Green, H. (1983) Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J. Biol. Chem. 258, 10083–10089 4 Hotamisligil, G. S., Johnson, R. S., Distel, R. J., Ellis, R., Papaioannou, V. E. and Spiegelman, B. M. (1996) Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein. Science 274, 1377–1379 5 Uysal, K. T., Scheja, L., Wiesbrock, S. M., Bonner-Weir, S. and Hotamisligil, G. S. (2000) Improved glucose and lipid metabolism in genetically obese mice lacking aP2. Endocrinology 141, 3388–3396 6 Shao, D. and Lazar, M. A. (1997) Peroxisome proliferator activated receptor γ , CCAAT/enhancer-binding protein α, and cell cycle status regulate the commitment to adipocyte differentiation. J. Biol. Chem. 272, 21473–21478 7 Hu, W., Zhou, X., Jiang, M., Duan, Y., Chen, Y., Li, X., Yin, Z., He, G. W., Yao, Z., Zhu, Y. et al. (2012) Statins synergize dexamethasone-induced adipocyte fatty acid binding protein expression in macrophages. Atherosclerosis 222, 434–443 8 Llaverias, G., Noe, V., Penuelas, S., Vazquez-Carrera, M., Sanchez, R. M., Laguna, J. C., Ciudad, C. J. and Alegret, M. (2004) Atorvastatin reduces CD68, FABP4, and HBP expression in oxLDL-treated human macrophages. Biochem. Biophys. Res. Commun. 318, 265–274 9 Fu, Y., Luo, N., Lopes-Virella, M. F. and Garvey, W. T. (2002) The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages. Atherosclerosis 165, 259–269 10 Boord, J. B., Maeda, K., Makowski, L., Babaev, V. R., Fazio, S., Linton, M. F. and Hotamisligil, G. S. (2002) Adipocyte fatty acid-binding protein, aP2, alters late atherosclerotic lesion formation in severe hypercholesterolemia. Arterioscler., Thromb., Vasc. Biol. 22, 1686–1691 11 Makowski, L., Boord, J. B., Maeda, K., Babaev, V. R., Uysal, K. T., Morgan, M. A., Parker, R. A., Suttles, J., Fazio, S., Hotamisligil, G. S. and Linton, M. F. (2001) Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis. Nat. Med. 7, 699–705 12 Perrella, M. A., Pellacani, A., Layne, M. D., Patel, A., Zhao, D., Schreiber, B. M., Storch, J., Feinberg, M. W., Hsieh, C. M., Haber, E. and Lee, M. E. (2001) Absence of adipocyte fatty acid binding protein prevents the development of accelerated atherosclerosis in hypercholesterolemic mice. FASEB J. 15, 1774–1776 13 Peeters, W., de Kleijn, D. P., Vink, A., van de Weg, S., Schoneveld, A. H., Sze, S. K., van der Spek, P. J., de Vries, J. P., Moll, F. L. and Pasterkamp, G. (2011) Adipocyte fatty acid binding protein in atherosclerotic plaques is associated with local vulnerability and is predictive for the occurrence of adverse cardiovascular events. Eur. Heart J. 32, 1758–1768 14 von Eynatten, M., Breitling, L. P., Roos, M., Baumann, M., Rothenbacher, D. and Brenner, H. (2012) Circulating adipocyte fatty acid-binding protein levels and cardiovascular morbidity and mortality in patients with coronary heart disease: a 10-year prospective study. Arterioscler., Thromb., Vasc. Biol. 32, 2327–2335 15 Desta, Z., Ward, B. A., Soukhova, N. V. and Flockhart, D. A. (2004) Comprehensive evaluation of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro : prominent roles for CYP3A and CYP2D6. J. Pharmacol. Exp. Ther. 310, 1062–1075  c The Authors Journal compilation  c 2013 Biochemical Society

16 Lin, S. X., Chen, J., Mazumdar, M., Poirier, D., Wang, C., Azzi, A. and Zhou, M. (2010) Molecular therapy of breast cancer: progress and future directions. Nat. Rev. Endrocrinol. 6, 485–493 17 Jordan, V. C. (2006) Tamoxifen (ICI46,474) as a targeted therapy to treat and prevent breast cancer. Br. J. Pharmacol. 147 Suppl. 1, S269–S276 18 Stamatelopoulos, K. S., Lekakis, J. P., Poulakaki, N. A., Papamichael, C. M., Venetsanou, K., Aznaouridis, K., Protogerou, A. D., Papaioannou, T. G., Kumar, S. and Stamatelopoulos, S. F. (2004) Tamoxifen improves endothelial function and reduces carotid intima-media thickness in postmenopausal women. Am. Heart J. 147, 1093–1099 19 Simon, T., Boutouyrie, P., Simon, J. M., Laloux, B., Tournigand, C., Tropeano, A. I., Laurent, S. and Jaillon, P. (2002) Influence of tamoxifen on carotid intima-media thickness in postmenopausal women. Circulation 106, 2925–2929 20 Clarke, S. C., Schofield, P. M., Grace, A. A., Metcalfe, J. C. and Kirschenlohr, H. L. (2001) Tamoxifen effects on endothelial function and cardiovascular risk factors in men with advanced atherosclerosis. Circulation 103, 1497–1502 21 Grainger, D. J., Witchell, C. M. and Metcalfe, J. C. (1995) Tamoxifen elevates transforming growth factor-β and suppresses diet-induced formation of lipid lesions in mouse aorta. Nat. Med. 1, 1067–1073 22 Reckless, J., Metcalfe, J. C. and Grainger, D. J. (1997) Tamoxifen decreases cholesterol sevenfold and abolishes lipid lesion development in apolipoprotein E knockout mice. Circulation 95, 1542–1548 23 Williams, J. K., Wagner, J. D., Li, Z., Golden, D. L. and Adams, M. R. (1997) Tamoxifen inhibits arterial accumulation of LDL degradation products and progression of coronary artery atherosclerosis in monkeys. Arterioscler., Thromb., Vasc. Biol. 17, 403–408 24 Ouimet, M. and Marcel, Y. L. (2012) Regulation of lipid droplet cholesterol efflux from macrophage foam cells. Arterioscler., Thromb., Vasc. Biol. 32, 575–581 25 Han, J., Parsons, M., Zhou, X., Nicholson, A. C., Gotto, Jr, A. M. and Hajjar, D. P. (2004) Functional interplay between the macrophage scavenger receptor class B type I and pitavastatin (NK-104). Circulation 110, 3472–3479 26 Sui, Y., Xu, J., Rios-Pilier, J. and Zhou, C. (2011) Deficiency of PXR decreases atherosclerosis in apoE-deficient mice. J. Lipid Res. 52, 1652–1659 27 Sun, L., Nicholson, A. C., Hajjar, D. P., Gotto, Jr, A. M. and Han, J. (2003) Adipogenic differentiating agents regulate expression of fatty acid binding protein and CD36 in the J744 macrophage cell line. J. Lipid Res. 44, 1877–1886 28 Chen, Y., Duan, Y., Kang, Y., Yang, X., Jiang, M., Zhang, L., Li, G., Yin, Z., Hu, W., Dong, P. et al. (2012) Activation of liver X receptor induces macrophage interleukin-5 expression. J. Biol. Chem. 287, 43340–43350 29 Pelton, P. D., Zhou, L., Demarest, K. T. and Burris, T. P. (1999) PPARγ activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes. Biochem. Biophys. Res. Commun. 261, 456–458 30 Fu, Y., Luo, N. and Lopes-Virella, M. F. (2000) Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages. J. Lipid Res. 41, 2017–2023 31 Nagy, L., Tontonoz, P., Alvarez, J. G., Chen, H. and Evans, R. M. (1998) Oxidized LDL regulates macrophage gene expression through ligand activation of PPARγ . Cell 93, 229–240 32 Murphy, E. (2011) Estrogen signaling and cardiovascular disease. Circ. Res. 109, 687–696 33 Ewer, M. S. and Gluck, S. (2009) A woman’s heart: the impact of adjuvant endocrine therapy on cardiovascular health. Cancer 115, 1813–1826 34 Santen, R. J. (2011) Clinical review: effect of endocrine therapies on bone in breast cancer patients. J. Clin. Endocrinol. Metab. 96, 308–319 35 Wiseman, H., Cannon, M., Arnstein, H. R. and Halliwell, B. (1993) Tamoxifen inhibits lipid peroxidation in cardiac microsomes. Comparison with liver microsomes and potential relevance to the cardiovascular benefits associated with cancer prevention and treatment by tamoxifen. Biochem. Pharmacol. 45, 1851–1855 36 Wiseman, H., Paganga, G., Rice-Evans, C. and Halliwell, B. (1993) Protective actions of tamoxifen and 4-hydroxytamoxifen against oxidative damage to human low-density lipoproteins: a mechanism accounting for the cardioprotective action of tamoxifen? Biochem. J. 292, 635–638 37 Holleran, A. L., Lindenthal, B., Aldaghlas, T. A. and Kelleher, J. K. (1998) Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes. Metab., Clin. Exp. 47, 1504–1513 38 Gylling, H., Mantyla, E. and Miettinen, T. A. (1992) Tamoxifen decreases serum cholesterol by inhibiting cholesterol synthesis. Atherosclerosis 96, 245–247 39 Suarez, Y., Fernandez, C., Gomez-Coronado, D., Ferruelo, A. J., Davalos, A., Martinez-Botas, J. and Lasuncion, M. A. (2004) Synergistic upregulation of low-density lipoprotein receptor activity by tamoxifen and lovastatin. Cardiovasc. Res. 64, 346–355

Tamoxifen inhibits macrophage FABP4 expression 40 Love, R. R., Wiebe, D. A., Newcomb, P. A., Cameron, L., Leventhal, H., Jordan, V. C., Feyzi, J. and DeMets, D. L. (1991) Effects of tamoxifen on cardiovascular risk factors in postmenopausal women. Ann. Intern. Med. 115, 860–864 41 Dewar, J. A., Horobin, J. M., Preece, P. E., Tavendale, R., Tunstall-Pedoe, H. and Wood, R. A. (1992) Long term effects of tamoxifen on blood lipid values in breast cancer. Br. Med. J. 305, 225–226

477

42 Dong, P., Xie, T., Zhou, X., Hu, W., Chen, Y., Duan, Y., Li, X. and Han, J. (2011) Induction of macrophage scavenger receptor type BI expression by tamoxifen and 4-hydroxytamoxifen. Atherosclerosis 218, 435–442 43 Cook, J. S., Lucas, J. J., Sibley, E., Bolanowski, M. A., Christy, R. J., Kelly, T. J. and Lane, M. D. (1988) Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP. Proc. Natl. Acad. Sci. U.S.A. 85, 2949–2953

Received 25 April 2013/24 June 2013; accepted 28 June 2013 Published as BJ Immediate Publication 28 June 2013, doi:10.1042/BJ20130580

 c The Authors Journal compilation  c 2013 Biochemical Society