Tamoxifen Inhibits The Expression Of FABP4in Macrophages Through The Combined Effects Of GR And PPARγ Pathways

Cardiovascular disease is the biggest threat to our health and life. Coronary heart disease (CHD) is a major disease in cardiovascular system. The atherosclerotic lesion is the one of major pathogens for CHD. Formation of atherosclerosis is a complex process including endothelial injury, monocyte adhesion, macrophage differentiation and accumulation, smooth muscle cells proliferation and production of a variety of chemotactic factors and cytokines in arteries. During the development of atherosclerosis, monocyte-derived macrophages express macrophage scavenger receptors which bind and internalize large amounts of low-density lipoprotein (LDL) and modified LDL. The accumulation of lipids in macrophages drives the formation of foam cell, the major component in lesions. Thus, the formation of foam cells is the initial and critical step in the lesion development.Fatty acid binding proteins (FABPs) is a family of cytoplasmic proteins with small molecular-weight (14-15kD) and capable of binding hydrophobic fatty acids with high affinity. Adipocyte fatty acid binding protein (FABP4, also known as A-FABP or aP2), is a member of the FABP family. FABP4is widely expressed by various tissues and cells with the highest leves in adipose tissues and macrophages. In addition to fatty aicd transport and modulation of intracellular lipid metabolism, FABP4also plays an important role in other biological processes, particularly in many aspects of metabolic syndrome. Genetic deletion of FABP4expression inhibits the high fat diet-induced atherosclerosis in mice, and over-expressed FABP4facilitates foam cell formation in macrophages. Thus, FABP4plays an important role in the development of atherosclerosis.Tamoxifen is a selective estrogen receptor modulator. It also functions as an anti-angiogenesis factor and protein kinase C inhibitor. By the action of cytochrome P4502D6(CYP2D6), tamoxifen is hydroxylated into4-hydroxytamoxifen, which has more potent modulator activity. Clinically, tamoxifen has been used for more than two decades to treat both the early and advanced ER-positive breast cancer. However, both pre-clinical and clinical studies also have suggested the cardioprotective effects of tamoxifen. For example, tamoxifen reduces the incidence of fatal myocardial infarction and the intima-media thickness of the common carotid artery in postmenopausal women. It also increases the endothelium-dependent flow-mediated dilation (ED-FMD) in men with advanced atherosclerosis. In animal models, tamoxifen reduces the high-fat diet induced atherosclerotic lesions in both male wild type and apoE deficient (apoE-/-) mice and surgically postmenopausal monkeys. In spite of the cardioprotective effects, molecular mechanisms by which tamoxifen functions have not been fully elucidated. In the study, we determined if tamoxifen inhibits the development of atherosclerosis is completed through the inhibition on macrophage FABP4expression. We also disclosed the involved mechanism of tamoxifen-inhibited macrophage FABP4expression.Firstly, we isolated peritoneal macrophages from both male and female mice. We treated the cells with tamoxifen and4-hydroxytamoxifen at different concentrations overnight. The results of Western blot indicate that tamoxifen or4-hydroxtamoxifen inhibited FABP4protein expression in macrophages isolated either from male or female mice incidating the inhibition of macrophage FABP4expression by tamoxifen is in a sex-independent manner. Our previous studies suggest that dexamethasone or dexamethasone plus pitavastatin or PPARy activation can induce macrophage FABP4expression. In this study, we observed that tamoxifen and4-hudroxytamoxifen are able to antagonize the above inductions. FABP4mRNA expression was also inhibited by tamoxifen and4-hydroxytamoxifen indicating the. regulation is at the transcriptional level. In deed, we found that tamoxifen and4-hydroxytamoxifen inhibited FABP4promoter activity. We further disclosed the GRE (nGRE/pGRE) and putative PPRE (PPRE1/PPRE2) in FABP4promoter and our study indicated that tamoxifen and4-hydroxytamoxifen inhibited FABP4transcriptuon by activating nGRE and inhibiting PPRE1activity. EMSA.indicated that tamoxifen and4-hydroxytamoxifen enhanced the binding of nGRE with nuclear proteins but reduced the affinity of PPRE1to nuclear proteins. In addition, we determined tamoxifen did not affecet GR expression but reduced dexamethasone-induced GR nuclear translocation by Western Blot and image analysis. To disclose the physiological relavence of inhibition of macrophage FABP4expression by tamoxifen, we initially determined the inhibitory effect of tamoxifen on FABP4expression in vivo. We fed apoE-/-female and male mice a high-fat diet (0.5%cholesterol and21%fat) or the diet containing tamoxifen (2mg/100g food) for one month. After treatment, the peritoneal macrophages and adipose tissuses were individually collected and used to determine changes in FABP4protein expression. We observed the similar inhibitory effects on macrophage or adipose FABP4expression in vivo between male and female mice further suggesting the regulation of FABP4expression by tamoxifen is sex-independent. We also determined the effects of tamoxifen and4-hydroxytamoxifen on FABP4protein expression in human monocyte-derived macrophages from a healthy donor’s blood. Similar to mouse macrophages and in vivo, tamoxifen and4-hydroxytamoxifen inhibited human macrophage FABP4protein expression.Finally, to disclose if the inhibition of FABP4expression by tamoxifen would impact the development of atherosclerosis in apoE-/-female mice (the effect on male mice has been reported), we fed the mice a high-fat diet or the diet containing tamoxifen (2mg/100g food) for16weeks. We observed that tamoxifen inhibited the development of atherosclerosis in en face aortas induced by the Western diet. By using FABP4siRNA, we observed that the inhibition of foam cells by tamoxifen was completed by inhibiting macrophage FABP4expression that can be attributed to the inhibitory properties of tamoxifen on atherosclerosis.Taken together, for the first time, our study suggests that tamoxifen inhibits macrophage FABP4expression by a GR and PPARy combinational pathway. Tamoxifen inhibits the development of atherosclerosis is partially related to its inhibition of macrophage FABP4expression. Also, our study may have implication for the clinical treatment of atherosclerosis.