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FGF21 protects against ox-LDL induced apoptosis through suppressing CHOP expression in THP1 macrophage derived foam cells
© Li et al. 2015
Received: 10 October 2014
Accepted: 24 July 2015
Published: 30 July 2015
FGF21,as a member of the fibroblast growth factor superfamily, is an important endogenous regulator to systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects with coronary heart disease and carotid artery plaques. The formation and apoptosis of foam cell, induced by ox-LDL and oxysterols, are key steps in the development of atherosclerosis.
In this study, THP1 derived macrophages were induced into foam cells by ox-LDL or sterols. The formation and apoptosis of foam cells treated with or without FGF21 were analyzed.
We demonstrated that the accumulation of cholesterol was decreased after FGF21 treatment in THP1 macrophage derived foam cells. Consistently, the apoptosis of macrophage was alleviated dramatically with FGF21 treatment. ERK1/2 knockdown didn’t abrogate the effect of FGF21 on THP1 macrophage derived foam cells. However, FGF21 suppressed the induced expression of CHOP and DR5 in THP1 macrophage derived foam cells.
FGF21 protects against the formation and apoptosis of THP1 macrophages derived foam cells through suppressing the expression of CHOP.
Fibroblast growth factor (FGF) superfamily,, has an important functions in metabolic processes. Among members of the FGF superfamily, FGF21 plays a very important regulatory role in glucose and lipid homeostasis [1–3].
The role of FGF21 in glucose and lipid metabolism has been well studied [4, 5]. Human recombinant FGF21 has been demonstrated to stimulate glucose incorporation in mouse and human adipocytes, and to lower blood glucose and triglyceride levels in diabetic and obese mice as well as diabetic monkeys . By contrast, FGF21 deficient mice showed mild weight gain, slightly impaired glucose homeostasis, and also developed hepatosteatosis and obvious impairments in ketogenesis and glucose control when raised on a ketogenic diet . These findings suggest that FGF21 is an important metabolic hormone in maintaining glucose and lipid homeostasis. Recently, it is reported that serum FGF21 levels are increased in coronary heart disease (CHD) and FGF21 is also found in carotid artery plaques [5, 8, 9]. In myocardial infarction, FGF21could attenuate pathological myocardial remodeling through the adiponectin-dependent mechanism . What’s more, FGF21 have also been found to attenuate hyperlipidemia and diabetes induced early-stage apoptosis . Based on these results, FGF21 has been proposed to be associated with arteriosclerosis. However, the role of FGF21 in arteriosclerosis remains unclear.
In normal macrophages, low density lipoprotein (LDL) cholesterol particles are loaded from late endosomes to the ER. In the ER, cholesterol is esterified and accumulated to form inert lipid droplets [12, 13]. In atherosclerotic macrophages, ER-mediated cholesterol reesterification is markedly impaired resulting in excessive intracellular deposits of nonesterified cholesterol and the formaition of foam cells , where intraluminal ER oxidoreductases oxidize cholesterol to 7-ketocholesterol (7-KC) and other oxysterols. Oxysterols are highly cytotoxic and may induce cell death through ROS-mediated oxidative damage . Prolonged ER stress contributes to apoptosis of lesional macrophages, which is associated with robust expression of C/EBP homologous protein (CHOP) in human lesions  and atherosclerotic plaques of apolipoprotein (apo) E-deficient mice . Inactivating CHOP in apoE-deficient mice slows down macrophage apoptosis and plaque necrosis [17–19]. CHOP contributes to ER stress-induced macrophage death by inducing Fas activation, depletion of ER-associated calcium stores, and release of apoptogens from mitochondria . Moreover, CHOP is found to induce cell apoptosis through activating death receptor 5 (DR5) in human carcinoma cells .
Isolation and oxidation of Low density lipoprotein
The native low-density lipoprotein (LDL) was obtained from Sigma. LDL was oxidized with CuSO4 at 37 °C for 18 h and transferred into ethylene diamine tetraacetic acid (EDTA; 200 mM) in phosphate-buffered saline (PBS) for 24 h at 4 °C to remove Cu2+. Subsequently, the product was dialyzed in PBS for 24 h at 4 °C to remove EDTA. LDL oxidation was confirmed by thiobarbituric acid reaction substances with malondialdehyde as the standard. The content of ox-LDL was 1.12 compared with 0.30 nmol/100 mg protein in the native LDL preparation (p < 0.01). The ox-LDL was then sterilized by filtration and stored at 4 °C as previously described .
The human THP-1 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640, Hyclone) containing 10 % fetal bovine serum and 2 mM L-glutamine. The cells were differentiated into macrophages by adding 100 ng/mL phorbol 12-myristate-13-acetate for 24 h, and the medium was then replaced with that containing ox-LDL (50 mg/mL) and human FGF21 (Peprotech, 20 nmol/L) for 24 h to obtain fully differentiated foam cells before use in experiments. And human FGF21 (Peprotech, 20 nmol/L) was added to cotreat the THP1 macrophage derived foam cell.
THP-1 cells were transfected with specific siRNA oligomers directed against Erk (80 nM) using Lipofectaminqe 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Negative control siRNA oligomers were used as a negative control. After transfection for 48 h, the cells were exposed to ox-LDL (50 mg/L) for 24 h. The silencing of target genes was validated by western blotting.
Total proteins and nuclear proteins from cells were extracted using RIPA lysis buffer and nuclear extraction kits, respectively, following the manufacturer’s instructions. Equal amounts of protein were separated by SDS-PAGE and probed with various primary antibodies as indicated. Immunoblots were visualized using ECL reagent, and the integrated optical density (IOD) of immunoreactive bands was measured using Image-Pro Plus software and normalized by house-keeping protein (GAPDH).
Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen). 2 μg of total RNA was reversely transcripted using MMLV Reverse Transcriptase (Invitrogen). Real-time PCR was performed on a Rotor-Gene Q real-time PCR cycler (Roche, Shanghai, China) using SYBR-green PCR master mix kits. The data were analyzed using the Rotor-Gene Q software (version 1.7, Qiagen), and relative mRNA levels were calculated using the 2--△△Ct method and normalized against 18S rRNA. The primers used for real-time PCR were synthesized by Sangon Biotech (Shanghai, China).
Analysis of variance was conducted to examine whether significant (P, 0.05) main treatment and time effects occurred. Additional post hoc comparisons of treatment means were conducted by using the Dunnett’s t-test (treatments vs. controls) and Bonferroni t-test (selected comparisons) as indicated. Data given represent meansstandard deviation.
FGF21 decreases the formation of foam cell fromTHP1 derived macrophages
FGF21 protects against ox-LDL and 7-KC induced macrophage apoptosis
The effect of FGF21 on macrophages is independent upon ERK signaling pathway
FGF21 suppresses CHOP expression induced by ox-LDL and 7-KC in macrophages
So, the conclusion is that the protection effect of FGF21 on macrophage apoptosis is through CHOP pathway suppression.
Discussion and conclusion
FGF21, as a member of hormone-like subgroup within the FGF superfamily, is emerging as a key regulator of energy homeostasis and a potential target for the treatment of diabetes, cardiovascular disease, and obesity [4, 8, 23]. A previous study showed that incubation of rodent cardiac microvascular endothelial cells (CMECs) with oxidized LDL led to increased FGF21 expression and inhibited CMEC apoptosis . These results supported the hypothesis that FGF21 function as an endogenous protective factor in the cardiovascular system can improve endothelial function during early stages of atherosclerosis. Moreover, elevated serum FGF21 levels have recently been reported in patients with CHD and are associated with the presence of carotid artery plaques [5, 8]. However, the role of FGF21 in atherosclerosis plaques remains unclear. In this study, we present that FGF21 can protect macrophage against foam cell formation and apoptosis.
In summary, FGF21 repressed the cholesterol accumulation and foam cell formation in THP1 derived macrophage induced by ox-LDL or 7-KC. And the apoptosis of foam cell was decreased with the treatment of FGF21. Moreover,the effect of FGF21 on THP1 derived macrophages was ERK1/2 MAPK pathway independent,and was mediated through suppressing the expression of CHOP and its downstream target DR5. These findings provide evidence for the role of FGF21 in arteriosclerosis and present the new direction for atherosclerosis prevention.
This work was supported by grants from science and technology projects in Henan province (No. 142102310109 and 122102310088). We are grateful to Prof. Zongfang Liu and Prof. Lihua Zhang for helpful discussions and technical assistance. We thank Prof. Jian Liguo for helpful comments.
- Cuevas-Ramos D, Aguilar-Salinas CA, Gomez-Perez FJ. Metabolic actions of fibroblast growth factor 21. Curr Opin Pediatr. 2012;24(4):523–9.View ArticlePubMedGoogle Scholar
- Ding X, Boney-Montoya J, Owen BM, Bookout AL, Coate KC, Mangelsdorf DJ, et al. betaKlotho is required for fibroblast growth factor 21 effects on growth and metabolism. Cell Metab. 2012;16(3):387–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Woo YC, Xu A, Wang Y, Lam KS. Fibroblast growth factor 21 as an emerging metabolic regulator: clinical perspectives. Clin Endocrinol (Oxf). 2013;78(4):489–96.View ArticleGoogle Scholar
- Habegger KM, Stemmer K, Cheng C, Muller TD, Heppner KM, Ottaway N, et al. Fibroblast growth factor 21 mediates specific glucagon actions. Diabetes. 2013;62(5):1453–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin Z, Wu Z, Yin X, Liu Y, Yan X, Lin S, et al. Serum levels of FGF-21 are increased in coronary heart disease patients and are independently associated with adverse lipid profile. PLoS One. 2010;5(12), e15534.View ArticlePubMedPubMed CentralGoogle Scholar
- Kharitonenkov A, Wroblewski VJ, Koester A, Chen YF, Clutinger CK, Tigno XT, et al. The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology. 2007;148(2):774–81.View ArticlePubMedGoogle Scholar
- Badman MK, Koester A, Flier JS, Kharitonenkov A, Maratos-Flier E. Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis. Endocrinology. 2009;150(11):4931–40.View ArticlePubMedPubMed CentralGoogle Scholar
- An SY, Lee MS, Yi SA, Ha ES, Han SJ, Kim HJ, et al. Serum fibroblast growth factor 21 was elevated in subjects with type 2 diabetes mellitus and was associated with the presence of carotid artery plaques. Diabetes Res Clin Pract. 2012;96(2):196–203.View ArticlePubMedGoogle Scholar
- Li G, Gu HM, Zhang DW. ATP-binding cassette transporters and cholesterol translocation. IUBMB Life. 2013;65(6):505–12.View ArticlePubMedGoogle Scholar
- Joki Y, Ohashi K, Yuasa D, Shibata R, Ito M, Matsuo K, et al. FGF21 attenuates pathological myocardial remodeling following myocardial infarction through the adiponectin-dependent mechanism. Biochem Biophys Res Commun. 2015;459(1):124–30.View ArticlePubMedGoogle Scholar
- Zhang C, Shao M, Yang H, Chen L, Yu L, Cong W, et al. Attenuation of hyperlipidemia- and diabetes-induced early-stage apoptosis and late-stage renal dysfunction via administration of fibroblast growth factor-21 is associated with suppression of renal inflammation. PLoS One. 2013;8(12), e82275.View ArticlePubMedPubMed CentralGoogle Scholar
- Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223–61.View ArticlePubMedGoogle Scholar
- Maxfield FR, Tabas I. Role of cholesterol and lipid organization in disease. Nature. 2005;438(7068):612–21.View ArticlePubMedGoogle Scholar
- Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, et al. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol. 2003;5(9):781–92.View ArticlePubMedGoogle Scholar
- Myoishi M, Hao H, Minamino T, Watanabe K, Nishihira K, Hatakeyama K, et al. Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome. Circulation. 2007;116(11):1226–33.View ArticlePubMedGoogle Scholar
- Zhou J, Lhotak S, Hilditch BA, Austin RC. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation. 2005;111(14):1814–21.View ArticlePubMedGoogle Scholar
- Thorp E, Li G, Seimon TA, Kuriakose G, Ron D, Tabas I. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe−/− and Ldlr−/− mice lacking CHOP. Cell Metab. 2009;9(5):474–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Tsukano H, Gotoh T, Endo M, Miyata K, Tazume H, Kadomatsu T, et al. The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2010;30(10):1925–32.View ArticlePubMedGoogle Scholar
- Ozcan L, Tabas I. Pivotal role of calcium/calmodulin-dependent protein kinase II in ER stress-induced apoptosis. Cell Cycle. 2010;9(2):223–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Fisher FM, Kleiner S, Douris N, Fox EC, Mepani RJ, Verdeguer F, et al. FGF21 regulates PGC-1alpha and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 2012;26(3):271–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Hu YW, Wang Q, Ma X, Li XX, Liu XH, Xiao J, et al. TGF-beta1 up-regulates expression of ABCA1, ABCG1 and SR-BI through liver X receptor alpha signaling pathway in THP-1 macrophage-derived foam cells. J Atheroscler Thromb. 2010;17(5):493–502.View ArticlePubMedGoogle Scholar
- Lu P, Yan J, Liu K, Garbacz WG, Wang P, Xu M, et al. Activation of aryl hydrocarbon receptor dissociates fatty liver from insulin resistance by inducing fibroblast growth factor 21. Hepatology. 2015.Google Scholar
- Jung UJ, Torrejon C, Chang CL, Hamai H, Worgall TS, Deckelbaum RJ. Fatty acids regulate endothelial lipase and inflammatory markers in macrophages and in mouse aorta: a role for PPARgamma. Arterioscler Thromb Vasc Biol. 2012;32(12):2929–37.View ArticlePubMedPubMed CentralGoogle Scholar
- Kosinski JR, Hubert J, Carrington PE, Chicchi GG, Mu J, Miller C, et al. The glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin. Obesity (Silver Spring). 2012;20(8):1566–71.View ArticleGoogle Scholar
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