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IL-1β pathway
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
A novel urotensin II receptor antagonist, KR-36676, prevents ABCA1 repression via ERK/IL-1β pathway Mi-Young Kima,1, Sattorov Ilyosbeka,1, Byung Ho Leeb, Kyu Yang Yib, Yi-Sook Junga,c, a b c
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College of Pharmacy, Ajou University, Worldcupro 206, Suwon 16499, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea Research Institute of Pharmaceutical Sciences and Technology, Ajou University, Suwon 16499, Republic of Korea
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Keywords: Atherosclerosis Urotensin II receptor antagonist KR-36676 ATP binding cassette transporter-A1 Interleukin-1β
Urotensin II (U-II), the most potent vasoconstrictor peptide known to date, is expressed at a high level in vascular smooth muscle cells (VSMC) and endothelial cells, whereas its receptor, urotensin (UT) receptor, is abundant in monocytes and macrophages of atherosclerotic lesions. U-II is highly present in the coronary arteries of the atherosclerotic patients compared to normal subjects. Recently, U-II was shown to down-regulate ATP binding cassette transporter-A1 (ABCA1) expression, which is responsible for reverse cholesterol transport in macrophages of atherosclerotic lesions. However, the mechanism of this observation was not clearly elucidated. Previous studies also revealed that the proinflammatory cytokine interleukin-1β (IL-1β) repressed ABCA1 expression. To clarify the signaling pathway involved with respect to U-II-induced ABCA1 downregulation, we investigated whether IL-1ß was involved. Our results provided that U-II repressed ABCA1 through an ERK/ IL-1ß pathway. We further demonstrated that U-II receptor antagonist KR-36676 decreased IL-1ß production and significantly led to a recovery of ABCA1 expression at both mRNA and protein levels. In previous investigations, U-II receptor antagonists have been shown to protect atherosclerosis in cell and animal models. Our results imply that U-II receptor antagonist KR-36676 might be a potent candidate for treating atherosclerosis, and leading to a recovery of ABCA1 expression, affected by the ERK/IL-1ß pathway.
1. Introduction Atherosclerosis is an inflammatory disease of the cardiovascular system, characterized by accumulation of lipids and collagen-like fibrous elements in the human arteries. In the initial stages of the resulting arterial lesions, formation of the foam cells is an early sign of atherosclerotic plaque progression (Lusis, 2000). Urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, is expressed at a high level in vascular smooth muscle cells (VSMC), endothelial cells of the cardiovascular system, motor neurons and cardiac fibroblasts, whereas urotensin (UT) receptor is abundant in monocytes and macrophages of atherosclerotic lesions (Bousette et al., 2004; Tolle and van der Giet, 2008). Patients with coronary artery disease have recorded higher U-II levels than normal subjects (Heringlake et al., 2004), and the role of U-II on atherosclerosis has been investigated in a number of studies. In the case of immune cells, U-II led to increased cytokines, TNF-α, IL-1β, and MIF, as they lead to the migration of monocytes into the endothelial layer of vascular system (Tomiyama et al., 2015).
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Furthermore, U-II increases the progression of foam cell formation by up-regulating acetyl-coenzyme A acetyltransferase 1 (ACAT-1) in human monocyte derived macrophages (Watanabe et al., 2005). Extra, unneeded “free” cholesterol is toxic to macrophages and can lead to activation of the unfolded protein response and ultimately to apoptosis (Cuchel and Rader, 2006). To maintain cholesterol homeostasis, ATP binding cassette transporter A1 (ABCA1), abundant in the membrane of macrophages, mediates cholesterol and phospholipid (PL) efflux to lipid-poor apoA1 and plays a pivotal role in reverse cholesterol transport (Oram and Lawn, 2001). In ABCA1 gene knocked out mice, multiple pale foci lesions of pulmonary parenchyma were increased by 30%, and the lesions mainly consisted of foamy type II pneumocytes, intraalveolar macrophages, and cholesterol clefts when observed microscopically (McLaren et al., 2011). Recently, a hidden role for U-II in reverse cholesterol transportation was revealed by repressing ABCA1 in THP-1 macrophages, consequently speeding the foam cell formation, which leads to the atherosclerotic lesion progression (Wang et al., 2014). However, the underlying mechanism was not clearly elucidated.
Corresponding author at: College of Pharmacy, Ajou University, Worldcupro 206, Suwon 16499, Republic of Korea. E-mail address: [email protected] (Y.-S. Jung). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2017.03.056 Received 12 February 2017; Received in revised form 24 March 2017; Accepted 27 March 2017 0014-2999/ © 2017 Published by Elsevier B.V.
Please cite this article as: Kim, M.-Y., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.03.056
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2.3. RNA isolation and reverse transcription- polymerase chain reaction
The peptide and non-peptide U-II antagonists have been shown to have protective effects on cardiac dysfunction (Bousette et al., 2006), renal ischemia (Clozel et al., 2004) and atherosclerosis. The potent U-II inhibitor, Urantide resulted in a reduction of atherosclerotic plaques and by diminishing expression of U-II and UT receptor (Zhao et al., 2013). Recently, we demonstrated the pharmacological properties of U-II antagonist KR-36676 (2-(6,7-dichloro-3-oxo-2H-benzo[b][1,4] oxazin-4(3H)-yl)-N-methyl-N-(2-(pyrrolidin-1-yl)-1-(4-(thiophen-3yl) phenyl) ethyl) acetamide) including its binding affinity to UT receptor and its inhibitory effects on cardiac hypertrophy (Oh et al., 2015). KR-36676 blocked U-II-induced stress fiber formation and significantly reduced LV hypertrophy and cardiac dysfunction by having a high binding affinity to and high potency against the UT receptor. In the present study, we investigated U-II-induced ABCA1 repression at the molecular level and the effects of U-II antagonist KR36676 in allowing recovery of ABCA1.
Total RNA was extracted from THP-1 cells by using RNA extraction solution manufactured by Nanohelix (Daejon, Korea). For the RT-PCR, 2 µg of total RNA was first reverse transcribed by using random hexamers and M-MLV Reverse Transcriptase. The cDNA was amplified by using Taq DNA polymerase and specific primers. The PCR conditions with annealing temperature and primer extension time were dependent on each individual primer pairs (Yun et al., 2016). The targeted genes and primer sequences used in this study were as follows: ABCA1 (forward) 5′ CAT GCC CAG GAG ACT GGT TT 3′ and (reverse) 5′ GAC AAA TGG GCA CAG GCT TC 3′; IL-1ß (forward) 5′ ACA GGC TGC TCT GGG ATT CT 3′ and (reverse) 5′ TGA AGC CCT TGC CCA GTG AAA 3′. The PCR products were loaded and run in to 1% agarose gel containing 5 µl/100 ml EtBr stain from Inclone Biotech (Yongin, South Korea).
2. Materials and methods
2.4. Western immunoblotting analysis
2.1. Chemicals and reagents
Cells were washed twice by ice cold PBS and lysed in a buffer containing 150 mM NaCl, 50 mM Tris-HCl, 1%NP-40, 0.5% Nadeoxycholate, 1 mM PMSF, 2 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 10 µg/ml aprotinin and 10 µM leupeptin. Proteins was loaded and run on 10% SDS-polyacrylamide electrophoresis gel (SDS-PAGE) and transferred to a PVDF membrane. The membranes were incubated with primary and following secondary antibodies (Ma et al., 2016). Immunoreactivity was detected by ECL and visualized using LAS1000 (Fuji Film, Tokyo Japan). Protein quantity was measured using the Image J software (NIH, Bethesda, MD, USA).
RPMI 1640 media was from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). The mouse monoclonal anti-ABCA1 antibody (AB.H10) from Abcam (Cambridge, MA, USA) and antiphospho ERK, anti-ERK antibodies were purchased from Cell Signaling (Beverly, MA, USA). U0126 and SN50 were bought from Calbiochem (San Diego, CA, USA). The neutralizing anti-IL-1β antibody was from Invitrogen (Carlsbad, CA, USA). Human U-II were obtained from Sigma-Aldrich (St. Louis, MO, USA) and KR-36676 was synthesized at the Bio-Organic Division of the Korean Research Institute of Chemical Technology (Daejon, South Korea).
2.5. Statistical analysis All data were presented as mean ± S.E.M. Results were analyzed by one-way analysis of variance and student's t-test. A P-values < 0.05 was considered statistically significance.
2.2. Cell culture THP-1 human monocyte cell line was purchased from Korean Cell Line Bank (Seoul, South Korea). They were propagated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C temperature and 5% CO2. For monocyte/ macrophage differentiation, the cells were seeded at 0.5– 1.0×106 cell/ml in 6-well plates and treated with PMA (200 nM/L) for 24 h. Medium was changed to the serum-free version and the cells were then used for further experiments (Kulkarni et al., 2015).
3. Results 3.1. KR-36676 recovered U-II-induced down-regulation of ABCA1 Previous investigators had shown that urotensin II down-regulated ABCA1 level in THP-1 macrophages. In the present study, THP-1 cells treated with U-II (50 nM) showed a similar result by having reduced
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Fig. 1. Effect of KR-36676 on U-II-induced ABCA1 expression. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with U-II (50 nM). Relative mRNA levels (A) and protein levels (B) of ABCA1 were assayed by RTPCR and Western blot, respectively. Quantification was via normalization by the actin message. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
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Fig. 2. Effect of KR-36676 on U-II-induced p-ERK phosphorylation. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were incubated following U-II (50 nM) for different time h (A). The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with U-II (50 nM, 4 h) (B). ERK phosphorylation was measured by using Western blot. Quantification was normalized by ERK expression. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
(10 µM). U-II-induced IL-1β induction was completely blocked by treatment with U0126, suggesting ERK1/2 as an upstream signaling molecule of IL-1β.
ABCA1 mRNA and protein levels, implying U-II affected ABCA1 at both the level of the transcript and the protein product. Furthermore, KR-36676 also affected ABCA1 mRNA and protein expression, as the U-II-induced down-regulation of ABCA1 mRNA and protein expression was restored by KR-36676 in a concentration-dependent manner (Figs. 1A and 2A).
4. Discussion In the present study, we found for the first time that U-II-induced ABCA1 repression was mediated through IL-1ß. In addition, we demonstrated the inhibitory effect of a novel UT receptor inhibitor KR-36676 on U-II-induced IL-1ß induction and ABCA1 repression. U-II, a cyclic peptide, is highly expressed in the coronary arteries of the atherosclerotic patients compared to normal subjects. U-II is localized with macrophage infiltration within the human coronary artery atherosclerotic lesion area (Maguire et al., 2004). Furthermore, oxidized LDL was shown to increase U-II mRNA expression in rat aortic smooth muscle cells, which could contribute to the overexpression of the U-II in atherosclerotic lesions (Wang et al., 2006). At the sites of injury, pro-inflammatory molecules such as IL-1ß and TNF-α and adhesion molecules including I-CAM1 and V-CAM1 were found to be highly present as part of an inflammatory cascade triggered by U-II and early release of cytokines in lipopolysaccharide/ D-galactosamine challenged mice (Kwon et al., 2016; Liu et al., 2015). We first examined whether U-II participated in a crosstalk in cytokine production. Our data indicated that human U-II up-regulated IL-1ß in THP-1 cells. These findings are consistent with previous EA.hy926 cell and mouse studies and support the recorded proinflammatory properties of U-II (Liu et al., 2015; Park et al., 2013). In addition, blockade of UT receptor with KR-36676 significantly inhibited U-II-induced IL-1ß. It is known that increased plasma concentrations of the U-II and cytokine, adhesion molecules at the damaged lesions of atherosclerosis had positive correlations in human and in in vivo studies. Studies on U-II and the UT receptor system in the immune cells of amphibians have implied that U-II and the UT receptor system play a pivotal role in the regulation of two major immune functions, namely migration of the monocytes to the endothelial cell layer and cytokine production (Tomiyama et al., 2015). Our study also supports the notion that U-II functions in the proinflammatory cytokine regulation of human immune cells. Reverse Cholesterol Transportation (RCT) promotes excess cholesterol efflux from peripheral tissues to the blood as part of the synthesis required for biliary excretion in hepatocytes. RCT also has an inhibitory effect on atherosclerotic lesion development by speeding lipid efflux from macrophages to apolipoprotein A1 (ApoA-1) (Lewis and Rader,
3.2. KR-36676 inhibited U-II-dependent phosphorylation of ERK1/2 Phosphorylation of extracellular signal-regulated kinase (ERK1/2) is a well-known downstream marker of U-II activation. As shown in Fig. 2A, ERK1/2 phosphorylation began to increase at 30 min after treatment with U-II and increased to about 5-fold after 2 h of U-II stimulus. It was then remarkably reduced after 4 h of U-II stimulus. To analyze whether KR-36676 could attenuate activation of ERK1/2, THP-1 cells were pre-incubated with KR-36676 (10, 30, 100 nM) 1 h before U-II exposure. Pre-treatment with KR-36676 significantly inhibited the phosphorylation of ERK1/2 induced by U-II at 2 h in a concentration-dependent manner (Fig. 2B). 3.3. U-II-induced IL-1ß expression was inhibited by KR-36676 Next, we examined effect of U-II on IL-1ß expression in THP-1 cells by RT-PCR. The level of IL-1ß mRNA was increased by U-II (50 nM) in a time dependent manner, peaking at 4 h as demonstrated in Fig. 3A. To investigate whether, blockage of U-II would decrease IL-1ß expression, we pretreated cells with different concentrations of KR-36676 as the U-II receptor antagonist and at various concentrations (10, 30, 100 nM), and checked for IL-1β expression. KR-36676 inhibited the UII-induced IL-1β expression in a concentration-dependent manner (Fig. 3B). 3.4. U-II-induced ABCA1 repression is mediated by IL-1β As shown in Fig. 4A and B, we investigated whether ERK1/2 and IL-1β regulated the U-II-induced down-regulation of ABCA1 by using a neutralizing anti-IL-1β antibody and U0126. ABCA1 mRNA and protein levels were down-regulated by U-II exposure, and this downregulation was completely restored by treatment with U0126 and neutralizing anti-IL-1β antibody. These results suggest a participation of ERK1/2 and IL-1β in the U-II-induced down-regulation of ABCA1 expression. Next, we examined whether ERK1/2 was involved in the UII-induced IL-1β induction by using the ERK1/2 inhibitor, U0126 3
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Fig. 3. Effect of KR-36676 on U-II-induced IL-1β expression. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were incubated following U-II (50 nM) treatment for different time periods (A). The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with UII (50 nM, 4 h) (B). Relative mRNA levels of IL-1ß were assayed by RT-PCR. Quantification was via normalization with the actin message. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
ERK1/2 inhibitor in presence of U-II. Our results demonstrated that inhibition of ERK1/2 completely suppressed the increase in IL-1ß message. Our results suggest that early activation and phosphorylation of ERK1/2 is main activator of IL-1ß in the U-II-induced ABCA1 repression pathway. In summary, the present study demonstrated that U-II downregulated ABCA1 expression in THP-1 cells via the ERK/IL-1ß pathway. These findings provide a new insight to urotensin II and cholesterol homeostasis in athero-pathogenic conditions. The lipid accumulation in macrophages with formation of foam cells was significantly increased in macrophage ABCA1 deficient C57/BL6 mice (Aiello et al., 2002). Our studies contribute to the view that U-II accelerates foam cell formation and by a novel mechanism. Furthermore, IL-1β also stimulates recruitment of monocytes to the endothelial layer and the apoptosis of macrophages, which contributes to plaque vulnerability (McLaren et al., 2011; Park et al., 2013). The use of the U-II antagonist KR-36676 significantly ameliorated IL-1ß and recovered ABCA1 reduction implying that a blocking in the UT receptor pathway may have beneficial effects in management of atherosclerosis.
2005). In previous studies, proatherogenic cytokine IL-1β, which is abundant in atherosclerotic lesions, was shown to inhibit the expression of ABCA1. Our data implies that U-II-induced ABCA1 downregulation might be associated with up-regulation of IL-1β. Recently, U-II down-regulated ABCA1 levels, and increased foam cell formation in THP-1 macrophages (Wang et al., 2014). ERK1/2 was involved in the U-II-induced ABCA1 repression; however, the mechanism was not clearly elucidated. Here, we demonstrated a significant recovery in ABCA1 mRNA and protein levels by KR-36676, suggestive of a positive effect in blocking the UT receptor in inhibition of atherosclerosis. Activation of ERK1/2 by U-II is an immediate response and causes vascular smooth muscle cell proliferation with subsequent vascular remodeling in rat aortic smooth muscle cells. These alterations in vascular smooth muscle cells contribute to the atherosclerotic lesion development (Tsai et al., 2009). We observed that ERK 1/2 was activated as an immediate response to U-II, and that this activation was blocked by KR-36676 in dose dependent manner in THP-1 cells. Furthermore, U-II mediated an increase in IL-8 levels and increased the proliferation of human umbilical vein endothelial (HUVEC) cells through p38 and the ERK1/2 MAPK kinase pathway. We investigated whether IL-1ß might be associated with the ERK pathway in downregulation of ABCA1 levels. We incubated THP-1 cells with U0126, an
Fig. 4. Link between IL-1β and ABCA1 during U-II stimulus. THP-1 cells were pre-incubated with U0126 (10 μM), SN-50 (10 μg/ml) for 1 h before treatment with U-II (50 nM) for the indicated times. The relative mRNA levels of ABCA1 (A) and IL-1ß (C) were assayed by RT-PCR. THP-1 cells were pre-incubated with U0126 (10 μM) and anti-IL-1β (10 μg/ml) for 1 h before treatment with U-II (50 nM, 24 h). ABCA1 protein expression was measured by Western blot (B). Quantification was via normalization by expression of actin. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
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Liu, L.M., Zhao, L., Liang, D.Y., Yu, F.P., Ye, C.G., Tu, W.J., Zhu, T., 2015. Effects of urotensin-II on cytokines in early acute liver failure in mice. World J. Gastroenterol. 21, 3239–3244. Lusis, A.J., 2000. Atherosclerosis. Nature 407, 233–241. Ma, J.W., Zhang, Y., Ye, J.C., Li, R., Wen, Y.L., Huang, J.X., Zhong, X.Y., 2016. Tetrandrine exerts a Radiosensitization Effect on human glioma through inhibiting proliferation by attenuating ERK phosphorylation. Biomol. Ther. (Seoul.) 1 (2), 24, (123-31). Maguire, J.J., Kuc, R.E., Wiley, K.E., Kleinz, M.J., Davenport, A.P., 2004. Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries. Peptides 25, 1767–1774. McLaren, J.E., Michael, D.R., Ashlin, T.G., Ramji, D.P., 2011. Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy. Prog. Lipid Res. 50, 331–347. Oh, K.S., Lee, J.H., Yi, K.Y., Lim, C.J., Lee, S., Park, C.H., Seo, H.W., Lee, B.H., 2015. The orally active urotensin receptor antagonist, KR36676, attenuates cellular and cardiac hypertrophy. Br. J. Pharmacol. 172, 2618–2633. Oram, J.F., Lawn, R.M., 2001. ABCA1. The gatekeeper for eliminating excess tissue cholesterol. J. Lipid Res. 42, 1173–1179. Park, S.L., Lee, B.K., Kim, Y.A., Lee, B.H., Jung, Y.S., 2013. Inhibitory effect of an urotensin II receptor antagonist on proinflammatory activation induced by urotensin II in human vascular endothelial cells. Biomol. Ther. (Seoul.) 21, 277–283. Tolle, M., van der Giet, M., 2008. Cardiorenovascular effects of urotensin II and the relevance of the UT receptor. Peptides 29, 743–763. Tomiyama, S., Nakamachi, T., Uchiyama, M., Matsuda, K., Konno, N., 2015. Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis. Gen. Comp. Endocrinol. 216, 54–63. Tsai, C.S., Loh, S.H., Liu, J.C., Lin, J.W., Chen, Y.L., Chen, C.H., Cheng, T.H., 2009. Urotensin II-induced endothelin-1 expression and cell proliferation via epidermal growth factor receptor transactivation in rat aortic smooth muscle cells. Atherosclerosis 206, 86–94. Wang, Y., Wu, J.F., Tang, Y.Y., Zhang, M., Li, Y., Chen, K., Zeng, M.Y., Yao, F., Xie, W., Zheng, X.L., Zeng, G.F., Tang, C.K., 2014. Urotensin II increases foam cell formation by repressing ABCA1 expression through the ERK/NF-kappaB pathway in THP-1 macrophages. Biochem. Biophys. Res. Commun. 452, 998–1003. Wang, Z.J., Ding, W.H., Shi, L.B., Bu, D.F., Ren, Z.W., Tang, C.S., 2006. [Effects of oxidized-low density lipoprotein on expression of urotensin II receptor GPR14 in rat aortic smooth muscle cells]. Zhonghua Yi Xue Za Zhi 86, 242–246. Watanabe, T., Suguro, T., Kanome, T., Sakamoto, Y., Kodate, S., Hagiwara, T., Hongo, S., Hirano, T., Adachi, M., Miyazaki, A., 2005. Human urotensin II accelerates foam cell formation in human monocyte-derived macrophages. Hypertension 46, 738–744. Yun, J., Kim, S.Y., Yoon, K.S., Shin, H., Jeong, H.S., Chung, H., Kim, Y.H., Shin, J., Cha, H.J., Han, K.M., Hyeon, S., Lee, T.H., Park, H.K., Kim, H.S., 2016. P21 (Cdc42/Rac)activated kinase 1 (pak1) is associated with cardiotoxicity induced by antihistamines. Arch. Pharm. Res. 39 (12), 1644–1652. Zhao, J., Yu, Q.X., Kong, W., Gao, H.C., Sun, B., Xie, Y.Q., Ren, L.Q., 2013. The urotensin II receptor antagonist, urantide, protects against atherosclerosis in rats. Exp. Ther. Med. 5, 1765–1769.
Acknowledgement This research was supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C2417, HI16C0992). This work was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01060069). References Aiello, R.J., Brees, D., Bourassa, P.A., Royer, L., Lindsey, S., Coskran, T., Haghpassand, M., Francone, O.L., 2002. Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages. Arterioscler. Thromb. Vasc. Biol. 22, 630–637. Bousette, N., Hu, F., Ohlstein, E.H., Dhanak, D., Douglas, S.A., Giaid, A., 2006. Urotensin-II blockade with SB-611812 attenuates cardiac dysfunction in a rat model of coronary artery ligation. J. Mol. Cell Cardiol. 41, 285–295. Bousette, N., Patel, L., Douglas, S.A., Ohlstein, E.H., Giaid, A., 2004. Increased expression of urotensin II and its cognate receptor GPR14 in atherosclerotic lesions of the human aorta. Atherosclerosis 176, 117–123. Clozel, M., Binkert, C., Birker-Robaczewska, M., Boukhadra, C., Ding, S.S., Fischli, W., Hess, P., Mathys, B., Morrison, K., Muller, C., Muller, C., Nayler, O., Qiu, C., Rey, M., Scherz, M.W., Velker, J., Weller, T., Xi, J.F., Ziltener, P., 2004. Pharmacology of the urotensin-II receptor antagonist palosuran (ACT-058362; 1-[2-(4-benzyl-4-hydroxypiperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt): first demonstration of a pathophysiological role of the urotensin System. J. Pharmacol. Exp. Ther. 311, 204–212. Cuchel, M., Rader, D.J., 2006. Macrophage reverse cholesterol transport: key to the regression of atherosclerosis? Circulation 113, 2548–2555. Heringlake, M., Kox, T., Uzun, O., Will, B., Bahlmann, L., Klaus, S., Eleftheriadis, S., Armbruster, F.P., Franz, N., Kraatz, E., 2004. The relationship between urotensin II plasma immunoreactivity and left ventricular filling pressures in coronary artery disease. Regul. Pept. 121, 129–136. Kulkarni, N.M., Muley, M.M., Jaji, M.S., Vijaykanth, G., Raghul, J., Reddy, N.K., Vishwakarma, S.L., Rajesh, N.B., Mookkan, J., Krishnan, U.M., Narayanan, S., 2015. Topical atorvastatin ameliorates 12-O-tetradecanoylphorbol-13-acetate induced skin inflammation by reducing cutaneous cytokine levels and NF-kB activation. Arch. Pharm. Res. 38 (6), 1238–1247. Kwon, I.S., Yim, J.H., Lee, H.K., Pyo, S., 2016. Lobaric acid inhibits VCAM-1 expression in TNF-alpha-stimulated vascular smooth muscle cells via modulation of NF-kappaB and MAPK signaling pathways. Biomol. Ther. (Seoul.) 24, 25–32. Lewis, G.F., Rader, D.J., 2005. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ. Res. 96, 1221–1232.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
A novel urotensin II receptor antagonist, KR-36676, prevents ABCA1 repression via ERK/IL-1β pathway Mi-Young Kima,1, Sattorov Ilyosbeka,1, Byung Ho Leeb, Kyu Yang Yib, Yi-Sook Junga,c, a b c
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College of Pharmacy, Ajou University, Worldcupro 206, Suwon 16499, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea Research Institute of Pharmaceutical Sciences and Technology, Ajou University, Suwon 16499, Republic of Korea
A R T I C L E I N F O
A BS T RAC T
Keywords: Atherosclerosis Urotensin II receptor antagonist KR-36676 ATP binding cassette transporter-A1 Interleukin-1β
Urotensin II (U-II), the most potent vasoconstrictor peptide known to date, is expressed at a high level in vascular smooth muscle cells (VSMC) and endothelial cells, whereas its receptor, urotensin (UT) receptor, is abundant in monocytes and macrophages of atherosclerotic lesions. U-II is highly present in the coronary arteries of the atherosclerotic patients compared to normal subjects. Recently, U-II was shown to down-regulate ATP binding cassette transporter-A1 (ABCA1) expression, which is responsible for reverse cholesterol transport in macrophages of atherosclerotic lesions. However, the mechanism of this observation was not clearly elucidated. Previous studies also revealed that the proinflammatory cytokine interleukin-1β (IL-1β) repressed ABCA1 expression. To clarify the signaling pathway involved with respect to U-II-induced ABCA1 downregulation, we investigated whether IL-1ß was involved. Our results provided that U-II repressed ABCA1 through an ERK/ IL-1ß pathway. We further demonstrated that U-II receptor antagonist KR-36676 decreased IL-1ß production and significantly led to a recovery of ABCA1 expression at both mRNA and protein levels. In previous investigations, U-II receptor antagonists have been shown to protect atherosclerosis in cell and animal models. Our results imply that U-II receptor antagonist KR-36676 might be a potent candidate for treating atherosclerosis, and leading to a recovery of ABCA1 expression, affected by the ERK/IL-1ß pathway.
1. Introduction Atherosclerosis is an inflammatory disease of the cardiovascular system, characterized by accumulation of lipids and collagen-like fibrous elements in the human arteries. In the initial stages of the resulting arterial lesions, formation of the foam cells is an early sign of atherosclerotic plaque progression (Lusis, 2000). Urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, is expressed at a high level in vascular smooth muscle cells (VSMC), endothelial cells of the cardiovascular system, motor neurons and cardiac fibroblasts, whereas urotensin (UT) receptor is abundant in monocytes and macrophages of atherosclerotic lesions (Bousette et al., 2004; Tolle and van der Giet, 2008). Patients with coronary artery disease have recorded higher U-II levels than normal subjects (Heringlake et al., 2004), and the role of U-II on atherosclerosis has been investigated in a number of studies. In the case of immune cells, U-II led to increased cytokines, TNF-α, IL-1β, and MIF, as they lead to the migration of monocytes into the endothelial layer of vascular system (Tomiyama et al., 2015).
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Furthermore, U-II increases the progression of foam cell formation by up-regulating acetyl-coenzyme A acetyltransferase 1 (ACAT-1) in human monocyte derived macrophages (Watanabe et al., 2005). Extra, unneeded “free” cholesterol is toxic to macrophages and can lead to activation of the unfolded protein response and ultimately to apoptosis (Cuchel and Rader, 2006). To maintain cholesterol homeostasis, ATP binding cassette transporter A1 (ABCA1), abundant in the membrane of macrophages, mediates cholesterol and phospholipid (PL) efflux to lipid-poor apoA1 and plays a pivotal role in reverse cholesterol transport (Oram and Lawn, 2001). In ABCA1 gene knocked out mice, multiple pale foci lesions of pulmonary parenchyma were increased by 30%, and the lesions mainly consisted of foamy type II pneumocytes, intraalveolar macrophages, and cholesterol clefts when observed microscopically (McLaren et al., 2011). Recently, a hidden role for U-II in reverse cholesterol transportation was revealed by repressing ABCA1 in THP-1 macrophages, consequently speeding the foam cell formation, which leads to the atherosclerotic lesion progression (Wang et al., 2014). However, the underlying mechanism was not clearly elucidated.
Corresponding author at: College of Pharmacy, Ajou University, Worldcupro 206, Suwon 16499, Republic of Korea. E-mail address: [email protected] (Y.-S. Jung). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2017.03.056 Received 12 February 2017; Received in revised form 24 March 2017; Accepted 27 March 2017 0014-2999/ © 2017 Published by Elsevier B.V.
Please cite this article as: Kim, M.-Y., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.03.056
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2.3. RNA isolation and reverse transcription- polymerase chain reaction
The peptide and non-peptide U-II antagonists have been shown to have protective effects on cardiac dysfunction (Bousette et al., 2006), renal ischemia (Clozel et al., 2004) and atherosclerosis. The potent U-II inhibitor, Urantide resulted in a reduction of atherosclerotic plaques and by diminishing expression of U-II and UT receptor (Zhao et al., 2013). Recently, we demonstrated the pharmacological properties of U-II antagonist KR-36676 (2-(6,7-dichloro-3-oxo-2H-benzo[b][1,4] oxazin-4(3H)-yl)-N-methyl-N-(2-(pyrrolidin-1-yl)-1-(4-(thiophen-3yl) phenyl) ethyl) acetamide) including its binding affinity to UT receptor and its inhibitory effects on cardiac hypertrophy (Oh et al., 2015). KR-36676 blocked U-II-induced stress fiber formation and significantly reduced LV hypertrophy and cardiac dysfunction by having a high binding affinity to and high potency against the UT receptor. In the present study, we investigated U-II-induced ABCA1 repression at the molecular level and the effects of U-II antagonist KR36676 in allowing recovery of ABCA1.
Total RNA was extracted from THP-1 cells by using RNA extraction solution manufactured by Nanohelix (Daejon, Korea). For the RT-PCR, 2 µg of total RNA was first reverse transcribed by using random hexamers and M-MLV Reverse Transcriptase. The cDNA was amplified by using Taq DNA polymerase and specific primers. The PCR conditions with annealing temperature and primer extension time were dependent on each individual primer pairs (Yun et al., 2016). The targeted genes and primer sequences used in this study were as follows: ABCA1 (forward) 5′ CAT GCC CAG GAG ACT GGT TT 3′ and (reverse) 5′ GAC AAA TGG GCA CAG GCT TC 3′; IL-1ß (forward) 5′ ACA GGC TGC TCT GGG ATT CT 3′ and (reverse) 5′ TGA AGC CCT TGC CCA GTG AAA 3′. The PCR products were loaded and run in to 1% agarose gel containing 5 µl/100 ml EtBr stain from Inclone Biotech (Yongin, South Korea).
2. Materials and methods
2.4. Western immunoblotting analysis
2.1. Chemicals and reagents
Cells were washed twice by ice cold PBS and lysed in a buffer containing 150 mM NaCl, 50 mM Tris-HCl, 1%NP-40, 0.5% Nadeoxycholate, 1 mM PMSF, 2 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 10 µg/ml aprotinin and 10 µM leupeptin. Proteins was loaded and run on 10% SDS-polyacrylamide electrophoresis gel (SDS-PAGE) and transferred to a PVDF membrane. The membranes were incubated with primary and following secondary antibodies (Ma et al., 2016). Immunoreactivity was detected by ECL and visualized using LAS1000 (Fuji Film, Tokyo Japan). Protein quantity was measured using the Image J software (NIH, Bethesda, MD, USA).
RPMI 1640 media was from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). The mouse monoclonal anti-ABCA1 antibody (AB.H10) from Abcam (Cambridge, MA, USA) and antiphospho ERK, anti-ERK antibodies were purchased from Cell Signaling (Beverly, MA, USA). U0126 and SN50 were bought from Calbiochem (San Diego, CA, USA). The neutralizing anti-IL-1β antibody was from Invitrogen (Carlsbad, CA, USA). Human U-II were obtained from Sigma-Aldrich (St. Louis, MO, USA) and KR-36676 was synthesized at the Bio-Organic Division of the Korean Research Institute of Chemical Technology (Daejon, South Korea).
2.5. Statistical analysis All data were presented as mean ± S.E.M. Results were analyzed by one-way analysis of variance and student's t-test. A P-values < 0.05 was considered statistically significance.
2.2. Cell culture THP-1 human monocyte cell line was purchased from Korean Cell Line Bank (Seoul, South Korea). They were propagated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C temperature and 5% CO2. For monocyte/ macrophage differentiation, the cells were seeded at 0.5– 1.0×106 cell/ml in 6-well plates and treated with PMA (200 nM/L) for 24 h. Medium was changed to the serum-free version and the cells were then used for further experiments (Kulkarni et al., 2015).
3. Results 3.1. KR-36676 recovered U-II-induced down-regulation of ABCA1 Previous investigators had shown that urotensin II down-regulated ABCA1 level in THP-1 macrophages. In the present study, THP-1 cells treated with U-II (50 nM) showed a similar result by having reduced
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Fig. 1. Effect of KR-36676 on U-II-induced ABCA1 expression. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with U-II (50 nM). Relative mRNA levels (A) and protein levels (B) of ABCA1 were assayed by RTPCR and Western blot, respectively. Quantification was via normalization by the actin message. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
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Fig. 2. Effect of KR-36676 on U-II-induced p-ERK phosphorylation. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were incubated following U-II (50 nM) for different time h (A). The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with U-II (50 nM, 4 h) (B). ERK phosphorylation was measured by using Western blot. Quantification was normalized by ERK expression. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
(10 µM). U-II-induced IL-1β induction was completely blocked by treatment with U0126, suggesting ERK1/2 as an upstream signaling molecule of IL-1β.
ABCA1 mRNA and protein levels, implying U-II affected ABCA1 at both the level of the transcript and the protein product. Furthermore, KR-36676 also affected ABCA1 mRNA and protein expression, as the U-II-induced down-regulation of ABCA1 mRNA and protein expression was restored by KR-36676 in a concentration-dependent manner (Figs. 1A and 2A).
4. Discussion In the present study, we found for the first time that U-II-induced ABCA1 repression was mediated through IL-1ß. In addition, we demonstrated the inhibitory effect of a novel UT receptor inhibitor KR-36676 on U-II-induced IL-1ß induction and ABCA1 repression. U-II, a cyclic peptide, is highly expressed in the coronary arteries of the atherosclerotic patients compared to normal subjects. U-II is localized with macrophage infiltration within the human coronary artery atherosclerotic lesion area (Maguire et al., 2004). Furthermore, oxidized LDL was shown to increase U-II mRNA expression in rat aortic smooth muscle cells, which could contribute to the overexpression of the U-II in atherosclerotic lesions (Wang et al., 2006). At the sites of injury, pro-inflammatory molecules such as IL-1ß and TNF-α and adhesion molecules including I-CAM1 and V-CAM1 were found to be highly present as part of an inflammatory cascade triggered by U-II and early release of cytokines in lipopolysaccharide/ D-galactosamine challenged mice (Kwon et al., 2016; Liu et al., 2015). We first examined whether U-II participated in a crosstalk in cytokine production. Our data indicated that human U-II up-regulated IL-1ß in THP-1 cells. These findings are consistent with previous EA.hy926 cell and mouse studies and support the recorded proinflammatory properties of U-II (Liu et al., 2015; Park et al., 2013). In addition, blockade of UT receptor with KR-36676 significantly inhibited U-II-induced IL-1ß. It is known that increased plasma concentrations of the U-II and cytokine, adhesion molecules at the damaged lesions of atherosclerosis had positive correlations in human and in in vivo studies. Studies on U-II and the UT receptor system in the immune cells of amphibians have implied that U-II and the UT receptor system play a pivotal role in the regulation of two major immune functions, namely migration of the monocytes to the endothelial cell layer and cytokine production (Tomiyama et al., 2015). Our study also supports the notion that U-II functions in the proinflammatory cytokine regulation of human immune cells. Reverse Cholesterol Transportation (RCT) promotes excess cholesterol efflux from peripheral tissues to the blood as part of the synthesis required for biliary excretion in hepatocytes. RCT also has an inhibitory effect on atherosclerotic lesion development by speeding lipid efflux from macrophages to apolipoprotein A1 (ApoA-1) (Lewis and Rader,
3.2. KR-36676 inhibited U-II-dependent phosphorylation of ERK1/2 Phosphorylation of extracellular signal-regulated kinase (ERK1/2) is a well-known downstream marker of U-II activation. As shown in Fig. 2A, ERK1/2 phosphorylation began to increase at 30 min after treatment with U-II and increased to about 5-fold after 2 h of U-II stimulus. It was then remarkably reduced after 4 h of U-II stimulus. To analyze whether KR-36676 could attenuate activation of ERK1/2, THP-1 cells were pre-incubated with KR-36676 (10, 30, 100 nM) 1 h before U-II exposure. Pre-treatment with KR-36676 significantly inhibited the phosphorylation of ERK1/2 induced by U-II at 2 h in a concentration-dependent manner (Fig. 2B). 3.3. U-II-induced IL-1ß expression was inhibited by KR-36676 Next, we examined effect of U-II on IL-1ß expression in THP-1 cells by RT-PCR. The level of IL-1ß mRNA was increased by U-II (50 nM) in a time dependent manner, peaking at 4 h as demonstrated in Fig. 3A. To investigate whether, blockage of U-II would decrease IL-1ß expression, we pretreated cells with different concentrations of KR-36676 as the U-II receptor antagonist and at various concentrations (10, 30, 100 nM), and checked for IL-1β expression. KR-36676 inhibited the UII-induced IL-1β expression in a concentration-dependent manner (Fig. 3B). 3.4. U-II-induced ABCA1 repression is mediated by IL-1β As shown in Fig. 4A and B, we investigated whether ERK1/2 and IL-1β regulated the U-II-induced down-regulation of ABCA1 by using a neutralizing anti-IL-1β antibody and U0126. ABCA1 mRNA and protein levels were down-regulated by U-II exposure, and this downregulation was completely restored by treatment with U0126 and neutralizing anti-IL-1β antibody. These results suggest a participation of ERK1/2 and IL-1β in the U-II-induced down-regulation of ABCA1 expression. Next, we examined whether ERK1/2 was involved in the UII-induced IL-1β induction by using the ERK1/2 inhibitor, U0126 3
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Fig. 3. Effect of KR-36676 on U-II-induced IL-1β expression. THP-1 monocytes were stimulated by PMA (200 nM) for 24 h to initiate monocyte/macrophage differentiation. The cells were incubated following U-II (50 nM) treatment for different time periods (A). The cells were pre-incubated with KR-36676 (10, 30, 100 nM) for 1 h before treatment with UII (50 nM, 4 h) (B). Relative mRNA levels of IL-1ß were assayed by RT-PCR. Quantification was via normalization with the actin message. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
ERK1/2 inhibitor in presence of U-II. Our results demonstrated that inhibition of ERK1/2 completely suppressed the increase in IL-1ß message. Our results suggest that early activation and phosphorylation of ERK1/2 is main activator of IL-1ß in the U-II-induced ABCA1 repression pathway. In summary, the present study demonstrated that U-II downregulated ABCA1 expression in THP-1 cells via the ERK/IL-1ß pathway. These findings provide a new insight to urotensin II and cholesterol homeostasis in athero-pathogenic conditions. The lipid accumulation in macrophages with formation of foam cells was significantly increased in macrophage ABCA1 deficient C57/BL6 mice (Aiello et al., 2002). Our studies contribute to the view that U-II accelerates foam cell formation and by a novel mechanism. Furthermore, IL-1β also stimulates recruitment of monocytes to the endothelial layer and the apoptosis of macrophages, which contributes to plaque vulnerability (McLaren et al., 2011; Park et al., 2013). The use of the U-II antagonist KR-36676 significantly ameliorated IL-1ß and recovered ABCA1 reduction implying that a blocking in the UT receptor pathway may have beneficial effects in management of atherosclerosis.
2005). In previous studies, proatherogenic cytokine IL-1β, which is abundant in atherosclerotic lesions, was shown to inhibit the expression of ABCA1. Our data implies that U-II-induced ABCA1 downregulation might be associated with up-regulation of IL-1β. Recently, U-II down-regulated ABCA1 levels, and increased foam cell formation in THP-1 macrophages (Wang et al., 2014). ERK1/2 was involved in the U-II-induced ABCA1 repression; however, the mechanism was not clearly elucidated. Here, we demonstrated a significant recovery in ABCA1 mRNA and protein levels by KR-36676, suggestive of a positive effect in blocking the UT receptor in inhibition of atherosclerosis. Activation of ERK1/2 by U-II is an immediate response and causes vascular smooth muscle cell proliferation with subsequent vascular remodeling in rat aortic smooth muscle cells. These alterations in vascular smooth muscle cells contribute to the atherosclerotic lesion development (Tsai et al., 2009). We observed that ERK 1/2 was activated as an immediate response to U-II, and that this activation was blocked by KR-36676 in dose dependent manner in THP-1 cells. Furthermore, U-II mediated an increase in IL-8 levels and increased the proliferation of human umbilical vein endothelial (HUVEC) cells through p38 and the ERK1/2 MAPK kinase pathway. We investigated whether IL-1ß might be associated with the ERK pathway in downregulation of ABCA1 levels. We incubated THP-1 cells with U0126, an
Fig. 4. Link between IL-1β and ABCA1 during U-II stimulus. THP-1 cells were pre-incubated with U0126 (10 μM), SN-50 (10 μg/ml) for 1 h before treatment with U-II (50 nM) for the indicated times. The relative mRNA levels of ABCA1 (A) and IL-1ß (C) were assayed by RT-PCR. THP-1 cells were pre-incubated with U0126 (10 μM) and anti-IL-1β (10 μg/ml) for 1 h before treatment with U-II (50 nM, 24 h). ABCA1 protein expression was measured by Western blot (B). Quantification was via normalization by expression of actin. The results are expressed as mean ± S.E.M. from three different experiments. *P < 0.05 vs. control (CTL); #P < 0.05 vs. vehicle (Veh).
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