PAPP-A negatively regulates ABCA1, ABCG1 and SR-B1 expression by inhibiting LXRα through the IGF-I-mediated signaling pathway

Atherosclerosis 222 (2012) 344–354

Contents lists available at SciVerse ScienceDirect

Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

PAPP-A negatively regulates ABCA1, ABCG1 and SR-B1 expression by inhibiting LXR␣ through the IGF-I-mediated signaling pathway Shi-Lin Tang a,b,1 , Wu-Jun Chen a,c,1 , Kai Yin a , Guo-Jun Zhao a , Zhong-Cheng Mo a , Yun-Cheng Lv a , Xin-Ping Ouyang a , Xiao-Hua Yu a , Huai-Jun Kuang a,d , Zhi-Sheng Jiang a , Yu-Chang Fu e , Chao-Ke Tang a,∗ a Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, Life Science Research Center, University of South China, Hengyang, Hunan 421001, China b Department of Intensive Care Unit, the First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, China c Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China d Department of Cardiovascular Medicine, Second Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, China e Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-0012, USA

a r t i c l e

i n f o

Article history: Received 2 October 2011 Received in revised form 16 February 2012 Accepted 6 March 2012 Available online 27 March 2012 Keywords: PAPP-A IGF-1/PI3-K/Akt signaling pathway ABCA1 ABCG1 SR-BI LXR␣ Atherosclerosis

a b s t r a c t Pregnancy-associated plasma protein-A (PAPP-A) has been involved in the atherosclerotic process through regulation of local expression of IGF-1 that mediates the activation of the phosphatidylinositol-3 (PI3-K) and Akt kinase (Akt) signaling cascades which lead to constitutive nitric oxide formation, with its attending vasodilator, antiplatelet and insulin-sensitizing actions. In addition, IGF-1 may decreased cholesterol efflux through reductions of expression in ABCA1 and SR-B1 by the PI3-K/Akt signaling pathway. In the current study, we examined whether PAPP-A was involved in LXR␣ regulation and in expression of ABCA1, ABCG1 or SR-B1 through the IGF-I-mediated signaling pathway (IGF/PI3-K/Akt). Results showed that PAPP-A significantly decreased expression of ABCA1, ABCG1 and SR-BI at both transcriptional and translational levels in a dose-dependent and time-dependent manner. Cellular cholesterol content was increased while cholesterol efflux was decreased by PAPP-A treatment. Moreover, LXR␣ which can regulate the expression of ABCA1, ABCG1 and SR-B1, was also down-regulated by PAPP-A treatment. LXR␣-specific activation by LXR␣ agonist almost rescued the down-regulation of ABCA1, ABCG1 and SR-B1 expression by PAPP-A. In addition, PAPP-A can induce the IGF-1/PI3-K/Akt pathway in macrophages. Furthermore, our results indicate that the decreased levels observed in LXR␣, ABCA1, ABCG1 and SR-B1 mRNA and protein levels upon treating cells with PAPP-A were strongly impaired with the PI3-K inhibitors or IGF-1R siRNA while the MAPK cascade inhibitor did not execute this effect, indicating that the process of ABCA1, ABCG1 and SR-BI degradation by PAPP-A involves the IGF-1/PI3-K/Akt pathway. In conclusion, PAPP-A may first down-regulate expression of LXR␣ through the IGF-1/PI3-K/Akt signaling pathway and then decrease expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells. Therefore, our study provided one of the mechanisms for understanding the critical effect of PAPP-A in pathogenesis of atherosclerosis. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: PAPP-A, pregnancy-associated plasma protein-A; IGF-1, insulinlike growth factor 1; IGF-1R, insulin-like growth factor 1 receptor; PI3-K, phosphatidylinositol 3-kinase; Akt, Akt kinase or protein kinase B (PKB); ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; SR-BI, scavenger receptor class B type I; LXR␣, liver X receptor ␣; RCT, reverse cholesterol transport; HDL, high density lipoprotein. ∗ Corresponding author at: Institute of Cardiovascular Research, University of South China, Hengyang, Hunan 421001, China. Tel.: +86 734 8281853; fax: +86 734 8281853. E-mail address: [email protected] (C.-K. Tang). 1 These authors contributed equally to this work. 0021-9150/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2012.03.005

Atherosclerosis, one of the major diseases of the world, is a complex inflammatory response to chronic injury which involves several cell types and associated cytokines, growth factors, and enzyme systems [1]. The characteristic component of the atherosclerotic plaque is the differentiation of monocytes to macrophages that accumulate lipoprotein-derived cholesterol to form foam cells within the arterial wall [2]. Moreover, intracellular free cholesterol (FC) can be toxic to the cell and this cholesterol laden macrophage is thought to derive foam cell. Therefore an efficient cholesterol efflux mechanism in the macrophage is mandatory to prevent cholesterol accumulation. High density lipoprotein (HDL) and its

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

apolipoproteins, especially apolipoprotein A1 (apoA1), are responsible for the transfer of cholesterol from peripheral tissues and cells to the liver for biliary excretion, the process of “reverse cholesterol transport” (RCT) [3,4]. Based on studies in RCT, ATP binding cassette transporter A1 (ABCA1), ATP binding cassette transporter G1 (ABCG1) and scavenger receptor B1 (SR-B1), play critical roles in preventing cholesterol accumulation in macrophages. Previous studies from our laboratory and others had also shown that increasing ABCA1, ABCG1 and SR-B1 activity in macrophages leads to promote cellular cholesterol efflux in vitro and a massive reduce in macrophage lipid accumulation in vivo [2–8]. Pregnancy-associated plasma protein-A (PAPP-A), a newly recognized metalloproteinase in the insulin-like growth factor (IGF) systems [9,10], has been recently shown to increase in the development of atherosclerosis and/or lesion progression or is simply a marker of the injury response [11–16]. Furthermore, PAPP-A has been detected in ruptured and eroded plaques from human arterial specimens, with the most intense immunospecific associated with activated macrophages and smooth muscle cells [17,18]. In addition to this, other laboratories have also shown that the increased PAPP-A activity in macrophages and smooth muscle cells results in increasing lipid accumulation [18–20]. However, the detailed mechanism for lipid accumulation induced by PAPPA is still unclear. In vitro, PAPP-A has been shown to cleave inhibitory IGF binding protein-4 (IGFBP-4) thereby increasing local IGF bioavailability and receptor activation [21,22]. Free IGF-1 can thus interact with its membrane receptor (IGF-1R), mediates the activation of the phosphatidylinositol-3 (PI3-K) and Akt kinase (Akt) signaling which leads to constitutive nitric oxide formation, with its attending vasodilator, antiplatelet and insulin-sensitizing [18–20,23]. In addition, IGF-1 may decrease cholesterol efflux through reduction in liver X receptor (LXR␣), ABCA1 and SR-B1 by the PI3-K/Akt signaling pathway [24–26]. LXR␣ responds to oxidized LDL and promotes cholesterol efflux through activation of the ABCA1, ABCG1 transporter and SR-B1 [27–29]. Based on these studies, we hypothesized that PAPP-A downregulate the expression of LXR␣ through the IGF-I-mediated signaling pathway (IGF/PI3K/Akt) and then decrease the expression of ABCA1, ABCG1 or SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells.

2. Experimental procedures 2.1. Reagents and antibodies PAPP-A (R&D Systems Inc., Minneapolis, MN, USA), LY294002, Raf1 kinase inhibitor I, were purchased from Calbiochem. TRIzol Reagent (Invitrogen, 1600 Faraday Ave, Carlsbad, USA), BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). ReverAidTM First Strand cDNA Synthesis Kit (#k1622) (Fermentas, 830 Harrington Court, Burlington, Ontario, Canada), DyNAmoTM SYBR® Green qPCR Kits (Finnzymes, keilaranta 16, 02150 Espoo, Finland) and immobilon-P transfer membranes (Millipore, 290 Concord Road, Massachusetts, USA) were obtained as indicated above. Human LDL (d = 1.019–1.063 g/mL) was isolated by density ultracentrifugation from EDTA-treated plasma. The LDL supernatant was dialyzed against isotonic saline to remove EDTA. Oxidation of LDL was obtained by incubating LDL with 5 ␮M CuSO4 at 37 ◦ C for 3 h, and then dialyzed overnight against saline prior to add to cells. The extent of oxidation of the LDL preparations was determined by measuring thiobarbituric acid reactive substance (TBARS) (PeroXOquant, Pierce, Rockford, IL). LDL processed in parallel but without CuSO4 was 1.7 TBARS/␮g protein and oxLDL was 15.6 TBARS/␮g protein. All other chemicals were of the best grade available from commercial sources.

345

2.2. Cells culture The human monocytic cell line THP-1 originally obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in RPMI-1640 supplemented with 0.1% nonessential amino acids, penicillin (100 U/mL), streptomycin (100 ␮g/mL), and 20% fetal bovine serum (FBS) at 37 ◦ C in 5% CO2 at a cell density of 0.2 to 1.0 × 106 /mL. After three to four days, cells were treated with PMA (160 nmol/L) for 24 h, and the medium was then replaced by a serum-free medium containing oxLDL (50 ␮g/mL) for 48 h in order for the cells to become fully differentiated macrophages before their use in experiments. 2.3. RNA isolation and real-time quantitative polymerase chain reaction analysis Total RNA from cells was extracted by using TRIzol reagent in accordance with the manufacture’s instructions. Real-time quantitative polymerase chain reaction (PCR), using SYBR Green detection chemistry, was performed on Roche LightCycler with a 5.32 realtime PCR system. Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. Quantitative measurements were determined using the  Ct method, and expression of ␤-actin was used as the internal control [6]. 2.4. Western blot analyses Cells were harvested and protein extracts prepared as previously described [2]. They were then subjected to Western blot analyses [8% SDS-PAGE; 30 ␮g protein per lane] using rabbit anti-ABCA1, anti-ABCG1 and anti-SR-BI-(Novus Biologicals Littleton, CO, USA), mouse anti-LXR␣ (PPMX, Tokyo, Japan) and ␤-actin (Santa Cruz, CA, USA)-specific antibodies. The proteins were visualized using a chemiluminescence method (enhanced chemiluminescence Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA). 2.5. ELISA assay Cells were plated in six-well plates and treated as described above. Culture supernatants were collected and stored at −20 ◦ C until analysis. The concentrations of IGF-1 in supernatants was measured by enzyme-linked immunosorbent assay (ELISA) (DuoSet ELISA Development System, R&D Systems, Abingdon, UK) following the manufacturers instructions. Quantitative determinations in three different experiments were performed. 2.6. Transfection for IGF-1R silencing Short-interfering RNA (siRNA) specific for human IGF-1R was from the Santa Cruz Biotechnology and nonsilencing as control siRNAs were synthesized by the Biology Engineering Corporation in Shanghai, China. THP-1 macrophage-derived foam cells (2 × 106 cells/well) were transfected using Lipofectamine 2000 (Invitrogen). After 48 h of the transfection, in comparison to the control siRNA, the siRNA of IGF-1R suppressed the expression of IGF-1R proteins by 78% according to Western blot analysis. 2.7. Cellular cholesterol efflux experiments Cells were cultured as indicated above. Then, they were labeled with 0.2 mCi/mL of [3 H] cholesterol. After 72 h, cells were subsequently washed with PBS and incubated overnight in RPMI 1640 medium containing 0.1% (wt/vol) BSA to allow equilibration of [3 H] cholesterol in all cellular pools. Equilibrated [3 H] cholesterollabeled cells were washed with PBS and incubated in 2 mL of efflux

346

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

medium containing RPMI 1640 medium and 0.1% BSA with or without 10 ␮g/mL of human plasma apoA-I or 50 ␮g/mL HDL. A 150-mL sample of efflux medium was obtained at the times designated and passed through a 0.45-mm filter to remove any floating cells. Monolayers were washed twice in PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3 H] cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation: [total media counts/(total cellular counts + total media counts)] × 100% [3]. 2.8. HPLC analysis of the lipid levels The sterol analyses were performed using a high performance liquid chromatography (HPLC) system (model 2790, controlled with Empower Pro software; Waters Corp, Milford, MA, USA). Sterols were detected using a photodiode array detector equipped with a 4 mL cell (model 996; Waters Corp.). Analysis of cholesterol and cholesteryl esters was performed after elution with acetonitrile–isopropanol 30:70 (v/v) 20 and detection by absorbance at 210 nm. 2.9. Statistical analysis Data are expressed as means ± S.D. Results were analyzed by one-way ANOVA and Student’s t test, using SPSS 13.0 software. A value of P < 0.05 was considered statistically being significant. All experiments were performed at least three times. 3. Results 3.1. PAPP-A inhibits ABCA1, ABCG1, SR-BI expression and reduces cholesterol efflux in THP-1 macrophage-derived foam cells Previously researches revealed that macrophage cholesteryl ester accumulation increased in cells after being treated by PAPPA [18,24]. In the present study, we first examined the effect of PAPP-A on the expression of ABCA1, ABCG1 and SR-BI in THP1 macrophage-derived foam cells by real-time quantitative PCR and western immunoblotting assays. As shown in Fig. 1, PAPP-A reduced ABCA1, ABCG1 and SR-B1 expression at both transcriptional (Fig. 1A and B) and translational (Fig. 1C and D) levels in a dose-dependent and time-dependent manner. ABCA1, ABCG1 and SR-BI are key performers in RCT and are critical in regulating cellular cholesterol homeostasis [6]. As ABCA1, ABCG1 and SR-BI were decreased by PAPP-A, we next examined the effect of PAPP-A on cholesterol content and apoA-I(ABCA1-dependent) or HDL-mediated (ABCG1/SR-B1-dependent) cholesterol efflux in THP-1 macrophage-derived foam cells by HPLC and liquid scintillation counting assays. Cellular cholesterol content was increased (Tables 1 and 2), whereas apoA-I- or HDL-mediated cholesterol efflux was decreased (Fig. 1E–H) when cells were treated with PAPP-A, suggesting that PAPP-A could decrease apoAI- or HDL-mediated cholesterol efflux which is possibly through a

Table 1 Effect of PAPP-A on cholesterol content in different concentration in THP-1 macrophage-derived foam cells. PAPP-A TC FC CE CE/TC (%)

0 ng/mL 500 ± 35 195 ± 27 305 ± 38 61.0

10 ng/mL

50 ng/mL

250 ng/mL

533 ± 39 206 ± 36 327 ± 47 61.3

656 ± 59* 257 ± 46* 399 ± 38* 60.8

769 ± 55* 309 ± 43* 470 ± 29* 61.1

THP-1 macrophage-derived foam cells were divided into four groups and cultured in medium at 37 ◦ C containing 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL PAPPA for 24 h respectively. Cellular cholesterol and cholesterol ester were extracted as described above. And HPLC was performed to determine the cellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE). The results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. * P < 0.05 vs 0 ng/mL group.

reducing expression of ABCA1, ABCG1 and SR-BI pathway in THP-1 macrophage-derived foam cells. 3.2. LXR˛ is involved in PAPP-A-induced down-regulation of ABCA1, ABCG1 and SR-BI in THP-1 macrophage-derived foam cells The LXR␣ has been shown to regulate the expression of ABCA1, ABCG1 and SR-BI, which serve as free-cholesterol and phospholipids translocators enabling cholesterol efflux from the macrophage to various acceptors, including nascent cholesterolpoor HDL, and thus have a central role in the regulation of reverse cholesterol transport [3,4,6]. To confirm whether the LXR␣ expression can be affected by PAPP-A, real-time quantitative PCR and western immunoblotting analysis were performed. As shown in Fig. 2, we observed that PAPP-A causes a marked decrease of LXR␣ mRNA (Fig. 2A and B) and protein levels (Fig. 2C and D). We then examined the effect of LXR␣ agonist 22(R)-Hch on the down-regulation of ABCA1, ABCG1 and SR-B1 induced by PAPP-A. As demonstrated in Fig. 2E, ABCA1, ABCG1 and SR-B1 mRNA expression were significantly up-regulated in cells treated by 22(R)-Hch. Down-regulation of ABCA1, ABCG1 and SR-B1 mRNA expression by PAPP-A was almost totally rescued by addition of 22(R)-Hch. Similar result was also found using western immunoblotting to determine the expression of ABCA1, ABCG1 and SR-B1 protein (Fig. 2F). At the same time, our results also indicated that decrease in cholesterol efflux with PAPP-A were strongly impaired with the LXR␣ agonist 22(R)-Hch (Fig. 2G and H). 3.3. PAPP-A promotes the IGF-1/PI3-K/Akt signaling pathway in THP-1 macrophage-derived foam cells. PAPP-A specifically degrades insulin-like growth factor binding proteins (IGFBPs)-4 and -5, thereby allowing active IGF-1 to bind to cell-surface IGF receptors [9,10]. Free IGF-1 can thus interact with its membrane receptor, causing activation of PI3-K/Akt signaling pathway [19,23]. Because IGF-1 has been found to stimulate IGFBP-5 mRNA expression in vitro and in vivo, increased IGFBP5 mRNA expression can be used as an indicator of increased IGF

Table 2 Effect of PAPP-A on cholesterol content on different time in THP-1 macrophage-derived foam cells. Time TC FC CE CE/TC (%)

24 h (BSA) 516 ± 47 197 ± 29 319 ± 28 61.8

0 h (PAPP-A) 508 ± 50 195 ± 27 313 ± 26 61.6

6 h (PAPP-A) 530 ± 56 205 ± 32 325 ± 32 61.3

12 h (PAPP-A) 665 ± 61* 249 ± 27* 416 ± 38* 62.5

24 h (PAPP-A) 745 ± 58* 275 ± 23* 470 ± 35* 63.0

THP-1 macrophage-derived foam cells were divided into five groups and cultured in medium at 37 ◦ C containing 5 mg/ml bovine serum albumin (BSA) for 24 h and cultured in medium at 37 ◦ C containing 250 ng/mL PAPP-A for 0 h, 6 h, 12 h, 24 h respectively. Cellular cholesterol and cholesterol ester were extracted as described above. And HPLC was performed to determine the cellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE). The results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. * P < 0.05 vs 0 h group.

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

347

Fig. 1. Dose-dependent and time-dependent effects of PAPP-A on ABCA1, ABCG1 and SR-BI expression and ABCA1/ABCG1/SR-B1-dependent cholesterol from THP-1 macrophage-derived foam cells. (A, C, E and G) Foam cells were treated with PAPP-A at 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL for 24 h respectively. (B, D, F and H) Cells were treated with 5 mg/mL BSA for 24 h or with 250 ng/mL PAPP-A for 0 h, 6 h, 12 h, 24 h respectively. A and B, ABCA1, ABCG1 and SR-BI gene were measured by real-time quantitative PCR. (C and D) ABCA1, ABCG1 and SR-BI protein expressions were measured by western immunoblotting assays. (E, F, G and H) Cellular apoA-I(ABCA1-dependent) or HDL-mediated (ABCG1/SR-B1-dependent) cholesterol efflux were analyzed by liquid scintillation counting assays as shown above. All the results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 vs baseline.

signaling through the IGF-IR. An advantage for in vitro assessment is that IGFBP-5 mRNA levels are upregulated for at least 24 h in response to IGF-1 receptor activation in contrast with the transient changes in intracellular signaling intermediates [30]. As shown in Fig. 3, PAPP-A increased expression of IGFBP-5 mRNA (Fig. 3A and B) in a dose-dependent and time-dependent manner

in THP-1 macrophage-derived foam cells. In addition, to exclude the possibility that increase in free IGF-1 concentration is due to increased IGF-1 production in response to PAPP-A treatment [31], we also estimated the free IGF-1 in the culture medium with ELISA. Free IGF-1 was below the sensitivity of the assay. PAPP-A overexpression significantly increased the free IGF-1

348

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

Fig. 2. LXR␣ is involved in the down-regulation of ABCA1, ABCG1 and SR-BI expression induced by PAPP-A in THP-1 macrophage-derived foam cells. (A, B, C and D) PAPP-A decreased the expression of LXR␣ at both mRNA and protein levels. Cells were treated with PAPP-A at 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL for 24 h respectively and cells were treated with 250 ng/mL for 0 h, 6 h, 12 h, 24 h respectively or with 5 mg/ml BSA for 24 h. A and B total RNA was extracted and real-time quantitative PCR was performed to determine the expression of LXR␣ mRNA. (C and D) Western immunoblotting assays using antibody against human LXR␣ and ␤-actin were conducted. (E, F, G and H) THP-1 macrophage-derived foam cells were treated with control or LXR␣ agonist 22(R)-Hch, and then incubated with PAPP-A (250 ng/mL) for 24 h. (E and F) mRNA and protein expression of ABCA1, ABCG1 and SR-BI were determined using real-time quantitative PCR and western immunoblotting assays. (G and H) Cellular apoA-I- (ABCA1-dependent) or HDL-mediated (ABCG1/SR-B1-dependent) cholesterol efflux from THP-1 macrophage-derived foam cells were analyzed by liquid scintillation counting assays as shown above. Similar results were obtained in three independent experiments. Data are mean ± S.D. *P < 0.05 vs baseline.

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

349

Fig. 3. PAPP-A induces the IGF-1/PI3-K/Akt pathway in macrophages. (A and B) PAPP-A increased the expression of IGFBP-5 mRNA levels. Cells were treated with PAPP-A at 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL for 24 h respectively and cells were treated with 250 ng/mL for 0 h, 6 h, 12 h, 24 h respectively or with 5 mg/ml BSA for 24 h. (A and B) Total RNA was extracted and real-time quantitative PCR was performed to determine the expression of IGFBP-5 mRNA. (C) Free IGF-1 concentrations in the culture medium was estimated with ELISA. (D) Quantitative real-time PCR analysis IGF-1 mRNA in THP-1 macrophage-derived foam cells. (E and F) THP-1 macrophage-derived foam cells were tranfected with control or IGF-1R siRNA, and then incubated with PAPP-A (250 ng/mL) for 24 h. (E) Protein samples were immunoblotted with anti-IGF-1R or anti-␤-actin antibodies. (F) Western analysis for phosphorylated Akt and PI3-K p85 subunit. Similar results were obtained in three independent experiments. Data are mean ± S.D. *P < 0.05 vs baseline.

concentration compared with control (Fig. 3C). Furthermore, quantitative real-time PCR analysis revealed that treatment of THP-1 macrophage-derived foam cells with PAPP-A did not affect the level of IGF-1 mRNA (Fig. 3D). These data suggest that PAPP-A increases free IGF-1 concentration and increased PAPP-A expression results in increased IGF-1 bioavailability. At the same time, we observed that PAPP-A increased the phosphorylation of Akt and of p85 regulatory subunit of PI3-K in THP-1 macrophages (Fig. 3F). On the other hand, treatment with siRNA for IGF-IR down-regulated IGF1R protein expression by 78% (Fig. 3E) and PAPP-A that mediates the activation of the PI3-K and Akt were significantly impaired with the

IGF-IR siRNA (Fig. 3F). All of our results indicated that PAPP-A can induce the IGF-1/PI3-K/Akt pathway in THP-1 macrophage-derived foam cells. 3.4. IGF-1/PI3-K/Akt signaling pathway is related to down-regulated expression of LXR˛, ABCA1, ABCG1 and SR-BI by PAPP-A in THP-1 macrophage-derived foam cells Previously researches revealed that Insulin and IGF-1 specifically down-regulates activity of ABCA1 and SR-B1 through PI3-K/Akt signaling pathway [24–26,32]. At the same time, IGF-1

350

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

Fig. 4. Negative regulation of LXR␣, ABCA1, ABCG1 and SR-B1 expression by PAPP-A is dependent on the IGF-1/PI3-K/Akt signaling pathway in THP-1 macrophage-derived foam cells. (A and B) Cells were incubated for 24 h at 37 ◦ C in the presence (+) or absence (−) of PAPP-A (250 ng/mL) with (+) and without (−) LY294002 or Raf1 kinase inhibitor I (10 ␮M each). Total RNA was extracted and real-time quantitative PCR was performed to determine the expression of LXR␣, ABCA1, ABCG1 and SR-BI mRNA (A), and then Western immunoblotting assays using antibody against human LXR␣, ABCA1, ABCG1, SR-BI and ␤-actin were conducted (B). (C and D) THP-1 macrophage-derived foam cells were tranfected with control or IGF-1R siRNA, and then incubated with PAPP-A (250 ng/mL) for 24 h. (C) For mRNA and (D) for protein expression of LXR␣, ABCA1, ABCG1 and SR-BI were determined using real-time quantitative PCR and western immunoblotting assays. Similar results were obtained in three independent experiments. Data are mean ± S.D. *P < 0.05 vs baseline.

also induces the MAPK signaling pathway in macrophages. These studies suggest that PAPP-A could inhibit expression of LXR␣, ABCA1, ABCG1 and SR-BI either through IGF-1/PI3-K/Akt or IGF1/MAPK signaling pathway in cells. Involvement of these pathways was examined in our current study by using inhibitors of each of these pathways. As show in Fig. 4, a selective PI3K inhibitor, LY294002, abated down-regulations of LXR␣, ABCA1, ABCG1 and SR-BI mRNAs and proteins by PAPP-A in THP-1 macrophagederived foam cells, while an inhibitor of the MAPK cascade, Raf1 kinase inhibitor I, did not have this effect (Fig. 4A and B), indicating that the down-regulation expression of LXR␣, ABCA1, ABCG1 and SR-BI by PAPP-A involves the PI3-K/Akt pathway only. On the other hand, the same effects were also observed by using IGF-1R siRNA. Treatment with siRNA for IGF-1R made the down-regulation of PAPP-A on LXR␣, ABCA1, ABCG1 and SR-BI expression completely reversed (Fig. 4C and D). Thus, the IGF-1/PI3-K/Akt signaling

pathway plays a critical role in the negative regulation of the LXR␣, ABCA1, ABCG1 and SR-BI expression by PAPP-A.

4. Discussion The current study established an active role for PAPP-A in the development of atherosclerosis, presented in vivo evidence that PAPP-A functions through regulation of IGF-1 systems (IGF1/PI3-K/Akt), and provided rationale for exploring novel strategies aimed at PAPP-A, a relatively accessible extracellular enzyme, to control atherosclerosis [1,9,33,34]. However, several studies show that PAPP-A is not a marker of the vulnerable atherosclerotic plaque [35–37]. Our findings support the notion that PAPP-A may contribute to plaque formation in atherosclerosis by decreasing cholesterol efflux, and increasing macrophage foam cell formation.

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

PAPP-A is highly expressed in atherosclerotic plaque and can increase lipid accumulation in cells [18–20] and is a proposed new marker of acute coronary syndromes [33,38]. However the detailed mechanism for lipid accumulation induced by PAPP-A, which reduced cholesterol efflux through down-regulated expression of cholesterol transports is still unclear. The results of this study are unequivocal in that PAPP-A downregulates the expression of LXR␣ through the IGF-1/PI3-K/Akt signaling pathway and then decreases expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells. It is important to assess lipid accumulation as a major source of PAPP-A in plaque, and long-term administration of PAPP-A may increase the development of experimental atherosclerosis in animals. Macrophages possess a number of mechanisms to regulate the balance between cholesterol uptake/synthesis and export. Of major importance are transport mechanisms that promote the efflux of excess cholesterol to extracellular acceptors, i.e., “reverse cholesterol transport” (RCT) [39]. Cellular cholesterol efflux, being the first step in RCT, plays an important role in reducing the accumulation of lipids in arterial wall and preventing the development of atherosclerosis. Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins, and cholesterol efflux from macrophage has recently been shown to be mainly mediated by ABCA1, ABCG1 and SR-BI [40]. Lipid-poor apolipoproteins serve as extracellular acceptors for ABCA1-mediated cellular phospholipid (PL) and cholesterol efflux [5,41]; Whereas ABCG1 appears to promote efflux by redistributing intracellular cholesterol to plasma membrane domains accessible for removal by HDL, but not lipid-poor apoA-I [2,42]; SR-BI expression enhanced both cell cholesterol efflux and cholesterol influx from HDL, but did not lead to altered cellular cholesterol mass. SR-BI-dependent efflux occurred to larger HDL particles but not to smaller HDL3 [6,43,44]. In the current study, we determined that PAPP-A-treated macrophages exhibited a significant decrease in cholesterol efflux by apoA-I or HDL. The decrease in apoA-I- or HDL- mediated efflux is consistent with down-regulations at both the transcriptional and translational levels in the amount of ABCA1, ABCG1 and SR-BI expression in PAPP-A-treated foam cells. Recently, others and our laboratory previous studies demonstrated that LXR can up-regulate the expression of ABCA1, ABCG1 and SR-BI through forming heterodimers with RXR [6,45–48]. Therefore, we tested the effect of PAPP-A on the expression of LXR␣ to find out whether the expression of LXR␣ is changed during the course. It turned out that PAPP-A markedly decreased the expression of LXR␣ at both mRNA and protein levels in THP1 macrophage-derived foam cells. LXR␣ agonist 22(R)-Hch were then used to see whether LXR␣ is involved in the down-regulation of ABCA1, ABCG1 and SR-B1 induced by PAPP-A. ABCA1, ABCG1, and SR-B1 mRNA and protein expression were significantly upregulated by 22(R)-Hch. Down-regulation of ABCA1, ABCG1 and SR-B1 expression by PAPP-A was almost totally blocked by addition of 22(R)-Hch. At the same time, our results also indicated that decrease in cholesterol efflux with PAPP-A were strongly impaired with the LXR␣ agonist 22(R)-Hch. These results suggested that LXR␣ was involved in the down-regulation of ABCA1, ABCG1 and SR-B1 induced by PAPP-A in THP-1 macrophagederived foam cells. These findings support the notion that PAPP-A may have increased the development of atherosclerosis properties by decreasing cholesterol efflux and increasing macrophage foam cell formation. PAPP-A has been shown to cleave inhibitory IGF binding protein-4 thereby increasing local IGF bioavailability and receptor activation [49]. At the molecular level, IGF-1 activates the PI3-K/Akt pathway, but also the synthesis of nitric oxide, with its multiple cardiovasculo-protective actions [33,50]. Macrophages also phagocytes its membrane-bound PAPP-A, which could restrain

351

further IGF action or have physiological consequences independent of IGFs [18]. In addition, expression of the IGF-I-responsive gene, IGFBP-5, has been used as an in vivo marker of IGF-1 receptormediated activity [30]. In the present study, we examined the activation of the IGF-1/PI3-K/Akt pathway (PI3-K and Akt activation) after PAPP-A stimulation in THP-1 macrophage-derived foam cells. As shown in our results, we observed that treatment of THP1 macrophages with PAPP-A increased IGFBP-5 expression levels in a dose-dependent and time-dependent manner, and PAPP-A overexpression significantly increased the free IGF-1 concentration compared with control. In addition, we observed that PAPP-A increased the phosphorylation of Akt and of p85 regulatory subunit of PI3-K. Our results also indicate that the increased levels observed in phosphorylation of Akt and PI3-K upon treating cells with PAPP-A were strongly impaired with the IGF-1R siRNA in foam cells. Altogether, these results emphasize a predominant role of the PI3-K/Akt pathway in mediating IGF-1 activation by PAPP-A in THP-1 macrophage-derived foam cells. Recently, several studies have demonstrated that PI3-K/Akt pathway participate in IGF-I-suppression of ABCA1 and SR-B1 expression, which suggests that the activation of Akt plays an important role in cholesterol metabolism in vivo [25,26]. In order to determine whether activation of the IGF-1/PI3-K/Akt pathway was involved in the down-regulation of LXR␣-dependent gene expression, we tested the effects of the PI3-K inhibitor LY294002 on the expression profile of downstream effectors, such as LXR␣, ABCA1, ABCG1 and SR-B1 in THP-1 macrophage. Our results indicate that the decreased levels observed in LXR␣, ABCA1, ABCG1 and SR-B1 mRNA and protein levels upon treating cells with PAPP-A were strongly impaired with the inhibition of PI3-K. We further investigated the effect of IGF-1R siRNA on LXR␣-dependent gene expression in PAPP-A-stimulated THP-1 macrophage-derived foam cells. As shown, treatment with siRNA for IGF-1R down-regulated IGF-1 protein expression by 78% and made the down-regulation of PAPP-A on LXR␣, ABCA1, ABCG1 and SR-BI expression completely reversed. Therefore, we concluded that the down-regulation of LXR␣-dependent gene metabolic cascade induced by PAPP-A was mediated by IGF-1/PI3-K/Akt signaling pathway. Interestingly, PI3-K inhibitors to reverse the decrease of LXR␣dependent gene by PAPP-A are remained while IGF-1R siRNA was completely, suggesting that IGF-1 was very important to PAPP-A function, and indicate that decrease of LXR␣-dependent gene by PAPP-A may have other pathways. IGF-1 also stimulates one other signaling pathway, mitogen-activated protein kinase (MAPK) cascades. Involvement of this pathway was examined by using inhibitor in our studies. But an inhibitor of the MAPK cascade, Raf1 kinase inhibitor I, did not influence the LXR␣, ABCA1, ABCG1 and SR-B1 expression in our experiments, indicating that the PAPP-A down-regulates ABCA1, ABCG1 and SR-B1 protein level in cultured cells through PI3-K but not by the MAPK cascade. Ten years ago, several groups demonstrated that the activation of PI3-K/Akt signaling, through different upstream pathways, could trigger the activation of NF-␬B system [51,52]. NF-␬B signaling has been recognized as one of the targets of PI3-K/Akt pathway. Although a number of studies have convincingly demonstrated that the PI3-K/Akt pathway activates the NF-␬B system, the molecular mechanisms are still unclear. IKK␣/␤ and NIK are the major upstream kinases activating NF-␬B signaling [53,54]. Recent studies revealed that through the IKK␣ kinase and IKK␤ kinase or NIK, the PI3-K/Akt signaling pathway can activate the nuclear translocation of NF-␬B complexes as well as potentiate their transactivation efficiency [55,56]. There is clear evidence that IGF-1 and the IGF-1 receptor signaling can target IKK/NF-␬B signaling pathway [57,58]. Several studies demonstrate that IGF-1 regulate the immunity and

352

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

Fig. 5. PAPP-A reduces ABCA1, ABCG1, SR-B1 expression and cholesterol efflux by inhibiting LXR␣ through the IGF-1/PI3-K/Akt signaling pathway in THP-1 macrophagederived foam cells. The results of the present study revealed the following scheme for the possible mechanism in which PAPP-A down-regulated of cholesterol efflux. When THP-1 macrophage-derived foam cells are treated with PAPP-A, PAPP-A releases IGF-1 from IGFBP-4, and then freed IGF-1 can thus interact with its membrane receptor, causing phosphorylation of PI3-K and Akt, activated the P13-K/Akt pathway. Thus the expression of LXR␣ is down-regulated, which in turn decrease the expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux. →: Activation; : inhibition.

immune responses via the NF-␬B signaling which is mediated by PI3-K/Akt signaling pathway [57–60]. The most potent stimulators of PAPP-A expression in these vascular cells are the proinflammatory, proatherogenic cytokines, interleukin-1␤ and tumor necrosis factor-␣ [61,62], and have been found that genetic ablation of PAPPA in mice results in resistance to the development of neointimal hyperplasia as an acute injury response [63] and to the progression of atherosclerosis as a chronic injury response [38]. Moreover, Our group and other laboratory found that IKK/NF-␬B signaling and LXR␣-independent pathway were involved in the downregulation of ABCA1 and ABCG1 or SR-B1 induced by LPS, IL-1␤, TNF-␣, IFN-␥, etc., then promote the accumulation of lipid and decrease cellular cholesterol efflux in THP-1 macrophage derived foam cells [64–67]. However, the exact mechanism of the apparent cooperation between PAPP-A/IGF-1 system and PI3-K/Akt/IKK/NF␬B signaling in the regulation of ABCA1 and ABCG1 or SR-B1 is unknown. Berfield et al. [24] demonstrated that rat glomerular mesangial cells have reduced expression of PPAR␤, suggesting that chronic exposure to IGF-1 might limit the normal TG-mediated downregulation of the VLDL receptor and reduce TG efflux, both of which would favor TG accumulation. The specificity of these effects was further supported by the absence of alterations in HMG-CoA reductase, LDL, or scavenger receptors that might have increased lipid accumulation. In addition to this, Demers et al. [68] have shown that the PI3-K/Akt pathway plays a prominent role in mediating ghrelin responsiveness of macrophages to activate the PPAR␥-LXR␣-ABC metabolic cascade. Clearly, additional studies are needed to further define the specific effects of PAPP-A by which IGF1 responds to enhanced PI3-K/Akt pathway in macrophages on fatty acid synthase, VLDL receptors, HMG-CoA reductase, LDL, scavenger receptors and the PPAR␤/␥-LXR␣-ABC metabolic cascade expression. In another study [69], Chen et al. study a novel pathway except for LXR␣ in regulating macrophage ABCA1 expression: treatment of macrophages with LDL increased PKC-␨ phosphorylation and

induced physical interaction of PKC-␨ and Sp1. Transfection of macrophages with a kinase dead PKC-␨ or treatment of the cells with a PKC inhibitor attenuated LDL-induced Sp1 phosphorylation, LXR␣ and ABCA1 promoter activity. Moreover, the authors observed that LDL-induced PKC-␨ phosphorylation was reduced by inhibition of PI3-K, which is a protein kinase that mediates PKC-␨ phosphorylation. It appears that activation of the PI3-K-PKC-␨ cascade is critical for LDL-induced Sp1 phosphorylation and ABCA1 expression. It is highly likely that LDL sequentially activates PI3-K and PKC-␨, which in turn phosphorylates Sp1, increasing its binding to the ABCA1 promoter and up-regulating ABCA1 transcription, and therefore enhances cholesterol efflux. These current study suggest that PAPP-A may activate the PI3-K-PKC-␨-Sp1 signaling cascade, which in turn down-regulates ABCA1, ABCG1, and SR-B1 expression by repressing LXR␣ expression, and therefore reduces cholesterol efflux. In summary, as shown in Fig. 5, this study addresses the missing link between PAPP-A treatment and decreasing of cholesterol efflux by providing evidence that down-regulating of PAPP-A is positively associated with expression of ABCA1, ABCG1 and SR-BI in THP-1 macrophage-derived foam cells, possibly by PAPP-A decreasing LXR␣ expression. At the same time, expression of LXR␣ is also inhibited while phosphorylation of Akt and p85 regulatory subunit of PI3-K are increased, which can be compensated by a PI3-K inhibitor LY294002 or IGF-1R siRNA. All these findings suggest that PAPP-A may first down-regulate the expression of LXR␣ through the IGF-1/PI3-K/Akt signaling pathway and then inhibit cholesterol efflux by decreasing the expression of ABCA1, ABCG1 and SR-BI in THP-1 macrophage-derived foam cells. The IGF-1/PI3-K/Akt pathway may play a critical role in the activation of the ABCA1, ABCG1 and SR-B1 metabolic cascade by PAPP-A. Therefore, our findings raise the possibility that future drugs being able to selectively modulate PAPP-A expression might provide a novel form of therapy that enhance the activity of this cholesterol-removal pathway and prevent atherosclerosis.

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Sciences Foundation of China (81170278, 81101213, 81100211, 81070220), the Heng Yang Joint Funds of The Hunan Provincial Natural Sciences Foundation of China (10JJ9019), and the Science and Technology Department Funds of Heng Yang of Hunan Province Grant (2010kj41). References [1] Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell 2001;104:503–16. [2] Chen SG, Xiao J, Liu XH, et al. Ibrolipim increases ABCA1/G1 expression by the LXRalpha signaling pathway in THP-1 macrophage-derived foam cells. Acta Pharmacol Sin 2010;31:1343–9. [3] Out R, Hoekstra M, Habets K, et al. Combined deletion of macrophage ABCA1 and ABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosis at relatively low plasma cholesterol levels. Arterioscler Thromb Vasc Biol 2008;28:258–64. [4] Dai XY, Ou X, Hao XR, et al. The effect of T0901317 on ATP-binding cassette transporter A1 and Niemann-Pick type C1 in apoE−/− mice. J Cardiovasc Pharmacol 2008;51:467–75. [5] Mo ZC, Xiao J, Liu XH, et al. AOPPs inhibits cholesterol efflux by down-regulating ABCA1 expression in a JAK/STAT signaling pathway-dependent manner. J Atheroscler Thromb 2011;18:796–7. [6] Hu YW, Wang Q, Ma X, 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;17:493–502. [7] Julve J, Llaverias G, Blanco-Vaca F, et al. Seeking novel targets for improving in vivo macrophage-specific reverse cholesterol transport: translating basic science into new therapies for the prevention and treatment of atherosclerosis. Curr Vasc Pharmacol 2011;9:220–37. [8] Ohgami N, Miyazaki A, Sakai M, et al. Advanced glycation end products (AGE) inhibit scavenger receptor class B type I-mediated reverse cholesterol transport: a new crossroad of AGE to cholesterol metabolism. J Atheroscler Thromb 2003;10:1–6. [9] Lawrence JB, Oxvig C, Overgaard MT, et al. The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A. Proc Natl Acad Sci U S A 1999;96:3149–53. [10] Boldt HB, Overgaard MT, Laursen LS, et al. Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin. Biochem J 2001;358:359–67. [11] Thorn EM, Khan IA. Pregnancy-associated plasma protein-A: an emerging cardiac biomarker. Int J Cardiol 2007;117:370–2. [12] Consuegra-Sanchez L, Fredericks S, Kaski JC. Pregnancy-associated plasma protein-A (PAPP-A) and cardiovascular risk. Atherosclerosis 2009;203:346–52. [13] Bayes-Genis A, Conover CA, Overgaard MT, et al. Pregnancy-associated plasma protein A as a marker of acute coronary syndromes. N Engl J Med 2001;345:1022–9. [14] Lund J, Qin QP, Ilva T, et al. Circulating pregnancy-associated plasma protein a predicts outcome in patients with acute coronary syndrome but no troponin I elevation. Circulation 2003;108:1924–6. [15] Heeschen C, Dimmeler S, Hamm CW, et al. Pregnancy-associated plasma protein-A levels in patients with acute coronary syndromes: comparison with markers of systemic inflammation, platelet activation, and myocardial necrosis. J Am Coll Cardiol 2005;45:229–37. [16] Bayes-Genis A, Schwartz RS, Lewis DA, et al. Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty. Arterioscler Thromb Vasc Biol 2001;21:335–41. [17] Sangiorgi G, Mauriello A, Bonanno E, et al. Pregnancy-associated plasma protein-a is markedly expressed by monocyte-macrophage cells in vulnerable and ruptured carotid atherosclerotic plaques: a link between inflammation and cerebrovascular events. J Am Coll Cardiol 2006;47:2201–11. [18] Conover CA, Harrington SC, Bale LK, et al. Surface association of pregnancyassociated plasma protein-A accounts for its colocalization with activated macrophages. Am J Physiol Heart Circ Physiol 2007;292:H994–1000. [19] Crea F, Andreotti F. Pregnancy associated plasma protein-A and coronary atherosclerosis: marker, friend, or foe? Eur Heart J 2005;26:2075–6. [20] Tchoukalova YD, Nathanielsz PW, Conover CA, et al. Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon. Biochem Biophys Res Commun 2009;380:679–83. [21] Ortiz CO, Chen BK, Bale LK, et al. Transforming growth factor-beta regulation of the insulin-like growth factor binding protein-4 protease system in cultured human osteoblasts. J Bone Miner Res 2003;18:1066–72. [22] Byun D, Mohan S, Yoo M, et al. Pregnancy-associated plasma protein-A accounts for the insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) proteolytic activity in human pregnancy serum and enhances the mitogenic activity of IGF by degrading IGFBP-4 in vitro. J Clin Endocrinol Metab 2001;86:847–54. [23] Jia D, Heersche JN. Pregnancy-associated plasma protein-A proteolytic activity in rat vertebral cell cultures: stimulation by dexamethasone—a potential mechanism for glucocorticoid regulation of osteoprogenitor proliferation and differentiation. J Cell Physiol 2005;204:848–58.

353

[24] Berfield AK, Chait A, Oram JF, et al. IGF-1 induces rat glomerular mesangial cells to accumulate triglyceride. Am J Physiol Renal Physiol 2006;290:F138–47. [25] Nonomura K, Arai Y, Mitani H, et al. Insulin down-regulates specific activity of ATP-binding cassette transporter A1 for high density lipoprotein biogenesis through its specific phosphorylation. Atherosclerosis 2011;216:334–41. [26] Cao WM, Murao K, Imachi H, et al. Insulin-like growth factor-i regulation of hepatic scavenger receptor class BI. Endocrinology 2004;145:5540–7. [27] Hao XR, Cao DL, Hu YW, et al. IFN-gamma down-regulates ABCA1 expression by inhibiting LXRalpha in a JAK/STAT signaling pathway-dependent manner. Atherosclerosis 2009;203:417–28. [28] Hu YW, Ma X, Li XX, et al. Eicosapentaenoic acid reduces ABCA1 serine phosphorylation and impairs ABCA1-dependent cholesterol efflux through cyclic AMP/protein kinase A signaling pathway in THP-1 macrophage-derived foam cells. Atherosclerosis 2009;204:e35–43. [29] Liu XH, Xiao J, Mo ZC, et al. Contribution of D4-F to ABCA1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells. J Cardiovasc Pharmacol 2010;56:309–19. [30] Conover CA, Mason MA, Bale LK, et al. Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development. Am J Physiol Heart Circ Physiol 2010;299:H284–91. [31] Kumar A, Mohan S, Newton J, et al. Pregnancy-associated plasma protein-A regulates myoblast proliferation and differentiation through an insulin-like growth factor-dependent mechanism. J Biol Chem 2005;280:37782–9. [32] Huwait EA, Greenow KR, Singh NN, et al. A novel role for c-Jun N-terminal kinase and phosphoinositide 3-kinase in the liver X receptor-mediated induction of macrophage gene expression. Cell Signal 2011;23:542–9. [33] Andreotti F, Rio T, Conti E. Role of PAPP-A in atherothrombosis: messages to take home. Atherosclerosis 2009;203:353–4. [34] Pellitero S, Reverter JL, Granada ML, et al. Association of the IGF1/pregnancyassociated plasma protein-A system and adipocytokine levels with the presence and the morphology of carotid plaques in type 2 diabetes mellitus patients with stable glycaemic control. Eur J Endocrinol 2009;160:925–32. [35] Iversen K, Teisner A, Dalager S, et al. Pregnancy associated plasma protein-A (PAPP-A) is not a marker of the vulnerable atherosclerotic plaque. Clin Biochem 2011;44:312–8. [36] Heider P, Pfaffle N, Pelisek J, et al. Is serum pregnancy-associated plasma protein A really a potential marker of atherosclerotic carotid plaque stability? Eur J Vasc Endovasc Surg 2010;39:668–75. [37] Consuegra-Sanchez L, Fredericks S, Kaski J, et al. Pregnancy-associated plasma protein A: Has this biomarker crossed the boundary from research to clinical practice? Drug News Perspect 2009;22:341–8. [38] Harrington SC, Simari RD, Conover CA, et al. Genetic deletion of pregnancyassociated plasma protein-A is associated with resistance to atherosclerotic lesion development in apolipoprotein E-deficient mice challenged with a highfat diet. Circ Res 2007;100:1696–702. [39] Jahangiri A, Vaughan AM, de Beer FC, et al. ATP binding cassette G1-dependent cholesterol efflux during inflammation. J Lipid Res 2011;52:345–53. [40] Adorni MP, Zimetti F, Billheimer JT, et al. The roles of different pathways in the release of cholesterol from macrophages. J Lipid Res 2007;48:2453–62. [41] Tang C, Kanter JE, Bornfeldt KE, et al. Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys. J Lipid Res 2010;51:1719–28. [42] Kobayashi A, Takanezawa Y, Hirata T, et al. Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1. J Lipid Res 2006;47:1791–2. [43] Ji A, Meyer JM, Cai L, Akinmusire A, et al. Scavenger receptor SR-BI in macrophage lipid metabolism. Atherosclerosis 2011;217:106–12. [44] El Bouhassani M, Gilibert S, Moreau M, et al. CETP expression partially attenuates the adverse effects of SR-BI deficiency on cholesterol metabolism and atherosclerosis. J Biol Chem 2011;286:17227–38. [45] Krycer JR, Brown AJ. Crosstalk between the androgen receptor and the liver X receptor: implications for cholesterol homeostasis. J Biol Chem 2011;286:20637–47. [46] Shen Q, Bai Y, Chang KC, et al. Liver X receptor-retinoid X receptor (LXR-RXR) heterodimer cistrome reveals coordination of LXR and AP1 signaling in keratinocytes. J Biol Chem 2011;286:14554–63. [47] El Hajjaji FZ, Oumeddour A, Pommier AJ, et al. Liver X receptors, lipids and their reproductive secrets in the male. Biochim Biophys Acta 2011;1812: 974–81. [48] Aron-Wisnewsky J, Julia Z, Poitou C, et al. Effect of bariatric surgery-induced weight loss on SR-BI, ABCG1-, and ABCA1-mediated cellular cholesterol efflux in obese women. J Clin Endocrinol Metab 2011;96:1151–9. [49] Iversen KK, Teisner B, Winkel P, et al. Pregnancy associated plasma protein-A as a marker for myocardial infarction and death in patients with stable coronary artery disease: a prognostic study within the CLARICOR Trial. Atherosclerosis 2011;214:203–8. [50] Conti E, Carrozza C, Capoluongo E, et al. Insulin-like growth factor-1 as a vascular protective factor. Circulation 2004;110:2260–5. [51] Kane LP, Shapiro VS, Stokoe D, et al. Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol 1999;9:601–4. [52] Ozes ON, Mayo LD, Gustin JA, et al. NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999;401:82–5. [53] Scheidereit C. Ikappa B kinase complexes: gateways to NF-kappaB activation and transcription. Oncogene 2006;25:6685–95. [54] Dejardin E. The alternative NF-kappaB pathway from biochemistry to biology: pitfalls and promises for future drug development. Biochem Pharmacol 2006;72:1161–79.

354

S.-L. Tang et al. / Atherosclerosis 222 (2012) 344–354

[55] Gustin JA, Korgaonkar CK, Pincheira R, et al. Akt regulates basal and induced processing of NF-kappaB2 (p100) to p52. J Biol Chem 2006;281:16473–81. [56] Tanaka H, Fujita N. T suruo T, 3-Phosphoinositide-dependent protein kinase1-mediated IkappaB kinase beta (IkkB) phosphorylation activates NF-kappaB signaling. J Biol Chem 2005;280:40965–73. [57] Kelley KW, Weigent DA, Kooijman R, et al. Protein hormones and immunity. Brain Behav Immum 2007;21:384–92. [58] Salminen A, Kaarniranta K. Insulin/IGF-1 paradox of aging: regulation via AKT/IKK/NF-kappaB signaling. Cell Signal 2010;22:573–7. [59] Che W, Lerner-Marmarosh N, Huang Q, et al. Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression. Circ Res 2002;90:1222–30. [60] Madrid LV, Mayo MW, Reuther JY, et al. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 2001;276:18934–40. [61] Conover CA, Bale LK, Harrington SC, et al. Cytokine stimulation of pregnancy-associated plasma protein A expression in human coronary artery smooth muscle cells: inhibition by resveratrol. Am J Physiol Cell Physiol 2006;290:C183–8. [62] Conover CA, Harrington SC, Bale LK. Differential regulation of pregnancy associated plasma protein-A in human coronary artery endothelial cells and smooth muscle cells. Growth Horm IGF Res 2008;18:213–20.

[63] Resch ZT, Simari RD, Conover CA. Targeted disruption of the pregnancyassociated plasma protein-A gene is associated with diminished smooth muscle cell response to insulin-like growth factor-I and resistance to neointimal hyperplasia after vascular injury. Endocrinology 2006;147: 5634–40. [64] Cao DL, Yin K, Mo ZC, et al. Lipopolysaccharide down regulates ABCA1 expression in foam cells in a nucleus factor B pathway dependent manner. Prog Biochem Biophys 2010;37:540–8. [65] Chen M, Li W, Wang N, et al. ROS and NF-kappaB but not LXR mediate IL-1beta signaling for the downregulation of ATP-binding cassette transporter A1. Am J Physiol Cell Physiol 2007;292:C1493–501. [66] Field FJ, Watt K, Mathur SN. TNF-alpha decreases ABCA1 expression and attenuates HDL cholesterol efflux in the human intestinal cell line Caco-2. J Lipid Res 2010;51:1407–15. [67] Xue JH, Yuan Z, Wu Y, et al. High glucose promotes intracellular lipid accumulation in vascular smooth muscle cells by impairing cholesterol influx and efflux balance. Cardiovasc Res 2010;86:141–50. [68] Demers A, Caron V, Rodrigue-Way A, et al. A concerted kinase interplay identifies PPARgamma as a molecular target of ghrelin signaling in macrophages. PLoS One 2009;4:e7728. [69] Chen X, Zhao Y, Guo Z, et al. Transcriptional regulation of ATP-binding cassette transporter A1 expression by a novel signaling pathway. J Biol Chem 2011;286:8917–23.