Hepatic Overexpression of Idol Increases Circulating...

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Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for atherosclerotic car-

diovascular diseases (CVD). LDL receptor (LDLR) binds then uptakes apolipoprotein (apo) B–containing lipoproteins, in particular LDL, thereby playing a pivotal role in regulat-ing both intracellular and extracellular cholesterol levels. Indeed, human LDLR mutations cause familial hypercho-lesterolemia (FH), a common genetic disorder character-ized by high LDL-C levels and substantially increased CVD risk. Furthermore, hydroxymethylglutaryl-CoA reductase

inhibitors, statins, reduce LDL-C levels by enhancing LDLR expression especially in the liver,1 the central organ in coordi-nating regulation of circulating cholesterol.

LDLR expression is reportedly regulated by transcriptional and post-translational mechanisms.2,3 The latter includes proprotein convertase subtilisin/kexin type 9 (PCSK9), which was discovered as a novel gene causing autosomal dominant FH.4 PCSK9, a secretory protein belonging to the proteinase K subfamily of the secretory subtilase family, is highly expressed and secreted from the liver, followed by

© 2014 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.113.302670

Objective—Low-density lipoprotein receptor (LDLR) is degraded by inducible degrader of LDLR (Idol) and protein convertase subtilisin/kexin type 9 (PCSK9), thereby regulating circulating LDL levels. However, it remains unclear whether, and if so how, these LDLR degraders affect each other. We therefore investigated effects of liver-specific expression of Idol on LDL/PCSK9 metabolism in mice and hamsters.

Approach and Results—Injection of adenoviral vector expressing Idol (Ad-Idol) induced a liver-specific reduction in LDLR expression which, in turn, increased very-low-density lipoprotein/LDL cholesterol levels in wild-type mice because of delayed LDL catabolism. Interestingly, hepatic Idol overexpression markedly increased plasma PCSK9 levels. In LDLR-deficient mice, plasma PCSK9 levels were already elevated at baseline and unchanged by Idol overexpression, which was comparable with the observation for Ad-Idol–injected wild-type mice, indicating that Idol-induced PCSK9 elevation depended on LDLR. In wild-type mice, but not in LDLR-deficient mice, Ad-Idol enhanced hepatic PCSK9 expression, with activation of sterol regulatory element–binding protein 2 and subsequently increased expression of its target genes. Supporting in vivo findings, Idol transactivated PCSK9/LDLR in sterol regulatory element–binding protein 2/LDLR-dependent manners in vitro. Furthermore, an in vivo kinetic study using 125I-labeled PCSK9 revealed delayed clearance of circulating PCSK9, which could be another mechanism. Finally, to extend these findings into cholesteryl ester transfer protein–expressing animals, we repeated the above in vivo experiments in hamsters and obtained similar results.

Conclusions—A vicious cycle in LDLR degradation might be generated by PCSK9 induced by hepatic Idol overexpression via dual mechanisms: sterol regulatory element–binding protein 2/LDLR. Furthermore, these effects would be independent of cholesteryl ester transfer protein expression. (Arterioscler Thromb Vasc Biol. 2014;34:1171-1178.)

Key Words: cholesterol ester transfer proteins ◼ Idol protein, mouse ◼ Pcsk9 protein, mouse ◼ receptors, LDL

Received on: October 7, 2013; final version accepted on: March 18, 2014.From the Division of Anti-aging and Vascular Medicine, Department of Internal Medicine (M.S., Y.T., M.A., H.U.-K., M.I., M.Y., S.T., E.Y., K.N., M.O.,

T.K., K.I.) and Department of Physiology (K.H.), National Defense Medical College, Tokorozawa, Japan.*These authors contributed equally to this article.The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302670/-/DC1.Correspondence to Makoto Ayaori, MD, PhD, Division of Anti-aging, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki,

Tokorozawa, Japan 359-8513. E-mail [email protected]

Hepatic Overexpression of Idol Increases Circulating Protein Convertase Subtilisin/Kexin Type 9 in Mice and Hamsters

via Dual MechanismsSterol Regulatory Element–Binding Protein 2 and Low-Density Lipoprotein

Receptor–Dependent Pathways

Makoto Sasaki,* Yoshio Terao,* Makoto Ayaori, Harumi Uto-Kondo, Maki Iizuka, Makiko Yogo, Kosuke Hagisawa, Shunichi Takiguchi, Emi Yakushiji, Kazuhiro Nakaya,

Masatsune Ogura, Tomohiro Komatsu, Katsunori Ikewaki

by guest on June 6, 2018http://atvb.ahajournals.org/

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mailto:[email protected]

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1172 Arterioscler Thromb Vasc Biol June 2014

binding and internalization in a LDLR-dependent fashion, resulting in LDLR degradation. Gain-of-function muta-tions in PCSK9 accelerate hepatic LDLR degradation and therefore raise LDL-C levels to increase CVD risk.4 In con-trast, loss-of-function mutations enhance LDLR expression, lower LDL-C levels, and are associated with a substantial reduction in the incidence of CVD events.5 Recently, induc-ible degrader of LDLR (Idol) has emerged as yet another molecule involved in LDLR protein degradation.6 Idol is an E3 ubiquitin ligase for LDLR and facilitates LDLR degradation in lysosomes via polyubiquitination of LDLR. In view of these observations, both PCSK9 and Idol have been considered to be promising therapeutic targets for hypercholesterolemia.

PCSK9 and Idol expressions are transcriptionally regu-lated by sterol regulatory element–binding protein (SREBP) and liver X receptor (LXR), respectively. Intracellular cho-lesterol accumulation attenuates nuclear translocation of SREBP2, resulting in transrepression of PCSK9 and other SREBP-target genes whereas in sharp contrast, such circum-stances induce an increase in cellular oxysterol levels, thereby leading to LXR activation, which in turn transactivates Idol. These observations raise the question why PCSK9 and Idol should be regulated by such opposing mechanisms depend-ing on intracellular cholesterol levels, despite their similar molecular function of degrading of LDLR, and this has yet to be fully answered. Furthermore, it remains unclear whether, and if so how, PCSK9 and Idol affect each other.

In vivo LDLR degradation by Idol has been demonstrated only in mice, which lack cholesteryl ester transfer protein (CETP), resulting in an high-density lipoprotein (HDL)–dominant lipoprotein profile. Considering the fact that in subjects with CETP deficiency, the profiles7 and kinetic char-acteristics8,9 of overall lipoproteins are different from those of their normal counterparts, the effects of Idol on LDL metabo-lism should be assessed in CETP-expressing animals, such as hamsters. Furthermore, a CETP transgene reportedly resulted in decreased expression of hepatic LDLR,10 and it is therefore possible that the effect of Idol on LDL-C levels is less robust in CETP-expressing animals.

The present study demonstrated that, in mice and hamsters, hepatic overexpression of Idol raised not only circulating LDL but also serum PCSK9 levels in a LDLR/SREBP-dependent fashion.

Materials and MethodsMaterials and Methods are available in the online-only Supplement.

ResultsHepatic Overexpression of Idol Increased LDL/Very-Low-Density Lipoprotein Levels by Delaying LDL CatabolismWe first assessed the effects of hepatic Idol overexpression on the lipoprotein profile in wild-type C57BL/6 mice. Figure 1A and 1B show that injection of adenoviral vector expressing Idol (Ad-Idol) resulted in a marked increase in Idol mRNA lev-els and a dramatic reduction in LDLR expression in the liver, but not peritoneal macrophages, demonstrating that systemic Ad-Idol injection yielded a high degree of Idol overexpres-sion in the liver. Idol reportedly degrades very-low-density lipoprotein (VLDL) receptor.11 Although Idol did not affect VLDL receptor expression levels in skeletal muscle and heart as shown in Figure A–C in the online-only Data Supplement, we could not detect VLDL receptor expression in the liver. As previously reported,6 decreased liver LDLR expression attributable to Idol translated into marked increases in VLDL/LDL-C levels (Figure 1C). In parallel with cholesterol levels, apoB levels in VLDL/LDL fractions were also raised in the Idol-expressing mice. The apoE levels in LDL/HDL frac-tions were also increased by Idol (Figure 1D). Zelcer et al6 previously demonstrated that systemic injection of adenovi-rus expressing C387A mutant Idol, a catalytic center of the RING domain essential for E3 ligase activity, did not affect VLDL/LDL-C levels in mice and as shown in Figure 1E, we observed a similar phenomenon in mutant Idol-expressing mice. To further investigate the metabolic background for the increase in VLDL/LDL-C levels attributable to Idol, we performed a kinetic study using 125I-labeled LDL. As shown in Figure 1F, hepatic Idol overexpression significantly decreased rates of LDL catabolism as compared with control (fractional catabolic rates in mice injected with adenoviral vector expressing luciferase [Ad-Luc], 0.12±0.01; Ad-Idol, 0.09±0.01 pool/hour; P<0.001).

Hepatic Overexpression of Idol Increased Circulating PCSK9 LevelsThe next issue we addressed was whether hepatic Idol over-expression modulated circulating PCSK9 levels. As shown in Figure 2A–2C, intravenous injection of Ad-Idol caused a dose-dependent increase in serum total cholesterol (up to 462 mg/dL at peak from 113 mg/dL at baseline, at high-est dose), triglycerides (up to 508 mg/dL at peak from 241 mg/dL at baseline, at highest dose), and apoB levels. Fast-protein liquid chromatography (FPLC) lipoprotein analy-ses revealed that less dramatic Idol overexpression using a lower dose of adenovirus produced a modest increase in VLDL/LDL cholesterol levels as compared with high dose adenovirus (Figure 2D). Figure 2E shows that hepatic Idol overexpression increased circulating PCSK9 levels (up to 1122 ng/dL at peak from 116 ng/dL at baseline, at high-est dose), in a dose-dependent fashion. Interestingly, serum PCSK9 levels increased more rapidly and peaked earlier as compared with serum total cholesterol/triglyceride levels.

Nonstandard Abbreviations and Acronyms

CETP cholesteryl ester transfer protein

CVD cardiovascular diseases

FH familial hypercholesterolemia

HDL high-density lipoprotein

LDL low-density lipoprotein

LDLR low-density lipoprotein receptor

LXR liver X receptor

PCSK9 proprotein convertase subtilisin/kexin type 9

SREBP sterol regulatory element–binding protein

VLDL very-low-density lipoprotein


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Idol-Induced Hepatic Expression of PCSK9 and SREBP2 Exhibited Different Time CoursesDuring 14 days of observation of hepatic LDLR, PCSK9, and SREBP2 expression after Idol injection, we noticed differential responses for these molecules between an early (4 days) and a late (14 days) time point. At the early time point, LDLR levels were reduced by Idol in a dose-dependent manner; however, PCSK9 and SREBP2 levels were unchanged (Figure 3A). In sharp contrast, 14 days after the injection, hepatic Idol over-expression dose dependently increased levels of PCSK9 and nuclear SREBP2 (Figure 3B), indicating enhanced SREBP2 activity. To further investigate underlying molecular mecha-nisms, we determined the expression levels of various molecules involved in LDL metabolism in the liver at these 2 time points. Findings obtained from quantitative real-time polymerase chain reaction (RT-PCR) supported the above observations in Western blots. Four days after the injection, we found that Ad-Idol was indeed dose dependently transduced in the liver (Figure 3C). Idol induced modest increases in the expressions of SREBP2-target genes such as LDLR, hydroxymethylglutaryl-CoA reduc-tase, Tmem97, and SREBP2 itself, but PCSK9 mRNA levels were unchanged. In contrast, at the late time point, injection of Ad-Idol, especially at the middle dose, increased expression of all SREBP2-target genes (Figure 3D), supporting the findings for PCSK9/SREBP2 protein levels (Figure 3B).

Hepatic Overexpression of Idol Increased Circulating Cholesterol and PCSK9 Levels in an LDLR-Dependent FashionGiven the stimulatory effect of hepatic Idol overexpres-sion on PCSK9 described above, we next assessed whether this phenomenon was dependent on presence of LDLR. As shown in Figure 4A, injection of Ad-Idol did not affect cho-lesterol levels among lipoprotein fractions in Ldlr−/− mice, a finding consistent with a previous study.6 Interestingly, PCSK9 levels in Ldlr−/− mice were higher than those in wild-type mice at baseline (Figure 4B, 916 versus 112 ng/mL, respectively) as previously reported.12 Also, PCSK9 levels in the latter were markedly increased (up to 1123 ng/mL at day 8) by adenovirus injection to levels comparable with those in Idol injected Ldlr−/− mice (1208 ng/mL at day 8). Furthermore, in Ldlr−/− mice, PCSK9 levels were identical between Idol and control. Collectively, these data indicated that Idol increased circulating PCSK9 levels in an LDLR-dependent fashion. Figure 4C and 4D shows that, in contrast to wild-type mice, hepatic levels of PCSK9/SREBP2 protein and mRNA levels of SREBP-target genes, PCSK9, hydroxymethylglutaryl-CoA reductase, and SREBP2 itself, were not affected by Idol overexpression, both at days 4 (Figure D and E in the online-only Data Supplement) and 14 (Figure 4C and 4D).

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* Figure 1. Hepatic overexpression of Idol increases low-density lipoprotein (LDL)/very levels of LDL by delaying LDL catabolism. A and B, Seven days after intravenous injection of adenoviral vector express-ing Idol (Ad-Idol) or Ad-Luc (2.5×108 pfu), C57BL/6 mice (n=5, each) were euthanized to harvest livers, peritoneal macrophages, and plasma. Real-time quantitative poly-merase chain reaction (RT-PCR) (A) and Western blot analyses (B) were performed as described in Methods. C and D, For lipoprotein fractionation analysis, equal volumes of plasma samples were pooled from mice for each group. Lipoproteins were fractionated by fast-protein liquid chromatography (FPLC). Fractions were collected and used for cholesterol mea-surement (C) and Western blot analysis (D). E, Seven days after intravenous injection of Ad-Idol C387A and adenoviral vector expressing luciferase (Ad-Luc) (n=5, each), blood samples were obtained from the mice and lipoprotein fractionation, and cholesterol determination were performed as describe above. F, Four days after intra-venous injection of Ad-Idol or Ad-Luc to C57BL/6J mice (n=6, each), blood samples were obtained at the indicated time points after injection of 125I-labeled LDL, and then the 125I remaining in the plasma was measured by a γ-counter. The fractional catabolic rate (FCR) was determined using the SAAMII program. Luc indicates lucif-erase. The results are representative of 3 or more experiments and are presented as mean±SD (SE in case of 125I-kinetic study). *P<0.001 vs control. HDL indicates high-density lipoprotein; and VLDL, very-low-density lipoprotein. by guest on June 6, 2018

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Idol Activated PCSK9/LDLR Promoter via SREBP/LDLR-Dependent PathwaysWe next performed promoter assays using HepG2 cells and luciferase constructs containing PCSK9 and LDLR promoter to further clarify the precise mechanism for the inducible effect of Idol on PCSK9. Figure 5A shows LDLR expression levels based on presence/absence of cholesterol (Chol/25-hydroxycholesterol (25HC), LDL) and Idol, condi-tions used in the promoter assays. Under cholesterol depletion media, addition of cholesterol by means of 25HC and LDL markedly reduced LDLR protein expression (Figure 5A), which was further reduced by Idol overexpression. Idol over-expression also dramatically reduced LDLR levels even under cholesterol depletion media (Figure 5A, left 2 lanes). As com-pared with cholesterol depletion media, cholesterol added by means of 25HC and LDL suppressed both PCSK9 and LDLR promoter activities (Figure 5B). Under cholesterol depletion, Idol significantly reduced the promoter activity of PCSK9, but not of LDLR. Under media with cholesterol added, however, Idol increased both PCSK9 and LDLR promoter activities only in the presence of LDL, but not cholesterol 25HC. Furthermore, when mutations were introduced into sterol-responsive elements, the above changes all disappeared. Overall, these results indicate that Idol overexpression acti-vates PCSK9/LDLR promoter via LDLR/SREBP2-dependent pathways, supporting the in vivo results.

LDLR Reduction Induced by Idol Overexpression Impaired Circulating PCSK9 ClearanceTo find out why PCSK9 levels were increased at the early time point despite no change in PCSK9 expression, we per-formed an in vivo radiotracer study on PCSK9. As shown by

the curves in Figure 5C, PCSK9 decay was slower for hepatic overexpression in Idol mice than in control mice (fractional catabolic rates in mice injected with Ad-Luc, 0.25±0.09; Ad-Idol, 0.21±0.04, P<0.05). This result was consistent with the previous observation: delayed clearance of PCSK9 in Ldlr−/− mice.13 Taken together, these results indicate that LDLR reduction induced by Idol overexpression impairs cir-culating PCSK9 clearance.

Hepatic Overexpression of Idol Increased Serum Cholesterol Levels by Delaying LDL Catabolism and Raised Circulating PCSK9 Levels in HamstersBecause mice lack CETP expression, HDL, rather than LDL, is the dominant lipoprotein (Figure 1C), which would be a major limitation in translating the findings of the present study into human circumstances. Therefore, we performed in vivo experiments using hamsters, which like humans are a CETP-expressing animal. As shown in Figure 6A, ham-sters had comparable cholesterol levels in VLDL/LDL/HDL fractions. Ad-Idol intravenous injection resulted in increased cholesterol levels in VLDL/LDL, but not HDL, which was consistent with the findings in mice (Figure 1C). Similar to mice, hepatic overexpression in hamsters increased apoB con-tent in VLDL/LDL fractions, and apoE in the HDL fraction (Figure 6B). Western blot analyses revealed that Idol over-expression in the liver reduced LDLR and raised PCSK9/SREBP2 levels (Figure 6C). The LDL kinetic study also confirmed the effects of Idol on LDL catabolism in hamsters (Figure 6D, fractional catabolic rates in hamsters injected with Ad-Luc, 0.14±0.02; Ad-Idol, 0.11±0.01 pool/hour; P<0.01). There was also an increase in serum PCSK9 levels after Ad-Idol injection in hamsters (Figure 6E, up to 169 ng/mL

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Figure 2. Hepatic overexpression of Idol increases circulating protein convertase sub-tilisin/kexin type 9 (PCSK9) levels. A−D, At the indicated days after injection of adenovi-ral vector expressing Idol (Ad-Idol) or Ad-Luc (2.5×108 pfu) to C57BL/6 mice (n=6, each), blood sampling was performed to obtain plasma, which was subjected to measure-ment of total cholesterol (A), triglycerides (B), and PCSK9 (E). Pooled plasma obtained 7 days after the injection was subjected to Western blot analysis using antiapoB antibodies (C) and FPLC (D). Fractions iso-lated by FPLC were collected and used for cholesterol measurement (D). The results are representative of 2 or more experi-ments and are presented as mean±SD. *P<0.05, †P<0.001 vs control. HDL indicates high-density lipoprotein; LDL, low density lipoprotein; and VLDL, very-low-density lipoprotein.


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at peak from 67 ng/mL at baseline). Overall, the results for hamsters were consistent with those for mice, indicating that the observed effects of Idol are CETP independent.

DiscussionIn the following, we summarize the putative mechanisms by which hepatic Idol overexpression increased circulating PCSK9 levels. At the early time point, Idol overexpression reduced LDLR expression and reduced LDL catabolism, resulting in LDL accumulation. Reduced LDLR expression attributable to Idol overexpression also impaired PCSK9 clearance, leading to PCSK9 accumulation. As a result, Idol overexpression positively modulated PCSK9, and the changes in both of them, in turn, accelerated LDLR degradation and increased LDL levels by decreasing LDL catabolism.

At the later time point, however, increased transcriptions of SREBP2 and PCSK9 were evident. We speculate that cellular cholesterol content in the liver decreased because of reduction of hepatic LDLR expression induced by Idol, leading to SREBP2 activation, which in turn increased PCSK9 transcription and secretion. Such PCSK9 regulation translates into the stimulatory effect of statin treatment on PCSK9 transcription and secretion as previously observed in vitro14 and in vivo15,16 experiments.

LDLR plays a central role in cellular cholesterol homeosta-sis. When cellular cholesterol levels are reduced, SREBP2 in the endoplasmic reticulum is cleaved and translocated to the nucleus, where it transactivates SREBP2-target genes, includ-ing LDLR. Under such circumstances, cellular oxysterol content is also decreased, leading to reduced Idol expression, which in turn upregulates LDLR expression. Thus, transcrip-tional regulation of Idol by LXR makes sense with regard to physiological cholesterol homeostasis. In contrast, PCSK9 regulation seems complex; activation of SREBP2 results in transactivation of both LDLR and PCSK9, which counteract increased LDLR expression. One possible explanation for this type of regulation is that promoted expression of PCSK9 under cholesterol depletion contributes to cholesterol homeo-stasis as a brake in advance, that is, an attempt made not to accumulate excess cholesterol via LDLR. Similar regulation is observed between interleukin-1 and interleukin-1 antago-nist, as a brake against inflammatory acceleration.17

In vitro promoter assays (Figure 5) revealed that Idol over-expression increased the promoter activities of PCSK9 and LDLR under the presence of extracellular LDL, which was, in general, consistent with the in vivo experiments (Figure 3). This observation can be interpreted as Idol-mediated LDLR reduction leading to reduced cellular cholesterol levels which in turn activates SREBP2 with consequent PCSK9/LDLR transactivation. If this is the case, this raises the question; why did SREBP2 activation only occur at the later time point? (Figure 3). Although speculative, we propose a putative mecha-nism in which time is needed for Idol-induced LDLR under the presence of extracellular cholesterol to cause a reduction in the cholesterol pool size in the liver. In the in vitro promoter assays (Figure 5B), only under cholesterol depletion did Idol attenu-ate the promoter activity of PCSK9, but not LDLR, and this phenomenon was independent of SREBP. The mechanisms for this remain unclear and thus deserve further studies.

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Figure 3. Idol induces increases in hepatic protein convertase subtilisin/kexin type 9 (PCSK9) levels and sterol regulatory element–binding protein 2 (SREBP2) activation at later time point. A–D, Four (A, C) and 14 (B, D) days after intravenous injection of adenoviral vector expressing Idol (Ad-Idol) or Ad-Luc at the indicated doses, C57BL/6J mice (n=6, each) were euthanized to harvest livers. Western blot analyses for low-density lipoprotein receptor (LDLR), PCSK9 (P, proprotein; CL, cleaved form), and SREBP2 (P, precursor; N, nuclear) and real-time quantitative RT-PCR (C, D, n=6, each group; HMGCR, HMG-CoA reductase) were performed as described in Meth-ods. The results are representative of 3 or more experiments and are presented as mean±SD. *P<0.05, †P<0.01, ‡P<0.001 vs control.


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However, some of the findings of the in vivo study need dis-cussion; as compared with the highest dose, the middle dose of Ad-Idol had a greater positive effect on the mRNA levels of SREBP-target genes (Figure 3D), whereas protein levels of SREBP2 and PCSK9 increased in an Ad-Idol dose-dependent manner (Figure 3B). Although we do not know the precise reasons for this phenomenon at present, unknown compensa-tory mechanisms counteracting the highest dose of Ad-Idol could be involved.

LXR activation in macrophages has recently been receiv-ing increased attention as a promising therapeutic target against atherosclerotic diseases because it directly stimulates

HDL-mediated cholesterol efflux via ATP-binding cassette transporter A1/G1 pathways.18 However, development of LXR agonists has been hampered by their induction of hepatic ste-atosis, which might be because of increased fatty acid bio-synthesis via increased expression of SREBP1-c.18 Besides, LXR agonists might also raise LDL-C levels. In this regard, Honzumi et al19 reported that administration of a synthetic LXR ligand, TO0901317, increased LDL-C levels at a dose that did not affect triglyceride levels in rabbits. They did not assess Idol and PCSK9 expressions but TO0901317 may enhance them. In the present study, although overexpression of Idol in mice does not represent physiological conditions, our observations

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B Figure 5. Idol overexpression activates protein con-vertase subtilisin/kexin type 9 (PCSK9)/LDLR pro-moter via LDLR/sterol regulatory element–binding protein 2 (SREBP2)-dependent pathways. A and B, HepG2 cells were transfected with mPCSK9 (SREmut)-Luc/hLDLR (SREmut)-Luc and phRL-TK. Ten hours after transfection, the medium was replaced with cholesterol depletion media (5% lipoprotein-deficient serum (LPDS), 50 μmol/L pravastatin, 10 μmol/L mevalonate) in the absence or presence of cholesterol (Chol)/25 hydroxy-cho-lesterol (25HC) or LDL. Simultaneously, Ad-Idol or adenoviral vector expressing β-galactosidase (Ad-LacZ, 30 multiplicity of infection [MOI]) was added to the cells, which were harvested after 24 hours of incubation, and samples subjected to Western blot analysis (A) and luciferase assays (B). The results are representative of 3 experiments and are pre-sented as mean±SD (n=4, each group). *P<0.01, †P<0.001 vs LacZ, ‡P<0.001 vs control (cholesterol depletion). C, Three days after injection of Ad-Idol or Ad-Luc (2.5×108 pfu) to C57BL/6 mice (n=6, each), blood samples were obtained at the indicated time points after injection of 125I-labeled PCSK9, and then the 125I remaining in the plasma was measured by a γ-counter. The fractional catabolic rate (FCR) was determined using the SAAMII program. The results are representative of 2 experiments and are pre-sented as mean±SE. *P<0.05 vs Luc. LacZ indicates β-galactosidase; and Luc, luciferase.

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Figure 4. Hepatic overexpression of Idol increases circulating cholesterol and protein convertase subtilisin/kexin type 9 (PCSK9) levels in an low-density lipoprotein receptor (LDLR)-dependent fashion. A and B, At the indicated days after intravenous injection of Ad-Idol or Ad-Luc (2.5×108 pfu), Ldlr−/− mice (n=5, each) were euthanized to obtain plasma, which was subjected to lipoprotein fraction-ation (8-day plasma), determination of choles-terol (A), PCSK9 (B) levels, and Western blot analysis for apolipoprotein B (apoB; C, 8-day plasma, n=3, each group) were performed as described in Methods. At the end of the above experiments, livers were obtained and real-time quantitative RT-PCR (D, n=6, each group) was performed as described in Methods. The results are representative of 2 or more experiments and presented as mean±SD. * P<0.001 vs Luc. HMGCR indi-cates HMG-CoA reductase; Luc, luciferase; SREBP2, sterol regulatory element–binding protein 2; and WT, wild-type.


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suggest the possibility that Idol plays an important role in LDL metabolism via a LXR pathway and could thus lead to a novel LXR-targeted therapy against atherosclerosis.

Another major finding of the present study is the interaction between Idol and PCSK9. Idol increased PCSK9 levels in serum and the liver, which together accelerated LDLR catabolism and increased LDL levels by delaying catabolism. PCSK9 report-edly facilitates LDLR protein degradation after internalization of LDLR primarily by binding to the extracellular domain,20,21 this mechanism differing from that of Idol, which works intra-cellularly.6 It has also been reported that the capability of PCSK9 to induce LDLR degradation is preserved even in cells lacking Idol,22 indicating that Idol and PCSK9 function in complemen-tary but independent pathways. Consistent with the findings of previous research,13 our PCSK9 kinetic experiment revealed that LDLR deletion or inhibition impaired PCSK9 catabolism (Figure 5C). The resultant increase in PCSK9 acted in concert with Idol to exacerbate hypercholesterolemia, including FH, in the mice. Raal et al16 recently demonstrated that PCSK9 levels were elevated in untreated FH patients, particularly in those with homozygous FH, as compared with the control subjects. PCSK9 levels in heterozygous FH were also higher than those in the controls; however, they seemed to be lower than those in homo-zygous FH. In the present study, we demonstrated that complete inhibition of LDLR expression by Idol overexpression at high dose resulted in a comparable increase in PCSK9 levels to that in Ldlr−/− mice (Figure 4B). In contrast, partial LDLR inhibi-tion yielded a modest increase in PCSK9 (Figure 2D). These observations support the above findings in homozygous and het-erozygous FH patients; namely LDLR-dependent regulation of PCSK9. Treatment of FH subjects with statins and antibodies

against PCSK9 reportedly exerted additive LDL-C lowering effects.23 Because PCSK9 inhibition or deletion might attenuate hypercholesterolemia in Idol-expressing mice, further studies using PCSK9 knockout mice or its inhibitors are needed.

Mice are the most commonly used research animal, but caution should be exercised in the interpretation of data and translation into the human setting because they lack CETP. Indeed, in subjects with CETP deficiency, the profiles7 and kinetic characteristics8,9 of overall lipoproteins are unique and substantially different from their normal counterparts. Although we should be cautious in translating the findings obtained from hamsters into the human setting, the hamster model is perhaps a better alternative to the mouse model in the sense that it possesses CETP activity. Therefore, we repeated experiments using hamsters to confirm the effects of hepatic Idol overexpression, and they yielded similar findings regard-ing lipoprotein profiles, LDL kinetics, and circulating PCSK9 concentrations, suggesting that the effects of Idol overexpres-sion are CETP independent.

In conclusion, the present study demonstrated for the first time that hepatic Idol overexpression increased PCSK9 via dual mechanisms, SREBP2, and LDLR-dependent pathways, which then act in concert to degrade LDLR. Furthermore, because these phenomena were independent of CETP expression, our observa-tions may provide the basis for novel Idol-PCSK9 targeted ther-apies against hypercholesterolemia and CVD in humans.

AcknowledgmentsWe thank Dr Takeshi Adachi and Dr Yasushi Miyahira for providing us with a fast protein liquid chromatography system and an isolation room for the adenovirus experiments, respectively.

1

10

100

0 10 20 30 40 50Perc

enta

ge re

mai

ning

(%)

Time after injection (hr)

A

B

D4-

67-

910

-12

13-1

516

-18

19-2

122

-24

VLDL

Fractions

ApoB

LDL HDL

4-6

7-9

10-1

213

-15

16-1

819

-21

22-2

4VLDL LDL HDL

ApoA-I

ApoE

Luc Idol

100

48

VLDL

LDL

HDL *

E

LDLR

C

Luc Idol

* *

-β actin

Figure 6. Hepatic overexpression of Idol increases serum cholesterol levels by delay-ing low-density lipoprotein (LDL) catabolism and raises circulating protein convertase sub-tilisin/kexin type 9 (PCSK9) levels in hamsters. A–C, Seven days after intravenous injection of adenoviral vector expressing Idol (Ad-Idol) or Ad-Luc (7.5×108 pfu) to Syrian Golden hamsters, blood and livers were obtained. FPLC-fractions from plasma were subjected to cholesterol measurement (A) and Western blot analyses (B and C). D, Four days after intravenous injection of Ad-Idol or Ad-Luc to Syrian Golden hamsters (7.5×108 pfu), blood samples were obtained at the indicated time points after injection of 125I-labeled LDL, and then the 125I remaining in the plasma was measured by a γ-counter. The fractional cata-bolic rate was determined using the SAAMII program. The results are representative of 2 or more experiments and are presented as mean±SE. *P<0.001 vs Luc. Apo indicates apolipoprotein.


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Sources of FundingThis work was supported by Foundation for Promotion of Defense Medicine.

DisclosuresNone.

References 1. Heber D, Koziol BJ, Henson LC. Low density lipoprotein receptor regula-

tion and the cellular basis of atherosclerosis: implications for nutritional and pharmacologic treatment of hypercholesterolemia. Am J Cardiol. 1987;60:4G–8G.

2. Brown MS, Goldstein JL. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A. 1979;76:3330–3337.

3. Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for mem-brane sterols. Cell. 2006;124:35–46.

4. Abifadel M, Varret M, Rabès JP, et al. Mutations in PCSK9 cause autoso-mal dominant hypercholesterolemia. Nat Genet. 2003;34:154–156.

5. Cohen JC, Boerwinkle E, Mosley TH Jr, Hobbs HH. Sequence varia-tions in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med. 2006;354:1264–1272.

6. Zelcer N, Hong C, Boyadjian R, Tontonoz P. LXR regulates choles-terol uptake through Idol-dependent ubiquitination of the LDL receptor. Science. 2009;325:100–104.

7. Nagano M, Yamashita S, Hirano K, Takano M, Maruyama T, Ishihara M, Sagehashi Y, Kujiraoka T, Tanaka K, Hattori H, Sakai N, Nakajima N, Egashira T, Matsuzawa Y. Molecular mechanisms of cholesteryl ester trans-fer protein deficiency in Japanese. J Atheroscler Thromb. 2004;11:110–121.

8. Ikewaki K, Nishiwaki M, Sakamoto T, Ishikawa T, Fairwell T, Zech LA, Nagano M, Nakamura H, Brewer HB Jr, Rader DJ. Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency. J Clin Invest. 1995;96:1573–1581.

9. Ikewaki K, Rader DJ, Sakamoto T, Nishiwaki M, Wakimoto N, Schaefer JR, Ishikawa T, Fairwell T, Zech LA, Nakamura H. Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency. J Clin Invest. 1993;92:1650–1658.

10. Jiang XC, Masucci-Magoulas L, Mar J, Lin M, Walsh A, Breslow JL, Tall A. Down-regulation of mRNA for the low density lipoprotein receptor in transgenic mice containing the gene for human cholesteryl ester transfer protein. Mechanism to explain accumulation of lipoprotein B particles. J Biol Chem. 1993;268:27406–27412.

11. Hong C, Duit S, Jalonen P, Out R, Scheer L, Sorrentino V, Boyadjian R, Rodenburg KW, Foley E, Korhonen L, Lindholm D, Nimpf J, van Berkel TJ, Tontonoz P, Zelcer N. The E3 ubiquitin ligase IDOL induces the deg-radation of the low density lipoprotein receptor family members VLDLR and ApoER2. J Biol Chem. 2010;285:19720–19726.

12. Tavori H, Fan D, Blakemore JL, Yancey PG, Ding L, Linton MF, Fazio S. Serum proprotein convertase subtilisin/kexin type 9 and cell surface low-density lipoprotein receptor: evidence for a reciprocal regulation. Circulation. 2013;127:2403–2413.

13. Grefhorst A, McNutt MC, Lagace TA, Horton JD. Plasma PCSK9 preferentially reduces liver LDL receptors in mice. J Lipid Res. 2008;49:1303–1311.

14. Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L, Prat A. Statins upregulate PCSK9, the gene encoding the proprotein conver-tase neural apoptosis-regulated convertase-1 implicated in familial hyper-cholesterolemia. Arterioscler Thromb Vasc Biol. 2004;24:1454–1459.

15. Careskey HE, Davis RA, Alborn WE, Troutt JS, Cao G, Konrad RJ. Atorvastatin increases human serum levels of proprotein convertase sub-tilisin/kexin type 9. J Lipid Res. 2008;49:394–398.

16. Raal F, Panz V, Immelman A, Pilcher G. Elevated PCSK9 levels in untreated patients with heterozygous or homozygous familial hypercho-lesterolemia and the response to high-dose statin therapy. J Am Heart Assoc. 2013;2:e000028.

17. Weber A, Wasiliew P, Kracht M. Interleukin-1 (IL-1) pathway. Sci Signal. 2010;3:cm1.

18. Im SS, Osborne TF. Liver x receptors in atherosclerosis and inflammation. Circ Res. 2011;108:996–1001.

19. Honzumi S, Shima A, Hiroshima A, Koieyama T, Terasaka N. Synthetic LXR agonist inhibits the development of atherosclerosis in New Zealand White rabbits. Biochim Biophys Acta. 2011;1811:1136–1145.

20. Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J. Molecular basis for LDL receptor recognition by PCSK9. Proc Natl Acad Sci U S A. 2008;105:1820–1825.

21. Zhang DW, Garuti R, Tang WJ, Cohen JC, Hobbs HH. Structural require-ments for PCSK9-mediated degradation of the low-density lipoprotein receptor. Proc Natl Acad Sci U S A. 2008;105:13045–13050.

22. Scotti E, Hong C, Yoshinaga Y, Tu Y, Hu Y, Zelcer N, Boyadjian R, de Jong PJ, Young SG, Fong LG, Tontonoz P. Targeted disruption of the idol gene alters cellular regulation of the low-density lipoprotein receptor by sterols and liver x receptor agonists. Mol Cell Biol. 2011;31:1885–1893.

23. Stein EA, Mellis S, Yancopoulos GD, Stahl N, Logan D, Smith WB, Lisbon E, Gutierrez M, Webb C, Wu R, Du Y, Kranz T, Gasparino E, Swergold GD. Effect of a monoclonal antibody to PCSK9 on LDL choles-terol. N Engl J Med. 2012;366:1108–1118.

Inducible degrader of low-density lipoprotein (LDL) receptor (Idol), together with protein convertase subtilisin/kexin type 9 (PCSK9), promotes LDL receptor degradation, thereby regulating circulating LDL levels. Idol and PCSK9 are transcriptionally regulated via liver X receptor and sterol regulatory element–binding protein pathways, respectively. However, it remains largely unknown whether, and if so how, these 2 af-fect each other. We demonstrated for the first time that hepatic Idol overexpression did increase circulating PCSK9 in mice and hamsters. We also showed that the interplay between them was mediated by dual mechanisms: sterol regulatory element–binding protein 2 and LDL receptor–dependent pathways, which then act in concert to degrade LDL receptor, leading to generation of a vicious cycle. Because these phenomena were independent of cholesteryl ester transfer protein expression, our observations may provide the basis for novel Idol-PCSK9 targeted therapies against hypercholesterolemia and cardiovascular diseases in humans.

Significance


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Tomohiro Komatsu and Katsunori IkewakiKosuke Hagisawa, Shunichi Takiguchi, Emi Yakushiji, Kazuhiro Nakaya, Masatsune Ogura,

Makoto Sasaki, Yoshio Terao, Makoto Ayaori, Harumi Uto-Kondo, Maki Iizuka, Makiko Yogo,Dependent Pathways−Protein 2 and Low-Density Lipoprotein Receptor

Binding−Type 9 in Mice and Hamsters via Dual Mechanisms: Sterol Regulatory ElementHepatic Overexpression of Idol Increases Circulating Protein Convertase Subtilisin/Kexin

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2014 American Heart Association, Inc. All rights reserved.

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1

Hepatic overexpression of Idol increases circulating PCSK9 in mice and hamsters via dual mechanisms: SREBP2 and LDL receptor dependent pathways

Sasaki et al, Idol degrades LDL receptor partially via PCSK9

Makoto Sasaki1*, MD; Yoshio Terao1*, MD, PhD; Makoto Ayaori1, MD, PhD; Harumi

Uto-Kondo1, PhD; Maki Iizuka1, PhD; Kosuke Hagisawa2, MD, PhD; Shunichi Takiguchi,

MD, PhD ;1, Emi Yakushiji1, MD, PhD; Makiko Yogo1, MD; Kazuhiro Nakaya1, MD, PhD;

Masatsune Ogura1, MD, PhD; Tomohiro Komatsu1, MD; Katsunori Ikewaki1, MD, PhD

1Division of Anti-aging and Vascular Medicine, Department of Internal Medicine, 2Department of Physiology, National Defense Medical College

*These authors equally contributed to this paper.

Correspondence to Makoto Ayaori

Division of Anti-aging, Department of Internal Medicine, National Defense Medical College

3-2 Namiki, Tokorozawa, Japan 359-8513

TEL:+81-4-2995-1617 FAX: +81-4-2996-5202

E-mail: [email protected]

2

Materials and Methods

Animals and Materials

C57BL/6 male mice and Syrian golden male hamsters were obtained from Clea Japan

(Tokyo, Japan) and fed a standard chow diet. Low-density lipoprotein (LDL) receptor

(LDLR)-deficient (Ldlr−/−) mice with the C57BL/6J background1 were purchased from the

Jackson Laboratory (Bar Harbor, ME). The experiments were performed on mice and

hamsters aged 6-8 weeks. Animals were handled according to the guidelines of National

Defense Medical College Institutional Animal Care and Use Committee. Pravastatin,

mevalonate and 25-hydroxycholesterol (25HC) were purchased from Sigma (St. Louis, MO)

Cloning and Generation of Recombinant Adenoviruses Encoding for Mouse Wild type

and Mutant Idol

A recombinant adenovirus expressing Flag-tagged mouse inducible degrader of LDLR

(Ad-Idol) was produced using the ViraPower Adenoviral Expression System (Invitrogen)

according to the manufacturer's instructions. Briefly, to generate an entry clone of the

Gateway system (Invitrogen), cloning of the open reading frame with a carboxy-terminal

Flag-tag into a pENTR/D-TOPO vector (Invitrogen) was carried out using first strand cDNA

derived from mouse liver as a template and the specific primers as follows: forward: 5′-CAC

CAT GCT GTG CTA TGT GAC GAG-3′; reverse: 5′-TTA CTT GTC ATC GTC GTC CTT

GTA GTC GAT GAC AGT CAG ATT GAG GAG-3′. To obtain a reporter construct with

mutations (C387A) in the RING domain of the mouse Idol, we performed site-directed

mutagenesis using a Quick Change II Site-Directed Mutagenesis Kit (Stratagene La Jolla,

CA, USA) and the primers (lower cases indicate mutated bases) indicated as follows:

forward: 5'- GGA AGC CAT GCT Ggc cAT GGC GTG CTG CGA GG-3' – reverse: 5'- CCT

CGC AGC ACG CCA Tgg cCA GCA TGG CTT CC -3'.

An expression clone for adenoviral vector was then generated by performing a LR

recombination reaction between the entry clone and a pAd/CMV/V5-DEST (Invitrogen)

according to the manufacturer's protocol. The recombinant adenoviral plasmid was purified,

and then transfected into 293A cells. After a sufficient cytopathic effect was observed in the

cells, the adenovirus was purified using the Adeno-X Virus Purification Kit (Clontech, Pao

Alto, CA, USA). Adenoviral vectors expressing luciferase (Ad-Luc) and β-galactosidase

(Ad-LacZ) were kindly donated by Dr. Santamarina-Fojo S of National Institute of Health,

and used as a control. The adenovirus titer in plaque-forming units (pfu) was determined by

a plaque formation assay following infection of 293A cells. The multiplicity of infection

(MOI) was defined as the ratio of the total number of plaque-forming units to the total

number of cells that were infected. Mice and hamsters were injected intravenously via the

tail vein with respectively 2.5×108 and 7.5×108 pfu of purified recombinant adenovirus on

day 0 of the study. Blood was obtained from mice tail veins and hamster jugular veins at

3

several time points after injection. Aliquots of serum were stored at -80○C for subsequent

lipid analysis. At the indicated days after adenovirus injection, mice and hamsters were

exsanguinated and livers were removed and stored at -80○C.

Isolation of Mouse Peritoneal Macrophages

Phosphate-buffered saline (PBS) was injected into the peritoneal cavity as previously

reported.2 Fluid was then carefully collected and centrifuged at 3000 rpm. The supernatant

was withdrawn, and the pellet was resuspended in Dulbecco’s modified Eagle’s medium

(DMEM). Then, the macrophages were plated onto a 6-well cell culture cluster for 1 hr,

harvested and subjected to Western blot analyses.

Western Blot Analyses

Protein extracts from the livers and mouse peritoneal macrophages were prepared with

T-Per (Pierce Chemical Co., Rockford, IL) in the presence of protease inhibitors (Roche

Applied Science, Barcelona, Spain), and subjected to Western blot analyses as previously

described.2, 3 They were then subjected to Western blot analyses (NuPAGE Novex 4-12%

Bis-Tris Gel, Invitrogen, Carlsbad, CA; 25 µg protein per lane) with rabbit anti LDLR

(Abcam, Cambridge, UK), rabbit anti proprotein convertase subtilisin/kexin type 9 (PCSK9,

Cayman Chemical, Ann Arbor, MI), gout anti very low-density lipoprotein receptor

(VLDLR, R&D, Minneapolis, MN), mouse anti- sterol regulatory element binding protein 2

(SREBP2, Santa Cruz, Santa Cruz CA), anti apolipoprotein (apo) B (Biodesign, Archamps,

France), rabbit anti apoA-I (Santa Cruz), goat anti apoE (Chemicon International, Temecula,

CA) and β-actin (Santa Cruz)-specific antibodies. The proteins were visualized by a

chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham

Biosciences, Foster City, CA).

Real-time Quantitative RT-PCR

Total RNA was extracted from the livers, and first-strand cDNA was synthesized from the

total RNA (250 ng) by placing in a Reverse Transcription Reagent (Applied Biosystems,

Foster City, CA). Quantitative PCR was performed with a Perkin–Elmer 7900 PCR machine,

TaqMan PCR master mix and FAM-labeled TaqMan probes (Assays-on-Demand, Applied

Biosystems) for mouse Idol, sterol SREBP-2, PCSK9, HMG-CoA reductase, Tmem97, and

glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression data were normalized for

GAPDH levels.

Determination of Plasma Lipids and PCSK9 levels and Lipoprotein fractionation

Blood sampling was performed at the indicated days after injection of Ad-Idol or Ad-Luc

into the mice or hamsters. Plasma total cholesterol (TC) and triglyceride (TG) levels were

4

measured using commercially available assay kits (Wako Pure Chemicals, Osaka, Japan).

Plasma PCSK9 levels were determined using an ELISA kit (Cyclex, Ina, Japan). For

lipoprotein fractionation analysis, equal volumes of plasma samples were pooled from mice

for each group in (a total volume of 400 µL). Lipoproteins were fractionated using a

Superose 6 10/300 GL FPLC column (Amersham Biosciences, Piscataway, NJ). Fractions

were collected and used for lipid measurement and Western blot analyses.

125I-LDL Turnover Study

Human LDL was isolated from pooled human plasmas by sequential ultracentrifugation

(density 1.019<density 1.063 g/ml), and then iodinated with 125I by the iodine monochloride

method4 to give a specific activity of 125I-LDL >200 cpm/ng protein. Mice and hamsters

received an intravenous bolus via the external jugular vein of 125I-labeled mouse LDL (5 µg

of protein for mice, 20 µg of protein for hamsters). Blood was collected from the tail vein at

the indicated timings. The blood was subjected to low-speed centrifugation (1800 g) and the

10% trichloroacetic acid (TCA)-precipitable radioactivity of the plasma was measured.

Fractional catabolic rates (FCR) were determined using the SAAMII program.5

Construction of Luciferase Reporter Plasmids

Luciferase reporter plasmids containing mouse PCSK9 spanning from -47 to +390 bp

(relative to transcription start site) and human LDLR spanning from -544 to 152

(mPSCK9-Luc and hLDLR-Luc, respectively) were generated. The DNA fragments of these

promoter lesions were PCR amplified from mouse or human genomic DNA with primers

indicated as follows: mPCSK9 – forward: 5′- CGA CGC GTC AGC ACG CCT CTG AGT

TGG CA -3′ – reverse: 5′- CCG CTC GAG CTC GGG AAG GAC ATG GAC -3′; hLDLR –

forward: 5′- CGA CGC GTT CCG TAC AAT TGA TTT TTC AGA TG-3′– reverse: 5′- CCG

CTC GAG TCA CGA CCT GCT GTG TCC TA-3′. The amplified products were cloned into

the MluI/XhoI site upstream of the firefly luciferase gene in pGL3 Basic Vector (Promega,

Madison, WI). To obtain a reporter construct with mutations in the sterol responsive element

(SRE) of the mouse PCSK9 6 and human LDLR 7 promoter, we performed site-directed

mutagenesis using a commercially available kit as described above and the primers (lower

cases indicate mutated bases) indicated as follows: mPCSK9 – forward: 5'- CCG ATG GGG

CTC GGG GTG GCG atc aCT CCC GGC CCC CAG GC -3' – reverse: 5'- GCC TGG GGG

CCG GGA Gtg atC GCC ACC CCG AGC CCC ATC GG -3'; hLDLR – forward: 5'- GAC

ATT TGA AAA TCA CCg CAC TGC AAA CTC CTC CC-3' – reverse: 5'- GGG AGG AGT

TTG CAG TGc GGT GAT TTT CAA ATG TC -3'.

Cell Cultures and Luciferase Assay

HepG2 cells (Riken Cell Bank, Tsukuba, Japan) were maintained in DMEM containing 5 %

5

fetal bovine serum (FBS). All cultures were kept in a humidified atmosphere of 5% CO2 and

95% air at 37C°. At 80-90% confluency, the media were replaced with cholesterol depletion

medium (DMEM containing 5% bovine lipoprotein-deficient serum (LPDS), 50 µmol/L of

pravastatin and 10 µmol/L of mevalonate) in the absence or presence of

25-hydroxycholesteol (25HC, 1 µmol/L)/cholesterol (100 µmol/L) or LDL (100 µg/mL).

Ad-Idol or Ad-LacZ was added to the cells and they were then transfected with 500 ng of

luciferase reporter plasmids and 12.5 ng of phRL-TK (Promega) per well using

Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s instructions. The

cells were incubated for an additional 24 hr, harvested and subjected to Western blot analysis

and luciferase as previously described.8, 9

PCSK9 Turnover Study

Recombinant mouse PCSK9 (Adipo Bioscience, Santa Clara, CA) was labeled with

sodium 125I,4 and 4 µg was injected into the right jugular vein of male C57BL/6 mice. The

specific activity of the 125I -labeled PCSK9 ranged from 900–1,000 cpm/ng protein. Aliquots

of blood were obtained from the tail vein at the indicated timings. Fractional catabolic rates

(FCR) were determined using the SAAMII program.5

Statistical Analysis

The Student’s t-test was performed as appropriate. To make a comparison among 4 groups

using data obtained from the time-course experiments, two-way repeated measure ANOVA

was used. A p value of less than 0.05 was considered to be statistically significant. Values

are expressed as mean ± SD (or SE for kinetic studies).

References

1. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J.

Hypercholesterolemia in low density lipoprotein receptor knockout mice and its

reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993;92:883-893.

2. Ogura M, Ayaori M, Terao Y, Hisada T, Iizuka M, Takiguchi S, Uto-Kondo H,

Yakushiji E, Nakaya K, Sasaki M, Komatsu T, Ozasa H, Ohsuzu F, Ikewaki K.

Proteasomal inhibition promotes ATP-binding cassette transporter A1 (ABCA1) and

ABCG1 expression and cholesterol efflux from macrophages in vitro and in vivo.

Arterioscler Thromb Vasc Biol. 2011;31:1980-1987.

3. Uto-Kondo H, Ayaori M, Ogura M, Nakaya K, Ito M, Suzuki A, Takiguchi S,

Yakushiji E, Terao Y, Ozasa H, Hisada T, Sasaki M, Ohsuzu F, Ikewaki K. Coffee

consumption enhances high-density lipoprotein-mediated cholesterol efflux in

6

macrophages. Circ Res. 2010;106:779-787.

4. Bilheimer DW, Eisenberg S, Levy RI. The metabolism of very low density

lipoprotein proteins. I. Preliminary in vitro and in vivo observations. Biochim

Biophys Acta. 1972;260:212-221.

5. Barrett PH, Bell BM, Cobelli C, Golde H, Schumitzky A, Vicini P, Foster DM.

SAAM II: Simulation, Analysis, and Modeling Software for tracer and

pharmacokinetic studies. Metabolism. 1998;47:484-492.

6. Jeong HJ, Lee HS, Kim KS, Kim YK, Yoon D, Park SW. Sterol-dependent regulation

of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory

element binding protein-2. J Lipid Res. 2008;49:399-409.

7. Gierens H, Nauck M, Roth M, Schinker R, Schurmann C, Scharnagl H, Neuhaus G,

Wieland H, Marz W. Interleukin-6 stimulates LDL receptor gene expression via

activation of sterol-responsive and Sp1 binding elements. Arterioscler Thromb Vasc

Biol. 2000;20:1777-1783.

8. Ayaori M, Sawada S, Yonemura A, Iwamoto N, Ogura M, Tanaka N, Nakaya K,

Kusuhara M, Nakamura H, Ohsuzu F. Glucocorticoid receptor regulates ATP-binding

cassette transporter-A1 expression and apolipoprotein-mediated cholesterol efflux

from macrophages. Arterioscler Thromb Vasc Biol. 2006;26:163-168.

9. Nakaya K, Ayaori M, Hisada T, Sawada S, Tanaka N, Iwamoto N, Ogura M,

Yakushiji E, Kusuhara M, Nakamura H, Ohsuzu F. Telmisartan enhances cholesterol

efflux from THP-1 macrophages by activating PPARgamma. J Atheroscler Thromb.

2007;14:133-141.

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