lipoprotein(a)catabolismisregulatedbyproprotein … · results: lp(a) catabolism in hepatoma cells...

Lipoprotein(a) Catabolism Is Regulated by ProproteinConvertase Subtilisin/Kexin Type 9 through the Low DensityLipoprotein Receptor*

Received for publication, September 14, 2014, and in revised form, February 21, 2015 Published, JBC Papers in Press, March 16, 2015, DOI 10.1074/jbc.M114.611988

Rocco Romagnuolo‡, Corey A. Scipione‡, Michael B. Boffa‡, Santica M. Marcovina§, Nabil G. Seidah¶,and Marlys L. Koschinsky‡1

From the ‡Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada, the §NorthwestLipid Metabolism and Diabetes Research Laboratories, University of Washington, Seattle, Washington 98109, and the ¶Laboratoryof Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada

Background: Plasma lipoprotein(a) (Lp(a)) levels can be reduced through proprotein convertase subtilisin/kexin type 9(PCSK9) through an unknown mechanism.Results: Lp(a) catabolism in hepatoma cells and primary fibroblasts is inhibited by PCSK9 via the low density lipoproteinreceptor (LDLR).Conclusion: LDLR mediates the effects of PCSK9 on Lp(a) internalization.Significance: Our results provide a mechanistic explanation for the effects of PCSK9 inhibitors on plasma Lp(a) levels.

Elevated levels of lipoprotein(a) (Lp(a)) have been identifiedas an independent risk factor for coronary heart disease. PlasmaLp(a) levels are reduced by monoclonal antibodies targetingproprotein convertase subtilisin/kexin type 9 (PCSK9). How-ever, the mechanism of Lp(a) catabolism in vivo and the role ofPCSK9 in this process are unknown. We report that Lp(a) inter-nalization by hepatic HepG2 cells and primary human fibro-blasts was effectively reduced by PCSK9. Overexpression of thelow density lipoprotein (LDL) receptor (LDLR) in HepG2 cellsdramatically increased the internalization of Lp(a). Internaliza-tion of Lp(a) was markedly reduced following treatment ofHepG2 cells with a function-blocking monoclonal antibodyagainst the LDLR or the use of primary human fibroblastsfrom an individual with familial hypercholesterolemia; inboth cases, Lp(a) internalization was not affected by PCSK9.Optimal Lp(a) internalization in both hepatic and primaryhuman fibroblasts was dependent on the LDL rather than theapolipoprotein(a) component of Lp(a). Lp(a) internalizationwas also dependent on clathrin-coated pits, and Lp(a) wastargeted for lysosomal and not proteasomal degradation. Ourdata provide strong evidence that the LDLR plays a role inLp(a) catabolism and that this process can be modulated byPCSK9. These results provide a direct mechanism underlyingthe therapeutic potential of PCSK9 in effectively loweringLp(a) levels.

Lipoprotein(a) (Lp(a))2 has been identified as an indepen-dent, causal risk factor for cardiovascular disease, includingcoronary heart disease (1, 2). Lp(a) is similar to low densitylipoprotein (LDL) in lipid core composition and the presence ofapolipoprotein B-100 (apoB) but also contains a unique glyco-protein, apolipoprotein(a) (apo(a)), that has strong structuralhomology with the fibrinolytic zymogen plasminogen (3).Apo(a) contains multiple copies of plasminogen-like kringle IV(KIV) sequences, followed by sequences closely resemblingplasminogen kringle V (KV) and an inactive protease domain(3, 4). The KIV domain can be further subdivided into ten types(KIV1–10) differing from each other in amino acid sequence. InLp(a) particles, apo(a) is disulfide-linked to the apoB compo-nent of the LDL-like moiety through a free cysteine residue inKIV9 (5); formation of Lp(a) requires initial non-covalent inter-actions between lysine residues in apoB with weak lysine bind-ing sites present in apo(a) KIV7 and KIV8 (6). Additionally,apo(a) contains a strong lysine binding site present in KIV10,which is important for its ability to interact with substrates suchas fibrin (7).

Apo(a) can contain from 3 to �40 identically repeated KIV2domains, which gives rise to the isoform size heterogeneityreported in the population (8). A general inverse relationshipbetween the size of apo(a) and Lp(a) plasma concentration hasbeen well established, with Lp(a) levels varying widely in thepopulation (8). It has been reported that the inverse correlationbetween size and concentration is primarily controlled by thelevel of synthesis rather than catabolism (9, 10). Up to 90% ofthe variation is genetically determined based on variation in* This work was supported by Canadian Institutes of Health Research Grants

126076 (to M. L. K. and M. B. B.) and 102741 (to N. G. S.), Heart and StrokeFoundation of Canada Grant G-13-0003091 (to M. L. K.), the Canada Foun-dation for Innovation/Ontario Ministry of Research and Innovation (toM. L. K. and M. B. B.), Canada Research Chair Program Grant 216684 (toN. G. S.), and Leducq Foundation Grant 13 CVD 03 (to N. G. S.). S. M. M. andM. L. K. are members of an advisory board to Sanofi SA.

1 To whom correspondence should be addressed: Dept. of Chemistry andBiochemistry, University of Windsor, 401 Sunset Ave., Windsor, OntarioN9B 3P4, Canada. Tel.: 519-253-3000 (ext. 3010); Fax: 519-973-7068; E-mail:[email protected].

2 The abbreviations used are: Lp(a), lipoprotein(a); apoB, apolipoproteinB-100; KIV and KV, kringle IV and V, respectively; FH, familial hypercholes-terolemia; PCSK9, proprotein convertase subtilisin/kexin type 9; GOF, gain-of-function; MEM, minimum essential medium; LPDS, lipoprotein-de-pleted serum; r-apo(a), recombinant apo(a); �-ACA, �-aminocaproic acid;LDLR, low density lipoprotein receptor; VLDLR, very low density lipopro-tein receptor; ARH, autosomal recessive hypercholesterolemia.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 18, pp. 11649 –11662, May 1, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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LPA, the gene encoding apo(a), including its size heterogeneity(11).

Many of the details of Lp(a) catabolism remain obscure. Var-ious receptors have been suggested to mediate Lp(a) catabolismby the liver, which include the LDL receptor (LDLR) (12–15),very low density lipoprotein receptor (VLDLR) (16), low den-sity lipoprotein receptor-related protein 1 (LRP1) (17), mega-lin/gp330 (18), scavenger receptor class B type 1 (SR-B1) (19),and plasminogen receptors (12). The role of the LDLR remainshighly controversial, however. Hofmann and co-workers (20)reported that Lp(a) clearance was significantly increased inmice overexpressing LDLR. Additionally, several other studiesboth in vitro and in vivo have shown that the LDLR is capable ofmediating Lp(a) binding and uptake (12–15). A recent cross-sectional analysis of 1,960 patients with familial hypercholes-terolemia (FH) revealed that Lp(a) levels were significantlyhigher in patients with a null LDLR allele compared with con-trol subjects (21), a finding that is in agreement with an earlierreport on this topic (22). Conversely, Cain et al. (23) reportedthat whereas plasma clearance of Lp(a) in mice occurs primarilythrough the liver and is mediated by apo(a), the catabolism ofLp(a) in Ldlr�/� mice was similar to that in wild-type mice.Similar results were observed in metabolism studies of Lp(a) inhuman subjects with FH (24). In addition, plasma Lp(a) concen-trations are largely insensitive to statins, which act by increas-ing the abundance of hepatic LDLR (1).

Recent studies have shown that Lp(a) levels in plasma can bereduced up to 30% using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-inhibitory monoclonal antibody (25–30).In patients treated with a PCSK9 monoclonal antibody, theextent of Lp(a) lowering correlated with the extent of LDL low-ering in some studies (27, 28) but not others (30); a more robusteffect was consistently observed for LDL levels, whichdecreased up to �70% (27, 28).

PCSK9 is an important regulator of hepatic LDLR numberand consists of a pro-domain, followed by a catalytic domain, ahinge region, and a carboxyl-terminal cysteine/histidine-richdomain (31–33). PCSK9 is synthesized as an inactive proen-zyme that undergoes intramolecular autocatalytic cleavage inthe endoplasmic reticulum (32, 33). The cleaved prosegmentremains associated with PCSK9, maintaining PCSK9 in a cata-lytically inactive form, and the complex is transported to theGolgi apparatus and subsequently secreted. PCSK9 acts as anendogenous regulator of LDLR levels and has been implicatedin some cases of FH due to the dominant gain-of-function(GOF) mutations identified in the population (34). GOF muta-tions lead to increased affinity of PCSK9 for the LDLR, whichresults in a more rapid degradation of the LDLR and thus higherplasma LDL (34). Conversely, loss-of-function mutations inPCSK9 result in dramatically lowered plasma LDL (34). It is notyet known whether PCSK9 mutations influence Lp(a) concen-trations. PCSK9 can target the LDLR for degradation as well asthe VLDLR, LRP1, and apolipoprotein E receptor 2 (apoER2;LRP8) (35, 36). However, plasma LDL is predominately clearedthrough the LDLR (37, 38).

In the current study, using a human hepatocellular carci-noma model system, we sought to understand the mechanisticbasis of the ability of PCSK9-inhibitory antibodies to lower

plasma Lp(a) concentrations, in the context of the ongoing con-troversy about the role of the LDLR in Lp(a) catabolism.

EXPERIMENTAL PROCEDURES

Cell Culture—Human embryonic kidney (HEK293) cellswere maintained in MEM (Life Technologies) containing 5%fetal bovine serum (FBS; Life Technologies) and 1% antibiotic-antimycotic (Life Technologies). Human hepatocellular carci-noma (HepG2) cells were obtained from the American TypeCulture Collection (ATCC) and maintained in MEM supple-mented with 10% FBS (ATCC) and 1% antibiotic-antimycotic(Life Technologies). Primary FH fibroblasts were obtained fromCoriell Institute (catalogue numbers GM01386, GM01355, andGM00701) and maintained in MEM containing 10% FBS(ATCC). Experiments with FH fibroblasts were performedbetween passages 5 and 20.

Construction, Expression, and Purification of RecombinantApo(a)—The construction of expression plasmids encoding thevarious recombinant apo(a) (r-apo(a)) variants utilized in thisstudy (17K, 17K�LBS10, and 17K�LBS7,8), their transfectioninto HEK293 cells, and the purification of r-apo(a) from condi-tioned medium were described previously (6). Briefly, the con-ditioned medium was subjected to lysine-Sepharose affinitychromatography, and r-apo(a) was eluted using the lysine ana-logue �-aminocaproic acid (�-ACA). Following concentrationand buffer exchange, protein concentrations were determinedspectrophotometrically. The purity of r-apo(a) was assessedusing SDS-PAGE followed by silver staining.

Construction, Expression, and Purification of RecombinantPCSK9 —PCSK9, and PCSK9 D374Y expression plasmids inpIRES2-EGFP (Clontech) were described previously (32, 33).The PCSK9 cDNAs were excised from pIRES2-EGFP usingAfeI and AgeI restriction endonucleases and ligated intopcDNA4C (Invitrogen), previously digested with EcoRV andAgeI, such that the expressed protein would contain a carboxyl-terminal His6 tag. HEK293 cells at 1.8 � 106 cells/well of a6-well plate were seeded and transfected 24 h later with 2 �g ofexpression plasmid using MegaTran version 1.0 (Origene) witha 3:1 ratio of reagent to DNA as per the manufacturer’s instruc-tions. Stable cells were selected with zeocin (150 �g/ml) 48 hpost-transfection. Stable cells were seeded into triple flaskswith Opti-MEM (Life Technologies), and conditioned mediumwas collected every 3 days with the addition of phenylmethyl-sulfonyl fluoride at a final concentration of 1 mM to the harvest.The harvested medium was adjusted to 50 mM phosphate bufferpH 8.0, 0.5 M NaCl, 1 mM �-mercaptoethanol, 5 mM imidazole,and 10% glycerol, applied to a nickel-Sepharose Excel (GEHealthcare) column, washed, and eluted with 15 mM and 400mM imidazole, respectively. The eluted pool (4 column vol-umes) was extensively dialyzed against PCSK9 storage buffer(25 mM HEPES, pH 7.9, 150 mM NaCl, 0.1 mM CaCl2, and 10%glycerol). The dialyzed samples were then concentrated withPEG 20,000 (Sigma) and dialyzed against storage buffer. Con-centrations were determined through a bicinchoninic acidassay (BCA assay; Pierce) using BSA as a standard. The purity ofPCSK9 was assessed through SDS-PAGE followed by silverstaining and stored in aliquots at �70 °C until use.

PCSK9 Regulates Lp(a) Catabolism by LDLR

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Labeling of PCSK9 —Purified PCSK9 was dialyzed against 0.1M Na2CO3, pH 8.6, 0.2 M NaCl. PCSK9 was then incubated witha 5-fold molar excess of Alexa Fluor 488 carboxylic acid, suc-cinimidyl ester mixed isomers dissolved in dimethyl sulfoxideat 10 mg/ml (Invitrogen). The reaction mixture was rocked for4 h at 4 °C to ensure complete labeling. The reaction wasquenched with the addition of 0.01 volumes of 1 M Tris, pH 8.0,followed by incubation for 30 min at 4 °C. Free dye was removedthrough extensive dialysis against 25 mM HEPES, pH 7.5, 300mM NaCl, 50 mM KH2PO4, 0.1 mM CaCl2, and 10% glycerol.PCSK9 was concentrated using an Amicon Ultra-4 centrifugalfilter with a 10 kDa membrane cut-off (Millipore). Concentra-tion was determined spectrophotometrically with a dye/pro-tein (mol/mol) ratio of 2.8.

Transient Transfection—HepG2 cells were transfected withclathrin heavy chain siRNA or scrambled control siRNA (SantaCruz Biotechnology, Inc.) at a concentration of 80 nM as per themanufacturer’s protocol. The transfection mixture was incu-bated on cells for 8 h, followed by the addition of completemedium. Cells were assayed 48 –72 h post-transfection. Thepercentage knockdown was determined using quantitative RT-PCR (see below). HepG2 cells were transiently transfected withv5 (empty vector), LDLR, or LDLR�CT (36) using MegaTranversion 1.0 (Origene) as per the manufacturer’s protocol.Briefly, HepG2 cells were seeded at a density of 2 � 105 cells/6-well plate in antibiotic-free medium and transfected 24 h laterwith 1.3 �g of cDNA with a 3:1 ratio of reagent to DNA. Cellswere assayed 72 h post-transfection.

Quantitative RT-PCR—Determination of clathrin heavy chainknockdown efficiency was determined through the iTaq one-stepRT-PCR kit with SYBR� Green (Bio-Rad). The following primerswere used: clathrin heavy chain forward, 5�-GGC CCA GAT TCTGCC AAT TCG TTT-3�; clathrin heavy chain reverse, 5�-TGATGG CGC TGT CTG CTG AAA TTG-3�; GAPDH forward,5�-GGA GCC AAA AGG GTC ATC ATC-3�; GAPDH reverse,5�-GTT CAC ACC CAT GAC GAA CAT G-3�.

Internalization Assays—HepG2 cells (in some cases stablytransfected with an expression vector for PCSK9) were seededat 2 � 105 cells/well in a 24-well plate (precoated with 1 mg/mlgelatin), in medium containing 10% lipoprotein-depletedserum (LPDS) for 16 h. Cells were washed twice with Opti-MEM (Gibco) and treated with Lp(a) purified from humanplasma (5–10 �g/ml) or r-apo(a) variants (100 –200 nM) in thepresence of 0, 1, 10, or 20 �g/ml purified recombinant PCSK9 inOpti-MEM for 4 h at 37 °C. For experiments using LDLR-blocking monoclonal antibodies, cells were pretreated for 30min with 50 �g/ml 5G2 or 7H2 followed by incubation withLp(a) or apo(a) in the continued presence of 50 �g/ml antibodyfor 2 h at 37 °C. In some experiments, cells were co-treated with10 �g/ml lactacystin (Cayman), 150 �g/ml E-64d (Cayman), orvehicle (dimethyl sulfoxide) along with Lp(a) or apo(a) for 4 h at37 °C. Concanamycin A (Cayman) was dissolved in 100% etha-nol and used at a final concentration of 50 nM for 16 h followedby co-treatment with Lp(a) for 4 h. For all internalization exper-iments, HepG2 cells were extensively washed: three times withPBS, 0.8% BSA; two times with PBS, BSA, 0.2 M �-ACA for 5 mineach; two times with 0.2 M acetic acid, pH 2.5, containing 0.5 M

NaCl for 10 min each; two times with PBS. The cells were then

lysed with lysis buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40,0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1%SDS, 1 mM PMSF, and 150 �g/ml benzamidine).

For experiments with fibroblasts, cells were seeded in a24-well plate at 1.4 � 105 cells/well in medium containing 10%LPDS for 16 h. Cells were washed twice with Opti-MEM andtreated with Lp(a) (5 �g/ml) or apo(a) (100 nM) in the presenceor absence of 20 �g/ml PCSK9 in Opti-MEM for 4 h at 37 °C.Cells were extensively washed (three times with PBS, 0.8% BSA;two times with PBS containing 10 �g/ml heparin for 10 min;one time with PBS, BSA, 0.2 M �-ACA for 5 min; two times with0.2 M acetic acid, pH 2.5, containing 0.5 M NaCl for 10 min; onetime with 0.5 M HEPES, pH 7.5, 100 mM NaCl for 10 min; andone time with PBS) and then lysed with lysis buffer. Concentra-tions of lysate samples were determined by BCA assay with BSAas a standard and analyzed by Western blotting.

Western Blotting—Cell lysates were subjected to SDS-PAGEon 5–15% (Lp(a)-treated cells) or 7–15% (apo(a)-treated cells)polyacrylamide gradient gels, respectively. The gels were trans-ferred onto PVDF membranes (Millipore) and immunoblottedwith either mouse anti-human apo(a) a5 antibody (39), mouse-anti human �-actin (Sigma), rabbit anti-human LDLR (Gene-Tex), or goat-anti-human clathrin heavy chain (Santa Cruz Bio-technology). After incubation with the appropriate horseradishperoxidase-linked secondary antibody, immunoreactive bandswere visualized with SuperSignal� West Femto Maximum Sen-sitivity Substrate (Thermo Scientific) and quantified usingAlpha View software (Alpha Innotech).

LDLR Degradation Assay—HepG2 cells were seeded at 2 �105 cells/well in a 24-well plate in medium containing 10%LPDS for 16 h. PCSK9 (20 �g/ml) with 0, 100, or 250 �g/mlplasma-purified Lp(a) or LDL or 0, 100, or 250 nM apo(a) wasadded in Opti-MEM, and the cells were incubated for 4 h. Cellswere washed three times with PBS and lysed. Concentrations ofsamples were determined by BCA assay and LDLR levels wereanalyzed by Western blotting.

Binding Study—Saturation binding curves were generated byincubating LDL or Lp(a), at 0.5 mg/ml, with increasing amountsof PCSK9-Alexa Fluor 488 (25–1200 nM) in binding buffer (25mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1% BSA) for 1 hat 37 °C. Glycerol was added to the samples to a final concen-tration of 10%, and the samples were subjected to electropho-resis on 0.7% agarose gels (UltraPure Agarose, Invitrogen) for2 h at 40 V in 90 mM Tris, pH 8.0, 80 mM borate, 2 mM calciumlactate. In-gel scanning and quantification of the amount oflabeled PCSK9 free and bound to Lp(a) or LDL was achievedwith a FluorChem Q imager (Alpha Innotech). Dissociationconstants (KD) were determined by fitting the data to an equa-tion describing a rectangular hyperbola by nonlinear regressionusing Graph Pad Prism version 6.

Purification of LDL and Lp(a) and Preparation of Lipopro-tein-deficient Serum (LPDS)—Blood was collected from ahealthy human volunteer (with written informed consent) withno detectable Lp(a) into BD Vacutainers containing sodiumpolyanethol sulfonate and acid citrate dextrose. The blood wascentrifuged at 2,000 � g for 15 min at 4 °C, and LDL was isolatedfrom plasma through sequential ultracentrifugation (1.02g/ml � d � 1.063 g/ml); the centrifugation steps were at




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45,000 � g for 18 h at 4 °C. The isolated LDL was extensivelydialyzed against 150 mM NaCl, 5.6 mM Na2HPO4, 1.1 mM

KH2PO4, 0.01% EDTA (pH 7.4). LPDS was prepared throughthe addition of NaBr to FBS (ATCC) to a final density of 1.21g/ml followed by ultracentrifugation as described above. Thetop fraction was removed, and the infranatant fraction contain-ing LPDS was extensively dialyzed against HEPES-bufferedsaline (20 mM HEPES, pH 7.4, 150 mM NaCl). Lp(a) was pre-pared from a single donor with high Lp(a) and a single 16-krin-gle apo(a) isoform as described previously (40). Concentrationsof LDL and Lp(a) were determined by a BCA assay using BSA asa standard.

Immunofluorescence—HepG2 cells were seeded on gelatin-coated coverslips in the wells of 24-well plates at 1.25 � 105

cells/well for 16 h in medium containing 10% LPDS. Cells werewashed twice with Opti-MEM (Gibco) and treated with Lp(a)purified from human plasma (5 �g/ml) in the presence of 20�g/ml purified recombinant PCSK9 in Opti-MEM for 4 h at37 °C. Cells were washed three times with PBS, 0.8% BSA; twotimes with PBS, BSA, 0.2 M �-ACA for 5 min each; and threetimes with PBS. The cells were then fixed with 3.7% paraformal-dehyde for 20 min at room temperature. Cells were permeabi-lized with 0.2% Triton X-100 in PBS for 5 min and blocked with5% normal goat serum containing 0.1% Triton X-100 (blockingbuffer) for 30 min. Mouse anti-human apo(a) (a5) antibody (39)(1:50) was incubated in blocking buffer for 45 min at 37 °C;washed three times for 5 min with PBS, 0.1% BSA; incubatedwith Alexa Fluor 595-conjugated goat anti-mouse IgG (0.5�g/ml) in blocking buffer for 30 min at 37 °C; and washed threetimes with PBS, 0.1% BSA with the final wash containing 4�,6-diamidino-2-phenylindole (DAPI). After this, coverslips weremounted to slides using anti-fade fluorescence mountingmedium (Dako). Immunofluorescence microscopy was per-formed with a Leica DMI6000B inverted fluorescence micro-scope with a �63.0 oil immersion objective with a numericalaperture of 1.4 and refractive index of 1.52. The microscope wasfitted with a Leica DFC 360FX camera using A4 (DAPI) and Txr(Alexa Fluor 595) filters. Images were acquired using LAS AFsoftware and processed with Corel Draw Graphics Suite X6.

Purification of LDLR-blocking Monoclonal Antibodies—Anti-human LDLR monoclonal antibodies 5G2 and 7H2 (a gift from Dr.Ross Milne, University of Ottawa Heart Institute) were purifiedfrom ascites fluid using Protein G-Sepharose 4 Fast Flow affinitychromatography according to the manufacturer’s recommenda-tions (GE Healthcare). Concentrations of antibodies were deter-mined using a BCA assay with BSA as a standard.

Statistical Methods—Comparisons between data sets wereperformed using a two-tailed Student’s t test assuming unequalvariances.

RESULTS

PCSK9 Inhibits Lp(a) and Apo(a) Internalization—PCSK9can target the LDLR for degradation in an intracellular pathwayby targeting the LDLR from the trans-Golgi network directly tolysosomes (41). Conversely, extracellular PCSK9 targets theLDLR for degradation through binding of PCSK9 to the EGF-Adomain of the LDLR and subsequently targeting the complex tolysosomes for degradation (42, 43).Herein, we evaluated the

role of both the intra- and extracellular PCSK9-mediated deg-radation of LDLR in Lp(a)/apo(a) internalization by HepG2cells. Overexpression of PCSK9, which would stimulate boththe intracellular and extracellular pathway of targeting theLDLR for degradation, resulted in a significant decrease in theamount of Lp(a) internalized by HepG2 cells (Fig. 1A). Similarresults were obtained for the internalization of a physiologicallyrelevant r-apo(a) species (17K) that contains eight identicallyrepeated KIV2 domains (Fig. 1B).

Interaction of apo(a) and Lp(a) with cell surface receptors hasbeen shown to be mediated, at least in part, by the binding oflysine-binding kringles in apo(a) to lysine-containing receptors(12, 44). The addition of a lysine analog, �-ACA, markedlyinhibited the uptake of both Lp(a) and r-apo(a) (Fig. 1, A and B),although PCSK9 still significantly reduced the uptake of Lp(a).Likewise, mutating the strong lysine binding site present inKIV10 of 17K (17K�LBS10 variant) results in a significantdecrease in its ability to be internalized (Fig. 1B). Interestingly,however, PCSK9 is able to significantly decrease internalizationof either 17K or 17K�LBS10 in the absence, but not in the pres-ence, of �-ACA (Fig. 1B). Because �-ACA cannot totally abolishthe ability of PCSK9 to decrease internalization of Lp(a), we canconclude that there must be a component of the binding andinternalization of Lp(a) that is not dependent on the binding tocell surface lysines.

To specifically determine the role of the extracellular PCSK9degradation pathway, HepG2 cells were exposed to exogenous,purified PCSK9 or a GOF mutant of PCSK9 (D374Y) in thepresence of Lp(a) or apo(a). Treatment of HepG2 cells withvarious concentrations of wild type (WT) PCSK9 resulted in asignificant decrease in Lp(a) and 17K internalization (Fig. 1, Cand D). The GOF mutant was found to have a more robusteffect on Lp(a) (at 1 �g/ml) and 17K (at 1 and 10 �g/ml) inter-nalization compared with WT PCSK9.

PCSK9 Does Not Bind to Lp(a)—It has been previouslyreported that PCSK9 can bind to LDL in vitro consistent with aone-site binding model with a KD of �325 nM (45). Further-more, the binding of PCSK9 to LDL inhibits its ability to targetthe LDLR for degradation in HuH7 human hepatoma cells (45).Hence, we determined whether Lp(a) can bind to PCSK9 invitro and if Lp(a)/apo(a) can inhibit the ability of exogenousPCSK9 to target the LDLR for degradation. We found that LDLcan bind to PCSK9 in vitro with a KD of �130 nM, a value closeto that reported previously (45) (Fig. 2A). On the other hand,little or no binding of Lp(a) to PCSK9 was detected (Fig. 2, A andC). Treatment of HepG2 cells with exogenous PCSK9 results ina substantial decrease in LDLR levels, whereas co-treatment ofPCSK9 with LDL results in recovery of LDLR levels (Fig. 2D).These findings are also in agreement with previously reporteddata (42). However, co-treatment of Lp(a) or 17K with PCSK9results in no significant recovery in LDLR levels (Fig. 2, E and F).Together, these results suggest that Lp(a) does not bind toPCSK9 and therefore cannot block the ability of PCSK9 to tar-get the LDLR for degradation.

Lp(a)/Apo(a) Internalization Involves Clathrin-mediatedEndocytosis and Internalized Lp(a)/Apo(a) Is Targeted toLysosomes—PCSK9 has been previously shown to target theLDLR for degradation via clathrin heavy chain-mediated endo-




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cytosis and subsequent targeting to lysosomes (46, 47). Wetherefore determined whether Lp(a) and/or apo(a) can undergothe same degradation pathway. Knockdown of clathrin heavychain in HepG2 cells results in a significant decrease in Lp(a)and apo(a) internalization (Fig. 3). In both cases, whereasPCSK9 treatment results in a dose-dependent decrease in inter-nalization in the absence of clathrin heavy chain knockdown,no further decrease resulting from PCSK9 is observed in thepresence of clathrin heavy chain knockdown (Fig. 3). Theseresults indicate that the PCSK9-regulated internalization ofLp(a)/apo(a) is dependent on clathrin-coated pits.

The degradation pathway that Lp(a)/apo(a) undergoes wasfurther evaluated through inhibitors of both the lysosomal andproteosomal pathway. Treatment of HepG2 cells with the lys-osomal inhibitor E-64d or concanamycin A resulted inincreased intracellular accumulation of Lp(a) and apo(a) (Fig.4). However, treatment with a proteosomal inhibitor, lactacys-tin, resulted in no change in intracellular accumulation of Lp(a)or apo(a). These results indicate that Lp(a)/apo(a) is internal-ized through clathrin-mediated endocytosis and is subse-quently targeted for lysosomal degradation.

PCSK9 Regulates Lp(a) Internalization through the LDLR—Previous studies have shown that apo(a) can be internalizedinto HepG2 cells through the LDLR or through lysine-depen-

dent interactions with plasminogen receptors (12). We there-fore wanted to examine which of these routes might be sensitiveto PCSK9, particularly in view of our findings that �-ACA haddifferent effects on the internalization of Lp(a) and apo(a) (Fig.1). Apo(a) is not itself a ligand for the LDLR, but r-apo(a) pres-ent in HepG2 cell medium binds non-covalently (and, ulti-mately, covalently) to apoB-containing lipoproteins secreted bythe HepG2 cells (5, 48), which allows the complex to be inter-nalized by the LDLR in a “piggyback” manner (12, 49). The weaklysine binding sites in KIV type 7 and 8 mediate these non-covalent interactions (6, 49), and therefore, for internalizationstudies, we utilized a r-apo(a) variant in which both of theselysine binding sites were mutated (17K�LBS7,8) (6). We foundthat 17K�LBS7,8 was poorly internalized in HepG2 cells (Fig.5A); although its internalization did not appear to be affected byPCSK9, this conclusion has to be tempered by the fact that theinternalization of this species is at our limit of detection. None-theless, it is clear that prevention of the association of apo(a)and apoB100-containing lipoproteins in the medium of HepG2cells decreases the ability of apo(a) to be internalized by thesecells.

To determine more directly whether the LDLR plays a role inLp(a)/apo(a) internalization, the LDLR or the LDLR lacking itscarboxyl tail (LDLR�CT) was overexpressed in HepG2 cells.

FIGURE 1. PCSK9 reduces the internalization of both Lp(a) and apo(a) in HepG2 cells. A and B, HepG2 cells stably transfected with empty vector or PCSK9-v5were grown for 16 h in LPDS medium followed by treatment with either 10 �g/ml Lp(a) (A) or 200 nM apo(a) (B) in the presence or absence of 200 mM �-ACA for4 h. The cells were extensively washed to remove any bound Lp(a)/apo(a) and lysed to determine the relative amount that was internalized, using �-actin as aninternal control. Error bars, S.E. with n � 3 independent experiments. *, p � 0.05; **, p � 0.01. C and D, HepG2 cells were grown in LPDS medium for 16 h followedby treatment with various concentrations of PCSK9 or PCSK9 D374Y in the presence of either 10 �g/ml Lp(a) (C) or 200 nM 17K (D). The cells were extensivelywashed to remove any bound Lp(a)/apo(a) and lysed to determine the relative amount that was internalized, using �-actin as an internal control. The datashown are the means of at least three independent experiments; error bars, S.E. Representative Western blots are shown. *, p � 0.05; **, p � 0.01.




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The �CT deletion occurs where the autosomal recessive hyper-cholesterolemia (ARH) adaptor protein binds and is importantfor recruiting the complex into clathrin-coated pits (50, 51).

Overexpression of LDLR in HepG2 cells results in a dramaticincrease in Lp(a) internalization (Fig. 5B) and only a modest andnot statistically significant increase in apo(a) internalization

FIGURE 2. PCSK9 binds to LDL in vitro but not to Lp(a). LDLR levels are recovered from PCSK9 degradation by treatment with LDL but not Lp(a) or apo(a).Various concentrations of PCSK9-Alexa Fluor 488 were incubated with 0.5 mg/ml purified LDL or Lp(a) for 1 h at 37 °C. Samples containing PCSK9 and either LDLor Lp(a) were resolved on 0.7% agarose gels. A, bound and free levels of PCSK9 were quantified and fit to a saturation curve by nonlinear regression usingGraphPad Prism 6 to give a mean KD for LDL of 128 40 nM (n 3). B and C, representative gel images for LDL (B) or Lp(a) (C). D–F, HepG2 cells were grown for16 h in LPDS medium followed by treatment with various concentrations of LDL (D), Lp(a) (E), or 17K r-apo(a) (F) in the presence or absence of 20 �g/ml PCSK9for 4 h. The relative LDLR levels were determined and normalized to �-actin. The data shown are the means of at least three independent experiments; errorbars, S.E. Representative Western blots are shown. *, p � 0.05; **, p � 0.01 compared with control in the absence or presence of PCSK9.




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(Fig. 5C). Treatment of the cells overexpressing the LDLR orLDLR�CT with PCSK9 leads to a significant decrease in Lp(a)internalization (Fig. 5B).

Treatment of HepG2 cells with a blocking monoclonal LDLRantibody was also utilized to confirm that the LDLR is involvedin Lp(a) catabolism and its regulation by PCSK9. Two LDLR-blocking monoclonal antibodies, 5G2 and 7H2, were used; bothwere previously shown to specifically block the binding of LDLto the LDLR in cultured human fibroblasts (52). Lp(a) internal-ization was markedly decreased by the addition of either anti-body, and PCSK9 had no effect on Lp(a) internalization in thepresence of the antibodies (Fig. 5D). Furthermore, we foundthat 7H2 likewise markedly decreased 17K internalization (Fig.5E). On the other hand, PCSK9 did not decrease 17K internal-ization in the presence of the antibody, and internalization of17K�LBS7,8 appeared to be insensitive to both PCSK9 and theantibody (Fig. 5E). These results indicate that the LDLR medi-ates internalization of Lp(a) through the LDL component andin a manner that is regulated by PCSK9.

The role for the LDLR was also explored using primary fibro-blasts isolated from individuals with or without FH. The threecell lines studied were GM01386 (fully functional LDLR),GM01355 (clinically affected with severe hypercholesterolemiawith LDLR activity found to be partially negative), andGM00701 (LDLR activity �1% compared with normal cells).Lp(a) internalization substantially decreases in cells with adefective LDLR, and the internalization was unaffected byPCSK9 in these cells (Fig. 6A). Conversely, no significantdifference in 17K internalization is observed between LDLR-defective and normal fibroblasts, and there is no effect ofPCSK9 on 17K internalization by any cell line (Fig. 6B).PCSK9 was able to dramatically decrease the LDLR contentof those fibroblasts that contained immunoreactive receptor(Fig. 6C). These findings underscore the requirement forapo(a) to couple to apoB-containing lipoproteins in order tointernalize through the LDLR in a PCSK9-regulable mannerbecause these fibroblasts do not express apoB-containinglipoproteins.

FIGURE 3. Lp(a) and apo(a) internalization is dependent on clathrin coated pits in HepG2 cells. A and B, HepG2 cells were transfected with control orclathrin heavy chain siRNA for 48 h followed by incubation of cells in LPDS medium for 16 h. HepG2 cells were then treated with 5 �g/ml Lp(a) (A) or 200 nM 17K(B) in the presence or absence of PCSK9 for 4 h. Internalization of Lp(a)/apo(a) was measured as described in the legend to Fig. 1. The data shown are the meansof at least three independent experiments; error bars, S.E. *, p � 0.05; **, p � 0.01. C and D, clathrin heavy chain mRNA was quantified using quantitative RT-PCR(C) as well as total clathrin heavy chain protein levels compared with �-actin (D), as determined through Western blot analysis, following clathrin heavy chainsiRNA treatment to determine knockdown efficiency. The data shown are the means of at least three independent experiments; error bars, S.E.




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DISCUSSION

Elevated plasma Lp(a) levels have been recently shown to beeffectively reduced with the use of two different monoclonalantibodies against PCSK9 (25–30). This therapy was conceivedto lower LDL levels because inhibition of PCSK9 leads to up-regulation of the LDLR. The ability of PCSK9-based therapiesto lower plasma Lp(a) challenges the existing dogma that theLDLR does not play a major, if any, role in Lp(a) catabolism.Indeed, we propose, based on our findings, that PCSK9 inhibi-tion leads to a combination of supraphysiological hepatic LDLRabundance and dramatic lowering of LDL that unmasks LDLRas a significant route of clearance of Lp(a) (Fig. 7).

We found that PCSK9 is indeed able to inhibit Lp(a) inter-nalization in HepG2 cells (Fig. 1). This effect was observedwhether PCSK9 was ectopically overexpressed (and henceactive both intracellularly and extracellularly) or added as apurified protein to the culture medium along with Lp(a) (henceacting exclusively extracellularly). Notably, we also found thatPCSK9 can stimulate internalization of apo(a) itself (Fig. 1).However, we conclude that the effect of PCSK9 on apo(a) inter-nalization is dependent on the ability of free apo(a) to associatewith apoB-containing lipoprotein particles in the culture

medium, with internalization of the resultant complex beingsensitive to PCSK9 (Fig. 7). This conclusion is based on the factthat internalization of apo(a) by fibroblasts, which do notexpress apoB, is insensitive to PCSK9 (Fig. 6). Moreover, inter-nalization of the 17K�LBS7,8 variant, which cannot associatenon-covalently with apoB-containing lipoproteins (6), appearsto be insensitive to the effects of PCSK9 (Fig. 5). Given thesefindings and a previous report that demonstrated that apoB-100, not apo(a), is the ligand in Lp(a) for the LDLR (12), wesuspected that the LDLR, the major target of PCSK9, was medi-ating the PCSK9-sensitive component of Lp(a) and apo(a)internalization.

Importantly, apo(a), due to its structural similarities to plas-minogen, may also potentially bind to and be internalized byplasminogen receptors, which contain carboxyl-terminal lysineresidues (12). Previous results have shown that removal of thestrong lysine binding site in r-apo(a) (the 17K�LBS10 variant)results in an inability to effectively bind to fibrin surfaces (7). Inthe current study, 17K�LBS10 internalization is significantlyreduced, but not abolished, compared with wild-type 17K inHepG2 cells (Fig. 1). Treatment of HepG2 cells with the lysineanalogue �-ACA resulted in a significant decrease in both Lp(a)

FIGURE 4. Lp(a) and apo(a) degradation in HepG2 cells occurs in lysosomes and not proteosomes. A and B, HepG2 cells were grown for 16 h in LPDSmedium followed by treatment with either DMSO, E-64d (150 �g/ml), or lactacystin (10 �M) in the presence or absence of 20 �g/ml PCSK9 as well as Lp(a) (A)or 17K (B) for 4 h. Internalization of Lp(a)/apo(a) was measured as described in the legend to Fig. 1. The data shown are the means of at least three independentexperiments; error bars, S.E. Representative Western blots are shown. C and D, HepG2 cells were grown for 16 h on gelatinized coverslips in LPDS medium witheither vehicle or concanamycin A (50 nM) followed by treatment with Lp(a) in the presence of either vehicle, lactacystin (10 �M), E-64d (150 �g/ml), orconcanamycin A (50 nM), in the presence or absence of 20 �g/ml PCSK9, for 4 h. The cells were fixed with PFA, permeabilized with Triton X-100, and stained forapo(a) (red) with a monoclonal anti-apo(a) a5 antibody followed by an Alexa Fluor 595-linked secondary antibody. Nuclei were stained (blue) using DAPI, andcells were visualized by fluorescence microscopy. Representative images are shown. Bar, 25 �m. E, quantification of fluorescence relative to total cell number.The data shown are the means of at least three independent experiments, in which apo(a) internalization was averaged across three different fields of view;error bars, S.E. *, p � 0.05; **, p � 0.01.




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and apo(a) internalization, and PCSK9 was still able to inhibitLp(a) (but not apo(a)) internalization in the presence of �-ACA.Thus, removal of the strong lysine binding site in apo(a) affectsits ability to be internalized through lysine-dependent plasmin-ogen receptors but not through non-covalent interactions withapoB and subsequent binding to LDLR. However, treatmentwith �-ACA abolishes the ability of both apo(a) and Lp(a) tobind to lysine-dependent plasminogen receptors as well as theability of apo(a) to couple to the apoB component of LDL (53)(Fig. 7); the latter effect accounts for the inability of PCSK9 toinhibit apo(a) uptake in the presence of �-ACA (Fig. 1B).

Recently, it has been reported that LDL can bind to PCSK9and inhibit its ability to target the LDLR for degradation (45).This effect may serve to limit the extent to which PCSK9 can actto lower hepatic LDLR abundance. We therefore analyzedwhether Lp(a) can bind to PCSK9 in order to determine if (i)less Lp(a) is being internalized due to its ability to bind toPCSK9 and thus prevent its internalization or (ii) Lp(a) bindingto PCSK9 leads to a reduced ability for PCSK9 to target theLDLR for degradation or other receptors, limiting the ability ofLp(a) to be internalized through those receptors. Through invitro binding experiments, we have shown that PCSK9 cannot

FIGURE 5. The LDLR contributes to Lp(a) and apo(a) internalization in HepG2 cells. A, HepG2 cells were grown in LPDS medium for 16 h followed bytreatment with various concentrations of PCSK9 in the presence of 17K or 17K�LBS7,8 (200 nM). Internalization of apo(a) was measured as described in thelegend to Fig. 1. Error bars, S.E. with n � 3 independent experiments. **, p � 0.01. B and C, HepG2 cells were transfected with expression plasmids encodingeither LDLR or LDLR�CT for 48 h and were then grown for 16 h in LPDS medium. The cells were incubated with 10 �g/ml Lp(a) (B) or 200 nM apo(a) (C) in thepresence or absence of 20 �g/ml PCSK9 for 4 h after which internalization of Lp(a)/apo(a) was measured. Error bars, S.E. with n � 3 independent experiments.*, p � 0.05; **, p � 0.01. D and E, HepG2 cells were grown in LPDS medium for 16 h followed by pretreatment with monoclonal LDLR blocking antibodies (mAb5G2 or 7H2; 50 �g/ml) for 30 min. Cells were then incubated with 5 �g/ml Lp(a) (D) or 100 nM apo(a) (E) in the presence or absence of 10 �g/ml PCSK9 and inthe continuing presence of mAbs for 2 h, after which internalization of Lp(a)/apo(a) was measured. The data shown are the means of at least four independentexperiments; error bars, S.E. *, p � 0.05; **, p � 0.01 for comparison of absence of antibody and PCSK9.




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bind to Lp(a) (Fig. 2). In addition, neither Lp(a) nor apo(a)inhibits the ability of PCSK9 to target the LDLR for degradationin HepG2 cells. Therefore, it is possible that the apo(a) compo-nent of Lp(a) is interfering with the interaction of PCSK9 andapoB. Notably, the exact site at which the apoB component ofLDL binds to PCSK9 is currently unknown.

We also explored the degradation pathway of Lpa(a)/apo(a)through PCSK9. Previous work has shown that PCSK9 can tar-get the LDLR for degradation through clathrin-mediated endo-cytosis and subsequent lysosomal degradation (42, 43). Weshow here, through knockdown of clathrin heavy chain, thatLp(a) and apo(a) are also internalized through clathrin-medi-

FIGURE 6. Lp(a), but not apo(a), internalization decreases in FH fibroblasts lacking LDLR. A and B, FH fibroblasts used were GM01386 (fully functionalLDLR), GM01355 (clinically affected with severe hypercholesterolemia with LDLR activity found to be partially negative), and GM00701 (LDLR activity �1%compared with normal cells). The FH fibroblasts were grown in LPDS medium for 16 h followed by incubation with 5 �g/ml Lp(a) (A) or 100 nM apo(a) (B) in thepresence or absence of 10 �g/ml PCSK9 for 4 h. Internalization of Lp(a)/apo(a) was measured as described in the legend to Fig. 1. The data shown are the meansof at least five independent experiments; error bars, S.E. Representative Western blots are shown. *, p � 0.05; **, p � 0.01 for comparison of control in theabsence of PCSK9 and �-ACA. C, the LDLR content of the respective fibroblast cell lines in the presence or absence of PCSK9 treatment was assessed by Westernblot analysis using a monoclonal antibody against LDLR.

FIGURE 7. Model for receptor-mediated catabolism of apo(a) and Lp(a). Apo(a) and apoB-containing lipoproteins are independently secreted from hepa-tocytes and form a non-covalent, and subsequently covalent, complex as Lp(a). Apo(a) can be internalized by plasminogen receptors, and the apo(a) compo-nent of Lp(a) mediates clearance of the particle by plasminogen receptors. Apo(a) can only be internalized by the LDL receptor when in a complex with LDL.�-ACA can inhibit binding of apo(a) or Lp(a) to plasminogen receptors as well as Lp(a) assembly, the latter through inhibition of non-covalent interactionsbetween apo(a) and apoB-100. However, �-ACA cannot prevent binding of pre-formed Lp(a) to the LDL receptor. In the presence of inhibitors of PCSK9, thereis a substantial increase in DL receptor number such that clearance of Lp(a) through this route is of a much greater magnitude.




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ated endocytosis. Treatment with PCSK9 results in no furtherdecrease in Lp(a)/apo(a) internalization following clathrinheavy chain knockdown. This indicates that the receptors thatinternalize Lp(a)/apo(a), which can be regulated by PCSK9, aredependent on clathrin-mediated endocytosis. Furthermore,treatment with the lysosomal inhibitor E-64d or concanamycinA, but not a proteosomal inhibitor, lactacystin, results in a sig-nificant accumulation of intracellular Lp(a) and apo(a) with orwithout PCSK9 treatment. E-64d and lactacystin are highlyselective, potent, and irreversible inhibitors of their respectivetarget proteases (54, 55). E-64d inhibits calpain and the cysteineproteases cathepsins F, K, B, H, and L and acts by forming athioether bond with the active site cysteine of target proteaseswithout affecting cysteine residues in other enzymes. Lactacys-tin covalently modifies the amino-terminal threonine of spe-cific catalytic subunits of the proteasome. Conversely, conca-namycin A specifically blocks lysosomal acidification throughinhibition of V-type ATPase (56). Taken together, these resultsindicate that Lp(a)/apo(a) internalization (whether regulatedby PCSK9 or not) occurs, in part, through clathrin-mediatedendocytosis and Lp(a)/apo(a) is subsequently targeted for lyso-somal degradation. These findings are consistent with a role forLDLR in PCSK9-regulated Lp(a) catabolism.

The contribution of the LDLR to Lp(a) catabolism has beencontroversial. Compared with LDL, plasma Lp(a) concentra-tions are much less responsive to conventional lipid-loweringtherapies, such as statins. Indeed, some studies have shown anincrease, no effect, or a decrease in plasma Lp(a) levels withstatins (57). More recent studies have found that statins mod-estly, but significantly, reduce Lp(a) in subjects with dyslipi-demia or heterozygous FH (58, 59). Moreover, in some studiesof FH kindreds with a null LDLR, elevated plasma Lp(a) levelsare observed in affected individuals (21, 22, 60), although thisresult has not been unanimously observed (22, 61, 62).Although overexpression of the LDLR in mice significantlyincreased Lp(a) clearance (20), plasma clearance studies inLdlr�/� mice and human FH patients reported no significantdifference in Lp(a) clearance compared with the presence ofnormal LDLR, although a non-significant decrease in fractionalcatabolic rate in the absence of the LDLR of about 10% wasreported in both studies (23, 24). Plausible evidence thereforeexists to indicate that the LDLR does participate in Lp(a) catab-olism, which may account for the ability of PCSK9 inhibitors tolower plasma Lp(a). Accordingly, we directly examined the roleof the LDLR in the regulation of Lp(a) catabolism by PCSK9.

The following lines of evidence from our study very stronglysupport the concept of the LDLR being a PCSK9-regulableclearance receptor for Lp(a). (i) The GOF PCSK9 mutantD374Y, which can target the LDLR for degradation more rap-idly, was more effective than WT PCSK9 in inhibiting bothLp(a) and apo(a) internalization in HepG2 cells (Fig. 1, C andD). (ii) Overexpression of LDLR (and LDLR�CT) dramaticallyincreases Lp(a) clearance (Fig. 5B). The addition of PCSK9 inthe context of LDLR overexpression decreased internalization,but the difference did not reach statistical significance. It ispossible that the dose of PCSK9 added was not sufficient toinfluence the very high concentrations of ectopically expressedLDLR. (iii) The addition of blocking monoclonal antibodies

against LDLR decreased Lp(a) internalization, and PCSK9 hadno effect in the setting of LDLR blockade with the antibodies(Fig. 5D). (iv) Human fibroblasts lacking the LDLR showeddecreased internalization of Lp(a) that was unaffected by theaddition of PCSK9 (Fig. 6A).

It is notable that the LDLR lacking the cytoplasmic tail, whichinteracts with the ARH adaptor protein to promote endocyto-sis, retains a considerable fraction of the wild-type receptor’sability to internalize Lp(a) (Fig. 5B). It has been previouslyshown that PCSK9 cannot target the LDLR for degradation inprimary hepatocytes isolated from Arh�/� mice (63). However,PCSK9 can target the LDLR for degradation upon removal ofthe cytoplasmic tail in CHO cells (64), and the ARH adaptorprotein is not necessary in PCSK9-mediated LDLR degradationin fibroblasts (47). These results suggest a potential PCSK9-interacting partner in mediating endocytosis of the LDLR-PCSK9 complex in HepG2 cells.

Less of an increase is observed with apo(a) internalizationfollowing LDLR overexpression (Fig. 5C), indicating therequirement for apo(a) coupling to apoB for recognition by thisreceptor. Although HepG2 cells were deprived of LDL bygrowth in LPDS, these cells do express apoB and secrete apoB-containing lipoprotein particles in the LDL density range (12).Formation of non-covalent complexes between these particlesand apo(a) could be a rate-limiting process and therefore mayaccount for why less of an increase in internalization isobserved for apo(a) compared with Lp(a) with LDLR overex-pression. Our results also show that Lp(a) internalization is sig-nificantly reduced in human FH fibroblasts with a defectiveLDLR compared with fibroblasts with WT LDLR function (Fig.6A). Fibroblasts do not express apoB, and therefore the inter-nalization of apo(a) cannot be affected by PCSK9 in either thecontrol or LDLR-defective fibroblast cells (Fig. 6B). Collec-tively, these results definitively indicate a role for the LDLR ininternalization of Lp(a) through the apoB component ratherthan apo(a).

PCSK9 has been reported to down-regulate other membersof the LDLR, specifically the VLDLR and the apoER2 receptor(35). It is not known if these are ligands for Lp(a) in vivo, but itdoes not appear that they are playing a role in Lp(a) internaliza-tion in our experiments, at least with respect to the PCSK9-de-pendent component. This conclusion stems from our observa-tions that Lp(a) internalization is insensitive to PCSK9 in thepresence of antibodies that block the LDLR (Fig. 5D) or in fibro-blasts lacking functional LDLR (Fig. 6A).

Previously reported clinical studies have shown that antibod-ies that target PCSK9 lower Lp(a) to a lesser extent (�30%decrease) than LDL (�70% decrease) (25–30). Because LDLconcentrations are higher than Lp(a) on a particle numberbasis, LDL can compete with Lp(a) for binding to the LDLR. It isnotable that all study subjects receiving PCSK9-inhibitory anti-bodies were also receiving an optimal dose of statin (27), and anLp(a) lowering effect was still observed, possibly because statinsincrease PCSK9 expression (65). Thus, by increasing hepaticLDLR to supraphysiological levels, possibly along with pro-found lowering of LDL levels, clearance of Lp(a) through theLDLR assumes a greater importance. This is validated by a pre-vious finding where overexpressing the LDLR in mice results in




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an increase in Lp(a) catabolism (20). Our results clearly suggestthat the effects of PCSK9-inhibitory antibodies on Lp(a) levelsin vivo are the consequence of greater LDLR-mediated catabo-lism of Lp(a) (Fig. 7). Therefore, although the LDLR may not bea major route of clearance of Lp(a) under most circumstances,its importance may increase in the setting of supraphysiologicallevels of the LDLR, such as is the case with the use of inhibitoryantibodies against PCSK9. Definitive proof of this concept willultimately require further studies in an in vivo setting.

Acknowledgment—We thank Dr. Ross Milne (University of OttawaHeart Institute) for the generous gift of the monoclonal antibodiesagainst LDLR.

REFERENCES1. Boffa, M. B., and Koschinsky, M. L (2013) Update on lipoprotein(a) as a

cardiovascular risk factor and mediator. Curr. Atheroscler. Rep. 15,360 –366

2. Tsimikas, S., and Hall, J. L. (2012) Lipoprotein(a) as a potential causalgenetic risk factor of CVD: a rationale for increased efforts to understandits pathophysiology and develop targeted therapies. J. Am. Coll. Cardiol.60, 716 –721

3. McLean, J. W., Tomlinson, J. E., Kuang, W. J., Eaton, D. L., Chen, E. Y.,Fless, G. M., Scanu, A. M., and Lawn, R. M. (1987) cDNA sequence ofhuman apolipoprotein(a) is homologous to plasminogen. Nature 330,132–137

4. Gabel, B. R., and Koschinsky, M. I. (1995) Analysis of the proteolytic ac-tivity of a recombinant form of apolipoprotein(a). Biochemistry 34,15777–15784

5. Koschinsky, M. L., Côté, G. P., Gabel, B., and van der Hoek, Y. Y. (1993)Identification of the cysteine residue in apolipoprotein(a) that mediatesextracellular coupling with apolipoprotein B-100. J. Biol. Chem. 268,19819 –19825

6. Becker, L., Cook, P. M., Wright, T. G., and Koschinsky, M. L. (2004) Quan-titative evaluation of the contribution of weak lysine-binding sites presentwithin apolipoprotein(a) kringle IV types 6 – 8 to lipoprotein(a) assembly.J. Biol. Chem. 279, 2679 –2688

7. Hancock, M. A., Boffa, M. B., Marcovina, S. M., Nesheim, M. E., andKoschinsky, M. L. (2003) Inhibition of plasminogen activation by lipopro-tein(a): critical domains in apolipoprotein(a) and mechanism of inhibitionon fibrin and degraded fibrin surfaces. J. Biol. Chem. 278, 23260 –23269

8. Marcovina, S. M., Albers, J. J., Wijsman, E., Zhang, Z., Chapman, N. H.,and Kennedy, H. (1996) Differences in Lp(a) concentrations and apo(a)polymorphs between black and white Americans. J. Lipid Res. 37,2569 –2585

9. Rader, D. J., Cain, W., Ikewaki, K., Talley, G., Zech, L. A., Usher, D., andBrewer, H. B., Jr. (1994) The inverse association of plasma lipoprotein(a)concentrations with apolipoprotein(a) isoform size is not due to differ-ences in Lp(a) catabolism but to differences in production rate. J. Clin.Invest. 93, 2758 –2763

10. White, A. L., Guerra, B., and Lanford, R. E. (1997) Influence of allelicvariation on apolipoprotein(a) folding in the endoplasmic reticulum.J. Biol. Chem. 272, 5048 –5055

11. Boerwinkle, E., Leffert, C. C., Lin, J., Lackner, C., Chiesa, G., and Hobbs,H. H. (1992) Apolipoprotein (a) gene accounts for greater than 90% of thevariation in plasma lipoprotein(a) concentrations. J. Clin. Invest. 90,52– 60

12. Tam, S. P., Zhang, X., and Koschinsky, M. L. (1996) Interaction of a re-combinant form of apolipoprotein[a] with human fibroblasts and with thehuman hepatoma cell line HepG2. J. Lipid Res. 37, 518 –533

13. Floren, C. H., Albers, J. J., and Bierman, E. L. (1981) Uptake of Lp[a]lipoprotein by cultured fibroblasts. Biochem. Biophys. Res. Commun. 102,636 – 639

14. Havekes, L., Vermeer, B. J., Brugman, T., and Emeis, J. (1981) Binding ofLp[a] to the low density lipoprotein receptor of human fibroblasts. FEBS

Lett. 132, 169 –17315. Krempler, F., Kostner, G. M., Roscher, A., Haslauer, F., Bolzano, K., and

Sandhofer, F. (1983) Studies on the role of specific cell surface receptors inthe removal of lipoprotein[a] in man. J. Clin. Invest. 71, 1431–1441

16. Argraves, K. M., Kozarsky, K. F., Fallon, J. T., Harpel, P. C., and Strickland,D. K. (1997) The atherogenic lipoprotein Lp(a) is internalized and de-graded in a process mediated by the VLDL receptor. J. Clin. Invest. 100,2170 –2181

17. März, W., Beckmann, A., Scharnagl, H., Siekmeier, R., Mondorf, U., Held,I., Schneider, W., Preissner, K. T., Curtiss, L. K., and Gross, W. (1993)Heterogeneous lipoprotein[a] isoforms differ by their interaction with thelow density lipoprotein receptor and the low density lipoprotein receptor-related protein/� 2-macroglobulin receptor. FEBS Lett. 325, 271–275

18. Niemeier, A., Willnow, T., Dieplinger, H., Jacobsen, C., Meyer, N., Hilpert,J., and Beisiegel, U. (1999) Identification of megalin/gp330 as a receptor forlipoprotein(a) in vitro. Arterioscler. Thromb. Vasc. Biol. 19, 552–561

19. Yang, X. P., Amar, M. J., Vaisman, B., Bocharov, A. V., Vishnyakova, T. G.,Freeman, L. A., Kurlander, R. J., Patterson, A. P., Becker, L. C., and Rema-ley, A. T. (2013) Scavenger receptor-BI is a receptor for lipoprotein(a). J.Lipid Res. 54, 2450 –2457

20. Hofmann, S. L., Eaton, D. L., Brown, M. S., McConathy, W. J., Goldstein,J. L., and Hammer, R. E. (1990) Overexpression of human low densitylipoprotein receptors leads to accelerated catabolism of Lp(a) lipoproteinin transgenic mice. J. Clin. Invest. 85, 1542–1547

21. Alonso, R., Andres, E., Mata, N., Fuentes-Jiménez, F., Badimón, L., López-Miranda, J., Padró, T., Muñiz, O., Díaz-Díaz, J. L., Mauri, M., Ordovás,J. M., and Mata, P., and SAFEHEART Investigators (2014) Lipoprotein(a)levels in familial hypercholesterolemia: an important predictor of cardio-vascular disease independent of the type of LDL receptor mutation. J. Am.Coll. Cardiol. 63, 1982–1989

22. Kraft, H. G., Lingenhel, A., Raal, F. J., Hohenegger, M., and Utermann, G.(2000) Lipoprotein(a) in homozygous familial hypercholesterolemia. Ar-terioscler. Thromb. Vasc. Biol. 20, 522–528

23. Cain, W. J., Millar, J. S., Himebauch, A. S., Tietge, U. J., Maugeais, C.,Usher, D., and Rader, D. J. (2005) Lipoprotein[a] is cleared from theplasma primarily by the liver in a process mediated by apolipoprotein[a]. J.Lipid Res. 46, 2681–2691

24. Rader, D. J., Mann, W. A., Cain, W., Kraft, H. G., Usher, D., Zech, L. A.,Hoeg, J. M., Davignon, J., Lupien, P., and Grossman, M. (1995) The lowdensity lipoprotein receptor is not required for normal catabolism of Lp(a)in humans. J. Clin. Invest. 95, 1403–1408

25. McKenney, J. M., Koren, M. J., Kereiakes, D. J., Hanotin, C., Ferrand, A. C.,and Stein, E. A. (2012) Safety and efficacy of a monoclonal antibody toproprotein convertase subtilisin/kexin type 9 serine protease,SAR236553/REGN727, in patients with primary hypercholesterolemia re-ceiving ongoing stable atorvastatin therapy. J. Am. Coll. Cardiol. 59,2344 –2353

26. Roth, E. M., McKenney, J. M., Hanotin, C., Asset, G., and Stein, E. A. (2012)Atorvastatin with or without an antibody to PCSK9 in primary hypercho-lesterolemia. N. Engl. J. Med. 367, 1891–1900

27. Desai, N. R., Kohli, P., Giugliano, R. P., O’Donoghue, M. L., Somaratne, R.,Zhou, J., Hoffman, E. B., Huang, F., Rogers, W. J., Wasserman, S. M., Scott,R., and Sabatine, M. S. (2013) AMG145, a monoclonal antibody againstproprotein convertase subtilisin kexin type 9, significantly reduces lipo-protein(a) in hypercholesterolemic patients receiving statin therapy: ananalysis from the LDL-C Assessment with Proprotein Convertase Subtil-isin Kexin Type 9 Monoclonal Antibody Inhibition Combined with StatinTherapy (LAPLACE)-Thrombolysis in Myocardial Infarction (TIMI) 57trial. Circulation 128, 962–969

28. Raal, F. J., Giugliano, R. P., Sabatine, M. S., Koren, M. J., Langslet, G., Bays,H., Blom, D., Eriksson, M., Dent, R., Wasserman, S. M., Huang, F., Xue, A.,Albizem, M., Scott, R., and Stein, E. A. (2014) Reduction in lipoprotein(a)with PCSK9 monoclonal antibody evolocumab (AMG 145): a pooled anal-ysis of more than 1,300 patients in 4 phase II trials. J. Am. Coll. Cardiol. 63,1278 –1288

29. Stein, E. A., Giugliano, R. P., Koren, M. J., Raal, F. J., Roth, E. M., Weiss, R.,Sullivan, D., Wasserman, S. M., Somaratne, R., Kim, J. B., Yang, J., Liu, T.,Albizem, M., Scott, R., Sabatine, M. S., and PROFICIO Investigators




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

(2014) Efficacy and safety of evolocumab (AMG 145), a fully humanmonoclonal antibody to PCSK9, in hyperlipidaemic patients on variousbackground lipid therapies: pooled analysis of 1359 patients in four phase2 trials. Eur. Heart J. 35, 2249 –2259

30. Gaudet, D., Kereiakes, D. J., McKenney, J. M., Roth, E. M., Hanotin, C.,Gipe, D., Du, Y., Ferrand, A. C., Ginsberg, H. N., and Stein, E. A. (2014)Effect of alirocumab, a monoclonal proprotein convertase subtilisin/kexin9 antibody, on lipoprotein(a) concentrations (a pooled analysis of 150 mgevery two weeks dosing from phase 2 trials). Am. J. Cardiol. 114, 711–715

31. Abifadel, M., Varret, M., Rabès, J. P., Allard, D., Ouguerram, K., Devillers,M., Cruaud, C., Benjannet, S., Wickham, L., Erlich, D., Derré, A., Villéger,L., Farnier, M., Beucler, I., Bruckert, E., Chambaz, J., Chanu, B., Lecerf,J. M., Luc, G., Moulin, P., Weissenbach, J., Prat, A., Krempf, M., Junien, C.,Seidah, N. G., and Boileau, C. (2003) Mutations in PCSK9 cause autosomaldominant hypercholesterolemia. Nat. Genet. 34, 154 –156

32. Seidah, N. G., Benjannet, S., Wickham, L., Marcinkiewicz, J., Jasmin, S. B.,Stifani, S., Basak, A., Prat, A., and Chretien, M. (2003) The secretory pro-protein convertase neural apoptosis-regulated convertase 1 (NARC-1):liver regeneration and neuronal differentiation. Proc. Natl. Acad. Sci.U.S.A. 100, 928 –933

33. Benjannet, S., Rhainds, D., Essalmani, R., Mayne, J., Wickham, L., Jin, W.,Asselin, M. C., Hamelin, J., Varret, M., Allard, D., Trillard, M., Abifadel,M., Tebon, A., Attie, A. D., Rader, D. J., Boileau, C., Brissette, L., Chrétien,M., Prat, A., and Seidah, N. G. (2004) NARC-1/PCSK9 and its naturalmutants: zymogen cleavage and effects on the low density lipoprotein(LDL) receptor and LDL cholesterol. J. Biol. Chem. 279, 48865– 48875

34. Abifadel, M., Rabès, J. P., Devillers, M., Munnich, A., Erlich, D., Junien, C.,Varret, M., and Boileau, C. (2009) Mutations and polymorphisms in theproprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol me-tabolism and disease. Hum. Mutat. 30, 520 –529

35. Poirier, S., Mayer, G., Benjannet, S., Bergeron, E., Marcinkiewicz, J., Nas-soury, N., Mayer, H., Nimpf, J., Prat, A., and Seidah, N. G. (2008) Theproprotein convertase PCSK9 induces the degradation of low density li-poprotein receptor (LDLR) and its closest family members VLDLR andApoER2. J. Biol. Chem. 283, 2363–2372

36. Canuel, M., Sun, X., Asselin, M. C., Paramithiotis, E., Prat, A., and Seidah,N. G. (2013) Proprotein convertase subtilisin/kexin type 9 (PCSK9) canmediate degradation of the low density lipoprotein receptor-related pro-tein 1 (LRP-1). PLoS One 8, e64145

37. Rohlmann, A., Gotthardt, M., Hammer, R. E., and Herz, J. (1998) Inducibleinactivation of hepatic LRP gene by cre-mediated recombination confirmsrole of LRP in clearance of chylomicron remnants. J. Clin. Invest. 101,689 – 695

38. Frykman, P. K., Brown, M. S., Yamamoto, T., Goldstein, J. L., and Herz, J.(1995) Normal plasma lipoproteins and fertility in gene-targeted micehomozygous for a disruption in the gene encoding very low density lipo-protein receptor. Proc. Natl. Acad. Sci. U.S.A. 92, 8453– 8457

39. Marcovina, S. M., Albers, J. J., Gabel, B., Koschinsky, M. L., and Gaur, V. P.(1995) Effect of the number of apolipoprotein(a) kringle 4 domains onimmunochemical measurements of lipoprotein(a). Clin. Chem. 41,246 –255

40. Romagnuolo, R., Marcovina, S. M., Boffa, M. B., Koschinsky, M. L. (2014)Inhibition of plasminogen activation by apo(a): role of carboxyl-terminallysines and identification of inhibitory domains in apo(a). J. Lipid Res. 55,625– 634

41. Poirier, S., Mayer, G., Poupon, V., McPherson, P. S., Desjardins, R., Ly, K.,Asselin, M. C., Day, R., Duclos, F. J., Witmer, M., Parker, R., Prat, A., andSeidah, N. G. (2009) Dissection of the endogenous cellular pathways ofPCSK9-induced low density lipoprotein receptor degradation: evidencefor an intracellular route. J. Biol. Chem. 284, 28856 –28864

42. Zhang, D. W., Lagace, T. A., Garuti, R., Zhao, Z., McDonald, M., Horton,J. D., Cohen, J. C., and Hobbs, H. H. (2007) Binding of proprotein conver-tase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of lowdensity lipoprotein receptor decreases receptor recycling and increasesdegradation. J. Biol. Chem. 282, 18602–18612

43. Kwon, H. J., Lagace, T. A., McNutt, M. C., Horton, J. D., and Deisenhofer,J. (2008) Molecular basis for LDL receptor recognition by PCSK9. Proc.Natl. Acad. Sci. U.S.A. 105, 1820 –1825

44. Miles, L. A., Fless, G. M., Scanu, A. M., Baynham, P., Sebald, M. T., Skocir,P., Curtiss, L. K., Levin, E. G., Hoover-Plow, J. L., and Plow, E. F. (1995)Interaction of Lp(a) with plasminogen binding sites on cells. Thromb.Haemost. 73, 458 – 465

45. Kosenko, T., Golder, M., Leblond, G., Weng, W., and Lagace, T. A. (2013)Low density lipoprotein binds to proprotein convertase subtilisin/kexintype-9 (PCSK9) in human plasma and inhibits PCSK9-mediated low den-sity lipoprotein receptor degradation. J. Biol. Chem. 288, 8279 – 8288

46. Nassoury, N., Blasiole, D. A., Tebon Oler, A., Benjannet, S., Hamelin, J.,Poupon, V., McPherson, P. S., Attie, A. D., Prat, A., and Seidah, N. G.(2007) The cellular trafficking of the secretory proprotein convertasePCSK9 and its dependence on the LDLR. Traffic 8, 718 –732

47. Wang, Y., Huang, Y., Hobbs, H. H., and Cohen, J. C. (2012) Molecularcharacterization of proprotein convertase subtilisin/kexin type 9-medi-ated degradation of the LDLR. J. Lipid Res. 53, 1932–1943

48. Ernst, A., Helmhold, M., Brunner, C., Pethö-Schramm, A., Armstrong,V. W., and Müller, H. J. (1995) Identification of two functionally distinctlysine-binding sites in kringle 37 and in kringles 32–36 of human apolipo-protein(a). J. Biol. Chem. 270, 6227– 6234

49. Theuerle, J. D. (2009) Analysis of Lipoprotein(a) Catabolism. M.S. thesis,Queen’s University, Kingston, Canada

50. Garcia, C. K., Wilund, K., Arca, M., Zuliani, G., Fellin, R., Maioli, M.,Calandra, S., Bertolini, S., Cossu, F., Grishin, N., Barnes, R., Cohen, J. C.,and Hobbs, H. H. (2001) Autosomal recessive hypercholesterolemiacaused by mutations in a putative LDL receptor adaptor protein. Science292, 1394 –1398

51. He, G., Gupta, S., Yi, M., Michaely, P., Hobbs, H. H., and Cohen, J. C.(2002) ARH is a modular adaptor protein that interacts with the LDLreceptor, clathrin, and AP-2. J. Biol. Chem. 277, 44044 – 44049

52. Nguyen, A. T., Hirama, T., Chauhan, V., Mackenzie, R., and Milne, R.(2006) Binding characteristics of a panel of monoclonal antibodiesagainst the ligand binding domain of the human LDLr. J. Lipid Res. 47,1399 –1405

53. Becker, L., Webb, B. A., Chitayat, S., Nesheim, M. E., and Koschinsky,M. L. (2003) A ligand-induced conformational change in apolipopro-tein(a) enhances covalent Lp(a) formation. J. Biol. Chem. 278,14074 –14081

54. Barrett, A. J., Kembhavi, A. A., Brown, M. A., Kirschke, H., Knight, C. G.,Tamai, M., and Hanada, K. (1982) L-trans-Epoxysuccinyl-leucylamide(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine pro-teinases including cathepsins B, H and L. Biochem. J. 201, 189 –198

55. Fenteany, G., and Schreiber, S. L. (1998) Lactacystin, proteasome function,and cell fate. J. Biol. Chem. 273, 8545– 8548

56. Dröse, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A., and Alten-dorf, K. (1993) Inhibitory effect of modified bafilomycins and concanamy-cins on P- and V-type adenosinetriphosphatases. Biochemistry 32,3902–3906

57. Tziomalos, K., Athyros, V. G., Wierzbicki, A. S., and Mikhailidis, D. P.(2009) Lipoprotein a: where are we now? Curr. Opin. Cardiol. 24, 351–357

58. Gonbert, S., Malinsky, S., Sposito, A. C., Laouenan, H., Doucet, C., Chap-man, M. J., and Thillet, J. (2002) Atorvastatin lowers lipoprotein(a) but notapolipoprotein(a) fragment levels in hypercholesterolemic subjects athigh cardiovascular risk. Atherosclerosis 164, 305–311

59. van Wissen, S., Smilde, T. J., Trip, M. D., de Boo, T., Kastelein, J. J., andStalenhoef, A. F. (2003) Long term statin treatment reduces lipoprotein(a)concentrations in heterozygous familial hypercholesterolaemia. Heart 89,893– 896

60. Bea, A. M., Mateo-Gallego, R., Jarauta, E., Villa-Pobo, R., Calmarza, P.,Lamiquiz-Moneo, I., Cenarro, A., Civeira, F. (2014) [Lipoprotein(a) is as-sociated to atherosclerosis in primary hypercholesterolemia.] Clin. Inves-tig. Arterioscler. 26, 176 –183

61. Soutar, A. K., McCarthy, S. N., Seed, M., and Knight, B. L. (1991) Relation-ship between apolipoprotein(a) phenotype, lipoprotein(a) concentrationin plasma, and low density lipoprotein receptor function in a large kindredwith familial hypercholesterolemia due to the Pro6643 Leu mutation inthe LDL receptor gene. J. Clin. Invest. 88, 483– 492

62. Ghiselli, G., Gaddi, A., Barozzi, G., Ciarrocchi, A., and Descovich, G.(1992) Plasma lipoprotein(a) concentration in familial hypercholester-




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

olemic patients without coronary artery disease. Metabolism 41, 833– 83863. Lagace, T. A., Curtis, D. E., Garuti, R., McNutt, M. C., Park, S. W., Prather,

H. B., Anderson, N. N., Ho, Y. K., Hammer, R. E., and Horton, J. D. (2006)Secreted PCSK9 decreases the number of LDL receptors in hepatocytesand in livers of parabiotic mice. J. Clin. Invest. 116, 2995–3005

64. Strøm, T. B., Holla, Ø. L., Tveten, K., Cameron, J., Berge, K. E., and Leren,T. P. (2010) Disrupted recycling of the low density lipoprotein receptor by

PCSK9 is not mediated by residues of the cytoplasmic domain. Mol. Genet.Metab. 101, 76 – 80

65. Dubuc, G., Chamberland, A., Wassef, H., Davignon, J., Seidah, N. G.,Bernier, L., and Prat, A. (2004) Statins upregulate PCSK9, the gene encod-ing the proprotein convertase neural apoptosis-regulated convertase-1implicated in familial hypercholesterolemia. Arterioscler. Thromb. Vasc.Biol. 24, 1454 –1459




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G. Seidah and Marlys L. KoschinskyRocco Romagnuolo, Corey A. Scipione, Michael B. Boffa, Santica M. Marcovina, Nabil

Type 9 through the Low Density Lipoprotein ReceptorLipoprotein(a) Catabolism Is Regulated by Proprotein Convertase Subtilisin/Kexin

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