Portal de Periódicos da CAPES

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

2007; Elsevier BV; Volume: 48; Issue: 7 Linguagem: Inglês

10.1194/jlr.m700071-jlr200

1539-7262

Yuewei Qian, Robert J. Schmidt, Youyan Zhang, Shaoyou Chu, Aimin Lin, He Wang, Xiliang Wang, Thomas P. Beyer, William R. Bensch, Weiming Li, Mariam Ehsani, Deshun Lu, Robert J. Konrad, Patrick I. Eacho, David E. Moller, Sotirios K. Karathanasis, Guoqing Cao,

Cellular transport and secretion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR. Increased plasma LDL cholesterol is a major risk factor for atherosclerotic cardiovascular disease. Importantly, recent studies have suggested further benefits of very aggressive LDL cholesterol lowering compared with the typical clinical targets in place today (1.Cannon C.P. The IDEAL cholesterol: lower is better.J. Am. Med. Assoc. 2005; 294: 2492-2494Crossref PubMed Scopus (41) Google Scholar, 2.Wiviott S.D. Cannon C.P. Morrow D.A. Ray K.K. Pfeffer M.A. Braunwald E. Can low-density lipoprotein be too low? The safety and efficacy of achieving very low low-density lipoprotein with intensive statin therapy: a PROVE IT-TIMI 22 substudy.J. Am. Coll. Cardiol. 2005; 46: 1411-1416Crossref PubMed Scopus (285) Google Scholar). Plasma LDL cholesterol is controlled primarily by hepatic cholesterol biosynthesis and hepatic low density lipoprotein receptor (LDLR) levels (3.Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4362) Google Scholar). The master transcription factor controlling mRNA levels that encode key enzymes involved in cholesterol biosynthesis and LDLR is sterol-responsive element binding protein 2 (SREBP2). When cellular cholesterol levels are low, SREBP2 is activated through sequential proteolytic cleavage by two proteases. The activated N terminus of the protein then enters the cell nucleus to mediate the transcription of genes that contain sterol response element(s) in their promoter or enhancer region (4.Brown M.S. Goldstein J.L. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.Proc. Natl. Acad. Sci. USA. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). Cellular cholesterol levels are thus tightly regulated through this feedback mechanism. Recent human genetic studies have revealed that proprotein convertase subtilisin/kexin type 9 (PCSK9) is a critically important additional mechanism that regulates cellular LDLR levels. Although the molecular basis has yet to be determined, multiple mutations in the PCSK9 gene have been described to result in reduced cellular LDLR levels and thus significantly increased plasma LDL cholesterol (5.Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2188) Google Scholar, 6.Timms K.M. Wagner S. Samuels M.E. Forbey K. Goldfine H. Jammulapati S. Skolnick M.H. Hopkins P.N. Hunt S.C. Shattuck D.M. A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree.Hum. Genet. 2004; 114: 349-353Crossref PubMed Scopus (270) Google Scholar, 7.Leren T.P. Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia.Clin. Genet. 2004; 65: 419-422Crossref PubMed Scopus (206) Google Scholar, 8.Cameron J. Holla O.L. Ranheim T. Kulseth M.A. Berge K.E. Leren T.P. Effect of mutations in the PCSK9 gene on the cell surface LDL receptors.Hum. Mol. Genet. 2006; 15: 1551-1558Crossref PubMed Scopus (226) Google Scholar). More importantly, opposite to these "gain-of-function" mutations, Cohen et al. (9.Cohen J.C. Boerwinkle E. Mosley Jr., T.H. Hobbs H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.N. Engl. J. Med. 2006; 354: 1264-1272Crossref PubMed Scopus (2428) Google Scholar, 10.Kotowski I.K. Pertsemlidis A. Luke A. Cooper R.S. Vega G.L. Cohen J.C. Hobbs H.H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.Am. J. Hum. Genet. 2006; 78: 410-422Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 11.Berge K.E. Ose L. Leren T.P. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1094-1100Crossref PubMed Scopus (207) Google Scholar, 12.Zhao Z. Tuakli-Wosornu Y. Lagace T.A. Kinch L. Grishin N.V. Horton J.D. Cohen J.C. Hobbs H.H. Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.Am. J. Hum. Genet. 2006; 79: 514-523Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 13.Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1077) Google Scholar) found apparent loss-of-function mutations that are presumed to lead to increased cellular LDLR protein levels. In humans carrying these mutations, plasma LDL cholesterol is reduced by 30–40% compared with controls. More strikingly, an apparently healthy human subject with mutations affecting both alleles of the PCSK9 gene was described with an LDL cholesterol level of 14 mg/dl. This finding further indicates a critical role for PCSK9 in modulating LDLR and plasma LDL cholesterol levels and provides evidence that loss of PCSK9 function in humans is not associated with apparent deleterious effects (12.Zhao Z. Tuakli-Wosornu Y. Lagace T.A. Kinch L. Grishin N.V. Horton J.D. Cohen J.C. Hobbs H.H. Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.Am. J. Hum. Genet. 2006; 79: 514-523Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). PCSK9 belongs to the proprotein convertase family and was only recently cloned from brain tissue as a secreted protein (14.Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.Proc. Natl. Acad. Sci. USA. 2003; 100: 928-933Crossref PubMed Scopus (925) Google Scholar). The closest homolog is site 1 protease, another protein intimately involved in cholesterol homeostasis (15.Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells.Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Beyond the function implied by human genetic studies, the physiological function of PCSK9 was not clear until the recent demonstration that hepatic overexpression in mice greatly reduced hepatic LDLR levels and led directly to increased plasma LDL cholesterol (16.Maxwell K.N. Breslow J.L. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.Proc. Natl. Acad. Sci. USA. 2004; 101: 7100-7105Crossref PubMed Scopus (509) Google Scholar, 17.Park S.W. Moon Y.A. Horton J.D. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Consistent with these observations, PCSK9 deficiency in mice resulted in significantly increased hepatic LDLR levels (18.Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.Proc. Natl. Acad. Sci. USA. 2005; 102: 5374-5379Crossref PubMed Scopus (563) Google Scholar). Furthermore, PCSK9 deficiency augmented statin-induced increase of LDLR protein levels and thus strongly suggested the value of PCSK9 as a pharmacological target for LDL cholesterol lowering (18.Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.Proc. Natl. Acad. Sci. USA. 2005; 102: 5374-5379Crossref PubMed Scopus (563) Google Scholar). Although the function of PCSK9 in reducing hepatic LDLR protein levels is firmly established, the precise molecular basis for this effect has remained elusive. To date, the direct substrates and the active form of the enzyme are not known. In this report, we have investigated the potential molecular mechanisms of PCSK9 activity. Human PCSK9 (accession number NM_174936) was cloned by PCR from human liver cDNA (BD Biosciences Co.) using Turbo pfu polymerase (Stratagene) and the following primers: 5′ primer, 5′-GCTCCTGAACTTCAGCTCCTGCACA-3′; 3′ primer, 5′-CTGAGAGAGGGACAAGTCGGAACCATTT-3′. The nucleotide sequences encoding full-length PCSK9 were inserted into a modified pJB02 vector with a C-terminal histidine (HIS) tag. The resulting construct was used to generate a HEK293 stable cell line overexpressing PCSK9. PCSK9 was purified from conditioned medium derived from this cell line by nickel-nitrilotriacetic acid agarose according to the manufacturer's instructions (Qiagen), followed by size-exclusion chromatography on a HiLoad 16/60 Superdex 200 column (Amersham) in the storage buffer (50 mM Tris, 150 mM NaCl, and 10% glycerol, pH 8.0). The final protein concentration was determined by Bradford assay using BSA as the standard. The identity of the purified PCSK9 protein was confirmed by N-terminal sequencing. The purified protein was stored at −80°C in small aliquots. Human LDLR (accession number g15680298) was cloned by PCR from human lung cDNA (BD Biosciences Co.) using Turbo pfu polymerase (Stratagene) and the following primers: 5′ primer, 5′-GCCTGGCAGAGGCTGCGAGCATG-3′; 3′ primer, 5′-TCACGCCACGTCATCCTCCAGACT-3′. The nucleotide sequences encoding the LDLR extracellular domain (amino acids 1–788) were inserted into modified pJB02 vector with a C-terminal Flag tag. The resulting construct was used to transfect 15 liters HEK293E suspension cells. Four days after the transfection, conditioned medium was collected and concentrated 10-fold for purification. The LDLR extracellular domain was purified by anti-Flag M2 affinity chromatography according to the manufacturer's instructions (Sigma), followed by size-exclusion chromatography on a HiLoad 16/60 Superdex 200 column (Amersham) in the storage buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, and 0.01% Brij-35, pH 7.5). The final protein concentration was determined by Bradford assay using BSA as the standard. The identity of the purified protein was confirmed by N-terminal sequencing, which starts at amino acid 25. The purified LDLR (25–788)-Flag protein was stored at −80°C in small aliquots. HEK293 cells were seeded at 1 × 106 cells/35 mm well and grown overnight in 3:1 DMEM/F12 + 10% FBS. After 24 h of growth, cells were washed in PBS, the medium was replaced with 3:1 DMEM/F12 + 5% lipoprotein deficient serum (LPDS), and PCSK9 was added at the indicated concentrations. After 18 h, the cells were washed twice in PBS without Mg/Ca2+. The cells were scraped into buffer A (10 mM HEPES, pH 7.6, 1.5 mM MgCl, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, and Complete protease inhibitor; Roche) and passed through a 221/2 gauge needle 15 times. The nuclei were pelleted by centrifugation at 1,000 g for 15 min at 4°C. The supernatant was then centrifuged at 55,000 rpm for 30 min at 4°C in a TL100 rotor to pellet cellular membranes. The membrane pellet was solubilized in 100 μl of buffer l (10 mM Tris, pH 7.6, 100 mM NaCl, and 1.0% SDS). Protein concentrations were determined by a BCA protein assay kit (Pierce). Ten micrograms of membrane proteins was denatured at 85°C for 4 min and separated on a Tris/Gly SDS gel (4–20%). Proteins were transferred to a 0.45 μm nitrocellulose membrane (Invitrogen) and blocked for 1 h at room temperature in Odyssey blocking buffer. The blot was then transferred to blocking buffer containing 0.1% Tween 20 + 0.1 μg/ml rabbit anti-human LDLR antibody (RDI-PRO-61099; Fitzgerald International) at 4°C overnight. The blot was washed in PBS + 0.1% Tween 20 (3 × 10 min) and placed in fresh blocking buffer containing Alexa Fluor 680 goat anti-rabbit IgG (0.3 ng/ml; Molecular Probes, Inc.). After 1 h, the blot was washed in PBS + 0.1% Tween 20 (3 × 10 min) and imaged using the Li-Cor infrared imaging system. For tissue samples, 50–100 mg of liver was homogenized in 1.0 ml of RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% SDS, and Complete protease inhibitors; Roche). The homogenate was rocked at 4°C for 20 min to solubilize membrane proteins. After centrifugation at 14,000 rpm to clear cellular debris, protein concentrations were determined using a Pierce BCA protein assay kit. After denaturation at 85°C for 4 min, 50 μg of total protein was separated on a 4–20% Tris/Gly SDS gel. Western blot analysis was performed as described above with chicken polyclonal antibody to LDLR (14056; Abcam) or rabbit anti-cyclophilin A (catalog No. 07-313; Upstate Laboratories) as primary antibodies and Alexa Fluor 680 goat anti-rabbit/chicken IgG (Molecular Probes) as secondary antibody. Seven week old male C57B6 mice were purchased from Harlan (Indianapolis, IN) and acclimated for 1 week before the start of the study. The mice were provided Purina 5001 chow ad libitum throughout the experiment. Recombinant human PCSK9 was dosed daily by tail vein injection into restrained animals at various doses per animal for 3 or 7 consecutive days. The animals were euthanized at 6 h after the final injection by CO2 asphyxiation. Blood samples for serum preparation were collected by cardiac puncture, and livers were collected and frozen in liquid nitrogen. C57B6 or C57B6 LDLR KO (Taconic) mice were anesthetized with Na-pentobarbital (275 mg/kg). After dissection, a cannula was placed in the portal vein superior to the liver. A complete cut was made through the vena cava inferior to the liver for perfusate drainage. Liver perfusion medium (17701; Invitrogen) was infused into the liver for 10 min at a rate of 6.5 ml/min. Liver digestion medium containing collagenase (17703; Invitrogen) was then infused into the liver at a rate of 8.0 ml/min for 10 min. The liver was then removed from the mouse and placed in a 100 mm tissue culture dish containing liver digestion medium. The capsule surrounding the liver was dissected, and liver cells were removed. The hepatocytes were triturated to reduce clumping and filtered through a nylon mesh into a 50 ml centrifuge tube. After a 10 min spin at 50 g, the cells were resuspended in Williams E + 10% FBS (Invitrogen). Centrifugation was repeated, and the cells were again resuspended in 40 ml of Williams E. Cells were counted using trypan blue dye to determine viability. The hepatocytes were plated to 12-well collagen-coated plates (356500; Biocoat) at a density of 10,000 viable cells/well in Williams E + 10% FBS. After overnight incubation, the cells were used for uptake and imaging studies. Purified PCSK9 was labeled using the Alexa Fluor 488 Protein Labeling kit (catalog No. A-10235; Molecular Probes) according to the manufacturer's recommendations. LDL or PCSK9 endocytosis was studied with confocal imaging of fluorescently labeled LDL (DiI-LDL or BODIPY-LDL; Molecular Probes) or PCSK9-Alexa 488. Imaging experiments were performed on cells grown on 96-well plates. For live cell imaging, we changed culture medium with Ringer's buffer (containing in mM: 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 20 HEPES, and 5.5 d-glucose, pH 7.4). For fixed cells, we used 3.7% formaldehyde in PBS for 30 min at 25°C, followed by three washes with PBS, and kept the cells in PBS at 100 μl/well. Cells were imaged with the Zeiss LSM 510 confocal imaging system outfitted with a 40× lens (numerical aperture 0.6). Laser excitation was at 543 nm and emission at 565–615 nm for LDL-DiI, or excitation was at 488 nm and emission at 505–550 nm for LDL-BODIPY or PCSK9-Alexa 488. Laser power, detector gain/offset, and pinhole size were fixed for all samples. To image the colocalization of LDL and PCSK9 uptake, we incubated cells to LDL-DiI/PCSK9-Alexa 488. The confocal settings were as mentioned above. To avoid fluorescent signal spillover between DiI and Alexa 488, we used the multi-track function in line scan mode of the LSM 510 with alternating 488 nm/505–550 nm (excitation/emission) and 543 nm/565–615 nm. Transmitted light images were collected simultaneously to identify cells during all confocal imaging experiments. Cells on 96-well plates with or without PCSK9 treatment were incubated with LDL-BODIPY. Nuclei were stained with propidium iodide. Plates were scanned with the Acumen Explorer (LabTech Limited), excited by 488 nm laser and measuring emission at 500–530 nm (LDL-BODIPY uptake) or 575–640 nm (nucleus). Total fluorescence of LDL-BODIPY per objector or per well was obtained for quantification. Lentivirus short hairpin RNA (shRNA) transduction particles were obtained from Sigma-Aldrich Corp. Knockdown for both LDLR and autosomal recessive hypercholesterolemia (ARH) genes was performed using three different lentiviral transduction particles. The sequences for LDLR and ARH are as follows: for LDLR, 5′-CCGGCCAGCGAAGATGCGAAGATATCTCGAGATATCTTCGCATCTTCGCTGGTTTTTG-3′, 5′-CCGGGCCGTCTTTGAGGACAAAGTACTCGAGTACTTTGTCCTCAAAGACGGCTTTTTG-3′, and 5′-CCGGCGGGAAATGCATCTCCTACAACTCGAGTTGTAGGAGATGCATTTCCCGTTTTTG-3′; for ARH, 5′-CCGGCGACAAGGTGTTTGCATACATCTCGAGATGTATGCAAACACCTTGTCGTTTTTG-3′, 5′-CCGGGTCCATATACAGGATCTCCTACTCGAGTAGGAGATCCTGTATATGGACTTTTTG-3′, and 5′-CCGGGAGAAAGAGAAGAGGGACAAACTCGAGTTTGTCCCTCTTCTCTTTCTCTTTTTG-3′. A 96-well dish was seeded at 3,000–5,000 cells/well and incubated at 37°C overnight in complete medium. At the time of transfections, hexadimethrine was added to a final concentration of 8 μg/ml. Virus particles were added to each well at a multiplicity of infection of 10 plaque-forming units/cell. The cells were exposed to virus for 6 h and washed with PBS. Fresh growth medium containing 10% FBS was then added, and incubation was continued for 72 h. Cells were then used for uptake studies, or RNA was prepared to determine the effect of lentiviral expression on LDLR or ARH levels. RNA was isolated for Taqman analysis in an ABI-6100 Nucleic Acid PrepStation. The effect of lentivirus RNA interference on LDLR and ARH levels was monitored by Taqman analysis with primer pairs for LDLR and ARH commercially supplied by ABI (HS00181192-ml and HS00296701-ml). For uptake studies, the medium was changed to 100 μl of fresh 5% LPDS in DMEM/F12 (3:1) with or without 10 μg/ml PCSK9, and cells were incubated at 37°C and 5% CO2 overnight. Alexa Fluor 488-labeled PCSK9 (100 ng/ml) and DiI-LDL (1 μg/ml) were added to the cells, and after a 4 h incubation at 5% CO2 and 37°C, cells were washed with PBS. Uptake of Alexa Fluor 488-labeled PCSK9 and/or DiI-LDL was analyzed using confocal fluorescence microscopy. Ten micrograms of LDLR (25–788)-Flag was mixed with either 10 μg of PCSK9-HIS or control protein glutathione-S-transferase (GST)-HIS in 50 μl of binding buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, and 0.01% Brij-35, pH 7.5) on ice for 1 h. The mixture was then incubated with 50 μl of anti-Flag M2 resin in 0.5 ml of binding buffer containing 1 mg/ml BSA at 4°C for 1 h. The resin was washed with 1 ml of binding buffer six times. The bound proteins were eluted with 100 μl of binding buffer containing 0.1 mg/ml Flag peptide and analyzed by immunoblot with either anti-PCSK9 antibody (Cayman Chemical Co.) or anti-GST antibody (Santa Cruz Biotechnology). HepG2 cells (1 × 106) were added into 5 ml polystyrene round-bottom tubes with 3 ml of 0.1% BSA/PBS. Cells were pelleted and resuspended with 100 μl of 0.1% BSA/PBS containing 500 ng of mouse anti-bovine LDLR monoclonal antibody (clone IgG-C7; Progen Biotechnik GmbH) and incubated on ice for 1 h. Three milliliters of 0.1% BSA/PBS was then added, and the cells were pelleted again. This was followed by resuspending the cells with 100 μl of 0.1% BSA/PBS containing 200 ng of Alexa Fluor 488 goat anti-mouse IgG antibody (Molecular Probes). After incubation on ice for 1 h, cells were washed again with 3 ml of 0.1% BSA/PBS. Cells were then resuspended with 600 μl of 0.1% BSA/PBS and analyzed using flow cytometry (FACSCalibur, CellQuest Pro; Becton Dickinson). Mouse IgG2b isotype (catalog No. 02-6300; Invitrogen) was used as the control. As a first step toward understanding how PCSK9 functions to reduce LDLR levels, we generated a HEK293 stable cell line that overexpressed human PCSK9. We engineered a six-HIS tag at the C terminus of the protein for purification purposes. Secreted PCSK9 in the medium was readily purified, and the protein was assessed to be >95% pure as judged by Coomassie blue staining (Fig. 1). N-terminal sequencing revealed the zymogen cleavage of the prosegment at the junction of glutamine and serine, consistent with the report by Benjannet et al. (19.Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.C. Hamelin J. Varret M. Allard D. et al.NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). The prosegment was copurified with the C-terminal domain, as revealed by the 14 kDa band on the denaturing gel (Fig. 1). To address whether secreted PCSK9 reduces cellular LDLR protein levels, we transferred the medium from PCSK9-overexpressing cells to control HEK293 cells expressing endogenous LDLR. As depicted in Fig. 2A, Western blot analysis of cellular LDLR revealed that LDLR protein levels (160 kDa mature form) were reduced dramatically after an overnight incubation, indicating that secreted PCSK9 was functional in degrading LDLR (lane 3). To confirm our hypothesis, purified PCSK9 was added in the medium to control cells at 5 or 10 μg/ml, and a similar level of LDLR reduction was observed (Fig. 2A, lanes 4–6). Detailed titration of PCSK9 suggested an estimated EC50 of ∼2 μg/ml for this effect of purified PCSK9 protein to reduce LDLR protein (Fig. 2B).Fig. 2.Reduction of cellular low density lipoprotein receptor (LDLR) by secreted recombinant PCSK9. A: Secreted PCSK9 is fully active in reducing cellular LDLR levels. Extracts from HEK293 cells that were treated overnight as described below were obtained, and cellular LDLR levels were monitored by Western blot analysis as described in Methods. Cells cultured in LPDS medium overnight were used as a control (lane 2). Lane 1 depicts LDLR protein levels in the HEK293 stable line overexpressing PCSK9, and lane 3 shows LDLR levels from cells exposed to the medium from these PCSK9-overexpressing cells after washing. Purified PCSK9 (10 μg/ml) was added to serum-free (SF) medium (lane 4), or 5 or 10 μg/ml PCSK9 was added to full-serum (FS) medium (lanes 5, 6) for overnight incubation with HEK293 control cells. B: Concentration-dependent reduction of cellular LDLR in HEK293 cells. HEK293 cells were cultured in lipoprotein deficient serum (LPDS) as a control, and various amounts of PCSK9 were added to cells. After overnight incubation, cellular LDLR levels were analyzed by Western blot as described in Methods. C: Time-dependent reduction of cellular LDLR in HEK293 cells. HEK293 cells were incubated with 2 μg/ml purified PCSK9 for the indicated times, and cellular LDLR levels were assessed by Western blot analysis as described. D: PCSK9 reduces cellular LDL uptake. DiI-LDL was added to HEK293 cells after overnight incubation with 10 μg/ml PCSK9 followed by washing, and uptake was analyzed by confocal microscopy as described in Methods. E: Concentration-dependent reduction in cellular LDL uptake. HEK293 cells were incubated with various amounts of purified PCSK9 overnight. DiI-LDL was then added to the medium after washing, and LDL uptake was quantified as described in Methods. *** P < 0.001, ** P < 0.01, * P < 0.05 versus no-PCSK9 control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2.Reduction of cellular low density lipoprotein receptor (LDLR) by secreted recombinant PCSK9. A: Secreted PCSK9 is fully active in reducing cellular LDLR levels. Extracts from HEK293 cells that were treated overnight as described below were obtained, and cellular LDLR levels were monitored by Western blot analysis as described in Methods. Cells cultured in LPDS medium overnight were used as a control (lane 2). Lane 1 depicts LDLR protein levels in the HEK293 stable line overexpressing PCSK9, and lane 3 shows LDLR levels from cells exposed to the medium from these PCSK9-overexpressing cells after washing. Purified PCSK9 (10 μg/ml) was added to serum-free (SF) medium (lane 4), or 5 or 10 μg/ml PCSK9 was added to full-serum (FS) medium (lanes 5, 6) for overnight incubation with HEK293 control cells. B: Concentration-dependent reduction of cellular LDLR in HEK293 cells. HEK293 cells were cultured in lipoprotein deficient serum (LPDS) as a control, and various amounts of PCSK9 were added to cells. After overnight incubation, cellular LDLR levels were analyzed by Western blot as described in Methods. C: Time-dependent reduction of cellular LDLR in HEK293 cells. HEK293 cells were incubated with 2 μg/ml purified PCSK9 for the indicated times, and cellular LDLR levels were assessed by Western blot analysis as described. D: PCSK9 reduces cellular LDL uptake. DiI-LDL was added to HEK293 cells after overnight incubation with 10 μg/ml PCSK9 followed by washing, and uptake was analyzed by confocal microscopy as described in Methods. E: Concentration-dependent reduction in cellular LDL uptake. HEK293 cells were incubated with various amounts of purified PCSK9 overnight. DiI-LDL was then added to the medium after washing, and LDL uptake was quantified as described in Methods. *** P < 0.001, ** P < 0.01, * P < 0.05 versus no-PCSK9 control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2.Reduction of cellular low density lipoprotein receptor (LDLR) by secreted recombinant PCSK9. A: Secreted PCSK9 is fully active in reducing cellular LDLR levels. Ex