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Post-transcriptional Regulation of Low Density Lipoprotein Receptor Protein by Proprotein Convertase Subtilisin/Kexin Type 9a in Mouse Liver

2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês

10.1074/jbc.m410077200

ISSN

1083-351X

Autores

Sahng Wook Park, Young-Ah Moon, Jay D. Horton,

Tópico(s)

Drug Transport and Resistance Mechanisms

Resumo

Lipid homeostasis is transcriptionally regulated by three DNA-binding proteins, designated sterol regulatory element-binding protein (SREBP)-1a, -1c, and -2. Oligonucleotide arrays hybridized with RNA made from livers of transgenic SREBP-1a, transgenic SREBP-2, and SREBP cleavage-activating protein knockout mice recently identified 33 genes regulated by SREBPs in liver, four of which had no known connection to lipid metabolism. One of the four genes was PCSK9, which encodes proprotein convertase subtilisin/kexin type 9a, a protein that belongs to the proteinase K subfamily of subtilases. Mutations in PCSK9 are associated with an autosomal dominant form of hypercholesterolemia. Here, we demonstrate that hepatic overexpression of either wild-type or mutant PCSK9 in mice results in hypercholesterolemia. The hypercholesterolemia is due to a post-transcriptional event causing a reduction in low density lipoprotein (LDL) receptor protein prior to the internalization and recycling of the receptor. Overexpression of PCSK9 in primary hepatocytes and in mice lacking the LDL receptor does not alter apolipoprotein B secretion. These data are consistent with PCSK9 affecting plasma LDL cholesterol levels by altering LDL receptor protein levels via a post-transcriptional mechanism. Lipid homeostasis is transcriptionally regulated by three DNA-binding proteins, designated sterol regulatory element-binding protein (SREBP)-1a, -1c, and -2. Oligonucleotide arrays hybridized with RNA made from livers of transgenic SREBP-1a, transgenic SREBP-2, and SREBP cleavage-activating protein knockout mice recently identified 33 genes regulated by SREBPs in liver, four of which had no known connection to lipid metabolism. One of the four genes was PCSK9, which encodes proprotein convertase subtilisin/kexin type 9a, a protein that belongs to the proteinase K subfamily of subtilases. Mutations in PCSK9 are associated with an autosomal dominant form of hypercholesterolemia. Here, we demonstrate that hepatic overexpression of either wild-type or mutant PCSK9 in mice results in hypercholesterolemia. The hypercholesterolemia is due to a post-transcriptional event causing a reduction in low density lipoprotein (LDL) receptor protein prior to the internalization and recycling of the receptor. Overexpression of PCSK9 in primary hepatocytes and in mice lacking the LDL receptor does not alter apolipoprotein B secretion. These data are consistent with PCSK9 affecting plasma LDL cholesterol levels by altering LDL receptor protein levels via a post-transcriptional mechanism. Plasma LDL 1The abbreviations used are: LDL, low density lipoprotein; ARH, autosomal recessive hypercholesterolemia; apo, apolipoprotein; ER, endoplasmic reticulum; FCS, fetal calf serum; NCLPDS, newborn calf lipoprotein-deficient serum; LDLR, low density lipoprotein receptor; LRP, LDL receptor-related protein; PCSK9, proprotein convertase subtilisin/kexin type 9a; S1P, site 1 protease; S2P, site 2 protease; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; FPLC, fast protein liquid chromatography; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; PFU, plaque-forming units; HSV, herpes simplex virus; RAP, receptor-associated protein.1The abbreviations used are: LDL, low density lipoprotein; ARH, autosomal recessive hypercholesterolemia; apo, apolipoprotein; ER, endoplasmic reticulum; FCS, fetal calf serum; NCLPDS, newborn calf lipoprotein-deficient serum; LDLR, low density lipoprotein receptor; LRP, LDL receptor-related protein; PCSK9, proprotein convertase subtilisin/kexin type 9a; S1P, site 1 protease; S2P, site 2 protease; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; FPLC, fast protein liquid chromatography; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; PFU, plaque-forming units; HSV, herpes simplex virus; RAP, receptor-associated protein. cholesterol concentrations are determined by the relative rates of VLDL and LDL production by the liver and the rate of LDL uptake via hepatic LDL receptors (LDLRs) (1Goldstein J.L. Brown M.S. Annu. Rev. Biochem. 1977; 46: 897-930Crossref PubMed Scopus (1598) Google Scholar, 2Dietschy J.M. Turley S.D. Spady D.K. J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar). VLDL secretion from hepatocytes is positively correlated with rates of hepatic lipid synthesis (3Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3685) Google Scholar). Genes required for cholesterol and triglyceride biosynthesis and, thus, VLDL production are regulated by three sterol regulatory element-binding proteins (SREBPs), SREBP-1a, SREBP-1c, and SREBP-2 (4Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 5Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1055) Google Scholar). SREBPs also are the principal transcriptional regulators of the LDL receptor gene, which clears apoB-containing lipoproteins, such as VLDL and LDL, from the plasma (5Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1055) Google Scholar).To identify genes regulated by SREBPs, we used oligonucleotide arrays hybridized with RNA from livers of mice that overexpressed SREBPs (transgenic for SREBP-1a or transgenic for SREBP-2) and that lacked all SREBPs as a result of deleting SCAP, an escort protein required for SREBP activation (5Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1055) Google Scholar). With this physiologic filter, 33 genes were identified that were increased in the transgenic livers and decreased in the SCAP-deficient livers. Four of these 33 genes had no known function. One of these four genes was Pcsk9, which encodes the proprotein convertase subtilisin/kexin type 9a, also designated NARC-1 (neural apoptosis-regulated convertase 1). Seidah et al. (6Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 928-933Crossref PubMed Scopus (896) Google Scholar) showed that PCSK9 belongs to the proteinase K subfamily of subtilases. PCSK9 is synthesized first as a soluble zymogen that undergoes autocatalytic intramolecular processing in the ER to produce a prosegment that remains associated with the secreted enzyme.A link between PCSK9 and cholesterol metabolism was established by Abifadel et al. (7Abifadel M. Varret M. Rabès J.-P. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. 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. Boileau C. Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2134) Google Scholar), who showed that two missense mutations in PCSK9 were associated with an autosomal dominant form of hypercholesterolemia. The first mutation results in the substitution of an arginine for serine (S127R) in a conserved residue located between the primary and secondary zymogen processing sites. The second missense mutation (F216L), also in a conserved amino acid, is in the catalytic domain of the enzyme. Subsequently, two additional missense mutations in the catalytic domain of PCSK9 (D374Y and N157K) were shown to segregate in families with elevated plasma LDL cholesterol concentrations (8Leren T.P. Clin. Genet. 2004; 65: 419-422Crossref PubMed Scopus (204) Google Scholar, 9Timms 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. Hum. Genet. 2004; 114: 349-353Crossref PubMed Scopus (265) Google Scholar).The clinical phenotype of subjects with these missense mutations in PCSK9 is indistinguishable from two other autosomal dominant forms of hypercholesterolemia, familial hypercholesterolemia, which is caused by mutations in the LDLR and familial defective apoB, due to mutations that interfere with LDL binding to the LDLR and clearance from the plasma (10Rader D.J. Cohen J. Hobbs H.H. J. Clin. Invest. 2003; 111: 1795-1803Crossref PubMed Scopus (467) Google Scholar). We hypothesized that mutations in PCSK9 cause hypercholesterolemia by altering SREBP expression, apoB synthesis/secretion, and/or LDLR expression. To distinguish between these mechanisms, a series of in vitro and in vivo studies with wild-type and mutant PCSK9 was performed.EXPERIMENTAL PROCEDURESGeneral Methods and Supplies—DNA manipulations were performed using standard molecular biology techniques (11Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, New York2001Google Scholar). The concentrations of cholesterol and triglycerides in plasma were measured as described previously (12Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar). Plasma lipoprotein fractions were separated by fast performance liquid chromatography (FPLC) gel filtration using a Superose 6 column, and cholesterol concentrations were measured as described (13Yokode M. Hammer R.E. Ishibashi S. Brown M.S. Goldstein J.L. Science. 1990; 250: 1273-1275Crossref PubMed Scopus (143) Google Scholar, 14Horton J.D. Shimano H. Hamilton R.L. Brown M.S. Goldstein J.L. J. Clin. Invest. 1999; 103: 1067-1076Crossref PubMed Scopus (158) Google Scholar). Protein concentrations were determined using a BCA kit (Pierce). Newborn calf lipoprotein-deficient serum (NCLPDS) (d > 1.215 mg/ml) was prepared as described (15Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). Other reagents otherwise not specified were obtained from Sigma.Expression plasmids pTK-HSV-BP1 and pTK-HSV-BP2, encoding wild-type HSV-tagged full-length human SREBP-1 or SREBP-2 (16Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and pCMV-S1P-Myc, encoding hamster S1P (17Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), were constructed as described in the indicated references.Antibodies and Immunoblot Analyses—The following monoclonal antibodies were used in the current studies: anti-HSV (IgG1) from EMD Biosciences (Novagen Brand, Madison, WI), anti-Myc (clone 9E10) from Roche Applied Science, anti-FLAG (M2) from Sigma, anti-human transferrin receptor from Zymed Laboratories (South San Francisco, CA), polyclonal anti-human cAMP-responsive element-binding protein from Cell Signaling Technology, Inc. (Beverly, MA), horseradish peroxidase-conjugated donkey anti-mouse IgG from Jackson ImmunoResearch Laboratories (West Grove, PA), and horseradish peroxidase-conjugated donkey anti-rabbit IgG (affinity-purified) from Amersham Biosciences. Polyclonal antibodies against the mouse LDLR, LDL receptor-related protein (LRP), receptor-associated protein (RAP), ARH, SREBP-1, and SREBP-2 were described previously (12Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar, 18Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 19Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (736) Google Scholar, 20Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar, 21Jones C. Hammer R.E. Li W.-P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (851) Google Scholar, 23Shimano H. Shimomura I. Hammer R.E. Goldstein J.L. Brown M.S. Horton J.D. J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (352) Google Scholar). Immunoblot analyses were performed using the SuperSignal West Pico Chemiluminescent Substrate System from Pierce.Construction of Wild-type and Mutant PCSK9 Expression Vectors—An expression vector that encodes amino acids 1–692 of human PCSK9 followed by a FLAG epitope tag (DYKDDDDK) under the control of the CMV promoter-enhancer (pCMV-PCSK9-FLAG) was constructed as follows. The human PCSK9 cDNA was amplified using nested PCR. First-strand cDNA was synthesized from total RNA prepared from cultured HepG2 cells (ATCC number HB-8065) using methods described previously (24Moon Y.-A. Horton J.D. J. Biol. Chem. 2003; 278: 7335-7343Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The following primers were then used for the amplification of the PCSK9 cDNA: 5′ primer (5′-GCAACCTCTCCCCTGGCCCTCATG-3′) and 3′ primer (5′-GCTTCCTGGCACCTCCACCTGGGG-3′) for the primary amplification and 5′ primer (5′-CCAGTGTGCTGGAATTCGCCACCATGGGCACCGTCAGCTCCAGG-3′) and 3′ primer (5′-CCCCTCGAGTCACTCACTTGTCATCGTCGTCCTTGTAGTCCTGGAG CTCCTGGGAGGCCTGCGCCAG-3′) for the secondary amplification. The 5′ primer sequence for the secondary amplification contained an EcoRI restriction site, a Kozak sequence, and the sequence encoding amino acids 1–7 of human PCSK9. The sequence of 3′ primer used for the secondary amplification consisted of nucleotides that encode amino acids 684–692 of human PCSK9, a FLAG epitope tag, two copies of a stop codon with an intervening G nucleotide, and an XhoI restriction site. The PCR-amplified cDNA for human PCSK9 was digested with EcoRI and XhoI prior to ligation into pcDNA3 (Invitrogen).To generate plasmids expressing mutant forms of human PCSK9, the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to change the nucleotide sequences of pCMV-PCSK9-FLAG using the following oligonucleotides: 5′-CTTCCTGGTGAAGATGAGAGGCGACCTGCTGGAGCTG-3′ for the S127R plasmid (pCMV-S127R), 5′-GAGGAGGACGGGACCCGCCTCCACAGACAGGCCAGCA-3′ for the F216L plasmid (pCMV-F216L), and 5′-ACAGAGTGGGACAGCCCAGGCTGCTGCCC-3′ for the S386A plasmid (pCMV-S386A). The integrity of expression plasmid sequences was confirmed by DNA sequencing.Cultured Cell Experiments—Cells were cultured in one of the following media. Medium A contained a 1:1 mixture (v/v) of Ham's F-12 medium and Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate. Medium B contained medium A supplemented with 5% (v/v) fetal calf serum (FCS), 5 μg/ml cholesterol, 1 mm sodium mevalonate, and 20 μm sodium oleate. Medium C contained medium A supplemented with 5% (v/v) NCLPDS, 50 μm sodium compactin, and 50 μm sodium mevalonate.SRD-12B cells that harbor a genetic deletion of S1P (25Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) were set up at 5 × 105 cells/60-mm dish in medium B on day 0. On day 1, cells were transfected with the indicated plasmids (2 μg of DNA/dish) using 6 μlof Fugene 6 (Roche Applied Science) as described (26Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). On day 2, cells were washed twice with phosphate-buffered saline and cultured for 1 h in medium C containing 1% (w/v) hydroxypropyl-β-cyclodextrin. After incubation at 37 °C for 1 h, cells were washed twice with phosphate-buffered saline and cultured in medium C in the absence or presence of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol added in a final concentration of 0.2% ethanol. After a 4-h incubation, N-acetyl-leucinal-leucinal-norleucinal (25 μg/ml) was added for 1 h, after which the cells were harvested and processed as described (27DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar).HepG2 cells (ATCC number HB-8065) were set up at 5 × 105 cells/60-mm dish in DMEM containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate (medium D) supplemented with 10% FCS on day 0. On day 1, cells were infected with the indicated adenovirus described below in 1 ml of medium D. After a 2-h incubation, 1 ml of medium D supplemented with 20% NCLPDS was added and incubated overnight at 37 °C. After an overnight incubation, the cells were harvested and processed as described above.Stable Transfection of Chinese Hamster Ovarian (CHO-K1) Cells— On day 0, CHO-K1 cells (ATCC number CCL-61) were plated at a density of 5 × 104 cells/100-mm dish in medium A supplemented with 5% (v/v) FCS. On day 1, cells were transfected with 5 μg of pCMV-PCSK9-FLAG, pCMV-S127R, or pCMV-F216L per dish using the Fugene 6 reagent in a final volume of 0.2 ml. To generate control stable cell lines, 1 μg of pcDNA3 was used to transfect CHO-K1 cells. On day 2, the medium was changed to medium A containing 700 μg/ml G418 supplemented with 5% FCS. The medium was changed daily for 12 days until individual colonies were visible. Single-cell clones that stably expressed wild-type or mutant PCSK9 protein were isolated by limiting dilution and screened for PCSK9 expression by immunoblotting using the anti-FLAG monoclonal antibody. Cell lines expressing equivalent levels of wild-type and mutant forms of PCSK9 were selected for further studies and maintained in medium A containing 500 μg of G418 supplemented with 5% FCS.Construction of Adenoviral Vectors Expressing Wild-type or Mutant PCSK9 Proteins—Adenoviruses that express wild-type, S127R, F216L, or S386A forms of human PCSK9 were constructed using AdEasy system (Qbiogene, Carlsbad, CA) according to the manufacturer's protocol. HindIII-XbaI fragments of pCMV-PCSK91-FLAG, pCMV-S127R, pCMV-F216L, and pCMV-S386A were ligated to a HindIII-XbaI-digested pShuttle-CMV vector. The resulting pShuttle constructs were co-transformed with the pAdEasy-1 vector into BJ5183 cells to produce recombinant adenoviral genomic constructs for wild-type and mutant PCSK9 proteins. The recombinant adenoviral genomic constructs were linearized with PacI and transfected into QBI-293A cells (Qbiogene) cultured in DMEM supplemented with 5% FCS. Cells were overlaid with 1.25% agarose/DMEM 20 h after transfection and further cultured for 14 days until discrete plaques were identified. The resulting viral plaques were assayed for PCSK9 expression by immunoblot analysis using anti-FLAG antibody (Sigma).Viruses expressing wild-type or mutant human PCSK9 proteins were subjected to four rounds of amplification before purification by CsCl-ultracentrifugation (28Green M. Wold W.S. Methods Enzymol. 1979; 58: 425-435Crossref PubMed Scopus (70) Google Scholar). All viruses were dialyzed against 10 mm Tris, pH 8.0, 2 mm MgCl2, 4% sucrose buffer (29Nyberg-Hoffman C. Aguilar-Cordova E. Nat. Med. 1999; 5: 955-957Crossref PubMed Scopus (67) Google Scholar) and stored at -80 °C. Virus titers were determined using a plaque-forming unit (PFU) assay in QBI-293A cells (28Green M. Wold W.S. Methods Enzymol. 1979; 58: 425-435Crossref PubMed Scopus (70) Google Scholar). For administration to mice, the indicated amounts of each recombinant adenovirus were injected as a single dose into tail veins of nonfasted mice. Three days after injection, mice were killed, and plasma and livers were harvested. Membrane fraction and nuclear extract from liver were prepared as described (30Engelking L.J. Kuriyama H. Hammer R.E. Horton J.D. Brown M.S. Goldstein J.L. Liang G. J. Clin. Invest. 2004; 113: 1168-1175Crossref PubMed Scopus (217) Google Scholar).Real Time Reverse Transcription-PCR—Total RNA was prepared using an RNA STAT-60 kit (Tel-Test, Friendswood, TX). DNase I treatment of total RNA was performed using a DNA-free kit (Ambion, Austin, TX). cDNA was synthesized from 2 μg of DNase-treated total RNA using a TaqMan reverse transcription reagent kit and random hexamer primers (Applied Biosystems, Foster City, CA). Specific primers for each gene were designed using PRIMER EXPRESS software (Applied Biosystems) and previously published (31Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar). The real time reverse transcription-PCR contained 20 ng of reverse-transcribed total RNA, a 167 nm concentration of the forward and reverse primers, and 10 μlof2× SYBR Green PCR Master Mix in a final volume of 20 μl. The PCRs were carried out using the Applied Biosystems Prism 7700 Sequence Detection System. All reactions were done in triplicate, and the relative amounts of all mRNAs were calculated by using the comparative CT method (46, Applied Biosystems (2001) User Bulletin 2, Foster City, CAGoogle Scholar). Cyclophilin mRNA was used as the invariant control.Mice and Diets—Studies using wild-type mice were performed in 10–12-week-old male C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). Mice with the genetic deletion of ARH (Arh-/-) (21Jones C. Hammer R.E. Li W.-P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) were generously provided by Drs. Joachim Herz and Helen Hobbs (University of Texas Southwestern Medical Center). Knockout mice that lack the LDLR were previously described (32Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1263) Google Scholar). All mice were maintained on 12-h dark/12-h light cycles and fed a chow diet that contained 4% (w/w) animal fat and <0.04% (w/w) cholesterol (Teklad 4% mouse/rat diet 7001 from Harlan Teklad Premier Laboratory Diets, Madison, WI).ApoB Synthesis and Secretion in Mouse Primary Hepatocytes— C57BL/6J male mice (8–10 weeks of age) were injected with the indicated adenovirus, and 3 days later primary hepatocytes were isolated as described previously (33Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar, 34Yang J. Goldstein J.L. Hammer R.E. Moon Y.-A. Brown M.S. Horton J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13607-13612Crossref PubMed Scopus (188) Google Scholar). The hepatocytes were allowed to attach on type I collagen-coated six-well plates for 2 h in Met/Cys-free DMEM (Sigma) supplemented with 5% NCLPDS, 4 mm l-glutamine, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate (medium E). The cells were then incubated with 200 μCi of [35S]Met/Cys (Easy Tag EXPRESS-[35S] PROTEIN LABELING MIX; PerkinElmer Life Sciences) in 1 ml of medium E for 2 h.After incubation, the medium was collected, and protease inhibitors (1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 5 mm EDTA, 10 mm HEPES, pH 8.0) were added. The cells were harvested and lysed in a buffer containing 0.15 m NaCl, 5 mm EDTA, 50 mm Tris-Cl, pH 7.4, 62.5 mm sucrose, 0.5% Triton X-100, 0.5% sodium deoxycholate, 10 μg/ml leupeptin, 5 μg/ml pepstatin A, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 25 μg/ml ALLN, and 1 mm benzamidine.To immunoprecipitate apoB, 500 μg of cell extract or 0.5 ml of medium was incubated with 10 μg/ml of purified rabbit anti-mouse apoB antibody (provided by Dr. Helen Hobbs) in 0.5× NET (0.15 m NaCl, 5 mm EDTA, 50 mm Tris-Cl, pH 7.4, 0.5% Triton X-100, 0.1% SDS). To immunoprecipitate albumin, 50 μg of cell extract protein or 50 μl of medium was incubated with 10 μg/ml of rabbit anti-mouse albumin antibody (Biodesign International, Saco, ME). After a 6-h incubation with the antibodies, 30 μl of protein A/G PLUS-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated at 4 °C for 16 h. The protein-antibody-protein A/G-agarose complexes were washed five times with 500 μl of NET. The immunoprecipitated proteins were eluted in the loading buffer (0.125 m Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) by boiling for 5 min and then separated on 4% SDS-PAGE gels (for apoB) or 8% gels (for albumin). The gels were dried and exposed to a PhosphorImager plate. The resulting signals were quantified using a PhosphorImager® Molecular Dynamics Storm 820 system (Amersham Biosciences). ApoB values were corrected for the amounts of labeled albumin synthesized and secreted from the hepatocytes. Values are reported in arbitrary units of relative counts for apoB/albumin.RESULTSTo study the mechanism by which mutations in PCSK9 cause elevated plasma LDL cholesterol concentrations, we first determined whether wild-type or mutant PSCK9 could proteolytically activate SREBPs in an aberrant fashion. SREBPs are synthesized as inactive precursors in the endoplasmic reticulum (ER). To be active, the NH2-terminal segment of SREBP must be released from the membrane to enter the nucleus (4Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar). SREBP activation requires SCAP, an escort protein that functions as a sterol sensor and transports SREBPs from the ER to the Golgi apparatus, and two proteases, designated site-1 protease (S1P) and site-2 protease (S2P), located in the Golgi (35Goldstein J.L. Rawson R.B. Brown M.S. Arch. Biochem. Biophys. 2002; 397: 139-148Crossref PubMed Scopus (193) Google Scholar). When ER membranes become depleted of cholesterol, SCAP escorts SREBP to the Golgi, where it undergoes two sequential proteolytic cleavage events mediated by S1P, a membrane-bound subtilase-like serine proteinase, and S2P, a membrane-bound zinc metalloproteinase. S1P belongs to the same family of subtilase-like serine proteinase as does PCSK9; therefore, mutations in PCSK9 could result in a gain of function that results in unregulated or aberrant cleavage of SREBPs, which in turn would increase lipid biosynthesis and VLDL production.To test this hypothesis, SRD-12B cells that harbor a genetic deletion of S1P and are thus incapable of cleaving SREBPs were transiently transfected with full-length SREBP-1a (Fig. 1A) or SREBP-2 (Fig. 1B) and either an empty vector, human S1P, wild-type PSCK9, or mutant PCSK9 (S127R). Cells were cultured either in the presence of sterols, conditions that normally suppress SREBP cleavage, or in the absence of sterols, conditions that induce SREBP cleavage. Cellular membranes and nuclear proteins were isolated, aliquots were separated by SDS-PAGE, and immunoblot analyses were performed to determine whether wild-type or mutant PCSK9 proteolytically cleaved the membrane-bound SREBP-1a or SREBP-2 precursor proteins.As shown in Fig. 1, S1P restores normal sterol-regulated cleavage of SREBP-1a (A) and SREBP-2 (B). The transfection of wild-type PCSK9 results in equal expression of the proprotein (P) and cleaved (C) PCSK9 (compare P and C in Fig. 1, A and B, lanes 6 and 7). Transfection of the S127R mutant PCSK9 resulted in significantly less cleaved PCSK9. However, neither wild-type PCSK9 nor mutant PCSK9 restored cleavage of SREBP-1a (Fig. 1A) or SREBP-2 (Fig. 1B) in the presence or absence of sterols. These results demonstrate that mutations in PCSK9 do not increase plasma LDL cholesterol levels by bypassing the role of S1P in processing SREBPs.To determine whether wild-type and mutant PCSK9 proteins alter LDLR expression or function, wild-type CHO-K1 cell lines were stably transfected with CMV-driven wild-type PCSK9 or mutant PCSK9 cDNAs encoding either the S127R mutation or the F216L mutation. The PCSK9 proteins contained a FLAG epitope tag at the COOH terminus. Three clones with equivalent levels of PCSK9 expression were identified, and the expression and function of the LDLR were assessed. As shown in Fig. 2A, the amount of wild-type and mutant PCSK9 proteins expressed was similar in the three cell lines, although the S127R mutation resulted in a reduction in the relative proportion of cleaved PCSK9 (A, lower band). The amounts of secreted wild-type and F216L mutant were also equivalent, whereas the amount of secreted S127R appeared to be slightly lower in amount. The LDLR protein immunoblots showed two bands. The lower band represents the precursor form that is present in the ER. The upper band represents the mature receptor that has undergone O-linked glycosylation in the Golgi (36Goldstein J.L. Brown M.S. Anderson R.G. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar). The amount of mature LDLR protein was unaffected by wild-type or mutant PCSK9 overexpression. The slight reduction in the amount of the precursor form of the LDLR observed in the transfected cells was not a consistent finding. In addition, assays of LDLR function that measured LDL binding and uptake were also not consistently different among the four immortalized hamster ovarian cell lines (data not shown).Fig. 2LDLR expression in CHO-K1 and HepG2 cells transfected with wild-type or mutant forms of PCSK9.A, CHO-K1 cells stably transfected with pcDNA3 (CHO-K1), pCMV-PCSK9 (WT-PCSK9), pCMV-S127R (S127R), or pCMV-F216L (F216L) were grown in monolayer cultures as described under “Experimental Procedures.” For immunoblot analysis, 30 μg of cell lysate protein was subjected to 8% SDS-PAGE, transf

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