Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease. Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease. High levels of circulating low-density lipoprotein (LDL) 3The abbreviations used are: LDLlow density lipoproteinARHautosomal recessive hypercholesterolemiaCLCclathrin light chaindiI-LDLLDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorateDMEMDulbecco's modified Eagle's mediumEGF-Aepidermal growth factor domain AEGF-ABepidermal growth factor domain ABEGFPenhanced green fluorescent proteinELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sortingFBSfetal bovine serumKDknockdownKOknock-outLDLRlow density lipoprotein receptorLE/Llate endosomes/lyososomesLPDSlipoprotein-deficient serummAbmonoclonal antibodyPBSphosphate-buffered salinePCSK9proprotein convertases subtilisin kexin 9shRNAsmall hairpin RNAsiRNAsmall interfering RNATfRtransferrin receptorWTwild type. 3The abbreviations used are: LDLlow density lipoproteinARHautosomal recessive hypercholesterolemiaCLCclathrin light chaindiI-LDLLDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorateDMEMDulbecco's modified Eagle's mediumEGF-Aepidermal growth factor domain AEGF-ABepidermal growth factor domain ABEGFPenhanced green fluorescent proteinELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sortingFBSfetal bovine serumKDknockdownKOknock-outLDLRlow density lipoprotein receptorLE/Llate endosomes/lyososomesLPDSlipoprotein-deficient serummAbmonoclonal antibodyPBSphosphate-buffered salinePCSK9proprotein convertases subtilisin kexin 9shRNAsmall hairpin RNAsiRNAsmall interfering RNATfRtransferrin receptorWTwild type.-cholesterol represent a major risk factor that leads to coronary heart disease, the main cause of death and morbidity worldwide (1Lloyd-Jones D. Adams R. Carnethon M. De Simone G. Ferguson T.B. Flegal K. Ford E. Furie K. Go A. Greenlund K. Haase N. Hailpern S. Ho M. Howard V. Kissela B. Kittner S. Lackland D. Lisabeth L. Marelli A. McDermott M. Meigs J. Mozaffarian D. Nichol G. O'Donnell C. Roger V. Rosamond W. Sacco R. Sorlie P. Stafford R. Steinberger J. Thom T. Wasserthiel-Smoller S. Wong N. Wylie-Rosett J. Hong Y. Circulation. 2009; 119: e21-e181Crossref PubMed Scopus (0) Google Scholar). LDL particles are cleared mainly from the bloodstream by the hepatic cell surface LDL receptor (LDLR) (2Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4307) Google Scholar). Genetics studies demonstrated that loss-of-function mutations in either LDLR or apolipoprotein B, the protein component of LDL that binds LDLR, result in familial hypercholesterolemia and premature coronary heart disease (3Varret M. Abifadel M. Rabès J.P. Boileau C. Clin. Genet. 2008; 73: 1-13Crossref PubMed Scopus (148) Google Scholar). More recently, the proprotein convertases subtilisin kexin 9 (PCSK9) gene (4Seidah N.G. Prat A. J. Mol. Med. 2007; 85: 685-696Crossref PubMed Scopus (133) Google Scholar), which is highly expressed in liver and small intestine (5Seidah 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), was identified as the third locus associated with familial hypercholesterolemia (6Abifadel 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. Boileau C. Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2134) Google Scholar). It is now clear that PCSK9 binds the LDLR and triggers its intracellular degradation in acidic compartments, resulting in increased circulating plasma cholesterol (7Benjannet 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. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 8Maxwell K.N. Fisher E.A. Breslow J.L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2069-2074Crossref PubMed Scopus (301) Google Scholar, 9Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 10Nassoury N. Blasiole D.A. Tebon O.A. Benjannet S. Hamelin J. Poupon V. McPherson P.S. Attie A.D. Prat A. Seidah N.G. Traffic. 2007; 8: 718-732Crossref PubMed Scopus (199) Google Scholar).After its autocatalytic cleavage, PCSK9 is secreted as a stable noncovalent complex with its prosegment (pro·PCSK9) (5Seidah 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, 7Benjannet 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. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). This cleavage results in a conformational change (11Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. Mansour M.N. McGrath K.M. Seddon A.P. Shenolikar S. Stutzman-Engwall K.J. Warren L.C. Xia D. Qiu X. Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (350) Google Scholar) that favors the binding of PCSK9 to the epidermal growth factor A domain (EGF-A) of the LDLR (12Kwon H.J. Lagace T.A. McNutt M.C. Horton J.D. Deisenhofer J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1820-1825Crossref PubMed Scopus (303) Google Scholar), with increased affinity at acidic pH values (11Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. Mansour M.N. McGrath K.M. Seddon A.P. Shenolikar S. Stutzman-Engwall K.J. Warren L.C. Xia D. Qiu X. Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (350) Google Scholar). Although the C-terminal Cys-His-rich domain of PCSK9 is a spatially separate domain (11Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. Mansour M.N. McGrath K.M. Seddon A.P. Shenolikar S. Stutzman-Engwall K.J. Warren L.C. Xia D. Qiu X. Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (350) Google Scholar) that does not participate directly in the PCSK9-EGF-A interaction (12Kwon H.J. Lagace T.A. McNutt M.C. Horton J.D. Deisenhofer J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1820-1825Crossref PubMed Scopus (303) Google Scholar), it is a critical determinant for the PCSK9-enhanced cellular degradation of the LDLR (13Zhang D.W. Garuti R. Tang W.J. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13045-13050Crossref PubMed Scopus (177) Google Scholar). In agreement, we recently demonstrated that annexin A2, which binds the Cys-His-rich domain of PCSK9, blocks its effect on LDLR degradation (14Mayer G. Poirier S. Seidah N.G. J. Biol. Chem. 2008; 283: 31791-31801Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).Overexpression studies in liver suggested that both intra- and extracellular PCSK9 target the LDLR (9Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 15Maxwell K.N. Breslow J.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7100-7105Crossref PubMed Scopus (497) Google Scholar, 16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar) toward degradation in late endosomes/lysosomes (LE/L) (7Benjannet 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. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 8Maxwell K.N. Fisher E.A. Breslow J.L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2069-2074Crossref PubMed Scopus (301) Google Scholar, 9Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 10Nassoury N. Blasiole D.A. Tebon O.A. Benjannet S. Hamelin J. Poupon V. McPherson P.S. Attie A.D. Prat A. Seidah N.G. Traffic. 2007; 8: 718-732Crossref PubMed Scopus (199) Google Scholar). It was shown that the adaptor protein ARH, which interacts with the cytosolic tail of the LDLR, is essential for the endocytosis and degradation of the cell surface PCSK9·LDLR complex in vivo (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar). However, hepatic LDLR protein levels were also reduced upon overexpression of PCSK9 in Arh −/− mice (9Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar), suggesting the presence of an ARH-independent intracellular pathway. Intriguingly, at endogenous levels of PCSK9, the absence of ARH did not affect hepatic LDLR subcellular localization in LE/L or protein levels (17Jones 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). This is not the expected result if PCSK9 mostly targets LDLR by the extracellular pathway (18McNutt M.C. Kwon H.J. Chen C. Chen J.R. Horton J.D. Lagace T.A. J. Biol. Chem. 2009; 284: 10561-10570Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), as one would have expected that in Arh−/− mice total LDLR levels should have been more elevated.In this study, we focused on the relative contribution of the intra- versus extracellular pathways of endogenous PCSK9-induced LDLR degradation. This information should guide the choice of therapeutic approaches that will best target the site of PCSK9-LDLR interaction to control hypercholesterolemia and coronary heart disease.DISCUSSIONThe cell surface internalization of PCSK9 depends primarily on the presence of LDLR and the adaptor protein ARH that binds the cytosolic tail of the LDLR (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar, 32Cameron J. Holla Ø.L. Ranheim T. Kulseth M.A. Berge K.E. Leren T.P. Hum. Mol. Genet. 2006; 15: 1551-1558Crossref PubMed Scopus (222) Google Scholar, 33Qian Y.W. Schmidt R.J. Zhang Y. Chu S. Lin A. Wang H. Wang X. Beyer T.P. Bensch W.R. Li W. Ehsani M.E. Lu D. Konrad R.J. Eacho P.I. Moller D.E. Karathanasis S.K. Cao G. J. Lipid Res. 2007; 48: 1488-1498Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In addition, we showed that LDLR and PCSK9 interact early in the secretory pathway (10Nassoury N. Blasiole D.A. Tebon O.A. Benjannet S. Hamelin J. Poupon V. McPherson P.S. Attie A.D. Prat A. Seidah N.G. Traffic. 2007; 8: 718-732Crossref PubMed Scopus (199) Google Scholar) and questioned whether some PCSK9·LDLR complex or PCSK9 alone can be targeted directly from the trans-Golgi network to LE/L, without cycling through the cell surface. Aside from the extracellular pathway, the existence of an intracellular one is supported by the ability of PCSK9 to degrade the LDLR in vivo in the absence of ARH (9Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) and on the capability of PCSK9-Lamp1 chimeras, which directly traffic to LE/L, to enhance the degradation of the LDLR, VLDLR and apo-ER2 efficiently (26Poirier S. Mayer G. Benjannet S. Bergeron E. Marcinkiewicz J. Nassoury N. Mayer H. Nimpf J. Prat A. Seidah N.G. J. Biol. Chem. 2008; 283: 2363-2372Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). In liver, ARH seems to be critical for the endocytosis of the extracellular PCSK9·LDLR complex (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar) but does not seem to play an important role in regulating total levels of endogenous LDLR (Fig. 1d) (17Jones 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, 34Harada-Shiba M. Takagi A. Marutsuka K. Moriguchi S. Yagyu H. Ishibashi S. Asada Y. Yokoyama S. Circ. Res. 2004; 95: 945-952Crossref PubMed Scopus (22) Google Scholar).Endogenous PCSK9 secreted from primary hepatocytes of WT mice has no effect on those derived from Pcsk9−/− mice, whereas incubation with 100 nm PCSK9 clearly decreased LDLR levels (Fig. 3). Thus, after 24-h incubations, only high, nonphysiological concentrations of WT PCSK9 can reduce LDLR levels in primary hepatocytes (Fig. 3) and in HepG2 cells (Figs. 1 and 5). Interestingly, we observed that in the plasma of Ldlr−/− mice the levels of circulating PCSK9 and its Furin-cleaved form (20Zaid A. Roubtsova A. Essalmani R. Marcinkiewicz J. Chamberland A. Hamelin J. Tremblay M. Jacques H. Jin W. Davignon J. Seidah N.G. Prat A. Hepatology. 2008; 48: 646-654Crossref PubMed Scopus (310) Google Scholar) are at least 10-fold higher than in WT mice (supplemental Fig. S1b), and yet it was reported that sharing the blood circulation (parabiosis) of Ldlr−/− mice with that of WT mice did not affect the levels of liver LDLR in the latter nor plasma LDL-cholesterol (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar). Accordingly, the physiological role of circulating WT PCSK9 and its Furin-truncated form remains undefined. Although hepatocyte-specific transgenic mice weakly overexpressing PCSK9 (3-fold) exhibit no significant change in circulating LDL-cholesterol, a ∼30-fold overexpression increased it by ∼5-fold (20Zaid A. Roubtsova A. Essalmani R. Marcinkiewicz J. Chamberland A. Hamelin J. Tremblay M. Jacques H. Jin W. Davignon J. Seidah N.G. Prat A. Hepatology. 2008; 48: 646-654Crossref PubMed Scopus (310) Google Scholar). Data from transgenic mice expressing very high levels of human PCSK9 in mouse kidney (35Luo Y. Warren L. Xia D. Jensen H. Sand T. Petras S. Qin W. Miller K.S. Hawkins J. J. Lipid Res. 2009; 50: 1581-1588Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) or liver (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar), or continuous infusions of recipient WT mice with recombinant PCSK9 (36Grefhorst A. McNutt M.C. Lagace T.A. Horton J.D. J. Lipid Res. 2008; 49: 1303-1311Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) indicated that >100 nm amounts of circulating PCSK9 are required to affect liver LDLR protein levels significantly, without affecting extrahepatic LDLR. In that context, we recently showed that annexin A2 inhibits the effect of extracellular PCSK9 on LDLR, and in view of its absence from hepatocytes in vivo, it may exert its inhibitory effect in extrahepatic tissues (14Mayer G. Poirier S. Seidah N.G. J. Biol. Chem. 2008; 283: 31791-31801Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The available compiled data suggest that only supraphysiological levels of circulating PCSK9 have an impact on liver LDLR. Several ELISAs evaluated the level of circulating human PCSK9 to be ∼2–4 nm (16Lagace 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. Horton J.D. J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (512) Google Scholar, 29Dubuc G. Tremblay M. Pare G. Jacques H. Hamelin J. Benjannet S. Boulet L. Genest J. Bernier L. Seidah N.G. Davignon J. J. Lipid Res. 2009; (in press)Google Scholar, 37Alborn W.E. Cao G. Careskey H.E. Qian Y.W. Subramaniam D.R. Davies J. Conner E.M. Konrad R.J. Clin. Chem. 2007; 53: 1814-1819Crossref PubMed Scopus (133) Google Scholar). However, because the local concentration of PCSK9 secreted from hepatocytes into the extracellular microenvironment is expected to be much higher than in plasma, PCSK9 may well have an autocrine/paracrine effect in vivo.Our data revealed that a 4-h incubation of HepG2 cells with an EGF-AB peptide readily inhibits the LDLR-degrading activity of exogenously added PCSK9 but had little effect on endogenous secreted PCSK9 (Fig. 2a). Interestingly, it took at least 24 h to detect an effect of EGF-AB on LDLR levels and activity (Fig. 2, b and c). Thus, it is possible that the extracellular PCSK9 effect is a relatively slow process and may require a long incubation time and/or a critical threshold concentration. This may be related to the lower binding affinity of PCSK9 to LDLR at neutral versus acidic pH values (11Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. Mansour M.N. McGrath K.M. Seddon A.P. Shenolikar S. Stutzman-Engwall K.J. Warren L.C. Xia D. Qiu X. Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (350) Google Scholar).A very recent report revealed that a monoclonal antibody (mAb) that neutralizes the interaction of PCSK9 and LDLR is able to reduce the extracellular activity of PCSK9 both in naïve HepG2 cells and in vivo in mouse and cynomolgus monkey (38Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. Di Y. Shetterly S. Arimura Z. Salomonis H. Romanow W.G. Thibault S.T. Zhang R. Cao P. Yang X.P. Yu T. Lu M. Retter M.W. Kwon G. Henne K. Pan O. Tsai M.M. Fuchslocher B. Yang E. Zhou L. Lee K.J. Daris M. Sheng J. Wang Y. Shen W.D. Yeh W.C. Emery M. Walker N.P. Shan B. Schwarz M. Jackson S.M. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9820-9825Crossref PubMed Scopus (326) Google Scholar). Similar to our data in HepG2 cells using EGF-AB as a neutralizing agent (Fig. 2), the mAb effect was only reported for times ≥24-h incubation. The latter seems to be due to a conformational change as the mAb does not completely mask the PCSK9-LDLR interaction surface (38Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. Di Y. Shetterly S. Arimura Z. Salomonis H. Romanow W.G. Thibault S.T. Zhang R. Cao P. Yang X.P. Yu T. Lu M. Retter M.W. Kwon G. Henne K. Pan O. Tsai M.M. Fuchslocher B. Yang E. Zhou L. Lee K.J. Daris M. Sheng J. Wang Y. Shen W.D. Yeh W.C. Emery M. Walker N.P. Shan B. Schwarz M. Jackson S.M. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9820-9825Crossref PubMed Scopus (326) Google Scholar). Because no evidence was provided for antibody-antigen cellular internalization, we presume that the mAb blocks the extracellular PCSK9 effect on LDLR.Here, we showed that overexpression of PCSK9 or its D374Y mutant in HepG2 cells effectively reduces cell surface LDLR levels within 24 h. In contrast, only the D374Y mutant affects the levels of LDLR in nontransfected neighboring cells (Fig. 4), in agreement with its reported enhanced LDLR-binding affinity at neutral pH (11Cunningham D. Danley D.E. Geoghegan K.F. Griffor M.C. Hawkins J.L. Subashi T.A. Varghese A.H. Ammirati M.J. Culp J.S. Hoth L.R. Mansour M.N. McGrath K.M. Seddon A.P. Shenolikar S. Stutzman-Engwall K.J. Warren L.C. Xia D. Qiu X. Nat. Struct. Mol. Biol. 2007; 14: 413-419Crossref PubMed Scopus (350) Google Scholar, 18McNutt M.C. Kwon H.J. Chen C. Chen J.R. Horton J.D. Lagace T.A. J. Biol. Chem. 2009; 284: 10561-10570Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 23Bottomley M.J. Cirillo A. Orsatti L. Ruggeri L. Fisher T.S. Santoro J.C. Cummings R.T. Cubbon R.M. Lo S.P. Calzetta A. Noto A. Baysarowich J. Mattu M. Talamo F. De Francesco R. Sparrow C.P. Sitlani A. Carfi A. J. Biol. Chem. 2009; 284: 1313-1323Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Concentrations of secreted PCSK9, up to 12 nm (HepG2 cells; ∼3× endogenous levels; Fig. 4) or 20 nm (HEK293 cells; supplemental Fig. S3), were unable to enhance the degradation of the LDLR on neighboring cells in 24 h. This suggests that within 24 h and at levels close to endogenous ones, PCSK9 exerts its function mostly intracellularly and/or in an autocrine fashion.Our previous work demonstrated the importance of clathrin heavy chain for the PCSK9-dependent degradation of the LDLR (10Nassoury N. Blasiole D.A. Tebon O.A. Benjannet S. Hamelin J. Poupon V. McPherson P.S. Attie A.D. Prat A. Seidah N.G. Traffic. 2007; 8: 718-732Crossref PubMed Scopus (199) Google Scholar). However, because clathrin-mediated membrane trafficking is needed for both direct sorting from the trans-Golgi network to LE/L and internalization of cargo by endocytosis, the route taken by the PCSK9·LDLR complex remained unresolved. In the present work, our data support the presence of a relatively rapid intracellular route for the PCSK9-induced degradation of LDLR at endogenous levels because blockade of the trans-Golgi network to lysosome vesicular trafficking by KDCLCs clearly up-regulated LDLR levels already after 4 h (supplemental Fig. S5), without affecting endocytosis (Fig. 5 and supplemental Fig. S4b). Even though the KDCLCs slows down the activation of procathepsin D (19Poupon V. Girard M. Legendre-Guillemin V. Thomas S. Bourbonniere L. Philie J. Bright N.A. McPherson P.S. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 168-173Crossref PubMed Scopus (65) Google Scholar), a lysosomal hydrolase that is targeted to LE/L, our results also showed that after KDCLCs, the cognate hydrolases retained their capacity to degrade the LDLR (Fig. 5e). In addition, we also demonstrated that the effect of KDCLCs is PCSK9-dependent because LDLR levels are no longer affected when PCSK9 is absent (Fig. 5f). Although the level of LDLR is increased, its intracellular and surface localization remained comparable in cells lacking CLCs (supplemental Fig. S4a). Similarly, intracellular and secreted levels as well as the localization of PCSK9 were not significantly altered by KDCLCs (data not shown). Thus, although the underlying mechanism of CLCs effect is presently not fully unraveled, it may rely on another factor that is essential for the direct intracellular trafficking of PCSK9 to the LDLR degradation compartment(s).PCSK9 has emerged as a viable attractive target to reduce coronary heart disease through control of dyslipidemias (39Seidah N.G. Expert. Opin. Ther. Targets. 2009; 13: 19-28Crossref PubMed Scopus (117) Google Scholar). The choice of the best approach to diminish the effect of PCSK9 on LDLR degradation in vivo effectively may be influenced by the relative contribution of both cellular pathways under physiological and pathological conditions. The combination of a statin with an agent that blocks either the extracellular pathway (38Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. Di Y. Shetterly S. Arimura Z. Salomonis H. Romanow W.G. Thibault S.T. Zhang R. Cao P. Yang X.P. Yu T. Lu M. Retter M.W. Kwon G. Henne K. Pan O. Tsai M.M. Fuchslocher B. Yang E. Zhou L. Lee K.J. Daris M. Sheng J. Wang Y. Shen W.D. Yeh W.C. Emery M. Walker N.P. Shan B. Schwarz M. Jackson S.M. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9820-9825Crossref PubMed Scopus (326) Google Scholar) or the intracellular one (present work) or both would constitute a powerful novel approach to control hypercholesterolemia. With the rapid pace of discoveries in the field, it is hoped that in a few years lead molecules reducing the level and/or activity of PCSK9 will be uncovered and that these will find their way in clinical trials to assess their potency and safety. High levels of circulating low-density lipoprotein (LDL) 3The abbreviations used are: LDLlow density lipoproteinARHautosomal recessive hypercholesterolemiaCLCclathrin light chaindiI-LDLLDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorateDMEMDulbecco's modified Eagle's mediumEGF-Aepidermal growth factor domain AEGF-ABepidermal growth factor domain ABEGFPenhanced green fluorescent proteinELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sortingFBSfetal bovine serumKDknockdownKOknock-outLDLRlow density lipoprotein receptorLE/Llate endosomes/lyososomesLPDSlipoprotein-deficient serummAbmonoclonal antibodyPBSphosphate-buffered salinePCSK9proprotein convertases subtilisin kexin 9shRNAsmall hairpin RNAsiRNAsmall interfering RNATfRtransferrin receptorWTwild type. 3The abbreviations used are: LDLlow density lipoproteinARHautosomal recessive hypercholesterolemiaCLCclathrin light chaindiI-LDLLDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorateDMEMDulbecco's modified Eagle's mediumEGF-Aepidermal growth factor domain AEGF-ABepidermal growth factor domain ABEGFPenhanced green fluorescent proteinELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sortingFBSfetal bovine serumKDknockdownKOknock-outLDLRlow density lipoprotein receptorLE/Llate endosomes/lyososomesLPDSlipoprotein-deficient serummAbmonoclonal antibodyPBSphosphate-buffered salinePCSK9proprotein convertases subtilisin kexin 9shRNAsmall hairpin RNAsiRNAsmall interfering RNATfRtransferrin receptorWTwild type.-cholesterol represent a major risk factor that leads to coronary heart disease, the main cause of death and morbidity worldwide (1Lloyd-Jones D. Adams R. Carnethon M. De Simone G. Ferguson T.B. Flegal K. Ford E. Furie K. Go A. Greenlund K. Haase N. Hailpern S. Ho M. Howard V. Kissela B. Kittner S. Lackland D. Lisabeth L. Marelli A. McDermott M. Meigs J. Mozaffarian D. Nichol G. O'Donnell C. Roger V. Rosamond W. Sacco R. Sorlie P. Stafford R. Steinberger J. Thom T. Wasserthiel-Smoller S. Wong N. Wylie-Rosett J. Hong Y. Circulation. 2009; 119: e21-e181Crossref PubMed Scopus (0) Google Scholar). LDL particles are cleared mainly from the bloodstream by the hepatic cell surface LDL receptor (LDLR) (2Brown M.S. Goldstein J.L. Science. 1986; 232: 34
