Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

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; PubMed Scopus Google which is in and small N.G. S. L. J. S. A. Prat A. M. PubMed Scopus Google the associated with familial hypercholesterolemia M. M. Rabès J.P. D. K. M. C. S. L. D. A. L. M. E. J. B. G. P. J. Prat A. M. C. N.G. Boileau C. Genet. PubMed Scopus Google Scholar). is that PCSK9 binds the LDLR and its intracellular degradation in in increased circulating plasma S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google J.L. PubMed Scopus Google J. PubMed Scopus Google N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google its PCSK9 is a with its N.G. S. L. J. S. A. Prat A. M. PubMed Scopus Google S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google Scholar). in a D. J.L. S. D. Mol. 2007; PubMed Scopus Google that the of PCSK9 to the epidermal growth factor A domain (EGF-A) of the LDLR J. 2008; PubMed Scopus Google with increased at D. J.L. S. D. Mol. 2007; PubMed Scopus Google Scholar). Although the domain of PCSK9 is a domain D. J.L. S. D. Mol. 2007; PubMed Scopus Google that not in the J. 2008; PubMed Scopus Google it is a for the degradation of the LDLR R. 2008; PubMed Scopus Google Scholar). In we demonstrated that which binds the domain of its on LDLR degradation G. S. N.G. J. 2008; PubMed Scopus Google studies in that both and extracellular PCSK9 target the LDLR J. PubMed Scopus Google J.L. PubMed Scopus Google R. Ho J. Clin. PubMed Scopus Google degradation in endosomes/lysosomes S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google J.L. PubMed Scopus Google J. PubMed Scopus Google N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). that the protein which with the of the LDLR, is for the and degradation of the cell surface in R. Ho J. Clin. PubMed Scopus Google Scholar). hepatic LDLR protein levels of PCSK9 in J. PubMed Scopus Google the of an intracellular at endogenous levels of the of did not affect hepatic LDLR in or protein levels C. J. J. PubMed Scopus Google Scholar). is not the result PCSK9 LDLR by the extracellular pathway C. J. 2009; PubMed Scopus Google would that in LDLR levels we on the of the extracellular of endogenous LDLR degradation. the of that target the of to hypercholesterolemia and coronary heart cell surface of PCSK9 on the of LDLR and the protein that binds the of the LDLR R. Ho J. Clin. PubMed Scopus Google J. T. Mol. Genet. PubMed Scopus Google Y. S. A. W. D. G. J. 2007; PubMed Scopus Google Scholar). In we that LDLR and PCSK9 in the pathway N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google and or PCSK9 be from the trans-Golgi network to without the cell from the extracellular pathway, the of an intracellular is by the ability of PCSK9 to the LDLR in in the of J. PubMed Scopus Google and on the of which to to enhance the degradation of the LDLR, and S. G. S. E. J. N. J. Prat A. N.G. J. 2008; PubMed Scopus Google Scholar). In seems to be for the of the extracellular R. Ho J. Clin. PubMed Scopus Google not to an in levels of endogenous LDLR C. J. J. PubMed Scopus Google M. A. K. S. S. Y. S. PubMed Scopus Google PCSK9 from primary hepatocytes of on from incubation with PCSK9 LDLR levels 24-h of PCSK9 reduce LDLR levels in primary hepatocytes and in HepG2 cells and we that in the plasma of the levels of circulating PCSK9 and its A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google are at least in and it that the of with that of did not affect the levels of LDLR in the plasma R. Ho J. Clin. PubMed Scopus Google Scholar). Accordingly, the of circulating PCSK9 and its Although PCSK9 in circulating a increased it by A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google Scholar). from levels of PCSK9 in mouse Y. L. D. T. S. W. J. J. 2009; PubMed Scopus Google or R. Ho J. Clin. PubMed Scopus Google or of with PCSK9 A. J. 2008; PubMed Scopus Google that of circulating PCSK9 are to affect LDLR protein levels without affecting In that we that the of extracellular PCSK9 on LDLR, and in of its from hepatocytes in it its in G. S. N.G. J. 2008; PubMed Scopus Google Scholar). The data that levels of circulating PCSK9 an on the of circulating PCSK9 to be R. Ho J. Clin. PubMed Scopus Google G. M. G. J. S. L. J. L. N.G. J. J. 2009; G. J. Clin. 2007; PubMed Scopus Google Scholar). the of PCSK9 from hepatocytes the extracellular is to be in PCSK9 an in data that a incubation of HepG2 cells with an EGF-AB peptide the activity of PCSK9 on endogenous PCSK9 it at least to an of EGF-AB on LDLR levels and activity and it is that the extracellular PCSK9 is a and a incubation and/or a be to the of PCSK9 to LDLR at D. J.L. S. D. Mol. 2007; PubMed Scopus Google that a that the of PCSK9 and LDLR is to reduce the extracellular activity of PCSK9 both in HepG2 cells and in in mouse and D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). to data in HepG2 cells EGF-AB a the for The seems to be to a the not the surface D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). for we that the the extracellular PCSK9 on we that of PCSK9 or its in HepG2 cells cell surface LDLR levels within In contrast, the the levels of LDLR in cells in with its enhanced at D. J.L. S. D. Mol. 2007; PubMed Scopus Google C. J. 2009; PubMed Scopus Google A. L. L. A. A. J. M. De R. A. A. J. 2009; PubMed Scopus Google Scholar). of to endogenous or to enhance the degradation of the LDLR on cells in that within and at levels to endogenous PCSK9 its and/or in an demonstrated the of clathrin chain for the PCSK9-dependent degradation of the LDLR N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). trafficking is for both from the trans-Golgi network to and of by the by the In the present data the of a intracellular for the degradation of LDLR at endogenous levels of the trans-Golgi network to trafficking by LDLR levels without affecting and the the of V. M. V. S. L. J. 2008; PubMed Scopus Google a that is to that the to the LDLR In we demonstrated that the of is PCSK9-dependent LDLR levels are PCSK9 is Although the of LDLR is its intracellular and surface in cells intracellular and levels the of PCSK9 not significantly by not the mechanism of is not fully it on factor that is for the intracellular trafficking of PCSK9 to the LDLR degradation a attractive target to reduce coronary heart disease of N.G. 2009; PubMed Scopus Google Scholar). The of the to the of PCSK9 on LDLR degradation in be by the of both and The of a with an that either the extracellular pathway D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google or the intracellular or both would a to the of in the it is that in a the and/or activity of PCSK9 be and that in to and 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; PubMed Scopus Google which is in and small N.G. S. L. J. S. A. Prat A. M. PubMed Scopus Google the associated with familial hypercholesterolemia M. M. Rabès J.P. D. K. M. C. S. L. D. A. L. M. E. J. B. G. P. J. Prat A. M. C. N.G. Boileau C. Genet. PubMed Scopus Google Scholar). is that PCSK9 binds the LDLR and its intracellular degradation in in increased circulating plasma S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google J.L. PubMed Scopus Google J. PubMed Scopus Google N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). low density lipoprotein autosomal recessive hypercholesterolemia clathrin light chain LDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine modified Eagle's epidermal growth factor domain A epidermal growth factor domain enhanced green fluorescent protein immunosorbent cell bovine knockdown low density lipoprotein receptor proprotein convertases subtilisin kexin 9 small hairpin small interfering receptor type. low density lipoprotein autosomal recessive hypercholesterolemia clathrin light chain LDL coupled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine modified Eagle's epidermal growth factor domain A epidermal growth factor domain enhanced green fluorescent protein immunosorbent cell bovine knockdown low density lipoprotein receptor proprotein convertases subtilisin kexin 9 small hairpin small interfering receptor type. its PCSK9 is a with its N.G. S. L. J. S. A. Prat A. M. PubMed Scopus Google S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google Scholar). in a D. J.L. S. D. Mol. 2007; PubMed Scopus Google that the of PCSK9 to the epidermal growth factor A domain (EGF-A) of the LDLR J. 2008; PubMed Scopus Google with increased at D. J.L. S. D. Mol. 2007; PubMed Scopus Google Scholar). Although the domain of PCSK9 is a domain D. J.L. S. D. Mol. 2007; PubMed Scopus Google that not in the J. 2008; PubMed Scopus Google it is a for the degradation of the LDLR R. 2008; PubMed Scopus Google Scholar). In we demonstrated that which binds the domain of its on LDLR degradation G. S. N.G. J. 2008; PubMed Scopus Google Scholar). studies in that both and extracellular PCSK9 target the LDLR J. PubMed Scopus Google J.L. PubMed Scopus Google R. Ho J. Clin. PubMed Scopus Google degradation in endosomes/lysosomes S. D. R. J. L. W. J. M. D. M. Abifadel M. A. Boileau C. L. M. Prat A. N.G. J. PubMed Scopus Google J.L. PubMed Scopus Google J. PubMed Scopus Google N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). that the protein which with the of the LDLR, is for the and degradation of the cell surface in R. Ho J. Clin. PubMed Scopus Google Scholar). hepatic LDLR protein levels of PCSK9 in J. PubMed Scopus Google the of an intracellular at endogenous levels of the of did not affect hepatic LDLR in or protein levels C. J. J. PubMed Scopus Google Scholar). is not the result PCSK9 LDLR by the extracellular pathway C. J. 2009; PubMed Scopus Google would that in LDLR levels In we on the of the extracellular of endogenous LDLR degradation. the of that target the of to hypercholesterolemia and coronary heart disease. cell surface of PCSK9 on the of LDLR and the protein that binds the of the LDLR R. Ho J. Clin. PubMed Scopus Google J. T. Mol. Genet. PubMed Scopus Google Y. S. A. W. D. G. J. 2007; PubMed Scopus Google Scholar). In we that LDLR and PCSK9 in the pathway N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google and or PCSK9 be from the trans-Golgi network to without the cell from the extracellular pathway, the of an intracellular is by the ability of PCSK9 to the LDLR in in the of J. PubMed Scopus Google and on the of which to to enhance the degradation of the LDLR, and S. G. S. E. J. N. J. Prat A. N.G. J. 2008; PubMed Scopus Google Scholar). In seems to be for the of the extracellular R. Ho J. Clin. PubMed Scopus Google not to an in levels of endogenous LDLR C. J. J. PubMed Scopus Google M. A. K. S. S. Y. S. PubMed Scopus Google PCSK9 from primary hepatocytes of on from incubation with PCSK9 LDLR levels 24-h of PCSK9 reduce LDLR levels in primary hepatocytes and in HepG2 cells and we that in the plasma of the levels of circulating PCSK9 and its A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google are at least in and it that the of with that of did not affect the levels of LDLR in the plasma R. Ho J. Clin. PubMed Scopus Google Scholar). Accordingly, the of circulating PCSK9 and its Although PCSK9 in circulating a increased it by A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google Scholar). from levels of PCSK9 in mouse Y. L. D. T. S. W. J. J. 2009; PubMed Scopus Google or R. Ho J. Clin. PubMed Scopus Google or of with PCSK9 A. J. 2008; PubMed Scopus Google that of circulating PCSK9 are to affect LDLR protein levels without affecting In that we that the of extracellular PCSK9 on LDLR, and in of its from hepatocytes in it its in G. S. N.G. J. 2008; PubMed Scopus Google Scholar). The data that levels of circulating PCSK9 an on the of circulating PCSK9 to be R. Ho J. Clin. PubMed Scopus Google G. M. G. J. S. L. J. L. N.G. J. J. 2009; G. J. Clin. 2007; PubMed Scopus Google Scholar). the of PCSK9 from hepatocytes the extracellular is to be in PCSK9 an in data that a incubation of HepG2 cells with an EGF-AB peptide the activity of PCSK9 on endogenous PCSK9 it at least to an of EGF-AB on LDLR levels and activity and it is that the extracellular PCSK9 is a and a incubation and/or a be to the of PCSK9 to LDLR at D. J.L. S. D. Mol. 2007; PubMed Scopus Google that a that the of PCSK9 and LDLR is to reduce the extracellular activity of PCSK9 both in HepG2 cells and in in mouse and D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). to data in HepG2 cells EGF-AB a the for The seems to be to a the not the surface D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). for we that the the extracellular PCSK9 on we that of PCSK9 or its in HepG2 cells cell surface LDLR levels within In contrast, the the levels of LDLR in cells in with its enhanced at D. J.L. S. D. Mol. 2007; PubMed Scopus Google C. J. 2009; PubMed Scopus Google A. L. L. A. A. J. M. De R. A. A. J. 2009; PubMed Scopus Google Scholar). of to endogenous or to enhance the degradation of the LDLR on cells in that within and at levels to endogenous PCSK9 its and/or in an demonstrated the of clathrin chain for the PCSK9-dependent degradation of the LDLR N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). trafficking is for both from the trans-Golgi network to and of by the by the In the present data the of a intracellular for the degradation of LDLR at endogenous levels of the trans-Golgi network to trafficking by LDLR levels without affecting and the the of V. M. V. S. L. J. 2008; PubMed Scopus Google a that is to that the to the LDLR In we demonstrated that the of is PCSK9-dependent LDLR levels are PCSK9 is Although the of LDLR is its intracellular and surface in cells intracellular and levels the of PCSK9 not significantly by not the mechanism of is not fully it on factor that is for the intracellular trafficking of PCSK9 to the LDLR degradation a attractive target to reduce coronary heart disease of N.G. 2009; PubMed Scopus Google Scholar). The of the to the of PCSK9 on LDLR degradation in be by the of both and The of a with an that either the extracellular pathway D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google or the intracellular or both would a to the of in the it is that in a the and/or activity of PCSK9 be and that in to and The cell surface of PCSK9 on the of LDLR and the protein that binds the of the LDLR R. Ho J. Clin. PubMed Scopus Google J. T. Mol. Genet. PubMed Scopus Google Y. S. A. W. D. G. J. 2007; PubMed Scopus Google Scholar). In we that LDLR and PCSK9 in the pathway N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google and or PCSK9 be from the trans-Golgi network to without the cell from the extracellular pathway, the of an intracellular is by the ability of PCSK9 to the LDLR in in the of J. PubMed Scopus Google and on the of which to to enhance the degradation of the LDLR, and S. G. S. E. J. N. J. Prat A. N.G. J. 2008; PubMed Scopus Google Scholar). In seems to be for the of the extracellular R. Ho J. Clin. PubMed Scopus Google not to an in levels of endogenous LDLR C. J. J. PubMed Scopus Google M. A. K. S. S. Y. S. PubMed Scopus Google Scholar). PCSK9 from primary hepatocytes of on from incubation with PCSK9 LDLR levels 24-h of PCSK9 reduce LDLR levels in primary hepatocytes and in HepG2 cells and we that in the plasma of the levels of circulating PCSK9 and its A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google are at least in and it that the of with that of did not affect the levels of LDLR in the plasma R. Ho J. Clin. PubMed Scopus Google Scholar). Accordingly, the of circulating PCSK9 and its Although PCSK9 in circulating a increased it by A. A. R. J. A. J. M. W. J. N.G. Prat A. 2008; PubMed Scopus Google Scholar). from levels of PCSK9 in mouse Y. L. D. T. S. W. J. J. 2009; PubMed Scopus Google or R. Ho J. Clin. PubMed Scopus Google or of with PCSK9 A. J. 2008; PubMed Scopus Google that of circulating PCSK9 are to affect LDLR protein levels without affecting In that we that the of extracellular PCSK9 on LDLR, and in of its from hepatocytes in it its in G. S. N.G. J. 2008; PubMed Scopus Google Scholar). The data that levels of circulating PCSK9 an on the of circulating PCSK9 to be R. Ho J. Clin. PubMed Scopus Google G. M. G. J. S. L. J. L. N.G. J. J. 2009; G. J. Clin. 2007; PubMed Scopus Google Scholar). the of PCSK9 from hepatocytes the extracellular is to be in PCSK9 an in data that a incubation of HepG2 cells with an EGF-AB peptide the activity of PCSK9 on endogenous PCSK9 it at least to an of EGF-AB on LDLR levels and activity and it is that the extracellular PCSK9 is a and a incubation and/or a be to the of PCSK9 to LDLR at D. J.L. S. D. Mol. 2007; PubMed Scopus Google Scholar). A that a that the of PCSK9 and LDLR is to reduce the extracellular activity of PCSK9 both in HepG2 cells and in in mouse and D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). to data in HepG2 cells EGF-AB a the for The seems to be to a the not the surface D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google Scholar). for we that the the extracellular PCSK9 on Here, we that of PCSK9 or its in HepG2 cells cell surface LDLR levels within In contrast, the the levels of LDLR in cells in with its enhanced at D. J.L. S. D. Mol. 2007; PubMed Scopus Google C. J. 2009; PubMed Scopus Google A. L. L. A. A. J. M. De R. A. A. J. 2009; PubMed Scopus Google Scholar). of to endogenous or to enhance the degradation of the LDLR on cells in that within and at levels to endogenous PCSK9 its and/or in an demonstrated the of clathrin chain for the PCSK9-dependent degradation of the LDLR N. S. J. V. Prat A. N.G. 2007; PubMed Scopus Google Scholar). trafficking is for both from the trans-Golgi network to and of by the by the In the present data the of a intracellular for the degradation of LDLR at endogenous levels of the trans-Golgi network to trafficking by LDLR levels without affecting and the the of V. M. V. S. L. J. 2008; PubMed Scopus Google a that is to that the to the LDLR In we demonstrated that the of is PCSK9-dependent LDLR levels are PCSK9 is Although the of LDLR is its intracellular and surface in cells intracellular and levels the of PCSK9 not significantly by not the mechanism of is not fully it on factor that is for the intracellular trafficking of PCSK9 to the LDLR degradation PCSK9 a attractive target to reduce coronary heart disease of N.G. 2009; PubMed Scopus Google Scholar). The of the to the of PCSK9 on LDLR degradation in be by the of both and The of a with an that either the extracellular pathway D. C. W. J. J. Y. S. R. P. T. M. G. K. B. E. L. M. J. Y. M. B. M. 2009; PubMed Scopus Google or the intracellular or both would a to the of in the it is that in a the and/or activity of PCSK9 be and that in to and R. S. M. E. D. and G. for and J. G. and M. for the and V. and Y. for on primary to of the for and to for with with