?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
Secreted PCSK9 decreases the number
of LDL receptors in hepatocytes and in
livers of parabiotic mice
Thomas A. Lagace,1 David E. Curtis,2 Rita Garuti,1 Markey C. McNutt,1 Sahng Wook Park,1
Heidi B. Prather,1 Norma N. Anderson,1 Y.K. Ho,1 Robert E. Hammer,3 and Jay D. Horton1,4
1Department of Molecular Genetics, 2Department of Surgery, 3Department of Biochemistry, and 4Department of Internal Medicine,
University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a mem-
ber of the proteinase K subfamily of subtilisin-related serine endo-
proteases. Like other members of this family, the PCSK9 protein
has a signal sequence, followed by a prodomain, and a catalytic
domain that contains a conserved triad of residues (D186, H226,
and S386) required for activity (1). PCSK9 is synthesized as a
soluble, approximately 74-kDa precursor that undergoes intra-
molecular autocatalytic cleavage in the ER, generating a 14-kDa
prodomain and an approximately 60-kDa catalytic fragment. The
site of intramolecular cleavage (VFAQ↓SIP) differs from that of
subtilisin and other family members that typically cleave after a
basic residue (2, 3). The cleaved prodomain remains associated
with the catalytic domain, forming a PCSK9-prodomain complex
that is transported to the Golgi complex, where it undergoes sul-
fation prior to secretion (1, 3).
Insights into the physiological function of PCSK9 initially came
from the identification of point mutations in PCSK9 that cause
an autosomal dominant form of hypercholesterolemia (4). These
mutations were later shown to be gain-of-function alleles that act
in a dominant fashion (3, 5). PCSK9 was independently identified
as a SREBP-regulated gene in liver, using microarrays hybridized
with RNA from livers of mice that either overexpressed or lacked
SREBPs (6, 7). SREBPs are a family of transcription factors that
increase the expression of many genes involved in cholesterol and
fatty acid synthesis, as well as the LDL receptor (LDLR) gene (6). The
induction of PCSK9 by SREBPs further suggested that this protein
was involved in lipid metabolism.
The biological activity of PCSK9 was revealed through overex-
pression studies in mice. Overexpression of PCSK9 posttranscrip-
tionally reduced the amount of LDLR protein in liver (3, 8–10).
Confirmation that PCSK9 functions normally to regulate LDLR
protein levels came from loss-of-function studies in humans and
mice. Individuals who are heterozygous for a nonsense mutation
in allele PCSK9 have significantly lower plasma LDL cholesterol
levels, suggesting that a reduction in PCSK9 activity leads to an
increase in LDLRs (11). These conclusions were supported by stud-
ies in PCSK9-knockout mice, which revealed that loss of PCSK9
resulted in increased numbers of LDLRs in hepatocytes, acceler-
ated plasma LDL clearance, and significantly lower plasma choles-
terol levels (12). In the most recent studies, humans heterozygous
for loss-of-function mutations in PCSK9 were shown to have a
significant reduction in the long-term risk of developing athero-
sclerotic heart disease (13).
The genetic data from humans and the in vivo studies in mice
demonstrate that one function of PCSK9 is to reduce the number
of the LDLRs and that this function is manifest in humans in the
basal state. The mechanism by which PCSK9 reduces the number
of LDLRs is still undetermined. For example, it is unclear whether
PCSK9 acts to destroy LDLRs in the secretory pathway or whether
it acts outside of the cell. In the current studies, we provide evi-
dence that extracellular PCSK9 can be internalized by cultured
liver cells and fibroblasts in a manner that is largely dependent on
LDLRs. Incubation with extracellular PCSK9 led to loss of LDLRs
Nonstandard?abbreviations?used: ARH, autosomal recessive hypercholesterolemia;
CI-MPR, cation-independent mannose-6-phosphate receptor; LDLR, LDL receptor;
LRP, LDLR-related protein; MEF, mouse embryonic fibroblast; PCSK9, proprotein
convertase subtilisin/kexin type 9.
Conflict?of?interest: J.D. Horton is a consultant for Alnylam Pharmaceuticals,
Aegerion Pharmaceuticals Inc., Metabasis, Merck, and Pfizer, and is on the Speakers’
Bureau of Merck, Merck/Schering-Plough Pharmaceuticals, Schering-Plough.
Citation?for?this?article: J. Clin. Invest. 116:2995–3005 (2006). doi:10.1172/JCI29383.
2996?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
from the cell surface and accelerated destruction of LDLRs in liver-
derived cells. Finally, we demonstrate that increased PCSK9 levels
in the circulation of mice leads to diminished liver LDLR protein
and increased plasma cholesterol levels.
PCSK9, like other subtilisin family proteases, is synthesized with
a prodomain (1, 3). In the other subtilisin family proteases, the
cleaved prodomain remains associated with the protein and acts
as an inhibitor of the cognate enzyme activity. The prosegment
often undergoes secondary proteolytic processing, which relieves
this inhibition and unmasks enzymatic activity (14). When PCSK9
is overexpressed in cultured cells, the protein is secreted with the
prodomain still attached (1, 3). This result raised the possibil-
ity that cellular PCSK9 remains inactive within the secretory
pathway and that PCSK9 may act on LDLRs after secretion,
either at the cell surface or after the LDLR is internalized into
the endosomal/lysosomal system.
To test this possibility, we assessed the rate and extent of
secretion of endogenous PCSK9 expressed at physiological lev-
els in cultured human hepatoma HepG2 cells. This study was
made possible by the development of antibodies against the
catalytic domain of PCSK9. Synthesis and secretion of PCSK9
were quantified by pulse-labeling the cells with [35S]methionine/
cysteine, which was followed by a chase period in isotope-free
medium. Radiolabeled PCSK9 was immunoprecipitated from cells
and the medium at different times. Figure 1 shows results of a rep-
resentative pulse-chase experiment. Two forms of the protein, cor-
responding to the uncleaved precursor (~74 kDa) and the cleaved
mature catalytic fragment (~60 kDa), were detected in cells at 0.5
and 1 hour of chase. After 2 hours, none of the labeled uncleaved
PCSK9 was detected in the cells. After 4 hours, nearly all of the
radiolabeled PCSK9 was recovered in the medium, demonstrating
that the cleaved form of PCSK9 is rapidly and efficiently secreted
from these cells.
We recently reported that PCSK9 can be detected in human
plasma by immunoprecipitation (15). To quantify the concentra-
tion and determine the physiological range of PCSK9 in human
plasma, we developed an ELISA that used an anti-human PCSK9
monoclonal antibody to capture PCSK9 and a polyclonal anti-
human PCSK9 antibody for detection. Plasma levels of PCSK9
were quantified in 72 volunteers. The plasma levels ranged from
approximately 50 to approximately 600 ng/ml (Supplemental
Figure 1; supplemental material available online with this article;
doi:10.1172/JCI29383DS1). These measurements demonstrate
that considerable amounts of PCSK9 circulate in plasma and pro-
vided a range of physiologically relevant PCSK9 concentrations.
We next determined whether the secreted form of PCSK9 can
reduce the number of LDLRs when added to the medium of
HepG2 cells cultured in sterol-depleted medium to induce LDLR
expression. Recombinant PCSK9 that contained a FLAG tag at the
carboxyl terminus was purified from HEK 293S cells as described
in Methods. SDS-PAGE and Coomassie blue staining analysis
showed that purified FLAG-tagged PCSK9 consisted of the cata-
lytic fragment and the cleaved prodomain that migrated with
apparent molecular masses of approximately 60 kDa and approxi-
mately 17 kDa, respectively (Supplemental Figure 2A). Gel filtra-
Pulse-chase analysis of endogenous PCSK9 secretion from HepG2
cells. Cells were cultured in sterol-depleting medium C for 18 hours
prior to labeling for 30 minutes with [35S]methionine/cysteine. After
washing, cells were incubated in chase medium containing unla-
beled methionine and cysteine for the indicated times. Cells were
lysed, and PCSK9 was immunoprecipitated from the medium and
cell lysates as described in Methods. Samples were subjected to
8% SDS-PAGE, and the gel was treated with Amplify fluorogenic
reagent prior to drying and exposure to film. P and C denote the
proprotein and cleaved forms of PCSK9, respectively. Similar results
were obtained in 4 independent experiments.
Reduction of endogenous LDLRs in HepG2 cells following the
addition of recombinant purified PCSK9 to the culture medium. (A)
Dose response of exogenous PCSK9-mediated LDLR degrada-
tion in HepG2 cells. Cells were cultured for 18 hours in medium C
prior to treatment for 4 hours with the indicated amounts of purified
human PCSK9. (B) Time course of exogenous PCSK9-mediated
LDLR degradation. HepG2 cells cultured as described above were
treated with 5 μg/ml of purified PCSK9 for the indicated times.
Total lysates were prepared following cell-surface biotinylation as
described in Methods. Proteins were subjected to SDS-PAGE for
immunoblot analysis of the LDLR using IgG-HL1 and anti–FLAG
M2 monoclonal antibody to detect purified PCSK9. The transferrin
receptor protein was detected as described in Methods and used
as a control for loading and nonspecific protein degradation. Simi-
lar results were obtained in 3 independent experiments.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
tion revealed that the protein elutes at a single peak volume corre-
sponding to an apparent molecular mass of approximately 70 kDa
(Supplemental Figure 2B). After incubation with purified PCSK9,
the surface proteins of HepG2 cells were covalently modified with
a cell-impermeable biotinylation reagent and isolated using strep-
tavidin beads. The total cellular LDLRs in whole-cell extracts and
cell-surface LDLRs were measured by SDS-PAGE immunoblotting
using a monoclonal antibody. The total cell PCSK9 accumulation
and cell-surface PCSK9 were also measured by SDS-PAGE and
immunoblotting, but with anti-FLAG antibody.
As shown in Figure 2A, the number of cell-surface LDLRs
declined by approximately 50% after incubation with the physi-
ologically relevant concentration of 0.5 μg/ml PCSK9 (lane 2) and
became nearly undetectable after exposure to 2.5 μg/ml PCSK9
(lane 4). Incubation of HepG2 cells for 4 hours with 5 or 10 μg/ml
PCSK9 decreased whole-cell LDLR protein levels by approximately
50% (lanes 11 and 12). FLAG-tagged PCSK9 was detected in whole-
cell extracts in a concentration-dependent manner (lanes 7–12),
but it was not detected among the biotin-labeled cell-surface
proteins (lanes 1–6), suggesting that most of the cell-associated
PCSK9 had been internalized.
To determine the time course of PCSK9 action, HepG2 cells were
incubated in the presence of 5 μg/ml PCSK9 and then harvested at
different intervals over a 4-hour period (Figure 2B). The.number
of cell-surface LDLRs declined noticeably at 2 hours (lane 4) and
was undetectable at 4 hours (lane 5). The amount of cell-associated
FLAG-tagged PCSK9 was maximal after 30 minutes (lane 7) and
decreased slightly at 4 hours (lane 10). PCSK9 had no effect on
whole-cell or cell-surface transferrin receptors (Figure 2, A and B).
As discussed in the Introduction, certain point mutations in
PCSK9 cause hypercholesterolemia. To determine whether one
such mutation increases the activity of PCSK9 in a cell-based
assay, varying amounts of wild-type PCSK9 and the PCSK9
mutant D374Y (4) were added to HepG2 cells, after which LDLR
protein levels were measured (Figure 3). The D374Y mutation was
chosen for study because individuals who harbor this mutation
have been shown to manifest severe hypercholesterolemia (16).
PCSK9(D374Y) was at least 10-fold more active than wild-type
PCSK9 in reducing cell surface LDLRs. Thus, PCSK9(D374Y) at
0.25 μg/ml was at least as effective as 2.5 μg/ml of wild-type PCSK9
(compare lanes 5 and 11). After incubation with wild-type PCSK9,
the number of LDLRs was significantly reduced in whole-cell
extracts, and similar results were obtained with 10-fold lower con-
centrations of PCSK9(D374Y) (lanes 13–24). Despite the different
concentrations employed, the amounts of wild-type and mutant
PCSK9 measured in the cell extracts were similar, indicating that
the mutant protein was taken up by the cell approximately 10-fold
more efficiently than the wild-type protein.
To determine whether cellular association/uptake of PCSK9 is
dependent upon LDLRs, we turned to mouse embryonic fibro-
blasts (MEFs), to exploit the availability of cells from gene knock-
out mice that lack the LDLR and the closely related receptor, the
LDLR-related protein (LRP). MEFs derived from wild-type, Ldlr–/–,
Lrp–/–, and Ldlr–/–Lrp–/– mice were incubated with varying amounts
of PCSK9 (Figure 4A). Immunoblots revealed that abundant
PCSK9 was associated with the wild-type (lanes 1–4) and Lrp–/– cells
(lanes 9–12). PCSK9 cellular association was markedly reduced in
Ldlr–/– MEFs (lanes 5–8); however, a small amount of PCSK9 was
detectable in cells incubated with the highest concentration of
PCSK9 (lane 8). This small amount of association was abolished
in cells that lacked both LDLR and LRP (lane 16). These data sug-
gest that exogenous PCSK9 associates with MEFs in a manner that
is almost totally dependent upon the LDLR and that LRP may play
a small role in uptake at high concentrations of PCSK9.
The uptake of PCSK9 was characterized morphologi-
cally using indirect immunofluorescence in MEFs. Double
immunofluorescence labeling of cells incubated with PCSK9 for 4
hours showed diffuse localization of the protein in small punctate
structures, possibly representing endocytic vesicles, which par-
tially overlapped with the LDLR staining pattern (Figure 4B). To
determine whether these structures were endosomal and whether
PCSK9 colocalized intracellularly with the LDLR, we incubated
MEFs in the presence of chloroquine, which raises the pH of acidic
cellular compartments and inhibits lysosomal hydrolases. In the
presence of chloroquine, the cation-independent mannose-6-
phosphate receptor (CI-MPR) accumulates in late endosomes and
was used as a marker of these structures (17). As shown in Figure
4C, PCSK9 became concentrated in large perinuclear vacuoles of
MEFs in the presence of chloroquine. These vacuoles were also
labeled with anti-LDLR and anti–CI-MPR antibodies. Considered
together, the data indicate that exogenous PCSK9 is taken up by
fibroblasts in a manner that is dependent upon the LDLR and
Increased cell association and LDLR degradation by addition of purified mutant PCSK9(D374Y) to the medium of HepG2 cells. Cells were
cultured for 18 hours in medium C and then incubated for 4 hours with the indicated amounts of purified human PCSK9 or PCSK9(D374Y).
Immunoblot analysis of LDLR, FLAG-tagged PCSK9, and transferrin receptor was carried out as described in the legend to Figure 2. The asterisk
indicates a nonspecific band. Similar results were obtained in 3 independent experiments.
2998? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
that the PCSK9 travels with the LDLR to the endosome/lysosome.
Despite this uptake, PCSK9 does not destroy the LDLR in fibro-
blasts at the concentrations tested, either in the absence or pres-
ence of chloroquine. A similar lack of destruction was observed
earlier when PCSK9 was overexpressed in fibroblasts and Chinese
hamster ovary cells by transfection (9).
The requirement of LDLRs for PCSK9 internalization suggests
that PCSK9 may bind to the LDLR. Moreover, PCSK9(D374Y) was
taken up with higher efficiency than wild-type PCSK9, suggest-
ing that the mutant protein may bind to the LDLR more avidly
than wild-type PCSK9. To test this possibility, coimmunoprecipi-
tation studies with wild-type PCSK9 or mutant PCSK9(D374Y)
and the LDLR were carried out in HepG2 cells. The cells were
incubated with wild-type and mutant PCSK9 proteins contain-
ing FLAG epitope tags. After a 1-hour incubation in the presence
of chloroquine, cells were solubilized, and exogenously added
PCSK9 was precipitated with an anti-FLAG antibody. As shown
in Figure 5A (lanes 1 and 5), the FLAG antibody efficiently pulled
down the wild-type and mutant PCSK9 from cell extracts, and the
immunoprecipitation was reduced in the presence of competing
FLAG octapeptide (lanes 2 and 6). In cells that were incubated with
wild-type PCSK9, no detectable LDLR was coimmunoprecipitated
with PCSK9. In contrast, a significant amount of LDLR was pulled
down from cells incubated with PCSK9(D374Y) (lanes 1 and 5).
When the LDLR antibody was used for the immunoprecipitation,
a small, but consistently detectable amount of wild-type PCSK9
was coimmunoprecipitated. The LDLR antibody pulled down
much larger amounts of PCSK9(D374Y) (lanes 3 and 7).
To determine whether the interaction of PCSK9 and the
LDLR was direct, ligand blotting was performed using purified
extracellular domain LDLR protein and PCSK9. First, we deter-
mined the migration of the purified LDLR under reduced and
nonreduced conditions (Figure 5B). Next, we determined whether
PCSK9 could bind to the extracellular domain of the LDLR in a
specific manner. The reduced and nonreduced LDLR protein was
resolved by SDS-PAGE and transferred to nitrocellulose. The fil-
ters were then incubated with 5 μg/ml of purified PCSK9 protein,
and bound PCSK9 was visualized using the monoclonal anti-
body IgG-15A6. As shown in Figure 5C, PCSK9 bound to nonre-
duced LDLR protein but not to LDLRs that had been reduced.
The binding of wild-type PCSK9 and PCSK9(D374Y) to increas-
ing amounts of the LDLR protein was then determined by ligand
blotting (Figure 5D). Both forms of purified PCSK9 bound to
the extracellular domain of the LDLR protein in a concentration-
dependent manner. Consistent with the coimmunoprecipitation,
the PCSK9(D374Y) mutant appeared to bind to the LDLR protein
with a greater affinity. Combined, the results of these studies indi-
cate that PCSK9(D374Y) binds to LDLRs with higher affinity than
does wild-type PCSK9, a finding that correlates with the enhanced
ability of the mutant PCSK9 to destroy LDLRs.
LDLR-dependent endocytosis of PCSK9 in MEFs. (A)
Immunoblot analysis of PCSK9 association with MEFs.
Immortalized MEFs derived from wild-type, Ldlr–/–, Lrp–/–,
and Ldlr–/–Lrp–/– mice were cultured for 18 hours in medi-
um F prior to treatment with purified human PCSK9 for 4
hours. Cell lysates (30 μg) were subjected to SDS-PAGE
and immunoblot analysis using IgG-15A6 to detect PCSK9
or a polyclonal antiserum (Ab 3143) that recognizes the
LDLR, as described in Methods. Immunoblots of receptor-
associated protein (RAP) were used as a loading control.
(B) Indirect immunofluorescence localization of PCSK9
and the LDLR in wild-type MEFs. MEFs were incubated
for 4 hours with 5 μg/ml purified human PCSK9 and pro-
cessed for double immunofluorescence of PCSK9 (green)
and the LDLR (red) as described in Methods. (C) Indirect
immunofluorescence localization of PCSK9, LDLR, and
the late-endosomal marker CI-MPR in MEFs cultured in
the presence of chloroquine. Wild-type MEFs were incu-
bated for 4 hours with 5 μg/ml purified human PCSK9 in
the presence of 0.1 mM chloroquine and processed for
double immunofluorescence of PCSK9 (green) and the
LDLR (red) or CI-MPR (red) as described in Methods. The
merged image shows areas of colocalization of PCSK9 with
the LDLR and CI-MPR. Magnification, ×630. Similar results
were obtained in 3 independent experiments.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
In hepatocytes, the internalization of LDLRs is dependent upon
functional autosomal recessive hypercholesterolemia (ARH), an
adaptor protein that binds to the intracellular domain of the
LDLR and to clathrin, thereby clustering the receptor in coated
vesicles (18, 19). To determine whether ARH was similarly required
for removal of LDLR from the cell surface, PCSK9 was added to
the medium of primary hepatocytes isolated from 2 types of genet-
ically engineered mice: those that express only the human LDLR
(LDLRh/hArh+/+) (20) and those that express the human LDLR
and lack ARH (LDLRh/hArh–/–) (21). In LDLRh/hArh+/+ hepatocytes,
LDLRs were visualized on the cell surface and in intracellular
vesicles concentrated in the cell periphery (Figure 6A, upper left).
Incubation with PCSK9 eliminated the surface LDLRs, and the
remaining LDLRs were concentrated in perinuclear vesicles (Fig-
ure 6A, upper right). In LDLRh/hArh–/– hepatocytes, the LDLRs were
visualized almost entirely on the cell surface (Figure 6A, lower left),
and PCSK9 did not reduce the amount of surface LDLRs in these
cells (Figure 6A, lower right).
To confirm that PCSK9 still associated with the cell in the
absence of ARH, total lysates and cell-surface proteins were isolat-
ed for immunoblot analysis from LDLRh/hArh+/+ and LDLRh/hArh–/–
primary hepatocytes incubated with increasing concentrations
of PCSK9 (Figure 6B). The level of LDLR protein was reduced
by exogenously added PCSK9 in a dose-dependent manner in
both whole-cell extracts and on the cell surface in hepatocytes
expressing ARH (lanes 1–3 and 9–11). In contrast, incubation of
LDLRh/hArh–/– hepatocytes with PCSK9 did not alter LDLR protein
levels in either fraction (lanes 4–6 and 12–14), despite similar lev-
els of PCSK9 cell association (lanes 1–3 and 4–6). In the absence
of ARH, significantly more PCSK9 was detected at the cell surface
(lanes 13–14). No PCSK9 was detected in hepatocytes isolated
from Ldlr–/– mice (lanes 7–8 and 15–16). PCSK9 had no effect on
whole-cell or cell-surface transferrin receptors in hepatocytes, and
cellular integrity was maintained during cell-surface biotinylation,
as indicated by a lack of detection of intracellular actin among
biotin-labeled proteins. These results demonstrate that PCSK9
can associate with the cell in the absence of ARH but that internal-
ization is required for PCSK9 to reduce the cell-surface expression
of LDLR protein. They also show that PCSK9 binding is largely
dependent upon LDLR expression in mouse hepatocytes.
To test whether PCSK9 could function as a secreted protein in vivo,
transgenic mice that express human PCSK9 in liver (TgPCSK9 mice)
were produced. The transgene was under the control of an apoE
promoter with a liver-specific enhancer (22). As shown in Figure 7A,
transgenic overexpression of human PCSK9 eliminated LDLR pro-
tein expression in liver and caused a marked increase in plasma LDL
cholesterol levels (Figure 7B). The increase in plasma LDL cholesterol
was similar to that measured in Ldlr–/– mice that lacked LDLRs in all
tissues. The effects of transgenic PCSK9 overexpression are consis-
tent with the results of previously published experiments in which
adenoviral vectors were used to overexpress PCSK9 in mice (3, 8–10).
The concentration of human PCSK9 in plasma from the transgenic
mice as measured using the ELISA assay ranged from approximately
146 to 440 μg/ml (Table 1, animals 9a, 10a, 11a, and 12a).
Although an apoE promoter fragment and a liver-specific
enhancer were used to express the human PCSK9 cDNA in mice,
Association of PCSK9 and PCSK9(D374Y) with the LDLR. (A)
Coimmunoprecipitation of the LDLR and exogenously added
wild-type PCSK9 or PCSK9(D374Y) protein. HepG2 cells were
cultured for 18 hours in medium C prior to treatment for 1 hour in
the presence of 0.1 mM chloroquine with 20 μg/ml or 2 μg/ml of
purified human PCSK9 or PCSK9(D374Y), respectively. Cells
were harvested and lysed and proteins immunoprecipitated
with the indicated antibodies. Pellets of the immunoprecipitation
were subjected to SDS-PAGE and immunoblot analysis.
Polyclonal antiserum (Ab 3143) was used to detect LDLR,
and IgG-15A6 was used to detect PCSK9. (B) Reduced (R)
or nonreduced (NR) LDLR extracellular domain protein (2 μg)
was resolved by SDS-PAGE. LDLR protein was detected with
Coomassie brilliant blue R-250 stain. (C) Binding of PCSK9 to
LDLR on ligand blots. LDLR protein (2 μg) was transferred to
nitrocellulose and incubated with 5 μg/ml of purified PCSK9 or
no addition (NA). Bound PCSK9 was detected by immunoblot
analysis as described in Methods. (D) The indicated amounts
of purified LDLR were subjected to nonreduced SDS-PAGE,
transferred to nitrocellulose, and incubated with 5 μg/ml of puri-
fied wild-type PCSK9, PCSK9(D374Y), or buffer control (no
addition). Bound PCSK9 was detected by immunoblot analysis
as described in Methods. pep., FLAG octapeptide.
3000? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
the transgene mRNA was detected at low levels in other tissues,
such as the adrenal gland (data not shown). This expression com-
promised analysis of the effect of plasma PCSK9 on LDLR protein
expression in these extrahepatic tissues. Therefore, TgPCSK9 mice
were parabiosed with wild-type mice to determine whether secret-
ed PCSK9 derived from the transgenic mouse was able to decrease
LDLR protein levels in liver when transferred via the shared circu-
lation to the recipient wild-type mouse. For these studies, a liver
biopsy was performed 1 week prior to parabiosis to obtain a liver
sample for quantifying basal levels of LDLR protein expression.
The mice were allowed to recover from the partial hepatectomy
and then were parabiosed for 2 weeks to permit the development
of shared circulation. To control for potential changes induced
by surgery, wild-type mice were parabiosed with wild-type mice.
TgPCSK9 mice manifest elevated plasma LDL cholesterol levels
that could independently alter LDLR protein expression in the
recipient wild-type mouse. To control for this effect, wild-type
mice were parabiosed with Ldlr–/– mice, which have lipoprotein
profiles similar to those of TgPCSK9 mice (Figure 7B).
As shown in Table 1, parabiosis itself slightly increased plasma
total cholesterol levels in most mouse pairs. Plasma cholesterol
concentrations in wild-type mice parabiosed with Ldlr–/– mice
(pairs 5–8) were not significantly higher than those measured in
the wild-type mouse parabiotic pairs (pairs 1–4). Concentrations
of human PCSK9 in parabiotic TgPCSK9:wild-type pairs ranged
from approximately 17 to 250 μg/ml (pairs 9–12), suggesting that
the protein was transferred from the plasma of transgenic mice
into the plasma of the parabiosed wild-type mouse with varying
efficiencies. There also appeared to be a correlation between the
plasma PCSK9 concentration and the degree of plasma cholesterol
elevation of the recipient wild-type mouse. The wild-type mouse
with the highest PCSK9 concentration (mouse 9b) had an approxi-
mately 10-fold increase in plasma total cholesterol, whereas the
wild-type mouse with the lowest PCSK9 level (mouse 12b) had an
approximately 2-fold increase in plasma cholesterol.
To confirm that the observed plasma cholesterol changes were a
result of alterations in LDLR protein levels, immunoblots of liver
protein were performed in all parabiotic pairs before and 2 weeks
after parabiosis (Figure 8). LDLR protein levels in livers of wild-
type mice parabiosed with wild-type mice were not different before
and after parabiosis (Figure 8A). Similarly, the LDLR protein levels
in wild-type mice parabiosed with Ldlr–/– mice were not different
after parabiosis, suggesting that the increased level of LDL choles-
terol present in the knockout mouse did not alter LDLR expres-
sion in the wild-type mouse liver (Figure 8B). In contrast, the
LDLR protein was essentially undetectable in livers of wild-type
PCSK9-mediated degradation of the LDLR is
dependent on ARH. Immunofluorescence (A) and
immunoblot (B) analyses of the PCSK9-medi-
ated changes of LDLR protein in mouse primary
hepatocytes derived from LDLRh/hArh+/+ (Arh+/+) or
LDLRh/hArh–/– (Arh–/–) mice. (A) Primary hepatocytes
were incubated with no PCSK9 (–PCSK9) or with
5 μg/ml purified PCSK9 (+PCSK9) for 4 hours and
processed for indirect immunofluorescence of LDLR
using IgG-C7 and Alexa Fluor 563–conjugated sec-
ondary antibody, as described in Methods. Images
were taken using a 63 × 1.3 objective using a con-
focal microscope (Leica TCS SP). Similar results
were obtained in 2 independent experiments. (B)
Immunoblot analysis of LDLR, FLAG-tagged PCSK9,
transferrin receptor, and actin proteins in primary
hepatocytes from mice of the indicated genotype
incubated with varying amounts of PCSK9.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
mice after they were parabiosed with TgPCSK9 mice (Figure 8C),
indicating that PCSK9 was active in mouse plasma.
To ensure that the reduction in LDLR protein levels measured in
livers of the recipient wild-type mice parabiosed to the TgPCSK9
mice was due to posttranscriptional changes, mRNA levels of the
LDLR and SREBP-2 were measured using real-time RT-PCR (Sup-
plemental Table 1). Although individual mRNAs varied in
amount from mouse to mouse, no consistent changes in the
mRNAs encoding these genes were measured before and after
parabiosis. Combined, the data suggested that secreted PCSK9
in plasma of mice reduced hepatic LDLR protein levels inde-
pendent of changes in mRNA levels.
In the current report, we demonstrate that endogenous
PCSK9 is rapidly secreted from cells and that secreted PCSK9
destroys LDLRs when added to the medium of cultured
HepG2 cells and mouse primary hepatocytes. The effective
concentration of PCSK9 required to reduce the number of
LDLRs in cultured cells was within the range of plasma con-
centrations measured in human plasma. The cell association
and uptake of PCSK9 occurred via binding to the LDLR,
and both proteins colocalized to a late endocytic/lysosomal
compartment. The internalization of PCSK9 with the LDLR
into an endosomal/lysosomal compartment was required for
PCSK9 to reduce LDLR protein levels, since this activity was
blocked in the absence of ARH. Finally, we show that PCSK9
was present in plasma of transgenic mice and that the secret-
ed protein was active in destroying hepatic LDLRs.
Insights into the mechanism of secreted PCSK9’s action
were derived from studies in MEFs and mouse hepatocytes,
which showed that LDLRs were required for the major-
ity of PCSK9 to associate with the cell surface (Figure 4A
and Figure 6B). These studies suggested that the LDLR
and PCSK9 may directly interact, which was confirmed by
coimmunoprecipitation and ligand blotting studies with
the LDLR and PCSK9 (Figure 5). The coimmunoprecipita-
tion studies with PCSK9(D374Y) and the LDLR suggested
that the interaction of the mutant protein with the LDLR was
much more avid than that of the wild-type PCSK9 (Figure 5A). In
addition, PCSK9(D374Y) was approximately 10-fold more effi-
cient in destroying cell-surface LDLRs when added exogenously
to HepG2 cells (Figure 3). Previous overexpression studies in
McArdle-7777 rat hepatoma cells with PCSK9(D374Y) suggested
that the mutant protein increased plasma LDL cholesterol levels
through the overproduction of apoB (5). Similar conclusions were
drawn from in vivo kinetic studies in humans that carry a differ-
ent point mutation in PCSK9 (S127R) (23). The current studies
with the D374Y mutant suggest an alternative mechanism that
involves enhanced LDLR association and degradation. Whether
the S127R and F216L point mutations in PCSK9 behave in a man-
ner similar to PCSK9(D374Y) will be tested in future studies. Our
observations that purified PCSK9(D374Y) added to cells reduced
the number of LDLRs extend the results from a publication that
appeared during the preparation of this manuscript demonstrat-
Total plasma cholesterol and PCSK9 concentrations in pre- and post-
parabiotic pairs of wild-type, Ldlr–/–, and TgPCSK9 (Tg) mice
Decreased amounts of hepatic LDLR protein and increased levels of
plasma LDL cholesterol in transgenic PCSK9 mice. (A) Whole-cell
lysates (30 μg) from livers of 5 wild-type and 5 TgPCSK9 mice were
subjected to SDS-PAGE and immunoblot analyses of human PCSK9,
mouse LDLR, and mouse ARH. (B) Plasma from the mice of each
genotype described in A and from 5 age-matched male Ldlr–/– mice
was pooled and subjected to gel filtration by fast performance liquid
chromatography. The concentration of total cholesterol in each fraction
was measured as described in Methods.
3002?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
ing that conditioned medium containing PCSK9(D374Y) reduced
the uptake of LDL (24).
A small amount of residual PCSK9 binding to LRP was detected
in LDLR-deficient MEFs (Figure 4B). We have previously shown that
hepatic LRP protein levels are not altered by the adenoviral over-
expression of PCSK9 in mice (9), and LRP protein levels were not
significantly different in livers of wild-type mice parabiosed with the
TgPCSK9 mice (data not shown). Thus, although a small amount
of PCSK9 may associate with LRP in vitro, it does not appear to sig-
nificantly regulate LRP protein levels. The residual LRP-dependent
cellular association of PCSK9 might be due to the sequence similar-
ity between LRP and the LDLR. LRP contains 4 copies of the LDLR
extracellular domain with similar charge distributions and may suf-
ficiently resemble LDLR to support PCSK9 binding (25).
Recent studies by Maxwell et al. (26) showed that overexpres-
sion of PCSK9 in HepG2 cells induced the degradation of LDLR
intracellularly in a post-ER compartment. These experiments
also suggested that PCSK9-mediated reduction in the number of
LDLRs was not attributable to the proteasome or lysosomal cysteine
proteases. In addition, our previous studies using adenoviral overex-
pression of PCSK9 in mice demonstrated that high levels of PCSK9
expression in liver eliminated LDLR protein expression in wild-type
and Arh–/– mice equally, indicating that functional ARH was not
required for PCSK9 activity (9). This result differs from the data
presented in Figure 6, which show that functional ARH is required
for PCSK9 to destroy LDLRs when added exogenously to primary
hepatocytes. Therefore, it is possible that PCSK9 overexpression
may result in the association of PCSK9 with the LDLR within the
cell and that this binding leads to intracellular LDLR protein deg-
radation through the pathway described by Maxwell et al., whereas
PCSK9’s action at the cell surface is mediated by a distinct mecha-
nism that is dependent upon ARH-mediated endocytosis.
Considered together, the available data now suggest that PCSK9
can function both extra- and intracellularly, but we do not know
which pathway predominates under normal and/or pathologic
conditions. Currently, all studies suggesting that the protein func-
tions intracellularly have been performed using PCSK9 overexpres-
sion via a strong CMV promoter. Overexpression may permit asso-
ciation of PCSK9 and the LDLR in an intracellular compartment
that does not occur physiologically. In the current studies, we were
able to demonstrate that physiologically relevant concentrations
of PCSK9 could significantly reduce the number of cell-surface
LDLRs when added to HepG2 cells (Figures 2 and 3). Addition-
al studies to determine the relative contribution of extracellular
PCSK9 in the regulation of LDLR protein levels will require deter-
mining whether the infusion of PCSK9 into the circulation of
PCSK9-knockout mice will decrease the number of LDLRs in liver
and raise plasma cholesterol levels.
The results of the parabiosis studies shown in Figure 8 provide
evidence that PCSK9 can function in plasma to destroy LDLRs
in liver. The liver is the principal site of PCSK9 expression, and
PCSK9 is transcriptionally regulated by SREBP-2 (6). SREBP-2 reg-
ulates all genes required for cholesterol synthesis, and its activity is
modulated by cellular sterol levels (6, 27). If the concentration of
PCSK9 in plasma is primarily determined by the liver, then plasma
PCSK9 levels may be a reflection of hepatic cholesterol stores, since
PCSK9 transcription is reduced under conditions where SREBP-2
is suppressed, i.e., high cellular cholesterol concentrations. Such a
regulatory event also raises the possibility that the liver indirectly
regulates the amount of LDLRs in peripheral tissues by determin-
ing the amount of PCSK9 secreted into plasma. It is currently not
known whether PCSK9 in plasma is capable of destroying LDLRs
in tissues other than liver. This could not be directly addressed in
the current studies, owing to the detection limits of the anti-LDLR
antibody in tissues other than liver.
The genetic data from humans with loss-of-function mutations
in PCSK9 combined with the studies in knockout mice that lack
PCSK9 clearly indicate that inhibitors of the protease would be
of therapeutic benefit for the treatment of hypercholesterolemia.
Inasmuch as overexpression of the catalytically inactive form of
PCSK9 in mice did not alter LDLR protein levels (9), an inhibi-
tor of PCSK9’s protease activity in the ER should be sufficient
to block its ability to reduce LDLR protein levels. If PCSK9 func-
tions as a secreted factor as suggested by the current data, then
additional approaches to neutralize its activity, including the
development of antibodies to block its interaction with the LDLR
or inhibitors to block its action in plasma, can be explored for the
treatment of hypercholesterolemia.
Amounts of LDLR protein in livers of parabiosed
mice. Three combinations of mice were parabiosed
for study: wild-type:wild-type (A); Ldlr–/–:wild-type
(B); and TgPCSK9:wild-type (C). Prior to parabiosis,
a liver sample (<100 mg) via a partial hepatectomy
and approximately 50 μl of blood via the tail vein were
obtained from each mouse. After a 7-day recovery
period, 12- to 20-week-old female mice of the indicat-
ed genotype were parabiosed as described in Meth-
ods. After 2 weeks of parabiosis, the animals were
killed, and blood and liver samples were collected for
analysis. Liver membrane protein (40 μg) obtained
from each mouse before (–) and after (+) parabiosis
was subjected to SDS-PAGE for immunoblot analy-
ses of LDLR and RAP as a loading control (9).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
Cultured cell experiments. HepG2, HEK 293 and MEF cells were cultured as
described in Supplemental Methods.
Tissue culture medium. Medium A contained DMEM (cellgro; Mediatech
Inc.) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin sul-
fate, and 1 g/l glucose. Medium B contained medium A supplemented with
10% (vol/vol) FCS. Medium C contained medium A with 5% (vol/vol) new-
born calf lipoprotein–deficient serum (NCLPDS), 10 μM sodium compac-
tin, and 50 μM sodium mevalonate. Medium D contained DMEM supple-
mented with 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, and 4.3
g/l glucose. Medium E contained medium D supplemented with 10% (vol/
vol) FCS. Medium F contained medium D supplemented with 5% (vol/vol)
NCLPDS, 10 μM sodium compactin, and 50 μM sodium mevalonate.
Purification of human wild-type and PCSK9(D374Y)-FLAG fusion proteins. HEK
293S cells stably expressing FLAG-tagged human PCSK9 or PCSK9(D374)
were cultured in suspension without CO2 in IS GRO serum-free medium
(Irvine Scientific) supplemented with 10% FCS, 10 mM l-glutamine,
100 U/ml penicillin, and 100 μg/ml streptomycin. PCSK9 was purified by
anti–FLAG M2 affinity gel chromatography from 500 ml of medium per
the manufacturer’s instructions, followed by size-exclusion chromatogra-
phy on a Tricorn Superose 6 10/300 fast-performance liquid chromatog-
raphy column (Amersham Biosciences). Fractions containing PCSK9 were
concentrated approximately 5-fold using a Centricon filter (10 kDa-MW
cut-off; Millipore). Protein purity was monitored by SDS-PAGE and Coo-
massie Brilliant Blue R-250 staining (Bio-Rad).
Antibodies. For the anti-human PCSK9 polyclonal antibody, the human
PCSK9 amino acid sequence was analyzed using Protean software (Lasergene;
DNAStar) for immunogenic regions. Amino acids 165–180 (RYRADEYQP-
PDGGSLV) and 220–240 (ASKCDSHGTHLAGVVSGRDAG) were synthe-
sized, conjugated to keyhole-limpet hemocyanin using the Imject Maleimide
Activated mcKLH kit (Pierce), and rabbits were injected with a mixture of the
peptides (20 μg each) as described previously (28). IgG fractions from sera
were purified using the ImmunoPure (A/G) IgG purification kit (Pierce).
Monoclonal antibodies that recognize human PCSK9 were generated by
fusion of Sp2/mIL-6 (CRL-2016; ATCC) mouse myeloma cells with splenic
B lymphocytes derived from a female BALB/c mouse injected with puri-
fied human PCSK9 protein (29). The resulting antibodies, 13D3 and 15A6,
belong to IgG subclass 1 and recognize epitopes in the catalytic domain
and the C-terminal region of PCSK9, respectively. Additional anitbodies
used are described in Supplemental Methods.
Immunoblot analysis. Whole-cell lysate protein extracts were subjected to
SDS-PAGE for immunoblot analysis as previously described (9). Biotinyl-
ation of cell-surface proteins of HepG2 cells was performed as previously
described (30). Following biotinylation, whole-cell lysates were prepared,
and protein concentration was determined. An aliquot of the whole-cell
lysate was reserved, and the remainder was transferred to a fresh tube con-
taining 50 μl of Ultralink NeutrAvidin Protein Plus (Pierce) (1:1 slurry).
After 18 hours of incubation with rotation at 4°C, beads were collected by
centrifugation at 300 g for 5 minutes and washed 3 times with lysis buffer.
Proteins were eluted from the beads by boiling in SDS-PAGE sample buffer
and subjected to 8% SDS-PAGE and immunoblot analysis (9).
PCSK9 “sandwich” ELISA. LumiNunc Maxisorp white assay plates (Nunc)
were coated with anti-human PCSK9 monoclonal antibody (IgG-13D3)
diluted to 5 μg/ml in 100 μl of buffer A (20 mM sodium phosphate
pH 7.5, 100 mM sodium chloride) by incubation overnight at 4°C. The
assay plates were then washed with 350 μl of PBS with Tween-20, pH 7.4
(PBSt) and blocked with 150 μl of 0.5% BSA in buffer A for 1 hour at room
temperature with shaking.
Sample preparation was carried out in a separate polypropylene microti-
ter sample plate. Standards were prepared in duplicate using dilutions of
purified PCSK9 protein in buffer A plus 0.5% BSA. Plasma samples were
prepared in triplicate by diluting 6 μl of plasma in 84 μl of buffer A plus
0.5% BSA. Thirty microliters of buffer B (buffer A, 0.5% BSA, 7.2 M urea,
and 0.68% Tween-20) was then added to each well, and the sample plate was
incubated at 46°C for 30 minutes. Aliquots (100 μl) were transferred to the
assay plate and incubated for 2 hours at room temperature with shaking.
The plate was then washed with PBSt and incubated for 1 hour at room
temperature with 100 μl of rabbit anti-human PCSK9 polyclonal antibody
(7.5 μg/ml in buffer A plus 0.5% BSA). After washing with PBSt, 100 μl of
donkey anti-rabbit IgG plus HRP (GE Healthcare) diluted 1:10,000 was
added for 1 hour at room temperature. After a final wash with PBSt, 100 μl
of SuperSignal ELISA Femto Substrate (Pierce) was added for 1 minute and
luminescence quantified using a Dynex MLX microtiter plate luminometer
(Dynex Technologies). To determine plasma PCSK9 concentrations, lumi-
nescence of the sample was compared with that generated in the standard
curve with purified PCSK9. Assay validation experiments were conducted
in accordance with current bioanalytical recommendations (31).
Human plasma was obtained from 72 volunteers (35 men and 37 women)
after an overnight fast. Informed consent and blood samples were obtained
for all participating subjects by institutional review board–approved proto-
cols.The age of the individuals ranged from 21 to 56, and none were taking
Pulse-chase analysis of PCSK9. HepG2 cells were cultured in medium C,
and the pulse-chase was carried out as previously described (12).
Immunoprecipitation of cell and medium extracts was performed using
the polyclonal anti-PCSK9 antibody (32). Immunoprecipitates were elut-
ed in SDS sample buffer and subjected to 8% SDS-PAGE. The gels were
fixed and then immersed for 15 minutes in Amplify fluorogenic reagent
(Amersham Biosciences), dried under vacuum, and exposed to film.
Coimmunoprecipitation of PCSK9 and the LDLR. HepG2 cells were cultured
for 18 hours in medium C prior to treatment with 20 μg/ml or 2 μg/ml of
purified PCSK9 or PCSK9(D374Y) for 1 hour in the presence of 0.1 mM
chloroquine. Pooled cell pellets from 6 dishes of cells were lysed in 2 ml
of buffer C (20 mM HEPES-KOH at pH 7.4, 150 mM NaCl, 1 mM MgCl2,
and protease inhibitors) containing 1% (wt/vol) digitonin. Cell lysates were
incubated with 50 μl anti–FLAG M2 agarose (Sigma-Aldrich) to immu-
noprecipitate FLAG-tagged PCSK9 (for competition assays, 0.1 mg/ml of
FLAG octapeptide was included); IgG-HL1 to immunoprecipitate LDLR;
or IgG-2001, a mouse monoclonal antibody to an irrelevant antigen (33).
Immunoprecipitates were washed 3 times with 500 μl buffer C containing
0.1% (wt/vol) digitonin, eluted in SDS sample buffer by boiling for 5 min-
utes, and subjected to 8% SDS-PAGE.
Ligand blotting. Purified human LDLR extracellular domain (amino acids
1–699) was kindly provided by J. Deisenhofer (University of Texas South-
western Medical Center) and was described previously (34). LDLR protein
was resolved on nonreducing SDS-PAGE and transferred to nitrocellu-
lose as described (35). LDLR protein was heated for 5 minutes at 96°C in
sample buffer containing 2.5% (vol/vol) 2-mercaptoethanol for reducing
conditions. Blots were blocked for 30 minutes in buffer D (50 mM Tris-
Cl at pH 7.4, 90 mM NaCl, 2 mM CaCl2, 5% [wt/vol] BSA) and incubated
sequentially for 60 minutes with gentle agitation in buffer D containing
purified PCSK9 (5 μg/ml), the monoclonal anti-PCSK9 antibody IgG-15A6
(5 μg/ml), and HRP-conjugated goat anti-mouse IgG. PCSK9 and antibody
incubations were followed by washes in buffer E (50 mM Tris-Cl at pH 7.4,
90 mM NaCl, 2 mM CaCl2, 0.5% [wt/vol] BSA). Blots were visualized by
immunoblotting as described above.
Immunofluorescence microscopy. Indirect immunofluorescence using anti-
bodies that detect the LDLR, PCSK9, and CI-MPR in MEFs was performed
as follows. MEFs cultured on glass coverslips to 50% confluence were fixed
in 4% (wt/vol) paraformaldehyde in buffer F (PBS containing 2 mM MgCl2),
3004?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
quenched with 5 mM NH4Cl in buffer F, permeabilized in buffer F con-
taining 0.05% (wt/vol) Triton X-100 (Pierce) for 10 minutes at –20°C, and
blocked for 30 minutes in buffer G (buffer F containing 1% [wt/vol] BSA).
For double-label staining, cells were incubated sequentially with the mono-
clonal anti-PCSK9 antibody IgG-15A6 (2.5 μg/ml), Alexa Fluor 488–conju-
gated goat anti-mouse IgG (0.6 μg/ml), polyclonal LDLR antiserum Ab 4548
(1:1,000) or polyclonal CI-MPR IgG (2 μg/ml), and Alexa Fluor 555–con-
jugated goat anti-rabbit IgG (0.6 μg/ml). Primary (16 hours at 4°C) and
secondary (1 hour at 20°C) antibody incubations were followed by three
20-minute washes with buffer G. Coverslips were mounted in VECTASHIELD
Mounting Medium (Vector Laboratories), and images were taken on a Zeiss
confocal microscope (model LSM510-META). Indirect immunofluorescence
for the LDLR in primary hepatocytes derived from LDLRh/hArh+/+ and
LDLRh/hArh–/– mice was carried out as previously described (12, 21).
TgPCSK9 mice. A cDNA encoding human PCSK9 was inserted into a
pLiv-11 vector that contains the constitutive human apoE promoter and
its hepatic control region (a gift from J. Taylor, Gladstone Institute of Car-
diovascular Disease, San Francisco, California, USA) (22). Transgenic mice
were generated by injecting linearized pLiv-11-hPCSK9 into the fertilized
eggs as described previously (28).
Liver biopsy and parabiosis. One week prior to parabiosis, a liver biopsy was
performed to obtain a liver sample for protein analysis. Mice were anesthe-
tized with pentobarbitol, hair over the abdomen shaved, and skin cleaned
with chlorhexidine. A 0.5-cm transverse incision was made in the upper
abdomen, and a small lobe of the liver (~50–100 mg) was tied off with a
5-0 silk suture and excised. The liver sample was placed in liquid nitro-
gen for storage. The incision was closed in 2 layers using a 5-0 silk suture
and wound clips. During sedation, 50 μl of blood was removed from the
tail vein for plasma cholesterol measurements. The mice were allowed to
recover from this surgery before the parabiosis was performed.
For parabiosis, mice were surgically joined following a previously
published protocol (36), with slight modifications. Matching skin inci-
sions were made from the shoulder to the hip joint of each mouse, and
a 10-mm incision was made in the muscles of the abdominal wall. The
muscular incisions were joined using a 5-0 silk suture. The scapulae
were attached by a 2-0 silk suture, and the skin was approximated using
wound clips and 5-0 silk suture.
Three genetically distinct groups of female mice were studied: wild-type
mice joined with wild-type mice; Ldlr–/– mice joined with wild-type mice;
and TgPCSK9 mice joined with wild-type mice. Shared circulation was
confirmed by injecting Evans blue into one of the parabiotic pairs prior
to study. Mice were killed by isoflurane overdose 2 weeks after parabiosis,
and blood and liver were harvested for analysis. Animal experiments were
approved by the Institutional Animal Care and Research Advisory Com-
mittee at the University of Texas Southwestern Medical Center.
This work was supported by grants from the Perot Family Foun-
dation and the NIH (HL-20948 and HL-38049). T.A. Lagace is
supported by a fellowship from the Natural Sciences and Engi-
neering Research Council of Canada. D.E. Curtis is supported
by the UT Southwestern Physician Scientist Training Program.
The authors wish to thank David W. Russell, Helen H. Hobbs,
Jonathan Cohen, Joseph L. Goldstein, and Michael S. Brown for
critical reading of the manuscript. Scott Clark, Amy Cox, Tuyet
Dang, Linda Donnelly, Richard Gibson, Anh Pho, and Judy San-
chez provided excellent technical assistance.
Received for publication June 14, 2006, and accepted in revised
form August 1, 2006.
Address correspondence to: Jay D. Horton, Departments of Molec-
ular Genetics and Internal Medicine, University of Texas South-
western Medical Center at Dallas, 5323 Harry Hines Blvd., Room
L5-238, Dallas, Texas 75390-9046, USA. Phone: (214) 648-9677;
Fax: (214) 648-8804; E-mail: firstname.lastname@example.org.
Sahng Wook Park’s present address is: Kwandong University
College of Medicine, Department of Biochemistry, Kangneung,
Republic of Korea.
1. Seidah, N.G., et al. 2003. The secretory proprotein
convertase neural apoptosis-regulated convertase 1
(NARC-1): liver regeneration and neuronal differ-
entiation. Proc. Natl. Acad. Sci. U. S. A. 100:928–933.
2. Naureckiene, S., et al. 2003. Functional character-
ization of Narc 1, a novel proteinase related to pro-
teinase K. Arch. Biochem. Biophys. 420:55–67.
3. Benjannet, S., et al. 2004. NARC-1/PCSK9 and its
natural mutants: zymogen cleavage and effects on
the low density lipoprotein (LDL) receptor and
LDL cholesterol. J. Biol. Chem. 279:48865–48875.
4. Abifadel, M., et al. 2003. Mutations in PCSK9 cause
autosomal dominant hypercholesterolemia. Nat.
5. Sun, X.-M., et al. 2005. Evidence for effect of
mutant PCSK9 on apolipoprotein B secretion as
the cause of unusually severe dominant hypercho-
lesterolaemia. Hum. Mol. Genet. 14:1161–1169.
6. Horton, J.D., et al. 2003. Combined analysis of oli-
gonucleotide microarray data from transgenic and
knockout mice identifies direct SREBP target genes.
Proc. Natl. Acad. Sci. U. S. A. 100:12027–12032.
7. Maxwell, K.N., Soccio, R.E., Duncan, E.M., Sehayek,
E., and Breslow, J.L. 2003. Novel putative SREBP
and LXR target genes identified by microarray
analysis in liver of cholesterol-fed mice. J. Lipid Res.
8. Maxwell, K.N., and Breslow, J.L. 2004. Adenoviral-
mediated expression of Pcsk9 in mice results in a
low-density lipoprotein receptor knockout pheno-
type. Proc. Natl. Acad. Sci. U. S. A. 101:7100–7105.
9. Park, S.W., Moon, Y.-A., and Horton, J.D. 2004.
Post-transcriptional regulation of low density lipo-
protein receptor protein by proprotein convertase
subtilisin/kexin type 9a in mouse liver. J. Biol. Chem.
10. Lalanne, F., et al. 2005. Wild-type PCSK9 inhibits
LDL clearance but does not affect apoB-contain-
ing lipoprotein production in mouse and cultured
cells. J. Lipid Res. 46:1312–1319.
11. Cohen, J., et al. 2005. Low LDL cholesterol in individ-
uals of African descent resulting from frequent non-
sense mutations in PCSK9. Nat. Genet. 37:161–165.
12. Rashid, S., et al. 2005. Decreased plasma cholester-
ol and hypersensitivity to statins in mice lacking
Pcsk9. Proc. Natl. Acad. Sci. U. S. A. 102:5374–5379.
13. Cohen, J.C., Boerwinkle, E., Moseley, T.H., and
Hobbs, H.H. 2006. Sequence variations in PCSK9,
low LDL, and protection against coronary heart
disease. N. Engl. J. Med. 354:1264–1272.
14. Basak, A. 2005. Inhibitors of proprotein conver-
tases. J. Mol. Med. 83:844–855.
15. Zhao, Z., et al. 2006. Molecular characterization of
loss-of-function mutations in PCSK9 and identi-
fication of a compound heterozygote. Am. J. Hum.
16. Naoumova, R.P., et al. 2005. Severe hypercholes-
terolemia in four British families with the D374Y
mutation in the PCSK9 gene: long-term follow-up
and treatment response. Arterioscler. Thromb. Vasc.
17. Brown, W.J., Goodhouse, J., and Farquhar, M.G.
1986. Mannose-6-phosphate receptors for lyso-
somal enzymes cycle between the Golgi complex
and endosomes. J. Cell Biol. 103:1235–1247.
18. Garcia, C.K., et al. 2001. Autosomal recessive
hypercholesterolemia caused by mutations in a
putative LDL receptor adaptor protein. Science.
19. He, G., et al. 2002. ARH is a modular adaptor pro-
tein that interacts with the LDL receptor, clathrin,
and AP-2. J. Biol. Chem. 277:44044–44049.
20. Knouff, C., Malloy, S., Wilder, J., Altenburg, M.K.,
and Maeda, N. 2001. Doubling expression of the
low density lipoprotein receptor by truncation of the
3′-untranslated region sequence ameliorates type III
hyperlipoproteinemia in mice expressing the human
apoe2 isoform. J. Biol. Chem. 276:3856–3862.
21. Jones, C., et al. 2003. Normal sorting but defective
endocytosis of the low density lipoprotein receptor
in mice with autosomal recessive hypercholesterol-
emia. J. Biol. Chem. 278:29024–29030.
22. Simonet, W.S., Bucay, N., Lauer, S.J., and Taylor,
J.M. 1993. A far-downstream hepatocyte-specific
control region directs expression of the linked
human apolipoprotein E and C-I genes in trans-
genic mice. J. Biol. Chem. 268:8221–8229.
23. Ouguerram, K., et al. 2004. Apolipoprotein B100
metabolism in autosomal-dominant hypercholes-
terolemia related to mutations in PCSK9. Arterio-
scler. Thromb. Vasc. Biol. 24:1448–1453.
24. Cameron, J., et al. 2006. Effect of mutations in the
PCSK9 gene on the cell surface LDL receptors.
research article Download full-text
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
Hum. Mol. Genet. 15:1551–1558.
25. Herz, J., and Bock, H.H. 2002. Lipoprotein recep-
tors in the nervous system. Annu. Rev. Biochem.
26. Maxwell, K.N., Fisher, E.A., and Breslow, J.L. 2005.
Overexpression of PCSK9 accelerates the degrada-
tion of the LDLR in a post-endoplasmic reticu-
lum compartment. Proc. Natl. Acad. Sci. U. S. A.
27. Brown, M.S., and Goldstein, J.L. 1997. The SREBP
pathway: regulation of cholesterol metabolism by
proteolysis of a membrane-bound transcription
factor. Cell. 89:331–340.
28. Shimano, H., et al. 1996. Overproduction of cho-
lesterol and fatty acids causes massive liver enlarge-
ment in transgenic mice expressing truncated
SREBP-1a. J. Clin. Invest. 98:1575–1584.
29. Herz, J., Kowal, R.C., Ho, Y.K., Brown, M.S., and
Goldstein, J.L. 1990. Low density lipoprotein recep-
tor-related protein mediates endocytosis of mono-
clonal antibodies in cultured cells and rabbit liver.
J. Biol. Chem. 265:21355–21362.
30. Hanwell, D., Ishikawa, T., Saleki, R., and Rotin,
D. 2002. Trafficking and cell surface stability of
the epithelial Na+ channel expressed in epithe-
lial Madin-Darby canine kidney cells. J. Biol. Chem.
31. DeSilva, B., et al. 2003. Recommendations for the
bioanalytical method validation of ligand-binding
assays to support pharmacokinetic assessments of
macromolecules. Pharm. Res. 20:1885–1900.
32. Sakai, J., et al. 1997. Identification of complexes
between the COOH-terminal domains of sterol
regulatory element-binding proteins (SREBPs) and
SREBP cleavage-activating protein. J. Biol. Chem.
33. Beisiegel, U., Schneider, W.J., Brown, M.S., and
Goldstein, J.L. 1982. Immunoblot analysis of low
density lipoprotein receptors in fibroblasts from
subjects with familial hypercholesterolemia. J. Biol.
34. Rudenko, G., et al. 2002. Structure of the LDL
receptor extracellular domain at endosomal pH.
35. Daniel, T.O., Schneider, W.J., Goldstein, J.L., and
Brown, M.S. 1983. Visualization of lipoprotein recep-
tors by ligand blotting. J. Biol. Chem. 258:4606–4611.
36. Wright, D.E., Wagers, A.J., Gulati, A.P., Johnson,
F.L., and Weissman, I.L. 2001. Physiological migra-
tion of hematopoietic stem and progenitor cells.