Retro-endocytosis of Low Density
Lipoprotein by Cultured Human
Skin Fibrobiasts
Teresa H. Aulinskas, John F. Oram, Edwin L. Bierman, Gerhard A. Coetzee,
Wieland Gevers, and Deneys R. van der Westhuyzen
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A fraction of the low-density lipoprotein (LDL) internalized by cells via receptormediated endocytosis follows a short-circuit pathway, termed "retro-endocytosis,"
that results in the rapid exocytosis of ligand. Results from the current study suggest
that retro-endocytosis of LDL in human fibrobiasts is caused by resurfacing of endocytotic vesicles that contain both free and receptor-bound ligand, resulting in discharge of vesicular contents and in spontaneous dissociation of LDL from its receptor. The bulk of the released LDL particles had the same size, density, and
immunogenic properties as native LDL, indicating that they were discharged intact.
Some of the retro-endocytosed LDL was larger than native LDL, and some exhibited
altered sedimentation properties. When fusion of endosomes with lysosomes was
inhibited by chilling cells to 18° C, the proportion of intraceliular LDL subsequently
released was unaffected, suggesting that retro-endocytosis does not require lysosomal participation. Furthermore, the shorter the internalization phase, the greater
was the proportion of LDL subsequently released, suggesting that LDL was discharged from compartments formed early in endocytosis. Retro-endocytosis of LDL
was stimulated by agents that neutralize acid intraceliular compartments, such as
ionophores (monensin) and weak bases (chloroquine and methylamine). Monensin
increased the proportion of intraceliular LDL released, suggesting that it had a direct
effect on retro-endocytosis. The effect of weak bases appeared to be secondary to
their ability to promote cellular accumulation of undegraded LDL. Thus, retro-endocytosis of LDL becomes a major pathway when intraceliular compartments fail to maintain a low pH or where the intraceliular concentration of LDL reaches abnormal levels.
(Arteriosclerosis 5:45-54, January/February 1985)
O
ne of the most extensively studied model systems for receptor-mediated endocytosis involves the cellular transport of low-density lipoprotein (LDL).1 Entry into the cell is mediated by binding
of LDL to receptors on the cell surface, clustering of
occupied receptors in clathrin-coated pits, and co-
internalization of receptor-LDL complexes into coated vesicles. The complexes are transported via multiple vesicle fusions to large tubular structures called
endosomes. Through the action of an ATP-driven
proton pump, the endosomes are acidified, causing
the ligand to dissociate from its receptor. The receptor then returns to the cell surface where it initiates
another cycle of endocytosis and the bulk of the LDL
is delivered to lysosomes where it is degraded. A
fraction of the internalized LDL appears to escape
lysosomal targeting and is rapidly discharged, apparently intact, into the extracellular medium by a
process we have termed "retro-endocytosis."2
From the Division of Metabolism and Endocrinology, Department of Medicine, University of Washington, Seattle, Washington
(Drs. Aulinskas, Oram, and Bierman), and MRC, UCT Muscle
Research Unit, Department of Medical Biochemistry, University of
Cape Town Medical School, Observatory 7925, Cape Town,
South Africa (Drs. Coetzee, Gevers, and van der Westhuyzen).
This study was supported in part by NIH Grants AM 02456, AG
02673, HL 18645, a grant from R.J. Reynolds Industries, Incorporated, the Medical Research Council of South Africa, the South
African Atomic Energy Board, and the Merrin Bequest.
Address for reprints: Teresa H. Aulinskas, Division of Metabolism and Endocrinology, Department of Medicine RG-26, University of Washington, Seattle, Washington 98195.
Received June 1,1984; revision accepted September 28,1984.
Dr. Godfrey S. Getz kindly acted as Guest Editor for this
manuscript.
The intraceliular movement of ligands to selective
regions of the cell depends on factors that regulate
interaction of the transport vesicles with specific
membrane components and upon sorting of vesicle
contents. The mechanisms involved in targeting,
sorting, and receptor recycling are poorly understood. The fate of vesicle contents appears to vary
with the cell type and the ligand. Endocytosed con45
46
ARTERIOSCLEROSIS
VOL 5, No 1, JANUARY/FEBRUARY 1985
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tents are usually delivered to lysosomes except in
certain specialized cell types where delivery to storage granules (fetal yolk sac),3 Golgi elements (neuroblastoma cells),4 or across cells (neonatal intestinal epithelial cells)5 has been observed. Recent
evidence has shown that a variety of ligands internalized by receptor-mediated endocytosis follow a short
circuit pathway that does not lead to degradation but
results in the rapid exocytosis of intact ligand.2fM>
The occurrence of exocytosis in cultured smooth
muscle cells and fibroblasts2'8 as well as isolated
hepatocytes,7 reticulocytes,8 and macrophages9
suggests that retro-endocytosis may be a general
phenomenon.
We have previously shown2 that retro-endocytosis
is a rapid, temperature-dependent process that occurs following the binding and internalization of LDL
by specific cell-surface receptors. The present study
is focused on the mechanism(s) of retro-endocytosis
and, in particular, on the following questions: (1) Is
the LDL structurally altered during retro-endocytosis? (2) From which intracellular compartment is the
internalized LDL released? (3) Can retro-endocytosis of LDL be modulated by agents that affect vesicular transport processes? Results suggest that most
of the released LDL particles were structurally similar
to native LDL. Receptor-ligand complexes, as well
as free ligand, appear to be cycling from prelysosomal intracellular compartments to cell-surface
locations. Flux through this short-circuit pathway is
facilitated by agents such as chloroquine and methylamine that induce excessive intracellular accumulation of undegraded LDL, as well as by agents like
monensin that raise the pH of endosomes.
Methods
Cells and Llpoprotelns
Human skin fibroblast strains previously characterized as LDL-receptor-normal (GM 3348) and LDLreceptor-internalization-defective (GM 2408A) were
obtained from the Human Genetic Mutant Cell Repository (Camden, New Jersey). In addition, skin fibroblasts were established from a skin biopsy obtained from the ventral surface of the thigh of
normocholesterolemic subjects and were confirmed
as LDL receptor-normal. There were no systematic
differences between the different normal skin fibroblast lines. The cells were maintained in culture as
described previously10 and were used between Passages 5-15. Cells were plated at 0.5 x 105 cells per
35 mm dish, and cultures in late logarithmic growth
(Days 5 to 7) were used in experiments.
Human LDL (density = 1.019-1.063 g/ml) and
lipoprotein-deficient serum (density >1.25 g/ml)
(LPDS) were prepared by discontinuous gradient
centrifugation in a vertical rotor, and the LDL was
labeled with 125I using the iodine monochloride method as modified for lipoprotein.11
Binding, Internalization, Retro-endocytosis and
Degradation of 125I-LDL
The retro-endocytosis of 125I-LDL was determined
by using a pulse-chase procedure as previously described.2 Before each experiment, the number of
LDL receptors was up-regulated by incubation of
cells with culture medium containing 10% human
LPDS. The cell layers then received lipoprotein-deficient culture medium containing the indicated concentration of 125I-LDL and were incubated at 37° C for
the indicated time (pulse period). When pulse incubations were performed at 4° C, the medium was
buffered with 10 mM N-2-hydroxy-ethyl-piperazineN^-ethanesulfonic acid (HEPES), pH 7.4. The
cells were chilled on ice and each layer was washed
four times with 0.2% bovine serum albumin in phosphate-buffered saline, (PBS) pH 7.4, and three times
with PBS at 4° C. Surface-bound LDL was removed
by incubation of the cell layers with PBS containing 4
mg/ml sodium heparin (Sigma) or 4 mg/ml dextran
sulfate at 4° C for 45 to 60 minutes. The cells were
rinsed three times with PBS and then incubated for
up to 2 hours at 37° C in medium containing 10%
LPDS and the indicated concentration of unlabeled
LDL (chase period). Where specified, the cells were
chase-incubated with serum-free medium containing
0.2% albumin without unlabeled LDL. The chase period culture medium was retained and the cells were
washed three times with PBS at 4° C. The 125l-radioactivity (intracellular LDL after chase) and protein
content were then determined in NaOH-solubilized
cell layers. The release of 125l-protein and 125l-degradation products from cells was determined after incubation of the chase medium with an antibody to LDL
(see immunoprecipitation of 125l-protein). As indicated, 30 nM chloroquine (Sigma), 5 mM methylamine
(Sigma), and/or 25 fiM monensin (CalbiochemBehring Corporation) were used. In experiments
where monensin dissolved in ethanol was used,
0.25% ethanol was added to dishes not receiving
monensin. Unless indicated otherwise, each data
point for the figures and tables represents the mean
of determinations on duplicate dishes.
Immunoprecipitation of
125
l-Proteln
Sheep immune serum raised against human LDL
was prepared as described by Albers et al.12 Gel
diffusion studies indicated that this antibody did not
react with HDL (free of apoprotein B), apoproteins
A-l, A-ll, C-l, C-ll, C-lll, D, E, or the d > 1.21 g/ml
plasma fraction. Immunoprecipitation of 125l-protein
was performed by adding 0.2 ml sheep immune serum to 1 ml chase medium containing 35 to 50 p,g/ml
carrier LDL protein followed by incubation at 37° C
for 1 hour and then 4° C for 18 to 24 hours. This
proportion of LDL to immune serum gave maximum
precipitation. The immune precipitate was isolated
by centrifugation, washed by recentrifugation in
PBS, and counted for 1 2 5 I. Trichloroacetic acid (TCA;
final concentration of 10%), was added to the super-
RETRO-ENDOCYTOSIS OF LOW DENSITY LIPOPROTEINS
natant and the amount of nonimmunoprecipitable
l-protein was determined by counting the washed
TCA precipitate for 1 2 5 I. An aliquot of the acid supernatant was counted to determine 12SI-LDL degraded
during the chase period. The extent of retro-endocytosis was determined as the amount of 125l-radioactivity precipitated by an antibody to LDL. Immunoprecipitation of chase medium using affinity purified
rabbit antihuman LDL gave similar results. The "retro-endocytosis index" is defined as the ratio of immunoprecipitable 125l-protein released during the
chase to the intracellular content of 125I before the
chase. As the total 125I in the cell at the end of the
pulse would represent both 125I in the form of native
LDL (immunoprecipitable) as well as some 125I that is
partially or completely degraded (may not be immunoprecipitable), the retro-endocytosis index would
represent a minimum value.
Immunological studies revealed that, although approximately 95% of the 125l-protein material present
during the pulse incubation reacted with an antibody
to LDL, only 50% to 70% of that released upon subsequent incubation in isotope-free chase medium
was immunoreactive. Particles released during the
chase that did not react with anti-LDL immune serum
were separated from particles that had the same size
and immunogenic properties as native LDL by gel
filtration (Biogel A-5m; see molecular sieve chromatography). Autoradiographic analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of nonimmunoreactive 125l-particles purified by
gel filtration yielded molecular weight species that
appeared identical with apolipoproteins A-l and C-l
through C-l 11. Isopycnic sucrose density gradient
centrifugation indicated that these nonimmunoreactive particles exhibited a density of greater than 1.12
g/ml. These particles were found to be present in the
125
I-LDL preparation (<1%). It appears that the release of nonimmunoreactive 125l-particles was the
result of reversible binding to the cell surface of a
radiolabeled HDL-like particle present in the original
125
I-LDL preparation.
12S
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Sedimentation Velocity Centrifugation
Ultracentrifugation of lipoprotein samples was carried out using a two-step KBr gradient. Samples containing 125l-protein in 2 ml medium containing 10%
lipoprotein-deficient serum were adjusted to a density of 1.080 g/ml with solid KBr and layered beneath
15 ml of a 1.045 g/ml solution. Tubes were centrifuged at 65,000 rpm for 5.5 hours at 10° C in a Sorval
865B vertical rotor. Gradients were fractionated by
drainage from the bottom of the tube. Fractions of 14
drops were collected and assayed to determine the
amount of immunoreactive 125l-protein by incubation
with anti-LDL immune serum (recovery >95%).
Molecular Sieve Chromatography
Chromatography of samples were carried out on a
Biogel A-5m column (200-400 mesh, 1.5 x 90 cm)
Aulinskas et al.
47
(Biorad), equilibrated with 100 mM Tris-HCI (pH 7.5)
containing 150 mM NaCI and 10 mM EDTA. Samples contained 12Sl-protein in medium containing
10% LPDS and 500 ^g unlabeled LDL. A sample
volume of 3 ml was applied and the column eluted
with the above buffer at a flow rate of 6-8 ml/h.
Fractions of 60 drops (approximately 2 ml) were collected and assayed to determine the amount of immunoreactive 125l-protein by incubation with anti-LDL
antibody (recovery >90%). Blue dextran and 12SI
were used for the location of the void and total bed
volume, respectively.
Results
To examine the possibility that structural modification of LDL could occur during retro-endocytosis, the
physical properties of retro-endocytosed LDL were
compared with those of native LDL. In these studies,
fibroblasts were pulse-labeled with 12SI-LDL at 37° C,
exposed to heparin to remove surface-bound 1 2 5 ILDL, and subsequently incubated in isotope-free
chase medium at 37° C. The extent of retro-endocytosis was determined as the amount of 125l-radioactivity present in the chase medium that was precipitated by an antibody to LDL. Molecular sieve
chromatography demonstrated that approximately
75% of the immunoreactive 12Sl-particles released
into the chase medium eluted at the same position as
pulse 125I-LDL and heparin-released 125I-LDL (Figure
1). The remainder were larger than native LDL, eluting in the void volume. Sedimentation velocity centrifugation indicated that approximately 70% of the
chase-released immunoprecipitable 125l-protein exhibited a distribution profile like that of native 1 2 S ILDL. The remaining particles either were retained in
the density 1.080 g/ml fraction at the bottom of the
tube or exhibited a flotation velocity greater than
LDL, floating to the top of the tube (Figure 2). It
appears that different lipoprotein fractions were released from fibroblasts: 1) intact LDL, 2) higher molecular weight particles, and 3) particles with altered
sedimentation properties.
Our earlier observations indicated that retro-endocytosis was a rapid, temperature-sensitive process
that occurred following the binding of LDL by specific
cell-surface receptors.2 Experiments in which heparin was used to release surface-bound LDL before
the chase period supported the concept that exocytosis of LDL was taking place, rather than the dissociation of surface-bound LDL. To establish this point,
the metabolism of 125I-LDL by a mutant fibroblast
strain, prevously characterized as LDL-receptor-internalization-defective (GM 2408A), was examined
(Table 1). The inability of the mutant fibroblast strain
to internalize receptor-bound LDL was evident from
the low intracellular concentration and rate of degradation of LDL in relation to the amount bound. The
LDL-receptor internalization-defective cell strain released less LDL than receptor-normal cells, even
48
ARTERIOSCLEROSIS
VOL 5, No 1, JANUARY/FEBRUARY 1985
though the mutant strain bound the same or more
LDL than the receptor-normal strain.
To obtain further support for the concept of LDL
exocytosis, the effects of agents that perturb vesicular transport but do not substantially alter the binding
of LDL to cell surface receptors were examined
(Table 2). Weak bases were utilized to impair the
delivery of LDL to lysosomes and increase organelle
pH.13 Fibroblasts pulse-labeled with 125I-LDL for 18
hours in the presence of chloroquine (30 /xM) or
methylamine (5 mM) retro-endocytosed two- to fourfold more LDL than control cells loaded in the ab-
Native LDL
Native LDL
80Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
10
20
30
Fraction Number
LDL Released in Chase
Heparm-released LDL
Figure 2. Sedimentation velocity centrifugation of retroendocytosed 125I-LDL. The pulse-chase protocol outlined
in the legend to Figure 1 was followed. Ultracentrifugation
of 125l-protein particles in the pulse and chase medium
was carried out as described in Methods. Fractions were
collected and assayed to determine the amount of immunoprecipitable 125l-protein. Fraction 1 represents the most
dense fraction (-1.080 g/ml), and fraction 40 represents
the least dense (-1.045 g/ml).
•o
0)
in
00-
(2)
80-
A
ft
o
0)
m
or
60-
c
V
2
a.
400
0
30
40
Fraction Number
Figure 1. Molecular sieve chromatography of retro-endocytosed 125I-LDL. After incubation for 48 hours in lipoprotein-deficient serum, human skin fibroblasts (2 x 75
cm flasks) were pulse-labeled for 4 hours at 37° C with 40
/xg/ml 125I-LDL. Cells were chilled to 0° C, extensively
washed, treated with heparin, and chase-incubated for 2
hours at 37° C in lipoprotein-deficient medium. To concentrate chase-released particles in as small a volume as
possible, the timing of the pulse-chase procedure on the
second flask was delayed and the chase medium from the
first flask was collected for use on the second flask. 1 2 5 I protein particles in the pulse, heparin, and chase media
were fractionated using a Biogel A-5 m column, and the
amount of immunoprecipitable 125l-protein was determined as described in Methods.
40
20-
0-
(i)
v
o
1 1
f 1
* 1
. i 1
kJ \
1
*>20
30
40
50
60
Fraction Number
Figure 3. Effect of chloroquine on the size of retro-endocytosed 125I-LDL. The protocol in Figure 1 was followed.
Fibroblasts (35 mm dishes) were pulse-labeled for 24
hours with 30 /AM chloroquine (•) or for 4 hours without
chloroquine (o). The cells were washed, treated with heparin, and subsequently chase-incubated at 37° C for 2
hours with (•) or without (o) chloroquine. The chase medium was analyzed by molecular sieve chromatography followed by immunoprecipitation of 125l-protein.
RETRO-ENDOCYTOSIS OF LOW DENSITY LIPOPROTEINS
Table 1. Retro-endocytosls of
I-LDL by Mutant Fibroblast Strains
Expt.
no. Cell strain
LDL-receptor-normal
LDL-receptorinternalization-defective
(GM 2408A)
2*
49
125
125
1
Aulinskas et al.
LDL-receptor-nonnal
(GM3348)
I-LDL (ng • mg- 1 cell protein)
Degraded
in pulse
Bound
before
chase
Retroendocytosed
in chase
Intracellular
after chase
4082 ±99
379 ± 5
74±8
2653 ±31
235±16
327 ± 7
22±1
130±2
2084
129
54
484
66
175
27
96
LDL-receptorinternalization defective
(GM 2408A)
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The indicated fibroblast strains were pulse-labeled for 4 hours with 20 /ug/ml 125I-LDL, treated with
heparin, and chase-incubated for 30 minutes in lipoprotein-deficient medium containing 20 ug/ml unlabeled LDL. The pulse-medium was analyzed for its content of noniodide TCA-soluble 25l-degradation
products. The 1 (-radioactivity released by heparin and the intracellular content of 125l-radioactivity after
the chase were also determined. The chase medium was analyzed for its content of immunoprecipitable
125
l-protein. Data are mean values of duplicates (Expt. 2) or mean values ± SEM of quadruplicate dishes
(Expt. 1).
*LDL receptor activity was up-regulated for 16 hours only.
sence of drugs. In view of the ability of chloroquine to
prevent down-regulation of LDL receptor activity,14
control cells were pulse-incubated for only 4 hours
so that the amount of 125I-LDL bound before the
chase in control and chloroquine-treated cells was
similar. Increased retro-endocytosis was associated
with a massive intracellular accumulation of LDL during the loading period. The addition of weak bases
directly to the chase medium had little effect on the
release of LDL, suggesting that their effect on retroendocytosis was secondary to their ability to promote
cellular accumulation of undegraded LDL. Molecular
sieve chromatography of lipoprotein particles released from fibroblasts revealed that loading in the
presence of chloroquine for 18 hours enhanced the
amount of particles that fractionated in a position
similar to native LDL, as well as those that appeared
to be larger than LDL (Figure 3).
Table 2.
To approach the question of whether LDL was
being retro-endocytosed before or after the fusion of
ligand-containing vesicles with lysosomes, the effect
of temperature was studied. At temperatures below
20° C, ligands are internalized by endocytosis, but
the fusion of endosomes with lysosomes is selectively inhibited.15 We have recently demonstrated16 in the
case of LDL, that endocytosis and receptor recycling
occur at temperatures (<20° C) at which LDL degradation is blocked. To test whether retro-endocytosis
can occur without lysosomal participation, fibroblasts were pulse-incubated at 37° C or 18° C with
125
I-LDL for various lengths of time, and retro-endocytosis was monitored by reincubation at the same
respective temperature for 2 hours (Figure 4). At 18°
C, 125I-LDL degradation was inhibited (Figure 4 A),
while intracellular accumulation still occurred, albeit
at a slower rate than at 37° C (Figure 4 B). At both
Effect of Lysosomotrophlc Inhibitors on Retro-endocytosis of
125
Pulse
Time (hr)i
4
18
4
Addition
Chase
Addition
None
None
Chloroquine
Methylamine
Chloroquine
Methylamine
Chloroquine
Methylamine
None
125
I-LDL
1
I-LDL (ng • mg" cell protein)
RetroBound
before endocytosed
in chase
chase
75±7
109±8
127±4
303 ±9
133±7
61 ±5
86±2
108±5
Degraded
in
chase
Intracellular
after
466 ±20
356±10
185±5
2409±117
302 ±8
353±8
chase
11,318±304
3751 ±73
2768 ±40
2676 ± 4
Fibroblasts were pulse-labeled with 40 ug/ml 125I-LDL for the indicated times, treated with heparin and
chase-incubated for 2 hours in lipoprotein-deficient medium containing 40 ug/ml unlabeled LDL. Where
indicated, 30 uM chloroquine and 5 mM methylamine were employed. The ^-radioactivity releasable by
heparin and the intracellular content of 125l-radioactivity after the chase incubation were determined. The
chase medium was analyzed for its content of immunoprecipitable 125l-protein and 125l-degradation
products. Data are mean values ± SEM of the triplicate dishes.
50
ARTERIOSCLEROSIS
VOL 5, No 1, JANUARY/FEBRUARY 1985
60- Released and Degraded
40o
Q.
20-
o
Intracellular after Pulse
c
8
O>
400-
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
200-
0Retro-endocytosis
Index
0Jr
120 240
0J
0
10
20
30
Pulse Time (min)
Figure 4. Dependence of retro-endocytosis of 125I-LDL
on time of exposure to 1Z5I-LDL at 37° C and 18° C. Fibroblasts were pulse-labeled at 37° C (closed symbols) or 18°
C (open symbols) for the indicated times with 40 ^g/m 1 2 5 I LDL, treated with heparin, and chase-incubated in lipoprotein-deficient medium containing 40 /xg/ml unlabeled LDL
for 2 hours at the same temperature used during the pulse
incubation. When incubations were performed at 18° C,
the medium was buffered with 10 mM HEPES (pH 7.4).
The amounts of immunoprecipitable 125l-protein (circles)
and 125l-degradation products (triangles, dotted line) released with the chase medium were determined and the
cell layer was analyzed for intracellular 125I-LDL after the
chase incubation. The intracellular content of 125l-radioactivity before the chase (squares) is the sum of the intracellular 125l-radioactivity remaining after the chase, plus
immunoprecipitable 12*l-protein, plus 125l-degradation
products released during the chase. The retro-endocytosis
index is the ratio of immunoprecipitable 12Sl-protein released in the chase to the intracellular content of 1 2 5 I radioactivity before the chase. Data in the inset is from a
separate experiment in which cells were chase-incubated
at 37° C for up to 4 hours.
temperatures, the amount of LDL retro-endocytosed
in 2 hours increased with increasing loading time
(Figure 4 A). The fact that retro-endocytosis occurred at 18° C suggested that fusion of endosomes
with lysosomes was not essential for the exocytosis
of intact LDL. The proportion of LDL retro-endocytosed also varied with the length of time cells were
exposed to LDL during the loading phase. The
shorter the loading phase, the greater was the proportion of intracellular LDL retro-endocytosed (Figure 4 C). Furthermore, the proportion of intracellular
LDL retro-endocytosed was the same at both temperatures. The ratio of released LDL to intracellular
LDL has been used as a convenient measure of the
efficiency of retro-endocytosis and will be referred to
as the "retro-endocytosis index". These results support the hypothesis that LDL was retro-endocytosed
from a prelysosomal compartment. Moreover, the
finding that the retro-endocytosis index was higher
after the first few minutes of exposure to LDL suggested that LDL is released from endosomes formed
early in the endocytic pathway.
Because rapid receptor recycling may present a
possible mechanism by which cells could release
LDL previously internalized by the LDL receptor
pathway, we evaluated the effect of monensin on
retro-endocytosis. Monensin has been shown to
raise the pH of normally acid intracellular compartments,17 inhibit receptor recycling,18 prevent dissociation of receptor-ligand complexes within endosomes,19 and inhibit transfer of ligand from
endosomes to lysosomes.20 That monensin was effective in reducing LDL receptor binding activity was
confirmed by the fact that heparin-releasable 1 2 5 I LDL was reduced by 65% when fibroblasts were
pulse-incubated for 30 minutes with 25 ^.M monensin (Table 3). The expected inhibition of LDL degradation was also observed (>90%). Despite a reduction in the amount of LDL endocytosed, the
ionophore increased retro-endocytosis (60% to
200%). With cells loaded in the absence of monensin, its inclusion in the chase phase of the experiment alone was sufficient to increase both the
amount of LDL released and the retro-endocytosis
index. Even with cells loaded for 18 hours in the
presence of chloroquine, the inclusion of monensin
in the chase medium stimulated retro-endocytosis.
This combined drug treatment dramatically increased the amount of LDL released (40-fold) and
the retro-endocytosis index (10-fold).
One possible mechanism to explain retro-endocytosis is that a proportion of the internalized LDLreceptor complexes recycle to the cell surface where
some of the LDL dissociates from its receptor and is
released into the medium. Agents that block acidification of endocytic vesicles, such as monensin and
chloroquine, may enhance the process by preventing intracellular LDL-receptor dissociation.19 To test
this possibility, preloaded fibroblasts were incubated
at 37° C in culture medium containing agents known
to promote the dissociation of LDL from its receptor,
RETRO-ENDOCYTOSIS OF LOW DENSITY LIPOPROTEINS
51
Aulinskas et al.
Table 3. Effect of Monensin on Retro-endocytosls of 12SI-LDL
125
Pulse
Expt Time
no. (hr)
1
0.5 None
Monensin
2
4
18
Bound
before
chase
Degraded
in
chase
(D)
None
324±15
65±1
1153 ±155
Intraceiluiar
after
chase
(IJ
662±16
Monensin
116±11
104±1
45±1
915±7
0.098
4664±86
4500±179
0.023
0.075
17,236 ±393
13,489 + 208
0.064
0.263
Time
(hr) Addition
0.5
Retroendocytosis
index
(RE/lb)
Retroendocytosed
in chase
(RE)
Chase
Addition
I-LDL (ng •mg~1 cell protein)
None
2
None
Monensin
207 ±20
131+31
386 ±21
807±15
253±6
Chloroquine
2
Chloroquine
Chloroquine
+ monensin
179 + 18
1194 ±17
4880±184
171 ±1
152±3
0.035
The conditions outlined in Table 2 were employed. Fibroblasts not exposed to monensin (25 /AM) received 0.25% ethanol.
The retro-endocytosis index is defined as the ratio of immunopredpitable 125l-protein released (RE) to the intraceiluiar
content of 125l-radioactivity before the chase (l b = l a + RE + D). Data are mean values + SEM of triplicate dishes.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
including dextran sulfate, sodium heparin, and
EGTA (Table 4). In each case, the amount of LDL
retro-endocytosed was considerably higher if the
chase medium contained a dissociating agent, suggesting that the return of LDL-receptor complexes.
Similar results were observed with cells treated with
chloroquine or monensin.
The observed increase in retro-endocytosis
caused by dissociating agents was not merely due to
the discharge of residual LDL from the cell surface.
Table 4. Effect of Agents that Dissociate LDL-Receptor Complexes on Retro-endocytosls of 125I-LDL
Pulse
Time
(hr)
4
Chase
Addition
Addition
None
Dextran sulfate
Heparin
EGTA
18
0.5
0.5
Chloroquine
121 ±6
293±14|
213±5f
316±24t
1035 ±67*
Dextran sulfate
65±1
205 ±4f
4:Monensin
Monensin +
dextran sulfate
4°C Pulse
37*C Pulse
P-
744±33
JChloroquine
Chloroquine +
dextran sulfate
None§
Monensin
Retroendocytosed
LDL (ng/mg
cell protein)
When cells were pulse incubated at 4° C, a temperature that allows binding but prevents internalization,
the inclusion of dextran sulfate in the chase medium
had no effect on the release of residual surfacebound LDL (Figure 5). In contrast, after loading at 37°
C, retro-endocytosis was increased by the addition
of dextran sulfate to the medium. Since the amount
of LDL bound to the cells was approximately the
same after the 37° C and 4° C loading periods, dextran sulfate must have stimulated release of LDL
from within the cell, suggesting the return of LDLreceptor complexes to the cell surface.
The time-course of the reappearance of LDL at the
cell surface was studied under conditions that dramatically increase retro-endocytosis, i.e., preloading
in the presence of chloroquine and subsequent exposure to monensin during the chase phase (Figure
6). The amount of LDL on the cell surface was assayed by exposing fibroblasts to a second 4° C hep-
/
I
104±1
253 ±5f
125
Fibroblasts were pulse-labeled with 40 ^g/ml I-LDL
for the indicated times, treated with heparin, and chaseincubated for 2 hours in serum-free medium containing
0.2% albumin. Where indicated, 30 i*.M chloroquine, 25
/iM monensin, 4 mg/ml dextran sulfate, 4 mg/ml heparin,
and 3 mM EGTA, pH 7.6, were employed. Other conditions
were the same as those described in Table 3.
*p < 0.02; tp < 0.001.
^Control value used for statistical evaluation (unpaired
comparison) for each condition of the pulse incubation.
§Dishes contained 0.25% ethanol.
• Control
o DSQr
°
=8
Jj
40
80
I2O
40
Chase Time (min)
80
120
Figure 5. Effect of dextran sulfate on the retro-endocytosis of 12SI-LDL. Fibroblasts were pulse-labeled with 30
^g/ml 125I-LDL for 2 hours at 37° C or 4° C, treated with
dextran sulfate at 4° C, and chase-incubated at 37° C for up
to 2 hours in serum-free medium containing albumin without (•) or with (o) 4mg/ml dextran sulfate. The chase medium was analyzed for TCA-precipitable, 125l-protein. The
amounts of i2fel-LDL bound after the 37° C and 4° C pulse
periods were 23.8 and 23.7 ng 125I-LDL protein/dish
respectively.
52
ARTERIOSCLEROSIS
VOL 5, No 1, JANUARY/FEBRUARY 1985
I50I00-
Retro-endocytosed
Degraded
Heporin-releasoble
(post-chase)
arin treatment after the pulse-chase procedure. To
estimate the relative number of receptors left on the
cell surface after the chase, another group of cells
was incubated with unlabeled LDL during the pulsechase procedure, exposed to heparin at 4° C, and
subsequently incubated with 125I-LDL at 4° C. Both
the amounts of LDL retro-endocytosed into the
chase medium (Figure 6 A), as well as the amount of
125
I-LDL released during the postchase heparin treatment (Figure 6 B), increased rapidly within the first
minutes of the chase. In contrast, the relative number of functionally active LDL receptors (Figure 6 C)
decreased during the chase period, as would be expected if chloroquine and monensin were reducing
the rate of receptor recycling. These data are consistent with the hypothesis that LDL-receptor complexes are returning from intracellular compartments to
the cell surface.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Discussion
Receptor-activity
(post-chase)
20-
10Ratio B/C
0J
i
0
0
'IP
'20
'30
10
20
Chase Time (min)
30
Figure 6. The increase in cell-surface 125I-LDL as a
function of chase time under conditions that enhance retroendocytosis. One set of fibroblasts were pulse-labeled
with 5 ^g/ml 125I-LDL for 18 hours in the presence of 30 ^ M
chloroquine. Monensin (25 juM) was present during the
final 15 minutes of the pulse period. The cells were treated
with heparin containing 10% LPDS at 4° C and chaseincubated at 37° C for the indicated time in serum-free
medium containing albumin with chloroquine and monensin as described in the Methods section. For the zero time
value, cold-chase medium was added and immediately
removed. The chase medium was analyzed for immunoprecipitable 125l-protein and 125l-degradation products (A).
After the chase, the cells were washed once with PBS
containing albumin, chilled, and retreated with heparin at
4° C for 1 hour. The medium was collected and analyzed
for its content of 125l-radioactivity (B). The same procedure
was followed using more fibroblasts, substituting unlabeled LDL for 125 I-LDL After the second treatment with
heparin, the cells were chilled, washed three times with
PBS containing albumin and then assayed for LDL-receptor-activtty by incubation at 4° C with lipoprotein-deficient
medium containing 5 /xg/ml 125I-LDL and 10 mM HEPES
(pH 7.4). The cells were washed seven times as decribed
in Methods, and the 12Sl-radioactivity associated with
NaOH-solubilized cell-layers measured (C).
We have previously demonstrated that a fraction
of the LDL internalized via receptor-mediated endocytosis escapes lysosomal degradation and instead
enters a short-circuit pathway, termed "retro-endocytosis," which results in the rapid discharge of LDL
from fibroblasts and smooth muscle cells.2 The present study was conducted to identify and characterize
the cellular mechanism(s) involved in the retro-endocytosis of LDL and evaluate the changes in the physical properties of LDL processed by this pathway.
When the possibility that structural modification of
LDL could occur during retro-endocytosis was examined, observations indicated that the bulk of the released LDL particles were structurally identical to
native LDL, having the same size, density and immunogenic properties. However, some of the released LDL (approximately 20%) was larger than
native LDL and some exhibited altered sedimentation properties, suggesting that cellular processing
had occurred. It is not known if these two populations
of altered LDL particles are related. Recently, Greenspan and St. Clair21 showed that some of the LDL
retro-endocytosed from monkey-skin fibroblasts had
a greater density than the parent monkey LDL. In
addition, they showed that the retro-endocytosed
material was less immunoreactive with monkey LDL
immune serum than native LDL. Our initial observations indicated that 30-50% of the 12sl-protein released from human fibroblasts was not recognized
by human LDL immune serum. Subsequent studies,
however, demonstrated that the release of the nonimmunoreactive 12Sl-particles was the result of reversible binding to the cell surface of a radiolabeled
HDL-like particle present in the original 125I-LDL
preparation (see Methods section).
Results with skin fibroblasts derived from a patient
with familial hypercholesterolemia confirmed that
retro-endocytosis of LDL is dependent upon the prior
internalization of LDL by the LDL receptor. The mutant fibroblast strain, previously characterized as
RETRO-ENDOCYTOSIS OF LOW DENSITY LIPOPROTEINS
Aulinskas et al.
53
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LDL-receptor-internalization defective, released less
LDL than receptor-normal fibroblasts, even though
the mutant strain had a normal ability to bind LDL.
These results support the concept that release is the
expression of retro-endocytosis from intraceiiuiar
sites rather than the dissociation of residual LDL
from surface sites. When considering the intraceiiuiar compartment(s) involved, several lines of evidence indicate that retro-endocytosis does not require the participation of lysosomes. First, the
particle does not appear to be degraded. Second,
kinetic studies indicate that retro-endocytosis occurs
rapidly, before LDL has reached the lysosomal compartment. Third, retro-endocytosis occurs at temperatures of 20° C or less, at which fusion of endosomes with lysosomes is inhibited.1522 We have
previously demonstrated16 that LDL endocytosis and
receptor recycling continue to occur at temperatures
of 20° C or less at which LDL degradation is blocked.
Current results indicate that the proportion of intraceiiuiar LDL retro-endocytosed was similar at 37° C
and 18° C. Furthermore, the shorter the intemalization phase, the greater the proportion of LDL that
was subsequently released, suggesting that LDL is
released from endosomes formed early in the endocytic pathway. Finally, lysosomotrophic inhibitors,
agents known to block LDL degradation, enhanced
the amount of LDL retro-endocytosed.
by treatment of cells with agents that specifically promote dissociation of LDL from its receptor, such as
dextran suflate, heparin, or EGTA. Addition of these
agents to the medium enhanced retro-endocytosis of
LDL. Furthermore, when LDL-loaded cells were
treated with heparin at 4° C to remove surface-bound
ligand, a new pool of heparin-releasable LDL appeared on the cell surface within minutes after warming the cells to 37° C. Since LDL does not bind to its
specific receptor below pH 6,24 resurfacing receptorbound LDL may be derived from vesicles that have
not yet undergone marked acidification. It is not
known what causes the release of LDL from these
resurfacing vesicles. It would be expected that, even
at neutral pH, some LDL would spontaneously dissociate from its receptor and be discharged from the
cell. It is also possible that the receptor-bound LDL
undergoes modification during transit through the
cell so that its affinity toward its receptor is reduced
and it dissociates more readily. Agents such as ionophores and weak bases, which block acidification of
endosomes, may enhance the resurfacing process
by increasing the relative proportion of vesicles in the
cell that maintain a neutral pH. Resurfacing of receptor-ligand complexes as a mechanism for exocytosis
of internalized ligand has been observed for transferr i n g ^ mannose-terminated glycoconjugates,9 and
galactose-terminated ligands.27'a
Insight into the cellular mechanism involved in retro-endocytosis was provided by studies using weak
bases and ionophores, two classes of agents with
similar modes of action. Both classes have been
shown to raise the pH of normally acidic compartments,1723 lower the rate of receptor recyling,18 prevent dissociation of receptor-ligand complexes within endosomes,19 and inhibit transfer of ligand from
endosomes to lysosomes.20 Results from the present study indicate that both types of agents stimulate
retro-endocytosis. Fibroblasts loaded with LDL in the
presence of chloroquine or methylamine retro-endocytosed two- to eightfold more LDL than control cells
loaded in the absence of drug. The proportion of
intraceiiuiar LDL retro-endocytosed (retro-endocytosis index) was not affected by weak bases, suggesting that their effect on retro-endocytosis was secondary to their ability to cause excessive intraceiiuiar
accumulation of undegraded LDL. In contrast, exposure to monensin increased the retro-endocytosis
index threefold, indicating that the ionophore was
directly influencing the mechanism of LDL release.
The combined effect of weak base and ionophore
action was additive, elevating the amount retro-endocytosed to 40-fold, as well as the retro-endocytosis index to 10-fold. Chloroquine and methylamine
have recently been shown21 to enhance the amount
of monkey LDL retro-endocytosed by monkey skin
fibroblasts.
In summary, results from the present study suggest that some of the LDL internalized by receptormediated endocytosis is rapidly returned to the cell
surface from intraceiiuiar compartments formed early in the endocytic pathway (see Figure 7, Routes 1 3). Receptor-ligand complexes may recycle from endosomes that have not yet undergone marked acidi-
Current results showed a close association between retro-endocytosis of LDL and the rapid resurfacing of LDL still bound to its receptor. Resurfacing
of LDL-receptor complexes could be demonstrated
LDL
RE-LDL
TO MVB AND LYSOSOMES
Figure 7. Model for retro-endocytosis of LDL by human
skin fibroblasts. Y = cell surface receptors, CV = coated
vesicles, CURL = compartment for uncoupling of receptor
and ligand, MVB = multivesicular body, RE-LDL = retroendocytosed LDL.
54
ARTERIOSCLEROSIS
VOL 5, No 1, JANUARY/FEBRUARY 1985
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fication (Routes 1 and 2). Once the pH of the endosomes becomes acidic, LDL irreversibly dissociates
from its receptor. The receptor then returns to the cell
surface (Figure 7, Route 3) and the bulk of the LDL is
transported to lysosomes for degradation (Figure 7,
Route 4). A fraction of LDL may be returned to the
cell surface free in vesicular fluid (Route 3). Recycling receptor-rich vesicles subsequently fuse with
the plasma membrane, discharging their contents
and allowing LDL to dissociate from its receptor.
Some of the LDL may be modified during this recycling process. The physiological significance of retro-endocytosis of lipoproteins by cells remains to be
elucidated. This process may be significant in atherogenesis, since it could contribute to the delivery of
LDL cholesterol to cells, and lead to chemical modification of LDL so that it is recognized by the "scavenger" receptor on macrophages.29 The present study
indicates that retro-endocytosis becomes a quantitatively important alternate pathway in the cellular
processing of LDL when the intracellular concentration of undegraded LDL reaches abnormal levels, as
in the case of lysosomal deficiencies, or when normally acid intracellular compartments fail to maintain
a low pH.
Acknowledgments
We thank Vilma Fernandez, Carole Brewer, Thomas Johnson,
Ingrid Demasius, and Alexandra Strumpfer for their excellent
technical assistance; Anne Ferguson for skillful manuscript preparation; and John J. Albers and Marion Cheung for providing the
LDL-immune serum.
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• LDL receptors
• endocytosis
• cultured fibroblasts
Retro-endocytosis of low density lipoprotein by cultured human skin fibroblasts.
T H Aulinskas, J F Oram, E L Bierman, G A Coetzee, W Gevers and D R van der Westhuyzen
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Arterioscler Thromb Vasc Biol. 1985;5:45-54
doi: 10.1161/01.ATV.5.1.45
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville
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