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INTRODUCTION |
ApoB is the major structural protein responsible for the assembly
of lipoproteins by the liver and intestine. Multiple forms of apoB,
designated as the percentage of the N terminus of the largest secretory
product apoB100 (4536 amino acids), are produced from a single gene
transcript by mRNA editing and proteolytic cleavage (reviewed in
Refs. 1-3). Overproduction of apoB-containing lipoproteins by the
liver is responsible for familial combined hyperlipidemia (4). In
addition, overproduction of triglyceride-rich lipoproteins is
responsible for the human disease familial hypertriglyceridemia (5). In
these patients, the secretion of triglyceride-rich lipoproteins varies
in parallel with the rate of bile acid synthesis (6-8). These findings
suggest that the secretion of very low density lipoprotein triglyceride
is linked to hepatic sterol metabolism via an as yet undefined
mechanism that is dependent upon genes that contribute to hypertriglyceridemia.
The rate of hepatic secretion of apoB is regulated
post-transcriptionally. Only a portion of de novo
synthesized apoB is secreted; the remaining portion is degraded
intracellularly (9). Interruption of apoB translocation is one of
several criteria that lead to increased intracellular degradation
(reviewed in Ref. 10). Both translocation and lipid addition require
the presence of microsomal triglyceride transfer protein
(MTP)1 in the ER (11-13).
MTP exists in the ER lumen as a heterodimer with protein-disulfide
isomerase (reviewed in Ref. 14). In the absence of either sufficient
lipid (15-17) or MTP lipid transfer activity (11-13), apoB
translocation and lipoprotein assembly are blocked. The C terminus of
resulting translocation-arrested apoB, which resides in the cytoplasm
(18), is rapidly degraded by a ubiquitin-dependent
proteasome process (19-21).
The essential requirement of MTP for apoB translocation and lipoprotein
assembly is exemplified by the finding that genetic loss of the
expression of the MTP gene is responsible for the human recessive
disorder abetalipoproteinemia (22, 23). Similar to the CHO cells used
in the studies reported here, the liver of abetalipoproteinemics lacks
the ability to fully translocate apoB into the ER (24) and to secrete
apoB-containing lipoproteins (25, 26). Recent studies in mice having
one MTP allele inactivated show significant reductions in MTP-lipid
transfer activity and the ability to secrete apoB-containing
lipoproteins by the liver (27). These findings raise the possibility
that MTP expression may contribute to the rate-limiting step in the
lipoprotein assembly/secretion pathway.
Cholesterol-7
-hydroxylase NADPH:oxygen oxidoreductase
(7
-hydroxylase) is a liver-specific gene product that controls bile acid synthesis, the major pathway responsible for eliminating cholesterol from the body (reviewed in Ref. 28). The level of expression of 7
-hydroxylase, which is highly variable in response to
diet, metabolic state, and diurnal cycle, has a marked influence on the
assembly and secretion of apoB-containing lipoproteins by cultured rat
hepatoma cells (29). Oxysterols, which are hydroxylated sterol
derivatives, antagonize 7
-hydroxylase-induced changes in the
assembly and secretion of apoB-containing lipoproteins (29). In this
study, we examined how expression of 7
-hydroxylase affected the ER
degradation of translocation-arrested apoB in CHO cells that lack MTP.
Due to a lack of MTP, CHO cells can not fully translocate apoB across
the ER, resulting in its rapid degradation (18, 30). Our results show
that the normally rapid proteasome degradation of
translocation-arrested apoB can be reversibly blocked by
7
-hydroxylase via a sterol-sensitive ubiquitin conjugation step in
the ER.
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EXPERIMENTAL PROCEDURES |
Materials--
An expression plasmid encoding rat
7
-hydroxylase driven by the CMV promoter was constructed and
transfected into CHO cells, as described (29). Another pcDNA3
plasmid containing the coding region of human 7
-hydroxylase driven
by the CMV promoter was a gift from Alan McClelland (Genetic Therapy,
Inc.). Plasmid pCW8 encoding dominant negative ubiquitin, Ub (K48R),
was generously provided by Ron Kopito. An affinity-purified rabbit
antibody against ubiquitin was generously provided by Arthur Haas.
Lactacystin was a generous gift from Ardythe McCracken.
Cell Culture--
All cells were cultured in modified Eagle's
medium (MEM; Life Technologies) containing 5% fetal bovine serum
(Gemni) and antibiotics (100 units/ml penicillin, 100 units/ml
streptomycin, and 500 µg/ml fungizone) and other antibiotics for
selection as indicated below. Within each cell type, there was no
observed difference in cell viability or growth rate between individual
clones used for the experiments. For most experiments, cells were grown
to 80% confluence in the absence of antibiotics unless indicated.
JF7 Cells--
B53 cells (CHO cells expressing apoB53 (18, 30)
were plated at 106 cells/100-mm plate and were
co-transfected with an expression plasmid encoding rat 7
-hydroxylase
driven by the CMV promoter and a plasmid conferring resistance to
hygromycin (31) at an 18:1 molar ratio. Cells were selected in 500 µg/ml hygromycin (for 7
-hydroxylase) and 400 µg/ml G418 (for
apoB). Resistant cells were characterized as described below.
B53-7
h Cells--
B53 cells were co-transfected with a
pcDNA3 plasmid containing the coding region of human
7
-hydroxylase driven by the CMV promoter and a plasmid conferring
resistance to hygromycin, as described above. Resistant cells were
characterized as described below.
B53 Cells Transfected with Dominant Negative Ubiquitin, Ub
(K48R), Plasmid--
The coding sequence for the dominant negative
form of ubiquitin Ub (K48R) (with a Lys
Arg mutation at amino acid
48 (32) was released from plasmid pCW8 using restriction enzyme
BamHI and cloned into expression vector pIND at the
BamHI site (Ecdysone-inducible expression kit, Invitrogen).
The pIND-K48R plasmid is then co-transfected with the pVgRXR plasmid at
a 1:19 molar ratio into B53 cells. Cells were selected in 500 µg/ml
Zeocin for Ub (K48R) and then maintained in 250 µg/ml Zeocin. Single
cell clones were obtained.
Northern Blotting--
Poly(A+) RNA was isolated
from cells using a modification of the guanidinium isothiocyanate
method, as described (33). The resulting mRNA (2-5 µg) was
separated on a 0.8% agarose gel, transferred to a nylon membrane, and
probed with nick-translated 32P-cDNA probes for
7
-hydroxylase, and
-actin was prepared from gel-purified inserts
as described (33).
Western Blotting--
Cells were harvested in phosphate-buffered
saline (PBS) containing a mixture of protease inhibitors (100 µg/ml
aprotinin, 100 µg/ml leupeptin, and 2 mM
phenylmethylsulfonyl fluoride). Western blotting was performed as
described (18). Following SDS-PAGE, the gels were electroblotted onto
nitrocellulose membranes. The nonspecific binding sites of the
membranes were blocked using 10% defatted dry milk, followed by the
addition of primary antibody. The relative amount of primary antibody
bound to the proteins in the nitrocellulose membranes was detected with
species-specific horseradish peroxidase-conjugated IgG. Blots were
developed using the ECL detection kit (Amersham Pharmacia Biotech).
Pulse-Chase Analysis and Immunoprecipitation--
B53 and JF7
cells were grown to 80% confluence on 60-mm plates, after which the
culture medium was changed to methionine-free MEM (Sigma). One hour
later, cells were pulsed with [35S]methionine (100 µCi/ml; DuPont) for 10 min, after which cells were chased with
culture medium containing a 1000-fold excess of unlabeled methionine.
At indicated chase time points, cells were lysed in 1 ml of TETN buffer
(25 mM Tris, pH 7.5, 5 mM EDTA, 250 mM NaCl, and 1% Triton X-100) containing a mixture of
protease inhibitors (100 µg/ml aprotinin, 100 µg/ml leupeptin, and
2 mM phenylmethylsulfonyl fluoride). Immunoprecipitation
was carried out as described (18). Solubilized proteins were precleared with Sepharose CL-4B. 5 µl of a rabbit antiserum specific for human
apoB was preincubated with 20 µl of protein A-Sepharose beads (dry
volume) in 1 ml of TETN buffer at 4 °C overnight. The beads were
then washed with TETN buffer three times. The antibody-bound protein
A-Sepharose conjugates were incubated overnight at 4 °C with the
cellular protein samples in an amount determined empirically to
completely bind the apoB present in each sample. Beads were recovered
by centrifugation in a microcentrifuge and were washed three times with
the TETN buffer. The immunoprecipitates were dissolved in sample buffer
containing SDS and
-mercaptoethanol and separated on a 1-20%
gradient SDS-PAGE. The protein content of the cell lysate was
determined by the Bradford assay (Bio-Rad).
Microsome Isolation and Digestion with Trypsin--
JF7 cells
cultured to 85% confluence were disrupted by nitrogen cavitation, and
microsomes were isolated by subsequent ultracentrifugation (9). Protein
content was analyzed by the Bradford protein assay (Bio-Rad). Trypsin
digestion was carried out as described (18). Each microsomal sample
(containing 50 µg of protein) was incubated with 15 µg/ml trypsin
in ST buffer (0.25 M sucrose, 10 mM Tris-HCl buffer, pH 7.4) for 30 min on ice. The digestion was stopped by adding
protease inhibitors (300 µg/ml soybean trypsin inhibitor, 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin,
and 100 µg/ml leupeptin each). Microsomes were recovered by
centrifugation at 45,000 rpm in a Beckman TLA 45 rotor for 2 h at
4 °C. The microsomal pellet was resuspended in ST buffer and
solubilized in sample buffer containing
-mercaptoethanol. The
samples were separated on a linear 1-20% polyacrylamide gradient
SDS-PAGE, Western blotted, and detected by using anti-human apoB
monoclonal Ab 1D1 and a rabbit antiserum against protein-disulfide
isomerase (a generous gift from Steve Fuller).
Indirect Immunofluorescent Microscopy--
Cells were grown on
coverslips in 100-mm plates to 60% confluence. Media were removed from
the plates, and the cells were washed three times with PBS before they
were fixed with 3% paraformaldehyde for 15 min. Cells were then
permeabilized with 1% Triton X-100 for 7 min. Nonspecific binding
sites in the cells were blocked by incubation with 3% bovine serum
albumin in PBS for 30 min. The cells were then incubated for 45 min
with 20 µl of affinity-purified antibodies. At the end of the
incubation, cells were washed four times with PBST (PBS containing
0.1% Tween 20) and then incubated with the appropriate
species-specific Texas Red-conjugated IgG. After washing with PBST, the
coverslips were examined using a Nikon microscope with a camera attached.
Detection of Ubiquitinated ApoB--
Cells were washed once with
ice-cold PBS. Cellular proteins were then solubilized in 1 ml of TETN
buffer containing a mixture of protease inhibitors (see "Pulse-Chase
Analysis and Immunoprecipitation"). ApoB was immunoprecipitated using
5 µl of a rabbit antiserum specific for human apoB preincubated with
20 µl of protein A-Sepharose beads. The immunoprecipitates were
separated on a 1-20% gradient SDS-PAGE and then electroblotted onto
nitrocellulose membranes. The ubiquitin conjugated on apoB was detected
by an affinity-purified rabbit antibody against ubiquitin, followed by
chemiluminescence (Amersham Pharmacia Biotech). Western blots were
scanned by a densitometer and analyzed by the ImageQuant program.
 |
RESULTS |
Expression of 7
-Hydroxylase mRNA and Protein in B53
Cells--
Previous results showed that when apoB53 is stably
expressed in CHO cells (B53 cells), it is rapidly degraded, producing
an N-terminal fragment that is secreted without a lipid core (18). The
proteolytic inhibitor ALLN blocks the formation of the N-terminal apoB
fragment and causes intact apoB53 to accumulate as a transmembrane protein in isolated microsomes. These cells, stably transfected with a
plasmid expressing rat 7
-hydroxylase, are hereafter referred to as
JF7 cells. The rate of growth of JF7 cells was indistinguishable from
B53 and wild-type CHO-K1 cells. In JF7 cells, a single band for
7
-hydroxylase mRNA was detected by Northern blot, whereas none
was detected in B53 cells (data not shown). Analysis of the content of
7
-hydroxylase protein by Western blot showed a single protein band,
identical in size to the native protein (data shown below). The enzyme
activity of 7
-hydroxylase in CHO cells transfected with the
CMV-driven plasmid containing the rat cDNA was ~10 pmol/mg protein/min.
Expression of 7
-Hydroxylase Causes Intact ApoB53 to Accumulate
by Blocking a Pathway That Is Also Inhibited by ALLN--
In the
absence of proteolytic inhibitors, intact apoB53 was nearly absent in
B53 cells (Fig. 1, lane
1). Treating B53 cells with ALLN caused intact apoB53 to
accumulate (Fig. 1, lane 2). Remarkably,
7
-hydroxylase expression caused intact apoB53 to accumulate (JF7
cells; Fig. 1, lane 3). The amount of apoB53 in JF7 and B53 cells treated with ALLN was similar (Fig. 1, compare lane 3 with lane 2).
Moreover, treating JF7 cells with ALLN did not cause further
accumulation of apoB53 (Fig. 1, lane 4). This experiment was performed at least five times (using different cell
preparations), and there were no significant differences in the content
of apoB53 in JF7 cells treated with or without ALLN. Additional studies
using [35S]methionine pulse labeling and
immunoprecipitation also showed that ALLN did not cause the
accumulation of intact apoB53 in JF7 cells (data not shown).
Interestingly, treating JF7 cells with ALLN did increase the amount of
smaller apoB fragments (Fig. 1, lane 4). These
data suggest that, in contrast to apoB53, the degradation of small apoB
peptides is blocked by ALLN in JF7 cells. Thus, JF7 cells are sensitive
to proteolytic block by ALLN. These combined data suggest that the
expression of 7
-hydroxylase caused intact apoB53 to accumulate by
blocking a proteolytic process that is blocked by ALLN in cells that do
not express 7
-hydroxylase (i.e. B53 cells; Fig. 1,
lane 2).

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Fig. 1.
Effect of 7 -hydroxylase and ALLN on the
cellular content of apoB in CHO cells. CHO cells expressing only
human apoB53 (B53 cells), apoB53 and rat 7 -hydroxylase (JF7 cells),
and apoB53 and human 7 -hydroxylase (B53-7 h) were treated with
(+) or without ( ) ALLN (50 µg/ml) for 18 h prior to harvest.
Cell extracts (100 µg of protein) were separated by SDS-PAGE, Western
blotted, and detected (using ECL) with monoclonal antibody 1D1 (34).
The migration of molecular weight standards is indicated.
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To exclude the possibility that the phenotype we observed was due to an
artifact of the individual plasmid, the transfection procedure, or the
site of genomic integration, we isolated three individual single cell
clones of B53 cells expressing rat 7
-hydroxylase. Each clone showed
a comparable level of intact apoB53 in the absence of ALLN. Moreover,
to rule out the possibility that the JF7 phenotype was due to a
phenomenon unique to the transfected plasmid, we used an entirely
different plasmid encoding human 7
-hydroxylase (B53-7
h cells).
Cells stably expressing human 7
-hydroxylase displayed an
accumulation of apoB53 that was similar to that of JF7 cells (Fig. 1,
lane 5). Additional experiments showed that B53
cells transfected with different vectors not expressing
7
-hydroxylase (e.g. luciferase and MTP) together with a
hygromycin resistance plasmid did not cause the JF7 phenotype
(i.e. the accumulation of intact apoB53; data not shown).
These data indicate that the accumulation of apoB53 in JF7 cells is due
to the expression of 7
-hydroxylase (rat or human) and cannot be
ascribed to a phenomenon caused by the transfection procedure,
metabolic selection, or a single type of expression plasmid. The
finding that inhibition of proteolysis by ALLN caused equivalent
amounts of intact apoB53 to accumulate in B53 cells and JF7 cells (Fig.
1, compare lanes 2 and 4) suggests
that both cells types synthesize similar amounts of apoB53.
Expression of 7
-Hydroxylase in CHO Cells Blocks the Degradation
of ApoB53--
The turnover of newly synthesized apoB53 was determined
in JF7 and B53 cells using pulse-chase analysis. In both groups of cells, maximal accumulation of [35S]methionine-labeled
apoB53 was detected after 30 min of chase (Fig.
2). Similar pulse-chase labeling of apoB
has been observed using rat hepatocytes (9) and human hepatoma cells
(16). The amount of maximally labeled apoB53 in JF7 cells at the 30-min chase time was 14-fold higher than that in B53 cells. These data are
consistent with the proposal that apoB53 was degraded in a manner that
appeared to be co-translational and that the expression of
7
-hydroxylase in JF7 cells blocked this process.

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Fig. 2.
Pulse-chase analysis of apoB53 turnover in
B53 and JF7 cells. B53 and JF7 cells were incubated in
methionine-free MEM culture medium for 1 h and then pulsed with
[35S]methionine (200 µCi/ml; DuPont) for 10 min, after
which cells were chased with medium containing a 1000-fold excess of
unlabeled methionine. Cells were harvested at the indicated time
points. An aliquot containing half of the total cell suspension was
immunoprecipitated with a rabbit antiserum specific for human apoB. The
immunoprecipitates were separated by SDS-PAGE, and radioactivity was
assayed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Each value is the mean of duplicate samples. Variation of the mean
values was <10%.
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Oxysterols Reverse the Inhibition of ApoB53 Degradation Caused by
7
-Hydroxylase--
We examined if oxysterols could reverse the
block in apoB53 degradation exhibited by JF7 cells. Adding
25-hydroxycholesterol and 7-ketocholesterol to JF7 cells caused a
marked increased in the rate of apoB53 degradation (Fig.
3). The oxysterol effect was observed
during the 30-min pulse period and the 30-min chase period. After the
30-min chase period, the rate of decay of apoB53 was similar to that of
untreated cells (Fig. 3). Additional data show that total protein
synthesis was indistinguishable between JF7, B53, and JF7 cells treated
with oxysterols (i.e. the incorporation of
[35S]methionine into trichloroacetic acid-precipitable
protein was similar in all groups; data not shown). The pulse-chase
experiments were performed three separate times (using different
preparations of B53 and JF7 cells). In all experiments, oxysterols
reversed the blocked degradation of intact apoB53. In two additional
experiments, JF7 cells were incubated with and without oxysterols for
24 h. Western blot analysis of the cell extracts showed a 70-95%
decrease in the cellular content of apoB53 in cells treated with
oxysterols (data not shown). The combined data indicate that the rapid
degradation of apoB that is blocked by expression of 7
-hydroxylase
can be reversed by oxysterols.

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Fig. 3.
Oxysterols reverse the blocked degradation of
apoB53 in JF7 cells. JF7 cells were incubated with and without
oxysterols (25-hydroxycholesterol and 7-ketocholesterol (5 µg/ml) for
14 h. The culture medium was changed to methionine-free MEM (with
and without oxysterols), and cells were incubated for 1 h and then
pulsed with [35S]methionine (200 µCi/ml; DuPont) for 30 min. The cultured medium was changed to MEM containing a 1000-fold
excess of unlabeled methionine. Cells were harvested at the indicated
time points. An aliquot containing half of the total cell suspension
was immunoprecipitated with a rabbit antiserum specific for human apoB.
The immunoprecipitates were separated by SDS-PAGE, and radioactivity
was assayed using a PhosphorImager (Molecular Dynamics). Each value is
the mean of duplicate samples. Variation of the mean values was
<10%.
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ApoB Accumulates as a Transmembrane Protein in JF7 Cells--
We
examined the localization of apoB53 that accumulated in JF7 cells
following subcellular membrane fractionation and isolation. Following
cell disruption and ultracentrifugation, >90% of the apoB53 in JF7
cells was isolated in the 100,000 × g pellet
(i.e. microsomes). Moreover, in microsomes prepared from JF7
cells essentially all apoB53 was susceptible to digestion with
exogenous trypsin (Fig. 4). The major and
smallest molecular weight proteolytic fragment of apoB produced by
trypsin digestion had a molecular mass of 69 kDa, which is identical to
the results obtained using B53 cells treated with ALLN (18). There were
two additional immunoreactive bands (of about 128 and 116 kDa) that
were detected in JF7 microsomes following trypsin treatment. These
immunoreactive bands showed markedly less chemiluminescence compared
with the 69-kDa band. The 69-kDa fragment contained a defined apoB
N-terminal epitope (residues 474-539) as demonstrated by its
recognition by monoclonal antibody 1D1 (34) (Fig. 4). In contrast to
the complete degradation of apoB53, the ER luminal protein,
protein-disulfide isomerase, was resistant to trypsin digestion (Fig.
4), indicating that the microsomes remained intact. The combined data
suggest that the majority of apoB53 that accumulates in JF7 cells
assumes a transmembrane orientation in which 69 kDa of the N terminus resides within the ER lumen and the remaining C terminus is exposed to
the cytoplasm.

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Fig. 4.
Trypsin digestion of apoB53 in microsomes
obtained from JF7 cells. Microsomes were isolated from JF7 cells
by nitrogen cavitation and subsequent ultracentrifugation (18).
Microsomes (containing 50 µg of protein) were incubated with 15 µg/ml of trypsin for 30 min on ice. The digestion was stopped by
adding a mixture of protease inhibitors. Microsomes were recovered by
ultracentrifugation and subjected to SDS-PAGE and Western blotting.
ApoB (monoclonal antibody 1D1 recognizing the N-terminal epitope of
apoB (34)) and protein-disulfide isomerase were detected using an ECL
kit (Amersham Pharmacia Biotech). The migration of molecular weight
standards is indicated.
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In JF7 cells, ApoB53 Accumulates in Segregated Membrane Domains of
the Secretory Pathway--
Indirect immunofluorescence was used to
examine the accumulation of apoB in JF7 cells. We used two
epitope-specific antibodies: monoclonal antibody 1D1, which recognizes
the N-terminal residues 474-539 (34), and a rabbit antiserum, which
recognizes a C-terminal epitope of apoB53 at residue 2140 (35). The
N-terminal epitope-specific antibody will recognize both intact apoB53
and N-terminal apoB peptides, whereas the C-terminal specific antiserum
will recognize only intact apoB53. In B53 cells, the N-terminal
specific antibody resulted in a diffuse reticular immunofluorescence
pattern (Fig. 5). In JF7 cells, the
N-terminal epitope-specific antibody showed a unique punctate
immunofluorescence pattern that overlaid a more diffuse reticular
pattern (Fig. 5). In contrast, the antiserum recognizing the C-terminal
epitope showed no specific immunofluorescence in B53 cells, while there
was a defined punctate immunofluorescence pattern in JF7 cells (Fig.
5). The punctate distribution of apoB53 detected by the C-terminal
epitope antibody in JF7 cells is similar to the pattern observed for
apoB100 in HepG2 cells treated with lactacystin (20). These data
suggest that in JF7 cells, intact apoB53 accumulates in isolated
domains of the secretory pathway (e.g. "Russell Bodies")
in a manner similar to that described for a mutant form of IgM that can
be neither degraded nor secreted (36). Sequestration of
translocation-arrested apoB may allow normal cell function and
viability to be maintained when the "quality control" proteasome
degradation pathway is evaded.

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Fig. 5.
Indirect immunofluorescence microscopy of
apoB in B53 and JF7 cells. Cells were grown on coverslips to 60%
confluence. After removing the media, cells were washed and fixed with
paraformaldehyde. Cells were then permeabilized with 1% Triton X-100.
Nonspecific binding sites in the cells were blocked by incubation with
3% bovine serum albumin. The cells were then incubated for 45 min with
affinity-purified antibodies: N-terminal specific (N-t)
monoclonal antibody 1D1, which recognizes an epitope residing between
amino acids 474 and 539 of human apoB (34) and C-terminal specific
(C-t) rabbit antibody made against a synthetic human apoB
peptide containing residue 2140 (35). The cells were washed and then
incubated with Texas Red-conjugated goat anti-mouse or anti-rabbit IgG.
The coverslips were examined using a Nikon microscope with an attached
camera.
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ApoB Ubiquitination Is Blocked in JF7 Cells--
To examine if the
expression of 7
-hydroxylase might have inhibited the proteasome
degradation of apoB53, cells were cultured with or without the
proteasome inhibitor lactacystin. Cells were harvested, and apoB was
immunoprecipitated using a polyclonal rabbit antibody against human
apoB. The immunoprecipitates were separated by an SDS-PAGE gel, blotted
onto membranes, and then reacted with an antibody against ubiquitin and
subsequently with monoclonal antibody 1D1, which recognizes the N
terminus of apoB. In B53 cells not treated with lactacystin, almost no
intact apoB53 was detected using the antibody against apoB (Fig.
6A, lane
1). There were a limited number of protein bands produced
with the anti-ubiquitin antiserum (Fig. 6B, lane
1). In marked contrast, treating B53 cells with lactacystin
caused immunoreactive apoB53 to accumulate (Fig. 6A,
lane 2). Moreover, the accumulated apoB53 contained polyubiquitin conjugates as shown by the molecular weight "ladder" detected by the anti-ubiquitin antibody (Fig.
6B, lane 2). Although there was a
large amount of apoB53 accumulated in untreated JF7 cells (Fig.
6A, lane 3), the amount of ubiquitin conjugates of apoB was similar to that in B53 cells without lactacystin treatment (Fig. 6B, compare lane 3 with lane 1). Treating JF7 cells with lactacystin
did not change the amount of apoB53 in the cell (Fig. 6A,
lane 4) or the amount of apoB ubiquitin
conjugates (Fig. 6B, lane 4).
Additional studies show that when the same cell extracts were treated
with preimmune control rabbit antiserum, there were no immunoreactive
bands that contained either the anti-apoB or the anti-ubiquitin
epitopes (data not shown). The lack of accumulation of
ubiquitin-conjugated apoB53 in JF7 cells (with and without lactacystin
treatment) suggests that 7
-hydroxylase blocks the ubiquitin
conjugation of apoB53.

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Fig. 6.
Effect of the proteasome inhibitor
lactacystin on the accumulation of ubiquitin-conjugated apoB and
7 -hydroxylase protein. B53 and JF7 cells were treated with or
without 50 µM lactacystin for 14 h. Cells were
harvested in TETN250 buffer. Equal portions of the cell lysate were
immunoprecipitated using a rabbit polyclonal anti-apoB antibody. The
immunoprecipitates were separated on an SDS-PAGE gel and then Western
blotted for apoB (A, using monoclonal antibody 1D1 (34)) and
ubiquitin (B, using an affinity-purified rabbit antibody).
In other experiments, JF7 cells were treated with and without 50 µM lactacystin for 14 h. Cells were harvested and
disrupted by nitrogen cavitation, and microsomes were isolated by
ultracentrifugation. Microsomes from treated and untreated cells were
Western blotted using an affinity-isolated antibody directed against
rat 7 -hydroxylase (C).
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The rate of turnover of 7
-hydroxylase in vivo is rapid
and is essential for the diurnal rise and fall in enzyme expression (37). We examined if the 7
-hydroxylase expressed in JF7 cells was
also degraded by the proteasome. The specific proteasome inhibitor lactacystin caused a marked increase in the accumulation of
7
-hydroxylase protein in JF7 cells (Fig. 6C). These
findings suggest that while expression of 7
-hydroxylase blocks the
proteasome degradation of apoB53, it does not block its own degradation
by the proteasome.
Recapitulation of the JF7 Cell Phenotype by Expressing a Dominant
Negative Form of Ubiquitin in B53 Cells--
To examine the hypothesis
that 7
-hydroxylase blocks apoB53 degradation by blocking its
conjugation with ubiquitin, we attempted to recapitulate the JF7 cell
phenotype by blocking ubiquitin conjugation using a dominant-negative
form of ubiquitin (32). The dominant-negative form of ubiquitin has a
lysine to arginine mutation that prevents polyubiquitination (Ub
(K48R)) (32). Two different cell lines expressing dominant negative
ubiquitin were derived from B53 cells transfected with either a plasmid
containing the constitutive CMV promoter or a plasmid system that is
ecdysone-inducible (38).
B53 cells were co-transfected with a plasmid expressing Ub K48R, which
contains a His tag and a c-myc epitope (32) and a plasmid
conferring puromycin resistance (to allow for metabolic selection in
the presence of puromycin 10 µg/ml). A single colony of
puromycin-resistant cells was obtained and characterized. These cells
grew at a rate indistinguishable from B53 and wild-type CHO-K1 cells
(data not shown). The following data indicate that these cells
expressed Ub (K48R) mRNA and protein (Fig.
7A): 1) a Northern blot showed
Ub (K48R) mRNA in Ub (K48R) cells but not from B53; 2) a protein
having the expected molecular weight of Ub (K48R) could be
affinity-isolated by a nickel column from the Ub (K48R) cells but not
from B53 cells. This protein displayed immunoreactivity to an
anti-ubiquitin antibody and to an anti-c-myc antibody.
Moreover, cells expressing Ub (K48R) showed an accumulation of intact
apoB53 (Fig. 7B) that was similar to that displayed by JF7
cells (Fig. 1). Furthermore, like JF7 cells, the apoB53 that
accumulated in cells expressing UB (K48R) showed no accumulation of
ubiquitin-conjugated apoB53 when treated with either ALLN or lactacystin (data not shown).

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Fig. 7.
Dominant negative ubiquitin blocks apoB
degradation. A, B53 cells and B53 cells stably
expressing Ub (K48R) were examined for the presence of Ub (K48R) as
follows: mRNA by Northern blots (top) and nickel
affinity column-isolated protein recognized by anti-ubiquitin antibody
following Western blotting (middle) and also recognized by
an anti-c-myc antibody (bottom). B,
B53 cells and B53 cells stably expressing Ub (K48R) were examined for
the presence of apoB53 by Western blotting using monoclonal antibody
1D1 (34). C, three individual clones of B53 cells stably
expressing dominant negative ubiquitin Ub (K48R) produced by an
ecdysone-inducible plasmid were incubated with or without muristerone
(1 µM) for 3 days and then with 5 µM
muristerone for 1 day. Cellular proteins were separated on
SDS-polyacrylamide gel and then Western blotted for apoB by monoclonal
antibody 1D1 (34). The migration of molecular weight standards is
indicated.
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To rule out the possibility that the selection for stable expression of
Ub (K48R) might have caused the accumulation of apoB53 via a mechanism
not directly related to impaired ubiquitin conjugation, we isolated
three separate clones of B53 cells expressing Ub (K48R) in a form that
can be reversibly induced by the insect hormone muristerone A. In the
absence of muristerone A treatment, B53 cells transfected with the
plasmid showed a phenotype similar to that of B53 cells
(i.e. no intact apoB53 was detected; Fig. 7C). In
contrast, following treatment with muristerone A, there was an
accumulation of apoB53 in all three independent single cell clones
(Fig. 7C). Analysis of the culture medium of these cells
treated with muristerone A showed that no intact apoB53 was secreted
(data not shown). Thus, the expression of the dominant-negative form of
ubiquitin recapitulated the phenotype of JF7 cells.
7
-Hydroxylase Increases the Cellular Content of Mature SREBP1
and Increases the Synthesis of Lipids--
The cellular content of
mature SREBP1 was greater in JF7 cells compared with B53 cells (Fig.
8A). Adding
25-hydroxycholesterol (1 µg/ml) with cholesterol (10 µg/ml) caused
the cellular content of mature SREBP1 to decrease in both JF7 and B53
cells. These data suggest that CHO cells, like McArdle rat hepatoma
cells (29) respond to the expression of 7
-hydroxylase (increasing)
and oxysterols (decreasing) by changing the cellular content of mature
SREBP1. The synthesis of all very low density lipoprotein lipids
(phospholipids, triglycerides, cholesterol, and cholesterol esters)
were all increased in JF7 cells compared with wild-type CHO and B53
cells (Fig. 8B).

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Fig. 8.
Cellular content of SREBP1 and lipid
synthesis in B53 and JF7 cells. B53 and JF7 cells were plated and
grown to 85% confluence. A, both groups of cells were then
cultured for 24 h in medium with or without 25-hydroxycholesterol
(1 µg/ml) with cholesterol (10 µg/ml). Cells were harvested and
fractionated into nuclei and membrane fractions, and the content of
SREBP1 was determined by Western blot analysis. B, CHO-K1,
B53, and JF7 cells were plated and grown to 85% confluence.
[2-14C]Acetate (3.3 µCi/ml) was added to the medium,
and cells were incubated for 2 h. Cells and medium were harvested
and extracted with chloroform/methanol, the radiolabeled lipids were
separated by TLC, and the radioactivity was quantitated by
-scintillation analysis. Values represent the means ±S.D. of three
separate cell extracts in each group of cells.
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DISCUSSION |
The signals responsible for initiating ubiquitin conjugation and
entrance into the "quality control" ubiquitin-dependent
proteasome pathway are not well understood. These signals may be
mediated by changes in protein structure caused by folding,
post-translational modifications, and/or association with other
molecules (e.g. ligands and substrates). The results of our
studies suggest that the signals regulating the entrance of apoB into
the ubiquitin-dependent proteasome degradation pathway are
intimately linked to sterol metabolism. Furthermore, sterol-sensitive
ubiquitin conjugation determines the two possible fates of apoB in CHO
cells: 1) rapid degradation by the proteasome or 2) sequestration as a
transmembrane protein in segregated domains of the ER.
Treating B53 cells with lactacystin caused the accumulation of intact
apoB53 and 7
-hydroxylase protein in JF7 cells (Fig. 6). Since
lactacystin is a specific inhibitor of the proteasome (39), it is
reasonable to assume that the proteasome is responsible for degrading
apoB and 7
-hydroxylase protein. Moreover, ubiquitin-conjugates of
apoB53 accumulated as a result of lactacystin-blocked proteasome function (Fig. 7). Thus, we conclude that in CHO cells
ubiquitin-conjugated apoB is the substrate degraded by the proteasome.
In human hepatoma cells, lactacystin also caused ubiquitin-conjugated
apoB to accumulate as a translocation-arrested form (19-21). These
results suggest that the degradation pathway of translocation-arrested
apoB is similar in hepatic and nonhepatic cells.
In marked contrast to the ubiquitin-conjugated intact apoB53 that
accumulates in cells treated with lactacystin, the intact apoB that
accumulates in JF7 cells is not ubiquitin-conjugated (Fig. 7). These
data provide compelling evidence showing that expression of
7
-hydroxylase markedly reduces the proteasome degradation of apoB53
by inhibiting ubiquitin conjugation. Moreover, the additional finding
that expression of a dominant negative ubiquitin plasmid in B53 cells
recapitulates the JF7 cell phenotype further suggests that the
ubiquitin conjugation step is the site where 7
-hydroxylase acts to
divert translocation-arrested apoB away from the proteasome-degradative pathway.
Our finding that B53 cells that stably express the dominant negative
ubiquitin, Ub (K48R), accumulate apoB53 and show similar rates of
viability and replication as CHO cells not expressing Ub (K48R)
suggests that essential processes thought to involve ubiquitin-dependent proteasome degradation (e.g.
those required for cell cycle (40)) still function or are replaced by
other processes. In order for the dominant negative Ub (K48R) to block ubiquitin conjugation, it must compete with the endogenous ubiquitin pool for entering a particular ubiquitin conjugation system. It is
possible that the ubiquitin-dependent proteasome
degradation of individual proteins may have different susceptibilities
to inhibition by the dominant negative ubiquitin. Thus, processes that
are essential for survival may be resistant to Ub (K48R) inhibition.
Alternatively, there may be other "ubiquitin-like" molecules that
can replace essential ubiquitin-dependent proteasome processes (e.g. sentrin (41, 42) and Smt3p (43)). The B53 cells stably expressing Ub (K48R) may be useful to examine these possibilities.
In JF7 cells, intact apoB53 accumulates in microsomal membranes in a
form that is susceptible to degradation by exogenous trypsin (Fig. 4).
These data suggest that in JF7 cells, intact apoB53 spans the membrane
bilayer. Moreover, trypsin digestion of microsomes from JF7 cells
produced a similar 69-kDa N-terminal fragment that was produced by
digestion of microsomes obtained from B53 cells treated with ALLN (18).
These data indicate that 7
-hydroxylase does not overcome the block
in apoB translocation exhibited by CHO cells. This conclusion is
further supported by the finding that JF7 cells do not secrete any
intact apoB53. We have proposed that the block in apoB translocation
exhibited by CHO cells is due to the lack of MTP expression (18).
Additional studies showing that expressing MTP in JF7 cells allows
intact apoB to be secreted as a lipoprotein particle (data not shown) support this hypothesis.
The findings showing that apoB can exist as a stable transmembrane ER
protein are nevertheless paradoxical, since apoB has no amphipathic
-helices that are sufficiently long to act as canonical
membrane-spanning domains (44-46). A similar situation exists for
prion protein, which like apoB contains no canonical membrane-spanning
domains but can exist as a stable transmembrane protein (47). It is
likely that in addition to amphipathic
-helices, alternative
structures allow proteins to stably span membrane bilayers.
Our findings showing that oxysterols reverse the blocked degradation of
apoB in JF7 cells (Fig. 3) provide further evidence that
7
-hydroxylase blocks the ubiquitin conjugation of apoB by altering
the sterol status of cells. There are other examples of proteins that
are proteolytically cleaved in the ER by processes that are regulated
by 7
-hydroxylase and/or oxysterols. Degradation of the ER enzyme
HMG-CoA reductase, which regulates the isoprenoid biosynthetic pathway,
is intimately linked to the sterol status of cells (48). The rate of
degradation of HMG-CoA reductase is accelerated by several nonsterol
and sterol metabolites (49-52). Similar to the results reported here
for apoB, lactacystin also blocks the rapid degradation of HMG-CoA
reductase (53). In yeast, disruption of a gene required for proteasome
assembly causes the degradation of HMG-CoA reductase protein to be
impaired (54). The identities of the signals and the mechanisms through
which sterols affect the proteolytic processing and/or degradation of HMG-CoA reductase remain unknown.
Our findings show that 7
-hydroxylase expression in CHO cells blocked
the ubiquitin conjugation of apoB in a manner that could be reversed by
oxysterols. These data suggest that sterols alter ubiquitin conjugation
of apoB. It is interesting to note that JF7 cells contain significantly
greater levels of mature SREBP1 than do B53 cells (Fig. 7A).
In addition, similar to the reversal of the blocked degradation of
apoB53, the increased content of mature SREBP1 displayed by JF7 cells
is also blocked by oxysterols, albeit with one potentially important
difference. The decrease in mature SREBP1 required only
25-hydroxycholesterol, whereas the reversal of apoB degradation
required both 7-ketocholesterol and 25-hydroxycholesterol.
7-Ketocholesterol is unique in its ability to block the enzymatic
activity of 7
-hydroxylase (55). Based on these findings, it is
reasonable to propose that the enzymatic activity of 7
-hydroxylase
is responsible for reducing the ubiquitin conjugation and subsequent
degradation of translocation-arrested apoB53. The combined data are
consistent with the proposal that 7
-hydroxylase expression acts via
changing sterol metabolism and that oxysterols antagonize this effect.
As a result, the ubiquitin conjugation of apoB is impaired, as is its
degradation by the proteasome. Concurrent with the blocked degradation
of apoB, there is an increase in the content of mature SREBP1 and
lipogenesis. Thus, expression of 7
-hydroxylase in both CHO cells and
McArdle rat hepatoma cells (29) causes similar phenotypic changes:
increased content of SREBP1, increased lipogenesis, and decreased
degradation of apoB. There is one important difference between these
two cell types: the presence of MTP in McArdle rat hepatoma cells
allows apoB to be translocated and assembled into a lipoprotein particle.
Overproduction of triglyceride-rich lipoproteins is responsible for the
human disease familial hypertriglyceridemia (5). In these patients, the
secretion of triglyceride-rich lipoproteins varies in parallel with the
rate of bile acid synthesis (6-8). The increased synthesis of bile
acids in hypertriglyceridemic patients has been ascribed to decreased
bile acid absorption (8). Interestingly, treatment of
hypertriglyceridemic patients with chenodeoxycholic acid, which would
be expected to decrease the activity of 7
-hydroxylase, reduces
fasting plasma triglyceride levels (56). Conversely, in many
hypertriglyceridemic patients, bile acid binding resins increase bile
acid synthesis and cause a transient exacerbation of their
hypertriglyceridemia due to increased hepatic production of very low
density lipoprotein (57). Our findings showing that 7
-hydroxylase
blocks the ubiquitin-dependent proteasome degradation of apoB may
provide insights toward understanding the complex relationships between
lipoprotein assembly/secretion and hepatic cholesterol/bile acid
metabolism and how these relationships may contribute to the disordered
lipid metabolism.