Effects of CYP7A1 overexpression on cholesterol and bile acid
homeostasis
W. M.
Pandak1,
C.
Schwarz1,
P. B.
Hylemon2,
D.
Mallonee2,
K.
Valerie1,
D. M.
Heuman1,
R. A.
Fisher3,
Kaye
Redford1, and
Z. R.
Vlahcevic1
Departments of 1 Medicine, 2 Microbiology, and
3 Surgery, Veterans Affairs Medical Center and Virginia
Commonwealth University, Richmond, Virginia 23249
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ABSTRACT |
The initial and rate-limiting step in the classic
pathway of bile acid biosynthesis is 7
-hydroxylation of cholesterol,
a reaction catalyzed by cholesterol 7
-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human
liver cells has not been examined. The specific aim of this study was
to determine the effects of overexpression of CYP7A1 on key regulatory
steps involved in hepatocellular cholesterol homeostasis, using primary
human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in
HepG2 cells and PHH was accomplished by using a recombinant adenovirus
encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted
in a marked activation of the classic pathway of bile acid biosynthesis
in both PHH and HepG2 cells. In response, there was decreased
HMG-CoA-reductase (HMGR) activity, decreased acyl
CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl
ester hydrolase (CEH) activity, and increased low-density lipoprotein
receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and
CEH mRNA levels paralleled changes in enzyme specific activities. More
specifically, LDLR expression, ACAT activity, and CEH activity appeared
responsive to an increase in cholesterol degradation after increased
CYP7A1 expression. Conversely, accumulation of the oxysterol
7
-hydroxycholesterol in the microsomes after CYP7A1 overexpression
was correlated with a decrease in HMGR activity.
acyl-coenzyme A:cholesterol acyltransferase; cholesterol
7
-hydroxylase; 3-hydroxy-3-methyglutaryl-CoA reductase; low-density
lipoprotein receptor; liver; neutral cholesterol ester hydrolase
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INTRODUCTION |
CHOLESTEROL IS
SYNTHESIZED in essentially all cells in the human body. It serves
as a structural component of cell membranes and as a precursor for
steroid hormones and bile acids. Cholesterol also plays a role as a
regulatory molecule for several enzymes in the pathways of cholesterol
metabolism in the liver (21). Bile acid synthesis from
cholesterol is a major pathway for elimination of cholesterol from the
body, occurring either via the classic (also called "neutral") or
alternative (also called "acidic") bile acid biosynthetic pathways
(54). Cholesterol 7
-hydroxylase (CYP7A1) and sterol
27-hydroxylase (CYP27) are initial and rate-determining enzymes in the
classic and alternative pathways, respectively. CYP7A1 and CYP27 are
subject to regulation by bile acids and certain hormones
(54). The contribution of the alternative pathway to bile
acid synthesis is not precisely known and may be species dependent. In
the rat, the estimated contribution may be as high as 50% (36,
45, 55), whereas in humans, the contribution may be <10%
(10, 15, 51).
Excess cholesterol accumulation in the body is associated with two
major diseases of Western civilization, i.e., atherosclerosis and
cholesterol gallstones. Cholesterol accumulation reflects excess
cholesterol input into the body, reduced cholesterol elimination, or
both. The effects of excessive cholesterol input via de novo cholesterol synthesis or via an increase in dietary cholesterol on
serum cholesterol levels are well known (21). The
importance of cholesterol degradative pathways, i.e., bile acid
synthesis, on overall cholesterol homeostasis and serum cholesterol
levels is less well understood. Different species differ widely in
their response to dietary cholesterol load. The rat responds to
cholesterol feeding with induction of CYP7A1 and bile acid synthesis
via the classic pathway (33) and is relatively resistant
to development of hypercholesterolemia. In contrast, increased dietary
cholesterol fails to induce CYP7A1 in rabbits (57), Green
monkeys (37), and hamsters (19, 32). In these
species, excess cholesterol leads to the development of
hypercholesterolemia and atherosclerosis. The ability of most humans to
respond to a cholesterol load is probably more akin to hamsters, but
some humans can process excessive dietary cholesterol by increasing
their bile acid synthesis rate (8, 25).
A seminal study by Spady et al. (42) demonstrated that
overexpression of CYP7A1 in hamsters fed a diet high in cholesterol resulted in enhanced bile acid synthesis and prevented an increase in
low-density lipoprotein (LDL) cholesterol levels. The results of this
study firmly established the importance of bile acid biosynthetic pathways in the pathogenesis of hypercholesterolemia and
atherosclerosis. Similar data were obtained in mice lacking the LDL
receptor gene in which increased expression of hepatic CYP7A1 also lead
to a 50% reduction in plasma LDL cholesterol concentrations
(43). No similar data are available in humans.
Cholesterol homeostasis in the liver is governed by several key
regulatory enzymes and receptors, including 1) HMG-CoA
reductase (HMGR), the rate-determining step in the cholesterol
biosynthetic pathway; 2) CYP7A1 and CYP27, the initial
enzymes in the classic and alternative pathways of bile acid
biosynthesis, respectively; 3) acyl-coenzyme A:cholesterol
acyltransferase (ACAT) and neutral cholesterol ester hydrolase (CEH),
the two enzymes that regulate the sizes of free and esterified
cholesterol compartments in the liver; and 4) cholesterol
uptake via LDL and other lipoprotein receptors (21). The
ability of each of these to respond to changes in a metabolically
active sterol pool within the liver is the basis for how cholesterol
homeostasis is maintained.
In the present study, we determined the effects of overexpression
of CYP7A1 on several parameters of bile acid and hepatic cholesterol
metabolism in primary human hepatocytes (PHH) and in HepG2 cells, a
well-characterized human hepatoblastoma cell line (34).
The data show that overexpression of CYP7A1 in cultured cells derived
from humans resulted in a marked increase of bile acid synthesis via
the "classic" pathway. Alterations of hepatic cholesterol
metabolism that have not been previously reported and that differ from
the changes observed in other species, were also noted.
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MATERIALS AND METHODS |
Materials.
All cell culture materials were obtained from GIBCO BRL (Grand Island,
NY) unless otherwise specified. William's E medium was purchased from
GIBCO BRL. The CsCl, agarose, and RNA ladder used to size CYP7A1 mRNA
were also purchased from GIBCO BRL. Taurocholate and cholesterol
oxidase were purchased from Calbiochem (San Diego, CA). All other
chemicals used were obtained from Sigma Chemical (St. Louis, MO) or
Bio-Rad Laboratories (Hercules, CA) unless otherwise specified.
-Cyclodextrin was purchased from Cylcodextrin Technologies
Development (Gainesville, FL). All solvents were obtained from Fisher
Scientific (Fair Lawn, NJ) unless otherwise indicated. All
radionucleotides, aquasol solution, and enhanced chemiluminescence reagents were purchased from DuPont NEN
(Boston, MA). Poly Attract mRNA isolation system II was obtained from
Promega (Madison, WI). The nick translation kit was obtained from
Bethesda Research Laboratories (Grand Island, NY), and Tri-Reagent was purchased from Molecular Research Center (Cincinnati, OH). Nylon membranes were purchased from Micron Separation (Westborough, MA).
Silica gel thin-layer chromatography plates (LK6 D) were from Whatman
(Clifton, NJ).
Generation of recombinant adenovirus encoding rat CYP7A1.
The AdCMV-CYP7A1 and AdCMV-
-gal adenovirus clones were constructed
in an AD5dl309 adenovirus strain (24) essentially as previously described (53). Briefly, for AdCMV-CYP7A1, a
2.6-kb rat cDNA encoding CYP7A1 was obtained from Dr. John Chiang
(7) and was cloned into the EcoR I site of
pADCMV. Plasmid pAdCMV (7.7 kb) was constructed from a pGEM4Z (Promega)
backbone and contained AD5dl309 sequence on either side of the CMV
promoter/multiple cloning site region of pcDNA3 (Invitrogen,
Carlsbad, CA). The resulting pAdCMV/CYP7A1 recombinant plasmid was
cotransfected with the right arm (XbaI digest) of Ad5dl309
into 293 cells (16). Adenovirus DNA isolated from
resulting plaques were screened by Southern blot for the presence of
the CYP7A1 insert.
Propagation of AdCMV-CYP7A1.
Large-scale production of recombinant virus was performed by infecting
confluent monolayers of human embryonic kidney 293 cells (American Type
Culture Collection, Rockville, MD) grown in 15-cm tissue culture dishes
with stock adenovirus at a multiplicity of 1 plaque-forming
units/cell. After 2 h of infection, the virus was removed
and replaced with DMEM with 2% fetal bovine serum (FBS). The
infected monolayers were harvested by scraping when >90% of the cells
showed cytopathic changes and were then centrifuged at 2,700 g, 4°C, for 10 min. The cellular pellet was
suspended in DMEM/2% FBS and subjected to five cycles of freeze-thaw
lysis to release the virus. Cell debris was removed by centrifugation at 7,700 g, 4°C, for 5 min. To purify, the crude
supernatant fluid was carefully layered over a two-step gradient
containing 3 ml of CsCl (density = 1.4 g/ml) in Tris dialysis
(TD) buffer (0.14 M NaCl, 5 mM KCl, 19 mM Tris pH 7.4, 0.7 mM
Na2 HPO4) layered over 3 ml of CsCl (d = 1.25 g/ml) in TD buffer, and centrifuged at 155,000 g,
20°C, 1 h. The viral band was removed, layered over 8 ml of CsCl
(d = 1.33 g/ml) in TD buffer and centrifuged at 155,000 g, 20°C, 18 h. The pure viral opalescent band was
removed and dialyzed against 10 mM Tris HCE pH 7.4, 1 mM
MgCl2, 10% glycerol, overnight at 4°C. The virus was
aliquoted and stored at
70°C. The virus titer (pfu) was determined
by plaque assay, and viral particles were determined by optical density
using spectrophotometry (
= 260).
Infection of HepG2 cells with AdCMV-CYP7A1.
HepG2 cells were grown in 162-cm2 (25-ml) tissue culture
flasks until they were 80-90% confluent as described previously
(34) in MEM containing nonessential amino acids, 0.03 M
NaCO3, 10% FBS, 1 mM L-glutamine, 1 mM sodium
pyruvate, and 1% Pen/Strep and were incubated at 37°C in 5%
CO2. Before infection, 15 ml of culture medium was
removed. Control flasks (i.e., no addition of adenovirus) were treated
in a similar manner. HepG2 cells were then infected with AdCMV-CYP7A1
with a multiplicity of infection of 1 using virus in a volume of
0.4-4 ml. The virus was allowed to dwell for 3 h. After an
additional 15 ml of fresh medium was added back to the flasks, they
were allowed to incubate at 37°C, 5% CO2 for 72 h.
Toxicity to cells was assessed as previously described
(34). Limited additional studies were done in the absence
of FBS (i.e., serum-free media). Of note is that each value designated
as N is the mean of duplicate-to-triplicate cultures for
each condition measured during the same setting.
Cells were divided in half and harvested in two distinct buffers as
previously described (34, 35). One-half of the cells was
used to isolate microsomes for measurement of HMGR and CYP7A1 specific
activities. The remainder was used to isolate microsomes and cytosol
for measurement of ACAT and CEH specific activities, respectively
(35). In brief, the cells were scraped and suspended in
the appropriate buffer. Cell disruption was performed by using a
Sonifier cell disruptor 350 (Branson Sonic Power) for 1 min at an
output of 3. Microsomes were isolated from cellular extracts by
centrifugation at 660 g. The supernatant fluid was
centrifuged at 105,000 g. The resulting microsomal pellets
were suspended in the appropriate buffer by using a hand-driven
homogenizer and were stored at
70°C until assayed for activity. The
microsomal protein concentration was determined by the Bradford
procedure (2), using BSA as a standard.
Isolation and culture of primary human hepatocytes.
Primary human hepatocytes were isolated according to the method of
Strom et al. (46, 47). Before plating, cells were
judged to be >90% viable by use of trypan blue exclusion. Cells were then plated onto 150-mm plastic Petri dishes previously coated with rat
tail collagen and were incubated in 20 ml of William's E medium
supplemented with insulin (0.25 units/ml) and penicillin (100 units/ml)
in a 5% CO2 atmosphere at 37°C. Cells were routinely harvested at 72 h of culture as previously described by our
laboratory (45). Unless otherwise indicated, culture
medium contained 0.1 µM dexamethasone and 1.0 µM
L-thyroxine. Toxicity was assessed as previously described
in the experiments using primary rat hepatocytes (45).
Primary human hepatocytes were plated in P150-cm2 plates to
confluency (~2.5 × 107 cells). Twenty-four hours
after plating, culture medium was removed, and 2.5 ml of fresh medium
were added. Cells were then infected with a replication defective
adenovirus containing CMV-CYP7A1 or control virus (replicative
defective adenovirus without CMV-CYP7A1) with an MOI of 10. The virus
was allowed to dwell for at least 2 h in minimal culture medium by
shaking the plates gently every 15 min, after which the medium
containing unbound virus was removed and 20 ml of fresh medium were
added back to the plates and allowed to incubate at 37°C, 5%
CO2 for an additional 48 h. Cells were then harvested
for the isolation of microsomes and RNA. Viral toxicity was determined
by trypan blue exclusion and lactic dehydrogenase release as previously
described in primary rat hepatocytes in culture (27, 45).
Proper approval for these studies was obtained through the university
institutional review board. More specifically, PHH cultures were
obtained from the following livers: 58-yr-old Hispanic man involved in
a motor vehicle accident (cells used for RNA and bile acid synthesis),
45-yr-old Caucasian man with an intracranial hemorrhage (cells used for
bile acid synthesis and CYP7A1 specific activity), and
15-yr-old African-American woman with drug intoxication (cells used for RNA).
Analysis of conjugated bile acids.
Conjugated bile acids were extracted according to the method of Folch
et al. (11). Conjugated bile acids in the culture medium
aspirated from HepG2 cells were analyzed by gas liquid chromatography
(GLC) by the methods of Setchell and Worthington (40). The conversion of [4-14C]cholesterol
into MeOH: H2O-soluble materials was used as an indication
of bile acid synthesis.
Determinations of enzyme specific activities.
The specific activity of CYP7A1 was determined in microsomes by using a
HPLC assay described previously (22). HMGR specific activity was assayed as described by Whitehead et al.
(56). Neutral cytosolic CEH specific activity was
determined according to the methods of Ghosh et al. (13).
ACAT specific activity was determined by the method of Burrier et al.
(4) and Pape et al. (35) with the following
modifications. ACAT reactions were carried out using 10 µg microsomal
protein with buffer conditions identical to those of Burrier et al.
Cholesterol was added as uniform vesicles in a
cholesterol-phosphatidylcholine (0.5 molar) ratio using an extruder.
After a 15-min incubation, 1-[14C]oleoyl-CoA was added to
a final concentration of (0.072 nM) (20 nCi). Fifteen minutes after
addition of 1-[14C]oleoyl-CoA, reactions were terminated
by direct application to silica gel TLC plates (Fisher Gel G 20 × 20 cm). Dried plates were developed in hexane-diethyl ether-acetic acid
85:15:1 by volume. 4-[14C]cholesteryl oleate formation
was detected using a PhosphorImager (Molecular Dynamics).
Microsomal free and total cholesterol were determined by HPLC analysis.
Free cholesterol was converted to cholest- 4-ene-3-one by using
cholesterol oxidase as previously described (22). Total cholesterol was measured as free cholesterol after saponification.
Experiments involving the exogenous addition of
7
-hydroxycholesterol.
In an attempt to further define the effects of 7
-hydroxycholesterol
on the specific activity of HMGR, 7
-hydroxycholesterol was added to
cell culture medium in molecusol (3 mg 7
-hydroxycholesterol to 1 ml
-cyclodextrin) to obtain final concentrations of 1, 25, and 50 µM.
Microsomes were harvested 24 h after the addition of the
7
-hydroxycholesterol, and endogenous microsomal
7
-hydroxycholesterol levels and HMG-R specific activities were then
determined. As reference, 7
-hydroxycholesterol serum levels have
been reported to be in the range of 0.01-2.2 µM in healthy
humans (3). In other studies, the addition of 6-12
µM concentrations of 7
-hydroxycholesterol have not been shown to
downregulate HMG-R activity in HepG2 cells (23).
7
-Hydroxycholesterol-induced cell toxicity was assessed as
previously described (34).
Quantitation of mRNA levels.
Methods for the isolation of RNA and the determination of mRNA levels
by Northern blotting have been previously described (33).
Rat cyclophilin cDNA was used as the internal loading standard. The
cDNA probes used were as follows: CYP7A1 (28); HMGR
(26); LDL receptor (LDLR; pLDLR3 was obtained from
American Type Culture Collection); ACAT (6); CEH
(13); CYP27 (48); and cyclophilin
(9).
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RESULTS |
Studies in HepG2 cells.
Shown in Fig. 1 is a representative
comparison of CYP7A1 mRNA levels in HepG2 cells infected with
AdCMV-CYP7A1 vs. control cells. Infection with this recombinant
adenovirus (AdCMV-CYP7A1) led to a marked increase in CYP7A1
mRNA levels (
1,107 ± 372%; P < 0.01)
compared with control cells. To more easily detect basal CYP7A1 mRNA
levels in control cells, it was necessary to isolate and probe poly A
mRNA (34). However, after infection with AdCMV-CYP7A1, CYP7A1 mRNA levels were easily detectable by use of total RNA. Analysis
of RNA isolated from AdCMV-CYP7A1 infected cells showed the 3.6-kb
CYP7A1 band was not detectable, whereas when poly A was probed from
control cells, the usual three bands at 3.6, 2.5, and 1.2 kb were
detected (34). To ensure that the adenovirus itself was
having no effect on CYP7A1 mRNA levels, HepG2 cells were also infected
with a recombinant adenovirus encoding
-galactosidase (AdCMV-
Gal). No increase in CYP7A1 mRNA was observed in those cells
(see Fig. 8). These data demonstrate that highly effective overexpression of CYP7A1 could be accomplished in HepG2 cells.

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Fig. 1.
Representative Northern blot of cholesterol
7 -hydroxylase (CYP7A1) mRNA levels in HepG2 cells after infection
with AdCMV-CYP7A1. HepG2 cells were grown until 80-90% confluent
under optimal culture conditions. Cells infected with AdCMV-CYP7A1 as
described under MATERIALS AND METHODS are compared with
control (CTRL) cells (no viral infection). Cells were harvested 72 h after infection, and mRNA levels for CYP7A1 and cyclophilin (loading
standard) were determined.
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Fig. 2 shows a marked increase in CYP7A1
specific activity in HepG2 cells after infection with AdCMV-CYP7A1
compared with control, noninfected cells. Activity in HepG2 cells
increased from undetectable levels in uninfected cells to 44.4 ± 2 nmol · h
1 · mg
1
for virus-infected cells. Preformed 7
-hydroxycholesterol was also
undetectable in uninfected cells, whereas significant levels were
detected in the infected cells (Table 1).

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Fig. 2.
Effect of infection with AdCMV-CYP7A1 on CYP7A1 specific
activity in HepG2 cells. HepG2 cells were grown until 80-90%
confluent under optimal culture conditions and then were infected with
AdCMV-CYP7A1 as described under MATERIALS AND METHODS.
Cells were harvested 72 h after infection, and specific activity
for CYP7A1 was determined. Data are expressed as means ± SE.
CYP7A1 specific activity was not detectable in control cells. Preformed
7 -hydroxycholesterol (endogenous) was also not found in control
cells, whereas endogenous microsomal 7 -hydroxycholesterol was easily
detected in infected cells before incubation (Table 1).
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To ensure that the recombinant adenovirus itself was not having a
stimulatory effect on CYP7A1 specific activity, specific activity was
determined in cells infected with AdCMV-
Gal, a representative control recombinant adenovirus. Shown in Fig.
3 are representative HPLC tracings
demonstrating levels of 7
-hydroxycholesterol in cells infected with
AdCMV-CYP7A1 (Fig. 3A) vs. AdCMV-
Gal (Fig. 3B). A large amount of 7
-hydroxycholesterol was observed
after infection with AdCMV-CYP7A1. No 7
-hydroxycholesterol was
detected after AdCMV-
Gal (i.e., no specific activity). Control
uninfected HepG2 cells also had no detectable CYP7A1 activity (see Fig.
2). These representative tracings demonstrate that control recombinant adenovirus had no stimulatory effect on endogenous CYP7A1 expression. Not shown are tracings demonstrating no stimulatory effect of control
virus (AdCMV-
Gal) on CYP7A1 activity compared with uninfected cells.

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Fig. 3.
Effect in HepG2 cells of infection with AdCMV-CYP7A1 vs.
infection with AdCMV- Gal on CYP7A1 specific activity. Representative
HPLC tracing (see MATERIALS AND METHODS) shows a large
amount of detectable 7 -hydroxycholesterol after infection with
recombinant adenovirus encoding CMV-CYP7A1 (A). No
7 -hydroxycholesterol was detectable after control virus infection
(B). Control uninfected HepG2 cells also showed no CYP7A1
specific activity (see Fig. 2).
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An approximately twofold increase in total bile acids synthesized by
HepG2 cells was observed in the culture medium after CYP7A1
overexpression. [14C]cholesterol conversion to
CH3OH/H2O extractable counts increased 105 ± 27% (P < 0.05) in HepG2 cells infected with
AdCMV-CYP7A1 compared with uninfected controls (Fig.
4A). The percent increase in
bile acid synthesis was in agreement with the increase in primary bile
acids (cholic and chenodeoxycholic acid) measured in the medium as
determined by GLC (Fig. 4B). Using GLC analysis, we found an increase of 98 ± 9.5% (P < 0.05) over
paired controls in bile acid concentration (i.e., synthesis) in the
medium in the cells infected with AdCMV-CYP7A1 (Fig. 4B).
The composition of the primary bile acids in HepG2 cell culture medium
was largely chenodeoxycholic acid in control cells, with approximately
equal proportions of cholic and chenodeoxycholic acid after CYP7A1
overexpression (Fig. 5). In limited
studies, HepG2 cells were grown in serum-free medium compared with the
serum-containing conditions employed above. As in HepG2 cells grown in
serum-containing medium, cells grown in serum-free medium increased
bile acid synthesis after CYP7A1 overexpression, but to a lesser degree
(
30%; P < 0.05). This lower rate of bile acid
synthesis after CYP7A1 overexpression in a growing cell line in the
absence of cholesterol-containing serum is most likely a function of a
decreased cholesterol pool availability for bile acid synthesis (i.e.,
absence of medium cholesterol uptake).

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Fig. 4.
Effect of overexpression of CYP7A1 on bile acid synthesis
in HepG2 cell cultures. HepG2 cells were grown until 80-90%
confluent under optimal culture conditions. Cells were infected with
AdCMV-CYP7A1 as described under MATERIALS AND METHODS.
A: bile acid synthesis as measured by conversion of
[14C]cholesterol to 14C-labeled bile acids.
B: bile acid synthesis as determined by GLC analysis of
culture medium.
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Fig. 5.
Effect of overexpression of CYP7A1 on relative
concentrations of cholic and chenodeoxycholic acids (Cholic and Cheno,
respectively) in HepG2 cell cultures. The relative concentrations of
these 2 primary bile acids secreted into HepG2 cell culture medium as
determined by GLC analysis. Data are expressed as means ± SE.
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The impact of overexpression of CYP7A1 on cellular cholesterol
homeostasis is reflected in changes in HMGR, ACAT, and CEH specific
activities. A decrease in HMGR specific activity (
34 ± 7%;
P < 0.01) was observed in AdCMV-CYP7A1-infected cells
compared with uninfected control cells (Fig.
6). Overexpression of CYP7A1 in HepG2
cells also perturbed hepatocellular cholesterol esterification and
storage, as can be seen by the changes in cytosolic CEH and microsomal
ACAT specific activities. Specific activity of neutral CEH increased
38 ± 5% (P < 0.01) after infection with
AdCYP7A1 compared with uninfected control cells (Fig. 6). A 56 ± 7% (P < 0.01) decrease in ACAT specific activity
occurred in HepG2 cells infected with AdCMV-CYP7A1.

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Fig. 6.
Response in enzymes involved in heptatic cholesterol
homeostatis after overexpession of CYP7A1 in HepG2 cell cultures. HMGR,
HMG-CoA reductase; ACAT, acyl coenzyme A/cholesterol acyltransferase;
CEH, cholesterol ester hydrolase. Data are expressed as percent of
paired control (means ± SE). Basal specific activities: HMGR = 2.9 ± 0.35 nmol · h 1 · mg 1 microsomal
protein; CEH = 15.8 ± 1.2 pmol · h 1 · mg 1 microsomal
protein; ACAT = 183.7 ± 21.5 pmol · min 1 · mg 1
microsomal protein.
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A significant increase in LDLR mRNA levels (41 ± 16%;
P < 0.01) was seen in HepG2 cells after infection with
AdCMV-CYP7A1 compared with uninfected controls (Fig.
7). LDLR mRNA levels were not affected in
cells infected with AdCMV-
Gal adenovirus lacking CYP7A1. HMGR and
ACAT mRNA levels were decreased 28 (n = 2; range
23-34%) and 25% (n = 2; range
17-34%), respectively. CEH mRNA levels were increased 19%
(n = 2; range
12-25%). A representative Northern blot comparing control HepG2 cell mRNA levels with recombinant control virus (AdCMV-
Gal) and AdCMV-CYP7A1 shown in Fig.
8 supports these cumulative results. With
the exception of LDLR, for which receptor activity was not
measured, changes in HMGR, ACAT, and CEH mRNA levels
correlated with changes observed in their respective specific
activities. In limited studies, HepG2 cells were also grown in
serum-free medium as opposed to the serum-containing conditions
employed above. As in HepG2 cells grown in serum-containing medium,
LDLR mRNA increased (
54 ± 27%; P < 0.05) in
cells grown in serum-free medium after CYP7A1 overexpression.

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Fig. 7.
Effect on steady-state mRNA levels in HepG2 cells after
overexpression of CYP7A1 mediated through infection with AdCMV-CYP7A1.
LDL-R, LDL receptor; ACAT-1, ACAT. Data expressed as a percent of
paired controls (means ± SE). By using laser densitometry, mRNA
levels were calculated as a ratio of mRNA of interest to that of
cyclophilin, employed as an internal loading standard.
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Fig. 8.
Representative Northern blot of mRNA levels in HepG2
cells after infection with AdCMV-CYP7A1 compared with control virus.
HepG2 cells were grown until 80-90% confluent under optimal
culture conditions. Cells were infected with AdCMV-CYP7A1 or
AdCMV- Gal as described under MATERIALS AND METHODS.
Cells were harvested 48 h after infection, and mRNA levels for
LDL-R, HMGR, CYP7A1, ACAT-1, CEH, and cyclophilin (loading standard)
were determined. Note: Despite the attempt to generate a blot with the
same RNA loading in all columns, mRNA levels in the Gal column were
less as determined by use of cyclophilin as an internal loading
standard.
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Overexpression of CYP7A1 also led to changes in microsomal free and
total cholesterol (Fig. 9). A 27 ± 1% (P < 0.01) decrease in microsomal free cholesterol
was seen after infection with AdCMV-CYP7A1 in HepG2 cells (control cell
microsomal free cholesterol levels, 200 nmol/mg microsomal protein). A
corresponding decrease (
45%; n = 2) in microsomal
total cholesterol was also noted with increased expression of CYP7A1.

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Fig. 9.
Effects of overexpression of CYP7A1 on microsomal free
and total cholesterol in HepG2 cells. Microsomal free and total
cholesterol were determined by HPLC analysis as described under
MATERIALS AND METHODS. Data are expressed as percent of
paired controls (means ± SE).
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After overexpression of CYP7A1, increased endogenous microsomal levels
of 7
-hydroxycholesterol were observed. As demonstrated (Fig. 6;
Table 1), the increase in these levels
was associated with lower HMGR specific activities. Previously, it has
been suggested that 7
-hydroxycholesterol may be one of several
oxysterols capable of downregulating HMGR. To further substantiate
this hypothesis, additional studies were performed in which
7
-hydroxycholesterol was added directly to culture medium (Table 1).
Concentrations attempting to approximate and exceed previously
documented human serum values were chosen. A range of 0.01-2.2
µM has previously been demonstrated in the serum of healthy humans
(3). A concentration of 6-12 µM of
7
-hydroxycholesterol had not previously been shown to downregulate
HMGR activity in HepG2 cells (23). In our studies, the
addition of 7
-hydroxycholesterol to medium to obtain a 1 µM
concentration had no effect on HMGR activity. However, 25 µM and 50 µM concentrations of 7
-hydroxycholesterol in the medium led to a
concentration-dependent decrease in HMGR activity (Table 1). Microsomal
7
-hydroxycholesterol concentrations after CYP7A1 overexpression were
between that of the 25 µM and 50 µM 7
-hydroxycholesterol medium
concentrations, with the decrease in HMGR most closely approximating
the effects of 25 µM (Table 1).
Studies in primary human hepatocytes.
Figure 10 shows a representative
Northern blot of CYP7A1 mRNA levels in PHH control cells (addition of
control adenovirus) compared with mRNA levels in cells infected with
AdCMV-CYP7A1. There is evidence for abundance of mRNA levels in
infected (PHH) compared with control cells in which the mRNA levels are
practically undetectable. Cyclophilin controls showed no changes in
either control or infected cells.

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Fig. 10.
Representative Northern blot of CYP7A1 mRNA levels in
primary human hepatocytes after infection with AdCMV-CYP7A1.
Hepatocytes were incubated for 24 h in culture medium containing
optimal concentrations of thyroxine (T4; 1.0 µM) and
dexamethasone (0.1 µM). At 24 h, cells were infected with
control virus or AdCMV-CYP7b1 as described under MATERIALS AND
METHODS. Cells were harvested 48 h after infection, and mRNA
levels for CYP7A1 and cyclophilin (loading standard) were
determined.
|
|
In Fig. 11, we show quantitative data
on CYP7A1 mRNA levels and the specific activities in PHH. In contrast
to control HepG2 cells in which CYP7A1 specific activity was
undetectable (Fig. 2), PHH controls had detectable CYP7A1 activity
(0.13 nmol · h
1 · mg
1
protein). After overexpression of CYP7A1, steady-state mRNA levels increased ~18-fold, whereas specific activities increased >10-fold. These data are consistent with significant augmentation of CYP7A1 after
infection with AdCMV-CYP7A1 virus.

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Fig. 11.
Effect on CYP7A1 mRNA levels and specific activity in
primary human hepatocytes after infection with AdCMV-CYP7A1.
Hepatocytes were incubated for 24 h in culture medium containing
optimal concentrations of T4 (1.0 µM) and dexamethasone
(0.1 µM). At 24 h cells were infected with control virus or
AdCMV-CYP7A1 as described under MATERIALS AND METHODS.
Cells were harvested 48 h after infection, and mRNA levels and
specific activity for CYP7A1 were determined. Data are expressed as
percent of paired controls.
|
|
Bile acid synthesis in primary human hepatocytes (Fig.
12) after infection with AdCMV-CYP7A1
increased 73% (n = 2; range
62-82%) at
22 h and 393% (n = 2; range
341-447%) at
48 h. These data show that the marked increase in CYP7A1 mRNA
levels and specific activities after CYP7A1 overexpression is
translated into a significant increase of bile acid synthesis and fully
supports the stimulation of the classic pathway of bile acid synthesis.

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Fig. 12.
Effect of overexpression of CYP7A1 using AdCMV-CYP7A1 on
bile acid synthesis in primary human hepatocyte cultures. Hepatocytes
were incubated for 24 h in culture medium containing optimal
concentrations of T4 (1.0 µM) and dexamethasone (0.1 µM). At 24 h cells were infected with control virus or
AdCMV-CYP7A1 as described under MATERIALS AND METHODS. Bile
acid synthesis was measured 22 and 48 h after infection as
conversion of [14C]cholesterol to 14C-labeled
bile acids. Data are expressed as percent of paired control (mean of 2 experiments).
|
|
Simultaneously, a significant increase occurred in LDLR mRNA levels
(Fig. 13) in PHH after infection with
AdCMV-CYP7A1 compared with paired controls. Cyclophilin levels did not
change, suggesting that unequal blot RNA loading could not account for
increases in LDLR mRNA levels. Similar to HepG2 cells, HMG-CoA
reductase activity decreased 67% (mean of 3 cultures;
n = 1) in PHH infected with AdCMV-CYP7A1.

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Fig. 13.
Representative Northern blot of LDLR mRNA levels in
primary human hepatocytes after infection with AdCMV-CYP7A1.
Hepatocytes were incubated for 24 h in culture medium containing
optimal concentrations of T4 (1.0 µM) and dexamethasone
(0.1 µM). At 24 h, cells were infected with control virus or
AdCMV-CYP7A1 as described under MATERIALS AND METHODS.
Cells were harvested 48 h after infection, and mRNA levels for
LDLR and cyclophilin (loading standard) were determined.
|
|
 |
DISCUSSION |
In the present paper, we provide evidence that overexpression of
CYP7A1 in HepG2 and in PHH results in an increase in bile acid
synthesis via the classic pathway coupled with suppression of
cholesterol synthesis. These findings are particularly noteworthy because they differ in several respects from responses observed in
other species. Most of the information on the mechanism by which
cholesterol homeostasis is maintained has been derived from experimental animals, which showed a great deal of species variation (19, 33, 37, 57). The rat is capable of inducing CYP7A1 and bile acid synthesis in response to a diet high in cholesterol (33). The resulting increase in cholesterol degradation
appears to protect this species from hypercholesterolemia and
atherosclerosis. In humans, the response to dietary cholesterol appears
to be heterogeneous. A trend toward increasing LDL cholesterol by
increasing dietary cholesterol is well established, but in a
significant number of humans, circulating LDL cholesterol does not
change in response to changes in dietary cholesterol (8,
25). In an interesting paper by Kern (25), a single
patient consuming 25 eggs per day for over 20 years had a marked
increase in the rate of bile acid synthesis coupled with near normal
serum cholesterol levels. This combination of findings suggests that,
in this particular individual, CYP7A1 was inducible by a diet high in
cholesterol in a manner similar to that of rat. Couture et al.
(8) studied the effect of A to C substitution at position
204 of the promoter of CYP7A1 gene and its association with
variations in plasma LDL cholesterol concentrations. At least in
humans, the C variant was associated with higher plasma LDL
cholesterol concentrations and an increase in the ratio of total
cholesterol to high-density lipoprotein. Although overexpression of
CYP7A1 in hamsters prevents hypercholesterolemia in animals fed a
Western diet (42), it is not certain whether such
observations can be extrapolated from other species to humans.
We have previously characterized HepG2 cells and found them a good
model to study the regulation of human CYP7A1 and other enzymes of the
bile acid biosynthetic pathways. HepG2 cells were shown to synthesize
primary bile acids and express enzymes and receptors involved in the
maintenance of cholesterol homeostasis (34). PHH have been
used to study numerous aspects of cholesterol and bile acid transport
and metabolism and have been shown to be a valid model to study the
physiological and metabolic events in humans. To further verify the
results in HepG2 cells, we also repeated some key experiments in PHH
for comparison purposes. The data in the two systems are consistent and
differ somewhat from those obtained in other species.
In AdCMV-CYP7A1-infected HepG2 cells, CYP7A1 mRNA levels and specific
activities increased severalfold. Bile acid synthesis increased
approximately twofold with the associated increase in CYP7A1
expression. No such increase occurred in control cells or cells
infected with a control adenovirus. After infection with the
AdCMV-CYP7A1, the concentration of cholic acid in the medium increased
from <10% to nearly 50% of total bile acid synthesis. This increase
in the proportion of cholic acid suggests that in humans the classic
pathway is mainly responsible for synthesis of cholic acid. Previous in
vitro and in vivo studies in the rat suggest that up to 50% of total
bile acid synthesis may occur via the alternative pathway and that the
predominant bile acid is chenodeoxycholic acid (36, 45,
55). How representative this finding is to other species is
uncertain. In contrast to findings in the rat, Duane and Javitt
(10) have recently reported that in healthy humans <10%
of total bile acid synthesis appeared to be synthesized via the
alternative pathway; their findings are consistent with previous
reports (15, 51). In our recent experiments,
overexpression of CYP27 in Chinese hamster ovary cells and
HepG2 cells led to a modest increase in 27-hydroxycholesterol and bile
acid synthesis, respectively (18). These data suggested that the alternative pathway may be induced in human-derived liver cell
lines. In the CYP7A1 knockout mouse, in which the classic pathway of
bile acid synthesis is absent, the alternative pathway can become
activated and produce sufficient bile acid synthesis to permit survival
without exogenous bile acid supplements (39).
A marked increase of CYP7A1 specific activity and bile acid synthesis
in both types of cells was coupled with compensatory changes in hepatic
cholesterol metabolism, including increased LDLR mRNA levels.
Surprisingly, the overexpression of CYP7A1 in HepG2 cells was
associated with a decrease in HMGR activity. This finding is different
from in vivo observations by Spady et al. (42), and in
vitro observations by Spitsen et al. (44) and Sudjana-Sugiaman et al. (50). In general, HMGR and CYP7A1
change in tandem, resulting in coordinate regulation of cholesterol and bile acid synthesis. The previous in vivo and in vitro studies (42, 44, 50) were carried out by infection of the CYP7A1 gene into hamsters or nonhuman, nonhepatic cell lines. HepG2 cells have
been shown to reflect in vivo events in humans, and because a decrease
in HMGR activity after overexpression of CYP7A1 was a reproducible
finding, one can conclude that the behavior of HMGR in HepG2 cells
(i.e., extrapolated to humans) may be different from that in the rat
and other species. Our limited findings in PHH in this study support of
this finding.
Although the decrease in HMGR in the face of markedly upregulated
CYP7A1 was not expected, a plausible explanation is available. The
ability of oxysterols to repress HMGR is well documented in several
studies (3, 30, 41). In our model, overexpression of
CYP7A1 results in formation of a large amount of
7
-hydroxycholesterol, which in turn may repress HMGR. This was
confirmed by adding 7
-hydroxycholesterol to the culture medium
(Table 1). These experiments, therefore, strongly suggest that
7
-hydroxycholesterol or a derivative may be responsible for the
observed feedback repression of HMGR under these experimental
circumstances. Increasing CYP7A1 activity directly in hepatocytes might
not only stimulate increased cholesterol catabolism, but also
simultaneously repress HMGR. This approach differs from the secondary
upregulation of CYP7A1 and HMGR associated with feeding the intestinal
bile acid-binding resin, cholestyramine. With cholestyramine feeding,
the increased intestinal loss of bile acids stimulates bile acid
synthesis via a reduction in negative bile acid biofeedback with a
subsequent increase in HMGR activity. Although the serum concentration
of 7
-hydroxycholesterol has been shown to increase with
cholestyramine feeding (1), the microsomal levels achieved
and the relationship to this study are not clearly defined. One
explanation for the differing findings in this study compared with
those of previous studies is the level of microsomal
7
-hydroxycholesterol.
The effect of 7
-hydroxycholesterol in the regulation of the LDLR is
even less clear, with previous effects clearly dependent on oxysterol
cellular concentration (29, 31). These results suggest a
differential effect of 7
-hydroxycholesterol on HMGR and LDLR
regulation. The fact that HMGR and LDLR usually also change in tandem
suggests that intracellular concentrations of 7
-hydroxycholesterol
can lead to a differential regulation in HMGR and LDLR. As
transcriptional control of HMGR and LDLR are believed to be controlled
by the same factor (22), one could postulate that
7
-hydroxycholesterol could be mediating its regulation of HMGR
through acceleration of mRNA degradation.
Our findings suggest that sufficiently increased CYP7A1 activity with a
subsequent increase in 7
-hydroxycholesterol microsomal concentrations will exhibit a cholesterol homeostatic response favorable to lowering serum cholesterol. The physiological significance of the large increase in CYP7A1 activity achieved in vitro compared with what can be achieved in vivo is uncertain. However, if this observation can be confirmed in in vivo experiments, it would suggest
that overexpression of CYP7A1 may be a highly effective strategy for
lowering serum cholesterol in humans.
The ability of ACAT to esterify cholesterol is important in maintaining
intracellular concentrations of free cholesterol as well as providing
cholesterol storage in the form of cholesterol esters. In the liver,
ACAT-derived cholesterol esters are secreted as a component of
very-low-density lipoproteins. ACAT activity is upregulated by LDL and
free cholesterol (49, 52). In general, as free cholesterol
substrate increases, so does ACAT activity, to maintain free
cholesterol in the narrow range. In the presence of decreased
cholesterol availability, the activity of ACAT has been shown to
decrease (49, 52). In the present study, increased cholesterol catabolism coupled with decreased microsomal free cholesterol levels was associated with a decrease in ACAT specific activity and ACAT-1 mRNA levels (ACAT-2 mRNA levels were not
determined). As with cholesterol, in vitro addition of
25-hydroxycholesterol has previously been shown to stimulate ACAT
activity (49). More recently, in isolated membranes
expressing ACAT activity, Cases et al. (5) found
27-hydroxycholesterol and 24(S),25-epoxycholesterol to be stimulators
of ACAT activity, with 7-hydroxycholesterol a potent suppressor. On the
basis of these observations, it has been suggested that oxysterols may
be important regulators of ACAT. In this study, a repression in ACAT
activity was associated with an increase in microsomal
7
-hydroxycholesterol, an increase also associated with
downregulation of HMGR. However, it is unclear in this study whether
the change in ACAT activity is in response to a decrease in the
regulatory cholesterol pool size in the liver or in response to the
increase in 7
-hydroxycholesterol. Transfection studies designed to
assess the role of cholesterol availability in the regulation of ACAT
specific activity, using the overexpression of ACAT in ACAT-deficient
cell lines, have suggested that ACAT regulation is governed at both the
transcriptional and posttranscriptional level (52). The
lesser decrease in steady-state mRNA levels compared with that observed
in specific activity in response to overexpression of CYP7A1 supports
this observation.
Recent findings have suggested that ACAT-2, primarily involved in
lipoprotein assembly and secretion, is the predominant liver ACAT
(38). However, HepG2 cells contain both ACAT-1 and ACAT-2. Therefore, the activities of two different gene products are reflected in the measured ACAT activity. Whether the ACAT-1 and -2 functions are
separate or can overlap in this cell line is uncertain. Only ACAT-1
mRNA levels were determined in this study. However, a decrease in mRNA
levels was correlated with decreased ACAT activity.
CEH is the key enzyme required for releasing the pool of metabolically
active free cholesterol from intracellular stores of cholesterol esters
(14). Thus neutral CEH is of great importance as an enzyme
that replenishes the pool of free cholesterol for bile acid synthesis
and biliary cholesterol secretion. This overexpression study of CYP7A1
clearly shows that enhanced bile acid synthesis is coupled with an
increase in CEH specific activity, a means by which the size of the
free cholesterol substrate pool is increased. The fact that an increase
in CEH specific activity was coupled with an increase in mRNA levels
also supports a transcriptional level of regulation of CEH. These data
are consistent with primary observations showing that a
cholesterol-enriched diet decreases CEH activity in rats and consistent
with an increase in activity in response to cholestyramine feeding
(12, 17).
In summary, increased expression of CYP7A1 in primary human hepatocytes
and in HepG2 cells resulted in increased bile acid synthesis via the
classic pathway. The cholesterol homeostatic response in HepG2 cells
included downregulation of HMGR, probably due to accumulation of
7
-hydroxycholesterol, and upregulation of LDLR. This response
suggests that overexpression of CYP7A1 in humans may be a useful
approach for lowering serum cholesterol. Other effects of
overexpression of CYP7A1 on ACAT and CEH are consistent with attempts
of the liver to maintain cholesterol homeostasis in the face of a
marked increase in cholesterol catabolism. The fact that the results in
HepG2 cells and PHH agree with each other adds confidence that the
results of this study in human cell lines may be extrapolated to an in
vivo situation.
 |
ACKNOWLEDGEMENTS |
We thank Tina Lucas, Li Zhao, Pat Bohdan, Melissa Thompson, and
Emily Gurley for technical assistance. We also thank Drs. J. Y. L. Chiang, S. Ghosh, W. Grogan, B. Kren, and T. Y. Chiang for providing cDNAs for CYP7A1, CEH, HMGR, and ACAT, respectively.
 |
FOOTNOTES |
This work was supported by grants from the Veterans Administration and
the National Institute of Diabetes and Digestive and Kidney Diseases
(P01 DK-38030).
Address for reprint requests and other correspondence: W. M. Pandak, Veterans Affairs Medical Center, Division of
Gastroenterology 111-N, 1201 Broad Rock Rd., Richmond, VA 23249.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 July 2000; accepted in final form 25 June 2001.
 |
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