Am J Physiol Gastrointest Liver Physiol
281: G878–G889, 2001.
Effects of CYP7A1 overexpression on cholesterol
and bile acid homeostasis
W. M. PANDAK,1 C. SCHWARZ,1 P. B. HYLEMON,2 D. MALLONEE,2 K. VALERIE,1
D. M. HEUMAN,1 R. A. FISHER,3 KAYE REDFORD,1 AND Z. R. VLAHCEVIC1
Departments of 1Medicine, 2Microbiology, and 3Surgery, Veterans Affairs Medical Center and
Virginia Commonwealth University, Richmond, Virginia 23249
Received 21 July 2000; accepted in final form 25 June 2001
acyl-coenzyme A:cholesterol acyltransferase; cholesterol 7␣hydroxylase; 3-hydroxy-3-methyglutaryl-CoA reductase; lowdensity lipoprotein receptor; liver; neutral cholesterol ester
hydrolase
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
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.
G878
the classic (also called “neutral”) or alternative (also
called “acidic”) bile acid biosynthetic pathways (54).
Cholesterol 7␣-hydroxylase (CYP7A1) and sterol 27hydroxylase (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
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Pandak, W. M., C. Schwarz, P. B. Hylemon, D. Mallonee, K. Valerie, D. M. Heuman, R. A. Fisher, Kaye
Redford, and Z. R. Vlahcevic. Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis. Am J
Physiol Gastrointest Liver Physiol 281: G878–G889,
2001.—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.
CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
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
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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 twostep 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
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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 wellcharacterized 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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CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
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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 cholest4-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 ␮M.
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 ␮M
in healthy humans (3). In other studies, the addition of 6–12
␮M 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).
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 (11,107 ⫾ 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 AdCMVCYP7A1 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.
Fig. 2 shows a marked increase in CYP7A1 specific
activity in HepG2 cells after infection with AdCMVCYP7A1 compared with control, noninfected cells. Activity in HepG2 cells increased from undetectable levels in uninfected cells to 44.4 ⫾ 2 nmol 䡠 h⫺1 䡠 mg⫺1 for
virus-infected cells. Preformed 7␣-hydroxycholesterol
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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 ␮M dexamethasone and 1.0 ␮M 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 CMVCYP7A1) 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 ␮g 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
CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
Fig. 1. Representative Northern blot of cholesterol 7␣-hydroxylase
(CYP7A1) mRNA levels in HepG2 cells after infection with AdCMVCYP7A1. 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. 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).
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 (130%; 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).
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 (234 ⫾ 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
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nificant levels were detected in the infected cells (Table
1).
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 AdCMVCYP7A1 (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.
An approximately twofold increase in total bile acids
synthesized by HepG2 cells was observed in the culture
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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 CMVCYP7A1 (A). No 7␣-hydroxycholesterol was detectable after control virus infection (B). Control
uninfected HepG2 cells also showed no CYP7A1 specific activity (see Fig. 2).
ties. 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.
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 223–34%) and 25% (n ⫽ 2;
range 217–34%), respectively. CEH mRNA levels
were increased 19% (n ⫽ 2; range 112–25%). A representative Northern blot comparing control HepG2
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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 (154 ⫾ 27%; P ⬍ 0.05) in cells
grown in serum-free medium after CYP7A1 overexpression.
Overexpression of CYP7A1 also led to changes in
microsomal free and total cholesterol (Fig. 9). A 27 ⫾
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CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
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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 corre-
sponding decrease (2 45%; n ⫽ 2) in microsomal total
cholesterol was also noted with increased expression of
CYP7A1.
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 ␮M has previously been demonstrated in the
serum of healthy humans (3). A concentration of 6–12
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.
Fig. 7. Effect on steady-state mRNA levels in HepG2 cells after
overexpression of CYP7A1 mediated through infection with AdCMVCYP7A1. 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.
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 AdCMVCYP7A1 as described under MATERIALS AND METHODS. A: bile acid
synthesis as measured by conversion of [14C]cholesterol to 14Clabeled bile acids. B: bile acid synthesis as determined by GLC
analysis of culture medium.
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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 䡠 h⫺1 䡠 mg⫺1 microsomal protein;
CEH ⫽ 15.8 ⫾ 1.2 pmol 䡠 h⫺1 䡠 mg⫺1 microsomal protein; ACAT ⫽
183.7 ⫾ 21.5 pmol 䡠 min⫺1 䡠 mg⫺1 microsomal protein.
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CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
Table 1. Effect of addition of 7␣-hydroxycholesterol
on HMGR specific activity
Condition
Infected
Additions
1 ␮M
25 ␮M
50 ␮M
Endogenous 7␣Hydroxycholesterol in
Microsomes, ␮M
HMGR Specific
Activity, % of
Control
213 ⫾ 81
66.3 ⫾ 7
No endogenous
85
272
96.7
54.9
36.3
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.
␮M 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 ␮M concentration had no
effect on HMGR activity. However, 25 ␮M and 50 ␮M
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 ␮M and 50 ␮M 7␣-hydroxycholesterol medium concentrations, with the decrease in
HMGR most closely approximating the effects of 25 ␮M
(Table 1).
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).
AJP-Gastrointest Liver Physiol • VOL
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.
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 䡠 h⫺1 䡠 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.
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 ␮M) and dexamethasone
(0.1 ␮M). At 24 h, cells were infected with control virus or AdCMVCYP7b1 as described under MATERIALS AND METHODS. Cells were
harvested 48 h after infection, and mRNA levels for CYP7A1 and
cyclophilin (loading standard) were determined.
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Infected: HepG2 cells infected with AdCMV-CYP7A1. Additions:
7␣-hydroxycholesterol was added to HepG2 cell culture medium to
achieve 1, 25, and 50 ␮M medium concentrations. Microsomes were
harvested 24 h after addition of the 7␣-hydroxycholesterol, and
endogenous 7␣-hydroxycholesterol microsomal concentrations were
determined (means ⫾ SE). HMGR, HMG-CoA reductase specific activity expressed as a percent of paired controls.
CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
Bile acid synthesis in primary human hepatocytes
(Fig. 12) after infection with AdCMV-CYP7A1 increased 73% (n ⫽ 2; range 162–82%) at 22 h and 393%
(n ⫽ 2; range 1341–447%) 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.
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 un-
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 ␮M) and dexamethasone (0.1 ␮M). 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).
AJP-Gastrointest Liver Physiol • VOL
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 ␮M) and dexamethasone (0.1 ␮M).
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.
equal blot RNA loading could not account for increases
in LDLR mRNA levels. Similar to HepG2 cells, HMGCoA reductase activity decreased 67% (mean of 3 cultures; n ⫽ 1) in PHH infected with AdCMV-CYP7A1.
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
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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 ␮M) and dexamethasone (0.1 ␮M).
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.
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CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
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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 ex281 • OCTOBER 2001 •
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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
CYP7A1 OVEREXPRESSION AND CHOLESTEROL HOMEOSTASIS
AJP-Gastrointest Liver Physiol • VOL
cellular 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.
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.
This work was supported by grants from the Veterans Administration and the National Institute of Diabetes and Digestive and
Kidney Diseases (P01 DK-38030).
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