(Received for publication, November 3, 1995; and in revised form, December 28, 1995)
From the
Regulation of squalene epoxidase (SE) gene expression was studied in comparison with those of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and low density lipoprotein (LDL) receptor. An increased expression of SE mRNA and protein content in mouse L929 cells grown in 10% lipoprotein-deficient fetal bovine serum (LPDS) for 48 h was found by performing immunoblot and Northern blot analyses when compared with the culture in the presence of fetal bovine serum (FBS). The same results in mRNA levels were seen using human cell lines HepG2, HeLa, and Chang liver cells. The increase of SE mRNA in HeLa cells grown in LPDS was preventable in a dose-dependent manner by feeding cells with 25-hydroxycholesterol or cholesterol. When an SE inhibitor, NB-598, was fed to HeLa cells grown in LPDS, it caused further increases in mRNA levels of SE, HMG-CoA reductase, and LDL receptor. In contrast, NB-598 had no effect on the message levels of these genes when fed to HeLa cells grown in FBS. These results suggest that sterol produced endogenously can also regulate SE expression at the level of transcription.
Since cholesterol is an essential structural component of
cytoplasmic membranes, it is crucial for cells to maintain
intracellular cholesterol homeostasis. Cells acquire cholesterol both
from the LDL ()receptor-mediated pathway (1) and the
biosynthetic pathway from acetyl-CoA(2, 3) . Brown and
Goldstein (4) demonstrated that both pathways are controlled by
end product repression by showing that sterol depletion resulted in
increased levels of mRNA for the LDL receptor and two sequential
enzymes in de novo cholesterol biosynthesis, HMG-CoA synthase,
and HMG-CoA reductase. Furthermore, restoration of sterols resulted in
decreased mRNA for these genes.
Although HMG-CoA reductase is considered to be the major regulatory enzyme in cholesterol biosynthesis, recent studies revealed that other enzymes involved in cholesterol biosynthesis, such as HMG-CoA synthase, farnesyl diphosphate synthase, and squalene synthase, are also regulated by sterols(5, 6) . HMG-CoA reductase inhibitors are widely used as agents for lowering plasma cholesterol levels. However, recent studies have revealed that mevalonate derived non-sterol metabolite(s), which play important roles in the regulation of normal cellular processes, are synthesized in a post-mevalonate pathway and that HMG-CoA reductase inhibitors cause the depletion of both mevalonate-derived non-sterol metabolite(s) as well as sterols(7, 8) . Since SE is situated after this branch point in the mevalonate pathway, cholesterol is the only end product for SE. Therefore, SE is considered to be a potential new target enzyme for anti-hyperlipidemic drugs(9, 10) .
SE is located in the endoplasmic reticulum and catalyzes the conversion of squalene to 2,3(S)-oxidosqualene, when coupled with a component of microsomal electron transport chain, NADPH-cytochrome P-450 reductase. SE seems to be an important rate-limiting enzyme in cholesterol biosynthesis, since it has an extremely low specific activity in comparison with HMG-CoA reductase or squalene synthase in HepG2 cells (11, 12) and since supplementation of exogenous cholesterol resulted in the accumulation of labeled squalene from precursor mevalonate in human renal carcinoma cells(13) . The activity of rat or human hepatic SE was shown to be regulated by dietary cholesterol or HMG-CoA reductase inhibitors(11, 12) . However, the regulation of SE protein and mRNA levels has not been directly investigated. We reported previously the isolation of rat and mouse SE cDNAs(14, 15) . In this report, we examine the regulation of SE transcription by sterols as well as inhibitors of SE and HMG-CoA reductase, and compare this with the regulation, by these agents, of the HMG-CoA reductase and LDL receptor genes.
Figure 1:
Expression of protein and
mRNA of SE in L929 cells. After confluent monolayers of L929 cells were
incubated for 48 h in either FBS or LPDS medium, cells were collected
by centrifugation, and crude microsome extracts and total RNAs were
prepared from the cells as described under ``Experimental
Procedures.'' A, 20 µg of crude microsome extracts
were subjected to electrophoresis on an SDS-polyacrylamide (10%) gel
and transferred to a nitrocellulose filter. The filter was incubated
with rabbit anti-recombinant rat SE serum (diluted 1:1000), followed by
incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG
(Life Technologies, Inc.), and visualized using Renaissance Western
blot chemiluminescence reagent (DuPont NEN). The filter was exposed to
Fuji RX film for 20 min at room temperature. We used a high range
prestained standard as a molecular weight marker (Bio-Rad). B,
20 µg of total RNAs from the cells cultured in FBS or LPDS medium
were electrophoresed on a 1% agarose gel and transferred to a nytran
membrane. The filter was hybridized with P-labeled rat SE
cDNA probe and exposed to Fuji RX film with an intensifying screen at
-70 °C for 10 days. The same filter was subsequently
hybridized with human
-actin probe and exposed to film for 4
h.
Figure 2:
Expression of SE mRNA in human cell lines.
Confluent monolayers of HepG2, HeLa, and Chang liver cells were
cultured for 48 h in FBS medium or LPDS medium, and then total RNA was
isolated from the cells. Twenty µg of the total RNA was subjected
to Northern blot analysis using a P-labeled human SE cDNA
probe. The same filter was subsequently hybridized with human
-actin probe. The signals were quantified using BAS 1000 system
and normalized to
-actin mRNA levels. These relative intensities
of SE mRNA signals were then plotted for both culture
condition.
Figure 3:
Time course of SE, HMG-CoA reductase, and
LDL receptor mRNA increase by LPDS in HeLa cells. After incubation of
various duration in LPDS medium, total RNA was prepared from the cells,
and Northern blot analysis was performed on 20 µg. The filter was
hybridized with SE probe. The same membrane was rehybridized with
HMG-CoA reductase, LDL receptor, and -actin probes. All mRNA
levels in this figure were determined by the BAS 1000 system and
normalized to
-actin mRNA levels. The average intensity (n = 2 experiments) of the mRNA bands was plotted relative to
the value for the mRNA at zero time. SE, squalene epoxidase; RED, HMG-CoA reductase; LDLR, LDL
receptor.
Figure 4: Effects of sterols on SE mRNA expression in HeLa cells. A, HeLa cells were cultured in LPDS medium with the following additions: ethanol vehicle alone (lane 2); 1, 10, 100, 1000, or 10,000 ng/ml of cholesterol (lanes 3-7); or 25-hydroxycholesterol (lanes 8-12), respectively. The cells in lane 1 were cultured in FBS medium. After 48-h incubation, total RNA was prepared from the cells, and 20 µg of RNA was subjected to Northern blot analysis. The filter was exposed to Fuji RX film with an intensifying screen at -70 °C for 10 days. B, the data in A were quantified and the average intensity (n = 2 experiments) of the SE mRNA band was plotted relative to the value for the SE mRNA band in FBS-cultured cells. All mRNA levels in this figure were determined as in Fig. 3. 25-OH chol., 25-hydroxycholesterol.
Figure 5:
Effects of NB-598 on expression of mRNAs
for SE, HMG-CoA reductase, and LDL receptor in HeLa cells. A,
HeLa cells were cultured in FBS medium (lanes 1-5) or
LPDS medium (lanes 6-10) containing NB-598 at 0 nM (ethanol vehicle alone) (lanes 1 and 6); 10
nM (lanes 2 and 7), 100 nM (lanes 3 and 8), 1 µM (lanes 4 and 9), or 10 µM (lanes 5 and 10). After a 48-h incubation, total RNA was prepared from the
cells, and 20 µg was subjected to Northern blot analysis. The
filter was exposed to Fuji RX film with an intensifying screen at
-70 °C for 10 days. B, HeLa cells were cultured in
FBS medium, FBS medium with 1 µM NB-598, or LPDS medium
with 1 µM NB-598. After a 48-h incubation, total RNA was
isolated from the cells. Twenty µg of the RNAs were subjected to
Northern blot analysis using a human SE cDNA probe, and the same filter
was rehybridized with human HMG-CoA reductase and LDL receptor cDNA
probes. The data were quantified, and the average intensity (n = 2 experiments) of the bands was plotted relative to the
values in the same cells cultured in FBS medium. All mRNA levels in
this figure were determined by BAS 1000 system and normalized to
-actin mRNA levels determined after rehybridization. SE,
squalene epoxidase; RED, HMG-CoA reductase; LDLR, LDL
receptor.
Figure 6:
Effects of lovastatin on expression of
mRNAs for SE, HMG-CoA reductase, and LDL receptor in HeLa cells. A, HeLa cells were cultured in FBS medium (lanes
1-5) or LPDS medium (lanes 6-10) containing
lovastatin at 0 nM (ethanol vehicle alone) (lanes 1 and 6), 10 nM (lanes 2 and 7),
100 nM (lanes 3 and 8), 1 µM (lanes 4 and 9), or 10 µM (lanes 5 and 10). After 48 h, total RNA was
prepared from the cells and 20 µg subjected to Northern blot
analysis. The filter was exposed to Fuji RX film with an intensifying
screen at -70 °C for 10 days. B, HeLa cells were
cultured in FBS medium, FBS medium with 1 µM lovastatin,
or LPDS medium with 1 µM lovastatin. After a 48-h
incubation, total RNA was isolated from the cells. Twenty µg of the
RNA were subjected to Northern blot analysis using human SE, HMG-CoA
reductase, and LDL receptor probes. The data were quantified, and the
average intensity (n = 2 experiments) of bands was
plotted relative to the values in the same cells cultured in FBS
medium. All mRNA levels in this figure were determined by BAS 1000
system and normalized to -actin mRNA levels determined after
rehybridization. SE, squalene epoxidase; RED, HMG-CoA
reductase; LDLR, LDL receptor.
Intracellular cholesterol homeostasis is maintained primarily through regulation of cholesterol biosynthetic and LDL receptor mediated pathways. Many genes involved in the biosynthetic pathway are coordinately controlled by sterols(4, 5, 6) . Previously, Hidaka et al.(11) reported that SE activity is controlled by endogenous and exogenous sterols. In this report, Western blot and Northern blot analyses of L929 cells (Fig. 1) clearly demonstrate that SE activity is regulated by changes in enzyme levels and that these changes occur mainly at the transcriptional level. In addition, we detected increased SE mRNA levels in the human cell lines HepG2, HeLa, and Chang liver cells when grown in LPDS medium, although the relative rates of increase are quite different in the three cell lines (Fig. 2). The increase of SE mRNA in LPDS medium is significantly greater than either HMG-CoA reductase or LDL receptor mRNAs (Fig. 3). On the basis of these and earlier studies (11, 12, 13) it seems likely that SE can serve as the rate-limiting enzyme in the post-mevalonate cholesterol biosynthetic pathway.
25-Hydroxycholesterol is known to down-regulate HMG-CoA reductase and LDL receptor transcription(4) . We demonstrate in Fig. 4that this same sterol, as well as cholesterol, suppresses the increase in SE mRNA normally observed in LPDS cultured HeLa cells. Although 25-hydroxycholesterol suppressed this increase more effectively than cholesterol, this difference may be due partly to the lower solubility of cholesterol. Since SE is involved in the pathway of synthesizing cholesterol alone after branch point, the fact that inhibition of SE activity by NB-598 causes an increase of SE, HMG-CoA reductase, and LDL receptor mRNA levels (Fig. 5) strongly suggests that the sterol produced endogenously can down-regulate these sterol-sensitive genes. We also observed that SE and LDL receptor mRNA levels are increased to a great extent by NB-598 than lovastatin, but the reverse is true for HMG-CoA reductase mRNA ( Fig. 5and Fig. 6). Since we recently demonstrated that purified recombinant SE catalyzes the conversion of squalene epoxide to squalene diepoxide(20) , which is thought to be a precursor of physiological oxysterol, epoxycholesterol(21) , it seems possible that NB-598 up-regulates transcription of the SE and LDL receptor genes higher than lovastatin by inhibiting oxysterol formation. Since lovastatin inhibits the formation of mevalonate-derived non-sterol metabolite(s) as well as cholesterol, the greater effect of lovastatin on HMG-CoA reductase mRNA levels can be explained if the HMG-CoA reductase gene is the only target of negative feedback regulation by the non-sterol metabolite(s) in the transcriptional level.
Recently, a cDNA for the sterol regulatory element-binding protein 1 was cloned, and this factor has been shown to up-regulate both HMG-CoA synthase and LDL receptor genes via octanucleotide sequence termed sterol regulatory element-1(22, 23) . Although the coordinated regulation of the mRNAs for LDL receptor, SE, HMG-CoA reductase, and other enzymes involved in cholesterol biosynthesis may be achieved by a common feedback mechanism, no sterol regulatory elements have yet been identified for the SE gene. Therefore, analysis of the SE gene promoter is in progress. The availability of SE expression systems provide a novel and important step toward elucidating the molecular mechanism(s) involved in the regulation of SE transcription by cholesterol.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D78129[GenBank].