(Received for publication, September 10, 1996, and in revised form, October 24, 1996)
From the Mammalian Cell and Molecular Biology Laboratory, Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182-0057
It is known that hepatic levels of reduced
glutathione correlate with the activity of the liver-specific enzyme
cholesterol-7-hydroxylase. We examined the possibility that
sulfhydryl reducing agents activate transcription of cholesterol
7
-hydroxylase. Adding dithiothreitol (DTT, 1 mM) and
dexamethasone to L35 hepatoma cells increased the content of
7
-hydroxylase mRNA 3-fold above the levels observed with
dexamethasone alone. Without dexamethasone, DTT had no affect. The
addition of reduced glutathione to L35 cells demonstrated a similar
potentiation of expression dependent on dexamethasone. Nuclear
run-on assays showed that in the presence of both dexamethasone and
DTT, the transcription of the 7
-hydroxylase gene was clearly increased. In contrast, by itself, dexamethasone did not cause a
detectable increase in the transcription of the 7
-hydroxylase gene.
Dexamethasone and DTT did not affect the transcription of
-actin,
suggesting a selective induction of the 7
-hydroxylase gene. DTT
reversed repression of 7
-hydroxylase expression by insulin but not
the repression by phorbol ester. Our data show for the first time that
the sulfhydryl redox potential of the hepatocyte (i.e.
level of reduced glutathione) has a marked influence on the
transcription and expression of the liver-specific gene 7
-hydroxylase.
The conversion of cholesterol to bile acids is the major
quantitative pathway through which cholesterol is removed from mammals (1, 2). The initial step controlling bile acid synthesis is catalyzed
by cholesterol 7-hydroxylase (EC 1.14.13.17). This cytochrome P-450
enzyme is expressed only in the liver (3). Hepatic expression of
7
-hydroxylase1 provides this organ with
the unique ability to take up cholesterol ester-rich lipoproteins from
the plasma and to excrete cholesterol into bile in the form of bile
acids and cholesterol (1, 2, 4). This liver-specific cholesterol
excretory pathway may help to maintain cholesterol homeostasis. Recent
studies of mice having a targeted deletion of a functional
7
-hydroxylase gene show that they have marked disruption of hepatic
function, lipid metabolism, and premature death (5, 6). Many of these
pathological effects could be relieved by supplementation with bile
acids and fat-soluble vitamins, providing evidence for the essential
function of bile acid production (5). Thus, it is clear that
7
-hydroxylase plays an essential role both in regulating cholesterol
homeostasis and in digestion and absorption of lipids including
fat-soluble nutrients.
Expression of 7-hydroxylase varies extensively in response to diet
(7, 8), hormones (9-11), diurnal variation, and the enterohepatic
circulation (1, 2). Recent studies indicate that the expression of
7
-hydroxylase is regulated mainly through changes in gene
transcription (7, 11-15).
We have used a unique line of rat hepatoma cells (L35 cells) to examine
the molecular mechanisms regulating 7-hydroxylase (9, 16). These
cells show the unique ability to express 7
-hydroxylase in cultured
cells at levels similar to those observed in vivo (9).
Expression of 7
-hydroxylase by L35 cells is sensitive to
dexamethasone (induced) and insulin (repressed)(16) in a manner similar
to that which occurs in vivo. In this study, we examine the
mechanism responsible for these changes. The results show that by
itself, dexamethasone induces 7
-hydroxylase mRNA expression mainly by a posttranscriptional mechanism (no detectable change in
transcription). However, when presented in combination with the
sulfhydryl reducing agent dithiothreitol (DTT), transcription of
7
-hydroxylase was increased, resulting in an increased mRNA expression that was greater than that observed with dexamethasone alone. Hepatic levels of reduced sulfhydryl reagents may have a
significant influence on 7
-hydroxylase expression.
All reagents used for biochemical techniques were purchased from
Sigma, VWR, or Fisher. Enzymes for restriction or
labeling of cDNA probes were purchased from New England Biolabs or
Boehringer Mannheim. Cell culture medium was obtained from Life
Technologies, Inc./BRL, and serum was from Gemini. DTT and glutathione
(obtained from Sigma) were stored as powdered forms (at 20 °C,
desiccated without exposure to light). Immediately prior to use, each
agent was dissolved in culture medium to a final concentration of 1 mM. This concentration of sulfhydryl reductant was chosen
based on previous studies by our laboratory and others showing that it
affects the sulfhydryl redox state of the cell as shown by changes in
gene expression, the secretion of specific proteins, and the expression
of 7
-hydroxylase without causing toxicity (see "Discussion").
The cDNA probes used for hybridizations have been described (3, 9,
16).
L35 rat hepatoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 8% serum, as described (9, 16). Prior to each experiment, the medium was changed to DMEM containing 0.5% fetal bovine serum and 1% Nutridoma HU for 3 days. The medium was then changed to DMEM lacking serum but containing the indicated concentrations of additives (as indicated in the figure legends). When used, dexamethasone, dissolved in ethanol, was added to DMEM to a final concentration of 0.1 mM (9, 16). Control cells received ethanol only.
RNA Isolation and QuantitationCells were harvested, at the times indicated in the figure legends, by removing the culture medium and adding guanidinium isothiocyanate (18), with modifications (16). Poly(A)-containing RNA was obtained using the miniscale oligo(dT) cellulose (Collaborative Biotech type 3) method as described (16).
RNA (3-10 µg of poly(A) RNA) was loaded onto 0.8% agarose, 3% formaldehyde gels (19) and subjected to electrophoresis. The gels were blotted onto Zetaprobe GT (Bio-Rad) nylon membranes and hybridized with nick translated cDNA probes in the conditions described for Zetaprobe by Bio-Rad. After hybridization and washing, Northern blots were exposed to storage phosphor screens of a Molecular Dynamics PhosphorImager, Kodak Biomax MS film, or to DuPont Reflection Autoradiography film.
Transcription Run-On Assays of NucleiTranscription run-on
assays were performed with modifications of the procedures outlined by
Ausubel et al. (17). L35 cells, cultured as described above,
were harvested by scraping the cells from the plate and pelleting the
cells in a Beckman GPR centrifuge at 1000 rpm using a swinging bucket
rotor for 5 min at 4 °C. The cell pellet was washed with cold,
sterile PBS, and 2.5 ml of hypotonic lysis buffer (10 mM
Tris, pH 7.4, 1 mM KCl, and 3.0 mM
MgCl2) were added and then brought up to 5.0 ml of
hypotonic lysis buffer with 0.3% Nonidet P-40. Then the tubes were
incubated on ice for 5 min. The resulting nuclei were pelleted at 500 rpm for 5 min at 4 °C. They were washed in 5 ml of hypotonic lysis
buffer with 0.3% Nonidet P-40 and pelleted for 5 min at 500 rpm; then
the supernatant was removed. The wash was repeated using hypotonic lysis buffer without Nonidet P-40. The nuclei were flash frozen in
liquid nitrogen and stored at 70 °C in 100 µl of nuclear storage buffer (40% glycerol, 50 mM Tris, 5 mM
MgCl2, and 0.1 mM EDTA, pH 8.3).
The nuclear run-on transcription buffer contained 30% glycerol, 2.5 mM DTT, 1.0 mM MgCl2, 70 mM KCl, 0.25 mM each of GTP, CTP, ATP, and 100 mCi of [32P]UTP (3000 Ci/mmol). The transcription
reaction was run for 15 min at 26 °C and stopped by adding EDTA,
tRNA, and guanidinium isothiocyanate-RNA isolation mix. After the
32P-labeled nuclear RNA was isolated and precipitated,
incorporation was estimated by binding to DE81 paper (Whatman), and
equal counts of each reaction hybridized to nylon-bound unlabeled
cDNAs for 7-hydroxylase and
-actin. Hybridization was
performed at 65 °C in 0.015 M Tris, 0.7 M
NaCl, 0.015 M EDTA, 1 × Denhardt's, 2% (w/v)
Na4P2O7, 0.2% SDS, and tRNA to a
final concentration of 1 mg/ml, pH 7.5. After washing, the blots were
exposed to DuPont Reflection Autoradiography film.
When incubated with L35
cells for 48 h together with DTT (1 mM), dexamethasone
increased the expression of 7-hydroxylase to levels that were
2-3-fold greater than the levels obtained by incubating with
dexamethasone alone (Fig. 1). In contrast, DTT by itself
had no effect on the expression of 7
-hydroxylase mRNA. The
increased abundance of 7
-hydroxylase mRNA caused by the
combination of dexamethasone and DTT was specific, as demonstrated by
no significant effect on the expression of
-actin (Fig. 1). Furthermore, the effect of DTT was not associated with any sign of
toxicity. Protein synthesis, cell viability, and growth were unaffected
(data not shown). These data suggest that the sulfhydryl reducing
agent, DTT, potentiates the induction of 7
-hydroxylase caused by
dexamethasone.
Induction of 7
Results similar to those
obtained using DTT were obtained using the natural sulfhydryl reducing
agent glutathione (Fig. 2). In the presence, but not in
the absence, of dexamethasone, reduced glutathione (1 mM)
induced the expression of 7-hydroxylase mRNA up to 4-fold
greater than the level obtained with dexamethasone alone. In the
absence of dexamethasone, reduced glutathione did not induce
7
-hydroxylase mRNA above that observed with culture medium alone
(Fig. 2). Furthermore, the increased abundance of 7
-hydroxylase
mRNA caused by glutathione was specific, as demonstrated by no
significant effect on the expression of
-actin
DTT Potentiates Dexamethasone Induction of 7
In cells cultured in the absence of either
dexamethasone or DTT, the transcription of 7-hydroxylase was barely
detectable, whereas the transcription of
-actin was clearly evident
(Fig. 3). Adding DTT by itself did not induce
7
-hydroxylase expression. Although dexamethasone by itself caused
dramatic increases in the expression of 7
-hydroxylase mRNA (Fig.
1), in three separate experiments performed on three separate
occasions, its rate of transcription was similar to that of control
cells. In marked contrast, adding DTT with dexamethasone increased the
rate of transcription of 7
-hydroxylase 4.5-fold above the level of
control cells and cells treated with only dexamethasone (Fig. 3). It
may be important to point out that in a fourth experiment of treating L35 cells with dexamethasone alone, the transcription of
7
-hydroxylase was slightly greater than control cells. However, this
rate of transcription was still less than 25% of cells treated with
dexamethasone and DTT (data not shown). Neither dexamethasone nor DTT
(separately or together) affected the transcription of
-actin.
DTT Blocks the Insulin Repression of 7
Previous studies show that once induced by dexamethasone,
L35 cells maintain an expression of 7-hydroxylase in the absence of
dexamethasone for at least 48 h, the longest time period examined (16). This apparently stable expression of 7
-hydroxylase can be
reversed by either insulin or phorbol esters (16). Studies by others
show that insulin (20) and phorbol esters (21) both repress
7
-hydroxylase by blocking transcription. In the absence of DTT,
insulin repressed the expression of 7
-hydroxylase but had no effect
on the expression of
-actin (Fig. 4). However, in the
presence of DTT, insulin had no significant effect on the expression of
7
-hydroxylase or
-actin (Fig. 4). Moreover, either in the
presence or absence of insulin, DTT in combination with dexamethasone
caused the same degree of induction of 7
-hydroxylase (Fig. 4). DTT
did not affect the ability of insulin to increase protein synthesis (as
determined by the incorporation of [35S]methionine into
trichloroacetic acid-precipitable protein (data not shown)). Similar
data were obtained using reduced glutathione (1 mM)(data
not shown).
In contrast to the blocking insulin repression of 7-hydroxylase, DTT
had no effect on the repression caused by phorbol esters (Fig.
5). Both in the absence and presence of DTT,
phorbol esters repressed the expression of 7
-hydroxylase (Fig.
5).
Our results show that DTT affects the ability of dexamethasone and
insulin to alter the expression of 7-hydroxylase. In contrast, DTT
by itself has no affect on 7
-hydroxylase expression. Furthermore, DTT did not affect the ability of phorbol esters to repress
7
-hydroxylase. Additional data demonstrated that the effect of DTT
could be recapitulated with the endogenous sulfhydryl reducing agent
glutathione (22). Together, our data indicate that in the unique
hepatoma cell line (L35 cells), the relative concentration of
sulfhydryl reducing agents selectively affects the ability of hormones
to alter the expression of 7
-hydroxylase mRNA. Previous studies
show that the changes in 7
-hydroxylase mRNA levels in L35 cells
correspond to parallel changes in enzyme activity (9). In addition, the changes in 7
-hydroxylase mRNA levels in L35 cells also
correspond to parallel changes in protein levels, determined by Western
blot analysis (data not shown). The question remains as to whether our
results obtained in L35 cells apply to other liver cell systems or to
the in vivo situation. Although our studies are the first to
show that sulfhydryl reductants affect 7
-hydroxylase gene transcription, there are several studies that show that sulfhydryl reductants affect the expression of 7
-hydroxylase enzyme activity, mRNA levels, and rates of bile acid synthesis in vivo
(rats)(23) and in human hepatoma cells (23-27).
It is well established that hepatic levels of the natural sulfhydryl
reductant glutathione are an important indicator of liver function
(22). Hepatic levels of glutathione change in response to liver injury,
inflammation, and disease. Our results showing a significant role of
sulfhydryl reductants in the expression of 7-hydroxylase may explain
the basis for decreased levels of expression in one or more of these
altered states. The level of glutathione in liver (23) and in HepG2
cells is positively correlated with bile acid synthesis (23) as well as
7
-hydroxylase mRNA levels (24, 25). Induction of
7
-hydroxylase was caused by altering glutathione levels (23) and by
increasing the level of L-cysteine (also a sulfhydryl
donor)(26). Increased cysteine levels were related to induction of bile
acid synthesis by taurine in HepG2 cells (27). Our data show that in
L35 cells, sulfhydryl reducing agents by themselves cannot increase
transcription of 7
-hydroxylase (Fig. 3). These cells require the
addition of dexamethasone to uncover the transcriptional activation of
the 7
-hydroxylase gene.
The redox state of the cell, or the addition of sulfhydryl reducing
agents such as DTT, can affect several components of the transcription
apparatus. The binding affinity of some transcription factors,
including the glucocorticoid receptor (28), SP1 (29), and c-Myb (30)
may be increased by the addition of DTT to cultured cells. Not only is
the DNA binding affinity of thyroid transcription factor I increased by
DTT addition (31) but so are its dimerization and activity in inducing
gene expression in FRTL-5 cells (31). Moreover, in lung adenocarcinoma
A549 cells, DTT can activate the transcription factor NF-B (32).
Interestingly, in these cells, DTT had no affect on SP1 (32). NF-
B
also responds to reduced glutathione levels and a number of different
thiols in T cells (33). These effects of reducing agents on gene
expression are thought to be related to physiologic responses, in at
least T cells (33) and the liver (29), via the relative levels and of
intracellular reduced glutathione. DTT and the cytokine, tumor necrosis
factor, were antagonistic to each other in the regulation of NF-
B
activity in HeLa cells, with tumor necrosis factor activating and DTT
inhibiting NF-
B (34). Regulation of NF-
B by sulfhydryl agents is
mediated via I-
B phosphorylation, dissociation, and proteolysis
(35-37).
The expression of 7-hydroxylase has been shown to be negatively
regulated by phorbol esters (16, 21) and various cytokines including
tumor necrosis factor and interleukin-1 (38). Phorbol esters also
decreased the level of intracellular thiols (35). Although based on
these findings, it might be possible that phorbol esters repress
7
-hydroxylase via a mechanism associated with reduced intracellular
sulfhydryl agents; however, our finding that DTT and glutathione could
not reverse the phorbol ester repression of 7
-hydroxylase in L35
cells argues against this.
An alternative mechanism that might account for reduced sulfhydryl
reagents affecting the transcription of 7-hydroxylase is through
an indirect effect mediated by the secretory pathway. The activation of
NF-
B occurs through proteolysis of the regulatory factor I-
B in
the endoplasmic reticulum (35-37). DTT can alter the retention,
degradation, and secretion of some proteins containing disulfide
bridges by interfering with their ability to fold properly (39).
Because NF-
B can be regulated by the level of unfolded proteins in
the endoplasmic reticulum (34), it is possible that DTT induction of
7
-hydroxylase might be mediated through a signal derived from the
secretory pathway. This signal could affect I-
B proteolysis and/or
NF-
B itself or the content of other transcription factors or gene
products. NF-
B does not always act positively on its target genes;
at least one gene that NF-
B represses has been identified (40).
Our findings showing that sulfhydryl reducing agents have a marked
influence on 7-hydroxylase transcription and expression are
analogous to those reported on the transcriptional regulation of CYP1A1
and CYP1A2 (41). In cultured hepatoma cells, all three hepatic
cytochrome 450s (CYP7, CYP1A1, and CYP1A2) are repressed by insulin in
a manner that is reversed by sulfhydryl reducing agents. CYP1A1 and
CYP1A2 are also repressed by inflammation (41). Previous studies in
C57BL/6 and BALB/c inbred mice show that CYP7 is repressed by feeding a
high fat diet containing cholic acid (4). In marked contrast, feeding
the same diet to inbred strain C3H does not cause a repression of CYP7
(4). Additional studies show that the high fat diet containing cholic
acid causes a NF-
B-induced inflammatory response in C57BL/6 and
BALB/c mice but not in C3H mice (42). This inflammatory response is
associated with reduced hepatic glutathione content (42).
Our findings showing that sulfhydryl reducing agents block the ability
of insulin to repress 7-hydroxylase may help to explain an apparent
enigma in regard to in vivo regulation. It is well established that the expression of 7
-hydroxylase in vivo
is greatest soon after food consumption (1). Normally, this is also
when insulin levels are greatest. Our findings would predict that in the context of normal hepatic function, the hepatic content of reduced
glutathione is sufficient to block insulin repression of
7
-hydroxylase in vivo. An appreciation of our finding
that sulfhydryl reducing agents have a marked influence on
7
-hydroxylase transcription may help to delineate the molecular
events through which the transcription of this important gene product
is altered in response to diet and physiologic state.
We gratefully acknowledge the technical support of Gina Moore and Karl Lewis. In addition, we thank Christian A. Drevon for reading the manuscript and giving helpful suggestions.