(Received for publication, August 9, 1996, and in revised form, February 17, 1997)
From the The intracellular dioxin (aryl hydrocarbon)
receptor mediates signal transduction by dioxin
(2,3,7,8-tetrachlorodibenzo-p-dioxin) and related
environmental pollutants and functions as a ligand-activated transcription factor. In this study we have examined the effects on
dioxin receptor function of a potentially novel ligand, omeprazole, which is widely clinically used as a gastric anti-ulcer drug. In
primary human hepatocytes omeprazole potently induced cytochrome P4501A1 mRNA expression, whereas this effect was not detected in
mouse primary hepatocytes. In human hepatoma cells omeprazole was found
to induce transcription of reporter genes via the xenobiotic response
element that is recognized by the ligand-activated dioxin receptor. In
contrast, the human dioxin receptor was not activated by omeprazole
upon expression in a receptor-deficient mouse hepatoma cell line. In a
reconstituted yeast (Saccharomyces cerevisiae) model
system, however, both the mouse and human dioxin receptors were
potently activated by omeprazole. Although omeprazole failed to
displace dioxin in in vitro ligand binding assays, a
residue within the ligand binding domain that is critical for dioxin
binding in vitro was also critical for omeprazole
responsiveness in vivo. Consistent with this observation,
both omeprazole and dioxin responsiveness of the dioxin receptor was
inhibited in mutant yeast cells expressing low levels of the molecular
chaperone hsp90 that is critical for ligand binding activity. The
sulfoxide group that is essential for formation of a planar conversion
product of omeprazole was found to be critical for dioxin receptor
activation. Taken together, these data suggest that omeprazole
represents a precursor for a novel class of dioxin receptor agonists
that are bona fide dioxin receptor ligands but generated in a strictly
species-specific manner.
The dioxin (also termed the aryl hydrocarbon) receptor is a
ubiquitous intracellular protein that mediates signal transduction by
the widespread and persistent environmental contaminant
2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin,
TCCD1; for recent reviews see Refs. 1 and
2). In various animal models, dioxins and structurally related
compounds elicit a broad spectrum of biological effects ranging from
hepato-, neuro-, and immunotoxic effects, reproductive and teratogenic
disorders to carcinogenic effects (3, 4). The dioxin receptor functions as a ligand-activated transcription factor that belongs to the basic
helix-loop-helix (bHLH) family of DNA binding proteins (5, 6). Within
this large family of proteins the dioxin receptor is distinguished by a
region of homology, the PAS domain, that is contiguous with the bHLH
motif and is common to the Drosophila circadian rhythm
regulatory protein period, the Drosophila developmental regulators trachealess and single-minded, the mammalian
hypoxia-inducible factor-1 Upon exposure to ligand the dioxin receptor acquires an increased
affinity for xenobiotic responsive elements (XREs) that confer dioxin
regulation on target genes, e.g. a battery of genes encoding
drug metabolizing enzymes such as cytochrome P4501A1 (11). Activation
of the receptor to an XRE binding form is accompanied by its apparent
translocation from the cytoplasm to the nucleus of target cells (12, and references therein). The receptor activation pathway is not fully
understood but is clearly a multistep process. In nonstimulated cells
the receptor is recovered as a complex with the molecular chaperone
hsp90. The ligand binding process induces release of hsp90 from the
receptor (13) and concomitant recruitment of the bHLH/PAS factor Arnt,
enabling both proteins to specifically recognize the XRE motif
(14-17). Individually neither the receptor nor Arnt show any
detectable affinity for this target sequence (15).
Targeted disruption of the dioxin receptor gene in mice has indicated
that the receptor may play a role in hepatic development (18, 19).
However, a physiological ligand of the dioxin receptor, if any, has not
yet been identified. Among the known xenobiotic dioxin receptor
ligands, all of which have a planar structure (3, 20), there seems to
exist a good correlation between the affinity of halogenated aromatic
hydrocarbons, their in vitro dioxin receptor activating
capacity (measured as in vitro induction of XRE binding
activity of the receptor in cellular extracts), and their potency to
induce transcription of the cytochrome P450IA1 gene in vivo
(21). Other classes of dioxin receptor ligands include polycyclic
aromatic hydrocarbons (e.g.
benzo[a]pyrene and 3-methylcholanthrene), flavonoids, and
indole derivatives, most notably indolo[3,2-b]carbazole, a
dioxin receptor agonist of dietary origin that binds to the receptor
with the same affinity as the prototypical dioxin TCDD (20, 22, 23, and
references therein). Within the latter three classes of ligands,
resistance to metabolism plays a very important role in the biological
potency of the compound (23, 24). Interestingly, omeprazole, a
benzimidazole derivative clinically used as a gastric proton pump
inhibitor (25-27 and references therein), has been demonstrated to
induce expression of the dioxin receptor target gene cytochrome P4501A1
in both primary human hepatocytes (28), human hepatoma cells (29), tissues of the human alimentary tract (30), and human colon adenocarcinoma cells (31). In this context, the mechanism of action of
omeprazole remains, however, unclear. Whereas omeprazole has been shown
to induce XRE binding activity of the dioxin receptor in nuclear
extracts of human hepatoma cells (29), the failure to detect any
binding of omeprazole to the dioxin receptor has been reported (32).
Thus, these data would argue against the simple model that omeprazole
represents a dioxin receptor ligand.
In the present study we have investigated regulation of dioxin receptor
function by omeprazole. We demonstrate that induction of cytochrome
P4501A1 mRNA expression by this compound, and activation of a
minimal XRE-driven reporter gene construct, was species-specific. Thus,
the omeprazole-induced activation response was only observed in human
but not mouse primary hepatocytes or hepatoma cell lines. In contrast,
omeprazole potently activated both the mouse and human dioxin receptors
in a reconstituted yeast model system. Strikingly, a specific point
mutation that abrogates dioxin binding in vitro by the
receptor inhibited the omeprazole activation response in yeast,
strongly suggesting that receptor activation was mediated by the ligand
binding domain. Moreover, omeprazole as well as dioxin responsiveness
in yeast was totally impaired at low levels of expression of hsp90 that
is a critical chaperone for ligand binding activity. We failed,
however, to observe any binding by omeprazole in in vitro
receptor ligand binding assays. In conclusion, dioxin receptor-mediated
signal transduction by omeprazole appears to be regulated at two
distinct levels. Although the mouse dioxin receptor is responsive to
omeprazole treatment per se, the omeprazole activation
response is repressed in mouse hepatocytes or hepatoma cells.
Furthermore, omeprazole appears to require chemical conversion or
metabolism in vivo to an active, planar principle of
transient nature that regulates dioxin receptor function via the ligand binding domain, possibly as a novel class of receptor ligand.
Primary hepatocytes were prepared from human hepatic
tissue fragments following lobectomy, or from C57BL/6 mouse liver as described (28, 33, 34), and maintained in culture in a 1:1 mixture of
Ham's F12 and Williams' E media, supplemented as described (35).
Mouse Hepa 1c1c7, Hepa 1c1c7 c12, and human HepG2 hepatoma cells were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
as described (13). For RNase protection experiments cells were exposed
to 100 µM Cytosolic
extracts were prepared from confluent Hepa 1c1c7 cells by
homogenization of untreated cells in 1 volume of TEG buffer (1 mM EDTA, 20 mM Tris-HCl, pH 7.4, 10% (v/v)
glycerol, 1 mM dithiothreitol), followed by centrifugation
at 120,000 × g for 45 min. The resulting supernatant
was taken as the cytosolic fraction and used either immediately or
frozen in small aliquots at pLBD-(230-421) containing the minimal
ligand binding domain of the mouse dioxin receptor (43) was used for
in vitro translation in rabbit reticulocyte lysate (Promega)
in the presence of either [35S]methionine (Amersham
Corp.) or unlabeled methionine, according to the manufacturer's
protocol. In vitro translation mixtures (50 µl) or Hepa
1c1c7 cell cytosolic extracts were incubated for 3 h at 0-4 °C
with 50 µl of TEG buffer containing 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and
[3H]TCDD (40 Ci/mmol; Chemsyn, Lenexa, KS) at a final
concentration of 2.5 nM in the absence or presence of a
400, 4,000, or 20,000-fold molar excess of competitor ligand. Ligand
binding activity was analyzed by centrifugation on 5-20% (w/v) or
10-40% (w/v) sucrose density gradients, respectively, prepared in TEG
buffer containing 25 mM KCl and 20 mM sodium
molybdate. Prior to sucrose gradient centrifugation, sodium molybdate
was added to the samples to a final concentration of 20 mM,
and the samples were treated with dextran-coated charcoal (13) for 5 min to remove unbound ligand. After rapid centrifugation the resulting
supernatants were layered onto sucrose gradients that were centrifuged
at 300,000 × g for 15 h to a cumulative
centrifugal force of 1.7 × 10 12 rad2/s in a Beckman
L8-60 ultracentrifuge. Fractions were collected by gravity flow,
starting from the bottom of the gradients, and analyzed for
radioactivity by scintillation counting.
Yeast strain GRS4 transformed with the mouse
pHG/GRDBD/mDR83-805 or human pHG/GRDBD/hDR84-848
glucocorticoid-dioxin receptor fusion proteins, respectively, has been
described previously (44). Yeast expression vectors for the full-length
mouse dioxin receptor (pH2GPD/mDR), human dioxin receptor (pH2GPD/hDR),
the human dioxin receptor carrying the specific point mutations
Val381 Single colonies were grown
to saturation in selective medium containing either 2% galactose or
2% glucose at 25 °C for the chimeric receptors and with 2% glucose
at 30 °C for the full-length dioxin receptor constructs. Cultures
were diluted 1:6 in fresh medium and grown overnight in the absence or
presence of either omeprazole or the high affinity dioxin receptor
ligand indolo[3,2-b]carbazole, synthesized as described
(48). Omeprazole analogues, timoprazole (TI), and desoxytimoprazole
(D-TI), were prepared as described (49). Yeast cells were collected
from the overnight culture by centrifugation and assayed for enzyme
activity as described previously (50, 51).
Yeast
cells expressing wild-type or point-mutated dioxin receptor forms were
grown at 30 °C in appropriate selection media containing 2%
glucose. Extracts were prepared as described previously (52). For
assaying expression levels of wild-type and mutant dioxin receptor
forms, 20 µg of total yeast extract protein was analyzed in
immunoblot experiments using a dioxin receptor antiserum as described
previously (15).
To investigate effects of omeprazole on dioxin
receptor function in vivo, we transiently transfected mouse
Hepa 1c1c7 and human HepG2 cells with the XRE-driven reporter gene
construct pXRE-MMTV-AP. As schematically represented in Fig.
1A, this reporter gene contains a minimal
mouse mammary tumor virus (MMTV) promoter in which endogenous
glucocorticoid responsive elements have been substituted with a single
XRE from the cytochrome P4501A1 gene (15). In Hepa 1c1c7 cells this
reporter gene shows very low basal activity in the absence of ligand
treatment and strong transcriptional activation following the addition
of 10 nM dioxin (15; Fig. 1B). Interestingly,
activation of the reporter gene was not detected upon addition of 50 µM omeprazole to Hepa 1c1c7 cells (Fig. 1B). In contrast, transcription of the XRE-driven reporter gene was induced
about 3-fold by omeprazole in human HepG2 cells. This induction
response represented only a fraction of the maximal activation response
(around 9-10-fold induction) produced by dioxin treatment (Fig.
1B), possibly due to rapid metabolism of the inducing compound during the prolonged (24-48 h) reporter gene assay.
Consistent with the modest reporter gene activation response observed
in the present study, omeprazole has been demonstrated to induce cytochrome P4501A1 mRNA levels in HepG2 cells to approximately 30%
of the response observed with dioxin (29).
Within the asymmetric
P4501A1 XRE core motif TGCGTG (53; E box
half-site shown in italics) a single point mutation (TGAGTG; mutation underlined) abrogates recognition of the
XRE target sequence by the ligand-activated dioxin receptor in in vitro DNA binding assays (42) and dioxin receptor function
in vivo (15). Given the XRE-driven reporter gene activation
response that was produced by omeprazole treatment of human HepG2
cells, we also examined the effect of omeprazole on the activity of a reporter gene driven by a single copy of a mutated XRE motif, XREmut (Fig. 1A), containing this single point
mutation. In HepG2 cells, omeprazole failed to induce reporter gene
activity from the XREmut-driven promoter (Fig.
1B), demonstrating that the XRE sequence motif was the
target for regulation and strongly implying the XRE binding dioxin
receptor-Arnt complex in mediating the omeprazole induction
response.
The failure to activate either the XRE-driven
reporter gene construct (Fig. 1B) or cytochrome P4501A1
mRNA levels (data not shown) by omeprazole treatment of mouse Hepa
1c1c7 cells prompted us to examine if the absence of this response was
due to the loss of a possible differentiation-sensitive cofactor. We
therefore compared by a sensitive RNase protection assay (37)
regulation of cytochrome P4501A1 mRNA expression by omeprazole in
human primary hepatocytes and primary hepatocytes from the dioxin- and
polycyclic aromatic hydrocarbon-responsive (3) mouse strain C57BL/6. In human or mouse primary hepatocytes treated with vehicle
(Me2SO) alone, no basal expression levels of cytochrome
P4501A1 mRNA were detected (Fig. 2, A and
B, lane 2). As demonstrated earlier (28, 33)
exposure of human primary hepatocytes to 50 µM omeprazole resulted in potent induction of cytochrome P4501A1 mRNA
steady-state levels. A similar induction response was produced by an
identical dose of the well characterized (3, 4) dioxin receptor ligand
To further examine
species-specific differences in omeprazole regulation of mouse
versus human dioxin receptor function, we transiently
expressed the human dioxin receptor in mutant mouse hepatoma C12 cells
that express very low levels of dioxin receptor and are functionally
dioxin-nonresponsive (1). Whereas this procedure reconstituted
induction of XRE-dependent reporter gene activity by
dioxin, no activation of the reporter gene was seen following
omeprazole treatment for 24 h (Fig. 3). Since
omeprazole is biologically unstable and rapidly and extensively
metabolized in both rodents and humans (54, 55, and references
therein), we also analyzed reporter gene activity after 8 and 12 h
of omeprazole treatment but failed to observe any induction response
(data not shown). These results demonstrate that activation of human
dioxin receptor function by omeprazole is repressed in a mouse cell
environment.
Given the rapid
metabolism of omeprazole, we next examined the effect of omeprazole on
dioxin receptor function in a cellular environment that is
metabolically more neutral than cells of hepatic origin. We first used
a reconstituted yeast (Saccharomyces cerevisiae) model
system where either the mouse or human full-length dioxin receptor was
expressed in the presence of Arnt and a
The yeast experiments described above demonstrated
that both the mouse and human dioxin receptor-Arnt complexes were
activated by omeprazole. However, this experimental system did not
provide an answer as to whether the dioxin receptor, Arnt, or both
transcription factors were regulated by omeprazole. To critically
examine specific regulation of dioxin receptor function by omeprazole,
we therefore used yeast strains that express chimeric dioxin receptor
constructs and, in the presence of galactose, wild-type levels of the
molecular chaperone hsp90 (44). As outlined in Fig.
5A, the chimeric receptors, GRDBD/mDR83-805
and GRDBD/hDR84-848, respectively, contain a heterologous DNA binding
domain, the zinc finger motif of the human glucocorticoid receptor,
fused to structures of the mouse or human dioxin receptors that lack
the bHLH dimerization and DNA binding motif but span regions important
for ligand binding and thus conditional regulation (56). These chimeric
receptors are functionally uncoupled from the ubiquitous bHLH/PAS
partner factor Arnt since they show ligand responsiveness in
Arnt-deficient mutant hepatoma cells (56) and in yeast cells that do
not express endogenous Arnt (44, 57).
Exposure to omeprazole did not alter the low levels of constitutive
functional activity produced by the glucocorticoid receptor DNA binding
domain on a glucocorticoid response element-driven The chimeric dioxin receptor constructs were next expressed
in yeast cells in the presence of glucose, i.e. conditions
yielding very low levels (about 5% of wild-type levels) of hsp90
expression (44, 58). Under these conditions, activation of mouse and human dioxin receptor function by omeprazole was totally abrogated (Fig. 5B). In a similar fashion, the receptor was
unresponsive to ICZ or It has
previously been reported that omeprazole does not bind to the dioxin
receptor in extracts from primary human hepatocytes in vitro
(32). This apparent contradiction to the present results prompted us to
further explore the possibility that omeprazole may bind to the dioxin
receptor. We initially performed ligand binding studies with cytosolic
extracts from mouse Hepa 1c1c7 cells. These extracts were labeled with
[3H]dioxin in the absence or presence of a 400-fold molar
excess of the unlabeled high affinity receptor ligand
indolo[3,2-b]carbazole, ICZ, or 4,000 to 20,000-fold molar
excesses of omeprazole, i.e. concentrations producing
maximal induction in the yeast model system. Upon sucrose density
gradient centrifugation the [3H]dioxin-labeled dioxin
receptor was recovered in the ~9 S region of the gradient (Fig.
6A), consistent with the sedimentation
properties of the ~300-kDa dioxin receptor-hsp90 complex (13). In
contrast to results obtained with ICZ, omeprazole did not displace
[3H]dioxin from the dioxin receptor (Fig. 6A).
We also used a fast hydroxylapatite adsorption assay to separate
protein-bound [3H]dioxin from free ligand. In this assay
incubation of the cytosolic extract from nontreated Hepa 1c1c7 cells
with [3H]dioxin in the absence or presence of an excess
of TCCD or ICZ demonstrated specific binding of
[3H]dioxin, but we observed no competition for binding by
omeprazole (data not shown).
In additional ligand binding experiments we expressed the minimal
ligand binding domain of the dioxin receptor, DR230-421 (43, 56, schematically represented in Fig. 6B), by in
vitro translation in reticulocyte lysate. Consistent with the
results obtained in crude cytosolic extracts from Hepa1c1c7 cells,
[3H]dioxin binding to the minimal ligand binding domain
was not displaced by an excess of omeprazole, as assessed by sucrose
gradient sedimentation (Fig. 6B). Thus, we did not detect
any affinity of omeprazole for the mouse dioxin receptor in a series of
different ligand binding assays. In agreement with the present results, omeprazole has also been reported to fail to displace
[3H]dioxin from the human dioxin receptor in
vitro (31). Moreover, incubation of Hepa1c1c7 cytosolic extracts
with increasing concentrations (up to 50 µM) of
omeprazole in vitro did not result in any induction of the
DNA binding activity of the dioxin receptor in gel mobility shift
experiments (data not shown). In conclusion, omeprazole was inert in
binding to the dioxin receptor and activating it to a DNA binding form
in vitro.
We have
demonstrated above that activation of the dioxin receptor by omeprazole
was mediated by structures spanning the ligand binding domain. In
contrast, omeprazole failed to bind the dioxin receptor and activate
its DNA binding activity in vitro. To further examine the
role of the ligand binding domain in omeprazole signaling, we expressed
in yeast cells either wild-type or point-mutated dioxin receptor
derivatives together with the Arnt partner factor. As schematically
represented in Fig. 7A, Val381
within the ligand binding domain of the human dioxin receptor has been
mutated to either Ala or Asp. The Val
Omeprazole has three important structural elements: a
substituted benzimidazole ring, a substituted pyridine ring, and a
sulfoxide-containing chain connecting the 2 positions of the two ring
systems (Fig. 8A). A highly labile
sulfenamide structure (Fig. 8A), generated by acid
decomposition of omeprazole, has been proposed to be the active gastric
acid secretion inhibitor formed in vivo (26). Interestingly,
this conversion product shows planarity, a structural hallmark of
dioxin receptor ligands (3, 20). This compound is, however, very labile
and of transient nature (26). Consistent with these properties, we have
failed to induce the DNA binding activity of the cytosolic mouse dioxin
receptor activity in a highly sensitive in vitro gel
mobility shift assay using extracts from human HepG2 cells pretreated
with 50 µM omeprazole for 2 and 4 h, respectively
(data not shown). To examine the importance of the hypothetical planar
sulfenamide for dioxin receptor activation, we therefore tested the
activity of omeprazole analogues containing either a sulfoxide
(timoprazole, TI) or a sulfide (desoxytimoprazole, D-TI) group (Fig.
8A) but lacking substituents. To achieve ring closure
omeprazole undergoes C-S bond cleavage and aromatization, and finally,
the oxygen of the sulfoxide is protonized forming sulfenic acid. The
subsequent formation of the sulfenamide is due to known reactions
between sulfenic acids and amines (26, and references therein). Because
of the loss of the oxygen atom the sulfide-containing form (D-TI) is
unable to form the ring structure.
In the reconstituted yeast model system, omeprazole and TI showed very
similar dose dependences in activating the dioxin receptor (Fig.
8B), demonstrating that the substituent groups were not important for generating this response. In excellent agreement with
this observation, lansoprazole, a gastric anti-ulcer drug that
chemically only differs from omeprazole with regard to substituents, potently activated dioxin receptor function in yeast cells (data not
shown) and induced cytochrome P4501A1 mRNA expression in
hepatocytes (33). Interestingly, however, D-TI was inert at the lowest
tested doses (5 and 10 µM) and showed activity only at
the highest tested dose: 50 µM (Fig. 8B).
Thus, the sulfoxide group is critical for dioxin receptor activation.
The ability of D-TI to activate the receptor at high doses is most
probably due to metabolism generating the sulfoxide form, thereby
enabling it to undergo the same reaction scheme as described for
omeprazole. In strict correlation with these results, the sulfoxide
chain has been reported to be essential for the antisecretory effect of
omeprazole, whereas the sulfide group only has a low activity (62, 63).
On the basis of these data we propose that the planar sulfenamide
structure is a possible ligand of the dioxin receptor that is formed
in vivo from omeprazole.
In the present report we have used a reconstituted yeast model
system to demonstrate that omeprazole potently activated both mouse and
human dioxin receptor function in a dose-dependent manner via a mechanism mediated by the ligand binding domain of the receptor. In agreement with these data, transfection experiments using minimal reporter gene constructs indicated that the omeprazole activation response in human hepatoma cells was mediated via the core XRE motif
that is recognized by the ligand-activated dioxin receptor-Arnt complex. Strikingly, however, we failed to observe omeprazole-induced reporter gene activation in mouse hepatoma cells or induction by
omeprazole of cytochrome P4501A1 mRNA steady-state levels in either
mouse primary hepatocytes or mouse hepatoma cells, suggesting that
omeprazole responsiveness in liver cells may be species-specific. In
strong support of this model, activation of the human dioxin receptor
by omeprazole was repressed following expression of this receptor in a
mutant mouse hepatoma cell line expressing very low levels of
endogenous mouse dioxin receptor.
It has not been
possible to demonstrate any affinity of omeprazole for either the human
(31, 32) or mouse (Fig. 6) dioxin receptors in a number of different
in vitro assays including ligand binding experiments or
assays monitoring induction of receptor-dependent DNA
binding activity, and it has recently been proposed that benzimidazole derivatives such as omeprazole may induce cytochrome P4501A1 expression by ligand-independent mechanisms (64). In contrast to this model, we
present here four independent lines of evidence based on in vivo experiments that, taken together, suggest that activation of
the dioxin receptor by omeprazole is mediated via ligand recognition by
the ligand binding domain. First, chimeric receptor experiments indicate that omeprazole responsiveness is mediated by the ligand binding domain of the receptor. Second, the omeprazole activation response was abrogated upon down-regulation in yeast cells of the
expression levels of the molecular chaperone hsp90 which has previously
been demonstrated to be required for folding of a high affinity ligand
binding conformation of the dioxin receptor in vitro (59,
60) and in vivo (44, 57). Third, point mutation experiments
demonstrated that omeprazole responsiveness was dependent on a residue,
Val381, within the ligand binding domain that is essential
for ligand binding activity in vitro (45). Whereas the
Val381 These data strongly argue that omeprazole activates the dioxin receptor
via a ligand binding mechanism in vivo. Since parental omeprazole is inert in binding to the receptor in vitro, a
specific metabolic pathway or chemical rearrangement appears to be
required to generate the active principle that regulates receptor
function, most likely by occupation of the ligand binding domain of the receptor in vivo. The present experiments indicate that the
putative ligand may be a planar sulfenamide structure, i.e.
the theoretical active gastric acid secretion inhibitor formed in
vivo from omeprazole (26, 63). Given the highly reactive and
transient nature of this compound, it has hitherto not been possible to
synthesize it or to "trap" it in conditioned medium or cells. In
summary, our results raise the possibility that benzimidazole
derivatives such as omeprazole and lansoprazole represent precursors of
a novel class of possibly biologically important dioxin receptor agonists. In view of the indicated role of the dioxin receptor in
normal liver growth and development (18, 19), it may be interesting to
examine if these clinically widely used compounds modulate
physiological regulatory pathways of the dioxin receptor in the liver
and other target tissues, e.g. within the gastrointestinal tract.
Interestingly, our results define two distinct levels of
regulation of omeprazole signaling. In addition to omeprazole possibly representing a novel class of dioxin receptor ligands with the ability
to activate both the mouse and human dioxin receptor forms, it is
striking that neither dioxin receptor function nor expression of the
dioxin receptor target gene cytochrome P4501A1 were induced by
omeprazole in primary C57BL/6 hepatocytes and Hepa 1c1c7 cells. Moreover, omeprazole responsiveness of the human dioxin receptor was
repressed in mouse Hepa-1 c12 cells. Thus, the omeprazole activation
response is also determined by cellular factors other than the
receptor. Since formation of the sulfenamide structure is critical for
the activation response, it is tempting to speculate that cellular
differences in the ability to chemically convert the parental compound
to the planar one could determine the inability of dioxin receptor to
respond to omeprazole in mouse hepatic cells. Alternatively, the very
labile sulfenamide structure is so transient in mouse hepatoma cells
that it fails to elict a response. In gastric parietal cells that
represent one of the few examples of the cells with a low pH value, the
sulfenamide structure is most likely generated by acid decomposition
(26, and references therein). However, there is a paucity of data on
other cellular processes generating this product. To understand the
complex biology of both omeprazole and the dioxin receptor, it is
therefore now important to not only identify the putative novel class
of ligands derived from omeprazole and structurally related compounds
but also to dissect the omeprazole signaling pathway to characterize molecular determinants of species specificity of omeprazole action in
hepatic tissues.
We thank Christine Kramer for assistance in
the preparation of primary hepatocytes, Dr. Anthony Wright for W303-1A
yeast cells, and Dr. Pekka Kallio (Karolinska Institute) for fruitful
discussions.
Department of Cell and Molecular Biology,
Department of
Chemistry, Faculty of Science, Tohoku University,
Sendai 980, Japan
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, the constitutively active transcription
factor Arnt, and a human putative Down syndrome-critical factor (7-10,
and references therein).
Cells, RNase Protection Assay, and Transient Transfection
Experiments
-naphthoflavone or 100 µM
omeprazole for 15 h. Total RNA was isolated as described (36) and
analyzed in RNase protection assays as described (37) using cytochrome P4501A1 antisense RNA probes in vitro synthesized from
cDNA spanning either exon 2 of human cytochrome P4501A1 or exon 3 of mouse cytochrome P4501A1. The wild-type or point-mutated reporter
gene constructs pXRE-MMTV-AP or pXMUT-MMTV-AP (15), respectively, were
transiently transfected into 60-80% confluent murine Hepa 1c1c7 or
human HepG2 cells using cationic liposomes
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate, Boehringer Mannheim) according to the manufacturer's protocol. After a 12-h transfection period, cells were incubated with
either 10 nM dioxin, 50 µM omeprazole (Glaxo
Research Group Ltd.), or vehicle alone (0.1% Me2SO) in
fresh medium for 48 h. Secreted placental alkaline phosphatase
levels produced by the reporter gene were determined by a colorimetric
assay (38). Briefly, cell supernatants were heated at 65 °C for 30 min and then incubated at 37 °C in a mixture containing 200 mM Tris, pH 8.8, 275 mM NaCl, 0.5 mM MgCl2, and 5 mM
p-nitrophenyl phosphate. Phosphatase levels were measured by
absorbance at 405 nm. The dioxin receptor was transiently expressed in
mutant hepatoma C12 cells (1) using pCMVDR (39), pTXDir (40) as an
XRE-dependent luciferase reporter gene, and cationic
liposomes (Lipofectin, Life Technologies, Inc.) as transfection
agent.
70 °C. DNA binding activity of the
dioxin receptor was monitored by a gel mobility shift assay performed
essentially as described previously (41) using a double-stranded XRE
oligonucleotide derived from the cytochrome P4501A1 gene as probe (42).
Briefly, DNA binding reactions were assembled in the presence of 1 µg
of nonspecific poly(dI-dC) (Pharmacia Biotech Inc.) in 10 mM Hepes, pH 7.9, 5% (w/v) glycerol, 0.5 mM dithiothreitol, 2.5 mM MgCl2, 1 mM
EDTA, 0.08 (w/v) Ficoll, in a final reaction volume of 20 µl, and at
a final concentration of 50 mM NaCl. The reactions were
incubated for 30 min at 25 °C, and protein-DNA complexes were
resolved on 4% (acrylamide/bis-acrylamide: 29/1) native
polyacrylamide gels at 30 mA and 25 °C using a Tris glycine EDTA
buffer (41).
Ala and Val381
Asp (45),
pH2GPD/hDRVal381
Ala and pH2GPD/hDRVal381
Asp, respectively, and wild-type Arnt (pTCAGPD/Arnt) were
transformed into the yeast strain W303-1A (46) together with the
reporter plasmid pXRE-CYC1-lacZ (containing two tandem synthetic XRE
elements in front of the CYC1 promoter and
lacZ)2 using a modification of the lithium
acetate method (47).
-Galactosidase Assays
Regulation of Dioxin-responsive Reporter Genes by
Omeprazole
Fig. 1.
Activation of reporter gene expression by
omeprazole is mediated by the cytochrome P4501A1 XRE motif.
A, schematic representation of the reporter gene constructs that
contain a single copy of wild-type or point-mutated
(XREmut) cytochrome P4501A1 XRE motif,
respectively, in front of the mouse mammary tumor virus promoter and
alkaline phosphatase (AP) reporter gene. B,
effect of omeprazole on XRE-dependent reporter gene
activity. Mouse Hepa1c1c7 cells or human HepG2 cells were transiently
transfected with the reporter gene constructs and incubated with
vehicle alone (dimethyl sulfoxide, DMSO), 10 nM
TCCD, or 50 µM omeprazole (OM), as
indicated. Results of a representative experiment are shown. Levels of
secreted alkaline phosphatase were measured by a colorimetric assay.
[View Larger Version of this Image (21K GIF file)]
-naphthoflavone (Fig. 2A, compare lanes 2-4).
In contrast, no induction of cytochrome P4501A1 mRNA levels was
detected upon incubation of mouse primary C57BL/6 hepatocytes with
increasing concentrations (up to 50 µM) of omeprazole. In
control experiments, cytochrome P4501A1 mRNA levels were maximally
induced in the mouse primary hepatocytes by 10 µM
-naphthoflavone (Fig. 2B, compare lanes 2-6).
Thus, although the XRE reporter gene experiments strongly implicated
the dioxin receptor in mediating the omeprazole activation response in
HepG2 cells, the dioxin receptor endogenous to mouse primary
hepatocytes or hepatoma cells was not activated by omeprazole, indicating striking species-specific differences in omeprazole sensitivity.
Fig. 2.
Species-specific differences in cytochrome
P4501A1 induction by omeprazole in primary hepatocytes. A,
human primary hepatocytes were incubated for 24 h in the absence
or presence of 50 µM of either -naphthoflavone
(BNF) (lane 3) or omeprazole (lane 4).
Total RNA was isolated and analyzed in an RNase protection assay with a
human cytochrome P4501A1 antisense probe. B, primary C57BL/6
mouse hepatocytes were treated for 17 h with vehicle alone (lane 2), 10 or 50 µM omeprazole (lanes
3 and 4), or 10 or 50 µM
-naphthoflavone (lanes 5 and 6). Total RNA was
isolated and analyzed by RNase protection using a mouse cytochrome
P4501A1 antisense probe. In both panels, lane 1 represents
undigested probe.
[View Larger Version of this Image (29K GIF file)]
Fig. 3.
Omeprazole responsiveness of the human dioxin
receptor is repressed in mouse hepatoma cells. A, schematic
representation of the reporter gene construct pTXDIR which contains two
direct repeats of the XRE in front of the tyrosine kinase
(TK) promoter and luciferase reporter gene. B,
mutant mouse hepatoma cells were transiently cotransfected with the
human dioxin receptor expression vector pCMVDR and the pTXDIR reporter
gene, and incubated with vehicle alone (dimethyl sulfoxide,
DMSO), 10 nM TCDD, or 50 µM omeprazole (OM), as indicated. Results of a representative
experiment are shown.
[View Larger Version of this Image (20K GIF file)]
-galactosidase reporter gene
driven by the yeast CYC1 promoter and dimerized copies of
the cytochrome P450 XRE sequence motif.2 These
reconstituted systems were highly inducible by the high affinity dioxin
receptor ligand indolo[3,2-b]carbazole (ICZ) with an
EC50 value estimated to be approximately 0.5 and 5 nM, respectively, for both the mouse (Fig.
4B) and human (Fig. 4C) dioxin
receptors. Thus, this yeast model system very faithfully reproduced the
high affinity (Kd
1 nM) with which
ICZ binds the dioxin receptor in ligand binding assays in
vitro (20, and references therein). In comparison, omeprazole also
potently activated function of both the mouse and human dioxin
receptor-Arnt complexes (Fig. 4A). This activation response
was mediated by the dioxin receptor-Arnt complex since it was not
observed in yeast cells only transformed with the reporter gene (Fig.
4A). Omeprazole yielded EC50 values of 5-10
µM for both the mouse (Fig. 4B) and human
(Fig. 4C) dioxin receptors. Thus, in striking contrast to
mouse primary hepatocytes or hepatoma cells, the mouse dioxin receptor
was omeprazole-responsive following expression in yeast cells. The
obtained EC50 values are at least 1 order of magnitude
lower than the EC50 value reported for cytochrome P4501A1
promoter activation in HepG2 cells (~100 µM; 29),
possibly due to a decreased rate of metabolism of omeprazole in yeast
cells.
Fig. 4.
Reconstitution of dioxin receptor function in
yeast. A, activation of full-length mouse or human dioxin
receptor function in yeast by omeprazole. Arnt was co-expressed with
either mouse or human dioxin receptor in yeast cells transformed with
lacZ reporter gene (XRE-CYC1-lacZ) driven by
tandem copies of the cytochrome P4501A1 XRE motif and the yeast CYC1
promoter. Yeast cultures were grown in medium containing 2% glucose
and incubated with 10 µM omeprazole (OM) or 10 nM ICZ and assayed for -galactosidase activity. The
effect of omeprazole and ICZ was also examined using yeast cells
transformed with the reporter gene alone, as indicated. Dose-dependent activation of mouse (B) and human
(C) dioxin receptors by omeprazole. Yeast cells transformed
with reporter gene, Arnt, and mouse or human dioxin receptor were
exposed to increasing concentrations of ICZ (squares) and
omeprazole (diamonds) and assayed for
-galactosidase
activity. Results of a representative experiment are shown.
[View Larger Version of this Image (26K GIF file)]
Fig. 5.
Activation of dioxin receptor function by
omeprazole requires the ligand binding domain of the receptor and the
molecular chaperone hsp90. A, schematic representation of
chimeric constructs spanning the DNA binding domain of the
glucocorticoid receptor, GRDBD (lacking the transactivation domain
located between amino acids 77 and 262), fused to mouse (amino acids
83-805) or human (amino acids 84-848) dioxin receptor (DR)
fragments lacking the bHLH motif. The PAS domain of the dioxin receptor
is indicated (hatched). B, omeprazole activates
the chimeric dioxin receptors in an hsp90-dependent
fashion. Yeast cells transformed with a glucocorticoid response
element-driven -galactosidase reporter gene together with either
GRDBD or the chimeric receptors GRDBD/mDR83-805 or GRDBD/hDR84-848 were
grown in a medium containing 2% galactose (yielding wild-type
expression levels of hsp90) or 2% glucose (resulting in low expression
levels of hsp90) and exposed to 50 µM omeprazole, 10 nM ICZ, or vehicle alone (dimethyl sulfoxide, DMSO), as indicated, and assayed for
-galactosidase
activity after 24 h of incubation. Results of a representative
experiment are shown.
[View Larger Version of this Image (25K GIF file)]
-galactosidase
reporter gene (Fig. 5B). However, when the yeast cells were
grown in the presence of galactose and thus expressed wild-type levels
of hsp90 (44), both the mouse and human dioxin-glucocorticoid receptor
fusion proteins were activated by omeprazole (Fig. 5B). As a
reference, ICZ treatment produced a strong activation response with
both chimeras (Fig. 5B). These experiments demonstrate that structures spanning the ligand binding domain of both the mouse and
human dioxin receptors mediate responsiveness to omeprazole.
-naphthoflavone in the presence of low levels
of hsp90 (44, 57, Fig. 5B). Since the molecular chaperone
hsp90 has been demonstrated to be required for ligand binding activity
of the dioxin receptor in vitro (59, 60), these results
imply that omeprazole regulates dioxin receptor function via a ligand
binding mechanism.
Fig. 6.
Failure of omeprazole to bind the dioxin
receptor in vitro. A, a cytosolic extract was
prepared from untreated mouse Hepa 1c1c7 cells and incubated in
vitro with 2.5 nM [3H]TCCD in the
absence (squares) or presence of a 400-fold molar excess of
unlabeled competitor, ICZ (diamonds), or a 4,000- (circles) or 20,000 (triangles) -fold molar
excess of omeprazole (OM). Ligand binding activity by the
receptor was analyzed by sedimentation through 10-40% (w/v) sucrose
density gradients, and bound [3H]TCDD was measured by
scintillation counting. B, the minimal ligand binding
domain, DR 230-421, was expressed by in vitro translation in rabbit reticulocyte lysate and labeled in vitro with 2.5 nM [3H]TCDD (squares) in the
absence or presence of a 400-fold molar excess of either ICZ
(diamonds) or omeprazole (circles), and analyzed for specifically bound [3H]TCDD on 5-20% (w/v) sucrose
density gradients, as described above. Results of a representative
experiment are shown.
[View Larger Version of this Image (21K GIF file)]
Asp mutation totally
abrogates ligand binding activity in vitro, whereas the Val
Ala mutation rather appears to result in a 2-5-fold increase in
ligand binding affinity (45). By analogy the corresponding residue
(375) in the C57BL/6 mouse dioxin receptor is Ala (45), and
importantly, this receptor binds dioxin in vitro with about 1 order of magnitude higher affinity than the DBA/2 mouse dioxin receptor (61) which contains Val in position 375 (45). Consistent with
these in vitro observations the Val381
Ala
mutation of the human dioxin receptor did not negatively affect the ICZ
activation response in the yeast reconstituted model system (Fig.
7B). In contrast, this activation response was abolished
when the dioxin receptor form containing the Val381
Asp
mutation was expressed in yeast (Fig. 7B). Surprisingly, the
effect of these mutations on omeprazole responsiveness mirrored those
observed with ICZ, suggesting that Val381 is a critical
residue not only for ICZ recognition but also for omeprazole signaling.
In fact, the Val381
Ala mutation appeared to increase
omeprazole responsiveness, in agreement with its increased ligand
binding affinity in vitro (45). Importantly, immunoblot
analysis demonstrated very similar expression levels of the wild-type
receptor relative to the responsive Val381
Ala mutant,
whereas the nonresponsive Val381
Asp mutant receptor
was expressed at slightly higher levels (Fig. 7C). Taken
together these data strongly suggest that activation of dioxin receptor
function by omeprazole is mediated by ligand recognition.
Fig. 7.
Effect of point mutations within the ligand
binding domain of the dioxin receptor on the omeprazole activation
response. A, schematic representation of human dioxin
receptor derivatives containing single point mutations within the
ligand binding domain (LBD). Val381 of the
wild-type LBD of the human dioxin receptor has been mutated to either
Ala (V/A) or Asp (V/D), as indicated.
B, ICZ and omeprazole (OM) responsiveness of
human dioxin receptor mutants containing amino acid substitutions at
position 381. Yeast cells transformed with the XRE-driven
-galactosidase reporter gene XRE-CYC1-lacZ, Arnt, and
wild-type (WT) or point-mutated human dioxin receptor forms
were grown in medium containing 2% glucose and incubated with vehicle
alone (
), ICZ (10 or 100 nM), or omeprazole
(OM; 5 or 50 µM) and assayed for
-galactosidase activity. Bars represent standard
deviation from three independent experiments. C, expression levels of dioxin receptor derivatives in yeast. Whole cell extracts were prepared from nontransformed yeast cells (W303) or
yeast cells transformed as described above and analyzed by
immunoblotting to monitor dioxin receptor (DR) expression
levels. As a positive control a cytosolic extract from Hepa 1c1c7 cells
was used (lane 1).
[View Larger Version of this Image (30K GIF file)]
Fig. 8.
Activation of the dioxin receptor by
omeprazole is structure-dependent. A, structures of
omeprazole, the sulfenamide, and analogues of omeprazole lacking
substituents and containing either a sulfoxide (timoprazole,
TI) or sulfide (desoxytimoprazole, D-TI) group.
B, activation of mouse or human dioxin receptor function by
omeprazole derivatives in yeast. Arnt was coexpressed with either
full-length mouse or human dioxin receptors in yeast cells together
with an XRE-driven -galactosidase reporter gene
(XRE-CYC-lacZ). Yeast cultures were grown in medium
containing 2% glucose and incubated separately with increasing
concentrations (5, 10, and 50 µM) of omeprazole
(OM), the various omeprazole derivatives, or vehicle alone
(dimethyl sulfoxide, DMSO), and assayed for
-galactosidase activity. Results of a representative experiment are
shown.
[View Larger Version of this Image (16K GIF file)]
Asp mutation inhibits ligand binding activity,
Val381
Ala increases the ligand binding affinity
in vitro (45). In excellent agreement with these in
vitro observations, the same mutations either inhibited or
enhanced in vivo responsiveness of the human dioxin receptor
to the classical high affinity receptor ligand ICZ. Importantly, the
effect of these mutations on ICZ responsiveness strictly correlated
with omeprazole responsiveness of the receptor. Finally,
structure-activity relationship experiments indicated that omeprazole
responsiveness of the dioxin receptor was dependent on the ability of
this compound to adopt planarity, a critical common property of dioxin
receptor ligands (3, 20).
*
This work was supported by the Swedish Cancer Society, the
Swedish Environmental Health Agency, and the Swedish Council for Work
Life Research.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.
**
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden. Tel.: 46-8 728 7330; Fax: 46-8 34 88 19;
E-mail: lorenz.poellinger{at}cmb.ki.se.
1
The abbreviations and trivial name used are:
dioxin, TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; bHLH,
basic helix-loop-helix; PAS, Per-Arnt-Sim homology domain; ICZ,
indolo[3,2-b]carbazole; hsp90, 90-kDa heat shock protein;
XREs, xenobiotic responsive elements; TI, timoprazole; D-TI,
desoxytimoprazole; MMTV, mouse mammary tumor virus.
2
J. McGuire, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.