(Received for publication, June 28, 1996, and in revised form, November 15, 1996)
From the Department of Biochemistry, School of
Medicine, University of Louisville, Louisville, Kentucky 40292 and
the § Department of Biochemistry and Molecular Biology,
University of South Dakota Medical School,
Vermillion, South Dakota 57069
We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes.
Both basal and PAH-dependent expression of a
chloramphenicol acetyltransferase expression plasmid containing
approximately 3.5 kilobase pairs of the 5-flanking region of
aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA
inhibitor H8 but not by the PKC inhibitor H7 (>20 µM).
cAMP analogs, activators of PKA activity, or protein phosphatase
inhibitors diminished expression of the reporter gene in a manner
identical to the native gene in cultured rat hepatocytes. Using
deletion analysis of the pALDH3.5CAT construct, we demonstrated the
existence of a negative regulatory region in the 5
-flanking region
between
1057 and
991 base pairs which appears to be responsible for
the cAMP-dependent regulation of this gene under both basal
and PAH-induced conditions. At least two apparently independent
mechanisms which involve protein phosphorylation regulate
aldh3 expression. One involves function of the
Ah receptor which requires PKC protein phosphorylation to
positively regulate both aldh3 and cyp1A1 gene
expression and the other a cAMP-responsive process which allows PKA
activity to negatively regulate expression of aldh3 under
either basal or inducible conditions.
The aldehyde dehydrogenases (ALDH,1
aldehyde NAD(P)+ oxidoreductase EC 1.2.1.3) are a family of
NAD(P)+-dependent enzymes that oxidize a broad
class of aldehydes to their carboxylic acids. The family is divided
into at least three classes based on sequence similarity, and the class
3 aldehyde dehydrogenase gene is expressed in a tissue-specific manner
in microsomal and cytosolic fractions (1, 2). The highest level of
basal expression of aldh32 is seen in corneal
epithelium, stomach, and heart, whereas PAH-induced expression is seen principally in liver, lung, bladder, colon, spleen,
and thymus of rodents. This gene is also expressed at high levels in
neoplastic tissue and some cell lines. Takimoto et al. (5)
and Xie et al. (6) have characterized the 5-flanking region
of the aldh3 gene and demonstrated that it contains at least
three major functional domains: a strong promoter proximal to the
transcription start site, an inhibitory region just upstream of the
promoter, and a PAH-responsive enhancer region. The transcription of
the aldh3 gene appears to be controlled by cooperation of at least these three functional domains.
Expression of this gene in liver is mediated by the arylhydrocarbon
(AhR) receptor, a cytosolic protein capable of binding PAH
as ligands (7). After ligand binding, the AhR-ligand complex is translocated into the nucleus, forms a heterodimeric complex with
the arylhydrocarbon nuclear transporter (ARNT), and they interact with
specific DNA sequences, designated xenobiotic responsive elements
(XREs), to alter the transcription of specific genes. The
AhR mediates induction of a number of xenobiotic
metabolizing enzymes (termed the Ah gene battery), including
cytochrome P4501A1, cytochrome P4501A2, glutathione
S-transferase Ya1 (GST1), NAD(P)H:quinone oxidoreductase, (QOR), UDP-glucuronosyltransferase 1.6 (UGT1A6), and
class 3 aldehyde dehydrogenase (8). The first gene whose activation was
shown to be directly mediated by the AhR was
cyp1A1 (7, 9); multiple XREs have been identified in the
5-upstream regulatory region of the cyp1A1 gene. Although
there are sequences of the aldh3 PAH-responsive enhancer
nearly identical to the XREs in the cyp1A1 flanking
sequences, one functional aldh3 XRE is located much farther
upstream than those seen in either cyp1A1, glutathione
S-transferase Ya1, or NADPH-quinone
oxidoreductase genes (5, 10, 11). A second feature of genes in the
Ah gene battery is the low levels of constitutive expression
that appears to be due to negative control mechanisms (12). Several different mechanisms have been described for the negative regulation of
cyp1A1, including putative regulatory genes on mouse
chromosome 7 (12), regulation by nucleosome structure (13), and
specific negative regulatory transcription factors (14).
Several studies have suggested that protein kinase C (PKC) plays an
important role in the regulation of cyp1A1 gene expression. Carrier et al. (15) and Reiners et al. (16) have
provided evidence that some step in transactivation by the
AhR is dependent upon phosphorylation by PKC. Our studies
(17) with cultured rat hepatocytes demonstrated that the PKC inhibitors
H7 and staurosporine concomitantly inhibited PAH induction of all five
genes we tested, including aldh3, cyp1A1, gst1,
qor, and ugt1.6 (ugt1A6).
This result suggests that PKC-dependent phosphorylation of
the AhR is required for PAH induction of all five xenobiotic
metabolizing enzymes. The protein kinase A inhibitors H8 and HA1004
were without effect on all of these genes at equivalent concentrations,
except PAH induction of ALDH-3 mRNA and protein which was
stimulated 2-4-fold (17). In this study, we sought to characterize the synergistic effects of the PAH, 1,2-benzanthracene, and protein kinase
A inhibitors, H8 and HA1004, on aldh3 gene expression and the mechanism whereby PAH regulates levels of the ALDH-3 enzyme in
cultured adult rat hepatocytes. Utilizing reporter genes containing the
5-flanking region of the aldh3 gene transfected into HepG2 tumor cells, we demonstrate the existence of a
cAMP-dependent negative cis-acting element.
Materials
Collagenase (type H) and chlorophenol
red--D-galactopyranoside were obtained from Boehringer
Mannheim. Benzaldehyde, 1,2-benzanthracene, chloramphenicol, cytochrome
c, dibutyryl cAMP, forskolin, Hank's modified balanced
salt, insulin-transferrin-sodium selenite medium supplement,
NADP+, NADPH, and L-ornithine were purchased
from the Sigma and arginine and arginine-free Eagle's
minimum essential medium from Life Technologies, Inc. Matrigel was
purchased from Collaborative Research Inc. (Bedford, MA) and
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), and N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide
dihydrochloride (H8) were obtained from Seikagaku America, Inc.
(Rockville, MD). Okadaic acid was obtained from LD Laboratories
(Woburn, MA). Resorufin was obtained from Aldrich, and
7-ethoxyresorufin was purchased from Pierce. Penicillin, streptomycin,
non-essential amino acids, Dulbecco's modified Eagle's medium (high
modified) were purchased from JRH BioSciences (Lexena, KS). Fetal
bovine serum and fungizone were obtained from Harlan Bioproducts for
Science (Indianapolis, IN). n-Butyryl-CoA was obtained from
Pharmacia Biotech Inc., and plasma purification kits were purchased
from Qiagen (Chatsworth, CA). [3H]Chloramphenicol and
32P-nucleotide triphosphates were obtained from DuPont
NEN.
Methods
Primary Hepatocyte Cell CultureHepatocytes were routinely
prepared from male adult Sprague-Dawley rats (180-250 g,
(Hsd:Sprague-Dawley SD), Harlan Sprague-Dawley, Indianapolis, IN) by
in situ liver collagenase perfusion (18, 19) with
modification (17). At 24 h in culture, inducing agents were added
to the fresh media. H7, H8, HA1004, dibutyryl cAMP, forskolin, and
okadaic acid were added 1 h prior to addition of BA. Equivalent
amounts (1.0% v/v) of solvent were added to control cells. At the
desired times, the media were removed from the dishes by aspiration,
the cells were washed with Dulbecco's phosphate-buffered saline
(2 × 1 ml), and cells were harvested (17). Protein concentrations were determined utilizing bicinchoninic acid with bovine serum albumin
as a protein standard (20). The samples were stored at
70 °C and
analyzed within 2-4 days for enzyme activity. The proteins were stable
for over 6 months at
70 °C when analyzed by Western immunoblot
analyses.
Cytochrome P4501A1 activity was determined using the specific substrate 7-ethoxyresorufin (21). Aldehyde dehydrogenase 3 activity was measured by monitoring the increase in absorbance at 340 nm caused by NADPH production during the oxidation of benzaldehyde as a substrate (22). Protein kinase A activity was measured as described by using the protein kinase A assay system obtained from Life Technologies, Inc.
Northern AnalysisTotal RNA was isolated by modification of
the method of Chomczynski and Sacchi (23) as described previously (17).
Northern blot experiments were performed after size-fractionation of
the denatured RNA (25 µg) on formaldehyde-containing 1% agarose gels and transfer of the RNAs to Zetaprobe membranes by diffusion (24). Hybridization was carried out at 43 °C overnight in 0.25 M sodium phosphate buffer, pH 7.2, containing 0.25 M sodium chloride, 50% formamide, 7% SDS, 1 mM EDTA, and 32P-labeled cDNA probe. The
hybridized membranes were washed three times for 15 min with 2 × SSC and 0.1% SDS, 0.5 × SSC, and 0.1% SDS or 0.1 × SSC
and 0.1% SDS at room temperature. The washed membrane was used to
expose x-ray film at 70 °C with an intensifying screen (DuPont
NEN). The exposed x-ray film was developed, and the optical density of
the relevant bands was quantitated by densitometry using a Bio-Rad
Model 620 video densitometer (La Jolla, CA).
Cytochrome P4501A1
mRNA was measured using a 635-bp PstI fragment of the
plasmid pA8 for cyp1A1 provided by R. N. Hines, Department of Pharmacology and Toxicology, Wayne State University (25). Aldehyde
dehydrogenase 3 mRNA was measured using a
EcoRI/HindIII cDNA fragment of the plasmid
pselALDHX, the aldh3-specific clone (1). As a control,
-actin mRNA levels were measured using the cDNA plasmid
encoding the mouse cytoskeletal
-actin (26). These nuclei acid
probes were labeled with [32P]dCTP using the random
primer labeling procedure (27).
A CAT construct (pALDH3.5CAT)
containing approximately 3.5 kb of 5-flanking region of the
aldh3 gene containing functional XREs (5) was used to test
for the effects of PAH and PKA inhibitors/activators. A series of
deletion mutants of the proximal 5
-flanking region of aldh3
were prepared by polymerase chain reaction as described previously (6),
namely pI-3, pI-4, pI-5, pI-6, and pI-8. Construct p
(
1054/
392)ALDH was produced by treating pALDH3.5 with the restriction enzyme PstI to remove a portion of the flanking
region from
1057 bp to
392 bp relative to the transcription start
site; the resulting plasmid was gel purified and religated to yield p
(
1057/
392)ALDH. Construct p
(
1057/
930) was prepared using polymerase chain reaction to generate a fragment from
374 bp to
930
bp (upstream primer 5
-GGAGGACAAAGTGTTGCTATG-3
; downstream primer,
5
-AGCTGCTGTTCTCTGAGTCC-3
). The polymerase chain reaction fragment was
incorporated into a pCRII vector and amplified in bacteria. The
PstI fragment was liberated from the pCRII vector prior to
religation into the PstI-cleaved vector,
p
(
1057/
392)CAT, to yield construct p
(
1057/
930) which was
identified by EcoRI restriction analysis.
HepG2 cells (ATCC, Bethesda)
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum, fungizone (2.5 µg/ml), penicillin (10 units/ml), and streptomycin (10 units/ml). The cells were grown at
37 °C in a 5% carbon dioxide, 95% air atmosphere. The cells were
grown to approximately 30-40% confluence, transfected with 2 µg of
pCMV-gal as a transfection control, and 4 µg of pALDH3.5CAT or
other reporter plasmid in T25 flasks using the calcium phosphate
precipitation/glycerol shock method and harvested as described by
Rushmore et al. (10). The chemicals used were prepared as
500 × stock solutions in dimethyl sulfoxide or water, and they
were added 24 h after the shock treatment. The chloramphenicol
acetyltransferase assay used was a modification of the method of Gorman
et al. (28) which includes a xylene extraction of the
products from [3H]chloramphenicol and subsequent
quantitation of products and reaction mixture by liquid scintillation
methods. The reactions were performed in 100-µl reaction mixtures
containing the cell extract (approximately 120 µg of protein) in 0.25 M Tris-HCl buffer, pH 7.5, containing 3.7 mM
chloramphenicol (25 nCi), and 5 µg n-butyryl-CoA. The
samples were extracted with 300 µl of xylene and back-extracted with
100 µl of Tris-HCl buffer. Aliquots of the xylene phases and the
original reaction mixture were assayed for tritium content.
-Galactosidase activity was measured spectrophotometrically using chlorophenol red
-galactopyranoside as substrate by measuring the
absorbance formed after 1 h at 595 nm on a Titretek Uniskan II
plate reader (Flow Laboratories, McLean, VA). Protein concentrations were determined using the method of Smith et al. (20). All
transient transfection experiments were performed three times in
triplicate. Although a single experiment is shown in the data
presented, the results seen in three separate transfection experiments
were similar.
All data were analyzed using a Student's t test.
Previous studies have demonstrated that protein
kinase C-mediated protein phosphorylation may be a determinant in the
regulation of PAH-dependent gene expression of aldh3,
cyp1A1, gst1, qor, and ugt1.6 (10, 11, 17). As we have
previously shown (17), the PKC inhibitor H7 (50 µM)
potently inhibited BA induction of both cyp1A1 and
aldh3 mRNA by over 80% in cultured rat hepatocytes (Fig. 1). In contrast, the PKA inhibitors H8 (50 µM) and HA1004 (50 µM) had little or no
effect on PAH induction of mRNA levels for cyp1A1; in
fact, they served as negative controls for inhibitor studies on PKC
activity since they only inhibit this activity at very high
concentrations (<200 µM). Surprisingly, the PKA
inhibitors H8 and HA1004 strikingly potentiated PAH induction of
mRNA for aldh3 by over 2-fold (Fig. 1). These results
were also seen in the levels of ALDH-3 and P4501A1 proteins (data not
shown), demonstrating that the changes seen are caused by the
regulation of pretranslational processes.
The time courses for induction of ALDH-3 and EROD (7-ethoxyresorufin
O-deethylase activity for P4501A1) activity in cultured rat
hepatocytes in the presence of 50 µM BA or BA plus 50 µM H8 were compared (Fig. 2). Maximal
induction of EROD activity by BA occurred at day 3, and H8 had no
observable effects on either BA-inducible or basal EROD activity. The
time courses of induction of ALDH-3 activity by BA or BA plus H8 were
very similar. Maximal induction occurred at 4 days, and H8 potentiated
PAH induction by 2-3-fold as early as 1 day and as late as 6 days in
culture. Basal ALDH-3 activity (6.2 nmol/min/mg protein) was observed
on day 0 but declined to 0.5 nmol/min/mg protein by day 3 in culture. H8 treatment alone at any concentration did not appreciably affect the
basal level of ALDH-3 enzyme activity. H8 potentiated PAH induction of
ALDH-3 activity over 3-fold in a concentration-dependent manner (Fig. 3) but had no effect on basal activity at
any concentration tested.
PKA activity was measured using a specific peptide substrate labeled
with a fluorescent dye, and an appropriate PKA standard with a known
activity was used to confirm the specificity of PKA activity (29).
Specific PKA enzyme activity in rat hepatocytes was inhibited (>80%)
in a concordant but opposite manner than was observed with the H8 and
HA1004 potentiation of PAH induction of ALDH-3 enzyme activity (Fig.
4). As a control, the same concentrations of a
structurally similar PKC inhibitor, H7, were observed to have little or
no effect on PKA activity. Since higher concentrations of H8 and HA1004
(>100 µM) also inhibit PKC activity (17), we routinely
used 50 µM concentrations of PKA inhibitors at which hepatocyte PKC activity was unaffected.
Effect of Elevated Intracellular cAMP Levels on PAH Induction and H8 Potentiation of PAH-mediated Induction of aldh3 Expression
The
level of intracellular cAMP, and therefore the activation of
cAMP-dependent protein kinase, was modulated in primary rat hepatocytes by addition of the cAMP analog, dibutyryl cAMP, or the PKA
activator, forskolin. After a period of 24 h in culture and
immediately prior to initiating PAH induction, hepatocytes were exposed
for 60 min to either dibutyryl cAMP or forskolin. PAH-mediated
induction of aldh3 mRNA and H8 potentiation of
PAH-mediated induction of aldh3 mRNA levels were
inhibited more than 60 and 70%, respectively, by either dibutyryl cAMP
or forskolin (Fig. 5). This treatment had no effect on
the PAH-mediated induction of levels of cyp1A1 mRNA.
Furthermore, the changes in levels of aldh3 mRNAs were
concordant with the changes observed for ALDH-3 enzyme activity (Fig.
6) and protein measured by Western immunoblot analysis
(data not shown). These results demonstrate that cAMP analogs and
activators of adenylyl cyclase led to repression of both PAH induction
and H8 potentiation of PAH induction of aldh3 gene
expression. This effect is specific for the aldh3 gene,
since these agents had little or no effect on expression of
cyp1A1.
Effect of Protein Phosphatase Inhibitors on the PAH Induction of ALDH-3 and on the H8 Potentiation of PAH Induction of ALDH-3
Okadaic acid is a potent and specific inhibitor of protein
phosphatases 1 and 2A and appears to exert its effects by preventing dephosphorylation of specific transcription factor proteins in cells.
Okadaic acid decreased both PAH induction and H8 potentiation of PAH
induction of ALDH-3 activity by over 50 and 80%, respectively, in a
concentration-dependent manner as expected (Fig.
7). Interestingly, H8 potentiation of PAH induction of
ALDH-3 enzyme activity was more sensitive to okadaic acid than was PAH
induction of ALDH-3 activity. For example, okadaic acid (15-20
nM) resulted in 50% inhibition of ALDH-3 activity induced
by BA alone, whereas 5-10 nM okadaic acid resulted in 50%
inhibition of ALDH-3 activity induced by treatment with BA plus H8
(Fig. 7).
To assess whether these effects of okadaic acid involve a
pretranslational mechanism of action, we performed Northern analysis of
mRNA isolated from cells treated with 50 µM H8, 50 µM BA, 20 nM okadaic acid, or combinations of
these agents (Fig. 8). These results document that the
decrease in aldh3 mRNA levels and enzyme activity by
okadaic acid are apparently caused by changes in pretranslational processes specific for aldh3 expression. Addition of okadaic
acid to cultured rat hepatocytes treated with BA or BA plus H8 had no
effect on PAH induction of cytochrome P4501A1 and levels of its
mRNA (data not shown).
Effect of PKA Inhibitors on Basal and PAH-inducible Expression of pALDH3.5 CAT
Lindahl and co-workers (1, 5, 6, 30) have developed
a highly inducible 5-flanking construct containing 3.5 kb of upstream
sequence of the rat aldh3 gene in a chloramphenicol acetyltransferase (CAT) reporter gene. Basal level expression of this
gene is cell type-specific, and liver displays very low basal
expression (1, 2). Unlike the native gene in rat primary hepatocyte
cultures, there is measurable basal expression of the pALDH3.5 plasmid
construct during transient transfection into HepG2 cells. We observed a
2-4-fold increase in pALDH3.5CAT expression in the presence of the PKA
inhibitor H8 in the absence (Fig. 9A) or
presence of 1,2-benzanthracene (Fig. 9B). In these
experiments, we noted that H7 and H8 also stimulated the expression of
the transfection control plasmid, pCMV-
gal. Therefore, we have
expressed the data relative to total cellular protein, since our
routine transfection efficiency with pCMV-
gal normally yields
-galactosidase activity which varied less than ±20% within a
single transfection experiment. The PKC inhibitor, H7, used as a
control for the structurally related H8 compound, had no effect on
basal expression at the concentrations used, suggesting that the
mechanism of regulation by PKA activators and inhibitors is independent
of the action of the AhR (ligand- or
phosphorylation-dependent) or PKC activation. Inhibition
was seen at higher concentrations of H7 and H8 (50 µM),
but this concentration of either agent was toxic to HepG2 cells in our
hands.
Subsequent transfection experiments demonstrated that forskolin,
dibutyryl cAMP, and okadaic acid inhibited the transient expression of
this reporter gene by 60% in the presence of BA and by 75% in the
presence of BA plus H8, similar to the manner they affect expression of
the native gene in cultured rat hepatocytes (Fig. 10).
In addition, both basal and PAH-induced reporter gene activity was
inhibited in a similar manner to the PKC inhibitor H7 (Fig. 9),
suggesting that the regulation of the aldh3 gene by PKA
activity functions independently of the AhR action.
Deletion Analysis to Define cis-Acting Elements Associated with Regulation by PKA
To map the region of the 5-flanking region of
the aldh3 gene responsible for a putative PKA-responsive
element, we tested a series of 5
-flanking CAT constructs derived from
the rat gene (5, 6). Of these constructs (Fig. 11),
only two CAT constructs were positively regulated by the PKA
inhibitor H8, namely pALDH3.5 and pI-8. The shorter deletion CAT
constructs pI-3, pI-4, pI-5, and pI-6 displayed higher basal level
expression than that observed with pALDH3.5CAT and pI-8CAT, suggesting
that a cis-acting negative regulatory element exists located
between nucleotides
1125 and
991 of the 5
-flanking region of
aldh3. Due to the effects of PKA activators and inhibitors
seen in experiments utilizing pALDH3.5, this element appears to be
regulated by a PKA-dependent phosphorylation event.
Since the expression of the pI-8 construct, but not pI-5, was affected
by H8, we prepared an internal deletion in the 3.5-kb construct around
the PstI site at 1057 bp. Specifically, the construct,
p
(
1057/
930)ALDH, was prepared from pALDH3.5 which had an
internal deletion between nucleotides
1057 and
930 of the flanking
region (Fig. 11). This construct had higher levels of basal expression
like the pI-5 or shorter constructs and was not regulated by the PKA
inhibitors, demonstrating loss of the PKA-regulated element from the
reporter gene. Given the results seen with the deletion analyses, we
propose that the PKA-responsive elements associated with regulation by
PKA lies within a 66-bp region from nucleotides
1057 and
991.
A number of extracellular signals regulate various intracellular processes via a complex cascade of receptors, transducers, and second messengers (31, 32). These second messengers, cyclic nucleotides including cAMP and cGMP, are well characterized biochemical systems (33, 34). The resulting activation of cyclic nucleotide-dependent protein kinases is the basis for many effects of cyclic nucleotides on cellular function. Recently, a number of in vivo and in vitro studies have been focused on the effects of protein kinase activation on the specific signal transduction pathways in the control of cyp1A1 gene expression. Notably, protein kinase C-mediated phosphorylation (15, 16) appears to be a determinant in the regulation of PAH-inducible cyp1A1 gene expression. Our past experiments suggested that PKC-dependent phosphorylation of AhR appears to be required for PAH induction of all of the xenobiotic metabolizing enzymes in the Ah gene battery, not just for cyp1A1 (17). Other studies have reported that P450-mediated biotransformation activities associated with phenobarbital-inducible isozymes, including cyp2B1, cyp2B2, and cyp3A1, were depressed by agents that elevated cAMP levels in rat liver and in rat hepatocytes (35), indicating a negative regulatory role for cAMP-dependent signal transduction pathway on gene induction by this barbiturate. This is not the case for cyp1A1, the control gene used in the studies reported herein.
In the current study, we used rat hepatocytes and transient transfection of reporter genes in HepG2 cells to investigate the regulatory control exerted by intracellular cAMP on basal and PAH-induced aldh3 gene expression. H8 and HA1004 are effective inhibitors of cyclic nucleotide-dependent protein kinases (29) and are especially effective as PKA and PKG inhibitors. Our results indicate that these inhibitors potentiate PAH induction of ALDH-3 activity in a concentration-dependent manner, but neither PKA inhibitor had any effect on PAH induction of P4501A1. Protein kinase A activity was shown to be effectively inhibited by H8 and HA1004 but not by H7 in cultured hepatocytes, demonstrating an inverse correlation between potentiation of PAH-dependent regulation of aldh3 gene expression and inhibition of specific PKA activity.
As H8 is also a potent protein kinase G inhibitor, we were concerned that the inhibition of this protein kinase (PKG) by H8 might be responsible for the H8 potentiation effect. However, elevation of intracellular cAMP levels, achieved through the use of a membrane-permeable cAMP analog, dibutyryl cAMP, also resulted in inhibition of PAH-mediated induction and H8 potentiation of PAH-mediated induction of aldh3 gene expression. Striking inhibition of BA induction and H8 potentiation of BA induction of aldh3 gene expression also was noted when intracellular cAMP levels were modulated via adenylate cyclase activation in hepatocytes with 25 µM forskolin. In recent studies by Bjornsson et al. (36) and Sidhu and Omiecinski (35), treatment of primary rat hepatocytes with forskolin results in dramatic stimulation of intracellular cAMP levels and decrease in expression of cyp2B. Finally, okadaic acid is a potent, selective inhibitor of protein phosphatase type 1 and 2A and apparently exerts its effects by inhibiting protein dephosphorylation in cells. Okadaic acid decreased PAH induction and H8 potentiation of PAH induction of ALDH-3 activity and mRNA levels in a concentration-dependent manner as expected. Dibutyryl cAMP, forskolin, and okadaic acid had no effect on the PAH induction of cyp1A1 gene expression under any condition. Since PKC inhibitors had pronounced effect on PAH induction of P4501A1, but okadaic acid did not, PKC-dependent phosphorylation of the AhR must be near maximal in cultured primary rat hepatocytes (17).
Transient transfection experiments utilizing a CAT construct containing
3.5 kb of the 5-flanking region of the aldh3 gene demonstrated that the effect of protein kinase A inhibitors, namely H8
and HA1004, is at the level of transcription. Furthermore, agents that
increase cAMP levels or prevent dephosphorylation of some critical
transcription factor act to inhibit the expression of this gene. The
results strongly suggest a specific and negative regulatory role for
the cAMP-dependent signal transduction pathway in basal and
PAH-inducible aldh3 gene expression.
Using deletion analysis of the regulatory 5-flanking region of this
gene, we have identified a region that likely contains a negative
regulatory element (
1057 and
991 bp; CTGCAGTGGC TGCTATGGCG CAAAGCCACC AGACAAGAGA AATTT CCAACTCCAT TGTAATC TTAT) that apparently regulates expression of this gene by a PKA-dependent
process. The mechanism for this decreased expression has not been fully established. In Saccharomyces cerevisiae, the aldehyde
dehydrogenase 2 (ALD2) gene (37) has been shown to be
negatively regulated by protein kinase A, and its 5
-flanking region
contains two classical cAMP-responsive elements. The 66-bp region
(
1057 to
991) of the rat aldh3 gene (Fig. 11) and that
of its immediate flanking regions contain no unique sequences with high
consensus to other canonical cAMP-responsive element binding protein
binding elements (classical or nonclassical, Ref. 38) or other elements
suggesting that the rat gene may be uniquely regulated by a novel
PKA-regulated transcription factor.
Our studies demonstrate that there are apparently two protein
phosphorylation mechanisms involved in the regulation of
aldh3 gene expression in hepatocytes (Fig.
12). One involves the AhR, which requires
PKC phosphorylation to positively regulate aldh3 gene in the
presence of PAH ligands. The second appears to involve a
cAMP-responsive process, which may require PKA phosphorylation to
negatively regulate aldh3 gene expression (Fig. 12). Future studies will characterize the cis- and
trans-acting elements involved in the negative regulation of
the class 3 aldehyde dehydrogenase gene of rat.
We express our thanks to Jaydev Dholakia and Michael Waterman for their useful input to this project and to Mary Pendleton for technical assistance.