Caffeine induction of Cyp6a2 and ...

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Gene 415 (2008) 49
–
59
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Gene
journal homepage: www.elsevier.com/locate/gene
Caffeine induction of Cyp6a2 and Cyp6a8 genes of Drosophila melanogaster is
modulated by cAMP and D-JUN protein levels
⁎
a
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996-0840, United States
b
Department of Biochemistry, Vanderbilt University School of Medicine, 23rd at Pierce Ave., Nashville, Tennessee 37232, United States
article info
abstract
Article history:
Received 28 September 2007
Received in revised form 6 January 2008
Accepted 17 February 2008
Available online 4 March 2008
Cytochrome P450 monooxygenases or CYPs, a family of endobiotics and xenobiotics metabolizing enzymes,
are found in all organisms. We reported earlier that the promoters of Drosophila Cyp6a2 and Cyp6a8 genes are
induced by caffeine. Since caffeine antagonizes adenosine receptor (AdoR) and inhibits cAMP
phosphodiesterase (PDE), we used luciferase reporter gene to examine whether in SL-2 cells and adult
Drosophila, induction of the two Cyp6 genes is mediated via AdoR and/or PDE pathway. Results showed that
AdoR is not involved because AdoR agonists or antagonists do not affect the Cyp6 promoter activities.
However, inhibition of PDE by speci
Received by I. King Jordan
c inhibitors including caffeine causes induction of both Cyp6 gene
Keywords:
Cell transfection
Transgenic
ies mutant for dunce gene coding for cAMP-PDE, have higher Cyp6a8
promoter activity than the wild-type
ies. We demonstrate that caffeine treatment increases intracellular
cAMP levels, and cAMP treatment induces the Cyp6 gene promoters. Since both Cyp6 genes have multiple
sites for JUN transcription factors, which generally play a positive role in cAMP pathway, effect of Drosophila
jun (D-jun) on the Cyp6a8 promoter activity was examined. Results showed that the expression of D-jun sense
plasmid causes downregulation rather than activation of the Cyp6a8 promoter. Conversely, expression of
antisense plasmid increased the promoter activity. Interestingly, caffeine treatment decreased the D-JUN
protein level in SL-2 cells as well as in adult
ies
cAMP
Reporter gene
Promoter assay
Gene regulation
ies. These results suggest that D-jun acts as a negative regulator,
and caffeine induction of Cyp6a8 and Cyp6a2 genes is mediated by the upregulation of cAMP pathway and
downregulation of the D-JUN protein level.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
feeding and acts as a pesticide (
Nathanson,1984
). Caffeine is also found
to be effective in killing or repelling slugs and snails when applied to
foliage (
Hollingsworth et al., 2002
). When adult Drosophila are allowed
to feed on caffeine containing food, a dose-dependent decrease in rest
was observed (
Shaw et al., 2000
). Interestingly, during normal wake or
active period, the expression of Cyp4e2 gene was found to be increased
about 2-fold higher compared to the rest or sleep period (
Shaw et al.,
2000
).
A modest number of studies have also been done to investigate the
effect of caffeine on gene expression. In one study, caffeine treatment
has been shown to increase CYP1A1 and CYP1A2 mRNA levels in rat
liver and kidney (
Goasduff et al., 1996
). In other studies, caffeine was
found to increase the levels of c-Fos, c-Jun, and junB mRNAs in rat
striatum (
Svenningsson et al., 1995b
), and sonic hedgehog mRNA in
primary murine neuronal and astroglial cells in culture (
Sahir et al.,
2004
). We also reported that caffeine increases the Cyp6a2 and
Cyp6a8 promoter activity both in adult females and in SL-2 cells. The
0.98- and 0.8-kb upstream DNA of Drosophila Cyp6a2 and Cyp6a8
genes respectively, have cis-regulatory elements that are involved in
high level of caffeine induction (
Bhaskara et al., 2006
). In a recent
study, caffeine treatment has been shown to increase the levels of
eleven Cyp gene transcripts in Drosophila larvae (
Willoughby et al.,
Caffeine, the most commonly used psychostimulant, is found in
coffee, tea, many soft drinks and several drug preparations. Because
of its widespread use, the effect of caffeine on various physiological
processes has been studied extensively and mostly in humans, rats,
mice and other mammals (
Svenningsson et al., 1995a; Lorist and
Tops, 2003; Porta et al., 2003
). Comparatively, very little is known
about the effect of caffeine in insects. In Drosophila melanogaster,
caffeine treatment has been shown to cause chromosome loss in the
larvae and lethality to the adult
⁎
Corresponding author. Tel.: +1 865 974 5148; fax: +1 865 974 6306.
E-mail address:
(R. Ganguly).
Abbreviations: AdoR, adenosine receptor; bp, base pair(s); cAMP, 3
cyclic
adenosine monophosphate; cAMP-PDE, cAMP phosphodiesterase; CYP, cytochrome
P450 monooxygenase; Cyp (or CYP), CYP-encoding gene; DDT, kb, kilobase(s) or
1000 bp; nt, nucleotide(s); LUC, Luciferase; luc, LUC-encoding gene; nt, nucleotide(s);
PDE, phosphodiesterase.
′
,5
′
see front matter © 2008 Elsevier B.V. All rights reserved.
doi:
–
Srividya Bhaskara
a
,
b
, Mahesh B. Chandrasekharan
b
, Ranjan Ganguly
a
,
promoters. We also found that
ies (
Clark and Clark, 1968;
Zimmering et al., 1977
). In Drosophila prosaltans, low dose of caffeine
treatment decreases both fecundity and longevity (
Itoyama et al.,
1998
). Caffeine that is naturally found in plants inhibits insect
0378-1119/$
50
S. Bhaskara et al. / Gene 415 (2008) 49
59
2006
). Although these studies demonstrate that caffeine can induce
the transcription of Cyp genes, the underlying mechanism of the
induction process is not known.
In mammals, caffeine exerts its action by antagonizing the A
1
and
A
2A
isoforms of adenosine receptor (AdoR) and/or by inhibiting the
cAMP phosphodiesterase enzyme (PDE) (
Poulsen and Quinn, 1998;
Fredholm et al., 1999; Fisone et al., 2004
). Antagonism of adenosine
receptor or PDE inhibition is known to elevate the intracellular cAMP
level and activation of cAMP-mediated pathway has been shown to
induce the expression of activator protein-1 (AP-1) family transcrip-
tion factors including c-Jun and c-Fos proteins (
Karin, 1995a; Fredholm
et al., 1999
). In fact, caffeine has been shown to increase the expression
of c-Fos, c-Jun, and junB mRNAs or protein in the rat brain striatum
(
Svenningsson et al., 1995b; Bennett and Semba, 1998a,b
). Sequence
analysis of the caffeine-responsive 0.8- and 0.98-kb upstream DNA of
the Cyp6a8 and Cyp6a2 genes, respectively, has identi
CATTCGTTTTATCGCCG-3
′
(
−
983/
−
965) and 5
′
ctcgtcgacTTTGCG-
TAGCTGCTCCC-3
′
(
−
1/
−
17). The sequences shown in lower cases at
17) primers,
have engineered sites for MluI (acgcgt) and SalI (gtcgac) restriction
enzymes. The PCR product was digested with MluI and SalI, and
cloned in front of the luc (luciferase) reporter gene between the MluI
and XhoI sites of the pGL3-Basic vector (Promega, Madison, WI).
Construction of 0.2luc-A8 and 0.8luc-A8 reporter plasmids have been
described previously (
Maitra et al., 2002
). All cloned PCR products
were veri
′
-ends of the distal (
−
983/
−
965) and proximal (
−
1/
−
ed by sequencing at the Molecular Biology Resource Facility
of the University of Tennessee.
2.3. Construction of D-jun promoter construct
ed the
presence of several putative AP1 binding motifs (
Bhaskara et al.,
2006
). It is possible that caffeine induction of Cyp6a2 and Cyp6a8
genes may be mediated via the signaling pathways described above
and the putative AP1 binding motifs. To test these possibilities and to
understand the mechanism of caffeine-mediated transcriptional
activation, we examined the effects of various antagonists and
agonists of AdoR and PDE on the Cyp6a2 and Cyp6a8 gene promoter
activities in SL-2 cells as well as in the transgenic
The organization of the D-jun gene of D. melanogaster has been
described previously (
Wang and Goldstein, 1994; Rousseau and
Goldstein, 2001
). Based on this report, and Drosophila genome
database, two PCR primers, D-jun-up (
−
540) and D-jun-up (+1),
540/+1 DNA of the D-jun gene. The
sequences of these distal and proximal upstream DNA primers
respectively were as follows: ggacgcgtTCCTTTCCTATTTACCGACGCC
and ggctcgagGGGTGGGAACTTTG. The MluI and XhoI sites added to the
5
−
-ends of the distal and proximal primers, respectively, are shown in
lower cases. These primers span
′
ies. We also
examined the effects of cAMP and Drosophila Jun protein (D-jun) on
Cyp promoter activity. Our results show that caffeine induces
Cyp6a2 and Cyp6a8 genes by activating the cAMP pathway and by
downregulating the D-jun protein level. Data show that D-jun acts as
a negative rather than a positive regulator for the expression of Cyp6
genes.
540 to +1 region of the D-jun gene
corresponding to the bases between 248772 and 249271 regions of
the Drosophila genomic scaffold. Genomic DNA isolated from
0.8luc110H-ry reporter transgenic line was used as a template to
amplify the upstream DNA of D-jun gene via PCR. The ampli
−
ed DNA
obtained for each gene was cloned into pGEMT-Easy vector (Promega,
Madison, WI) and sequenced. Using the BLAST program, the
sequences were compared with the upstream DNA sequences
reported in the database for each gene. The PCR products were then
excised by cutting with MluI and XhoI, and cloned into MluI/XhoI cut
pGL2 (N) basic vector, which is the pGL2-basic vector (Promega, WI)
with SalI site modi
2. Materials and methods
2.1. Fly stocks, cell culture and chemicals
ed to NotI (
Maitra et al., 2002
).
Two reporter transgenic lines of D. melanogaster, 0.2-luc4H-ry and
0.8-luc110H-ry, dnc
1
mutant strain and Schneider line SL-2 cells
(
Schneider, 1972
) were used in the present investigation. The dnc
1
mutant strain was obtained from Indrani Ganguly of Neuroscience
laboratory, San Diego (CA). The SL-2 cells were obtained from
Invitrogen (Carlsbad, CA) and maintained at 25 °C in Schneider's Dro-
sophila medium (Invitrogen) supplemented with 10% heat-inactivated
bovine calf serum and 0.01% penicillin
2.4. Construction of D-jun expression plasmids
streptomycin (Sigma, St. Louis,
MO). Every fourth day, the cells were transferred to fresh media. The
reporter transgenic lines were homozygous for single copy of 0.2-luc or
0.8-luc reporter transgene located on the second chromosome in ry
506
background (
Maitra et al., 2002; Bhaskara et al., 2006
). These trans-
genes were created by cloning a 0.2- (
–
For the D-jun plasmid construction, several plasmids were
obtained from different sources. D-jun/pBSK-carrying full-length D-
jun with 6× his-tag at the C-terminal end was kindly provided by Dirk
Bohmann, University of Rochester. FJF/pCasper4 plasmid carrying
tubulin (Tub) promoter was obtained from Laura Ciapponi, EMBL,
Heidelberg. D-jun/HS plasmid carrying D-jun cDNA in sense (S) (D-jun
(S)/HS) or antisense (AS) (D-jun (AS)/HS) orientations driven by Hsp70
promoter in pCasper plasmid was obtained from Nobert Perrimon,
Harvard University.
To create the D-jun cDNA plasmid in sense and antisense
orientation, the FJF/pCasper4 plasmid carrying tubulin (Tub) promoter
was
761) kb
upstream DNA of Cyp6a8 gene of the DDT resistant 91-R strain in front
of the
−
11 /
−
199) or 0.8- (
−
11 /
−
y luciferase (luc) gene. The details of the construction of
reporter plasmids and germ line transformation into ry
506
host strain
have been previously described (
Maitra et al., 2002
). All
re
rst modi
ed. This plasmid has unique KpnI and XbaI sites that
ies were
-
overhang was removed by digesting with S1 nuclease. The blunt-
ended plasmid was then ligated with XbaI linker (New England
Biolabs, Ipswich, MA) according to the manufacturer's instructions.
The converted plasmid was then used to transform DH5
′
raised on standard cornmeal
agar
–
molasses medium andmaintained
light cycle.
Caffeine, adenosine, 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX),
N
6
cyclopentyl xanthine (CPA), theophylline, rolipram and dibutyryl
cAMP were purchased from Sigma (St. Louis, MO). Caffeine, theophyl-
line and dibutyryl cAMP were dissolved in sterile water, whereas CPA,
DPCPX and rolipram were dissolved in DMSO. Adenosine was
dissolved in 250 mM Tris pH 8.0.
–
α
E. coli
ed and the FJF insert was removed by
cutting with XbaI. The remaining part of the plasmid was then self-
ligated to create pCasper4-Tub vector with a unique XbaI site
downstream of the tubulin promoter. To isolate the D-jun cDNA as
an XbaI fragment, the D-jun/pBSK-plasmid was used. This plasmid has
D-jun cDNA
2.2. Construction of reporter plasmids
anked by a unique XbaI site at the 5
′
-end and a HindIII
-
overhangs were repaired by Klenow polymerase. The resulting blunt-
ended plasmid was the ligated with XbaI linker used to transform
DH5
′
-end. The plasmid was cut with HindIII and the 5
′
To create 0.9luc-A2 chimeric reporter plasmid, a 0.9-kb upstream
DNA of Cyp6a2 between positions
−
983 and
−
1 (ATG at +1) was
ampli
ed by PCR using the following primer pairs: 5
-ctcacgcgtTT-
α
E. coli bacteria. The puri
ed plasmid was cut with XbaI, the D-
–
the 5
were designed to amplify the
ank the FJF insert (
Ciapponi et al., 2001
). To be able to remove the FJF
insert as an XbaI fragment, the plasmid was cut with KpnI and the 3
–
at 25 °C under 12 h dark
bacteria. The plasmid was puri
site at the 3
′
S. Bhaskara et al. / Gene 415 (2008) 49
–
59
51
jun cDNA insert was isolated and cloned into the unique XbaI site of
pCasper4-Tub vector in sense or antisense orientation. The resulting
recombinant plasmids were named D-jun(S)/Tub and D-jun (AS)/Tub,
respectively.
y luciferase (F-luc) activity. Immediately after recording
the F-luc activity, 100
re
l Stop and Glo Reagent was added, vortexed
gently and the Renilla luciferase (R-luc) activity was measured within
10 s. R-LUC activity was used to normalize the data, which were
expressed as the ratio of F-LUC to R-LUC activity.
Transfection of SL-2 cells with sense and antisense plasmid of D-
jun was done with 0.5
μ
2.5. Treatment of
ies,
y extract preparation and luciferase assay
g of D-jun expression plasmid. Empty vectors
were used to equalize total DNA quantity in all transfections. After a
24 h recovery period following transfection, cells were resuspended in
8 mM caffeine containing media. Cells transfected with tubulin ex-
pression plasmid (D-jun(S)/Tub) were harvested 24 h later and ex-
tracts prepared from these cells were assayed for luciferase activity.
Cells transfected with heat-shock plasmids (D-jun(S)/HS or D-jun
(AS)/HS) were heat-shocked in three 30 min pulses at 37 °C as soon as
they were transferred to 8 mM caffeine or control media. The cells
were allowed to recover for 30 min at 25 °C between heat-shock
pulses. After the third heat-shock cells were allowed to recover for
12 h before the extracts were prepared and assayed for luciferase
activity.
μ
10 day old females were used. Except for
dibutyryl cAMP, concentrations of other chemicals to be used for
treatment were determined by dose
–
–
response experiments (Supple-
6). For cAMP treatment 4 mM concentration was
chosen because similar concentration (5 mM) was used to treat adult
ies (
Chyb et al., 1999
) and 4 mM concentration was found to be
effective for S2 cell treatment (Supplementary Fig. 13). Flies to be
treated with a chemical or to be used as a control were always
collected from the same culture bottle, but three different culture
bottles were used to collect three different batches of
–
ies. Typically,
ies into individual
vials containing normal food. After overnight recovery from ether
shock,
y food
reconstituted with 8 mM caffeine, 8 mM adenosine, 16 mM theophyl-
line, or 4 mM dibutyryl cAMP solution. The control
2.7. Phosphodiesterase assay
ies for caffeine,
theophylline and dibutyryl cAMP treatments were transferred to food
made with water. In case of adenosine treatment, control
ies were
ies or SL-2 cells, scintillation proximity assay (SPA) was
done using a kit obtained fromAmershamBiosciences (Piscataway, NJ).
Briey, ten female ies or 0.5×10
6
SL-2 cells were homogenized in
KHEM buffer (50 mM KCl, 10 mM EGTA, 1.92 mM MgCl
2
,1mM
dithiothreitol, 50 mM HEPES, pH 7.2) containing P8340 protease (1
ies with
1 mM DPCPX, 16 mM CPA or 500
μ
M rolipram, solutions of these
chemicals made in DMSO were dripped evenly on the surface of
y
food in individual vials. Food dripped with solvent alone was used as
control. These vials were left overnight at room temperature for DMSO
to dry. Next morning, sorted live
μ
l/
l cell lysis buffer) inhibitor cocktail (Sigma, St. Louis) and cen-
trifuged at 13,000 rpm in an Eppendorf microcentrifuge for 5 min at
4 °C. The supernatants were collected and assayed for protein con-
centration using the Bradford protein assay kit (Bio-Rad, Hercules, CA).
For PDE assay,
μ
ies were directly transferred to
these vials. All treatments with chemicals or control solvents were
done for 24 h at room temperature in the dark. After treatments, the
ies were immobilized by keeping the vials on ice for 10 min, extracts
were prepared and stored at
y or cell extract, equivalent to 20
μ
gor40
μ
g
80 C until used for luciferase assay
using reagents obtained from Promega (Madison, WI). Total protein in
the
−
–
HCl pH 7.5, 8.3 mM MgCl
2
and 1.7 mM EGTA. Caffeine (concentrations
as mentioned in
Fig. 5
) and 0.05
ed using BCA Protein assay kit (Pierce). The
details of the procedures for extract preparation and protein assay
have been described before (
Maitra et al., 2000
). All experiments were
done in triplicate as explained above. Assays for each set of ex-
periment were done on the same day although the extracts were not
always prepared on the same day.
μ
ci of [5
,8-
3
H] adenosine 3
,5
′
-
cyclic phosphate (30Ci/mmol) were added, and the
nal volume of the
l. After incubating at 30 °C for
10 min, the reaction was terminated by adding 50
μ
l (1 mg) yttrium
silicate beads, which were provided in the SPA kit. A complex ion
chelationmechanismenables the linear nucleotide (5
μ
-AMP) to bind to
the bead and allow radiation from the tritium to excite the scintillant
within the beads. Following addition of the SPA beads, the reaction
mixtures were incubated at room temperature for 20 min to facilitate
the beads to settle down to the bottom of the tube. The radioactivity in
the beads was measured with a liquid scintillation counter and PDE
activity was expressed as counts per minute per
′
2.6. SL-2 cell transfection, extract preparation and dual luciferase assay
For cell transfection, plasmid DNAs were reconstituted in 0.1MCaCl
2
and then mixed with equal volume of 2× HEPES buffer, pH 7.5
(Invitrogen, Carlsbad, CA). Cells to be transfected were resuspended in
fresh SL-2 medium at 1×10
6
cells/ml density. To each well of a 48-well
microtiter plate, 0.5×10
6
cells were dispensed and 84
μ
g protein (CPM/
μ
g
protein). The assays were done in triplicate.
μ
lofcalcium
DNA mix was added in a drop-wise manner. After 18 h
incubation, cells werewashedwith SL-2media, resuspended in 500
–
2.8. Measurement of intracellular cAMP level
lof
fresh media and allowed to recover for 24 h at 25 °C prior to treatment
with any chemical. All treatments were done for 24 h unless otherwise
mentioned. Concentrations of different chemicals to be used for
treatment were determined by dose
μ
–
response experiments (Supple-
Intracellular cAMP level was measured by using the Biotrack cAMP
enzyme immunoassay or EIA kit (Amersham Biosciences, Piscataway,
NJ). Approximately, 10
5
cells suspended in SL-2mediawere transferred
to individual wells of a 96-well microtiter plate. The cells were treated
with caffeine for 24 h then lysed in the lysis reagent (Biotrack EIA kit).
The cell lysate (100
13). Following treatment, cells in the 48-well microtiter
plate were pelleted and lysed by adding 250
–
l 1× passive lysis buffer
(Promega, Madison, WI) to eachwell. The lysatewas collected fromeach
well and assayed for luciferase activity using reagents obtained from
Promega (Madison, WI). To normalize the data, protein content in the
extracts was determined by using BCA protein assay kit.
Experiments in which SL-2 cells were co-transfected with Renilla
luciferase plasmids (Promega, Madison, WI) as an internal control, cell
lysates were assayed by using a dual luciferase assay kit obtained from
Promega (Madison, WI). To 20
μ
l) was transferred to the wells of a microtiter plate
pre-coated with donkey anti-rabbit IgG (Biotrack EIA kit) and 100
μ
l
rabbit anti-cAMP antibody was then added to each well. After
incubating at 4 °C for 2 h, 50
μ
l of cAMP-peroxidase conjugate was
added to each well and incubated again at 4 °C for 1 h. The reaction
mixture was discarded and wells were gently washed with a wash
buffer (Biotrack EIA kit), and 150
μ
-tetramethyl benzidine
(TMB) was added to each well as substrate. Following 1 h incubation at
room temperature, the reactionwas terminated by adding 100
μ
lof3,3
,5,5
′
l luciferase assay reagent
II was added, mixed, and placed in a luminometer after 10 s to mea-
μ
l lysate, 100
μ
lof1M
sulphuric acid. Within 30 min, absorbance at 450 nm was measured
μ
sure the
For all experiments 5
mentary Figs. 1
ies were etherized and sorted into groups of ten
ies were transferred live to individual vials containing
Tomeasure total phosphodiesterase (PDE) activity in the extracts of
transgenic
transferred to food made with 250 mM Tris, pH 8.0. To treat
100
protein, was adjusted to 1× PDE assay buffer containing 50 mM Tris
y extracts was quanti
′
′
reaction mixture was adjusted to 100
phosphate
mentary Figs. 7
′
52
S. Bhaskara et al. / Gene 415 (2008) 49
59
using a plate reader (Labsystem Multiskan). Data were expressed as
fmol cAMP/10
5
cells. Two independent experiments, each in duplicate,
with different batches of cells were performed.
kara et al., 2006
), we used chemicals that are known to affect
adenosine receptors and the PDE enzyme. Since it is not known
whether the homologues of mammalian AdoR are present in Droso-
phila,we
rst examined the effect of adenosine on the Cyp6a8 and
Cyp6a2 promoter activity. If caffeine induction of Cyp6a2 and Cyp6a8
promoters is mediated via antagonism of adenosine receptor,
adenosine being an agonist of AdoR should show an opposite effect
and decrease the promoter activity. To test this hypothesis, SL-2 cells
transfected with 0.2luc-A8 or with 0.8luc-A8 reporter plasmids were
treated with various concentrations of adenosine or Tris buffer, and
extracts prepared from these cells were assayed for luciferase activity.
The results (
Fig. 1
A) showed that even at the highest concentration
(100
μ
M) adenosine did not have any signicant effect on the Cyp6a8-
luc activity. Adenosine also did not have any effect on the Cyp6a2
promoter activity in SL-2 cells (
Fig. 1
A). Furthermore, no effect of
adenosine was observed on the 0.2- and 0.8-kb promoter DNAs
driving the luciferase reporter gene in 0.2-luc4H-ry and 0.8-luc110H-ry
transgenic
2.9. Northern blot hybridization
Northern blots were prepared for three independent RNA samples
and hybridized using the procedures described in detail previously
(
Maitra et al., 2002; Bhaskara et al., 2006
). Total RNAwas isolated from
0.8luc110H-ry, 91R and 91C strains using Tri-reagent (Sigma, MO). The
cDNA fragment of D-junwas excised from the D-jun(s)/HS and used as
a probe for D-jun expression. RP49 was used as the internal control to
correct for loading errors. Hybridization signals were quantied by
scanning the blots with a Packard Instant imager.
2.10. Western blot hybridization
Extracts prepared from SL-2 cells transfected with expression
plasmids and from whole
ies (
Fig. 1
B).
Like caffeine, dipropyl cyclopentyl xanthine (DPCPX) and cyclo-
pentyl adenosine (CPA) also interact with adenosine receptor (for
review see
Poulsen and Quinn, 1998; Fredholm et al., 1999; Moreau
and Huber, 1999; Klinger et al., 2002
). DPCPX is a selective antagonist
for the inhibitory adenosine (A1) receptor and CPA is a selective
agonist for A1 receptor. Since adenosine has intermediary af
ies were examined for D-jun expression.
Extracts were prepared with Tris
NaCl (50 mM Tris pH 7.8,
150 mM NaCl, 1% v/v NP-40) lysis buffer. P8340, a cocktail of protease
inhibitor from Sigma was added just before making the extracts (1
–
NP40
–
μ
l/
80 °C until used.
Protein concentration was determined using D
C
protein assay kit
obtained from Bio-Rad (Hercules, CA).
SL-2 cell extracts with equivalent amounts of total proteins were
fractionated on SDS-polyacrylamide gel (12%) (SDS from Bio-Rad
Hercules, CA, Acrylamide from Sigma, TEMED from Sigma, St. Louis,
MO) and transferred to the polyvinylidene di
μ
l cell lysis buffer). All extracts were stored at
−
nity for
the A1 adenosine receptor, the effect of adenosine may not be as
strong as expected for CPA, which has much higher afnity for the
ouride (PVDF) mem-
brane (Millipore, Billerica, MA), which were then blocked by
incubating for 12 h with continuous shaking at 4 °C in Tris-buffered
Saline (TBS) 5% non-fat dry milk. The membranes were then incubated
at room temperature in D-jun (1/5000 dilution) or D-Fos (1/500
dilution) primary antibody solution for 1 h with rocking. The
membranes were washed with TBST (TBS+0.1% Tween-20) and
incubated for 1 h at room temperature in horseradish peroxidase-
conjugated anti-rabbit antibody (1/7500 dilution) solution. Following
incubation, membranes were again washed with TBST for two times,
15 min each. For chemiluminescence detection, ECL detection kit from
Amersham Biosciences (Piscataway, NJ) was used. ECL reagent was
added to the blots as per manufacturer's instructions and chemilu-
minescence was detected after 5 min by exposing the membrane to an
X-ray
software
(Bio-Rad). Rabbit polyclonal anti-D-jun and anti-D-Fos antibodies
were generous gifts from Dirk Bohmann, University of Rochester.
Horse-radish peroxidase (HRP)-conjugated anti-rabbit secondary
antibody was obtained from Amersham Biosciences (Piscataway, NJ).
lm. The blots were quanti
ed using
Quantity One
’
2.11. Statistical analysis
ies or S2 cells. Data obtained for each set of experiment
were analyzed by two-tailed t-test and the p values are indicated in
the
gure legends.
3. Results
3.1. Adenosine receptor antagonist and agonist have no effect on Cyp6a8
and Cyp6a2 promoter activities
ies. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-A8 or
0.9luc-A2 and pRL null plasmids, were treated for 24 h with either 8 mM caffeine (caff)
or with 8 mM adenosine (Ado). Plain media and 250 mM Tris, pH 8.0 used as solvent for
caffeine and adenosine, respectively were used as controls. (B) Adult 0.2-luc4H-ry and
0.8-luc110H-ry females were treated (T) with 16 mM caffeine or with 8 mM adenosine
for 24 h. Flies allowed to feed on media made with water or 250 mM Tris (pH 8.0) were
used as untreated (UT) controls. Values represent mean±S.E.M of triplicate samples. The
p values for treated (T) and untreated (UT) samples compared are: a, 0.001; b, 1.0;
c, 0.0002; d, 0.1; e, 0.0003; f, 0.08; g, 0.003; h, 0.18; i, 0.005; and j, 0.98.
Caffeine is known to antagonize both inhibitory (A
1
)and
stimulatory (A
2A
) adenosine receptor (AdoR) and inhibit cAMP
phosphodiesterase (PDE) (
Svenningsson et al., 1995a; Poulsen and
Quinn, 1998; Fredholm et al., 1999
). In order to determine which
signaling pathway is involved in caffeine-induced transcriptional
activation of Cyp6a8 and Cyp6a2 promoter reported recently (
Bhas-
–
100
‘
All experiments were done in triplicate with three batches of
transgenic
Fig. 1.
Effect of caffeine and adenosine on Cyp6a8 and Cyp6a2 promoter activities in SL-2
cells and transgenic
S. Bhaskara et al. / Gene 415 (2008) 49
–
59
53
c PDE (
Ye et al., 2001
). Thus, to examine whether PDE inhibitors
can induce Cyp6a2 and Cyp6a8 promoter activities, SL-2 cells
transfected with Cyp6a8 reporter plasmids, 0.2luc-A8 or with 0.8luc-
A8, were treated with rolipram or theophylline. While rolipram is a
PDE4-selective inhibitor, theophylline is a nonselective PDE inhibitor
(
Belibi et al., 2002
). Results showed that both PDE inhibitors induce
0.2-luc and 0.8-luc promoter activities in SL-2 cells (
Fig. 3
A). The
Cyp6a2 promoter was also induced by these PDE inhibitors in SL-2
cells (
Fig. 3
A). Induction of the luc reporter gene was also observed
when 0.2luc4H-ry and 0.8luc110H-ry transgenic
ies were treated
with 16 mM rolipram or with 16 mM theophylline (
Fig. 3
B).
3.3. Mutation of cAMP phosphodiesterase gene induces the Cyp6a8
promoter in absence of caffeine
Since PDE inhibitors induced the Cyp6a8 promoter activity, the
effect of loss of function mutation of PDE on Cyp6a8 promoter activity
in vivo was tested. For this purpose, we used dnc
1
, a hypomorphic
allele of a Drosophila X-linked gene called dunce, which is involved in
learning and memory, and codes for cAMP phosphodiesterase (
Dudai
et al., 1976; Davis and Kiger, 1981
). It has been shown previously that
compared to the wild-type strains, dnc
1
mutant has lower level of
cAMP-phosphodiesterase and higher amounts of intracellular cAMP
(
Davis and Kiger, 1981
). To examine the effect of partial loss of
endogenous PDE activity on the Cyp6a8 promoter activity, dnc
1
/dnc
1
;
+/+ females were crossed to the males of 0.8-luc110H-ry transgenic
line which is homozygous for a 2nd chromosome-linked 0.8luc-A8
M
CPA made in DMSO. Cells treated with DMSO were used as controls (UT). (B) Adult 0.2-
luc4H-ry and 0.8-luc110H-ry females were treated with 16 mM CPA or 1 mM DPCPX (T).
DMSO treated
M DPCPX or 500
μ
ies were used as untreated control (UT). Values represent mean±S.E.M
of triplicate samples. The p values for treated and untreated samples compared are:
a, 0.84; b, 0.29; c, 0.18; d, 0.93; e, 0.85; f, 0.07; g, 0.28; h, 0.13; I, 0.117; and j, 0.26.
nity for the A1 receptor than caffeine. Thus it was thought that
DPCPX or CPA might affect the Cyp6a8 and Cyp6a2 promoter activities,
as they are more potent and speci
c ligands. However, SL-2 cells
transfected with 0.2luc-A8 or 0.8luc-A8 plasmids did not show any
signi
cant change in the luc activity when treated with DPCPX or CPA
(
Fig. 2
A). It is possible that SL-2 cells may be devoid of the putative A1
receptor and for that reason the agonists and antagonist for the
receptor failed to show any effect. Therefore, luciferase activity was
measured in 0.2luc4-H-ry and 0.8luc-110H-ry transgenic
ies following
24 h DPCPX or CPA treatment. The results showed that neither DPCPX
nor CPA had any signi
cant effect on the Cyp6a8 promoter driving the
luc reporter transgene (
Fig. 2
B). These results together with those
obtained in the adenosine treatment experiments suggest that neither
Cyp6a8 nor Cyp6a2 promoter activity is affected by AdoR-mediated
signaling. It is to be noted that although the batches of S2 cells and
transgenic
ies used for these experiments did not respond to DPCPX
or CPA, in these batches of cells and
ies 0.8luc-A8 construct always
showed higher constitutive expression than the 0.2luc-A8 construct as
observed before (
Maitra et al., 2002; Bhaskara et al., 2006
). This
suggests that the cells and transgenic
ies maintained their biological
characteristics with respect to Cyp6a8 promoter activity.
Fig. 3.
Effect of phosphodiesterase inhibitors on Cyp6a2 and Cyp6a8 promoter activities
in SL-2 cells and in transgenic
M rolipram (RP)
or with 8mM theophylline (TP). Cells incubated in DMSO containing or plainmediawere
used as untreated (UT) controls for rolipram and theophylline, respectively. (B) Adult
females from 0.2-luc4H-ry and 0.8-luc110H-ry reporter strains were treated with 16 mM
theophylline or with 16 mM rolipram. Water or DMSO treated
μ
3.2. Phosphodiesterase inhibition induces Cyp6a2 and Cyp6a8 promoter
activities
ies served as controls for
theophylline and rolipram treatments, respectively. Values represent mean±S.E.M of
triplicate samples. The p values for treated and untreated samples compared are:
a, 0.0002; b, 0.003; c, 0.009; d, 0.001; e, 0.0014; f, 0.003; g, 0.0001; h, 0.0001; I, 0.0004;
and j, 0.006.
A high level of caffeine has been shown to inhibit cAMP
phosphodiesterase or PDE that hydrolyzes cAMP (
Svenningsson
et al., 1995a
). In mammals, caffeine inhibits PDE4, which is a cAMP-
speci
Fig. 2.
Effect of A1 adenosine receptor agonist (CPA) and antagonist (DPCPX) on Cyp6a2
and Cyp6a8 promoter activity. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-A8 or
0.9luc-A2 and pRL null plasmids, were treated (T) for 24 h with 5
μ
receptor (
Poulsen and Quinn, 1998
). Similarly, DPCPX has much higher
af
ies. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-
A8 or 0.9luc-A2 and pRL null plasmids, were treated for 24 h with 500
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