DHEA, dehydroepiandrosterone

Transcription

0026-895X196/010067-08$3.OO/O
Copyright
All rights
© by The American
Society
for Pharmacology
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in any form reserved.
PHARMACOLOGY,
50:67-74
(1996).
MOLECULAR
and
Experimental
Therapeutics
Peroxisome
Proliferator-Activated
Receptor
a Required
Gene Induction
by Dehydroepiandrosterone-3f3-sulfate
JEFFREY
M. PETERS,
DAVID J. WAXMAN
YUAN-CHUN
ZHOU,
PRABHA
A. RAM,
SUSANNA
Laborato,y
of Molecular
Carcinogenesis,
National
Cancer
Institute,
National
Institutes
(J.M.P., S.S.T.L.,
F.J.G.), and Department
of Biology,
Division of Cell and Molecular
Massachusetts
02215 (Y.-C.Z., PAR.,
D.J.W.)
Received
March
1 2, 1 996;
Accepted
S. T. LEE,
FRANK
for
J. GONZALEZ,
and
of Health,
Bethesda,
Maryland
20892
Biology,
Boston University,
Boston,
April 2, 1996
Peroxisome
proliferator-activated
receptor
a (PPARa)
mediates
the effects of foreign chemical
peroxisome
proliferators
on liver
and kidney,
including
the induction
of peroxisomal,
mitochondrial, and microsomal
enzymes
involved
in p3-oxidation
of fatty
acids.
However,
the role of this receptor
in the peroxisome
proliferative
effects
of the endogenous
steroid
dehydroepiandrosterone
(DHEA) is not known. DHEA-3 3-sulfate
(DHEA-S)
is shown to induce a liver peroxisome
proliferative
response
in
rats in vivo at a dose at which DHEA is much less active, which
is consistent
with cultured
hepatocyte
studies
indicating
a requirement
for sulfation
for the activation
of DHEA. Transient
transfection
experiments
demonstrated
that in contrast
to the
prototypic
foreign
chemical
peroxisome
proliferator
pirinixic
acid, DHEA-S
and its 1 7j3-reduced
metabolite,
5-androstene3 ,1 73-diol-3 -sulfate,
are inactive
in mediating
trans-activation by PPARa
in COS-1 cells.
Two other mammalian
PPAR
isoforms,
y and 6/Nucl , were also inactive
with respect
to
The
sor
adrenal
of both
from
steroid
DHEA
androgens
other
classes
anticarcinogemc
of
and
properties
dramatic
increase
somes
rodents
present
at high
panied
by
and
fatty
der
conditions
estrogens
(1-5).
in both
This
ES07381
by
also
the
striking
enzymes
can
stimulate
a
of peroxi-
(6-10)
response
transcription
work was
to D.J.W.
via
and
un-
( 10),
supported
the
in part
cellular
by National
involving
mechanisms
Institutes
activation
of
underlying
of Health
as
other
known to be associated
by
belonging
to the
on results
from
function
these
suggested
through
steroids
activity,
was
which
xenobiotic
Despite
DHEA
DHEA
the
PPARa
able
to induce
fatty
or DHEA-S
ABBREVIATIONS:
DHEA, dehydroepiandrosterone;
DHEA-S,
dehydroepiandrosterone-3f3-sulfate;
fate; PPAR, peroxisome
receptor;
RXR, retinoid X receptor;
Wy-14,643,
acid
observations,
mediates
pirinixic
an
receptor
superfamily
transfection!
PPAR
however,
its effects
(15,
ofthis
such
as
17,
18).
of
gene
receptor
Wy-14,643,
it is still
possible
through
other
ADIOL-S,
5-androstene-3j3,1
acid; CYP, cytochrome
not
neither
in reporter
activation
analogues
recepmay
because
increase
and
intracel-
DHEA-S
receptor
to the
fatty
drugs
transient
proliferators
by
(13,
endogenous
PPARa,
and
In
mac-
metabolized
or chimenc
an
12).
both
fibrate
steroid
intact
Ref.
3f3-sulfates
by
in vitro
that
is in contrast
and
first
by
with
peroxisome
these
are
diverse
is mediated
assays
was
see
ADIOL
activated
structurally
chemicals,
Based
a review,
corresponding
proliferation
trans-activation
it
and
unless
to the
as
receptor
16).
(for
DHEA
proliferators
well
foreign
lular
by
understood
peroxisome
clofibrate,
Grant
poorly
hepatocytes,
Peroxisome
tors,
and
its 17f3-reduced
meperoxisome
proliferator-
a mechanism
14).
(15,
to hepato-
as well as six mRNAs
sulfotransferase
acids,
( 1 1).
DHEA
a classic
as
steroid
p-oxidation
lead
are
rat
tive
administered
to
response
is accom-
in peroxisomal
effect
primary
and
number
when
administration
its
P450 4A proteins
with the peroxisomal
proliferative
response
in wild-type
mice. In
contrast,
neither DHEA-S
nor clofibrate
induced
these hepatic
proteins
and mRNAs
in PPARa-deficient
mice. Clofibrate-induced expression
of kidney CYP4A mRNAs was also blocked
in the PPARa gene knockout
mice. Thus, despite
its unresponsiveness
to steroidal
peroxisome
proliferators
in transfection
assays,
PPARa
is obligatory
for DHEA-S-stimulated
hepatic
peroxisomal
gene induction.
DHEA-S,
or one of its metabolites,
may thus serve as an important
endogenous
regulator
of liver
peroxisomal
enzyme
expression
via a PPARa-mediated
pathway.
this
precur-
distinguished
chemoprotective
can
and
CYP4A
of chronic
Although
it is clear
that
tabolite
ADIOL
can induce
gene
size
increase
acid-metabolizing
carcinogenesis
activated
other
in liver
and
kidney
doses.
This
proliferative
a substantial
is
steroids
its
DHEA
the
occurring
that
endogenous
activities
therapeutic
is a naturally
and
DHEA-S
trans-activation.
To test whether
PPARa
mediates
peroxisomal
gene induction
by DHEA-S
in intact animals,
we
administered
DHEA-S
or clofibrate
to mice lacking a functional
PPARa
gene.
Both
peroxisome
proliferators
markedly
increased
hepatic
expression
of two microsomal
cytochrome
that
related
7j3-diol-3f3-sul-
P450.
67
Downloaded from molpharm.aspetjournals.org by Gary Perdew on December 12, 2008
SUMMARY
68
Peters
receptors,
et aL
specifically,
Alternatively,
for DHEA
sufficiently
or may
PPARy
the
in vitro
and/or
sensitive
lack
metabolic
a mouse
associated
some
its
line
proliferators
apparent
may
or other
(22).
inactivity
mediate)
oxisomal
the
key
the
These
transfection
to test
present
in vivo
PPARa
receptor
and
to foreign
chemical
peroxi-
PPARa
establish
that
is required
of DHEA-S
on
its
despite
for
hepatic
(and
Transfections
were
performed
Methods
with
the following
amounts
of
plasmid
DNAs/well
(3.83 cm2) ofa 12-well tissue culture
plate: 0.4 .tg
of reporter
construct
(pLuc-4A6-880),
either
8 ng of mouse
PPARa
expression
plasmid
(pCMV-mPPARa
or pCMV-mPPARa-G),
0.6 p.g
of mouse
PPAR y
expression
plasmid
or 0.6 g of human
PPARh/
Nuci expression
plasmid,
and 0.16 ig of f3-galacthsidase
expression
plasmid
as an internal
control.
pCMX-mRXRa
was included
at 80
ng/well
where
indicated.
The total amount
of DNA was adjusted
to
0.96 tg/well
through
the addition
of pBluescript
DNA as required
(Stratagene).
The PPAR
test activators
Wy-14,643
(Wyeth-Ayerst,
Princeton,
NJ), DHEA-S, ADIOL-S,
and DHEA (Sigma;
99%
purity
by thin layer
chromatography),
7-keto-DHEA
(Steraloids,
Wilton,
NH;
98%
purity
by thin layer
chromatography),
and LY171883
(Biomol
Research
Labs, Plymouth
Meeting,
PA) were obtained
from
the sources
indicated.
PPA.R test activators
were each diluted
from a
1000-fold
stock in dimethylsulfoxide
and were added
to the cells in
fresh
media
at the concentrations
indicated
24 hr after cells were
washed
to remove
the DNA precipitates.
Control
cells received
an
equal
amount
ofdimethylsulfoxide.
At 24 hr after the addition
of the
PPAR test activators,
cells were washed
twice with cold phosphatebufFered
saline
and
then
extracted
in lysis
solution
(100
ma
KPi, pH
7.8, 0.2% Triton
X-100, with 1 mM dithiothreitol
added before use; 80
.tl/well)
for
15 mm
at 4#{176}.
The cell extract
was then
scraped
and
transferred
to a centrifuge
tube
for
removal
of insoluble
cell
debris
use
of j3-galactosidase
activity
values
induced
peroxisome
proliferative
responses
are minimally
detectable
at the lower dosage
(10), in which
case it may be possible
to detect
differences
between
the effectiveness
ofDHEA
and DHEA-S
that are
not apparent
at higher
doses. Animals
were killed 24 hr after the last
injection.
Rat liver RNA was prepared
from tissue frozen at -80#{176}
and
then analyzed
for CYP4A1
and CYP4A3
mRNA
through
Northern
with
viously
Plasm.ids.
Reporter
plasmid
pLuc-4A6-880,
containing
880 nt of
5’-flanking
DNA ofthe
rabbit
CYP4A6 gene cloned into pl9Luc,
and
the mouse
PPARa
expression
plasmids
pCMV-PPARa
and pCMVPPA.Ra-G
(23) were kindly
provided
by Dr. E. Johnson
(The Scripps
Research
Institute,
La Jolla,
CA). A plasmid
expressing
full-length
human
PPARWNuc1
cloned
into pJ3w
(21) was kindly
provided
by
Dr. A. Schmidt
(Merck
Research
Labs,
West
Point,
PA). Mouse
PPAR y
cloned
into pSV-Sportl
(19) was provided
by Dr. J. Reddy
(Northwestern
University
Medical
School,
Chicago,
IL), and mouse
RXRa expression
plasmid
pCMX-mRXRa
(24) was provided
by Dr. R.
Evans
(Salk Institute,
San Diego,
CA). j3-Galactosidase
expression
plasmid
(pSV-13-gal)
was purchased
from Promega.
Transfection
studies.
Transfection
of COS-1
cells grown onto
12-well
tissue
culture
plates
was carried
out according
to a calcium
phosphate
precipitation
method
(25). At 9 hr after addition
of DNA,
cells were washed
and then incubated
in Dulbecco’s
modified
Eagle’s
medium
(GIBCO)
containing
10% charcoal-stripped,
delipidated
bovine calfserum
(catalog
No. C-1696,
Sigma
Chemical
Co., St. Louis,
MO).
the
in
an Eppendorfcentrifuge
(model
5415C;
4 mm at 14,000 rpm). Luciferase
activity
was measured
with the use of an assay
kit and a
Monolight
2010 Luminometer
instrument
(Analytical
Luminescence
Laboratory,
Ann Arbor,
MI). 13-Galactosidase
activity
was determined
with a Galacto-light
chemiluminescent
reporter
kit (Tropix,
Bedford,
MA) scaled down to 6.7 jtl ofextract,
67 tl ofreagent
A, and
100 .d of reagent
B. Luciferase
activity
values
were normalized
for
gene-specific
oligonucleotide
probes
as
described
pre-
(26).
PPAR
gene
knockout
mice.
Male PPARa
(-I-)
mice
or (+1+)
(F3 homozygotes
or wild-type;
hybrids
ofC57BL/6N
x Sv/129 genetic
background;
10-12 weeks ofage)
(22) were housed
in plastic
cages in
a temperatureand light-controlled
environment.
Groups
of mice
(three
mice/group)
were injected
with either
DHEA-S
or clofibrate
(Sigma
Chemical
Co.) at 15 mg/100
g body weight
or corn oil (vehicle
control)
for 4 consecutive
days
by intraperitoneal
injection.
This
saturating
dose of DHEA-S
was chosen
to allow detection
of even a
low level peroxisome
proliferative
response
in the PPARa-deficient
mice.
Body
weights
were
measured
daily.
Twenty-four
hours
the final injection,
mice were killed through
carbon
dioxide
iation,
and the liver and kidneys
were removed
and rapidly
liquid
nitrogen.
Tissues
were stored
at - 80#{176}
until
they
were
isolation
of microsomes
or
after
asphyxfrozen in
used
for
RNA.
Analysis
of microsomal
CYP4A
protein
expression.
Liver
and kidney
microsomes
were prepared
from frozen
mouse
tissues
through
differential
centrifugation
and then analyzed
through
Western blotting
with polyclonal
anti-rat
CYP4A
antibody
raised
to a
di(2-ethylhexyl)phthalate-inducible
rat liver CYP4A
protein
related
to CYP4A1.
This
antibody
has been characterized
as being
crossreactive
with multiple
rodent
CYP4A
proteins
(27) and was kindly
provided
by
WA).
Mouse
were
Dr.
R. T. Okita
liver
obtained
guanidine
and
kidney
after
Polytron
thiocyanate
(Washington
mRNA
sonic
and
State
analysis.
disruption
subsequent
sium trifluoroacetate.
Samples
were
standard
spectrophotometric
analysis.
University,
Total RNA samples
of frozen
tissue
in
centrifugation
quantified
Samples
Pullman,
through
through
the
(10 jig) oftotal
ce-
use of
RNA
from each individual
animal
were electrophoresed
on a 1.0% agarose
gel containing
2.2 M formaldehyde
and
transferred
onto
a Genescreen
Plus (DuPont)
nylon
membrane
in 20x standard
saline
citrate (3 M NaC1, 0.15 M sodium
citrate,
pH 7.0). RNA was fixed to the
membrane
by being baked
at 80#{176}
for 2 hr in a vacuum
oven. Membranes
were then prehybridized
in a hybridization
buffer containing
50% formamide,
5X standard
saline/phosphatefEDTA
buffer
(lx
SSPE
=
0.18 M NaCl,
10 nns NaH2PO4,
1 m t EDTA,
pH 7.7), 5x
Denhardt’s
solution,
200 tg of salmon
sperm
DNA/ml,
0.1% sodium
dodecyl
sulfate,
and 10% dextran
sulfate
for 2 hr. Hybridization
probes
were labeled
with [32PJdCTP
through
random
priming
(Pharmacia,
Piscataway,
NJ). Seven
cDNA probes,
described
previously
(22), were used for sequential
Northern
blot analysis.
These
cDNA
probes
encode
rat peroxisomal
acyl-CoA
oxidase,
rat peroxisomal
bifunctional
enzyme,
rat peroxisomal
3-ketoacyl-CoA
thiolase,
rat
CYP4A1,
rat CYP4A3,
mouse
liver fatty
acid-binding
protein,
and
mouse
f3-actin
as a control.
The
sizes
ofthese
probes
were
1037,
1094,
1017, 2200,
1800, 450, and 1150 bp, respectively.
After
overnight
hybridization
at 42#{176},
the nylon membranes
were washed
three times
for 20 mm with 2x standard
saline
citrate
with 0.5% sodium
dodecyl
sulfate
and subjected
to autoradiography
through
exposure
of membranes
the
to X-ray
signal
film
strength.
for
periods
ranging
from
2 to 24 hr
depending
on
and
with
from the same preparation
of cell lysate.
DHEA-S
and DHEA
induction
ofrat
liver CYP4A.
Adult male
Fischer
344 rats (Taconic,
Germantown,
NY) were given daily intraperitoneal
injections
of DHEA or DHEA-S
at either
1 mg/100
g body
weight
or 6 mg/100
g body weight
for 4 consecutive
days.
DHEA-
blotting
per-
proliferation.
Materials
efficiency
determined
of these
posthe role of
proliferation
studies
in vitro,
in vivo effects
factors
several
evaluate
peroxisome
lacks
(21).
used
of PPARa
may
be inactivation
by the steroid
examine
We also
response
PPAR6INuc1
systems
We
study.
that
pleiotropic
or
capacity
in DHEA-S-induced
using
20)
DHEA-S
activation
to detect
weak
only in the intact
animal.
sibilities
in the present
PPARa
(19,
transfection
Activation
DHEA-S
Statistical
analysis.
were
assessed
view
II ver.
Differences
through
1.03;
the
use
Abacus
in mean
of two-way
PPA.R
liver and body weights
analysis
of variance
Concepts).
Results
DHEA-S-induced
ing
that
CYP4A
DHEA
is
administered
mal or CYP4A
suggests
metabolite,
sponse.
cyte
DHEA
which
Because,
we
is also
DHEA-S
low
dose
[10
substantially
than
trols
in lanes
equally
effective
higher
The
steroids,
with
PPARa
porter
expression
ofreporter
gene
concentrations
later.
can
creased
expression
are
at the
at
higher
related
COS-1
(Fig.
tM
cells
observed
activation
after
or ADIOL-S
with
(20 p.M)
activity
no reproducible
the
addition
when
of
tested
at
2A).
heterodimers
of PPAR
the
re-
of the
with
to peroxisome
RXR,
leading
proliferator
and
enhanced
transcriptional
whether
RXR
is required
for
to inresponse
activation
(28).
DHEA-S-induced
activity
To
a low
et al.
(23)
4Al
the
same
expression
plasmid.
by
a 30-fold
tivity
3
4
5
6
7
8
9
10
II
12
13
14 15
16
lanes
3-5).
a 6-fold
shown.
of the
DHEA-S,
or
the
PPARa-G
basal
PPA.R
despite
for
the
the
activity,
level
greater
in
sensi-
detection
absence
of
in
control
this
cells
in either
cloned
use
reporter
increase
in reporter
treated
with
DHEA,
presence
activity
increase
vehicle
2B
does
PPARa
net
the
designated
Fig.
Consequently,
However,
or ADIOL-S
shown)
that
is greatly
with
wild-type
above
PPAR-G-transfected
DHEA-S.
mutant,
substitution.
of luciferase
increase
by
reported
greater
Wy-14,643
overall
activation,
no reproducible
could
be detected
in cells
of PPAR
gene
activity
7-keto-DHEA,
(Fig.
of cotransfected
2B and
RXR
data
(data
not
shown).
To address
be
the
involved
in
responses,
ence
possibility
that
and
agreement
earlier
and
(100
results
carried
plasmids
PPARy
by Wy-14,643
with
subtypes
out
in
were
Furthermore,
the
LY171883
DHEA-S,
ADIOL-S
did
from
duced
induce
significant
peroxisome
gene
and
knockout
liver
(+1+)
vehicle-injected
body
weights
compared
(-I-)
Northern
same
mice
of animals
blot
mice.
analysis
acyl-CoA
oxidase,
thiolase
in
DHEA-S
either
observed
with
higher
have
or
been
(+1+)
compared
mice
with
or DHEA-S
in
independent
of
prepared
in the
bifunctional
prolifor final
no difference
mice,
RNA
increases
rethis
gene
in initial
was
(-I-)
in
than
peroxisome
clofibrate
there
of liver
strong
acyl-CoA
or
with
for
prolif-
or DHEA-S
increases
Also,
(+1+)
shown).
encoding
brate
kidney.
tested
significantly
were
or
DHEA-S-inand
were
clofibrate
injected
control
revealed
PPAR’y
peroxisome
Similar
treated
body
weight
between
treatment
(data
not
(20)
and
wild-type
mice
(31). However,
mass
was not observed
in PPARa
4). No differences
with
liver
mice
were
with
controls.
mice
(Fig.
wild-type
weights
treated
for DHEA-treated
in mean
liver
erator
on
in
to DHEA-S-induced
Relative
mice
responses
not shown).
knockout
proliferation
responsiveness
ported
increase
data
gene
is in
PPAR-y
and
leukotnene
D41E4
antagonist
activated
PPARy.
However,
DHEA,
(Fig.
3 and
of PPARa
pres-
3), which
activator
selectively
not
with
the
PPAR&’Nucl
(Fig.
tM)
(30).
may
proliferator
were
expression
RXRa.
activated
PPAR
peroxisome
experiments
PPAR6INuc1
of cotransfected
weakly
other
DHEA-S-dependent
cotransfection
PPAR-y
knockout
2
in
sample
Conceivably,
control
sam-
activator
lower
using
experiment
in
1
a significantly
transfections
wild-type
Fig. 1. Induction of liver CYP4A
mRNAs by DHEA and DHEA-S administered
to adult male rats. Shown
is a Northern
blot of total liver RNA
samples
prepared
from individual
rats that were untreated
(lanes 1 and
2) or were induced
with
DHEA
(lanes 3-9) or DHEA-S
(lanes 10-16)
(intraperitoneal
injections
at 1 0 or 60 mg/kg/day
for 4 days, as indicated). Blot was probed
sequentially
with oligonucleotide
probes
specific
for CYP4A1
(A) and CYP4A3
(B) (26). DHEA-S is seen to be much more
effective
than DHEA with regard to induction
of CYP4A3
(and, to a
lesser
extent, CYP4A1)
at the 10 mg/kg dose (compare
lanes 10-12
(29)
cells
actiPPARa
-PPARa
PPAR
of COS-1
exper-
PPAR
of PPAR
et al.
(endogenous)
resulted
eration.
with
in
fold-induction
their
4A3
the
ac-
DHEA,
in these
Fig.
2B.
of the vehicle
activation
Hsu
cells
in
with
in
with
PPARa-G
PPARa
B.
level
transfection
result
cells
seen
comparing
control
activity
and
basal
PPA.R6fNucl
Influence
A.
by
basal
in
increase
of the
or ADIOL-S.
reporter
activity
seen
mask
that
not
further
treatment
2A,
2-3-fold
however,
when
using
a PPARa
that
has a Glu282-to-glycine
shows
into
no
expression
Fig.
in the absence
of exogenous
on the presence
of transfected
as
of this
reduced,
PPARa-G,
the
cells
and
could
Muerhoff
giving
in DHEA-S-acti-
Treatment
was
is
after
in
increased
however,
DHEA-S,
luciferase
compared
a
dose
and
detected
DNA,
indeed
sulfotransferases.
element-containing
DHEA-S,
form
binding
elements
investigate
DHEA-S
pCMV-mPPARa
In contrast,
of 100
PPARa
con-
administered
of PPARa
plasmid
expreswith
3-5
DHEA-S
response
7-keto-DHEA,
DHEA,
CYP4A3
RXRa;
RXRa
shown
was
the
+PPARa/vehicle
endogenous
activator
ple
is a
As
activity
(i.e., activity
was dependent
with
this
of
DHEA-S
peroxisome
proliferator
Wy-14,643
15-fold
increase
in luciferase
reporter
24 hr
with
plasmid
that
when
a compara-
we cotransfected
pLuc-4A6--880.
the
prototypic
resulted
in a
measured
role
hepato-
and
liver
by
inducer
in rat
activity
and
when
by
the
proliferator
plasmid
of liver
of DHEA
proliferation,
transfected
iments
vator)
active
re-
active
(10)],
10-12
DHEA
inducers
when
1 shows
to rats
at
4 days
inducer
PPAR
To investigate
the
for
sulfation
of
peroxisome
peroxisome
Fig.
effect
to its
trans-Activation
vated
in vivo.
administered
CYP4A
doses.
due
a preferential
lanes
2). In contrast,
as
is an
expression
(compare
1 and
reporter
7-keto-DHEA,
The basal
find-
to a more
proliferative
whether
effective
is DHEA
presumably
DHEA-S
mg/kg/day
more
sion
6-fold
evident
are
The
activate
peroxisohepatocytes
(13)
metabolism
mediates
the
enzyme
examined
vivo.
proliferator
but cannot
in cultured
DHEA,
luciferase
was
a mouse
pCMV-PPARa.
from
hepatic
enzyme,
these
mRNAs
and
3-keto-
injected
with
either
clofi-
control
mice
of the
same
tively
peroxisome
undergoes
in turn
peroxisomal
cultures,
the sulfate
DHEA
and
active
unlike
and
in
we cotransfected
with
tivity
induction
to intact
animals
gene
expression
that
of CYP4A
an
activation,
plasmid
(Stat-
69
of PPAR
70
Peters
et aL
A.
4000
0
0
Vehicle
.
Wyl4,643(20j.tM)
13000
Fig. 2. Activation
of PPARa by peroxisome
proliferators: transient
trans-activation
assay.
A, Comparison between
PPARa
alone and PPARa
cotransfected with RXRa.
B, Comparison
of the activation
of
PPARa
with that of PPARa-G.
Cotransfection
of
PPARa
or PPARs-G
expression
plasmids
with
P4504A6
promoter-luciferase
reporter
construct
in
the presence
or absence
of an RXRa expression
plasmid
was carried
out in COS-1
monkey
kidney
cells with a calcium
phosphate
precipitation
method.
Control
DHEA(lOOliM)
2000
III
DHEA-S
7-keto
(100pM)
DHEA
(100pM)
Cells were treated with the indicated
l000
ADIOL-S
0
PPARct
PPARct+RXRct
B.
600-
0
0
Vehicle
.
Wy14,643
D
DHEA(lOOliM)
Control
(20
tM)
400-
JJ;LL
D
‘B
III
200
C
z
-
genetic
1-3).
and
PPAR
cx
background
mRNAs
administered
mRNAs
CYP4A
compared
liferator-treated
were
5A,
compare
for
forms
with
a
(Fig.
Similarly,
two
+ PPAR
liver
were
+ PPAR
a-G
lanes
4-9
fatty
also
with
acid-binding
present
at
higher
( -I-
by
) mice
either
as
clofibrate
assessed
or
with
the
DHEA-S
use
7-keto
DHEA
from
analysis
the
ducible
when
of North-
with
lanes
Vehicle
.
Wy14,643(lOOpM)
0
LY171883
z
PPAR
#{246}/Nuc 1
+
RXRa
PPAR
y
+
RXR a
studies
5A,
internal
± range
(20
M)
standard
at higher
steroid
13-18).
The
lanes
were
of (+1+)
clofibrate
similar
13-17,
bands
5-15-
signals
(<2-fold
activity
concentrations.
obtained
difference)
for
not
A and
(-I-)
(Fig.
mice
5B,
after
treatment
compare
B). A constitutively
lanes
4-9
expressed
Control
(100pM)
Fig.
3. DHEA-S
Nuci
in
DHEA-S
(100 iM)
cotransfected
DHEA-S
(250pM)
plasmid
treated
ADIOL-S
but
or DHEA-S
sion
z
mean
by Wy-14,643
of f3-galactosidase
probe
in livers
either
-C
0
induced
were
(Fig.
-actin
with
0
N
are
all groups.
These
findings
were
confirmed
at the protein
level
in the case
of CYP4A:
two CYP4A
proteins
were
highly
in-
proliver
a)
shown
experiments.
Concentrations
of steroids
up to 250 tM were tested
and also found to be
inactive.
Solubility
problems
or, in some cases,
inhi-
(100pM)
blot
em
Data
in different
precluded
levels
0
C
standard.
activities
bition
lanes
U
a)
(5OliM)
fold
protein
controls
in the livers
of the peroxisome
( +1+ ) mice.
In contrast,
none
of these
induced
DHEA-S
internal
for duplicate
determinations
and are representative
of three or more independent
experiments.
Error
bars not visible for several of the treatment
groups
are too small to be seen. COS-1 cells exhibited
a low
level of endogenous
receptor
activity (-PPARa,
first
bar in B) but a comparatively
high level of endogenous
PPARa
activator
(+PPARa,
vehicle
control).
This endogenous
activator
activity
was greatly
reduced
when the mouse
PPARa-G
mutant was used
(B). Both receptors
were strongly
activated
by Wy14,643 but not by DHEA-S,
DHEA, 7-keto-DHEA,
or
ADIOL-3p-sulfate
(not shown).
Normalized
luciferase
(100pM)
pounds
does
not
activate
PPAR7
trans-activation
assays.
COS-1
with
PPAR-y
or PPARWNuc
plasmids
and
with
and
in the
luciferase
presence
reporter
the indicated
PPAR
analyzed
as described
of RXRa
plasmid
activator
in legend
or PPAR&’
cells
were
1 expres-
expression
and then
test comto Fig. 2.
an
0
PPAR activa-
tors for a 24-hr period beginning
24 hr after washing
of the
cells
to remove
the calcium
phosphate
DNA
precipitates.
Luciferase
reporter
activity
of cell extracts was then determined,
and the data
were normalized to a f3-galactosidase
reporter
(pSV-f3-gal)
as
(100pM)
Activation
DHEA-S
50
full-length,
b
b,c
a
a
a
naturally
when
using
ously
.
29),
which
with
respect
PPARy
and
may
not
carried
for
and
the
on
relative
liver weights.
four daily intra-
studies,
by
DHEA-S.
liferative
responses
such
or DHEA-S
given to
PPARa (+/+)
mice or PPARa (-/-)
mice. Values represent
mean ±
standard
error for three mice per group.
Differences
in mean values
were assessed
with two-way
analysis
of variance.
Bars marked
with
different
letters are significantly different at p
0.05. Body weights
did
not differ significantly
between
the groups.
PPARa.
mediator
Although
of the
CYP4A-immunoreactive
and
protein
molecular
weight
level
was
unaffected
the
PPA.Ra
these
lished.
was
by
knockout
of
In
contrast
to
samples
brate
enzyme,
nor
either
phenotype.
pendent
these
the
controls
similar
Fig. 6A with
Fig.
induce
two kidney
with
controls,
(Fig.
between
lanes
13-15).
prepared
from
the
served,
inactive
blot
same
immunoreactive
for
that
suggesting
the CYP4A1
in Fig. 6A.
this
protein
is distinct
was
mice
from
(Fig.
those
obGB),
encoded
mRNAs
by
The
finding
from
the
DHEA
in vivo with
is consistent
required
to activate
the
primary
rat
DHEA
in
DHEA-S
was
transient
COS-1
also
current
respect
with
inactive
earlier
inactive
DHEA,
in this
DHEA-S
proliferative
(13,
respect
assays
that
to
14).
metabolite,
transfection
assay,
both
when
act
PPAR
cultured
using
or other
were
the
of this
transfection
to Wy-14,643
peroxisome
that
may
but
assays.
P450-dependent
further
need
may
for
Alternatively,
serves
could
that
entry
need
to undergo
(37) but
may
a transporter
the
be
is
absence
of a
in kidney
under
conditions
gene
induction
(see Fig. 6A).
further
cultured
This
metabolism
metabolism
that
occurs
cell
used
in
hydroxylation
metabolism
of
membrane
lines
could
involve
reactions
of DHEA-S
(38,
to yield
sulfatide,
a diglyceride
ester
of DHEA-S
that
can
inant
form
of the
steroid
in plasma
(40).
Finally,
may
of
proximal
the
plasma
explain
as
steroid
production
as a more
is
for
was
such
the
to stimulate
in the
conjugation
neces-
accessory
activity,
in hepatocytes
Indeed,
if such
this
be
proliferation
trans-activa-
other
PPAR
a specific
require
not
a
possibility
response
stimulate
beDHEA-S
include
a requirement
than
RXR (28), which
of DHEA-S-stimulated
that
require
cells,
involve
or
third
may
kidney
may
discrepancy
chemical
factors
tissues
lacking
chrome
the
foreign
chemical
DHEA-S
by
a novel
to mediate
unresponsiveness
in transient
(36).
is present
cell types.
trans-activation
for
modulate
COUP-TF
cells
the
to PPAR6/
it is responsive
2A),
that
other
in
FFAR,
similar
PPA.Ra
more
might
transporter
absent
in
of
in
or
(Fig.
A
into
potential
in
where
diverse
that
activator.
Fourth,
this,
account
for
one
in liver
Thus,
made
induction
of the peroxisome
may
be absent
in the in vitro
or
receptor
33).
as
is 88%
the apparent
to DHEA-S
endogenous
in hepatocytes
ADIOL-S
could
activity
another
latter
negligible.
requirement
in vivo
in the
studies
also
responses
of
forms,
PPAR7
32,
such
that
on
itself
is a
of DHEA,
it
proliferation
tion
system.
These
factors
could
heterodimerization
partners
other
ineffective
with
respect
to restoration
might
(20,
receptors,
foreign
identified
of the
tissue
to
dependent
PPA.Ra
effects
presence
receptor
DHEA-S
(35)
and
requirement
peroxisome
other
First,
is
activation
out
and
proliferators.
HNF-4
DHEA
obligatorily
likely
that
proliferative
the
conditions
factors
major
both
are
liver
structurally
sulfation
Despite
PPA.R
carried
7-keto
than
to
PPARa
in
significant
DHEA-S
where
clofibrate
does
is more
proliferation
findings
hepatocytes
with
its
that
peroxisome
trans-activation
cells.
study
to peroxisome
under
protein
required
re-
in vivo and
receptor
sary
for
response
is nevertheless
pro-
hepatic
clear
other,
This
finding
to DHEA-S
peroxisome
mechanisms
the
only
clofi-
the
is probably
DHEA-S
detected
Discussion
potent
(34),
reporter
CYP4A-
induction
cross-hybridizing
in
(Fig.
microsomes
a single
no
in-
however,
wild-type
acid-activated
Nuci
not
to
Accordingly,
the
probably
Exmice
proliferation
despite
fatty
and
of
The
mice
and
assays
three
inde-
regard.
ofkidney
these,
isorecep-
peroxisome
level
to
PPAR
PPARa
response
is an indirect
one.
The
current
the
peroxisome
proliferative
be mediated
by two other
PPA.R
contribution
in
also
these
to DHEA-S.
is unresponsive
receptor
it seems
peroxisome
a significant
activity
was
a hepatic
clofibrate
that
detection
other
that
mice
lacking
proliferative
of liver
as
PPARSINuc1,
at
activation
steroid-like
levels
(compare
( -I-)
however,
CYP4A3
of all
mice,
observed,
study
that
cannot
Several
in
this
possible
currect
indicate
DHEA
tween
clofi-
clofibrate
did
mice
compared
revealed
clofibrate-treated
and
levels
(-I-)
is also
hepatic
although
(+1+)
analysis
animals
protein;
even
in
was not
clofibrate-treated
Western
estab-
neither
constitutive
than
in liver
Furthermore,
mRNAs
was
be
in
kidney,
6A).
Basal
(+1+)
and
CYP4A
DHEA-S
not
of
oxidase,
bifunctional
mRNAs
compared
with
Furthermore,
higher
in kidney
5A).
or by
observed
the
its
identifies
could
acyl-CoA
thiolase
in CYP4A
expression
RNA
samples
from
crease
kidney
in
apparent
C), but
precise
effects
) mice,
lower
band
proliferator
The
inductive
(+1+
of treatment.
mRNAs
were
SB,
bands
from
vehicle-treated
mRNAs
were
(Fig.
peroxisome
DHEA-S
induced
3-ketoacyl-CoA
or
slightly
detected
CYP4A-immunoreactive
tissue
6A,
also
the
in the
DHEA
response.
PPARa
gene
knockout
this
of
chemicals
after
involved
out
in
but also do not respond
that
although
PPARa
induction
sponse
DHEA-S
be
of two
suggesting
however,
that
peroxisome
in transfection
DHEA-S
PPA.R&Nucl,
and
previ-
to fatty
acid
ester
the
cyto-
39)
or
DHEAbe a domDHEA-S
deriva-
of clofibrate
for
to activation
TREATMENT
4. Influence
allow
forms,
0
Fig.
could
ineffective
brate
(22)
establishes
Shown are liver-to-body
weight
ratios determined
peritoneal injections of either corn oil, clofibrate,
in principle
(-I-)
demonstrated,
lack
the hepatic
CLOFIBRATE
receptors
identified
(+1+)
tors
also
periments
Oil
PPARa
mutant
due to its low background
PPAR
activators.
DHEA-S
bO
Corn
mouse
a receptor
oflow
DHEA-S
activity
the absence
of exogenous
a
0
(23,
occurring
PPA.Ra-G,
71
of PPAR
72
et aL
Peters
B.
A.
Genotype:
(+1+)
Treatment
Corn
oil
(-I-)
Clofibrate
DHEA.S
Corn
oil
Genotype:
aofibrate
DHEA-S
I 2 3 4 5 6 7 8 9101112131415161718
Probe
1
AXO
(-I-)
(+1+)
om
rreatment:
ctfibmte
ii
2
3
4
5
DHEA-S
6
7
corn oil
8
10
9
11
clofibrate
12
13
DHEA-S
14
15
16
17
CYP4A
BIEN
THIOL
LFABP
CYP4AI
CYP4A3
Fig. 5. Evaluation
of DHEA-S
induction
of a liver peroxisome
proliferator
response
in PPARa
(+/+)
and PPARO (-I-)
mice. A, Northern
blot
analysis
of wild-type
(+/+) and PPARa
(-/-)
mice treated
with corn oil (lanes 1-3 and 10-12),
clofibrate
(lanes 4-6 and 13-15),
or DHEA-S (lanes
7-9
and 16-18)
as described
in Materials
and Methods.
Total RNA (1 0 .tg) isolated
from individual
livers was electrophoresed
on a 1%
formaldehyde-agarose
gel and then transferred
to a nylon membrane
before probing
with each of the seven cDNA probes
indicated.
AXO,
acyl-CoA
oxidase;
BIEN,
bifunctional
enzyme;
THIOL, 3-ketoacyl-CoA
thiolase;
LFABP,
liver fatty acid-binding
protein.
B, Liver microsomes
(10
.tg of protein in lane 1 ; 1 5 j.tg of protein in all other lanes)
were prepared from tissue samples corresponding
to the samples shown in A and then
analyzed for immunoreactive
P450 4A proteins by Western blotting. P450 4A-immunoreactive
proteins marked A and B are induced by clofibrate
and DHEA-S
but only in PPARa
(+1+)
mice, whereas band C corresponds
to a constitutively
expressed protein that is unaffected
by the inducers
or the gene knockout.
A.
B.
Genotype:
(+1+)
Treatment
Corn
Probe
oil
2
3
(-I-)
Clofibrat.
4
5
Corn oil
p5P .
6
7
8
Genotype:
DHEA.S
Clofibrate
9 101112131415161718
1
AXO
(-I.)
(+1+)
corn
Treatment:
clottrate
oil
2
3
4
5
DHEA-5
6
7
om
8
9
oil csosb ale
1011
12
13
DHEA-5
14
15
16
17
CYP4A
BIEN
THIOL
CYP4AI
CYP4A3
3-Actin
Fig.
6. Influence
of PPARa gene knockout
on induction
of kidney mRNAs and proteins.
A, Northern
blot analysis of wild-type
(+/+)
and PPARa
mice treated
with corn oil (lanes 1-3 and 10-12),
clofibrate
(lanes
4-6
and 13-15),
or DHEA-S
(lanes 7-9 and 16-18)
was carried
out as
described
in legend to Fig. 5A for liver samples
from the same animals.
Probe designations
were as detailed
in legend to Fig. 5. B, Kidney
microsomes
prepared
from the same tissues analyzed
in A were analyzed
for immunoreactive
CYP4A proteins
by Western
blotting.
A single
constitutively
expressed
kidney CYP4A protein
that was not inducible
by clofibrate
or DHEA-S
was revealed
through
these
analyses.
(-/-)
tives,
which
(41, 42), and
anism
as the
readily
form
this metabolism
for transport
more
proximal
Compared
sponsive
with
into
liver,
to peroxisome
a mild
induction
sured
with
the
use
in plasma
might,
the cell
peroxisome
the
offatty
acids
proliferators.
kidney
proliferators
of peroxisomal
ofWestern
from
serum
lipoproteins
in turn,
provide
a mech-
blot
bifunctional
analysis
after
exposure
several
other
to a high
dose
enzyme
activities
oxisome
unaffected
response
in kidney
(8, 9). Similarly,
could
serve
DHEA-S
crease
in
treatment
kidney
to
43).
be
enzyme
has been
of DHEA
involved
less
Indeed,
reonly
as meareported
(300 mg/kg),
in the per-
in liver
are
in our study,
relatively
clofibrate
did
treatment.
Because
the
clofibrate
is an
important
target
some
proliferation.
suggested
the
for
by
and
clofibrate
absent
PPARa
in
also
in kidney.
in kidney,
liver,
but
The
into
or perhaps
the
mediabsence
even
not the
in wildkidney,
DHEA-S-regulated
transport
above,
in-
peroxisome
CYP4A1was
that
response
tissue
a noticeable
induced
induction
response
that
the
DHEA-S
as
both
it is apparent
induction
DHEA-S
suggests
inefficient,
latter
in
with
although
were
this
mice,
of a substantial
type
(+1+)
mice,
be
result
response,
mRNAs
PPA.Ra-deflcient
ates
not
associated
mRNAS
CYP4A3-related
is known
(8-10,
to occur
whereas
that
and
peroxi-
kidney
cells
enzymes
may
nec-
l-Actin
Activation
DHEA-S
for conversion
of DHEA-S
to
are limited
to the liver or present
in
low to allow for peroxisomal
or CYP
In conclusion,
our data establish
for the hepatic
gene induction
effects
more active
the kidney
essary
enous
regulator
mal
of hepatic
enzymes
reactions.
endogenous
steroidal
tinct
from
PPAR
the
fatty
acid-
whether
activators
use
anism,
such
two
or
activate
as
has
classes
perhaps
one
that
will
be necessary
endogenous
of endogenous
via
a direct
of
an
or
(13,
47).
to determine
enzyme
contributes
expression
class
of endogenous
to
chemoprotective
case
Further
whether
its metabolites
to serve
fatty
acid
metabolism
beneficial
the
the
its
mech-
of PPA.R-y
(30),
indirect
mechanism,
tial of DHEA-S
and
modulators
of liver
other
and
binding
in
activator
via an
dependent
path-
DHEA-S
demonstrated
is Ca 2
PPAR
activation
mechanisms.
been
dis-
It is presently
cellular
PPARa
an
is structurally
(44-46).
distinct
thiazolidinedione
may activate
PPARa
tigation
that
to
inves-
this
poten-
20. Kliewer,
and
21.
Schmidt,
steroids.
sion
thank
Drs.
provision
E. Johnson,
of plasmid
of anti-CYP4A
DNAS
A. Schmidt,
and
Dr.
J. Reddy,
R. T.
Okita
and
for
R.
provi-
23. Muerhoff,
antibody.
Regul.
Schwartz,
26:355-382
A. G.,
Pashko.
cancer
and P. Talalay.
Modulation
by dehydroepiandrosterone.
J.
(1987).
M. Whitcomb,
J.
Dehydroepiandrosterone
and
W.
Nyce,
M.
structural
L.
ofgrowth,
differ-
Adv.
Lewbar,
analogs:
and
a new
L.
M. A., R Hasegawa,
Hirose,
N. Ito, and
and indomethacin
55:4870-4874
T. Shirai.
in a rat
K. Imaida,
A. Hagiwara,
class
of
K. Ogawa,
Chemoprevention
by dehydroepiandrosterone
multiorgan
carcinogenesis
model.
Cancer
Res.
H. Q., B. J. Masset,
D. J. Tweedie,
L. Milewich,
R. A. Frenkel,
R. W. Estabrook,
and R. A. Prough.
Induction
of microNADPH-cytochrome
7.
Frenkel,
R. A.,
P-450
reductase
by
Cancer
Res.
C. A. Slaughter,
8.
Yamada,
J.,
M. Sakuma,
T. Ikeda,
of dehydroepiandrosterone
as a
in
49:2337-2343
K. Orth,
Snyder,
M. Bennett,
R. A. Prough,
isome
proliferation
and induction
rat liver by dehydroepiandrosterone
342 (1990).
and
cytochrome
P-4501VA1
rats:
a possible
peroxS. H. Hicks,
of the
oxisome
(1994).
brate
K. Fukuda,
peroxisome
hepatocarcinogenesis
proliferator,
caused
in
male
F-344
and T. Suga.
Characteristics
proliferator.
Biochim.
Biophys
by dehydroepiandrosterone,
rats.
Carcinogenesis
a per-
15:2215-2219
Borgmeyer,
D. J.
Differential
expression
(1994).
D. Shinar,
steroid
hormone
proliferator
receptor
and
and
recep-
G. A. Rodan.
receptor
and
fatty
superfamacids.
Mol.
F.
and E. F. Johnson.
mediates
w-hydroxylase,
D. J.,
Kakizuka,
and
the
induction
by clofibric
U. Borgmeyer,
and
acid.
The peroxisome
of CYP4A6,
J. Biol.
prolif-
a cytochrome
Chem.
R. A. Heyman,
J. Y. Zhou,
Characterization
R. M. Evans.
inducibility
of cytochrome
P450
4A
fatty
male
specificity
of liver and kidney
CYP4A2
regulation
by growth
hormone
and testosterone.
3921 (1992).
Okita,
R. T., and J. R. Okita.
Characterization
di(2-ethylhexyl)phthalate-treated
rats
which
Arch.
Biochem.
Biophys.
294:475-481
(1992).
27.
267:19051E. S. Ong, A.
of three
RXR
of a cytochrome
hydroxylates
29.
31.
32.
Lehmann,
Willson,
affinity
J. M., L. B. Moore,
and
S. A. Kliewer.
ligand
for peroxisome
An
R. A. Heyman,
and
P450 from
fatty
acids.
Convergence
of 9-cis retinoic
acid and peroxisome
proliferator
signalling
pathways
through
heterodimer
formation
of their
receptors.
Nature
(Lond.)
358:771-774
(1992).
Hsu, M. H., C. N. A. Palmer,
K J. Griffin,
and E. F. Johnson.
A single
amino acid change
in the mouse
peroxisome
receptor
a alters
transcriptional
responses
to peroxisome
proliferators.
Mol. Pharmacol.
48:559-567
(1995).
30.
D. J. Noonan,
omega-hydroxylases:
Kliewer,
\
S. A., K. Umesono,
acid
mRNA
and tissue-specific
J. Biol. Chem.
267:3915-
28.
J. M.
R. S. Putnam,
and L. Milewich.
Peroxof peroxisomal
enzymes
in mouse
and
feeding.
J. Steroid
Biochem.
35:333-
Acta 1092:233-243
(1991).
9. Rao, M. S., B. Reid, H. Ide, V. Subbarao,
and J. K. Reddy.
Dehydroepiandrosterone-induced
peroxisome
proliferation
in the rat: evaluation
of sex
differences.
Proc. Soc. Exp. Biol. Med. 207:186-190
(1994).
10. Prough,
R. A., S. J. Webb,
H. Q. Wu, D. P. Lapenson,
and D. J. Waxman.
Induction
of microsomal
and peroxisomal
enzymes
by dehydroepiandrosthrone
and its reduced
metabolite
in rats.
Cancer
Res. 54:2878-2886
(1994).
11. Hayashi,
F., H. Tamura,
J. Yamada,
H. Kasai,
and T. Suga.
Characteristics
26.
(1989).
C. R. Moomaw,
is
induc-
that
mediate
the action
of 9-cis
retinoic
acid.
Genes Dev. 6:329-344
(1992).
Chen, C., and H. Okayama.
Calcium
phosphate-mediated
gene transfer:
a
highly
efficient
system
for stably
transforming
cells with plasmid
DNA.
Biotechniques
6:632-638
(1988).
Sundseth,
S. S., and D. J. Waxman.
Sex-dependent
expression
and clofi-
25.
(1995).
(P-450LA
omega)
isomal
proliferator.
E. S. Ong, U.
R. Vogel,
Westphal,
A. S., K J. Griffin,
Mangelsdorf,
E. Oro,
A.
M.
w. C. Martin,
somal
pathway:
genes
L.
sterone
of the development
of mammary
carcinoma
induced
by 7,12dimethylbenz(a)anthracene
(DMBA)
in the rat. Breast
Cancer
Res. Treat.
29: 203-217
(1994).
4. Gordon,
G. B., J. K. Helzlsouer,
and G. W. Comstock.
Serum
levels
of
and its sulfate
and the risk of developing
bladder
cancer. Cancer Res. 51:1366-1369
(1991).
5. Shibata,
24.
Enzyme
chemopreventive
agents. Ado. Cancer Res. 51:391-424
(1988).
3. Li, S., X. Yan, A. Belanger,
and F. Labrie.
Prevention
by dehydroepiandro-
6. Wu,
S. J. Rutledge,
H.
erator-activated
G. B., L. M. Shantz,
and carcinogenesis
entiation
B. Blumberg,
of a new member
of the
activated
by a peroxisome
P450 fatty acid
19053 (1992).
1. Gordon,
proliferation
J. W. Owens,
D. L. Kroetz,
P. M.
J. Gonzalez.
Targeted
disruption of the alpha
isoform
of the peroxisome
receptor
gene in mice results
in abolishment
of the pleiotropic
effects
of peroxisome
proliferators.
Mol. Cell. Biol. 15:3012-3022
(1995).
References
2.
A., N. Endo,
Fernandez-Salguero,
authors
for
peroxisome
press.
3 beta-sulfate
Endocrinol.
6:1634-1641
(1992).
Lee, S. S., T. Pineau,
J. Drago,
E. J. Lee,
Acknowledgments
The
of the
K. Umesono,
and R. M. Evans.
of a family
of murine
peroxisome
Nati. Acad.
Sci. USA 91:7355-7359
Identification
ily that
is
intriguing
22.
Evans
activator
S. A., B. M. Forman,
Mangelsdorf,
activation
tars.
Proc.
anticarcinogenic
of this
endogenous
in
ofcytochrome
P-450 4A and acyl-CoA
oxidase
mRNAs
in primary
rat
culture
and inhibitory
effects
of Ca 2
channel
blockers.
Biochem.
J. 301:753-758
(1994).
14. Yamada,
J., M. Sakuma,
T. Ikeda,
and T. Suga. Activation
as a peroxisome
proliferator
by sulfate
conjugation.
Arch.
Biochem.
Biophys.
313:379-381
(1994).
15. Issemann,
I., and S. Green.
Activation
ofa member
ofthe
steroid
hormone
receptor superfamily
by peroxisome
proliferators.
Nature
(Lond.)
347:645650 (1990).
16. Wahui,
W., 0. Braissant,
and B. Desvergne.
Peroxisome
proliferator
activated receptors:
transcriptional
regulators
of adipogenesis,
lipid metabolism and more. Chem.
Biol. 2:261-266
(1995).
17. Gottlicher,
M., E. Widmark,
Q. Li, and J. A. Gustafsson.
Fatty
acids
activate
a chimera
ofthe
clofibric
acid-activated
receptor
and the glucocorticoid receptor.
Proc. Nat. Acad.
Sci USA 89:4653-4657
(1992).
18. Issemann,
I., R. A. Prince,
J. D. Tugwood,
and S. Green.
The peroxisome
receptor:
retinoid
X receptor
heterodimer
is activated by fatty acids and fibrate
hypolipidaemic
drugs.
J. Mol. Endocrinol.
11:37-47
(1993).
19. Zhu, Y., K. Alvares,
Q. Huang, M. S. Rao, and J. K. Reddy. Cloning
of a
new member
of the peroxisome
receptor
gene family
from mouse
liver. J. Biol. Chem.
268:26817-26820
(1993).
as physiological
and
peroxisomal
properties
of dehydroepiandro,
hepatocyte
w-hy-
correspond
in the activation
R. M. Evans.
T. A. Smith-Oliver,
W. 0. Wilkison,
T. M.
antidiabetic
thiazolidinedione
is a high
receptor
gamma
(PPARgamma).
J. Biol.
Sakuma,
M., J. Yamada,
Chem.
270:12953-12956
and T. Suga. Comparison
(1995).
ofthe
on
proliferation-associated
hepatic
peroxisome
inducing
effect
of
enzymes
in several
rodent
species:
a short-term
administration
study.
Biochem.
Pharmacol.
43:1269-1273
(1992).
Jones,
P. S., R. Savory,
P. Barratt,
A. R. Bell, T. J. B. Gray, N. A. Jenkins,
D. J. Gilbert,
N. G. Copeland,
and D. R. Bell. Chromosomal
localisation,
inducibility,
tissue-specific
expression
rine peroxisome-proliferator-activated-receptor
233:219-226
and
strain
differences
genes.
Eur.
in three
J.
mu-
Biochem.
(1995).
0., F. Foufelle,
C. Scotto,
M. Dauca,
and W. Wahli.
Differential
expression
of peroxisome
receptors
(PPA.Rs):
tissue
distribution
of PPAR-alpha,
-beta, and -gamma
in the adult
rat. Endocrinology
137:354-366
(1996).
34. Amri,
E. Z., F. Bonino,
G. Ailhaud,
N. A. Abumrad,
and P. A. Grimaldi.
33. Braissant,
antidiabetic
DHEA-S
may
D. J. Role of metabolism
sterone
as a peroxisome
proliferator.
J. Endocrinol.
13. Ram, P. A., and D. J. Waxman.
Dehydroepiandrosterone
tion
microso-
and
eicosanoid-derived
activation
may
thus
12. Waxman,
an
is required
an endog-
CYP4A
p-oxidation
recently
same
molecular
metabolites
and
these
the
and
acid
activator
identified
unknown
and
DHEA-S
PPAR
activators
ways
in fatty
induction.
that PPARa
of DHEA-S,
peroxisomal
involved
droxylation
enzyme
metabolites
at levels
too
73
of PPAR
74
Peters
et aL
Cloning
of a protein
that mediates
transcriptional
effects
of fatty acids
preadipocytes.
J. Biol. Chem.
270:2367-2371
(1995).
35. Winrow, C. J., S. L. Marcus,
K S. Miyata,
B. Zhang,
J. P. Capone,
and
A. Rachubinski.
Transactivation
of the peroxisome
receptor
Expr.
is differentially
4:53-62
36. Miyata,
modulated
by
hepatocyte
nuclear
42.
R.
Gene
43.
(1994).
K S., B. Zhang,
S. L. Marcus,
J. P. Capone,
and R. A. Rachubinski.
Chicken
ovalbumin
upstream
promoter
binds
to a peroxisome
proliferator-responsive
peroxisome
factor-4.
in
transcription
element
signaling.
J. Biol.
proliferator-mediated
factor
and
Chem.
(COUP-TF)
antagonizes
of expression
44.
268:19169-
19172 (1993).
37.
Reuter,
38.
droepiandrosterone
sulphate
into rat hepatocytes.
J. Steroid.
Biochem.
Mol. Biol. 54:227-235
(1995).
Waxman,
D. J. Interactions
of hepatic
cytochromes
P-450
with steroid
hormones:
regioselectivity
and stereospecificity
of steroid
metabolism
and
hormonal
regulation
ofrat
P-450 enzyme
expression.
Biochem.
Pharmacol.
39.
S., and
37:71-84
Iwasaki,
(1988).
M., D.
Multiple
steroid-binding
hydroepiandrosterone
40.
G.
Davis,
Transport
T. A.
orientations:
Darden,
L. G.
alteration
Pedersen,
and
and
of regiospecificity
M.
dehy-
Negishi.
of de-
2- and 7-hydroxylase
activities
of cytochrome
P-450
2a-5 by mutation
of residue
209. Biochem.
J. 306:29-33
(1995).
Pashko,
L. L., A. G. Schwartz,
M. Abou-Gharbia,
and D. Swern.
Inhibition
of DNA synthesis
in mouse
epidermis
and breast
epithelium
by dehydroepiandrosterone
and related steroids. Carcinogenesis
2:717-721
(1981).
Roy, R., and A. Belanger.
ZR-75-1
breast
cancer
cells generate
nonconjugated steroids from low density
lipoprotein-incorporated
lipoidal
dehydroepiandrosterone.
Endocrinology
133:683-689
(1993).
of peroxisomal
beta-oxidation
and
catalase
genes
Gomez,
unconMetab.
Osumi,
levels
in liver
and
extrahepatic
tissues
of rat. Cancer
Res. 48:5316-5324
(1988).
Yu, K., W. Bayona,
C. B. Kallen,
H. P. Harding,
C. P. Ravera,
G. McMahon,
M. Brown,
and M. A. Lazar.
Differential
activation
of peroxisome
receptors
by eicosanoids.
J. Biol. Chem.
270:23975-23983
(1995).
45.
Forman,
B. M., P. Tontonoz,
J. Chen, R. P. Brun, B. M. Spiegelman,
and R.
15-Deoxy-delta-12,14-prostaglandin
J2 is a ligand
for the adipocyte
determination
factor
PPAR gamma.
Cell 83: 803-812
(1995).
46. Kliewer, S. A., J. M. Lenhard,
T. M. Willson,
I. Patel,
D. C. Morris,
and J.
M. Lehmann.
A prostaglandin
J2 metabolite
binds peroxisome
proliferatar-activated
receptorgamma
and promotes adipocyte differentiation.
Cell
83:813-819
(1995).
M. Evans.
47.
Itoga,
H.,
suppressive
Biochim.
Send
Boston
H.
reprint
University,
[email protected]
Tamura,
effect
Biophys.
T. Watanabe,
of mcardipine
Acta 1051:21-28
requests
to:
5
Dr.
Cummington
David
and
T.
Suga.
on peroxisome
(1990).
J. Waxman,
Street,
Boston,
Characteristics
induction
of the
in
Department
MA
rat
liver.
of Biology,
02215.
E-mail:
41.
D. Mayer.
Belanger,
A., B. Candas,
A. Dupont,
L. Cusan,
P. Diamond,
J. L.
and F. Labrie.
Changes
in serum
concentrations
of conjugated
and
jugated
steroids
in 40- to 80-year-old
men. J. Clin. Endocrinol.
79:1086-1090
(1994).
Nemali,
M. R., N. Usuda,
M. K. Reddy,
K Oyasu,
T. Hashimoto,
T.
M. S. Rao, and J. K Reddy.
Comparison
ofconstitutive
and inducible

DHEA, dehydroepiandrosterone

Transcription

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