477
S-16-3
Retinoid S.KATO,
Status
and
H.MANO,
RARs
Expression
R.KOJIMA,
and
Y.YOSHIZAWA,
S.MASUSHIGE
Department of Agricultural Chemistry, 1-1-1, Sakuragaoka, Setagayaku, Tokyo
Tokyo University 156 (Japan)
of
Agriculture,
I . INTRODUCTION Vitamin A (retinol) and its derivative retinoic acid are well known to have profound effects on a wide range of biological functions such as vision, reproduction, growth and cellular differentiation in adult animals [1]. Moreover, recent studies have shown that retinoic acid is profoundly involved in the vertebrate development and embryogenesis [2]. The RARƒÀ
discovery
and
of
RARƒË7)
mechanisms
of of
transcription
for
parts
Therefore, role
on
the
the
differ
in
intact
spatial
animals,
them
the
latter
and
temporal
dependent
investigating
on the
of
RAR
genes
of
the
tissues
it
has
patterns
it
retinoid
are
action.
retinoid
case,
although
cellular
the
has
been
binding
proteins
retinoid
ligand
influence
of
the
the
steroid/thyroid can
at
has
a
themselves in
adult
already
is
activate
nuclear
gene
the
RARs In
expression
of
and
there
ligand
and that [5].
tissue-specific
status,
central
shown
expression that
least
animals
been
described
retinoid
RARs
expression
of
RARƒ¿,
ligand-inducible
of gene
specificity In
members
(
underlying
As
as
RAR-mediated
expression
target
embryogenesis.
[3,4].
other
of
receptors of
genes
the
some
acid
understanding
trunsduction
like
superfamily,
account
retinoic
an
retinoid-inducible
factors
receptor
nuclear
provided
retinoid
transcription
the
three
has
has
status
some
been on
no
of
report
RAR
expression. As genes, mRNAs
a in in
rats
as
II.
first
as
out genes
mRNAs of
transcripts
for
regulating
the retinoid-deficient,
the
levels
excess
of
expression
of
RAR a, ƒÀ and retinoid-repleted
RAR T
retinoids.
THE ƒ¿, ƒÀ
AND
y
RAR
mRNA
IN
RART
cDNA
probes,
mRNA
extracted
conditions. expressed
in and
results action.
and
poly(A)
expressed
3.8Kb (3.1
RARII
or
stringent
ubiqutiously
RAR
retinoid
,
RNA
under
and
These the
RAR
total
differentialy
were 2.8
tissue-specific. on
of
carried
transcripts
roles
mouse
analysis
receptor RARot
factor
given
OF
[321‚o]-labeled
was
three
animals
EXPRESSION
blot
tissues
a
RAT.
Using Northern
know
we investigated tissues of
normal
DIFFERENTIAL OF
to
study various
well
TISSUES
The
step
this the
all
As in
various
with
tissues
two
examined.
3.6Kb)
and
suggest
that
RART each
in
rat
rat
Fig.
1A
tissues.
distinct In
(3.3Kb) RAR
from
shown
contrast,
the
were may
have
distinct
IV
Symposium (16)
478
M.
VITAMIN To
A
DEFICIENCY
investigate
retinoid
in
investigated
by
tissues
of
periods.
fed
retinol-deficient and
After of
with
diet,
and
mRNA
analysis
levels
of
or
RARa
of
mRNAs
from
by was
various
for
various
feeding
judged
a
by
measuring
in
various
HPLC.
was
cleary
Fig.
reduced compare
1.].
various
not
the
The
lanes
significant
tissues
was
Fig.
were
were
extracted
state[ in
by
diets
with
in
mRNA.
affected
transcripts
achieved
autoradiographs(
RARy
RAR
RARs
was
RARƒÀ
levels
RARI3 is
deficiency
of
THE
retinol-deficient
content
of
and
levels
rats
retinol
level
OF
of
in
retinol the
A
expression
analysis
normal retinoid
RARII
densitometric
the
in a retinol-deficient "D"(defici ency)
and of
animals,
of
days,
rats
reduction
gene
blot
hepatic
35
tissues "C"(control)
DECREASE
RAR
Northern
rats
serum
THE
the
whole
Depletion
the
CAUSES
whether
status
Vitamin
confirmed
1B).
affected
In
by
the
contrast,
the
by
retinol-deficiency. To RARi3
determine
days
up
to
days
After
already
decreased(
retinol
was 50%
of
of
control
.
it
OF To
due
a
recover
30%
decrease
rule to
on
days
a
RETINOL
the
possibility
irreversible
test
was
RARƒÀ that
killed
serum RAR
retinol
day
the
precise
lung
completely
of
threshold
of
serum decreased
were
maintenance
were
though
retinol
transcripts the
in
0),
serum
10
were
gene
to
of
every
the
serum
normal retinol
25 ug/dl).
CAUSED
out
the
when
20,
require
were
compared
likely
mRNAS
an
and for
when
most may
OF
RARƒÀ
mRNAs
reduced.
is
reduction
diets
transcripts
(approximately
ADMINISTRATION
be
RAR$ the
mRNAs
concentrations
LEVEL
the
days,
level
RAR3
retinol-deficiency-induced
experimental
significantly
Thus,
level
of
fed
and 10
not
abolished.
onset
rats 30
analysed.
to
the
transcripts,
AND BY
that malfunction
performed.
RETINOIC
VITAMIN
ACID A
RESTORED
THE
REDUCED
DEFICIENCY.
the suffered
decrease
of from
RARi3 retinol
mRNA deficiency,
might
S.KATO
et
al.
479
Administration of retinoic acid to retinol-deficient rats rapidly restored the level of RAR mRNA in various tissues within 4hr. Retinol also induced a transient RAR gene expression, but in limited tissues. Moreover, the magnitude of the induction was less and the kinetics differd from retinoic acid.
To investigate at which level the induction of RAR by retinol or retinoic acid is achived, specific inhibitors of transcription ( actinomycin D) or translation(cycroheximide) were injected lhr before administration of retinoids. Actinomycin D completely blocked the 4hr-induction of RAR gene expression by retinoids, whereas cycloheximide did not affect the induction. Thus, the induction of RAR gene expression by retinoid may occur at the transcriptional level without de novo protein synthesis.
V.
RETINOIC
ACID
From
the
retinoic of acid
acid
rats
in and
result,
can normal
Thus, autoregulation
the
of
the
compared
RARƒÀ
level
of For
RAR
to
gene
clearly in
intact
RAR the
PAR this
GENE. question
transcripts purpose, to
13
normal
results
OF
addressed
intragastrically
only
present
we
status.
given
that when
the
OVEREXPRESSION
study,
enhance
was
found
overexpressed
THE
retinoid
retinol we
INDUCES
replenishment
transcripts rats(Fig. demonstrated
lmg
normal
whether
the
of
retinoic
rats.
were 2A
of in
As
tissues
a
significantly and
2B).
the
existence
of
animals.
VI. THE INFLUENCE OF RETINOID STATUS ON RAR ISOFORMS. More recently, each subtype of RAR gene has been shown to transcribe a group of different mRNAs, designated as RAR isoforms [6]. The analysis of the gene structure suggested that the gene expression of the isoforms is controled by different promoters. To get a better comprehending of retinoid transduction, currently we are investigating how the retinoid ligand status and synthetic retinoids affect the levels of each RAR isoform, as well as retinoid binding protein mRNAs.
Symposium 8
480
Vitamin A
VII. CONCLUSION From these results, we clearly demonstrated that the retinoid ligand status affects the RAR13 mRNA level in intact animals. The alterd levels of RARO by hypo- and hyper-vitaminosis A may affect retinoid-induccible gene expression, thereby affecting a wide range retinoid action.
REFERENCES [1]
Wolf,
Physiol. [2]
G.
(1984):
Rev.
64,
Eichele,
G.
formation. [3] A
Petkovich,
M.
Zelent,
novel
retinoic Proc.
levels
444-450.
A.
Petkovich,
and
limb
Chambon,
belongs
M. and ƒÀ
predominantly
to
pattern
P. the
Ph.
retinoic
acid in
(1987):
family
Kastner,
expressed
in
y
of A.
and
receptor
and
of
nuclear
Chambon,
P.
receptors
and
skin.
Nano, on various
acid
the
with
USA
Nature C. (1990):
P.
specific
tissues.
M.
Stoner,
expression
receptors
mouse.
87,
H. Kumazawa, the ƒ¿, ƒÀ rat
Petkovich,
Differential
Mendelsohn, Chambon,
'y Sci.
Ph.
(1989):
retinoic
limbs Krust, A.
Acad. S. status
Kastner, P.
and
Staub, acid
E.
Chambon,
P.
Natl.
[7] Kato, retinoid
vertrbrate
A.
which
330,
Robert,
and
developing
Kastner, P.
Krust,
murine ƒ¿ I
a, ƒÀ
the
[6] Leroy,
of
P. U.
encoding in
A.
714-717.
Dolle,
Gudas,
N.
Krust,
receptor 339,
Vitamin
246-251.
receptor
A.
of
and
5,
Brand,
Cloning
Nature [5]
Retinoids
Nature
(1989):
functions
Genet
retinoicacid
receptors.
a
(1989):
Trends
human
[4]
Multiple 873-937.
and
genes
CRABP
342,
702-705.
Gamier, Murine patterns
CM. of
JM. Zelent, isoforms of
A. of
expression.
2700-2704. T. and
and y
Masushige, retinoic
[submitted]
S.: acid
Effect receptor
of mRNA
of
S-16-3
Retinoid S.KATO,
Status
and
H.MANO,
RARs
Expression
R.KOJIMA,
and
Y.YOSHIZAWA,
S.MASUSHIGE
Department of Agricultural Chemistry, 1-1-1, Sakuragaoka, Setagayaku, Tokyo
Tokyo University 156 (Japan)
of
Agriculture,
I . INTRODUCTION Vitamin A (retinol) and its derivative retinoic acid are well known to have profound effects on a wide range of biological functions such as vision, reproduction, growth and cellular differentiation in adult animals [1]. Moreover, recent studies have shown that retinoic acid is profoundly involved in the vertebrate development and embryogenesis [2]. The RARƒÀ
discovery
and
of
RARƒË7)
mechanisms
of of
transcription
for
parts
Therefore, role
on
the
the
differ
in
intact
spatial
animals,
them
the
latter
and
temporal
dependent
investigating
on the
of
RAR
genes
of
the
tissues
it
has
patterns
it
retinoid
are
action.
retinoid
case,
although
cellular
the
has
been
binding
proteins
retinoid
ligand
influence
of
the
the
steroid/thyroid can
at
has
a
themselves in
adult
already
is
activate
nuclear
gene
the
RARs In
expression
of
and
there
ligand
and that [5].
tissue-specific
status,
central
shown
expression that
least
animals
been
described
retinoid
RARs
expression
of
RARƒ¿,
ligand-inducible
of gene
specificity In
members
(
underlying
As
as
RAR-mediated
expression
target
embryogenesis.
[3,4].
other
of
receptors of
genes
the
some
acid
understanding
trunsduction
like
superfamily,
account
retinoic
an
retinoid-inducible
factors
receptor
nuclear
provided
retinoid
transcription
the
three
has
has
status
some
been on
no
of
report
RAR
expression. As genes, mRNAs
a in in
rats
as
II.
first
as
out genes
mRNAs of
transcripts
for
regulating
the retinoid-deficient,
the
levels
excess
of
expression
of
RAR a, ƒÀ and retinoid-repleted
RAR T
retinoids.
THE ƒ¿, ƒÀ
AND
y
RAR
mRNA
IN
RART
cDNA
probes,
mRNA
extracted
conditions. expressed
in and
results action.
and
poly(A)
expressed
3.8Kb (3.1
RARII
or
stringent
ubiqutiously
RAR
retinoid
,
RNA
under
and
These the
RAR
total
differentialy
were 2.8
tissue-specific. on
of
carried
transcripts
roles
mouse
analysis
receptor RARot
factor
given
OF
[321‚o]-labeled
was
three
animals
EXPRESSION
blot
tissues
a
RAT.
Using Northern
know
we investigated tissues of
normal
DIFFERENTIAL OF
to
study various
well
TISSUES
The
step
this the
all
As in
various
with
tissues
two
examined.
3.6Kb)
and
suggest
that
RART each
in
rat
rat
Fig.
1A
tissues.
distinct In
(3.3Kb) RAR
from
shown
contrast,
the
were may
have
distinct
IV
Symposium (16)
478
M.
VITAMIN To
A
DEFICIENCY
investigate
retinoid
in
investigated
by
tissues
of
periods.
fed
retinol-deficient and
After of
with
diet,
and
mRNA
analysis
levels
of
or
RARa
of
mRNAs
from
by was
various
for
various
feeding
judged
a
by
measuring
in
various
HPLC.
was
cleary
Fig.
reduced compare
1.].
various
not
the
The
lanes
significant
tissues
was
Fig.
were
were
extracted
state[ in
by
diets
with
in
mRNA.
affected
transcripts
achieved
autoradiographs(
RARy
RAR
RARs
was
RARƒÀ
levels
RARI3 is
deficiency
of
THE
retinol-deficient
content
of
and
levels
rats
retinol
level
OF
of
in
retinol the
A
expression
analysis
normal retinoid
RARII
densitometric
the
in a retinol-deficient "D"(defici ency)
and of
animals,
of
days,
rats
reduction
gene
blot
hepatic
35
tissues "C"(control)
DECREASE
RAR
Northern
rats
serum
THE
the
whole
Depletion
the
CAUSES
whether
status
Vitamin
confirmed
1B).
affected
In
by
the
contrast,
the
by
retinol-deficiency. To RARi3
determine
days
up
to
days
After
already
decreased(
retinol
was 50%
of
of
control
.
it
OF To
due
a
recover
30%
decrease
rule to
on
days
a
RETINOL
the
possibility
irreversible
test
was
RARƒÀ that
killed
serum RAR
retinol
day
the
precise
lung
completely
of
threshold
of
serum decreased
were
maintenance
were
though
retinol
transcripts the
in
0),
serum
10
were
gene
to
of
every
the
serum
normal retinol
25 ug/dl).
CAUSED
out
the
when
20,
require
were
compared
likely
mRNAS
an
and for
when
most may
OF
RARƒÀ
mRNAs
reduced.
is
reduction
diets
transcripts
(approximately
ADMINISTRATION
be
RAR$ the
mRNAs
concentrations
LEVEL
the
days,
level
RAR3
retinol-deficiency-induced
experimental
significantly
Thus,
level
of
fed
and 10
not
abolished.
onset
rats 30
analysed.
to
the
transcripts,
AND BY
that malfunction
performed.
RETINOIC
VITAMIN
ACID A
RESTORED
THE
REDUCED
DEFICIENCY.
the suffered
decrease
of from
RARi3 retinol
mRNA deficiency,
might
S.KATO
et
al.
479
Administration of retinoic acid to retinol-deficient rats rapidly restored the level of RAR mRNA in various tissues within 4hr. Retinol also induced a transient RAR gene expression, but in limited tissues. Moreover, the magnitude of the induction was less and the kinetics differd from retinoic acid.
To investigate at which level the induction of RAR by retinol or retinoic acid is achived, specific inhibitors of transcription ( actinomycin D) or translation(cycroheximide) were injected lhr before administration of retinoids. Actinomycin D completely blocked the 4hr-induction of RAR gene expression by retinoids, whereas cycloheximide did not affect the induction. Thus, the induction of RAR gene expression by retinoid may occur at the transcriptional level without de novo protein synthesis.
V.
RETINOIC
ACID
From
the
retinoic of acid
acid
rats
in and
result,
can normal
Thus, autoregulation
the
of
the
compared
RARƒÀ
level
of For
RAR
to
gene
clearly in
intact
RAR the
PAR this
GENE. question
transcripts purpose, to
13
normal
results
OF
addressed
intragastrically
only
present
we
status.
given
that when
the
OVEREXPRESSION
study,
enhance
was
found
overexpressed
THE
retinoid
retinol we
INDUCES
replenishment
transcripts rats(Fig. demonstrated
lmg
normal
whether
the
of
retinoic
rats.
were 2A
of in
As
tissues
a
significantly and
2B).
the
existence
of
animals.
VI. THE INFLUENCE OF RETINOID STATUS ON RAR ISOFORMS. More recently, each subtype of RAR gene has been shown to transcribe a group of different mRNAs, designated as RAR isoforms [6]. The analysis of the gene structure suggested that the gene expression of the isoforms is controled by different promoters. To get a better comprehending of retinoid transduction, currently we are investigating how the retinoid ligand status and synthetic retinoids affect the levels of each RAR isoform, as well as retinoid binding protein mRNAs.
Symposium 8
480
Vitamin A
VII. CONCLUSION From these results, we clearly demonstrated that the retinoid ligand status affects the RAR13 mRNA level in intact animals. The alterd levels of RARO by hypo- and hyper-vitaminosis A may affect retinoid-induccible gene expression, thereby affecting a wide range retinoid action.
REFERENCES [1]
Wolf,
Physiol. [2]
G.
(1984):
Rev.
64,
Eichele,
G.
formation. [3] A
Petkovich,
M.
Zelent,
novel
retinoic Proc.
levels
444-450.
A.
Petkovich,
and
limb
Chambon,
belongs
M. and ƒÀ
predominantly
to
pattern
P. the
Ph.
retinoic
acid in
(1987):
family
Kastner,
expressed
in
y
of A.
and
receptor
and
of
nuclear
Chambon,
P.
receptors
and
skin.
Nano, on various
acid
the
with
USA
Nature C. (1990):
P.
specific
tissues.
M.
Stoner,
expression
receptors
mouse.
87,
H. Kumazawa, the ƒ¿, ƒÀ rat
Petkovich,
Differential
Mendelsohn, Chambon,
'y Sci.
Ph.
(1989):
retinoic
limbs Krust, A.
Acad. S. status
Kastner, P.
and
Staub, acid
E.
Chambon,
P.
Natl.
[7] Kato, retinoid
vertrbrate
A.
which
330,
Robert,
and
developing
Kastner, P.
Krust,
murine ƒ¿ I
a, ƒÀ
the
[6] Leroy,
of
P. U.
encoding in
A.
714-717.
Dolle,
Gudas,
N.
Krust,
receptor 339,
Vitamin
246-251.
receptor
A.
of
and
5,
Brand,
Cloning
Nature [5]
Retinoids
Nature
(1989):
functions
Genet
retinoicacid
receptors.
a
(1989):
Trends
human
[4]
Multiple 873-937.
and
genes
CRABP
342,
702-705.
Gamier, Murine patterns
CM. of
JM. Zelent, isoforms of
A. of
expression.
2700-2704. T. and
and y
Masushige, retinoic
[submitted]
S.: acid
Effect receptor
of mRNA
of
