Differential Expression of gas and gadd Genes at Distinct Growth

Vol.
6,
154 1- 1547,
December
1995
Cell
Differential
Expression of gas and gadd
Growth
Arrest Points during Adipocyte
Erika C. Shugart, Anait
and Robert M. Umekt
Department
22903
of Biology,
S. Levenson,2
Cara M. Constance,
University
of Virginia,
Charlottesville,
3T3-L1
(2),
promoters
Virginia
& Differentiation
1541
Genes at Distinct
Development’
differentiate,
C/EBPa4
Growth
which
cells
has
of several
require
been
the transcription
shown
factor
to trans-activate
adipocyte-specific
the
genes (3). Further-
more, forced expression
of C/EBPa
in logarithmically
growing adipoblasts
results in mitotic
growth
arrest independent
of adipogenesis,
whereas
its premature
expression
after ex-
Abstract
The characterization
of growth
arrest-associated
genes
has revealed
that cells actively
suppress mitotic
growth
in response
to extracellular
signals. Mouse 3T3-L1
cells
growth
arrest
at multiple
distinct points during terminal
posure to adipogenic
hormones
hastens differentiation
(4).
These observations
lead us to speculate that C/EBPa may be
a component
of a signal transduction
pathway
that coordinates
mitotic
growth
To better
cellular
serum-starved
observed to be coordinately
induced at growth arrest (5-8).
We report here that the gas/gadd
genes are differentially
expressed
throughout
adipocyte
development.
Certain of
these growth
arrest-associated
genes,
such as gas5,
are
induced by serum starvation
and contact inhibition.
Others,
including
gasl gas3, and gaddl 53, are expressed
in serumstarved adipoblasts,
contact-inhibited
adipoblasts,
and in
postmitotic
adipocytes.
In differentiated
cells,
gasl
and
gas3 appear to be induced
in response to nutrient depriva-
contact-inhibited
adipoblasts,
an exception
growth
to the general
arrest and gas/gadd
preferentially
expressed
concordance
expression
during
of mitotic
in that gas6
the clonal
expansion
is
of
postconfluent
adipoblasts.
Combined,
the expression
patterns
indicate
that growth
arrest-associated
genes are
regulated by numerous signal transduction
pathways
throughout
a discrete developmental
transition.
Introduction
Adipose
conversion
of cultured
3T3-L1
cells provides
a
model
system for examining
the relationship
between
diffenentiation
and cellular
growth
control.
In the standard
differentiation
protocol,
adipoblasts
are cultured
to postconfluence,
exposed
to adipogenic
hormones
to initiate
differentiation,
and then maintained
in differentiation
media to promote
the accumulation
of cytophasmic
fat droplets
(reviewed
in Ref. 1). The addition
of the adipogenic
hormones stimulates
the growth-arrested,
postconfhuent
cells to
reenter
the mitotic
cell cycle,
which
the cells exit again
upon withdrawal
of the hormones.
Thus, a discrete
period
of mitotic
division
separates
postconfhuent
adipoblasts
from
phenotypically
differentiated,
postmitotic
adipocytes.
To
Received
8/1 4/95;
revised
9/22/95;
accepted
9/27/95.
This work
was supported
by Grant
BE-183
from
the American
Cancer
Society
(to R. M. U.).
2 Present
address:
Robert
H. Lurie Cancer
Center,
Northwestern
University
Medical
School,
303 East Chicago
Avenue,
Olson
Pavilion
#8300,
Chicago,
IL 60611.
3 To
whom
requests
for reprints
should
be addressed,
at Department
of
Biology,
Gilmer
Hall,
University
of Virginia,
Charlottesville,
VA 22903.
Phone:
(804) 982-581
5; Fax: (804) 982-5626,
e-mail:
[email protected].
and adipocyte
differentiation.
between
we have charactenized the expression
of growth arrest-specific
( gas) and
growth
arrestand DNA
damage-inducible
(gadd)
genes
during
adipose
conversion
of 3T3-L1
cells. The gas genes
were isolated
by virtue of their preferential
accumulation
in
serum-starved
NIH-3T3
cells (5), whereas
the gadd family
was originally
described
as genes induced
in response
to
DNA damage
in Chinese
hamster
ovary cells (6). In general,
within
each family,
the gas and gadd
genes
have been
adipoblasts,
control
and phenotypic
the relationship
differentiation
to adipocytes.
We examined the
expression of growth arrest-specific
( gas) and growth
arrest- and DNA damage-inducible
(gadd)
genes as a
function
of 3T3-L1 growth arrest and adipocyte
development.
These growth arrest-associated
genes are
differentially
expressed throughout
adipocyte
development.
Some of the gas/gadd genes are
preferentially
expressed in a subset of growth arrest
states. In contrast,
gasl and gas3 are expressed in
and post-mitotic
adipocytes.
However,
in post-mitotic
adipocytes,
gasl and gas3 are induced in response to
nutrient deprivation,
not altered growth status. ga
is
growth
control
understand
development,
,
tion of the medium,
an expression
profile
reported
previously for gaddl 53/CHOP
(9). gas6 mRNA is exceptional
by
virtue
of its accumulation
in growing
cells.
Specifically,
gas6 accumulates
in response to the addition
of adipogenic
hormones
that stimulate
the postconfhuent
cells to reenter
the cell division
cycle.
The results
indicate
that growth
arrest-associated
genes
are targets
of multiple
signaling
pathways
and play diverse
Results
Expression
begin
roles in cellular
physiology.
of gas and gadd
Genes in 3T3-L1 Cells. To
growth
arrest states in 313-Li
cells, we
gas/gadd
mRNAs
are elevated
in ne-
characterizing
determined
whether
sponse to serum deprivation
of logarithmically
adipoblasts.
313-Li
cells are typically
propagated
calf serum
(i 0). We
and gaddmRNAs
examined
the expression
after switching
growing
in 10%
of several
cells to 0.2% serum.
gas
Fig. 1
shows the results of a typical
Northern
blot analysis.
Within
24 h incubation
in 0.2% serum,
gasi, gas3, and gaddls3
transcripts
are noticeably
induced.
gasl is undetectable
in
logarithmically
growing
cells and increases
throughout
the
5-day
period
examined.
gas3 mRNA
levels
are also elevated
in serum-starved
cells.
However,
unlike
gasi , gas3
mRNA
is detectable
in the logarithmically
growing
cells
4
The
abbreviation
used
is: C/EBPa,
CCAAT/enhancer
binding
protein
a.
1542
ga,/gadd
Expression
during
hrs
Adipose
Conversion
hrs
in 0.2%
0 2448120
#{149}‘.
a
gasl
-
.
.
a.
after
10%
12 24 365dy
gaS3.$
CHOP
(gaddl53)
.
‘
#{149}
..
$ !
.
.
.
-
.
. .
.
:.
.
,$
tubuhin
Fig. 1. Accumulation
01 gas and
gacid transcripts
in serum-starved
3T3-L1
adipoblasts.
Logarithmically
growing
3T3-L1
cells (1 x 10 in a 10-cm dish(
were seeded
into growth
media
containing
1 0/
calf serum,
and total RNA
was preparedi
24 h later (0 h in 0.2/
serum)
and 24, 48, and 120 h after
shifting
the cells to medium
containing
0.2/
serum.
Separately,
after 24 h in
0.2/
serum,
cells were
returned
to 1 0”/,, serum
for 1 2, 24, and 36 h to
stimulate
the cells to resume growth.
Total RNA was also prepared
from each
time point after the return to 10/
serum.
Note that the sample
labeled
0 h
after
10/,
is the same
RNA sample
labeled
24 h in 0.2%
serum.
RNA
prepared
from cells 5 days after seeding
and propagation
in 10/ serum (Sdy(
is shown
for comparison.
Ten j.tg of total RNA was separated
by formaldehy( l(’-,Ig,Iros(’
gel (‘((‘( frol)horPsis,
fransk’rr(’(l
to sol id support,
and prohe(1
svith 1 r,Idi )l,II)(’lI’(I
I ( I )NA tr,Ignl(’nt . I hI’ l)lot was soc ((‘ssiv(’ly
stripjx’d
.10(1 prol)t’(l
SOIl) ( I )NA’
of
1, ( 1 l( )P ( ga(I(ll
‘3 t(, and tuhulin.
the level
plateaus
after 24 h (Fig. 2). The 4add1 53
hon)ologue
CHOP
also exhibits
elevated
levels in response
lo serum
(k’privation,
but the induced
levels are lower than
the maximal
levels
seen it other growth
arrest points
in
3T3-L1 cells (below).
‘A/hen 24-h serum-deprived
cells are
SwiI(he(l
to 10/
serum,
niRNA
levels for all three transcripts exarnine(I
decline
to preincluction
levels within
12 h
(Fig. 1 ). The accumulation
of the as1
and ,‘as3 niRNAs
in
serun)-starve(l
adipoblasts
is similar
to their expression
profile in serum-deprived
NIH 3T3 cells. Specifically,
as1 and
14,1S3
niessages
(ire induced
in repne
to serum deprivatictfl
(113(1 (k’Cline
when growth-inhibite(l
cells are stimulated
to reenter
the cell division
cycle
(5). Two other
mRNAs,
LJS5 ,1t’Kl is6,
are also induced
in serum-deprived
adipoblasts (onlI)aral)le
to their induction
in serum-dlepnived
uibroblasts
(data not shown).
Specifically,
as5
induction
is
similar
to ,L4J51 , whereas
a.c6 exhibits
the least induction
niOt)g
the
genes
in serum-deprived
cells
(5). j,’ackl
mRNAs
have been shown
to be induced
in Chinese
hamster
ovary
(CHO)
(‘ellS in response
to several
growth
arrest
signals,
including
serum
deprivation
(7). Our results denionstrate
that the ,‘.idd1 .53 homologue
CHOP
is induced
during
3T3-L1 growth
arrest in response
to serum depnivation
(Fig.
1 ). Two
other
,‘add
transcripts,
add34
and
,‘,1(Jd45,
were not detected
(data not shown).
We next examined
the expression
of ,‘as/,’add
mRNAs
as
a function
of adipose
conversion
of 3T3-L1
cells. This developmental
transition
includes
several
separate
periods
of
niitotic growth
arrest (1 ). In the standard
differentiation
Jtl(l
k)gdnithniiCll
ly growing
3T3-L 1 adipoblasts
are
a post(’otlfluent
tiionolayer,
resu lting in growth
arrest
(lue to (ontact
inhibition.
The cells are then treated
with (lifferentiation
indllcers,
which
results in an increase
in
cell number
during
a period
of clonal
expansion
(1). The
differentiation
inducens
are subsequently
removed,
and the
cell number
again plateaus
and remains
constant
throughout phenotypic
differentiation,
the accumulation
of cytoplasmic
fat droplets.
We collected
RNA from 313-Li
cells
throughout
this developmental
transition
and analyzed
gas
and gadd expression
by Northern
blots. The results of that
analysis
are shown
in Fig. 2A. All ofthe
growth
arrest genes
examined,
except
gaddl 53, are detectable
in loganithmically growing,
subconfluent
adipoblasts.
gasi accumulates
to maximal
levels in the postconfluent,
day-0 cells, declines
during
the period
of clonal
expansion,
is induced
again on
day 3, and is expressed
at low levels during
phenotypic
differentiation
(days 4-8).
gas3 accumulates
on days 0 and
3, similar to gasi, but gas3 is also expressed at maximal
levels on alternate
days in the phenotypically
differentiated
cells (days 4-8).
gas5 exhibits
yet another
expression
pattern in that it accumulates
to maximal
levels on day 0 only.
Finally,
consistent
with
analyses
published
previously
of
gaddl 53 expression
(9), we find that CHOP
accumulates
to
maximal
levels on alternate
days in phenotypically
differentiated
cells. However,
we also detect
CHOP
expression
in 3T3-L1
cells that are growth
arrested
in response
to
contact
inhibition
(Fig. 2A, day 0). gas6 is a notable
exception to the general
observation
that the gas and gadd genes
are expressed
at growth
arrest points
during
adipocyte
development.
Specifically,
gas6 is maximally
expressed
on
days 1 and 2, coincident
with
the exposure
of cells
to
differentiation
inducens
and the period
of clonal
expansion.
We monitored
adipocyte
differentiation
by the appearance of fat droplets
(data not shown)
and the accumulation
of the 422 mRNA
that encodes
an intracellular
fatty acidbinding protein (1 1 ). The pattern of 422 expression
(Fig. 2A)
is similar
to that observed
previously
(i 1 ). To monitor
equivalent
loading
of RNA samples,
we have hybridized
the
same blots to detect two different
mRNAs
for normalization.
The pALl 5 cDNA
encodes
a mitochondnial
rihosomal
protein subunit5
whose
mRNA
abundance
is constant
from
days 0-8
(1 2). However,
we reproducibly
find
pALl 5
mRNA
to be less abundant
in semiconfluent
and confluent
cells (e.g., Fig. 2A). We have also probed
the blots to detect
a ubiquitously
expressed
tubulin
isoform
mRNA
(1 3). Tuhulin
mRNA
levels decline
throughout
phenotypic
differentiation
but are constant
at earlier
time points
(i4).
The
expression
profiles
of gasi , tas6,
,t,’as3, and gaddi 53 are
shown
graphically
for days 0-8
in Fig. 2B. mRNA
abundance is expressed
as a percentage
of the maximum
expression of each species
after normalization
to pALl 5. The top
graph contrasts
the profiles
of gasl and ,‘as6 during
clonal
expansion
(days 1 and 2), while
the bottom
graph
reveals
the coordinate
fluctuation
of ,t,’as3 and t,’addi 53 levels during days 5-8.
The mRNA
levels of gasi , as3,
and ,gas5 decline
during
clonal
expansion
(Fig. 2, CE). This reduction
in gas mRNA
levels
is coincident
with
the exposure
to differentiation
inducens
and the accompanying
increase
in cell number
(1).
In contrast
to the decline
in asi , gas3, and ,#{231}’asS
mRNA
on
days 1 and 2, gas6 mRNA
levels
rise dramatically
at the
same time (Fig. 2). To more
precisely
correlate
gas/gadd
expression
and mitotic
growth
arrest points throughout
ad.
ipogenesis,
we analyzed
3T3-Li
cells
for DNA
content
I)rotocol,
gnwti
to
,,
p. (rnelius
01(1 1). Lane,
I)(’rsonal
(oninlunRation.
Cell
5_
CE
DAYscc
0
12
Growth
& Differentiation
125
3
4
5
6
7
8
100
gasl
75
gas5
%
maximum
r#{149}#{149}tN#{248}a#{149}
gas3
25
-0--0 -
\
I
50
gust
gus6
J
gas6
0
.
CHOP
(gaddl
012345619
.
p
1 lb
53)
100
422
7
I-a--
gas3
#{149}1
CHOP
%
maximum
50
tubuhin
25\
o.-!#{176}’
pALI5
.
;
days
Fig. 2.
Expression
Profiles
ofgas
and gaddtranscripts
during
adipose
conversion
of3T3-L1
cells. A, total RNA was harvested
from 3T3-t.1
cells throughout
the
conversion
from ,idiI)ol)lasts
to adipocytes
using a standard
protocol
(10(. Beginning
with a semiconfluent
sc)
population
of adipoblasts,
the cells were grown
to confluence
((1, incubated
48 h postconf(uence
(day 0(. exposed
to differentiation
inducers
for 48 h (days 1 and 2(, and refed differentiation
media
every 48
h thereafter
((lays 3-8). The periods
of growth,
including
logarithmic
growth
(LG) and clonal
expansion
(CE( are indicated.
Ten g of total RNA was analyzed
at each stel) l)y Northern
blot analysis
as in Fig. 1 . Membranes
were successively
probed,
stripped,
and reprobed
with cDNAs
of gasl
gas3, gas5, gash, or CHOP
( gaddl
5fl and 422 (encoding
a fatty
acid-binding
protein),
tuhulin,
and pALl S (a ribosomal
subunit
encoding
mRNA(.
Each blot was analyzed
for tuhulin
and
pALl 5 expression
to normalize
the expression
profiles.
B, the relative
abundance
of each transcript
was determined
by quantification
of the hand
intensity
(fniageQuant
software,
Molecular
Dynamics(;
subsequent
normalization
to the pAL15
signal was obtained
for the same sample
on the same membrane.
The
,
sample
expressing
a given
mRNA
at the highest
level
on days
0-8
was
set at 100%,
throughout
the developmental
transition.
Fig. 3 shows
the
result
of a flow
cytometnic
analysis
of cells harvested
at
various
time
points
throughout
adipocyte
conversion
of
3T3-L1
cells. The resulting
area graph reveals
that the adipoblast
are an asynchronous
population
at semiconfluence
that growth
arrest in G1 as they progress
from confluence
to
postconfluence
(day 0). The cells reenter
the mitotic
division
cycle
in response
to the addition
of differentiation
inducers,
as evidenced
by the decline
in the percentage
of
cells in G.
Twenty-four
h after the withdrawal
of adipogenic hormones
(day 3), the distribution
is similar
to that of
the contact-inhibited
cells (day 0) and remains
so throughout phenotypic
differentiation.
Combined
with the results
presented
in Fig. 2, the flow cytometric
analysis
reveals that
the induction
of gasl,
gas3,
and gaddls3
coincide
with
exits
from
the mitotic
division
cycle,
both
in response
to contact
inhibition
and the removal
of differentiation
i nducers.
Regulation
of gas and gadd Genes in Post-Mitotic
Adipocytes.
We noticed
that gas3 accumulates
to maximal
levels on alternate
days in post-mitotic,
phenotypically
differentiated
3T3-L1
adipocytes
(Fig. 2), an expression
pattern
shared
by CHOP/gaddl53
(Ref. 9 and Fig. 2). gasi
levels
also fluctuate
during
this period
(Fig. 2). It has been shown
that
maximal
expression
of gaddl 53 coincides
with
and
all other
samples
are reported
relative
to that
amount.
medium
replenishment
of cultured
adipocytes
and is likely
a consequence
of nutrient
deprivation
(9). Canlson
et a!. (9)
demonstrated
that
frequent
med ium
replenishment
throughout
phenotypic
differentiation
abrogates
the alternate day fluctuations
in gaddl 53 expression.
We wondered
whether
gas3
and gasl
are also regulated
by nutrient
changes
in post-mitotic
adipocytes.
To test this possibility,
we differentiated
3T3-L1
cells with
frequent
medium
replenishment
according
to Carlson
et a!. (9). Specifically,
313-Li
cells
were
differentiated
as above
except,
after
switching
cells to differentiation
medium
on day 2, the
medium
was replenished
every 8 h instead of every 48 h as
in the standard
protocol.
RNA was prepared
for Northern
analysis
from cells throughout
the modified
differentiation.
A Northern
blot analysis
of gasi
and gas3,
along
with
gaddl 53, throughout
adipose
conversion
with frequent
medium replenishment
is shown
in Fig. 4A. Quantification
of
the mRNA
levels after normalization
to pALl 5 reveals
that
CHOP
(gaddls3)
is not as abundant
in these adipocytes
(compare
day 8 in Fig. 4B versus
Fig. 2B) non do the levels
fluctuate
throughout
phenotypic
differentiation
(Fig. 4B), in
agreementwith
Carlson
eta/. (9). Furthermore,
a coordinate
change
in the expression
of gas3 and gasi
along
with
CHOP
is evident
in the cells differentiated
with frequent
medium
neplenishments
(Fig. 4). Thus,
gas3
and gasi
1543
1544
gas/gadd
Expression
(luring
Adipose
Conversion
I 00.
A
75,
DAYsc
gasl
50
0
C
0
1
2
0
3
4
i
5
6
7
8
.,.
#{149}
gas3
25
0
,
CHOP
I-
I
I
I
I
I
Sc
c
0
0.5
1
1.5
2
2.5
3
4
D%S
tubuhin
m
‘1O..
422
DAYS
[ D%G1
“
Ia
II%G2+M
Fig.
1.
Flow
cytometric
analysis
of DNA
content
during
adipogenesis.
No-
dci were prepared
from 3T3-L1
cells at the time points
described
in Fig. 2
and a(Iditionally
at half-day
time points from days 0.5 to 2.5. Becton
Dickson
Cell Fit software
was used to calculate
the percentage
of cells in G,, S, and
G-M
phases of the cell cycle,
and the resulting
data were plotted
using the
Cricket
Graph.
The initial
exit from the cell cycle
in response
to contact
inhibition
(day 0), reentry
in response
to the presence
of adipogenic
horniones
(days 0-2).
and the final
exit from
the cell cycle
after hormone
withdrawal
(day 3( are all evident
in the profile.
The profile
is unchanged
from days 4-8
(data not shown).
The sottware
assigns
any cells with
a
staining
intensity
between
the 2N and 4N median
as S-phase
cells.
The
(TI-Li
line has variable
ploidy,
with
the reported
chromosome
number
ranging
from 28-i
44, and concentrated
from 30-70
(American
Type Culture
Collection(.
The small population
of cells reported
as S phase on days 0 and
(-8
are likely
cells in G with large chromosomal
content.
appear
vation
to be regulated
both in response
to nutrient
depniin post-mitotic
adipocytes
(Fig. 3) and in response
to
exit
from
the mitotic
division
cycle through
serum stanvation (Fig. 1 ) or contact
inhibition
(Fig. 2) of adipoblasts.
We noticed
a slight lag in the kinetics
of differentiation
by
morphological
criteria
in cultures
that were differentiated
with frequent
medium
replenishment
compared
to the standand protocol
(data not shown).
The lag is evident
in the
Northern
blot analysis
in that plateau
of the 422 mRNA
levels by day 3 in the standard
protocol
(Fig. 2A) but not
until day 4 with frequent
medium
replenishment
(Fig. 4A).
The finding
prompted
us to determine
whether
there are
differences
in the final exit from the cell division
cycle for
adipocytes
differentiated
with frequent
medium
replenishments compared
to the standard
protocol.
To evaluate
that
possibility,
we differentiated
313-Li
cells in parallel
by the
standard
protocol
or with frequent
medium
replenishments
and monitored
DNA content
by flow cytometry
throughout
the differentiation
process.
The result of that analysis
are
presented
in Fig. 5. Medium
neplenishments
began on day
2 when the differentiation
inducers
were removed
(1) and
continued
at 48-h (standard,
S) or 8-h intervals
(frequent,
FMR). Comparison
of the area graphs
in Fig. 5 reveals
no
significant
differences
in DNA
content
at various
times
throughout
adipogenesis.
Specifically,
by day 3, 24 h
after the removal
of differentiation
inducens,
both populations
are comprised
almost
exclusively
of cells with a
G DNA content,
and both remain
so throughout
phenotypic
differentiation.
pahl5
B
*
gasl
-0-
100
%
gas3
#{149}1
maximum
-0
-
CHOP
25
;
days
Fig. 4.
Coordinate
regulation
CHOP
( gaddi 53) gas3 and gasi
in postmitotic
adipocytes.
3T3-L1
cells were differentiated
to adipocytes
with frequent media
replenishments
after day 2 to maintain
elevated
nutrient
levels
in the media
(9). A, total RNA was harvested
from cells throughout
adipogenesis,
and 10 g were analyzed
by Northern
blot as described
in Fig. 1.
The accumulation
of 422 mRNA
indicates
the progression
of phenotypic
differentiation.
Note that the tubulin
probe reveals that the semiconfluent
(sd
and day 1 samples
are overloaded
with respect
to other samples.
B, relative
abundance
was determined
and plotted
as in Fig. 2B.
gas/gadd Genes Examined
Are Not Targets of C/EBPa
in
Differentiated
Adipocytes.
C/EBPa
transactivates
numenous adipocyte-specific
genes (3) and is essential
for adipose
conversion
of 313-Li
(2). We have shown
previously
that
temporal
misexpression
of C/EBPa
in undifferentiated
adipoblasts
results in a cessation
of mitotic
growth
(4). Specifically,
we expressed
a steroid
hormone-regulated
fusion
protein
of C/EBPa and the estrogen
receptor
hormone-binding domain,
C/EBP-ER,
in 3T3-Li
cells and observed
estrogen-dependent
growth
arrest. We have used this cell line to
ask whether
any of the gas on gadd genes expressed
in
differentiated
adipocytes
are regulated
by C/EBPa.
The
C/EBP-ER-expnessing
3T3-L1
cell line was differentiated
in
Cell
Standard
Differentiation
DAYcOI2
Growth
& Differentiation
4
6
I 00
gasl
75
gas3
1U#{149}#{149}#{149}
50
CHOP
(gadd
25
0
153)
tbl’422
Frequent
Media
.0.1.
Replenishment
I 00
UUlfl
75
pALI5
50
25
Fig. 6.
Expression
of growth
arrest-associated
transcripts
in adipocytes
cxpressing
a steroid
hormone-regulated
form of C/EBPr.
An adipoblast
cell line
expressing
an estrogen-regulated
form of C/EBPa,
C/EBP-ER
(4), was differentiated
as described
previously,
except
that estrogen
(+( or ethanol
vehicle
(-(
was present
in the differentiation
media
from day 2. Total
RNA was
harvested
from C/EBP-ER-expressing
cells at confluence
(c(, and days 0, 1,
and 2 (prior to addition
of estrogen)
as well as days 4 and 6 from cells in the
presence
or absence
of estrogen
since day 2. Ten jig of total RNA were
0
DAYS
D%G1
D%S
analyzed
mRNA
apparent
in
by Northern
blot analysis.
the presence
of estrogen
on days 4 and 6.
The preferential
as opposed
to
accumulation
its absence
of 422
is readily
#{149}%G2+M
Fig. 5.
Frequent
media
replenishment
during
phenotypic
differentiation
does not alter the exit from the mitotic
division
cycle.
Nuclei
were prepared
from
cells differentiated
using the standard
protocol
or with frequent
media
replenishment,
stained
with propidium
iodide,
and analyzed
by flow cytometry; the resulting
data were plotted
as described
in Fig. 3. The profiles
of
both sanples
are levels from clays 4-8
(data not shown(.
Note that 0.5, 1.5,
and 2.5 of the standard
differentiation
are the same data shown
in Fig. 3.
the presence
or absence
of estrogen
throughout
phenotypic
differentiation.
RNA was harvested
throughout
differentiation, and the expression
of the gas and gadd genes was
quantified
by Northern
blot analysis.
The results are presented in Fig. 6. In agreement
with our previous
findings
(4),
422, a phenotype-associated
target gene of C/EBPa,
is elevated
on days 4 and 6 in the presence
of estrogen
as
compared
to its absence.
However,
none of the gas/gadd
genes expressed
in post-mitotic
adipocytes
(Fig. 2) accumulate to greater levels in response
to estrogen
stimulation.
We
conclude
that the gas/gadd
genes examined
are not targets
of C/EBPa
regulation
in post-mitotic
adipocytes.
The expression
profiles
of the gas and gadd genes are notably
different in the C/EBP-ER
cells compared
to the parental
3T3-L3
cells (Fig. 2). These differences
can most likely
be
attributed
to the low efficiency
of differentiation
for the
C/EBP-ER
cells as determined
by the number
of cells in the
population
containing
fat droplets
visible
by phase contrast
microscopy
(2-S%;
data not shown).
For example,
these
cells do not exhibit
reduced
tubulin
levels as seen for the
parental
3T3-Li
cells (compare
Figs. 6 and 2). We reason
that the cells do not significantly
deplete
the media and thus
do not significantly
induce
CHOP
( gaddi 53). Furthermore,
the majority
ofthe
cells on the plate are not phenotypically
differentiated
and may be in a growth-arrest
state companable to day 0 on 3 of 313-Li
differentiation,
characterized
by elevated
levels of gasi and gas3 (Fig. 2).
Discussion
The results presented
here demonstrate
the uncoordinated
expression
of growth
arrest-associated
genes during
a discrete
developmental
transition:
adipose
conversion
of
313-Li
cells.
Differential
induction
during
multiple
exits
from the mitotic
division
cycle combined
with differential
expression
in post-mitotic
adipocytes
accounts
for most of
the variance.
gas genes were discovered
by virtue of their
coordinate
accumulation
in serum-starved
NIH-3T3
cells
(5). In adipoblasts,
the gas genes examined
are coordinately
induced
in response
to serum deprivation,
contact
inhibition, and the initiation
of differentiation
(with the exception
ofgas6).
However,
in post-mitotic
adipocytes,
expression
of
the gas genes varies. gasi and gas5 levels decline
relative
to
contact-inhibited
adipoblasts,
similar
to gas5 expression
in
differentiated
Friend
leukemic
cells (1 5), whereas
gas3 is
1545
1546
gas/gadd
Expression
during
Adipose
Conversion
expressed at high levels in response to nutrient deprivation.
gaddi 53 is also induced
by nutrient deprivation
of adipocytes
(9). To our knowledge,
this is the first observation
of
coordinate
regulation
of a gadd gene and a gas gene independent
of growth
control.
Perhaps
these genes cooperate
to inhibit
adipogenesis.
gaddi 53 (CHOP)
can heterodimer-
ize with
C/EBPa
and act as a repressor
of C/EBPa-dniven
gene transcription
(1 6). It has been suggested
that gaddl 53
accumulation
in response
to nutrient
deprivation
opposes
adipogenesis
by inhibiting
C/EBPa
stimulation
of adipocyte-specific
genes (6). We speculate
that the coordinate
regulation
of gas3 and gasi , which
encode
plasma
membrane proteins,
along with gaddi 53 (CHOP),
reflects a need
to coordinate
activities
within
the nucleus
and at the cell
membrane
to
inhibit
adipogenesis.
The
regulation
of
i 0% calf serum. For serum starvation,
cells were seeded at
a density of i05 cells/mI on a 10-cm dish with DMEM plus
i 0% calf serum
(CIBCO)
for 24 h then the media
changed
to DMEM
plus 0.2% calf serum.
Differentiation
was
of
313-Li
cells was as described
previously
(10). The stable
cell line expressing
C/EBP-ER was described
previously
(4).
For differentiation,
C/EBP-ER
cells were incubated
to 2 days
postconfluence
in DMEM
with 1 5% charcoal-stripped
calf
serum
(4), then switched
to DMEM
without
phenol
red
(CIBCO) and 10% fetal bovine serum (Hychone)
dexamethasone
(1 x i 0_6 M), isobutylmethylxanthine
ng/ml),
and
insulin
(1 00 ng/ml).
After
2 days,
containing
the media
(1 15
was
changed to DMEM without phenol red plus 1 5% charcoalstripped fetal bovine serum, insulin (100 ng/mh), and estrogen (i X 106
M), or ethanol
vehicle.
The frequent
media
gaddl 53, gas3, and gasi in response
to nutrient
deprivation
(Fig. 4) seems to be distinct
from gaddi53
stimulation in
response
to genomic
stress (7) on gasi and gas3 induction
in
response
to serum deprivation
and contact
inhibition
(Figs.
1 and 2; Ref. 5). These regulatory
properties
suggest
that
gaddi 53 and gas3 may be targets
of distinct
signaling
mechanisms
in post-mitotic
cells versus growing
cells.
gas6 expression
is a notable
exception
to the general
finding
that gas and gadd genes are expressed
at growtharrest states during
adipogenesis.
gas6 accumulates
to maximal
levels
during
the time that adipoblasts
reenter
the
mitotic
division
cycle in response
to differentiation
inducens. Recent characterization
of the gas6 gene product
suggests potential roles for this protein in overcoming
contactinhibited
growth
arrest. The gas6 protein
is a higand for the
ax! tyrosine
kinase growth
factor receptor
(i 7). In addition,
gas6 is a secreted
protein
related to human
protein
S (i 8), a
component
of a protease
cascade
that negatively
regulates
replenishment
the modified
a!. (9). RNA
blood
(1 8) was amplified
by PCR using a sense primer
5’-CCTCCTCCACTCCACCATCTT-3’
(nucheotides
1 81 1 to 1832)
and
an antisense
primer
5’-ACCCTCCTCGACTCTTC-
coagulation.
postconfluent
asone,
Our
preliminary
adipoblasts,
gas6
one of the adipogenic
results
is regulated
hormones.6
reveal
that
in
by dexameth-
We speculate
that
gas6 plays an important
role in initiating
the cascade
of
events that result in adipose
conversion
of 313-Li
cells.
We began our characterization
of the gas and gadd genes
presuming
to identify
targets of C/EBPa stimulation.
C/EBPa
is required
for adipocyte
differentiation
(2) and induces
mitotic
growth
arrest when
prematurely
expressed
in adipoblasts
(4). However,
none of the gas/gadd
genes exammed here are stimulated
by C/EBPa
(Fig. 6). The expression
patterns
ofthe gas/gaddgenes
during adipogenesis
provides
a likely explanation
for this observation.
gasi , gas5, and
gas6 are not expressed
at maximal
levels in phenotypically
differentiated
adipocytes
(Fig. 2), the time that C/EBPa
accumulates
(i 9). gas3 and CHOP
( gaddi 53) are expressed
in
differentiated
adipocytes
(Fig. 2) but likely antagonize
adipogenesis
(Ref. 9 and this report),
whereas
C/EBPa
stimuhates adipogenesis
(3, 19). It seems
unlikely
that C/EBPa
would
stimulate
the transcription
of genes involved
in an
opposing
metabolic
response.
We are conducting
molecuIan screens to identify
novel growth
arrest-associated
genes
that
are targets
of C/EBPa
stimulation
in post-mitotic
adipocytes.
Materials
Tissue
lection)
313-Li
maintained
R. M.
Umek,
to
et
‘
tially
denatured
94#{176}C
for 45
at 94#{176}C
for 2 mm,
5,
followed
by 35 cycles
45#{176}C
for 45 s, and 72#{176}C
for 2 mm.
was constructed
by subcloning
the gas3 fragment
Xhol site of the Bluescniptll
KS plasmid
(pBS). gas5
amplified
by PCR using
a sense
primer
of
pBSgas3
into the
(1 5) was
5’-ACCCTTTCC-
CATCCTCTCCCCCAT-3’
(nucleotides
i to 23) and an antisense
primer
TTT’
(nucheotides
425 to 446), which
yielded
a 446-hp
fragment.
The gas5 fragment
was subchoned
into pBS at the BamHl
site to give the plasmid
CACCA-3’
temperature
pBSgas5.
A 482-hp
fragment
of gas6
(nucleotides
2771 to 2292) with the annealing
at 55#{176}C.
pBSgas6 was constructed
by subclon-
ing the gas6 fragment
into the XhoI
fragments
were amplified
from cDNA
using the cDNA
cycle Kit (Invitrogen)
from 313-Li
cells grown
in DMEM
with
site of pBS. The gas
that was synthesized
from mRNA
isolated
0.2% calf serum for
72 h. A fragment
of CHOP
cDNA
(i 6) corresponding
to bp
2-772
was amplified
by PCR using
a sense primer
5’CCCAATTCCACAACCAACTCCAT-3
‘
(nucleotides
3 to
26) and an antisense
primer
5’-CCCAATTCACTTTACTC-
CACATC-3’
sized from
(nucleotides
mRNA pooled
751 to 772) from cDNA
from days 6, 8, 9, and
synthei 0 of a
standard
differentiation
of 313-Li
cells. pBSCH-i0full
was
constructed
by subchoning
the CHOP
fragment
into the
EcoRl site in pBS. gasi
(5) cDNA
was provided
by L.
Phihipson
(European
Molecular
Biology
Laboratory,
Heildehberg,
Cermany).
gadd4s
cDNA was provided
by N.
Holbrook
(National
636-hp 422 cDNA
Hopkins
Institute on Aging, Baltimore,
MD). The
(1 1 ) was provided
by M. D. Lane (Johns
University,
buhin cDNA
cells
in
(American
DMEM
Type Culture
supplemented
Colwith
Baltimore,
(1 3) was provided
MD),
and
the
1 600-hp
by D. Cleveland
pALi5
cDNA
(12). cDNA
probes
[32
P]dCTP accordi ng to manufacturer’s
using
tu-
(John Hop-
unpublished
results.
ported
were
labeled
with
recommendations
a random-primed
phosphonimager
and
according
by Carlson
was collected
as described
previously
(20).
DNA Probes. An 1 1 08-hp
fragment
of gas3 (21 ) was
amplified
by reverse transcniption-PCR
using a sense primer
5 ‘-CTCCACCCACTCCACTTTCTC-3
(nucleotides
79 to
99) and an antisense
primer 5’-CTCCATACTCCACCTAAATCCAC-3’
(nucleotides
1i66 to i187). cDNA was mi-
Northern
6 E. C. Shugart
conducted
described
kins University,
Baltimore,
MD). P. Cornelius
(John Hopkins
University,
Baltimore,
MD) and M. D. Lane provided
the
and Methods
Culture.
were
differentiation
was
313-Li
differentiation
Blot
labeling
kit (Pharmacia).
Images
were obtained
(Molecular
Dynamics
or Fuji)
Imaging.
to Photoshop
(Adobe)
as 8-bit
TIFF files.
with
a
and ex-
The
im-
Cell
ported
TIFF files were adjusted
for brightness
and contrast
automatically,
despeckled
once,
and blurred
once.
The
enhanced
images
were saved as TIFF files, imported
into
Canvas
(Deneba)
to be decorated
with text, and printed
on
a dye-sublimation
printer.
Original
unmolested
8-bit TIFF
files were archived.
Flow Cytometry
Analysis. Cells from a 1 0-cm dish were
scraped
into 1 0 ml of PBS, pelleted
in a centrifuge
(1 500 X
gfor 3 mm), resuspended
in 1 .5 ml PBS, and stored in liquid
nitrogen.
For analysis,
cells were thawed,
pelleted
at room
temperature
(7000
x g 30 5), and resuspended
in 1 .5 ml
propidium
iodide staining solution (0.1% sodium
citrate,
0.3% NP4O, 100 j.tml
RNase A, and 50 Wml
propidium
iodide). The nuclei were incubated in staining solution for
30 mm on ice. The stained
cells were analyzed
in a Becton
Dickinson
FACScan
analyzer
using
Cell Fit software.
A
particle
size gate was defined
using area versus width, and
2 X 1
gated events were scored
for each sample.
Acknowledgments
We thank
Lennart
Philipson,
Nikki
Holbrook,
viding
plasmids.
We are also grateful
to Daniel
the manuscript
and valuable
discussions.
and M. Daniel
Lane for proBurke for critical
reading
of
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