Differential Expression of gas and gadd Genes at

Vol. 6, 1541-1547, December 1995
Cell Growth & Differentiation
Differential Expression of gas and gadd Genes at Distinct
Growth Arrest Points during Adipocyte Development '
Erika C. Shugart, Anait S . Levenson,' Cara M. Constance ,
and Robert M . Umek3
Department of Biology, University of Virginia, Charlottesville, Virgini a
22903
Abstract
The characterization of growth arrest-associated gene s
has revealed that cells actively suppress mitotic growt h
in response to extracellular signals . Mouse 3T3-L1 cells
growth arrest at multiple distinct points during termina l
differentiation to adipocytes. We examined th e
expression of growth arrest-specific ( gas) and growt h
arrest- and DNA damage-inducible (gadd) genes as a
function of 3T3-L1 growth arrest and adipocyt e
development. These growth arrest-associated genes ar e
differentially expressed throughout adipocyte
development . Some of the gas/gadd genes are
preferentially expressed in a subset of growth arres t
states. In contrast, gasl and gas3 are expressed i n
serum-starved adipoblasts, contact-inhibited adipoblasts ,
and post-mitotic adipocytes . However, in post-mitoti c
adipocytes, gasl and gas3 are induced in response t o
nutrient deprivation, not altered growth status . gash i s
an exception to the general concordance of mitoti c
growth arrest and gas/gadd expression in that gas6 i s
preferentially expressed during the clonal expansion o f
postconfluent adipoblasts. Combined, the expressio n
patterns indicate that growth arrest-associated genes ar e
regulated by numerous signal transduction pathways
throughout a discrete developmental transition .
Introductio n
Adipose conversion of cultured 3T3-L1 cells provides a
model system for examining the relationship between differentiation and cellular growth control . In the standard
differentiation protocol, adipoblasts are cultured to post confluence, exposed to adipogenic hormones to initiat e
differentiation, and then maintained in differentiation media to promote the accumulation of cytoplasmic fat droplet s
(reviewed in Ref . 1) . The addition of the adipogenic hormones stimulates the growth-arrested, postconfluent cells t o
reenter the mitotic cell cycle, which the cells exit agai n
upon withdrawal of the hormones . Thus, a discrete perio d
of mitotic division separates postconfluent adipoblasts fro m
phenotypically differentiated, postmitotic adipocytes . To
Received 8/14/95 ; revised 9/22/95 ; accepted 9/27/95 .
This work was supported by Grant BE-183 from the American Cance r
Society (to R . M . U .) .
z Present address : Robert H . Lurie Cancer Center, Northwestern University
Medical School, 303 East Chicago Avenue, Olson Pavilion #8300, Chicago ,
IL 60611 .
s To whom requests for reprints should be addressed, at Department o f
Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903 .
Phone : (804) 982-5815 ; Fax : (804) 982-5626, e-mail : rmu4b@virginia .edu .
1
differentiate, 3T3-L1 cells require the transcription facto r
C/EBPa4 (2), which has been shown to trans-activate th e
promoters of several adipocyte-specific genes (3) . Further more, forced expression of C/EBPa in logarithmically growing adipoblasts results in mitotic growth arrest independen t
of adipogenesis, whereas its premature expression after exposure to adipogenic hormones hastens differentiation (4) .
These observations lead us to speculate that C/EBPa may b e
a component of a signal transduction pathway that coordinates mitotic growth control and phenotypic differentiation .
To better understand the relationship between cellula r
growth control and adipocyte development, we have characterized the expression of growth arrest-specific (gas) an d
growth arrest- and DNA damage-inducible (gadd) gene s
during adipose conversion of 3T3-L1 cells . The gas gene s
were isolated by virtue of their preferential accumulation i n
serum-starved NIH-3T3 cells (5), whereas the gadd famil y
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
observed to be coordinately induced at growth arrest (5-8) .
We report here that the gas/gadd genes are differentiall y
expressed throughout adipocyte development . Certain of
these growth arrest-associated genes, such as gas5, ar e
induced by serum starvation and contact inhibition . Others,
including gasl, gas3, and gaddl 53, are expressed in serum starved adipoblasts, contact-inhibited adipoblasts, and i n
postmitotic adipocytes . In differentiated cells, gasl an d
gas3 appear to be induced in response to nutrient deprivation 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 adipogeni c
hormones that stimulate the postconfluent cells to reente r
the cell division cycle . The results indicate that growt h
arrest-associated genes are targets of multiple signalin g
pathways and play diverse roles in cellular physiology .
Result s
Expression of gas and gadd Genes in 3T3-L1 Cells . To
begin characterizing growth arrest states in 3T3-L1 cells, w e
determined whether gas/gadd mRNAs are elevated in response to serum deprivation of logarithmically growin g
adipoblasts. 3T3-L1 cells are typically propagated in 10 %
calf serum (10) . We examined the expression of several ga s
and gadd mRNAs after switching cells to 0 .2% serum . Fig . 1
shows the results of a typical Northern blot analysis . Withi n
24 h incubation in 0 .2% serum, gasl, gas3, and gadd15 3
transcripts are noticeably induced . gasl is undetectable i n
logarithmically growing cells and increases throughout th e
5-day period examined . gas3 mRNA levels are also elevated in serum-starved cells . However, unlike gasl , gas3
mRNA is detectable in the logarithmically growing cell s
4 The abbreviation used is : C/EBPa, CCAAT/enhancer binding protein a .
154 1
1542
gas/gadd Expression during Adipose Conversion
hrs in 0 .2 %
0 24 4812 0
hrs after 10 %
0 12 24 36 5dy
tubulin
Fig . 1 . Accumulation of gas and gadd transcripts in serum-starved 3T3-L 1
adipoblasts . Logarithmically growing 3T3-L1 cells (1 x 10 s in a 10-cm dish )
were seeded into growth media containing 10% calf serum, and total RN A
was prepared 24 h later (0 h in 0 .2% serum) and 24, 48, and 120 h afte r
shifting the cells to medium containing 0 .2% serum . Separately, after 24 h i n
0.2% serum, cells were returned to 10% serum for 12, 24, and 36 h t o
stimulate the cells to resume growth . Total RNA was also prepared from eac h
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 . RN A
prepared from cells 5 days after seeding and propagation in 10% serum (5dy)
is shown for comparison . Ten µg of total RNA was separated by formaldehyde-agarose gel electrophoresis, transferred to solid support, and probe d
with a radiolabeled gasl cDNA fragment . The blot was successively strippe d
and probed with cDNAs of gas3, CHOP (gadd153), and tubulin .
and the level plateaus after 24 h (Fig . 2) . The gaddl 5 3
homologue CHOP also exhibits elevated levels in respons e
to serum deprivation, but the induced levels are lower tha n
the maximal levels seen at other growth arrest points i n
3T3-L1 cells (below) . When 24-h serum-deprived cells ar e
switched to 10% serum, mRNA levels for all three transcripts examined decline to preinduction levels within 12 h
(Fig . 1) . The accumulation of the gasl and gas3 mRNAs i n
serum-starved adipoblasts is similar to their expression pro file in serum-deprived NIH 3T3 cells . Specifically, gasl an d
gas3 messages are induced in response to serum depriva tion and decline when growth-inhibited cells are stimulate d
to reenter the cell division cycle (5) . Two other mRNAs ,
gas5 and gas6, are also induced in serum-deprived adipoblasts comparable to their induction in serum-deprived fibroblasts (data not shown) . Specifically, gas5 induction i s
similar to gasl, whereas gas6 exhibits the least inductio n
among the gas genes in serum-deprived cells (5) . gadd
mRNAs have been shown to be induced in Chinese hamste r
ovary (CHO) cells in response to several growth arres t
signals, including serum deprivation (7) . Our results demonstrate that the gadd153 homologue CHOP is induced
during 3T3-L1 growth arrest in response to serum deprivation (Fig . 1) . Two other gadd transcripts, gadd34 an d
gadd45, were not detected (data not shown) .
We next examined the expression of gas/gadd mRNAs a s
a function of adipose conversion of 3T3-L1 cells . This developmental transition includes several separate periods o f
mitotic growth arrest (1) . In the standard differentiatio n
protocol, logarithmically growing 3T3-L1 adipoblasts ar e
grown to a postconfluent monolayer, resulting in growt h
arrest due to contact inhibition . The cells are then treated
with differentiation inducers, which results in an increase in
cell number during a period of clonal expansion (1) . Th e
differentiation inducers are subsequently removed, and th e
cell number again plateaus and remains constant through out phenotypic differentiation, the accumulation of cytoplasmic fat droplets . We collected RNA from 3T3-L1 cell s
throughout this developmental transition and analyzed ga s
and gadd expression by Northern blots . The results of tha t
analysis are shown in Fig . 2A . All of the growth arrest gene s
examined, except gaddl 53, are detectable in logarithmically growing, subconfluent adipoblasts . gasl accumulate s
to maximal levels in the postconfluent, day-0 cells, decline s
during the period of clonal expansion, is induced again o n
day 3, and is expressed at low levels during phenotypi c
differentiation (days 4—8) . gas3 accumulates on days 0 an d
3, similar to gasl, but gas3 is also expressed at maxima l
levels on alternate days in the phenotypically differentiate d
cells (days 4—8) . gas5 exhibits yet another expression pat tern in that it accumulates to maximal levels on day 0 only .
Finally, consistent with analyses published previously o f
gaddl 53 expression (9), we find that CHOP accumulates to
maximal levels on alternate days in phenotypically differentiated cells . However, we also detect CHOP expressio n
in 3T3-L1 cells that are growth arrested in response to
contact inhibition (Fig . 2A, day 0) . gas6 is a notable excep tion to the general observation that the gas and gadd gene s
are expressed at growth arrest points during adipocyte development . Specifically, gas6 is maximally expressed o n
days 1 and 2, coincident with the exposure of cells t o
differentiation inducers and the period of clonal expansion .
We monitored adipocyte differentiation by the appearance of fat droplets (data not shown) and the accumulatio n
of the 422 mRNA that encodes an intracellular fatty acid binding protein (11) . The pattern of 422 expression (Fig . 2A )
is similar to that observed previously (11) . To monito r
equivalent loading of RNA samples, we have hybridized th e
same blots to detect two different mRNAs for normalization .
The pAL15 cDNA encodes a mitochondrial ribosomal protein subunits whose mRNA abundance is constant fro m
days 0—8 (12) . However, we reproducibly find pAL1 5
mRNA to be less abundant in semiconfluent and confluen t
cells (e .g., Fig . 2A) . We have also probed the blots to detec t
a ubiquitously expressed tubulin isoform mRNA (13) . Tubulin mRNA levels decline throughout phenotypic differentiation but are constant at earlier time points (14) . Th e
expression profiles of gasl, gas6, gas3, and gadd153 ar e
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 pAL15 . The to p
graph contrasts the profiles of gasl and gas6 during clona l
expansion (days 1 and 2), while the bottom graph reveal s
the coordinate fluctuation of gas3 and gaddl 53 levels during days 5–8 .
The mRNA levels of gasl , gas3, and gas5 decline durin g
clonal expansion (Fig . 2, CE) . This reduction in gas mRN A
levels is coincident with the exposure to differentiatio n
inducers and the accompanying increase in cell number (1) .
In contrast to the decline in gasl , gas3, and gas5 mRNA o n
days 1 and 2, gas6 mRNA levels rise dramatically at th e
same time (Fig . 2) . To more precisely correlate gas/gadd
expression and mitotic growth arrest points throughout adipogenesis, we analyzed 3T3-L1 cells for DNA conten t
5
P. Cornelius and D . Lane, personal communication .
Cell Growth & Differentiation
LG
CE
DAY sc c 0 1 2 3 4 5 6
7
8
gas 1
—o-- gas l
gas 5
- -o — gush
gas 3
gas 6
0 -
1
I
8 1 2 3 4 5 6 7
CHO P
8
(gaddl 53)
42 2
gas3
%
CHOP
maximu m
tubuli n
pAL15
days
Fig . 2. Expression profiles of gas and gadd transcripts during adipose conversion of 3T3-L1 cells . A, total RNA was harvested from 3T3-L1 cells throughout th e
conversion from adipoblasts to adipocytes using a standard protocol (10) . Beginning with a semiconfluent (sc) population of adipoblasts, the cells were grown
to confluence (c), incubated 48 h postconfluence (day 0), exposed to differentiation inducers for 48 h (days 1 and 2), and refed differentiation media every 4 8
h thereafter (days 3–8) . The periods of growth, including logarithmic growth (LG) and clonal expansion (CE) are indicated . Ten µg of total RNA was analyzed
at each step by Northern blot analysis as in Fig . 1 . Membranes were successively probed, stripped, and reprobed with cDNAs of gas .] , gas3, gas5, gas6, or CHO P
(pc/di 53) and 422 (encoding a fatty acid-binding protein), tubulin, and pAL15 (a ribosomal subunit encoding mRNA) . Each blot was analyzed for tubulin an d
pAL15 expression to normalize the expression profiles. B, the relative abundance of each transcript was determined by quantification of the band intensit y
(ImageQuant 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%, and all other samples are reported relative to that amount .
throughout the developmental transition . Fig . 3 shows th e
result of a flow cytometric analysis of cells harvested a t
various time points throughout adipocyte conversion o f
3T3-L1 cells . The resulting area graph reveals that the adipoblast are an asynchronous population at semiconfluenc e
that growth arrest in G, as they progress from confluence t o
postconfluence (day 0) . The cells reenter the mitotic division cycle in response to the addition of differentiatio n
inducers, as evidenced by the decline in the percentage o f
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 through out phenotypic differentiation . Combined with the results
presented in Fig . 2, the flow cytometric analysis reveals tha t
the induction of gasl, gas3, and gadd153 coincide wit h
exits from the mitotic division cycle, both in respons e
to contact inhibition and the removal of differentiatio n
inducers .
Regulation of gas and gadd Genes in Post-Mitotic Adipocytes. We noticed that gas3 accumulates to maxima l
levels on alternate days in post-mitotic, phenotypically differentiated 3T3-L1 adipocytes (Fig . 2), an expression patter n
shared by CHOP/gadd153 (Ref . 9 and Fig . 2) . gasl level s
also fluctuate during this period (Fig . 2) . It has been show n
that maximal expression of gaddl 53 coincides with
medium replenishment of cultured adipocytes and is likel y
a consequence of nutrient deprivation (9) . Carlson et al . (9 )
demonstrated that frequent medium replenishmen t
throughout phenotypic differentiation abrogates the alternate day fluctuations in gaddl 53 expression . We wondered
whether gas3 and gasl are also regulated by nutrien t
changes in post-mitotic adipocytes . To test this possibility ,
we differentiated 3T3-L1 cells with frequent medium replenishment according to Carlson et al . (9) . Specifically ,
3T3-L1 cells were differentiated as above except, afte r
switching cells to differentiation medium on day 2, th e
medium was replenished every 8 h instead of every 48 h a s
in the standard protocol . RNA was prepared for Norther n
analysis from cells throughout the modified differentiation .
A Northern blot analysis of gasl and gas3, along wit h
gaddl 53, throughout adipose conversion with frequent medium replenishment is shown in Fig . 4A . Quantification o f
the mRNA levels after normalization to pAL15 reveals tha t
CHOP (gadd153) is not as abundant in these adipocyte s
(compares day 8 in Fig . 48 versus Fig . 2B) nor do the level s
fluctuate throughout phenotypic differentiation (Fig . 4B), i n
agreement with Carlson et al. (9) . Furthermore, a coordinate
change in the expression of gas3 and gasl along wit h
CHOP is evident in the cells differentiated with frequen t
medium replenishments (Fig . 4) . Thus, gas3 and gasl
1543
1544
gas/gadd Expression during Adipose Conversio n
A
111111111
sc
c
0 0 .5 1
1 .5 2 2 .5 3
1
4
DAYS
%G1
O% S
n%G2+ M
Fig. 3 .
Flow cytometric analysis of DNA content during adipogenesis . Nuclei were prepared from 3T3-L1 cells at the time points described in Fig . 2
and additionally 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, an d
G 2 -M phases of the cell cycle, and the resulting data were plotted using th e
Cricket Graph . The initial exit from the cell cycle in response to contac t
inhibition (day 0), reentry in response to the presence of adipogenic hormones (days 0-2), and the final exit from the cell cycle after hormon e
withdrawal (day 3) are all evident in the profile . The profile is unchanged
from days 4-8 (data not shown) . The software assigns any cells with a
staining intensity between the 2N and 4N median as S-phase cells . The
3T3-L1 line has variable ploidy, with the reported chromosome numbe r
ranging from 28-144, and concentrated from 30-70 (American Type Cultur e
Collection) . The small population of cells reported as S phase on days 0 an d
3-8 are likely cells in G, with large chromosomal content .
appear to be regulated both in response to nutrient deprivation in post-mitotic adipocytes (Fig . 3) and in response t o
exit from the mitotic division cycle through serum starvation (Fig . 1) or contact inhibition (Fig . 2) of adipoblasts .
We noticed a slight lag in the kinetics of differentiation b y
morphological criteria in cultures that were differentiate d
with frequent medium replenishment compared to the standard protocol (data not shown) . The lag is evident in th e
Northern blot analysis in that plateau of the 422 mRN A
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 ar e
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 3T3-L1 cells in parallel by th e
standard protocol or with frequent medium replenishment s
and monitored DNA content by flow cytometry throughou t
the differentiation process . The result of that analysis are
presented in Fig . 5 . Medium replenishments began on da y
2 when the differentiation inducers were removed (1) an d
continued at 48-h (standard, S) or 8-h intervals (frequent ,
FMR) . Comparison of the area graphs in Fig . 5 reveals n o
significant differences in DNA content at various time s
throughout adipogenesis . Specifically, by day 3, 24 h
after the removal of differentiation inducers, both populations are comprised almost exclusively of cells with a
G, DNA content, and both remain so throughout phenotypic differentiation .
B
—~
gas l
-0-
gas3
—o — CHO P
25-
0
0
1
2 3
4
5 6 7
8
days
Fig . 4. Coordinate regulation CHOP (gadd153) gas3 and gasl in post mitotic adipocytes. 3T3-L1 cells were differentiated to adipocytes with frequent media replenishments after day 2 to maintain elevated nutrient level s
in the media (9) . A, total RNA was harvested from cells throughout adipogenesis, and 10 µg 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 (Sc )
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 i n
Differentiated Adipocytes. C/EBPa transactivates numer ous adipocyte-specific genes (3) and is essential for adipos e
conversion of 3T3-L1 (2) . We have shown previously tha t
temporal misexpression of C/EBP« in undifferentiated adipoblasts results in a cessation of mitotic growth (4) . Specifically, we expressed a steroid hormone-regulated fusio n
protein of C/EBPa and the estrogen receptor hormone-bind ing domain, C/EBP-ER, in 3T3-L1 cells and observed estro gen-dependent growth arrest . We have used this cell line t o
ask whether any of the gas or gadd genes expressed i n
differentiated adipocytes are regulated by C/EBPa . Th e
C/EBP-ER-expressing 3T3-L1 cell line was differentiated i n
Cell Growth & Differentiation
Standard Differentiatio n
DAY c 0 1
2
100
gasl
75 -
gas3
50 -
CHOP
25 -
(gadd
0
153 )
422
Frequent Media Replenishment
10 0
tubulin
75 -
pAL1 5
50 -
25 01
1
0
0 .5
1
1 .5
2
2.5
3
3 .5
4
DAYS
G1
q% S
n%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 medi a
replenishment, stained with propidium iodide, and analyzed by flow cytometry; the resulting data were plotted as described in Fig . 3 . The profiles of
both samples are levels from days 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 phenotypi c
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 ele vated on days 4 and 6 in the presence of estrogen a s
compared to its absence . However, none of the gas/gadd
genes expressed in post-mitotic adipocytes (Fig . 2) accumu late 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 notabl y
different in the C/EBP-ER cells compared to the parenta l
3T3-L3 cells (Fig. 2) . These differences can most likely b e
attributed to the low efficiency of differentiation for th e
C/EBP-ER cells as determined by the number of cells in th e
population containing fat droplets visible by phase contrast
Fig . 6. Expression of growth arrest-associated transcripts in adipocytes ex pressing a steroid hormone-regulated form of C/EBPa . 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 vehicl e
(–) 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 th e
presence or absence of estrogen since day 2 . Ten µg of total RNA were
analyzed by Northern blot analysis . The preferential accumulation of 42 2
mRNA in the presence of estrogen as opposed to its absence is readil y
apparent on days 4 and 6 .
microscopy (2—5% ; data not shown) . For example, these
cells do not exhibit reduced tubulin levels as seen for th e
parental 3T3-L1 cells (compare Figs . 6 and 2) . We reaso n
that the cells do not significantly deplete the media and thu s
do not significantly induce CHOP (gaddl 53) . Furthermore,
the majority of the cells on the plate are not phenotypicall y
differentiated and may be in a growth-arrest state comparable to day 0 or 3 of 3T3-L1 differentiation, characterize d
by elevated levels of gasl and gas3 (Fig . 2) .
Discussio n
The results presented here demonstrate the uncoordinate d
expression of growth arrest-associated genes during a discrete developmental transition : adipose conversion o f
3T3-L1 cells . Differential induction during multiple exit s
from the mitotic division cycle combined with differentia l
expression in post-mitotic adipocytes accounts for most o f
the variance . gas genes were discovered by virtue of thei r
coordinate accumulation in serum-starved NIH-3T3 cell s
(5) . In adipoblasts, the gas genes examined are coordinatel y
induced in response to serum deprivation, contact inhibition, and the initiation of differentiation (with the exceptio n
of gash) . However, in post-mitotic adipocytes, expression o f
the gas genes varies . gasl and gas5 levels decline relative to
contact-inhibited adipoblasts, similar to gas5 expression i n
differentiated Friend leukemic cells (15), whereas gas3 i s
1545
1546
gas/gadd Expression during Adipose Conversio n
expressed at high levels in response to nutrient deprivation .
gaddl 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 cooperat e
to inhibit adipogenesis . gaddl 53 (CHOP) can heterodimerize with C/EBPa and act as a repressor of C/EBPa-drive n
gene transcription (16) . It has been suggested that gaddl 5 3
accumulation in response to nutrient deprivation opposes
adipogenesis by inhibiting C/EBPa stimulation of adipocyte-specific genes (6) . We speculate that the coordinat e
regulation of gas3 and gasl, which encode plasma membrane proteins, along with gaddl 53 (CHOP), reflects a nee d
to coordinate activities within the nucleus and at the cel l
membrane to inhibit adipogenesis . The regulation of
gaddl 53, gas3, and gasl in response to nutrient deprivatio n
(Fig . 4) seems to be distinct from gadd153 stimulation i n
response to genomic stress (7) or gasl and gas3 induction i n
response to serum deprivation and contact inhibition (Figs .
1 and 2 ; Ref. 5) . These regulatory properties suggest tha t
gadd153 and gas3 may be targets of distinct signalin g
mechanisms in post-mitotic cells versus growing cells .
gas6 expression is a notable exception to the genera l
finding that gas and gadd genes are expressed at growth arrest states during adipogenesis . gas6 accumulates to max imal levels during the time that adipoblasts reenter the
mitotic division cycle in response to differentiation inducers . Recent characterization of the gas6 gene product suggests potential roles for this protein in overcoming contactinhibited growth arrest. The gas6 protein is a ligand for the
axl tyrosine kinase growth factor receptor (17) . In addition ,
gas6 is a secreted protein related to human protein S (18), a
component of a protease cascade that negatively regulate s
blood coagulation . Our preliminary results reveal that i n
postconfluent adipoblasts, gas6 is regulated by dexamethasone, one of the adipogenic hormones . 6 We speculate that
gas6 plays an important role in initiating the cascade o f
events that result in adipose conversion of 3T3-L1 cells .
We began our characterization of the gas and gadd genes
presuming to identify targets of C/EBPa stimulation . C/EBP a
is required for adipocyte differentiation (2) and induce s
mitotic growth arrest when prematurely expressed in adipoblasts (4) . However, none of the gas/gadd genes examined here are stimulated by C/EBPa (Fig . 6) . The expression
patterns of the gas/gadd genes during adipogenesis provides
a likely explanation for this observation . gasl, gas5, an d
gas6 are not expressed at maximal levels in phenotypicall y
differentiated adipocytes (Fig . 2), the time that C/EBPa ac cumulates (19) . gas3 and CHOP (gaddl 53) are expressed i n
differentiated adipocytes (Fig . 2) but likely antagonize adipogenesis (Ref . 9 and this report), whereas C/EBPa stimulates adipogenesis (3, 19) . It seems unlikely that C/EBP a
would stimulate the transcription of genes involved in a n
opposing metabolic response . We are conducting molecular screens to identify novel growth arrest-associated gene s
that are targets of C/EBPa stimulation in post-mitoti c
adipocytes .
Materials and Methods
Tissue Culture . 3T3-L1 cells (American Type Culture Collection) were maintained in DMEM supplemented wit h
6
E . C . Shugart and R . M . Umek, unpublished results .
10% calf serum . For serum starvation, cells were seeded a t
a density of 10 5 cells/ml on a 10-cm dish with DMEM plus
10% calf serum (GIBCO) for 24 h then the media wa s
changed to DMEM plus 0 .2% calf serum . Differentiation of
3T3-L1 cells was as described previously (10) . The stabl e
cell line expressing C/EBP-ER was described previously (4) .
For differentiation, C/EBP-ER cells were incubated to 2 day s
postconfluence in DMEM with 15%° charcoal-stripped cal f
serum (4), then switched to DMEM without phenol re d
(GIBCO) and 10% fetal bovine serum (Hyclone) containin g
dexamethasone (1 x 10 -6 M), isobutylmethylxanthine (11 5
ng/ml), and insulin (100 ng/ml) . After 2 days, the media wa s
changed to DMEM without phenol red plus 15% charcoal stripped fetal bovine serum, insulin (100 ng/ml), and estrogen (1 x 10 -6 M), or ethanol vehicle . The frequent medi a
replenishment differentiation was conducted according t o
the modified 3T3-L1 differentiation described by Carlson et
al. (9) . RNA was collected as described previously (20) .
DNA Probes . An 1108-bp fragment of gas3 (21) wa s
amplified by reverse transcription-PCR using a sense prime r
5'-GTGGACGCACTCGAGTTTGTG-3' (nucleotides 79 to
99) and an antisense primer 5'-GTGGATACTCGAGCTAAATGCAC-3 ' (nucleotides 1166 to 1187) . cDNA was initially denatured at 94°C for 2 min, followed by 35 cycles o f
94°C for 45 s, 45°C for 45 s, and 72°C for 2 min . pBSgas 3
was constructed by subcloning the gas3 fragment into th e
Xhol site of the Bluescriptll KS plasmid (pBS) . gas5 (15) wa s
amplified by PCR using a sense primer 5'-AGCCTTTCGGATCCTGTGCGGCAT-3' (nucleotides 1 to 23) and an antisense primer 5'-ATTTGAGCCTGGATCCAGGCAC-3 '
(nucleotides 425 to 446), which yielded a 446-bp fragment .
The gas5 fragment was subcloned into pBS at the BamH l
site to give the plasmid pBSgass . A 482-bp fragment of gas 6
(18) was amplified by PCR using a sense primer 5'-GGTCCTGGACTCGAGGATGTT-3 ' (nucleotides 1811 to 1832 )
and an antisense primer 5'-ACGCTGCTCGAGTGTTGCAGGA-3 ' (nucleotides 2771 to 2292) with the annealin g
temperature at 55°C . pBSgas6 was constructed by subcloning the gas6 fragment into the Xhol site of pBS . The ga s
fragments were amplified from cDNA that was synthesized
using the cDNA cycle Kit (Invitrogen) from mRNA isolate d
from 3T3-L1 cells grown in DMEM with 0 .2% calf serum fo r
72 h . A fragment of CHOP cDNA (16) corresponding to b p
2-772 was amplified by PCR using a sense primer 5 ' GGGAATTCCAGAAGGAAGTGCAT-3' (nucleotides 3 t o
26) and an antisense primer 5 ' -GGGAATTCACTTTACTGGACATG-3' (nucleotides 751 to 772) from cDNA synthesized from mRNA pooled from days 6, 8, 9, and 10 of a
standard differentiation of 3T3-L1 cells . pBSCH-10full wa s
constructed by subcloning the CHOP fragment into th e
EcoRl site in pBS . gasl (5) cDNA was provided by L .
Philipson (European Molecular Biology Laboratory ,
Heildelberg, Germany) . gadd45 cDNA was provided by N .
Holbrook (National Institute on Aging, Baltimore, MD) . Th e
636-bp 422 cDNA (11) was provided by M . D . Lane (John s
Hopkins University, Baltimore, MD), and the 1600-bp tubulin cDNA (13) was provided by D . Cleveland (John Hopkins University, Baltimore, MD) . P . Cornelius (John Hopkin s
University, Baltimore, MD) and M . D . Lane provided th e
pAL105 cDNA (12) . cDNA probes were labeled wit h
1 32 P]dCTP according to manufacturer's recommendation s
using a random-primed labeling kit (Pharmacia) .
Northern Blot Imaging. Images were obtained with a
phosphorimager (Molecular Dynamics or Fuji) and exported to Photoshop (Adobe) as 8-bit TIFF files . The im-
Cell Growth & Differentiation
ported TIFF files were adjusted for brightness and contrast
automatically, despeckled once, and blurred once . Th e
enhanced images were saved as TIFF files, imported int o
Canvas (Deneba) to be decorated with text, and printed o n
a dye-sublimation printer . Original unmolested 8-bit TIF F
files were archived .
Flow Cytometry Analysis . Cells from a 10-cm dish wer e
scraped into 10 ml of PBS, pelleted in a centrifuge (1500 X
gfor 3 min), resuspended in 1 .5 ml PBS, and stored in liqui d
nitrogen . For analysis, cells were thawed, pelleted at roo m
temperature (7000 X g 30 s), and resuspended in 1 .5 m l
propidium iodide staining solution (0 .1% sodium citrate ,
0 .3% NP40, 100 µg/ml RNase A, and 50 p,g/ml propidiu m
iodide) . The nuclei were incubated in staining solution fo r
30 min on ice . The stained cells were analyzed in a Becto n
Dickinson FACScan analyzer using Cell Fit software . A
particle size gate was defined using area versus width, an d
2 x 10 4 gated events were scored for each sample .
Acknowledgments
We thank Lennart Philipson, Nikki Holbrook, and M . Daniel Lane for providing plasmids . We are also grateful to Daniel Burke for critical reading o f
the manuscript and valuable discussions .
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