 |
INTRODUCTION |
It is well documented that global gene transcription and
translation dramatically decrease as cells exit the cell cycle and enter the quiescent G0 growth arrest state (1, 2). However, a small subset of genes become activated, and these gene products function in the initiation and maintenance of G0 growth
arrest (3, 4). Currently, little is known about the regulation and
function of G0 growth arrest-specific genes in cell
biology. Recently, an increase in expression and activity of the
retinoblastoma (Rb) family member p130 was observed in the
initiation and maintenance of G0 growth arrest (5).
Formation of the p130-E2F complex sequesters members of the E2F family
to repress the expression of genes necessary for cellular
proliferation. Alterations in the structure and function of
G0 growth arrest genes are linked to some types of cancers.
For example, germ line mutations found within the von Hippel-Lindau
tumor suppressor gene have been linked to hemangioblastomas of the
retina and central nervous system and renal carcinogenesis (6). The von
Hippel-Lindau protein functions in cell cycle control in a variety of
ways, including up-regulating the cyclin-dependent kinase
inhibitor p27 (7).
Previous reports from our laboratory (8-13) demonstrate that
CCAAT/enhancer-binding protein
(C/EBP
)1 functions in the
initiation and maintenance of G0 growth arrest in mouse
mammary epithelial cells (MECs). C/EBP
mRNA, protein, and DNA
binding activity increase during mouse MEC G0 growth arrest (11-13). STAT3 activation/phosphorylation is necessary for C/EBP
transcription in G0 growth-arrested and cytokine-treated
mouse MECs (10). In addition, the C/EBP
promoter exhibits
autoregulation during G0 growth arrest (12).
C/EBPs are a widely expressed, highly conserved family of leucine
zipper (bZIP)-type transcription factors (14, 15). Most C/EBPs are
encoded by intronless genes and exhibit a high degree of homology in
the basic and bZIP regions (14, 15). To date, six family members are
characterized including C/EBP
, C/EBP
(also called CRP2, NF-IL6,
LAP, AGP/EBP, IL6BP, or NF-M), C/EBP
(also called CRP3, NF-IL6b, and
CELF), C/EBP
, C/EBP
, and C/EBP-Homologous Protein10 (GADD153)
(15). C/EBPs bind to DNA as homodimers or as heterodimers with other
C/EBP family members or other bZIP proteins such as c-Fos and CREB/ATF
(14, 15). Functional C/EBP-binding sites are present in the promoters
of genes that function in cell growth arrest (gadd45
),
cell growth (c-fos), and differentiation (phosphoenolpyruvate carboxykinase and
-casein) (16-19).
C/EBPs are directly involved in the regulation of cell fate
determination (20-26). Early reports (20, 27) demonstrate that the
sequential expression of C/EBP
, C/EBP
, and C/EBP
is required for optimal adipocyte differentiation. Further studies (28-30) have
identified additional roles for C/EBP
in hepatocyte metabolism and
granulocyte differentiation. C/EBP
also plays an essential role in
ovarian granulosa cell biology and the development and differentiation
of the mammary gland (22-25, 31). Furthermore, C/EBP
functions in
the development and differentiation of neutrophils and eosinophils (32,
33).
Control of gene expression can occur at the transcriptional,
posttranscriptional, or posttranslational level (34-37). At the posttranscriptional level, mRNA stability is emerging as a key regulatory mechanism in cell cycle control and DNA damage repair (34,
35, 38-40). For example, the stability of growth arrest and DNA
damage-inducible mRNA increases after exposure to DNA-damaging agents or other growth arrest treatments (40). In addition, the growth
arrest-specific gene 5 (gas-5) exhibits a marked increase in
mRNA stability in density-arrested NIH 3T3 cells versus
exponentially growing and differentiating cells (41). Alterations in
the posttranscriptional regulation of genes that function in cell
growth control and cell cycle progression can play a crucial role in
tumorigenesis (42, 43). For example, alterations of trans-acting
factors that function in c-myc and c-myb mRNA
turnover results in increased c-myc and c-myb
mRNA stability, which is linked to acute myeloid leukemia (44).
Additionally, an increase in the basic fibroblast growth factor
mRNA half-life, due to defects in posttranscriptional regulation, has been implicated in a variety of human tumors (45). Although several
reports demonstrate that C/EBP
is regulated at the transcriptional level (12, 13), posttranscriptional control of C/EBP
has not been
investigated extensively.
In addition to posttranscriptional control, several genes that play
critical roles in cell cycle control are regulated posttranslationally, at the level of protein degradation (46-49). For example, the rate of
p27 protein degradation decreases in response to growth arrest, which
results in an accumulation of p27 protein (50, 51). Blocking
ubiquitination-dependent protein degradation increases p27
protein half-life and demonstrates that the p27 protein is degraded via
the ubiquitin/proteasome pathway (52, 53). In addition, increased p53
protein stability occurs during cellular genotoxic stress (54). This is
accomplished by N-terminal phosphorylation of the p53 protein, which
decreases the degree of ubiquitination and increases protein stability
(54). Accumulating evidence indicates that cellular proteins may be
degraded by ubiquitin-mediated mechanisms localized to either the
nucleus or cytoplasm (55, 56). Nuclear localized ubiquitin-mediated
degradation appears to provide a rapid mechanism for the disposal of
nuclear cell cycle regulatory proteins (57). For example, the tumor
suppressor protein product, p53, is degraded within the nucleus via a
ubiquitin-proteasome pathway during post-stress recovery (57).
The overall goal of this study was to investigate the
posttranscriptional and posttranslational regulation of C/EBP
in
G0 growth-arrested mouse MECs. Our laboratory has reported
previously (12) that C/EBP
exhibits increased transcription and
growth suppressor activity in G0 growth-arrested mouse
MECs. However, the posttranscriptional and posttranslational regulation
of C/EBP
has not been systematically investigated. Previous studies
(35) have demonstrated that key cell cycle regulatory proteins are encoded by unstable mRNAs. Because G0 growth arrest is
associated with a period of decreased cellular activity, we
hypothesized that both the C/EBP
mRNA and protein would exhibit
extended half-lives in G0 growth-arrested MECs.
Unexpectedly, the results demonstrate that C/EBP
mRNA exhibited
a novel short mRNA half-life in G0 growth-arrested
mouse MECs (t1/2 ~35 min) and contained a
relatively short poly(A) tail of ~100 nucleotides. In addition, the
C/EBP
protein also exhibited a short half-life in G0
growth-arrested mouse MECs (t1/2 ~120 min).
Furthermore, ubiquitination inhibitor studies indicated that C/EBP
protein degradation is ubiquitin-dependent and occurs predominantly within the nucleus. The results of these studies demonstrate that the C/EBP
mRNA and protein are under tight
regulation in G0 growth-arrested MECs, suggesting that
C/EBP
plays a key role in mouse MEC growth control.
 |
MATERIALS AND METHODS |
Cell Culture--
The nontransformed HC11 mouse MEC line (a kind
gift from Dr. Wolfgang Doppler, Universitat Innsbruck, Austria) was
cultured in complete growth media (CGM) consisting of RPMI 1640 (4.5 g/liter glucose) supplemented with 10% fetal bovine serum, 10 ng/ml
epidermal growth factor, 10 µg/ml insulin, 100 units/ml penicillin,
100 µg/ml streptomycin, and 500 ng/ml Fungizone (Invitrogen). NIH 3T3
mouse fibroblast cells (ATCC, Manassas, VA) were cultured in growth
media consisting of Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 µg/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, and
500 ng/ml Fungizone (Invitrogen). C/EBP
overexpression HC11 MECs
were maintained as described previously (13). Selection media included
the addition of G418 (350 µg/ml) (Invitrogen). For growth arrest
experiments, MECs were grown to 80% confluence, washed with serum-free
media, and cultured in growth arrest media (GAM) supplemented with
0.1% FBS.
Inhibitors--
Transcriptional inhibitor studies utilized both
actinomycin D (5 µg/ml) and 5,6-dichlorobenzimidazole
1-
-D-ribofuranoside (DRB) (5 µg/ml) (Sigma), and
translational inhibitor studies utilized anisomycin (10 µg/ml)
(Sigma). Ubiquitination inhibitor studies utilized MG-132 (5 µg/ml)
and N-Acetyl-Leu-Leu-Norleu-Al (LLnL) (50 µg/ml) (Sigma).
Northern Blot Analysis--
Total RNA was isolated using RNAzol
B (Tel-Test, Friendswood, TX). Thirty µg of total RNA was analyzed by
Northern blot analysis as described previously (11). The following
[
-32P]dCTP-labeled cDNAs were used as probes:
C/EBP
, C/EBP
(kind gifts from Dr. Steven McKnight, University of
Texas Southwestern Medical School, Dallas), gas-1,
c-fos (ATCC, Manassas, VA), bovine growth hormone, and
cyclophilin (Continental Laboratory Products Inc., San Diego, CA).
Membranes were visualized by autoradiography or by PhosphorImager
(Amersham Biosciences) analysis. Northern blot quantification was
performed with AlphaImager 2000 Documentation & Analysis System
software (Alpha Innotech, San Leandro, CA). To determine the mRNA
half-life of C/EBP
, C/EBP
, and gas-1, values obtained
from densitometric analysis were converted to a percentage of the
control time ("0" min) and graphs were plotted as "% mRNA
remaining versus time."
Western Blot Analysis--
MECs were washed twice in cold 1%
phosphate-buffered saline, transferred to microcentrifuge tubes,
pelleted by centrifugation, and subjected to whole cell protein
isolation using a RIPA buffer containing 20 mM Tris (pH
8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1%
SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM
PMSF, 1× Complete Protease Inhibitors (Roche Molecular Biochemicals). In addition, the following kinase and phosphatase inhibitors were added: 100 mM NaF, 100 mM NaVO3,
100 mM Na2MnO4, and 1 µM okadaic acid. Samples were placed in an Eppendorf
shaker for 30 min at 4 °C, and the supernatant was recovered after
centrifugation. Equal amounts of protein were subjected to
electrophoresis on 12.5% denaturing SDS-polyacrylamide gels and then
transferred to polyvinylidene difluoride membranes (Millipore, Bedford,
MA) at 150 V for 2 h. Blots were blocked for 60 min in 1×
phosphate-buffered saline and 0.5% Tween 20 (PBST) containing 10%
non-fat dry milk. Blots were subsequently incubated for 60 min in PBST
containing 5% non-fat dry milk and primary antisera against C/EBP
(Santa Cruz Biochemicals, Santa Cruz, CA), p27 (Transduction
Laboratories, Lexington, KY), Bcl-x (Santa Cruz Biochemicals), and
actin (Santa Cruz Biochemicals) (1:1000). Blots were washed in PBST,
incubated with horseradish peroxidase-linked secondary antibodies
(1:2000) (New England Biolabs, Beverly, MA), and visualized using
enhanced chemiluminescence (ECL) (Amersham Biosciences). For nuclear
and cytoplasmic protein isolation, MECs were isolated in Dignam buffers (nuclear buffer: 20 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 0.42 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 1×
complete protease inhibitors, 100 mM NaF, 100 mM NaVO3, 100 mM
Na2MnO4, and 1 µM okadaic acid;
cytoplasmic buffer: 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF, 1× complete protease, 100 mM NaF, 100 mM NaVO3, 100 mM Na2MnO4, and 1 µM
okadaic acid). Nuclear and cytoplasmic protein lysates were analyzed as
described for whole cell protein preparations. Western blot
quantification was performed with AlphaImager 2000 Documentation & Analysis System software (Alpha Innotech). To determine protein
half-life, C/EBP
values obtained from densitometric analysis were
converted to a percentage of the control time (0 min) and graphs
were plotted as "% protein remaining versus time."
Rapid Amplification of cDNA Ends-Poly(A) Test
(RACE-PAT)--
Five µg of total RNA was used to synthesize C/EBP
cDNA using the Superscript First-strand Synthesis for RT-PCR kit
(Invitrogen). C/EBP
cDNA was subsequently used to set up
standard PCR (5 min at 93 °C, followed by 30 cycles of 30 s at
93 °C, 30 s at 60 °C, 1 min at 72 °C, and a 7-min
extension at 72 °C) using an end-labeled [
-32P]dATP
C/EBP
upstream primer (5'-CCATTGCAGCTAAGGTACAT-3') and an oligo(dT)
anchor downstream primer (5'-GGGGATCCGCGGTTTTTTTTTTTT-3') (58). Labeled PCR products were run on 5% polyacrylamide gels at
4 °C for 5 h at 100 V in 0.5% TBE buffer. After
electrophoresis, gels were dried and visualized by autoradiography.
Oligo/RNase H Cleavage Northern Blot Analysis--
Ten µg of
total RNA and oligomers complementary to the 3' end of the C/EBP
mRNA (5'-CCAAAGAAACTAGCGATTCGGG-3') to the
-globin mRNA
(5'-GATCCACGTGCAGCTTGTCA-3') or the poly(A) tail
(poly(dT)12-18) were denatured at 65 °C for 10 min. A
digestion mixture (4 µl of 5× buffer (200 mM HEPES (pH
7.9), 50 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol (DTT)), 1 unit of RNase H (Invitrogen)
and 1 µl of RNasin (40 units/µl) (Promega, Madison, WI), in 20-µl reactions) was added to each sample and incubated at 37 °C for 30 min (58). Reactions were stopped with 1 µl of 0.5 M EDTA and precipitated with 0.1 volume of 3 M NaOAc and 2.5 volumes of 100% ethanol. Samples were pelleted by centrifugation,
resuspended in Northern blot tracking dye, and subsequently analyzed by
Northern blot analysis as described previously (11).
 |
RESULTS |
C/EBP
mRNA Exhibits a Short Half-life in
G0 Growth-arrested Mouse HC11 MECs--
To investigate the
posttranscriptional regulation of the C/EBP
mRNA in mouse HC11
MECs, we utilized transcriptional inhibitors followed by Northern blot
analysis. Briefly, confluent HC11 MECs were G0
growth-arrested by serum and growth factor withdrawal. After 48 h,
HC11 MECs were either maintained in growth arrest medium alone (GAM
)
or in GAM with the addition of the transcriptional inhibitor,
actinomycin D (GAM+) for the indicated times. mRNA half-life was
analyzed for a panel of cellular mRNAs including cyclophilin (cp),
which was used to confirm equal loading. Consistent with previous
reports from our laboratory, C/EBP
mRNA was detected in
G0 growth-arrested HC11 MECs (Fig.
1A, lanes 1-4).
C/EBP
mRNA levels rapidly declined with a half-life of ~35 min
(t1/2 ~35 min) following actinomycin D treatment
(Fig. 1A, lanes 5-8 and Fig. 1C).
After 60 min of actinomycin D treatment, C/EBP
mRNA was
undetectable (Fig. 1A, lane 7). Similar results
were observed with a second transcriptional inhibitor, DRB (Fig.
1B).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
C/EBP mRNA exhibits a short half-life
in G0 growth-arrested mouse HC11 MECs, actinomycin D
(A) and DRB (B) studies. Northern blot
analysis was performed with 30 µg of total RNA isolated from
untreated and actinomycin D or DRB-treated G0
growth-arrested HC11 MECs at the indicated time points. Northern blots
were sequentially probed with [ -32P]dCTP-labeled
C/EBP , C/EBP , gas1, c-fos, and cp
cDNAs. cp was used as a loading control. A,
lanes 1-4, RNA from G0 growth-arrested MECs
(GAM ); lanes 5-8, RNA from G0
growth-arrested MECs treated with actinomycin D (GAM+).
B, lanes 1-4, RNA from G0
growth-arrested MECs; lanes 5-8, RNA from G0
growth-arrested MECs treated with DRB. Results are representative of
three independent experiments. C, summary of mRNA
half-life data obtained from Northern blot/actinomycin D
(Act.D) analysis. Signals were quantified, and the relative
amount of each mRNA is expressed as a percentage of the "0-min"
control time, which was set at 100%. Graphs are plotted as % mRNA
remaining versus time. Filled triangles, RNA from
actinomycin D-treated MECs; filled circles, RNA from
non-treated MECs.
|
|
In addition to C/EBP
, we also investigated the mRNA level of
another C/EBP family member, C/EBP
. Reports from a number of laboratories, including our own, demonstrate that C/EBP
plays a
significant role in mammary gland growth and differentiation (22, 24,
25, 31). C/EBP
mRNA was detected in G0
growth-arrested HC11 MECs, suggesting that C/EBP
also plays a role
in G0 growth arrest (Fig. 1A, lanes
1-4). C/EBP
mRNA levels declined with a half-life of ~45
min following the addition of actinomycin D (Fig. 1A,
lanes 5-8, and Fig. 1C).
gas-1 mRNA levels are known to be induced during
G0 growth arrest of NIH 3T3 fibroblast cells (59-61). In
this report, gas-1 mRNA was also detected in
G0 growth-arrested HC11 MECs (Fig. 1A, lanes 1-4). Addition of actinomycin D reduced
gas-1 mRNA levels (t1/2 ~75 min)
(Fig. 1A, lanes 5-8, and Fig.
1C). Compared with C/EBP
, gas-1 mRNA was
relatively stable during G0 growth arrest. In agreement
with previous work, c-fos, an immediate early gene that is
induced at the G0/G1 transition, was
undetectable at the mRNA level in G0 growth-arrested
HC11 MECs (Fig. 1A, lanes 1-4).
In summary, results of transcriptional inhibitor studies indicate that
C/EBP
mRNA is highly unstable during G0 growth
arrest in HC11 MECs. C/EBP
, which has been associated previously
with cellular proliferation and differentiation, also exhibits a
relatively short mRNA half-life in G0 growth-arrested
HC11 MECs. In contrast, gas-1 mRNA is more stable during
G0 growth arrest. Overall, the results suggest that the
C/EBP
mRNA is undergoing rapid turnover despite the general
decline in global gene expression and biosynthetic activity during
G0 growth arrest.
C/EBP
mRNA Half-life during Cell Cycle Re-entry
in HC11 MECs--
Cell cycle re-entry requires coordination between
the inactivation and/or disposal of G0-specific proteins
and the expression of early G1 genes, such as
c-fos and c-myc. To investigate the posttranscriptional regulation of C/EBP
mRNA during early
G1, mRNA levels were analyzed from HC11 MECs upon
addition of complete growth media alone (CGM
) or CGM plus actinomycin
D (CGM+) at the indicated time points. In agreement with previous
reports from our laboratory, C/EBP
mRNA levels decline with the
onset of G1 and the initiation of the cell cycle (Fig.
2, lanes 1-4) (41). C/EBP
mRNA levels also decreased in early G1 following actinomycin D treatment; however, the cell cycle-induced decline of
C/EBP
mRNA was delayed compared with the decline observed in
G0 growth arrest (Fig. 2, lanes 5-8).

View larger version (94K):
[in this window]
[in a new window]
|
Fig. 2.
C/EBP mRNA
stability increases during the G0/G1 transition
upon transcriptional inhibitor treatment in HC11 MECs. RNA
isolated from untreated and actinomycin D-treated serum and growth
factor stimulated (G0/G1 transition) HC11 MECs
was analyzed by Northern blot analysis as described in Fig. 1.
Lanes 1-4, RNA from serum and growth factor-stimulated
(G0/G1 transition) MECs (CGM );
lanes 5-8, RNA from serum and growth factor-stimulated
(G0/G1 transition) MECs treated with
actinomycin D (CGM+). Results are representative of three
independent experiments.
|
|
When G0 growth-arrested HC11 MECs were induced to re-enter
the cell cycle by CGM addition, C/EBP
mRNA levels increased
~10-fold within the first 90 min (Fig. 2, lane 1-4). This
induction of C/EBP
mRNA levels during early G1 is
consistent with a growth-promoting role for C/EBP
. The addition of
actinomycin D blocked the growth-stimulated induction of C/EBP
mRNA, which suggests that C/EBP
transcription plays a major role
in the increase in C/EBP
mRNA levels during early G1
in HC11 MECs (Fig. 2, lanes 5-8).
gas-1 mRNA levels also declined after G0
growth-arrested HC11 MECs were induced to re-enter the cell cycle by
refeeding with CGM (Fig. 2, lanes 1-4). Interestingly,
addition of CGM and actinomycin D stabilized the gas-1
mRNA, resulting in high levels of gas-1 mRNA even at
90 min (Fig. 2, lanes 5-8). In agreement with previous reports, c-fos mRNA was transiently induced following
the addition of CGM and the initiation of the cell cycle (Fig. 2,
lanes 1-4). The induction of c-fos mRNA was
blocked by actinomycin D treatment, indicating that c-fos
gene transcription is required for the increase in c-fos
mRNA during early G1 (Fig. 2, lanes
5-8).
The results of the transcriptional inhibitor studies during cell cycle
re-entry demonstrate that the C/EBP
and gas-1 mRNAs are more stable during the G0/G1 transition
compared with G0 growth arrest. This suggests that both
C/EBP
and gas-1 mRNA degradation during cell cycle
re-entry is dependent on the transcription of gene product(s) important
for mRNA decay during the G0/G1 transition. In addition, the increase in C/EBP
and c-fos mRNA
levels during the G0/G1 transition are
inhibited by actinomycin D treatment, indicating that the
increase in these immediate early mRNAs is transcription-dependent.
C/EBP
mRNA Is More Stable in G0
Growth-arrested NIH 3T3 Cells Compared with HC11 MECs--
NIH 3T3
cells have been utilized extensively as a model system to investigate
mechanisms of cell growth control. We reported previously (11) that
C/EBP
mRNA is present in NIH 3T3 cells regardless of growth
status. To investigate the posttranscriptional control of the C/EBP
mRNA in NIH 3T3 cells, transcriptional inhibitor/Northern blot
analysis was performed. In agreement with our previous results, C/EBP
mRNA was detected in G0 growth-arrested
(GAM
) NIH 3T3 cells (Fig.
3A, lanes 1-4).
C/EBP
mRNA levels declined following actinomycin D treatment,
although the rate of decline is slower than that observed in
G0 growth-arrested HC11 MECs (t1/2 >100
min for NIH 3T3 cells versus t1/2 ~35
min for HC11 MECs) (Fig. 3A, lanes 5-8 and Fig.
3B). Following cell cycle re-entry, C/EBP
mRNA levels
decreased by 90 min (Fig. 3A, lanes 9-12). The
addition of actinomycin D stabilized C/EBP
mRNA (Fig.
3A, lanes 13-16), paralleling the results from
experiments in HC11 MECs.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
C/EBP mRNA
stability during G0 growth arrest and the
G0/G1 transition in NIH 3T3 cells. RNA
isolated from untreated and actinomycin D-treated G0
growth-arrested or serum and growth factor-stimulated
(G0/G1 transition) NIH 3T3 cells was analyzed
by Northern blot as described in Fig. 1. A, lanes
1-4, RNA from G0 growth-arrested MECs
(GAM ); lanes 5-8, RNA from G0
growth-arrested MECs treated with actinomycin D (GAM+);
lanes 9-12, RNA from serum- and growth factor-stimulated
(G0/G1 transition) MECs (CGM );
lanes 13-16, RNA from serum- and growth factor-stimulated
(G0/G1 transition) MECs treated with
actinomycin D (CGM+). Results are representative of three
independent experiments. B, summary of mRNA
half-life data obtained from Northern blot/actinomycin D
(Act.D) analysis as determine in Fig. 1. Filled
triangles, RNA from actinomycin D-treated MECs; filled
circles, RNA from non-treated MECs.
|
|
Consistent with results from mouse MECs, the C/EBP
mRNA was
detected in G0 growth-arrested NIH 3T3 cells (Fig.
3A, lanes 1-4). Upon addition of actinomycin D,
a steady decline in the C/EBP
mRNA content was observed
(t1/2 >100 min) (Fig. 3A, lanes
5-8, and Fig. 3B). C/EBP
mRNA levels increased (15-fold after 90 min) following cell cycle re-entry (Fig.
3A, lanes 9-12). This cell cycle-induced
increase in C/EBP
mRNA levels was blocked by actinomycin D
treatment (Fig. 3A, lanes 13-16). In agreement
with previous reports (59, 62), elevated levels of gas-1
mRNA was detected in G0 growth-arrested NIH 3T3 cells
(Fig. 3A, lanes 1-4). Addition of actinomycin D
resulted in a decline in gas-1 mRNA levels
(t1/2 ~75 min) (Fig. 3A, lanes
5-8, and Fig. 3B). Following the addition of complete
growth media and the initiation of the cell cycle, gas-1
mRNA content exhibited a decline by 90 min (Fig. 3A,
lanes 9-12). However, the addition of actinomycin D
stabilized the gas-1 mRNA (Fig. 3A,
lanes 13-16). Finally, c-fos mRNA was
undetectable in G0 growth-arrested NIH 3T3 cells at all
time points taken (Fig. 3A, lanes 1-8).
c-fos mRNA levels rapidly increased following cell cycle
re-entry and peaked 60 min after the addition of complete growth media
(Fig. 3A, lanes 9-12). Cell cycle-induced
increase in the c-fos mRNA level was blocked by
actinomycin D treatment, paralleling the results from experiments with
HC11 MECs (Fig. 3A, lanes 13-16).
The extended C/EBP
mRNA half-life detected in G0
growth-arrested NIH 3T3 cells suggests that C/EBP
is under less
stringent control in mouse fibroblast-derived cells compared with mouse mammary epithelial-derived cells. In contrast, posttranscriptional regulation of C/EBP
mRNA during cell cycle re-entry is similar between HC11 MECs and NIH 3T3 cells. The posttranscriptional regulation of C/EBP
, gas-1, and c-fos in both
G0 growth arrest and cell cycle re-entry is comparable
between HC11 MECs and NIH 3T3 cells.
C/EBP
mRNA Contains a Short Poly(A)
Tail--
Sequences present within mRNAs influence processing,
stability, and transport (34, 35, 39, 63). To investigate the role of
the poly(A) tail on C/EBP
mRNA stability, we utilized an
oligo/RNase H cleavage Northern blot analysis (58). In this analysis, a
C/EBP
-specific oligomer complimentary to the C/EBP
mRNA within the 3'-untranslated region (UTR) (oligo 1193) was used
to form a DNA/RNA heteroduplex that is cleaved by RNase H, producing
C/EBP
5' and 3' mRNA fragments (Fig.
4A). A
[
-32P]dCTP-labeled C/EBP
3'-UTR-specific probe that
spanned the oligo/RNase H digestion site was used to detect both of
the oligo/RNase H-generated C/EBP
fragments: the 5' C/EBP
cleavage product (~1.4 kb) composed of the C/EBP
mRNA
5'-untranslated region (UTR), coding sequence, and partial 3'-UTR and
the 3' C/EBP
mRNA cleavage product composed of the remaining
3'-UTR (~260 bp) plus the length of the poly(A) tail.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
C/EBP mRNA
contains a short poly(A) tail in mouse HC11 MECs. Oligo/RNase H
cleavage Northern blot analysis was performed on 20 µg of total RNA
isolated from untreated and actinomycin D-treated G0
growth-arrested or serum- and growth factor-stimulated
(G0/G1 transition) HC11 MECs at the indicated
time points. A, schematic of the oligo/RNase H cleavage
protocol. RNA samples were incubated with a 3'-UTR C/EBP
mRNA-specific oligomer (1193), treated with RNase H, and separated
by agarose gel electrophoresis. Both 5' and 3' cleavage fragments were
detected by using a [ -32P]dCTP-labeled probe that
spanned the C/EBP 3'-UTR. B, lanes 1-3,
RNA from G0 growth-arrested MECs (GAM );
lanes 4 and 5, RNA from G0
growth-arrested MECs treated with actinomycin D (GAM+);
lanes 6-8, RNA from serum- and growth factor-stimulated
(G0/G1 transition) MECs (CGM );
lanes 9-11, RNA from serum- and growth factor-stimulated
(G0/G1 transition) MECs treated with
actinomycin D (CGM+); lane 12, RNA from
G0 growth-arrested MECs treated with oligo(dT) and RNase H;
lane 13, untreated full-length C/EBP mRNA from
G0 growth-arrested MECs. Results are representative of
three independent experiments.
|
|
Initially, we performed the oligo/RNase H cleavage analysis on RNA from
G0 growth-arrested (GAM
) HC11 MECs, and Northern blot
analysis detected two cleavage products of ~1.4 kb and 370 bp (Fig.
4B, lanes 1-3). The 3'-UTR cleavage product
contains 260 bp from the 3'-UTR and reveals a poly(A) tail of ~100
nucleotides. To investigate the mechanism of C/EBP
mRNA
degradation, mRNA was isolated from actinomycin D-treated
G0 growth-arrested (GAM+) HC11 MECs. After 30 min of
actinomycin D treatment, both cleavage products were detected (Fig.
4B, lane 4). However, after 60 min of actinomycin
D treatment only the 3' oligo/RNase H C/EBP
mRNA cleavage
product was detected (Fig. 4B, lane 5). These
results confirm the short half-life of the C/EBP
mRNA in
G0 growth-arrested HC11 MECs.
We next investigated the C/EBP
mRNA poly(A) tail length during
the G0/G1 transition. G0
growth-arrested HC11 MECs were induced to re-enter the cell cycle by
the addition of CGM alone (CGM
) or CGM plus actinomycin D (CGM+).
Upon addition of CGM, the reduction of C/EBP
mRNA oligo/RNase H
cleavage products was observed, consistent with a decrease in the
C/EBP
mRNA content upon the onset of early G1 (Fig.
4B, lanes 6-8). After addition of actinomycin D,
the reduction of C/EBP
mRNA oligo/RNase H cleavage products was
slightly delayed compared with GAM+, and a more complex pattern of
C/EBP
mRNA degradation was detected (Fig. 4B,
lanes 9-11). The results of the CGM and actinomycin D
experiment suggest that transcription of gene products important for
mRNA decay is required for efficient C/EBP
mRNA degradation
during cell cycle re-entry. Finally, the estimated size of all the
C/EBP
3' oligo/RNase H cleavage products is consistent with a
poly(A) tail length of ~100 nucleotides. To confirm the length of the
poly(A) tail, we utilized an oligo(dT) in the oligo/RNase H experiment,
which generates a C/EBP
mRNA product lacking a poly(A) tail
(Fig. 4B, lane 12). The results reveal that the
mobility of this mRNA product compared with full-length C/EBP
mRNA (Fig. 4B, lane 13) is consistent with a
poly(A) tail length of ~100 nucleotides. Overall, the results
demonstrate that the C/EBP
mRNA contains a relatively short
poly(A) tail that is not shortened during mRNA degradation in HC11
G0 growth arrest and cell cycle re-entry.
RACE-PAT Analysis Confirms C/EBP
mRNA Poly(A)
Tail Length--
To investigate further the C/EBP
mRNA poly(A)
tail length, we utilized a rapid amplification of cDNA ends-poly(A)
test (RACE-PAT) (58). Initially, mRNA was obtained from
G0 growth-arrested HC11 MECs; cDNA was synthesized, and
PCR was performed utilizing a radiolabeled C/EBP
3'-UTR
upstream-specific primer and an oligo(dT) downstream primer. The PCR
produced multiple products varying in length from 170 to 270 bp (Fig.
5, lanes 1-3), which is
consistent with a C/EBP
poly(A) tail length of ~100 nucleotides.
Following addition of actinomycin D, C/EBP
mRNA levels declined
rapidly as observed previously in the transcriptional
inhibitor/Northern blot analysis (Fig. 5, lanes 4-6). This
decline is reflected in the decrease in the amount of RACE-PAT product
obtained. RACE-PAT analysis was also performed on mRNA obtained
from HC11 MECs refed with CGM, which also indicated that the C/EBP
mRNA poly(A) length is approximately ~100 nucleotides (Fig. 5,
lanes 7-9). Additionally, actinomycin D treatment resulted
in a more stable C/EBP
mRNA but had no apparent effect on
poly(A) tail length (Fig. 5, lanes 10-12). The results
confirm that the C/EBP
mRNA contains a poly(A) tail of ~100
nucleotides in G0 growth arrest and during the
G0/G1 transition and parallel previous mRNA
half-life results from experiments in HC11 MECs (Figs. 1A
and 2A).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5.
The short C/EBP
mRNA poly(A) tail is confirmed by RACE-PAT analysis.
RACE-PAT was performed on 5 µg of total RNA isolated from untreated
and actinomycin D-treated G0 growth-arrested or serum- and
growth factor-stimulated (G0/G1 transition)
HC11 MECs at the indicated time points. cDNA was synthesized and
subjected to PCR, and products were visualized by PAGE and
autoradiography. Lanes 1-3, G0 growth-arrested
MECs (GAM ); lanes 4-6, G0
growth-arrested MECs treated with actinomycin D (GAM+);
lanes 7-9, serum and growth factor stimulated
(G0/G1 transition) MECs (CGM );
lanes 10-12, serum and growth factor stimulated
(G0/G1 transition) MECs treated with
actinomycin D (CGM+). Results are representative of three
independent experiments.
|
|
The 3'-Untranslated Region Influences C/EBP
mRNA
Stability--
Numerous studies (34, 35, 39) have demonstrated that
specific sequences within the 3'-UTR regulate mRNA stability. To investigate the potential role of the C/EBP
3'-UTR in mRNA
stability, transcriptional inhibitor/Northern blot analysis was
repeated utilizing a stably transfected C/EBP
overexpression HC11
MEC line previously developed in our laboratory (13). This HC11 MEC
line expresses an exogenous C/EBP
mRNA that contains a bovine growth hormone (BGH) 3'-UTR in place of the C/EBP
3'-UTR. The cells
were growth-arrested for 48 h and maintained in GAM in the presence or absence of actinomycin D. The C/EBP
/BGH 3'-UTR mRNA levels were compared with the endogenous C/EBP
mRNA levels.
Consistent with previous results, the endogenous C/EBP
mRNA
levels were detected in G0 growth-arrested cells (Fig.
6, lanes 1-4). Similarly, the
C/EBP
/BGH 3'-UTR mRNA was also detected in G0
growth-arrested HC11 MECs at all indicated time points (Fig. 6,
lanes 1-4). Consistent with previous results, the addition
of actinomycin D resulted in a rapid reduction of endogenous C/EBP
mRNA levels (t1/2 ~35 min) (Fig. 6,
lanes 5-7). Interestingly, the C/EBP
/BGH 3'-UTR mRNA
levels did not decline after actinomycin D treatment (Fig. 6,
lanes 5-7). These results indicate that the presence of the BGH 3'-UTR downstream of the C/EBP
coding region stabilizes the C/EBP
mRNA compared with the endogenous C/EBP
mRNA and
suggests that the C/EBP
3'-UTR plays a role in mRNA stability
during G0 growth arrest.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 6.
The C/EBP 3'-UTR
regulates C/EBP mRNA stability in
G0 growth-arrested HC11 MECs. RNA isolated from
untreated and actinomycin D-treated G0 growth-arrested
C/EBP overexpression HC11 MECs was analyzed by Northern blot using
either a BGH 3'-UTR, C/EBP 3'-UTR, or a cp probe as described in
Fig. 1. Lanes 1-4, RNA from G0 growth-arrested
MECs (GAM ); lanes 5-7, RNA from G0
growth-arrested MECs treated with actinomycin D (GAM+).
Results are representative of three independent experiments.
|
|
C/EBP
Protein Exhibits a Short Half-life in
G0 Growth-arrested Mouse MECs--
Posttranslational
control is a major mechanism by which cells regulate the level of cell
cycle control proteins (46-49). To investigate the posttranslational
regulation of the C/EBP
protein, HC11 MECs were G0
growth-arrested for 48 h and maintained in GAM in the presence or
absence of the translational inhibitor anisomycin. Protein half-life
was analyzed for a panel of cellular proteins by Western blot analysis
including actin, which was used to confirm equal loading. Consistent
with previous reports from our laboratory (11-13), C/EBP
protein
was detected in G0 growth-arrested HC11 MECs (Fig.
7A, lanes 1-6).
Following anisomycin treatment, C/EBP
protein levels declined with a
half-life of ~120 min (Fig. 7A, lanes
7-12, and Fig. 7B). As a control, the stability
of p27, a growth arrest-specific protein that is regulated
predominantly at the posttranslational level, was assessed (50, 51). As expected, p27 protein was detected during G0 growth arrest
of HC11 MECs (Fig. 7A, lanes 1-6). In contrast
to the C/EBP
protein decay kinetics, the p27 protein is relatively
stable in G0 growth-arrested HC11 MECs even after treatment
with anisomycin (Fig. 7A, lanes 7-12). The
results demonstrate that the C/EBP
protein exhibits a shorter
half-life compared with p27, which suggests that C/EBP
protein is
tightly regulated in G0 growth-arrested mouse MECs.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 7.
C/EBP protein
exhibits a short half-life in G0 growth-arrested HC11
MECs. Western blot analysis was performed on 50 µg of whole cell
protein isolated from untreated and anisomycin-treated G0
growth-arrested HC11 MECs at the indicated time points. Western blots
were sequentially probed with C/EBP , p27, and actin antibodies.
Actin was used as a loading control. A, lanes
1-6, protein from growth-arrested MECs (GAM );
lanes 7-12, protein from growth-arrested MECs treated with
anisomycin (GAM+). Results are representative of three
independent experiments. B, summary of protein
half-life data obtained from Western blot/anisomycin analysis. Signals
were quantified, and the relative amount of each protein is expressed
as a percentage of the 0-min control time, which was set at 100%.
Graphs are plotted as % protein remaining versus time.
Filled triangles, protein from anisomycin-treated MECs;
filled circles, protein from non-treated MECs.
|
|
C/EBP
Protein Is Degraded via a
Ubiquitin-dependent Pathway in G0
Growth-arrested Mouse MECs--
To investigate the protein degradation
pathway utilized by the C/EBP
protein in G0
growth-arrested mouse MECs, we used the ubiquitination inhibitor,
MG-132. HC11 MECs were growth-arrested by addition of GAM for 48 h
and subsequently maintained in either growth arrest media alone
(control), GAM plus a vehicle control (+Me2SO), or GAM plus
the ubiquitination inhibitor (+MG-132). C/EBP
protein was detected
in both G0 growth-arrested control and Me2SO
samples (Fig. 8A, lanes
1-4 and 5-7, respectively). Interestingly, C/EBP
protein content dramatically increased by 60 min of MG-132 treatment
and continued to be elevated up to 180 min (Fig. 8A,
lanes 8-10). Similar results were obtained with a second
ubiquitination inhibitor, LLnL, in G0 growth-arrested HC11
MECs (Fig. 8B).

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 8.
C/EBP protein
undergoes ubiquitination in G0 growth-arrested MECs.
Western blot analysis was performed on 50 µg of whole cell protein
isolated from untreated and MG-132 or LLnL-treated G0
growth-arrested mouse MECs at the indicated time points as described in
Fig. 7. A, mouse HC11 MECs treated with MG-132.
Lanes 1-4, protein from G0 growth-arrested
MECs (control); lanes 5-7, protein from
G0 growth-arrested MECs treated with vehicle
(+DMSO); lanes 8-10, protein from G0
growth-arrested MECs treated with ubiquitination inhibitor
(+MG-132). B, mouse HC11 MECs treated with
LLnL. Lanes 1-4, protein from G0
growth-arrested MECs (control); lanes 5-7,
protein from G0 growth-arrested MECs treated with vehicle
(+DMSO); lanes 8-10, protein from G0
growth-arrested MECs treated with ubiquitination inhibitor
(+LLnL). Results are representative of three independent
experiments.
|
|
As a control, p27 protein levels were monitored in both mouse and human
MECs after MG-132 treatment. Previous work (52, 53) in vitro
and in vivo demonstrates that the ubiquitin-proteasome pathway regulates the p27 protein level. p27 protein was detected in
both G0 growth-arrested control and Me2SO
samples (Fig. 8, A and B). As expected, a modest
increase in p27 protein level is detected after MG-132 treatment in
HC11 MECs (Fig. 8A, lanes 8-10). Taken together,
these results demonstrate that the C/EBP
protein is degraded via a
ubiquitin-proteasome-dependent pathway that is conserved in
both mouse MECs.
Ubiquitination of the C/EBP
Protein Is Localized
Predominantly to the Nuclear Compartment--
To determine whether
C/EBP
protein degradation occurs in the nucleus and/or cytoplasm,
nuclear and cytoplasmic protein was analyzed from G0
growth-arrested HC11 MECs. HC11 MECs were growth-arrested by addition
of GAM for 48 h and subsequently maintained in either growth
arrest media alone (control), GAM plus a vehicle control (+Me2SO), or GAM plus the ubiquitination inhibitor
(+MG-132). Nuclear and cytoplasmic protein fractions were isolated at
the indicated times. C/EBP
protein was detected in the nuclear
protein fraction but not the cytoplasmic protein fraction in both the G0 growth-arrested control and Me2SO samples
(Fig. 9, lanes 1-6 and
lanes 7-10, respectively). In agreement with previous
results (Fig. 8), C/EBP
protein content increased after
MG-132 treatment (Fig. 9, lanes 11-14). Interestingly, the
increase in C/EBP
protein is restricted to the nuclear compartment
(Fig. 9, lanes 11 and 13).

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 9.
C/EBP protein
ubiquitination is localized to the nuclear compartment. Western
blot analysis was performed on 25 µg of nuclear and cytoplasmic
protein isolated from untreated or MG-132-treated G0
growth-arrested mouse HC11 MECs at the indicated time points as
described in Fig. 7. Lanes 1, 3, and
5, nuclear protein from G0 growth-arrested MECs
(control); lanes 2, 4, and
6, cytoplasmic protein from G0 growth-arrested
MECs (control); lanes 7 and 9, nuclear
protein from G0 growth-arrested MECs treated with vehicle
(+DMSO); lanes 8 and 10, cytoplasmic
protein from G0 growth-arrested MECs treated with vehicle
(+DMSO); lanes 11 and 13, nuclear
protein from G0 growth-arrested MECs treated with
ubiquitination inhibitor (+MG-132); lanes 12 and
14, cytoplasmic protein from G0 growth-arrested
MECs treated with ubiquitination inhibitor (+MG-132).
Results are representative of three independent experiments.
|
|
Nuclear and cytoplasmic p27 protein levels were also monitored in
MG-132-treated G0 growth-arrested HC11 MECs. Similar to the
previous study, p27 protein was detected in G0
growth-arrested control and Me2SO samples (Fig. 9,
lanes 1-6 and lanes 7-10, respectively). In
contrast to C/EBP
protein subcellular localization, p27 protein was
found in both the nuclear and cytoplasmic compartments. Detection of
the p27 protein increased slightly in both compartments after MG-132
treatment (Fig. 9, lanes 11-14).
As a control for our nuclear and cytoplasmic protein fractionation, we
monitored the subcellular localization of Bcl-x, which is known to be
localized to the cytoplasmic compartment (64). As expected, the
majority of the Bcl-x protein content was localized within the
cytoplasmic compartment in both G0 growth-arrested control
and Me2SO samples (Fig. 9, lanes 1-6 and
lanes 7-10, respectively). Importantly, upon MG-132
treatment, no change in Bcl-x localization was detected (Fig. 9,
lanes 11-14). Taken together, these results demonstrate
that the C/EBP
protein is absent from the cytoplasmic compartment,
which suggests a nuclear localized ubiquitin-mediated degradation pathway.
 |
DISCUSSION |
Although most cells in the adult animal exist in a G0
growth arrest state, little is known about the regulation and function of genes expressed during G0 (1-4). This study
investigated the posttranscriptional and posttranslational regulation
of C/EBP
in G0 growth-arrested mouse MECs in
vitro. Previous reports from our laboratory (11-13) have shown
that C/EBP
gene expression and DNA binding activity increase in
G0 growth-arrested MECs. The G0-specific
increase in C/EBP
gene expression is STAT3-dependent (10). In addition, overexpression of C/EBP
in MECs accelerated G0 growth arrest and apoptosis in response to serum and
growth factor withdrawal (13). In contrast, reducing C/EBP
levels by
antisense RNA delayed MEC G0 growth arrest and apoptosis
after serum and growth factor withdrawal (13). In this report, we demonstrated that C/EBP
mRNA exhibits a novel short half-life during G0 growth arrest in mouse MECs
(t1/2 ~35 min). Interestingly, mRNAs encoding
several important cell cycle control proteins, growth factors,
lymphokines, cytokines, and proto-oncogenes also exhibit short
half-lives (35). For example, the cytokine interleukin 6, which is
important in the inflammatory response, has an mRNA half-life of
~20 min (65). We suggest that the short C/EBP
mRNA half-life
in G0 growth-arrested MECs allows the cells to respond
rapidly to potential growth stimuli. Interestingly, the short half-life
of C/EBP
mRNA observed in mouse MECs appears to be a property of
mammary epithelial derived cell lines. For example, in G0
growth-arrested NIH 3T3 cells, the C/EBP
mRNA half-life is
~2-3-fold longer. This suggests that tight regulation of the
C/EBP
mRNA is important in the initiation and maintenance of
G0 growth arrest in mouse MECs. Similar to our studies in
mouse HC11 MECs, the C/EBP
mRNA exhibited a relatively short
half-life of ~40 min in G0 growth-arrested human MCF-12A MECs (data not shown). This suggests a conservation of mRNA decay kinetics for C/EBP
in both mouse and human MEC systems.
Cell cycle re-entry (G0/G1 transition) is
associated with dramatic changes in gene expression. Transcription of
growth arrest genes is known to decrease with cell cycle re-entry,
although mRNAs encoding growth arrest-specific proteins could
persist and may delay or interfere with cell cycle re-entry. The
disposal of G0-specific mRNAs and proteins during MEC
cell cycle re-entry is not well characterized. This study sought to
determine whether or not the decay kinetics of C/EBP
mRNA were
similar between G0 growth arrest and the
G0/G1 transition. Results of transcriptional inhibitor/Northern blot studies upon cycle re-entry demonstrate that
C/EBP
mRNA has a longer half-life during the
G0/G1 transition compared with G0
growth-arrested HC11 MECs. In fact, both C/EBP
and gas-1
mRNAs exhibited stabilization during cell cycle re-entry in
response to transcriptional inhibitors in two mouse MEC lines, HC11 and
COMMA D (later data not shown). Posttranscriptional control is known to
play a major role in the regulation of gas family members in many cell
types (61, 66, 67). Our results parallel previous work that shows an
increase in gas-1 and gas-6 mRNA stability after cell cycle re-entry and treatment with actinomycin D of fibroblastic cell lines (61, 66, 67). Furthermore, actinomycin D
treatment of Schwann cells during cell cycle re-entry stabilized the
gas-3 mRNA (68). The difference in C/EBP
mRNA
half-life in G0 growth arrest and the
G0/G1 transition suggests that there is a
specific mRNA degradation pathway for C/EBP
during the
G0/G1 transition that differs from
G0 growth arrest. In addition, increased stabilization of
the C/EBP
mRNA suggests that the synthesis of a trans-acting
factor(s) or RNA is required to degrade the C/EBP
mRNA upon cell
cycle re-entry.
The mechanism underlying C/EBP
mRNA degradation is currently not
known, although numerous studies have demonstrated that the length of
the poly(A) tail is a major factor in the stability of eukaryotic
mRNAs (i.e. a decrease in poly(A) tail length results in
an decrease in mRNA stability) (34, 35, 39, 63). In this report,
analysis of poly(A) tail length by oligo/RNase H cleavage and RACE-PAT
demonstrated that the C/EBP
mRNA has a short poly(A) tail of
~100 nucleotides. This is somewhat shorter than the average
eukaryotic mRNA that contains a poly(A) tail of ~200 nucleotides
(69).
Structural elements found within the 5'-UTR, coding region, and the
3'-UTR are known to be involved in regulating mRNA stability (34,
35, 39). For example, the 3'-UTR of many labile mRNAs, such as
cytokine and oncoprotein mRNAs, contain multiple copies of A/U-rich
elements (AREs) (34, 35, 39). These cis-acting elements interact with
trans-acting factors to destabilize the mRNA. Analysis of ARE
sequences from 12 transcription factor-encoding mRNAs that exhibit
early G1 instability classified two distinct groups of
mRNAs: 1) mRNAs with 3'-UTRs that contain one or more copies of
the well recognized "AUUUA" sequence and 2) mRNAs with 3'-UTRs
that contain one or more copies of a "non-AUUUA" sequence (70). An
example of a non-AUUUA mRNA is c-jun, which contains "U"-rich regions that confer G1 instability (70).
Interestingly, analysis of the C/EBP
3'-UTR revealed a single AUUUA
element and two U-rich regions (region 1, 18 uracils/32 nucleotides;
region 2, 17 uracils/26 nucleotides). This indicates that the C/EBP
mRNA has characteristics of both AUUUA and non-AUUUA AREs.
Mutational analysis is ongoing to characterize further the role of
these instability elements in C/EBP
mRNA decay.
Analysis of another C/EBP family member, C/EBP
, demonstrated similar
mRNA decay kinetics as C/EBP
during mouse MEC G0
growth arrest. Like C/EBP
, C/EBP
mRNA displayed a short
half-life of ~45 min. The results suggest a conserved mRNA decay
pathway shared between C/EBP
and C/EBP
in G0
growth-arrested mouse MECs. Although the homology between the C/EBP
and C/EBP
3'-UTRs is ~30%, both 3'-UTR sequences contain multiple
U-rich elements that may regulate mRNA degradation.
Because the C/EBP
mRNA was shown to have a short
half-life in G0 growth-arrested MECs, we hypothesized that
the C/EBP
protein would exhibit a similar short biological half-life
(39). A yeast genome-wide analysis has demonstrated that unstable
mRNAs encode for unstable proteins (71). Examples include
translation initiation factors, termination factors, and proteins of
the mating pheromone signal transduction pathway (71). The results in
this report established that the half-life of the C/EBP
protein is
shorter (t1/2 ~120 min) than the tumor suppressor, p27 (t1/2 >150 min). The short half-life of the
C/EBP
protein in G0 growth-arrested MECs suggests that C/EBP
function is tightly regulated during MEC quiescence, which may
allow MECs to respond rapidly to growth signals and re-enter the cell
cycle when necessary.
The ubiquitin-proteasome pathway is a major selective decay mechanism
of short-lived regulatory proteins (49). Cell cycle regulatory proteins
that are degraded by the ubiquitin-proteasome pathway include the tumor
suppressors, p21 (t1/2 ~30 min) (72), p53
(t1/2 ~20 min) (73), and p27
(t1/2 ~150 min) (52). The results in this report
established that the C/EBP
protein is also degraded via the
ubiquitin-proteasome pathway in growth-arrested MECs
(t1/2 ~120 min). It has yet to be determined
whether phosphorylation of the C/EBP
protein precedes
ubiquitination, which has been observed in the regulation of p27
protein decay.
It has been known for sometime that mammalian proteasome complexes are
localized throughout the cell including the nucleus, cytoplasm, and
within the endoplasmic reticulum membrane network (55, 56). Proteasomes
localized within the nucleus have been shown to be
responsible for the turnover of short lived proteins important for many
critical cellular processes. Some proteins that undergo ubiquitination
within the nuclear compartment include the large subunit of RNA
polymerase II (74), the progesterone receptor (75), the Xenopus
laevis kinase inhibitor, p27Xic1 (76), and the p53 protein (57).
It is speculated that cells are able to rid themselves of nuclear
proteins that are no longer necessary by ubiquitination within the
nucleus (57). Results of this study demonstrate that C/EBP
protein
ubiquitination is localized to the nucleus. We speculate that nuclear
protein degradation provides a mechanistic explanation for the
relatively short half-life of the C/EBP
protein during MEC
G0 growth arrest and allows for proper cell cycle
progression during the G0/G1 transition.
In summary, the data presented establish that the C/EBP
mRNA has
a short half-life in G0 growth-arrested MECs. The C/EBP
mRNA has a relatively short poly(A) tail (~100 nucleotides) that does not vary in length during decay in G0 growth arrest or
the G0/G1 transition. It is proposed that the
C/EBP
mRNA is degraded by a mechanism involving endonucleolytic
cleavage during G0 growth arrest. Additionally, the
C/EBP
protein has a relatively short half-life in G0
growth-arrested MECs and is degraded by the ubiquitin-proteasome pathway within the nuclear compartment. This study suggests that despite the decrease in cellular activity during G0 growth
arrest, C/EBP
mRNA and protein are tightly regulated in MECs. We
predict that this tight regulation allows G0
growth-arrested MECs to proliferate in response to growth stimuli.
Studies investigating possible instability elements in the C/EBP
mRNA 3'-UTR and characterization of trans-acting factors important
in C/EBP
mRNA degradation are currently underway.