(Received for publication, October 17, 1996)
From the Ohio State University Biochemistry Program
and the § Department of Veterinary Biosciences, Ohio
State University, Columbus, Ohio 43210
CCAAT/enhancer binding proteins (C/EBPs) are a
highly conserved family of DNA-binding proteins that regulate cell
growth and differentiation in a highly tissue-specific manner. These
experiments investigated the influence of the cell cycle on C/EBP
isoform expression in mammary epithelial cells (COMMA D) and
fibroblasts (NIH3T3). C/EBP- gene expression is induced in COMMA D
cells arrested in G0 by serum and growth factor withdrawal
or contact inhibition. C/EBP-
mRNA, nuclear protein content, and
DNA binding activity increase during G0 growth arrest and
decrease after cell cycle induction in COMMA D cells. Growth arrest is
markedly delayed in COMMA D cells expressing a C/EBP-
antisense
construct. C/EBP-
is induced during G1 of the cell
cycle. In contrast to COMMA D cells, C/EBP-
and C/EBP-
mRNA
levels remain relatively constant in growth-arrested and cell
cycle-induced NIH3T3 cells. However, C/EBP homologous protein (CHOP10)
mRNA levels markedly increase in growth-arrested NIH3T3 cells. Both
COMMA D and NIH3T3 cells express growth arrest-specific (gas1) and JunD
during G0. These results demonstrate that COMMA D and
NIH3T3 cells achieve a common growth arrest (G0) state by
cell-specific strategies that involve the induction of different C/EBP
isoforms.
CCAAT/enhancer-binding proteins
(C/EBP)1 are a highly conserved family of
leucine zipper type (bZIP) DNA-binding proteins (1). C/EBP-, the
first leucine zipper protein described, plays an important role in
growth control and differentiation of adipocytes and hepatocytes
(2-9). Additional C/EBP isoforms (C/EBP-
, C/EBP-
, C/EBP-
,
C/EBP homologous protein (CHOP10)) have subsequently been described in
a variety of species and cell types (1). C/EBP isoforms are generally
characterized by an intronless gene structure, a high degree of
sequence homology in the leucine zipper and basic regions, and the
capacity to form homo- and heterodimers (1-3). Heterodimers may form
among C/EBP family members, between C/EBP family members and other bZIP
proteins (such as Fos and CREB/ATF (1-3)). C/EBPs may also interact
with other transcription factors and cell cycle regulatory proteins.
For example, NF-IL6 (C/EBP-
) is coinduced and cooperatively
interacts with NF-
B during the acute phase response in liver (10).
NF-IL6 (C/EBP-
) also binds to the hypophosphorylated form of the
retinoblastoma protein (RB) (11).
C/EBP gene expression is regulated at multiple levels. A variety of
external stimuli, including cytokines, hormones, and growth factors
influence C/EBP isoform transcription rates (5, 10, 12). In addition,
C/EBP- and C/EBP-
mRNAs undergo alternative AUG initiation
codon usage, resulting in the translation of two protein products with
different biological activities from a single mRNA (13, 14).
Post-translational control by phosphorylation influences the
subcellular localization and DNA binding activity of C/EBP isoforms
(15-17).
C/EBPs have been implicated in the regulation of growth and
differentiation of a wide variety of cells including hepatocytes, adipocytes, intestinal epithelial cells, and muscle and myelomonocytic cells (2-10, 18-21). C/EBP- induces growth arrest and the
expression of differentiation-specific genes in hepatocytes and 3T3-L1
cells (4-6, 8, 9). C/EBP-
(NF-IL6, LAP) has been implicated in
growth control of hepatocytes and the direct conversion of NIH3T3 cells
to adipocytes (20, 21). Another C/EBP family member,
/EBP
mologous
rotein (CHOP10), functions in
G0 and G1/S phase growth control in NIH3T3
cells (22). Translocations involving CHOP10 have been identified in a
high percentage of human mixoid liposarcomas (23). C/EBP-
has not
been previously associated with growth arrest (20); however, C/EBP-
has been linked to early events in the hormone-dependent
differentiation of 3T3-L1 cells and to the acute phase response in
liver (3, 10, 12).
The overall goal of this study was to investigate the influence of the cell cycle on C/EBP isoform expression in COMMA D mammary epithelial cells. The mammary gland differs from most tissues in that it attains full functional capacity relatively late in life, at sexual maturation (24, 25). In the adult female, the mammary gland retains the capacity to undergo hormone-induced proliferation, differentiation, and involution throughout the reproductive years (24, 25). The growth control mechanisms underlying this unique biology are poorly understood.
Much of our current understanding of growth control and the mammalian
cell cycle is derived from studies with fibroblast cell lines (26).
Epithelial cells, however, utilize unique transcriptional control
mechanisms (27) and express some early growth response genes that
differ from fibroblasts and lymphocytes (28). Epithelial cell-specific
differences in the expression of growth response genes may also extend
to growth arrest response genes (29). Epithelial cells are commonly
implicated in human cancers (28). Understanding the growth arrest
response in mammary epithelial cells is of particular significance as
breast cancer is the leading cause of cancer and the second leading
cause of cancer deaths among women in the United States (30). Our
results demonstrate that growth arrest is associated with the
expression of different C/EBP isoforms in mammary epithelial cells and
fibroblasts. The specific induction of C/EBP- in growth-arrested
COMMA D mammary epithelial cells and its disappearance during the cell
cycle suggest a novel tissue-specific growth regulatory role for this
C/EBP isoform in mammary epithelial cells. This regulatory role is
supported by a delay in growth arrest of COMMA D cells expressing
C/EBP-
antisense.
COMMA D, a nontransformed line of mammary
epithelial cells (31, 32), were cultured in complete growth medium
containing Dulbecco's modified Eagle's medium with 4.5 g/liter
D-glucose and supplemented with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 µg/ml bovine insulin, 5 units/ml
penicillin, and 5 µg/ml streptomycin. NIH3T3 cells (ATCC CRL 1658)
were cultured in similar complete growth media without the addition of
epidermal growth factor. Cell cycle experiments were carried out by
incubating near-confluent cultures for 72 h in growth arrest media
containing Dulbecco's modified Eagle's medium with 4.5 g/liter
D-glucose, 0.5% fetal bovine serum, 5 units/ml penicillin,
and 5 µg/ml streptomycin. After reintroducing complete media, cells
were harvested at the designated times for RNA or protein analysis.
Growth arrest experiments were carried out by switching near-confluent
cultures from complete growth media to growth arrest media and
harvesting at the designated times. To produce stable antisense cell
lines COMMA D cells were transfected with a C/EBP- 400-base pair
partial cDNA fragment (antisense orientation) and vector controls
(pcDNA3, Invitrogen, Palo Alto, CA). Stable cell lines were
drug-selected, and single cell clones were isolated and expanded as
described (33).
Total RNA was isolated
using RNAzol B (Tel-Test, Inc., Friendswood TX). Northern blots were
carried as described (34). Blots were probed with the following random
primer labeled cDNAs: C/EBP-, C/EBP-
, C/EBP-
, CHOP10, gas1
cDNA, and histone 2B (Oncor, Gaithersburg, MD). Cyclophilin
receptor protein partial cDNA was used as a constitutive probe
(34). Results are representative of 2-4 experiments. Western blots
were performed by standard procedures (35). Rabbit antisera against a
C/EBP-
peptide (ARGPLKREPDWGDGDA (2)) was used to detect C/EBP-
.
Rabbit anti-C/EBP-
and anti-proliferating cell nuclear antigen
antisera were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.
A goat anti-rabbit antibody was used as a secondary antibody, and the
signal was developed with the ECL system (Amersham).
Nuclear pellets were
isolated and EMSA were carried out essentially as described by Ray
et al. (10, 16). C/EBP consensus (5-TGCAGATTGCGCAATCTGCA-3
) and mutant binding sites (Santa Cruz, Palo
Alto, CA) were 5
end-labeled with [
-32P]ATP using T4
polynucleotide kinase. For antibody interaction studies, appropriate
dilutions of specific antisera to C/EBP-
and control (preimmune)
sera were added to the EMSA reactions.
Cultures were synchronized in a growth-arrested (G0)
state by serum and growth factor withdrawal for 72 h, and the cell
cycle was initiated by the introduction of complete growth media.
Growth arrest (G0) and synchronous cell cycle progression
were verified by [3H]thymidine incorporation (data not
shown). C/EBP- mRNA content was elevated in growth-arrested
(G0) COMMA D cells after 72 h of serum and growth
factor withdrawal (Fig. 1). C/EBP-
mRNA content decreases rapidly (within 1 h of cell cycle induction), increases slightly during G1/early S phase (4-12 h after cell cycle
induction), and then declines during the remainder of the cell cycle.
In contrast, C/EBP-
mRNA content increases during early
G1/S phase, then gradually declines to base-line
levels. Growth arrest-specific 1 (gas1) mRNA, originally
characterized in growth-arrested NIH3T3 cells (36, 37), is highly
induced in quiescent (G0) COMMA D cells, declines during
G1, and then increases somewhat through S phase (36, 37).
Histone 2B (H2B) mRNA content was minimal after 72 h of growth
arrest, began to accumulate 8 h after cell cycle induction, peaked
at 18-20 h, and then declined.
Growth arrest (G0) and cell cycle progression had little
effect on C/EBP- or C/EBP-
mRNA levels in NIH3T3 cells. gas1
mRNA levels exhibited a growth-regulated biphasic pattern (high in G0, low in G1, moderate in S), similar to that
observed in COMMA D cells and previous reports from NIH3T3 cells (36,
37). H2B mRNA began to accumulate to significant levels in NIH3T3
cells 8-12 h after cell cycle induction, consistent with a synchronous cell cycle.
To investigate the association between C/EBP- and the onset of
growth arrest, time points were taken 1, 12, and 24 h after growth
arrest induction by serum and growth factor withdrawal. In COMMA D
cells, C/EBP-
mRNA levels began to accumulate after 12 h
and persisted after 24 h of growth arrest induction (Fig. 2). CHOP10 mRNA content decreased, and C/EBP-
and
JunD mRNA levels increased slightly after 24 h of growth
arrest induction.
In NIH3T3 cells, C/EBP- and C/EBP-
mRNA levels remain
relatively unchanged 24 h after growth arrest induction (Fig. 2). In contrast to COMMA D, CHOP10 mRNA content was markedly increased 24 h after growth arrest induction in NIH3T3 cells. JunD, a
previously described negative growth regulator in NIH3T3 cells (38),
increased slightly after 24 h of growth arrest in NIH3T3
cells.
The growth arrest response initiated by serum and growth factor
withdrawal may differ from density-dependent growth arrest, even though both induce a G0 state (39). To investigate the influence of density-dependent growth arrest on C/EBP-
expression, RNA was isolated from COMMA D cells after high density
plating (day 0) and grown to confluence in complete media (days 1 and 2). H2B mRNA content decreased as COMMA D cells reached confluence (day 2) (Fig. 3A). C/EBP-
mRNA content
was low in growing and near-confluent cells (days 0 and 1). C/EBP-
mRNA content increased in concert with the initiation of
density-dependent growth arrest and the decline in H2B
mRNA content (day 2). Day 2 COMMA D cultures were refed fresh
growth media, and one synchronous round of the cell cycle was initiated
as evidenced by an increase in H2B mRNA content and a decrease in
C/EBP-
mRNA content (day 3). This was followed by decreasing H2B
mRNA and increasing C/EBP-
mRNA levels (day 4 and 5) as
COMMA D cells re-enter a density-dependent, growth-arrested state.
Histone 2B mRNA content declined as NIH3T3 cells entered
density-dependent growth arrest (Fig. 3B).
C/EBP- mRNA content, however, remained relatively high in NIH3T3
cells regardless of cell density or growth status. C/EBP-
mRNA
was also induced in a second density-dependent
growth-arrested mouse mammary epithelial cell line, NMuMG cells (data
not shown).
COMMA D C/EBP- protein content declines during the cell cycle.
Nuclear C/EBP-
protein levels are elevated in 72-h growth-arrested COMMA D cells, then decline as cells synchronously progress through the
cell cycle (Fig. 4A). The identity and
potential function of a second lower molecular weight band in the
nuclear, but not the cytoplasmic, compartment is unknown. As COMMA D
cultures synchronously progress through the cell cycle, the nuclear
C/EBP-
protein content declines, and the cytoplasmic C/EBP-
content increases slightly. This suggests some degree of subcellular
localization of C/EBP-
in growing versus growth-arrested
COMMA D cells. C/EBP-
protein content, which is predominately
localized to the nucleus in COMMA D cells, increases slightly 6 and
12 h after cell cycle induction (data not shown). Proliferating
cell nuclear antigen is detectable in the nucleus 12 h after cell
cycle induction, consistent with normal cell cycle progression
(Fig. 4B).
In contrast to the cell cycle, C/EBP- nuclear protein content
increases in growth-arrested COMMA D cells. Low levels of C/EBP-
protein are detectable in the cytoplasm, but not the nucleus, of
growing COMMA D cells (Fig. 5A, lanes
1 and 2). C/EBP-
nuclear protein content increases
significantly after 48 h of growth arrest (Fig. 5A,
lanes 3 and 4). Increases in COMMA D C/EBP-
nuclear protein content are detectable as early as 8 h after the
withdrawal of serum and growth factors (data not shown). C/EBP-
protein, which is also primarily localized to the nucleus in COMMA D
cells, was relatively unchanged after 2 days of growth arrest (Fig.
5B, lanes 1-4). Specific binding to a C/EBP
consensus site increases in nuclear extracts from 2- and 4-day
growth-arrested COMMA D cells compared to day 0 (growing) cells (Fig.
6A). No binding was detected to a C/EBP
mutant site (Fig. 6A). Preincubation of COMMA D nuclear
extracts with anti C/EBP-
antisera, but not preimmune sera,
decreased C/EBP binding activity (Fig. 6, B and
C).
To more directly address the role of C/EBP- in COMMA D cells, stable
cell lines expressing a C/EBP-
antisense construct were produced.
Growth arrest induces C/EBP-
protein levels in control
(vector-transfected) COMMA D cells, but not in the C/EBP-
antisense
expressing cell line,
3 (Fig. 7A). The
influence of the C/EBP-
antisense expression on the growth arrest
response of
3 cells was assessed. C/EBP-
antisense expression in
3 cells significantly delays the
3 growth arrest response
compared with controls. [3H]thymidine incorporation
(indicator of DNA replication) rapidly declines (within 12 h) in
control cells after serum and growth factor withdrawal of
near-confluent cultures (Fig. 7B).
[3H]Thymidine incorporation continues to decline to near
base-line levels after 48 h of growth arrest in control cells. In
3 cells expressing C/EBP-
antisense, [3H]thymidine
incorporation remains elevated for 12-18 h, then begins to decline for
the first 30 h of the growth arrest response. In addition,
C/EBP-
antisense expression in
3 cells results in a slower
decline in
3 histone 2B mRNA content and a slower accumulation of
3 gas1 mRNA over the first 30 h of the growth arrest
response.
The mammary gland exhibits a unique tissue-specific pattern of
growth and development that is well-characterized at the hormonal level
but poorly understood at the molecular level. These results implicate
C/EBP- in the growth arrest response of COMMA D cells, a mouse
mammary epithelial cell line. The results also demonstrate differences
between the expression and potential function of C/EBP-
in COMMA D
cells compared with fibroblast-derived cells. Earlier reports with
3T3-L1 cells indicated that C/EBP-
expression peaks during active
cell division and decreases as 3T3-L1 cells withdraw from the cell
cycle and terminally differentiate (40). A relatively similar temporal
pattern of C/EBP-
expression occurs in differentiating myelomonocytic cells (19). The tissue-specific functions of C/EBP-
are also apparent in liver, where C/EBP-
expression increases early
in the proliferative response following partial hepatectomy and during
the acute inflammatory response (9, 10, 16, 41). In response to
inflammation, C/EBP-
transactivates the human complement component 3 and serum amyloid A1 genes (10, 16, 41). These results indicate that
C/EBP-
is expressed in a wide range of tissues, but its pattern of
induction and potential role in cell function, particularly growth
regulation, is highly tissue-specific.
The association between C/EBP- and growth arrest in mammary
epithelial cells may be specific to treatments that induce withdrawal from the cell cycle, or G0. For example, serum/growth
factor withdrawal and contact inhibition both induce G0 and
both induce C/EBP-
. If C/EBP-
is blocked by antisense RNA
expression, G0 induction by serum and growth factor removal
is markedly delayed. In addition, cell lines expressing C/EBP-
antisense RNA grow to a higher cell density than the parental COMMA D
cells and vector-transfected controls (data not shown). This suggests
that the removal or inactivation of C/EBP-
inhibits the initiation
of G0 by contact inhibition. Finally, drugs that induce
blocks within the cell cycle, such as hydroxyurea (G1/S
block) or nocodazole (G2 block), do not induce C/EBP-
(data not shown). Similar perturbations of the growth arrest response
occur in hepatoma cells expressing C/EBP-
antisense RNA, indicating
that C/EBP isoforms may function in the growth arrest response of a
variety of tissues (42). It is noteworthy that cells expressing
antisense RNA to the known tumor suppressor genes BRCA1 and Waf1 are
also aberrant in G0 induction and maintenance (43, 44).
The in vitro induction of C/EBP- in mammary epithelial
cells after serum and growth factor withdrawal parallels in
vivo observations during the immediate postweaning
period.2 Mammary gland C/EBP-
gene
expression is induced early in the postweaning period in conjunction
with the decline in lactogenic hormones and preceding the onset of
apoptosis in the mammary epithelial compartment (45). These results
suggest that C/EBP-
may play a role in reprogramming the mammary
epithelial cell after the removal of hormone and growth factor
stimulation. The specific gene(s) transactivated by C/EBP-
during
mammary epithelial cell growth arrest are unknown. However, C/EBP
isoforms have been implicated in the transactivation of the
-casein
gene during lactation (46, 47).
The rapid decline in C/EBP- mRNA following cell cycle initiation
suggests that C/EBP-
expression is tightly linked to the growth-arrested state in COMMA D cells. Similar results have been obtained in HC11 cells, a subline of the COMMA D cell line that retains
the capacity for differentiation (data not shown). Preliminary experiments indicate that C/EBP-
mRNA has a relatively short half-life, about 30 min, in both growing and quiescent COMMA D cells
(data not shown). This suggests that the rapid decrease in C/EBP-
mRNA content (10-fold decrease within 1 h of cell cycle induction) is due to a decrease in C/EBP-
gene transcription.
The role of C/EBP- in mammary epithelial cell growth control may be
tissue-specific. C/EBP-
has been consistently associated with growth
arrest in adipocytes and hepatocytes (4, 8, 9). Under the conditions
employed in these experiments, neither cell type expressed significant
amounts of C/EBP-
(3-9). Although C/EBP-
has been shown to
inhibit hepatoma growth, transient increases in C/EBP-
gene
expression, nuclear localization, and DNA binding activity occur during
the early G1 phase of the cell cycle and after partial
hepatectomy (9, 48, 49). In mammary epithelial cell cultures, C/EBP-
mRNA and nuclear protein content also transiently increase during
G1. This is consistent with a role for C/EBP-
in the
induction of immediate early gene promoters (48, 49). Mammary gland
C/EBP-
mRNA and protein levels are elevated during the
proliferative phase of pregnancy/lactation (45). Murine CHOP10 shares
extensive sequence homology with the hamster growth arrest DNA damage
(GADD 153) gene (22, 50, 51). CHOP10 mRNA is constitutively
expressed in adult skeletal muscle, liver, heart, and brain (22).
CHOP10 mRNA is also induced by growth arrest and DNA damage,
although the mechanisms of induction under these different conditions
may vary (22). Microinjection experiments suggest a role for CHOP10 at
the G1/S checkpoint (50). The rapid and persistent
elevation in CHOP10 mRNA content in response to serum and growth
factor withdrawal in NIH3T3 cells further supports a role for CHOP10 in
the initiation and maintenance of the growth arrest response in
fibroblasts. In COMMA D cells, CHOP10 mRNA content declines
somewhat early in the growth arrest response, then increases in long
term growth arrest, possibly as a result of glucose deprivation in long
term cultures (data not shown). Although CHOP10 induction patterns
differ between growth-arrested COMMA D and NIH3T3 cells, CHOP10 is
induced in both cell types in response to DNA damaging agents (data not
shown). Growth arrest-specific (gas) genes, which were originally
described in NIH3T3 cells, are induced during growth arrest and
down-regulated during G1 (36, 37). Although previous
reports suggested that gas genes were not expressed in epithelial cells
(29), we observed biphasic cell cycle-regulated gas1 mRNA patterns
in both COMMA D and NIH3T3 cells (Fig. 1). These results indicate that
gas1 plays a general role in growth control in both fibroblasts and
epithelial cells. It is of interest that gas1 and C/EBP-
are
representative of a small group of genes that are induced during
G0. The novel induction of C/EBP-
during G0
in mammary epithelial cells suggests that understanding its regulation
may have important implications in aberrantly growing cells or in
breast cancer.
Both C/EBP- and CHOP10 form dimers with other bZip proteins and
function as DNA-binding proteins (3, 10, 16, 51). Studies are in
progress to identify C/EBP-
dimerization partners and the genes
controlled by these transcription factor complexes during mammary
epithelial cell growth arrest. Studies are also in progress to define
the molecular mechanisms underlying C/EBP-
induction during growth
arrest. Since the C/EBP-
gene exhibits transcriptional
autoregulation, similar regulatory mechanisms may also influence the
expression of other C/EBP family members, such as C/EBP-
(5). Primer
extension analysis indicates that the C/EBP-
transcription
initiation site is similar in growing COMMA D cells, growth-arrested
COMMA D cells, involuting mammary gland, and NIH3T3 cells (data not
shown). This suggests that divergent biological signals may converge
into a single, or limited number, of transcriptional activators of the
C/EBP-
gene.
We thank Dr. Dan Medina, Baylor College of
Medicine, Houston, TX for the COMMA D cells; Dr. Steven McKnight,
Tularik, San Francisco, CA for the C/EBP-, C/EBP-
, and C/EBP-
cDNAs; Dr. David Ron, New York University, New York, NY for the
CHOP10 partial cDNA; and Dr. Claudio Schneider, Cosorzio
Interuniversitario Biotecnologie, Trieste, Italy for the gas1 cDNA.
We also thank Dr. Steven McKnight (Tularik, San Francisco, CA) and Dr.
Richard Hanson (Case Western Reserve University, Cleveland, OH) for
helpful comments on the manuscript.