(Received for publication, February 1, 1995; and in revised form, June 9, 1995)
From the
Nuclear factor B (NF-
B) is a pleiotropic transcription
factor which regulates the expression of a large number of cellular and
viral genes. Induction of NF-
B has been shown previously to occur
during cell cycle transition from G
to G
, but
the relationship of cytokine induction of this transcription factor to
cell cycling has not been directly addressed. Here we examine the
induction of NF-
B in serum-deprived and cycling cells in response
to tumor necrosis factor-
(TNF-
). In 3T3 fibroblasts deprived
of serum, and in the temperature-sensitive G
phase mutant
carcinoma line FT210, we find that NF-
B DNA binding activity is
rapidly induced upon addition of TNF-
. In addition, NF-
B
induction in cycling cells occurs without a significant change in cell
cycle distribution. These data reveal that NF-
B is rapidly induced
by TNF-
in both proliferating and arrested cells and suggest that
distinct activation pathways can lead to cell cycle-dependent or
-independent induction of NF-
B.
Nuclear factor B (NF-
B) (
)is comprised of a
family of at least five transcription
factors(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) related to and including the Rel
oncoprotein(12, 13, 14, 15) . First
identified as an activity in B cells which binds to a 10-base pair
conserved element in the immunoglobulin light chain
enhancer(16) , NF-
B is now known to be widely expressed in
mammalian cells. The best characterized form of NF-
B is a
heterodimer composed of 50- and 65-kDa subunits (15) ; however,
both homodimeric and heterodimeric forms of NF-
B/Rel in most
combinations are able to bind, albeit with different affinities, to
B-like regulatory elements found in a diverse array of cellular
and viral
genes(9, 10, 17, 18, 19) .
In most unstimulated cells NF-
B is thought to reside in the
cytoplasm bound to an inhibitor protein, designated
I
B(20, 21, 22) ; upon stimulation or
activation of the cell by
cytokines(23, 24, 25, 26) , phorbol
esters(27) , and viral
trans-activators(28, 29) , NF-
B is able to
rapidly dissociate from I
B and translocate into the nucleus, an
event which involves the phosphorylation of
I
B(30, 31) .
In order for most nontransformed
cells to progress through the cell cycle, appropriate extracellular
signals are required(32) . In cultured cells, these signals are
usually provided by the presence of growth factors contained within
serum added to the
media(33, 34, 35, 36) . Deprivation
of these factors has been shown to result in the cessation of both cell
growth and nuclear division, and the cells become arrested in a
quiescent state known as G(36) . Cells arrested in
this manner may remain in G
for days or weeks without a
significant decrease in viability. The subsequent addition of serum to
such cells allows re-entry into G
, which is followed by
continued growth and cell division(32) . Many of the growth
factors contained within serum have been characterized, as well as the
cellular factors which they
induce(37, 38, 39, 40) . In many
cases, the addition of serum results in the induction of mRNAs encoding
transcription factors, such as c-myc, c-fos, and
c-jun(41, 42, 43, 44, 45, 46, 47, 48) .
Activation of these factors is probably necessary to induce the
expression of sets of genes whose products are essential for
progression through the cell cycle.
Previous studies have revealed
that NF-B also fits into this category of serum-induced or
immediate-early genes(49) ; the addition of serum to
G
-arrested 3T3 cells has been shown to induce both nuclear
translocation of NF-
B and
B-directed transcriptional
activation. However, while NF-
B is induced at the G
to
G
transition, it is unknown whether this is the only stage
of the cell cycle at which NF-
B induction can occur. In addition,
it is unclear whether induction of NF-
B is associated with changes
in the distribution of cells at different points of the cell cycle.
Here we show, using cell cycle analysis of 3T3 cells and the G
mutant mouse mammary carcinoma line, FT210(50) , that
NF-
B may be activated with equivalent efficiency in both
proliferating and arrested cells. Furthermore, we show that activation
of NF-
B has no effect on the rate of progression or checkpoint
arrest throughout cell cycle. We conclude that NF-
B may be induced
in both a cell cycle-dependent and -independent manner and suggest a
model in which distinct signaling mechanisms can lead to NF-
B
activation.
Figure 1:
Effects of serum addition and TNF-
treatment on cycling versus growth-arrested 3T3 cells. 3T3
cells were transfected with multimerized
B reporter plasmid (A) or a
B mutant control (B), as described
under ``Materials and Methods.'' Cells were either
serum starved or mock treated 12 h after transfection. Serum or
TNF-
addition was performed 36 h post-transfection, and cells were
harvested 48 h after transfection. Transfected cells were harvested,
protein extracts made, and CAT assays performed as described under
``Materials and Methods.'' The results shown are
representative of at least two independent
experiments.
Figure 2:
Evidence that TNF- can induce nuclear
NF-
B activity in both cycling and G
-arrested cells.
3T3 cells were seeded at 50-70% confluence and maintained for 24
h in medium containing 10% serum (lanes 1 and 2) or
0.5% serum (lanes 3-7), followed by the addition of
TNF-
(lanes 2, 4-7) for 2 h. Nuclear
extracts were prepared and analyzed by EMSA as described under
``Materials and Methods.'' Lanes 4-6 show the results of a competition experiment in which reactions
additionally contained 10 ng of unlabeled double-stranded
oligonucleotide encompassing either the wild type
B site (lane
6) or a mutated
B control (lane
7).
Figure 3:
Cell cycle analysis of 3T3 cells. Aliquots
of the transfected 3T3 cells were taken at the same time as the harvest
for CAT assay as described in the legend to Fig.1, washed with
phosphate-buffered saline, resuspended in nuclear staining solution,
and analyzed by flow cytometry as described under ``Materials and
Methods.'' Representative samples are shown depicting
cycling cells, mock treated with 10% serum (a), cycling cells
treated with 20% serum for 24 h (b), cycling cells treated
with TNF- for 12 h (c), serum-starved cells, mock treated
with 0.5% serum for 12 h (d), serum-starved cells treated with
20% serum for 12 h (e), and serum-starved cells treated with
TNF-
for 12 h (f). The regions between the vertical
lines from left to right represent cells in
G
/G
, S, and G
/M,
respectively.
Figure 4:
Activation of B-directed CAT
expression in G
/M-arrested FT210 cells. FT210 cells (A) and the parental line, FM3A (B) maintained at 33
°C, were transfected with either the wild type (wt) or
mutant
B reporter plasmids as described under ``Materials and
Methods.'' Each sample was split equally between two
flasks 4 h after transfection; one sample was maintained at 33 °C
while the other was incubated at 39 °C, as indicated. Preparation
of cellular extracts and CAT analyses were performed as described under
``Materials and Methods.'' TNF-
addition was
performed 24 h after transfection, and cells were harvested 48 h
post-transfection. C, cell cycle analysis of FM3A and FT210
cells. FT210 and FM3A cells were incubated at 33 or 39 °C as
indicated, for 24 h, and analyzed by flow cytometry as described under
``Materials and Methods.''
Figure 5:
Induction of NF-B activity by
TNF-
in G
/M-arrested FT210 cells. FT210 cells were
incubated for 24 h at 39 °C and stimulated with 10 ng/ml PMA or 200
units/ml TNF-
for 2 h. Preparation of nuclear extracts and EMSA
were performed as described under ``Materials and Methods.''
The arrow indicates the position of the inducible,
B-specific nucleoprotein complex.
Cellular growth and differentiation is regulated by a
combination of growth factors which are present in serum at nanomolar
to picomolar concentrations. Addition of serum growth factors to
G-arrested cells induces a set of genes, often encoding
transcription factors, whose products are required for progression into
G
and continued cell growth. Previous studies have
demonstrated that NF-
B represents an immediate-early response gene (49) . Like other immediate-early response genes, induction of
NF-
B occurs in the presence of protein synthesis
inhibitors(49, 53) presumably due to its presence as
a covert, cytoplasmic complex with I
B. As shown
previously(49) , we find that NF-
B is inducible following
serum addition to serum-deprived cells. However, we also find that
NF-
B induction in response to TNF-
occurs in a cell-cycle
independent fashion.
The data reveal that NF-B can be induced
in serum-deprived cells by cytokines ( Fig.1and Fig. 2).
At least two possibilities existed to account for these effects. First,
the activation of NF-
B might itself be sufficient to induce
checkpoint release, leading to re-entry into the cell cycle. Second,
the activation of NF-
B by cytokines might occur independently of
cell cycle, predicting that NF-
B induction alone would not change
the cell cycle profile. The data presented in Fig.3appear to
fit the second model; induction of NF-
B in G
-arrested
cells by TNF-
did not dramatically affect the cell cycle profile,
while in the same experiment the addition of serum almost completely
relieved the G
arrest. Under some conditions, TNF-
stimulation has been shown to shorten the duration of S
phase(54) , but this finding was not observed in our
experimental system, using a different cell type and alternative
conditions of growth arrest (Fig.3, Table 1). The
addition of TNF-
to G
/M-arrested FT210 cells induced
NF-
B ( Fig.4and Fig. 5), providing further support
for this model.
Interestingly, in both 3T3 and FT210 cells, a
significant degree of cell death was observed after TNF- addition
to arrested cells, starting 2-3 h following addition of cytokine. (
)This effect was not observed with IL-1 which, at least in
3T3 cells, induced NF-
B to the same extent as TNF-
(data not
shown). This result suggests that the signaling events leading to cell
death by TNF-
are mediated by the Fas-related domains (55) and is therefore a consequence of apoptosis rather than
induction of NF-
B per se.
Considering the rapid
kinetics of induction (seconds to minutes), as well as its ability to
be induced in the presence of protein synthesis
inhibitors(53, 56) , NF-B is a good candidate as
both an immediate-early, cell cycle-responsive factor, and a
cytokine-induced, cell cycle-independent factor. It is most likely that
alternative signal transduction pathways are utilized in these two
different events. In support of this hypothesis, much evidence has
accumulated suggesting the involvement of serine-threonine kinases,
including protein kinase C, in the cytokine-induced (cell
cycle-independent) activation of
NF-
B(30, 57, 58) . However, the
observation that PMA was unable to induce NF-
B in FT210 cells
suggests that protein kinase C-independent pathways of NF-
B
activation might also exist, as described previously in another cell
type(23) . Analysis of cytoplasmic extracts following cellular
stimulation has revealed that only a fraction of NF-
B relocates to
the nucleus.
This suggests that there may be distinct
cytoplasmic stores of NF-
B, perhaps associated with different
isoforms of I
B. These forms could be subject to modification by
distinct kinases and phosphatases, allowing for regulation by distinct
signaling pathways. Furthermore, several studies have raised the
possibility of distinct pathways being involved in the cell
cycle-dependent induction of NF-
B. The first of these is the
implication that the addition of platelet-derived growth factor, an
event which activates the intrinsic tyrosine kinase activity of the
platelet-derived growth factor receptor, to serum-starved 3T3 cells can
induce NF-
B(49) . Also, stimulation of the T cell
costimulatory molecule, CD28, has been shown to induce
NF-
B(59) . Finally, induction of NF-
B by some stimuli
can be blocked by the tyrosine kinase inhibitor, herbimycin A, but this
reagent is unable to inhibit NF-
B induction by
TNF-
(60) . We therefore speculate that independent
signaling pathways converge to activate similar NF-
B family
members. It remains to be determined whether this convergence occurs
upstream of I
B, for example, by regulation of a kinase of
I
B(61) , downstream by modification of the NF-
B
family members themselves (62) or at the level of I
B
itself, perhaps by differential phosphorylation or degradation. The
dissection of these signaling events, as well as the levels at which
they converge, will provide insight into the cell cycle-dependent and
-independent mechanisms of cellular and viral gene regulation.