Received 22 January 1999/Returned for modification 3 March
1999/Accepted 3 May 1999
 |
INTRODUCTION |
NF-
B belongs to the Rel family of
transcription factors which regulate genes involved in immune and
inflammatory responses (3, 5, 70). In mammals, the Rel
family is composed of RelA/p65, c-Rel, RelB, p50 (NF-
B1), and p52
(NF-
B2), which have sequence similarity over approximately 300 amino
acids in the amino-terminal half of the protein. NF-
B subunits are
able to homo- or heterodimerize to form transcription factor complexes with a range of DNA-binding and activation potentials. Although all Rel
members bind DNA, only RelA/p65 (hereafter referred to as p65), c-Rel,
and RelB have extended carboxy termini harboring transactivation
function (70). The most widely studied form of NF-
B is a
heterodimer of the p50 and p65 subunits and is a potent activator of
gene transcription (56).
In most cells, NF-
B is found sequestered in the cytoplasm bound in
an inactive complex with its natural biological inhibitor I
B
(3, 70). The I
B family members include I
B
,
I
B
, p105/I
B
(precursor of p50), p100 (precursor of p52),
and I
B
(41, 74). Each has in common a series of
ankyrin repeats which interact with the DNA-binding domain and the
nuclear localization signal of NF-
B, thus maintaining the
transcription factor as an inactive complex. Activation of NF-
B is
induced by a variety of diverse stimuli including inflammatory
cytokines, phorbol esters, bacterial toxins (such as
lipopolysaccharide) viruses, UV light, and a variety of mitogens
(4, 5). Treatment of cells with these stimuli activate the
recently discovered I
B kinase complex, leading to the
phosphorylation of serines 32 and 36 of I
B
or serines 19 and 23 of I
B
(19, 46, 52, 77). This phosphorylation event
targets I
B for ubiquitin-dependent degradation through the 26S
proteasome complex, resulting in the release and nuclear translocation
of NF-
B (22, 68).
In addition to its well-established role in activating the
transcription of genes involved in immunological responses, studies indicate that NF-
B also functions in promoting cell growth. For instance, lymphocytes from mice lacking p50, p65, or c-Rel are defective in mitogenic responses (20, 38, 58, 65), and p50/p52 double-knockout animals fail to generate mature osteoclasts and
B cells (25, 35). Recent reports also demonstrate the expression of NF-
B/Rel proteins in the proliferative zone of the
developing avian limb bud and the requirement of NF-
B for the proper
growth of this tissue (15, 36). In addition, deregulated NF-
B activity has been associated with oncogenesis, since reports show elevated NF-
B/Rel levels in primary breast cancers (18, 66). NF-
B is activated by oncogenic Ras and is required by Ras
to induce foci in NIH 3T3 cells (23). Similarly, the
chimeric oncoprotein Bcr-Abl, implicated in acute lymphoblastic and
chronic myelogenous leukemias, also requires NF-
B to induce cellular transformation (54). Consistent with this latter study,
Hodgkin's lymphoma cells depleted of NF-
B activity revealed
strongly impaired tumor growth in mice (7). The ability of
NF-
B to protect cells against chemotherapeutic drugs or TNF-mediated
apoptosis function (9, 69, 72, 75), suggests that
NF-
B-regulated growth control may be related to its cell survival
properties. In fact, inhibition of NF-
B led to apoptosis in cells
expressing oncogenic forms of Ras (45). Finally, recent
demonstrations that cellular proliferation defects, attributed to the
absence of NF-
B, are associated with a delay in cell cycle
progression in G1 (7, 28), in addition to the
previously described physical association with NF-
B and CBP/p300
(26, 50), establishes a link between NF-
B and regulators
of the cell cycle. Although the above cited reports strongly suggest a
role for NF-
B in cell growth control, the molecular mechanism(s)
underlying this regulation remains unclear.
To gain insight into the role of NF-
B in regulating cell growth, we
first used a well-established skeletal myogenesis model, which is
characterized by the maturation of precursor myoblasts into
differentiated contractile myotubes. This cellular process is dependent
on the activation or induction of the myogenic basic helix-loop-helix
(bHLH) and MEF2 families of transcription factors that stimulate
tissue-specific gene expression, as well as changes in cell cycle
regulators that cause myoblasts to undergo irreversible growth arrest
(39, 48, 71). The latter process is regulated by a balance
in activities of cyclin-cyclin-dependent kinase (cdk) complexes and
their respective known kinase inhibitors. The signal to induce
differentiation, achieved most commonly in tissue culture by removing
growth factor-rich medium, causes the downregulated expression of
cyclins A and D1 and kinases cdk2 and cdc2, with an induced synthesis
of the cdk inhibitors p18 and p21 and stabilization of the p27 protein
(71). This regulated switch in activities leads to the
dephosphorylation of the product of the retinoblastoma susceptibility
gene (pRb), which maintains cells in a G1-arrested state by
inhibiting the E2F-DP1 transcription factor complex. Exit from the cell
cycle, therefore, is critical for myogenic transcription and completion
of the differentiation program.
Using the murine C2C12 skeletal muscle cell line, we demonstrate that
NF-
B functions in proliferating myoblasts to inhibit their
differentiation process. This was determined by showing that C2C12
myoblasts contain NF-
B in their nuclei and that NF-
B DNA-binding
activity and transactivation function are reduced during myogenesis. In
addition, myoblasts generated to lack NF-
B activity are greatly
accelerated in their differentiation program. Transfections in 10T1/2
cells showed that NF-
B strongly blocks the ability of the myogenic
transcription factor, MyoD, to induce myogenesis. Furthermore, this
latter regulation is specific to the transactivation-competent p65
subunit of NF-
B, arguing that NF-
B inhibits myogenic
differentiation through its activation of gene expression. The
observation that C2C12 cells lacking NF-
B display a reduction in
their proliferation rate and exit the cell cycle faster than do control
cells suggests that inhibition of myogenesis by NF-
B is in part
related to its growth-promoting activity. Importantly, these cells also
exhibit a marked reduction in cyclin D1 protein and mRNA levels. The
results of experiments performed with 10T1/2 fibroblasts indicate that
NF-
B regulation of cyclin D1 is one mechanism by which this
transcription factor inhibits myogenic differentiation. From this
differentiation model, we expanded our study to identify the level at
which NF-
B regulated cyclin D1. The results show that this
regulation occurs at the transcriptional level and is mediated by
several authentic NF-
B DNA-binding sites in the cyclin D1 promoter.
Furthermore, by using diploid fibroblasts, we addressed the potential
relevance of NF-
B regulation of cyclin D1 with respect to the cell
cycle. Our data show that in cells stimulated to reenter the cell
cycle, NF-
B activity is required for cyclin D1 transcriptional
initiation and hyperphosphorylation of pRb, leading to progression into
S phase. Similar to what was observed in C2C12 cells, embryonic fibroblasts lacking NF-
B activity also exhibit a reduction in proliferation, in conjunction with lower levels of cyclin D1. Taken
together, these data establish that the ability of NF-
B to control
cellular proliferation and differentiation are processes tightly
coupled to its ability to transcriptionally regulate cyclin D1.
 |
MATERIALS AND METHODS |
Cell culture.
Murine C2C12 myoblast cells obtained from the
American Type Culture Collection and primary murine myoblasts (a
generous gift from J. Samulski) were cultured at 37°C in Dulbecco's
modified Eagle's medium with high glucose (DMEM-H), supplemented with
20% fetal bovine serum (FBS) and antibiotics (Life Technologies). The
cells were grown at subconfluency and passaged every 2 to 3 days. To
induce differentiation, the cells were grown overnight to 60 to 70%
confluency in growth medium (GM), washed once with phosphate-buffered
saline (PBS), and then switched to DMEM-H supplemented with 2% horse
serum and 10 µg of insulin per ml plus antibiotics (DM). C3H10T1/2
clone 8 mouse embryo fibroblasts (10T1/2), also obtained from American
Type Culture Collection, were cultured in DMEM-H containing 15% FBS
plus antibiotics and passaged every 2 to 3 days. HeLa cells were
cultured in DMEM-H with 5% FBS and 5% calf serum, NIH 3T3 cells were
grown in DMEM-H with 10% Colorado calf serum, and for mouse embryo
fibroblasts (MEFs), cells were grown in DMEM-H plus 10% FBS.
Plasmids.
For reporter plasmids, 3x
B-Luc or 3x
Bmut-Luc
contain three tandem repeats of the wild-type or mutated
B site,
from the major histocompatibility complex (MHC) class I enhancer,
respectively, fused to the luciferase reporter gene (obtained from B. Sugden, University of Wisconsin, Madison, Wis.). TnI-Luc contains a
muscle-specific enhancer in the troponin I gene fused to luciferase,
and 4RTK-Luc contains four E boxes from the muscle creatine kinase
enhancer (gifts of S. Konieczny, Purdue University). Cyclin D1 promoter reporter constructs were described earlier (1). For
expression plasmids, p50 and p65 subunits of NF-
B were expressed
from a cytomegalovirus (CMV)-driven promoter as previously described (10). The mutant I
B
plasmid, designated I
B
SR,
was a gift of D. Ballard (Vanderbilt University). A cyclin D1
expression plasmid was generated by removing a 1,300-bp
EcoRI fragment containing the mouse cyclin D1 cDNA from the
pBSSK plasmid and inserting it into the EcoRI site of pCMV5.
pCDNA3-D3 plasmid expressing human cyclin D3 was a gift of C. Sherr
(St. Jude Children's Research Hospital). pEMC11s plasmid expressing
MyoD was obtained from the H. Weintraub laboratory (University of
Washington). Oncogenic ras was expressed from the
H-ras (V-12) plasmid as previously described
(45).
EMSAs.
Nuclear extracts for electrophoretic mobility shift
assays (EMSAs) were prepared as previously described (16),
except that 0.25% Nonidet P-40 was used to extract nuclei. A 5-µg
portion of extract were preincubated with 1 mM phenylmethylsulfonyl
fluoride and 1 µg of poly(dI-dC)-poly(dI-dC) in a volume of 12 µl
for 10 min. This mixture was subsequently incubated in a total volume of 20 µl at room temperature for 20 min with 2 × 104 cpm of a 32P-labeled oligonucleotide probe
containing a
B site (underlined) from the class I MHC promoter
(5'-CAG GGC TGG GGA TTC CCC ATC TCC ACA GTT TCA CTT C-3').
The buffer consisted of 10 mM Tris-HCl (pH 7.7), 50 mM NaCl, 0.5 mM
EDTA, 1 mM dithiothreitol, and 10% glycerol. Complexes were resolved
on a 5% polyacrylamide gel in Tris-glycine buffer (25 mM Tris, 190 mM
glycine, 1 mM EDTA) at 25 mA for 2 to 3 h at room temperature. The
gels were dried and exposed on film for approximately 1 to 3 days. For
supershift EMSAs, antibodies against specific NF-
B were added to the
nuclear extract and incubated for 10 min prior to the addition of
phenylmethylsulfonyl fluoride and poly(dI-dC)-poly(dI-dC). The
antibodies used for this portion of the study were p65 (Rockland), p50
(NLS; Santa Cruz Biotechnology), c-Rel (C; Santa Cruz Biotechnology),
and RelB (C-19; Santa Cruz Biotechnology). NF-
B-binding sites in the
cyclin D1 promoter were determined by generating a series of
oligonucleotides corresponding to both wild-type and mutant (M)
putative NF-
B sites within the human cyclin D1 promoter
(47). The oligonucleotides have the following sequences
(NF-
B wild-type and mutated sites are underlined):
858, 5'-GTG CAG
TTG GGG ACC CCC GCA AGG ACC GAC TGG TCA A-3';
858(M),
5'-GTG CAG TTC CCG ACC CCC GCA AGG ACC GAC TGG TCA A-3';
749, 5'-ACC ATC TTG GGC TGC TGC TGG AAT TTT CGG GCA TTT
A-3';
749(M), 5'-ACC ATC TTG GGC TGC TGC TCC CCT TTT CGG
GCA TTT A-3';
39, 5'-GGA CTA CAG GGG AGT TTT GTT GAA GTT
GCA AAG TCC T-3'; and
39(M), 5'-GGA CTA CAC CCC AGT TTT GTT GAA GTT GCA AAG TCC T-3'.
Transfections and viral infections.
To generate C2C12 cells
with stably integrated luciferase reporter plasmids, cells were seeded
at a density of 2 × 105 cells in 6-cm dishes 24 h prior to transfection. Cotransfections with 4 µg of reporter
plasmid 3x
B-Luc or 3x
Bmut-Luc and 1 µg of pcDNA3 (Invitrogen)
containing the neomycin resistance marker were performed with Superfect
reagent as recommended by the manufacturer (Qiagen). At 48 h
posttransfection, the cells were trypsinized and cultured at 1/30 their
density in 1 mg of Geneticin (G418; Life Technologies) per ml. Mixed
populations containing either wild-type or mutant versions of the
B
sites were allowed to expand under selection, and from the wild-type
population an individual clone expressing similar basal promoter
activity was selected. Cell extracts were prepared and luciferase
assays were performed as previously described (16).
C2C12 cells stably expressing I
B
SR or empty vector were generated
by retroviral infections as previously described (54). The
cells were seeded under identical conditions to those stated above.
Helper-free virus infection was performed in 1 ml of culture in the
presence of 2 µg of Polybrene for 3 h. Culture medium was aspirated, and fresh medium was added for 48 h. The cells were then trypsinized and replated in G418-containing medium at 1 mg/ml at a
ratio of 0.7 cell/well in a 96-well plate. The selection medium was
replaced every 4 to 5 days, and individual clones were expanded for
further study. To produce MEFs stably expressing I
B
SR, retrovirus
infections were performed as described above. The cells were then
placed under a 3-day selection of G418 at 400 µg/ml and subsequently
expanded as a mixed population for further study.
Transient transfections into 10T1/2 fibroblasts were performed by
seeding 5 × 105 cells in a 6-cm dish and growing the
cells overnight in complete medium. The following day, a total of 2.5 µg of plasmid DNA was incubated with Superfect as recommended by the
manufacturer (Qiagen). This mixture was subsequently added to the cells
with 1 ml of complete medium for approximately 3 h. The cells were
rinsed with PBS and then refed with 4 ml of complete medium overnight.
At this point, the cells were again rinsed once with PBS and then transferred to DM for a 48-h period. Cell extracts were prepared and
luciferase activity was monitored as previously described (16).
For adenovirus infections in cycling cells, HeLa cells and MEFs were
plated overnight in 10-cm culture dishes. The following day,
replication-defective adenovirus (Ad5) expressing the I
B
SR or
empty vector (CMV) were diluted in 2.5 ml of complete medium and placed
on the cells for 1 h. The volume was then raised to 10 ml, and
infections were allowed to proceed for an additional 48 h, at
which time the cells were harvested and total RNA was prepared. For
infections performed in quiescent cells, MEFs were plated overnight in
10-cm culture plates at 50 to 60% confluency and then switched for
48 h to medium containing 0.2% FBS. Infections were performed as
explained above, except that the cells were maintained quiescent for
24 h before being stimulated back into the cell cycle by the
addition of complete medium.
Western blot analysis.
Cells were harvested in PBS, and
whole-cell lysates were prepared by resuspending cell pellets in
ice-cold RIPA buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% sodium
dodecyl sulfate [SDS], 1% Nonidet P-40), and incubating the
suspension on ice for 20 to 30 min. For pRb Western blots, lysis buffer
included phosphatase inhibitors (50 mM sodium fluoride, 1 mM
orthovanadate, 50 mM
-glycerophosphate, 80 µM cantharidin).
Supernatant lysates were collected following high-speed centrifugation
for 20 min at 4°C. Equal amounts of extract were subjected to
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes (Schleicher & Schuell). Blocking was performed
in 5% nonfat dry milk-1× TBST (25 mM Tris-HCl [pH 8.0], 125 mM
NaCl, 0.1% Tween 20). Primary and secondary antibodies were diluted in
0.5% nonfat dry milk-1× TBST, and the incubations proceeded for 30 min at room temperature, with the exception of pRb blots, where the
primary antibody was incubated for 1 h. Washes were performed in
1× TBST for 5 to 10 min and repeated five times. Specific proteins
were visualized by enhanced chemiluminescence (Amersham Life Science).
Antibodies to I
B
(C-21), myogenin (M-225), cyclin D1 (R-124),
cyclin A (C-19), MyoD (M-318), and p21 (C-19) were obtained from Santa
Cruz Biotechnology, pRb antibody was obtained from Pharmingen, cyclin
D3, cdk4, and p27 antibodies were generous gifts of Y. Xiong
(University of North Carolina).
Immunofluorescence.
All immunofluorescence experiments were
performed directly in 12-well plates. Myoblasts were grown to
subconfluency in GM and then switched to DM for 48 h. For 10T1/2
fibroblasts, cells were grown overnight in complete medium following
transfections then switched to DM for 72 h. All steps were
performed at room temperature. The cells were washed in PBS and fixed
in a 2% formaldehyde-1× PBS solution for 30 min. They were
permeabilized with 0.5% Nonidet P-40-1× PBS for 5 min and then
blocked with horse serum (1:100) in PBS for 30 min. They were washed
with PBS, incubated for 1 h with anti-skeletal myosin heavy chain
(MY-32; Sigma) diluted 1:500 in 3% bovine serum albumin-1× PBS,
washed three times with PBS, and incubated for 1 h in the dark
with fluorescein isothiocyanate (FITC)-conjugated anti-mouse
immunoglobulin G (IgG; Sigma) diluted at 1:250. The cells were washed
in PBS and photographed with an Olympus inverted microscope equipped
with phase-contrast and UV illumination through an FITC filter. For
bromodeoxyuridine (BrdU) staining, MEFs grown on glass coverslips were
washed once in PBS, fixed for 5 min at room temperature with 100% cold
methanol, washed again in PBS, and permeabilized with 0.25% Triton
X-100 in PBS for 5 min at room temperature. Following a PBS wash, the
cells were treated for 10 min with 1.5 M HCl, washed four times with PBS, and incubated for 30 min with an FITC-conjugated anti-BrdU antibody (1:10 dilution in 1% bovine serum albumin-PBS [Becton Dickinson]). After several washes in PBS, the cells were
counterstained for 3 min with a 1-µg/ml solution of Hoechst dye 33258 (Sigma) in PBS. Following two additional washes in PBS, the coverslips were mounted on glass slides and examined on a Zeiss microscope.
Northern blot analysis.
Total RNA was isolated with Trizol
reagent as recommended by the manufacturer (Life Technologies). RNA
samples were fractionated on an agarose gel and transferred overnight
onto a nylon filter. The next day, RNA was cross-linked with a UV
cross-linker (Stratagene). For detection of I
B
, Id-1, Hes-1,
cyclin D3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNAs
blots were hybridized in QuickHyb buffer supplemented with 100 µg of
salmon sperm DNA as recommended by the manufacturer (Stratagene). For
detection of cyclin D1 mRNA, blots were hybridized overnight at 42°C
in 50% formamide-5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate)-1× PE (50 mM Tris-HCl [pH 7.5], 0.1% sodium
pyrophosphate, 1% SDS, 0.25% polyvinylpyrrolidene, 0.25% Ficoll, 5 mM EDTA)-150 µg of salmon sperm DNA. All the probes were generated
with a random-primed labeling kit (Life Technologies) in the presence
of [
-32P]dCTP (NEN-Dupont). The DNA products were
purified over micro-G-50 Sephadex columns (Life Technologies), boiled,
and added to the hybridization mixture. Washes were performed twice in
2× SSC-0.1% SDS for 10 min at room temperature and then twice in
0.1× SSC-0.1% SDS for 20 min at 42°C for cyclin D1 detection and
65°C for detection of all other mRNAs.
 |
RESULTS |
Loss of NF-
B activity correlates with myogenic
differentiation.
To analyze the role of NF-
B in cell growth
control, we initiated our study by using the C2C12 myogenic
differentiation model, since in this cell culture system the gene
products involved in regulating terminal cell cycle arrest are largely
known (39, 48, 71). First, we analyzed the relative levels
and subunit composition of NF-
B/Rel complexes found in
undifferentiated and differentiated C2C12 cell cultures. EMSAs
performed with nuclear extracts prepared from C2C12 cells identified
four potential NF-
B-containing complexes in proliferating myoblasts
(Fig. 1A, lane 1). The levels of
complexes I, II, and III decreased as cells became terminally differentiated (Fig. 1A, lanes 1 to 4). The level of complex IV initially decreased but was elevated 72 h following initiation of
the differentiation process. EMSA supershifts performed to identify
which Rel proteins were contained in these complexes revealed the
presence of the p65 subunit in complex III and p50 in complex IV in
both undifferentiated and differentiated cells (Fig. 1B, lanes 2 and 3 and lanes 7 and 8). Although the antibody against p50 was not able to
supershift complex III, the migration pattern of this complex
nevertheless resembled that of the classical p50-p65 heterodimer. None
of the antisera specific for NF-
B/Rel proteins caused a supershift
in complexes I and II, suggesting that NF-
B is most probably not a
component of these higher-molecular-weight complexes. To address
whether these effects were specific to C2C12 cells, EMSA and supershift
analyses were repeated with nuclear extracts prepared from primary
myoblasts. As seen with the C2C12 cells, loss of p50 and p65 NF-
B
DNA-binding activity was observed in primary myoblasts undergoing
differentiation (Fig. 1C), indicating that a similar reduction of
NF-
B may occur in naturally developing skeletal muscle.

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FIG. 1.
Loss of NF- B binding activity during myogenic
differentiation. (A) Proliferating C2C12 myoblasts (GM) were induced to
differentiate (DM), and at the indicated times nuclear extracts were
prepared and EMSA was performed with a radiolabeled oligonucleotide
containing an NF- B-binding site. (B) C2C12 cells were maintained in
GM or switched to DM for 72 h. Supershift EMSA was performed with
nuclear extracts preincubated with either no antibody (lanes 1 and 6)
or antisera specific for p65 (lanes 2 and 7), p50 (lanes 3 and 8),
c-Rel (lanes 4 and 9), or RelB (lanes 5 and 10). NF- B complexes
containing p50 and p65 subunits are shown. (C) EMSA and supershift EMSA
were performed as described above with nuclear extracts prepared from
primary myoblasts undergoing differentiation. (D) Supershift EMSA was
performed with nuclear extract prepared from WEHI-231 cells,
preincubated either with no antibody or with an antiserum specific to
the c-Rel subunit of NF- B.
|
|
Earlier reports established that upregulated expression and increased
DNA-binding activity of the NF-
B subunit c-Rel were associated with
B-cell development (27). By EMSA, we were unable to detect
c-Rel binding in skeletal muscle cells (Fig. 1B and C), suggesting that
the role of NF-
B subunits, with respect to differentiation, may be
tissue specific. To ensure that the oligonucleotide probe used in our
EMSAs did not favor p65 binding, we tested it with extract prepared
from the mature B-cell line WEHI-231, known to contain high levels of
c-Rel-binding activity (42). Supershift analysis showed that
the probe was readily bound by c-Rel in these cells (Fig. 1D),
supporting the notion that, unlike what is found in lymphocytes, c-Rel
binding is not induced during myogenic differentiation.
To determine whether loss of NF-
B binding activity correlated with
its transactivation potential, a population of C2C12 cells were
generated that stably integrated a luciferase reporter gene fused to
three tandem repeats of the
B site from the MHC class I enhancer
(3x
B-Luc). Upon differentiation of these cells, NF-
B transcriptional activity was reduced nearly 50% (Fig.
2A), a level which is likely to
underrepresent the total loss of
B-dependent transcriptional
activity, since not all C2C12 cells reach terminal differentiation
under these culture conditions. Similar results were obtained when
B-dependent reporter activity was tested in an isolated clone
undergoing differentiation (Fig. 2A). The functionality of the
B-dependent reporter was demonstrated by showing that the promoter
was responsive to the NF-
B-activating cytokine tumor necrosis factor
alpha (TNF-
) whereas a similar promoter containing a mutated version
of the
B sites (3x
Bmut-Luc) lacked basal activity and was
unresponsive to cytokine treatment (Fig. 2A). To further investigate
NF-
B transcriptional activity in myogenesis, Northern blot analysis
was performed to probe for I
B
, whose transcription is known to be
regulated by NF-
B (5). The results showed that a
significant reduction in the level of I
B
mRNA was associated with
differentiating C2C12 cells (Fig. 2B). Thus, the combined data from
EMSAs, reporter assays, and Northern blotting confirmed that NF-
B
activity, most probably represented by the classical, transcriptionally
active p50-p65 heterodimer, is reduced in differentiating skeletal
muscle cells.

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FIG. 2.
Myogenic differentiation correlates with loss of NF- B
transactivation function. (A) C2C12 myoblasts stably containing a
3x B-Luc reporter plasmid were propagated as either a mixed
population or a clonal isolate. Cells were plated in triplicate
overnight in 6-cm culture dishes, and on the following day they were
maintained in GM or switched to DM for 48 h. At that time, cell
extracts were prepared, and relative luciferase units were determined
by normalizing to total protein (RLU). Promoter activities were also
determined for 3x B-Luc and 3x Bmut-Luc populations that were
treated or not treated with 10 ng of TNF- per ml for 24 h. (B)
C2C12 cells were maintained in GM or differentiated in DM for up to
72 h. At the indicated times, total RNA was prepared and 10 µg
of sample was used for Northern blot analysis. The blot was hybridized
with an I B -specific probe, and RNA loading was normalized by
stripping the blot and reprobing for GAPDH mRNA.
|
|
NF-
B functions as a negative regulator of myogenesis in the
C2C12 model.
The results described above suggested a functional
role for NF-
B in precursor myoblasts. To test this hypothesis, C2C12
cells were generated to express a mutant form of the I
B
inhibitor for which serines at positions 32 and 36 had been changed to alanines. The resulting protein (referred to as I
B
superrepressor, or I
B
SR) is no longer subject to phosphorylation and subsequent proteasome degradation following an NF-
B-activating stimulus and
therefore functions as a potent and specific inhibitor of NF-
B
activity (13). The absence of endogenous I
B
protein in
I
B
SR-containing myoblasts (Fig.
3A), as well as the previously described
sensitivity of I
B
SR-expressing cells or p65
/
cells to TNF-induced killing (9, 69, 72) (Fig. 3B),
demonstrated that I
B
SR was functioning properly to block NF-
B
activity. The inhibitory activity of the I
B
SR was confirmed by
EMSA, which showed that only I
B
SR-expressing myoblasts were
blocked in their ability to activate NF-
B in response to TNF
treatment (data not shown). Parental, vector control, or
I
B
SR-expressing myoblasts were examined for the rate at which
they underwent differentiation. At 48 h following serum
withdrawal, less than 10% of the parental or vector control cells had
undergone differentiation, as determined by their myotube phenotype and
by their ability to express the late differentiation marker, myosin
heavy chain (Fig. 3C). In sharp contrast, nearly all the
I
B
SR-expressing cells had become fully differentiated by this
time (Fig. 3C). Similar enhanced rates of differentiation were observed
in pooled clones of I
B
SR-containing cells, demonstrating that
these effects were not due to clonal variations (Fig. 3C). An
examination of a second temporally regulated myogenic differentiation
marker, myogenin, demonstrated greatly accelerated expression of this
myogenic transcription factor in C2C12-I
B
SR cells compared to
vector control cells (Fig. 3D). These data suggested that NF-
B
activity was required in proliferating myoblasts to block myogenic
differentiation.

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FIG. 3.
C2C12 cells lacking NF- B activity have an accelerated
rate of differentiation. (A) Whole-cell lysates were prepared from
C2C12 parental, vector control, or I B SR proliferating myoblasts,
and 50 µg of sample was used for Western blot analysis. I B and
I B SR proteins were detected with an I B polyclonal antibody
(C-19; Santa Cruz Biotechnology) at a 1:1,500 dilution. The I B SR
protein is FLAG tagged and therefore migrates at a slightly higher
mobility compared to the endogenous protein. (B) I B SR-expressing
myoblasts were seeded in triplicate overnight in 12-well plates, and
the following day cells were treated with increasing concentrations of
TNF- for 48 h. Cell viability was scored by trypsinization and
the trypan blue exclusion method. Cells not treated with TNF- were
designated 100% viable. (C) C2C12 parental cells, vector control, an
I B SR clone, or five pooled I B SR clones were differentiated
in DM for 48 h, at which time the cells were prepared for
immunofluorescence to detect for the myosin heavy chain. (D) C2C12
vector control or I B SR cells were induced to differentiate in DM
for up to 72 h. At the indicated times, lysates were prepared and
Western blot analysis was performed probing for myogenin expression. V
(P) and I (P) denote myogenin expression from five pooled vector
control or I B SR clones, respectively, that were differentiated
for 48 h.
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To verify that overexpression of I
B
SR in C2C12 cells did not
introduce nonspecific effects leading to false interpretation of
NF-
B function, we tested the ability of NF-
B to regulate the
expression of a muscle-specific gene. One of the hallmark features of
MyoD, as well as other members of the myogenic bHLH family, myogenin,
myf5, and MRF4, is their ability to induce myogenic differentiation in
nonmuscle cells (39). We therefore performed cotransfection
experiments with murine embryonic C3H10T1/2 (10T1/2) fibroblasts and a
reporter plasmid containing the troponin I enhancer and promoter
(TnI-Luc), along with expression plasmids for MyoD, NF-
B, and/or
I
B
SR. As expected, MyoD strongly activated the troponin I
reporter when cells were placed in differentiation conditions (Fig.
4A). However, coexpression of the p50 and
p65 NF-
B subunits strongly repressed the activation by MyoD, at
levels comparable to those for oncogenic H-Ras, a known potent negative regulator of myogenesis (37, 40, 49). Viability assays
determined that the reduction in MyoD transactivation by NF-
B was
not due to cell death resulting from transfection conditions (data not shown). Similar inhibition levels were produced when the p65 subunit, but not p50, was cotransfected along with MyoD, suggesting that the
transcriptional activation function of NF-
B is required to mediate
this regulation. It was possible that expression of NF-
B subunits
sequestered a cofactor required for MyoD transcriptional function.
Therefore, we asked whether inhibition of NF-
B function would
enhance MyoD transcriptional activity. Expression of I
B
SR enhanced MyoD transcriptional activation of troponin I over that of
MyoD alone (Fig. 4A), reaffirming the notion that NF-
B functions as
an inhibitor of differentiation. We also observed that expression of
I
B
SR partially overcame the ability of Ras to inhibit MyoD transcriptional activity (data not shown), confirming that NF-
B is a
Ras-responsive transcription factor which also can mediate antidifferentiation. Similar results to the ones described above were
obtained when transfections were repeated with a reporter plasmid
containing four E-box sites from the muscle creatine kinase enhancer
(4RTK-Luc) (Fig. 4B), which is also known to be strongly regulated by
myogenic bHLH proteins (39). These results argue that
negative regulation on myogenesis by NF-
B is not specific to the
troponin gene. As an additional approach to assay the regulatory potential of NF-
B on differentiation, cotransfections with MyoD and
NF-
B subunits were performed with 10T1/2 fibroblasts and analyzed
for their effects on myotube formation and myosin heavy-chain expression. As can be seen in Fig. 4C, the expression of MyoD in 10T1/2
cells caused the formation of small myotubes 72 h following serum
withdrawal but addition of NF-
B was sufficient to completely abolish
this myogenic event. In contrast, the expression of I
B
SR with
MyoD enhanced both the overall number (quantitatively approximated to
be threefold over that of MyoD alone) and size of myotubes formed in
the culture wells (Fig. 4C). These findings correlated strongly with
the reporter assay data and confirmed that NF-
B functions as a
negative regulator of myogenic differentiation in vitro.


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FIG. 4.
NF- B inhibits MyoD-induced myogenesis in 10T1/2
fibroblasts. Cells were maintained in growth medium containing 15% FBS
and differentiated in DM. The cells were seeded in triplicate overnight
in 6-cm dishes, and the following day, cotransfections were performed
with Superfect (Qiagen). DNA consisted of 1 µg of a troponin-I-Luc
reporter plasmid (TnI-Luc) (A) or 1 µg of the 4RTK-Luc plasmid (B),
along with 0.25 µg of an expression plasmid for MyoD alone or in
combination with 0.5 µg of expression plasmids for either the
activated form of oncogenic ras (H-ras V-12),
p65, p50, or 1 µg of I B SR. DNA was standardized to 2.5 µg by
the addition of Bluescript plasmid (Stratagene). Cells were maintained
in growth medium for 24 h following transfections and then
switched to DM for 48 h, at which time cell extracts were prepared
and relative light units (RLU) were determined, by normalizing values
to total protein. (C) For immunofluorescence analysis, 10T1/2 cells
were seeded overnight and the next day similar transfections were
performed as described above, except that one-third of the amount of
DNA was used. At 24 h following transfections, the cells were
switched to DM for 72 h, at which time the cells were fixed and
probed for the myosin heavy chain. To score for the number of myotubes
formed, cells expressing myosin were counted and averaged from a
minimum of 10 randomly selected fields.
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NF-
B inhibits C2C12 myogenesis through its growth-promoting
activity and regulation of cyclin D1.
To determine the mechanism
by which NF-
B regulated myogenesis in vitro, we first examined
whether NF-
B had any effect on the expression of Id-1 or Hes-1,
which are known inhibitors of differentiation (12, 55).
Northern analysis performed with mRNA isolated from proliferating C2C12
parental, vector control, or I
B
SR-expressing myoblasts revealed
that the steady-state levels of Hes-1 was only slightly altered whereas
Id-1 was more strongly altered in cells lacking NF-
B activity (Fig.
5A). This result may indicate that these
genes could be involved in NF-
B-mediated inhibition of myogenic
differentiation. Relevant to the regulatory mechanism of NF-
B was
that C2C12-I
B
SR cells maintained in growth medium exhibited a
substantial increase in cell doubling time compared to parental or
vector control cells (Fig. 5B). Since successful progression of
myogenic differentiation is highly dependent on the ability of cells to
irreversibly exit the cell cycle in the G1 state
(71), we asked whether the effect on cell growth as a result
of the absence of NF-
B activity translated to changes in cell cycle
progression. Exit from cell cycle was assessed by immunoblot analysis
probing for the phosphorylation status of pRb. The results showed that
while no obvious differences in pRb states were observed in either
vector control or I
B
SR cells maintained in fresh growth medium
(t = 0 h), once cells were induced to differentiate
(t = 12 and 24 h) pRb hypophosphorylation occurred much faster in I
B
SR cells than in control cells (Fig. 5C). These data imply that in immortalized C2C12 cells, inhibition of NF-
B accelerates the rate at which cells exit the cell cycle.

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FIG. 5.
C2C12 cells lacking NF- B exhibit a growth defect and
changes in pRb phosphorylation. (A) Total RNA was prepared from C2C12
parental, vector control, or I B SR proliferating myoblasts, and
Northern analysis was performed to detect the expression of Hes-1 or
Id-1 genes. (B) C2C12 parental, vector control, or I B SR myoblasts
were plated in triplicate in 10-cm plates and maintained in GM for 3 days. Every 24 h, the total cell number was determined by
trypsinization and trypan blue exclusion. (C) C2C12 vector control (V)
or I B SR cells (I) were induced to differentiate for up to 24 h. At the indicated times, cell lysates were prepared with lysis buffer
containing phosphatase inhibitors and Western blotting was performed to
probe for the hypo- and hyperphosphorylated forms of Rb with a
monoclonal antibody (14001A; Pharmigen).
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The above finding prompted us to search for other cell cycle regulatory
factors that function upstream of pRb and that could potentially be
regulated by NF-
B. Immunoblotting results showed that under
proliferating conditions, I
B
SR-expressing myoblasts exhibited a
striking reduction in cyclin D1 levels compared to vector control cells
(Fig. 6A). In addition, cyclin D1 levels decreased more rapidly in differentiating I
B
SR cells than in control cells. Potential regulation by NF-
B for other cell cycle regulators, cyclin D3, cyclin A, and p21, was observed but was less
dramatic than the effect on cyclin D1 and may therefore be due to
secondary effects of changes on cyclin D1. Although basal levels of p21
were reduced in C2C12 cells lacking NF-
B (Fig. 6A), inhibition of
NF-
B did not block the accumulation of p21, which is associated with
myogenic differentiation (29, 30). The lack of any
significant regulation of cdk4, as well as for the cdk inhibitor
protein p27, also indicated that effects of NF-
B on cyclin D1
expression were specific. Consistent with immunoblotting results,
steady-state levels of cyclin D1 mRNA were also significantly lower in
proliferating I
B
SR-expressing myoblasts than in either parental
or vector control cells (Fig. 6B). In comparison, we detected only a
slight effect on cyclin D3 mRNA expression in the NF-
B-inhibited
cells, consistent with observations showing that cyclin D3 levels
increase during skeletal muscle differentiation (51). These
results therefore demonstrate that NF-
B is a specific regulator of
cyclin D1.

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FIG. 6.
C2C12 cells lacking NF- B are downregulated for cyclin
D1 protein and mRNA. (A) C2C12 vector control or I B SR cells were
differentiated in DM for up to 72 h. At the indicated times,
whole-cell lysates were prepared and 50 µg was used in Western blot
analyses to probe for various cell cycle proteins. (B) Total RNA was
prepared from proliferating parental (P), vector control (V), or
I B SR (I) myoblasts, and 10 µg of sample was used in Northern
blotting to probe for cyclin D1 or cyclin D3 mRNA.
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As shown in Fig. 6A and consistent with previous reports (51,
64), downregulated expression of cyclin D1 correlates with myogenic differentiation. One mechanism by which cyclin D1 is thought
to function as a negative regulator of this cellular process is by
blocking the transactivation function of MyoD (51, 64). Indeed, in agreement with these previous reports, cotransfections in
10T1/2 fibroblasts showed that while cyclin D1 strongly blocked the
ability of MyoD to activate the troponin I gene, cyclin D3 was only
partially inhibitory toward MyoD (Fig.
7A). To examine whether NF-
B inhibits
myogenesis through its regulation of cyclin D1, NF-
B activity was
inhibited in 10T1/2 cells through expression of I
B
SR and cyclin
D1 was reexpressed to determine if the block on MyoD transcriptional
function could be restored. The inhibitory potential of the I
B
SR
plasmid in these cells was confirmed on an NF-
B-responsive promoter
(data not shown). The results showed that increasing the amounts of the
cyclin D1 expression plasmid could restore the inhibition of MyoD
activity in cells where NF-
B was inhibited (Fig. 7A). In comparison,
similar cotransfections with the cyclin D3 expression vector were
significantly less effective at inhibiting MyoD function. Importantly,
similar results were obtained when myogenesis was assessed by scoring
for myotube formation (Fig. 7B), demonstrating that these effects were
not specific to the troponin I gene.

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FIG. 7.
NF- B inhibits MyoD transactivation function through
the regulation of cyclin D1. (A) 10T1/2 fibroblasts were seeded in
triplicate overnight in 6-cm culture dishes, and the next day
cotransfection was performed with DNA consisting of 1 µg of the
troponin-I reporter plasmid (TnI-Luc) and 0.25 µg of a MyoD
expression plasmid, along with either 0.25 µg of an I B SR
expression plasmid or the indicated amounts of cyclin D1 or cyclin D3
expression plasmid. DNA was normalized by the addition of Bluescript
plasmid (Stratagene). Transfected cells were maintained in growth
medium overnight and on the following day were switched to DM for
48 h, at which time extracts were prepared and a luciferase assay
was performed. The level of troponin-I activation by MyoD alone was set
to a value of 100. (Below) To verify the expression of proteins,
parallel transfections were performed and immunoblot analysis was
performed to probe for MyoD, cyclin D1, and cyclin D3. (B) Similar
transfections were performed in 10T1/2 fibroblasts with the following
amounts of expression plasmids: 0.25 µg of MyoD, 1 µg of
I B SR, and 2 µg of cyclin D1 or cyclin D3. The cells were
differentiated as described above for 72 h, at which point
myogenesis was quantitated by counting myotubes from a minimum of 10 fields of cells.
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To further explore whether cyclin D1 is involved in NF-
B regulation
of myogenesis, NF-
B was activated by TNF-
while C2C12 myoblasts
were induced to undergo differentiation. The results showed that TNF
treatment almost completely repressed the ability of C2C12 cells to
form myotubes, again supporting the claim that NF-
B functions as a
negative regulator of myogenesis (Fig.
8). In addition, while the levels of
cyclin D1 were diminished in untreated differentiating cells, cyclin D1
was stabilized in differentiating C2C12 cells activated for NF-
B.
Although it is possible that these effects of TNF occur independently
of NF-
B activation, this result, taken together with previous data,
strongly suggests that one mechanism by which NF-
B inhibits skeletal
muscle differentiation is through the regulation of cyclin D1
expression.

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FIG. 8.
TNF- inhibits C2C12 myogenesis and stabilizes cyclin
D1. C2C12 cells were induced to differentiate in the absence or
presence of 20 ng of TNF- per ml. Cytokine addition was repeated at
6 h and every additional 12 h after the induction of
differentiation. At 72 h, the cells were washed with PBS, fixed
for 10 min at room temperature with 4% paraformaldehyde, and
photographed by phase-contrast microscopy. In parallel treatment
cultures, cells were harvested at 24 and 48 h and whole-cell
lysates were prepared for Western blot analysis. A 50-µg portion of
total protein was fractionated by SDS-polyacrylamide gel
electrophoresis, and immunoblotting was performed to probe for cyclin
D1. The blot was subsequently stripped and reprobed for cdk4, used as
an internal control.
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NF-
B is a direct transcriptional activator of cyclin D1.
The use of the C2C12 myogenesis model allowed us to demonstrate that
NF-
B regulates cyclin D1 expression. Next, we were interested in
determining whether this regulation was tissue specific and at what
level it was controlled. To address the first of these points, an
adenoviral delivery system was used to transiently overexpress
I
B
SR in HeLa cells and MEFs. Northern analysis showed that,
similar to what was seen in C2C12 cells stably expressing I
B
SR,
transient inhibition of NF-
B activity led to a decrease in
steady-state levels of cyclin D1 mRNA (Fig.
9A). This result demonstrated that this
regulation was therefore not unique to skeletal muscle cells and,
perhaps equally important, did not result from a clonally selectable
process. Conversely, transfections in fibroblasts with a plasmid
expressing the p65 subunit of NF-
B led to increased levels of
endogenous cyclin D1 (Fig. 9B), thus underscoring the specificity of
the I
B
SR protein and demonstrating that expression of p65 is
sufficient to induce cyclin D1 expression.

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FIG. 9.
Regulation of cyclin D1 is specific to NF- B occurring
in multiple cell types. (A) HeLa cells or MEFs were infected with
adenovirus containing either the I B SR or empty vector (AdCMV) at
a multiplicity of infection of 50 or 200, respectively. Total RNA was
prepared 48 h postinfection, and Northern analysis was performed
to detect cyclin D1 mRNA expression. Western blot analysis with an
I B -specific antibody is shown to demonstrate the expression of
the I B SR in HeLa cells and MEFs by using the adenovirus delivery
system. (B) Fibroblasts were transfected with either empty vector or an
expression plasmid expressing the p65 subunit of NF- B. The following
day, cells were switched to serum-deprived conditions for a 48-h
period. Subsequently, whole-cell extracts were prepared and 50 µg was
used in immunoblot analysis probing for cyclin D1. The loading
efficiency was normalized by reprobing the blot for cdk4 expression.
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Second, to investigate the mechanism of this regulation,
cotransfections were performed with reporter plasmids containing different lengths of the human cyclin D1 promoter and an expression plasmid for p65. These experiments showed that p65 strongly activated cyclin D1 gene expression and that this regulation mapped to regions from
963 to
630 and
66 to
22 within the promoter (Fig.
10A). Transcriptional activation of the
cyclin D1 promoter was also observed when c-Rel was expressed in these
cells (data not shown), indicating that this regulation is not specific
to the p65 subunit of NF-
B. Examination of the sequence from the
human cyclin D1 promoter (47) identified potential
NF-
B-binding sites at positions
858,
749, and
39 that matched
the NF-
B consensus binding sequence, GGG(G/A)NNYYCC, and
that mapped to the regions we had identified to be regulated by p65.
Oligonucleotides to these sites were generated and tested by EMSA to
determine whether NF-
B binding occurred. With nuclear extracts
prepared from both NIH 3T3 and HeLa cells, complexes were formed at all
three sites (Fig. 10B). TNF treatment, which activates nuclear
translocation of NF-
B, displayed increased complex formation,
providing greater evidence that NF-
B was the component bound to
these sites. Supershift analysis determined that NF-
B complexes were
predominantly represented by the p65 subunit and that the
39 site
contained both the p50 homodimer and p50-p65 heterodimer complexes,
since at this site both antibodies either supershifted or blocked
complex formations (Fig. 10C). Furthermore, oligonucleotide competition
analysis established that the binding of these complexes was specific
to
B sites (Fig. 10C). Finally, site-directed changes made in the
NF-
B site located at position
39, within the
66CD1-Luc reporter
plasmid, demonstrated the requirement of this regulatory site for
cyclin D1 transcriptional activation relative to expression of the
NF-
B p65 subunit (Fig. 10D). Taken together, these data conclusively
demonstrate that NF-
B regulation of cyclin D1 occurs at the
transcriptional level mediated by direct binding of NF-
B to
potentially multiple regions within the cyclin D1 promoter.

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FIG. 10.
NF- B regulation of cyclin D1 occurs at the
transcriptional level. (A) NIH 3T3 cells were plated in triplicate in
12-well plates. Transfections were performed on the following day with
Lipofectamine reagent (Life Technologies) mixed with DNA consisting of
0.2 µg of reporter plasmids containing various 5' deletions of the
human cyclin D1 promoter, along with 0.15 µg of a p65 expression
plasmid. DNA remained on the cells for 3 h in serum-free medium,
and the cells were then switched for 48 h to complete medium
containing 10% calf serum, at which time the luciferase activity was
determined. Values were normalized to basal levels of promoter activity
obtained by transfecting cyclin D1 promoter constructs in the absence
of p65. (B) EMSAs were performed with nuclear extracts prepared from
either NIH 3T3 cells or HeLa cells treated (+) or not treated ( ) with
TNF- for 30 min. Putative NF- B-binding sites, located at
positions 858, 749, and 39 in the human cyclin D1 promoter, are
indicated within the oligonucleotide sequences used for EMSAs. (C)
Supershift EMSAs were performed with nuclear extracts prepared from
HeLa cells treated with TNF, which were preincubated either with no
addition (lanes 1, 6, and 11) or with addition of antisera specific for
p50 (lanes 2, 7, and 12), or the p65 subunit (lanes 3, 8, and 13).
Arrows denote p65-containing complexes. For competition EMSAs, extracts
were preincubated with either a 100-fold molar excess of unlabeled
oligonucleotides containing wild-type (xs wt) NF- B-binding sites
(lanes 4, 9, and 14), or a 100-fold molar excess of unlabeled
oligonucleotides containing mutations in the NF- B sites (xs mut)
(lanes 5, 10, and 15). The mutations are shown in the boxed regions
above the NF- B-binding sites. (D) The same mutation at position 39
was made in the NF- B-binding site within the 66CD1-Luc reporter
plasmid. Both the wild-type and mutant ( 66CD1- Bmut-Luc) reporter
plasmids were separately cotransfected along with a p65 expression
plasmid in NIH 3T3 cells under the same transfection conditions as
described for panel A.
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NF-
B is required in early G1 for cyclin D1
activation and progression into S phase.
Having established that
cyclin D1 is a novel transcriptional target of NF-
B and provided
evidence to show how this regulation is important for controlling the
differentiation of skeletal muscle cells, we focused the last part of
our study on examining the relevance of this regulatory mechanism to
cell cycle progression and cell growth. Cyclin D1 was originally
identified as a suppressor of G1 arrest in yeast cells and
as a gene that is inducible in the G1 phase of the cell
cycle (44, 76). The expression of cyclin D1 in
G1 is important for cell cycle progression (60, 61), and results demonstrating that acceleration into S phase by
cyclin D1 is greater for synchronized cultures emerging from quiescence
than for asynchronous cycling cells suggest a specialized role for this
D-type cyclin in the G0/G1 transition
(53). Interestingly, earlier results from our laboratory
showed that NF-
B was strongly activated when growth-arrested
fibroblasts were stimulated by serum to re-enter cell cycle
(6). This finding prompted us to investigate whether NF-
B
was required for cyclin D1 induction in cells reinitiating cell cycle.
Since differentiated C2C12 cells are not capable of reinitiating a cell
cycle, we instead turned to the use of diploid fibroblasts. Cotransfections were performed in quiescent fibroblasts with a reporter
plasmid containing the cyclin D1 promoter and the I
B
SR expression
plasmid, and cells were subsequently stimulated back into the cell
cycle with the addition of serum. The results showed that in the
absence of the I
B
SR plasmid, cyclin D1 promoter activity was
induced approximately fourfold in response to mitogen addition (Fig.
11A). In contrast, inhibition of
NF-
B by I
B
SR expression blocked this induction in a
dose-dependent manner, indicating that NF-
B is required for cyclin
D1 transcriptional activation in early G1. To examine
whether NF-
B regulation of cyclin D1 was also important for the
G1-to-S progression, quiescent MEFs were infected with
adenovirus containing either empty vector (CMV) or I
B
SR and cells
were allowed to reinitiate the cell cycle. G1-to-S
progression was first assessed by examining the phosphorylation status
of pRb. The result of this experiment showed that cells lacking NF-
B
contained a substantially lower level of hyperphosphorylated pRb than
did CMV control cells (Fig. 11B). Importantly, the decrease in pRb
hyperphosphorylation in these cells correlated with a marked reduction
of cyclin D1, supporting the notion that the Rb protein is a direct
target of the cyclin D1-cdk4 complex. By using similar infection
conditions, MEFs lacking NF-
B activity were also shown to be
significantly impaired for entry into S phase, as assessed by BrdU
incorporation (Fig. 11C). Importantly, the defect in S-phase entry in
these cells could be restored by addition of cyclin D1, arguing that
NF-
B activation of cyclin D1 is important for G1-to-S
progression.

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FIG. 11.
Requirement for NF- B activity in the early
G1 phase of the cell cycle. (A) NIH 3T3 cells were made
quiescent by being switched for 48 h from complete medium
containing 10% serum to medium containing 0.25% calf serum.
Serum-deprived cells were cotransfected either with a cyclin D1
promoter reporter plasmid alone ( 963CD1-Luc) (2.5 µg) or in
combination with indicated amounts of I B SR plasmid. The cells
were maintained in quiescence or switched to complete medium to induce
cyclin D1 transcription and reentry into the cell cycle. Extracts were
prepared, and relative luciferase activity was determined by
standardizing to total cellular protein. (B) MEFs were made quiescent
by culturing cells in 0.1% FBS for 48 h. The cells were then
infected under serum-deprived conditions with adenovirus containing
either empty vector (CMV) or I B SR at an MOI of 200. At 24 h
postinfections, MEFs were either maintained under serum-deprived
conditions or induced to reenter the cell cycle by the addition of 10%
FBS. Whole-cell lysates were prepared 24 h later, and immunoblot
analysis was performed to probe for both pRb and cyclin D1 (under these
culture conditions, we determined that pRb hyperphosphorylation in MEFs
is maximally detectable between 20 and 24 h following serum
stimulation). (C) Subconfluent MEFs were grown on coverslips overnight
and were rendered quiescent on the following day. Cells were infected
with either control adenovirus (CMV), I B SR, or a combination of
I B SR and cyclin D1 viruses (both at a multiplicity of infection
of 200). At 24 h following infections, the cells were maintained
quiescent or switched for 12 h to growth medium containing 10%
serum, at which point the medium was supplemented with 100 µM BrdU
(Sigma) for an additional 10 h. The cells were fixed and prepared
for immunofluorescence analysis as described in Materials and Methods.
The percentage of cells in the S phase was calculated by determining
the number of BrdU-positive cells with respect to the total number of
cells in a given field.
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Finally, based on the above findings, we asked whether NF-
B
regulation of cyclin D1 was a relevant mechanism involved in the
proliferation of primary fibroblasts. To investigate this, we used a
retrovirus delivery system to stably generate a mixed population of
MEFs expressing either empty vector or the I
B
SR protein. Again,
immunoblot analysis showing that endogenous levels of I
B
were
downregulated in I
B
SR-expressing fibroblasts (as shown in Fig.
3A) confirmed that NF-
B activity was effectively blocked (data not
shown). When growth rates were monitored, the results showed that over
an extended number of passage doublings, fibroblasts devoid of NF-
B
activity exhibited a significant defect in cellular proliferation (Fig.
12). Importantly, NF-
B inhibition in
these cells again led to a persistent decrease in the levels of cyclin
D1 (Fig. 12, inset), suggestive that NF-
B growth-promoting activity
is tightly coupled to its transcriptional regulation of cyclin D1.