1 Department of Molecular and Cell Biology and 2 Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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The transcription factor nuclear
factor (NF)-B regulates the expression of genes that can influence
cell proliferation and death. Here we analyze the contribution of
NF-
B to the regulation of epithelial cell turnover in the colon.
Immunohistochemical, immunoblot, and DNA binding analyses indicate that
NF-
B complexes change as colonocytes mature: p65-p50 complexes
predominate in proliferating epithelial cells of the colon, whereas the
p50-p50 dimer is prevalent in mature epithelial cells. NF-
B1 (p50)
knockout mice were used to study the role of NF-
B in regulating
epithelial cell turnover. Knockout animals lacked detectable NF-
B
DNA binding activity in isolated epithelial cells and had significantly
longer crypts with a more extensive proliferative zone than their
wild-type counterparts (as determined by proliferating cell nuclear
antigen staining and in vivo bromodeoxyuridine labeling). Gene
expression profiling reveals that the NF-
B1 knockout mice express
the potentially growth-enhancing tumor necrosis factor (TNF)-
and
nerve growth factor-
genes at elevated levels, with in situ
hybridization localizing some of the TNF-
expression to epithelial
cells. TNF-
is NF-
B regulated, and its upregulation in NF-
B1
knockouts may result from an alleviation of p50-p50 repression. NF-
B
complexes may therefore influence cell proliferation in the colon
through their ability to selectively activate and/or repress gene expression.
nuclear factor-B; cell proliferation; tumor necrosis factor-
; nerve growth factor-
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INTRODUCTION |
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THE TRANSCRIPTION FACTOR
nuclear factor (NF)-B is a dimeric protein generated from the
association of five possible subunits: p50, p52, RelA (p65), RelB, and
c-Rel (5). The Rel homology domain found in each of these
proteins functions in DNA binding, dimerization, and interaction with
various inhibitory proteins referred to as I
Bs (27,
57). I
B proteins include I
B
, I
B
, I
B
, p105,
and p100. The p105 and p100 proteins are alternative products of the
same genes that encode p50 and p52 (NF-
B1 and NF-
B2,
respectively). I
Bs contain multiple copies of the ankyrin repeat at
their COOH terminals, and this repeat is required for binding to, and
inhibition of, NF-
B subunits. NF-
B activation entails the
signal-induced phosphorylation and degradation of I
B molecules,
which in turn releases NF-
B to translocate into the nucleus and bind
to response elements of target promoters (11, 19, 30, 42, 47, 53,
69). The various NF-
B subunits and I
Bs enable the cell to
modulate NF-
B responses in a highly controlled manner. For example,
I
Bs are degraded at different rates, which can control the kinetics
of an NF-
B response (59). I
Bs also display certain
stimuli-specific responses (59). The subunit composition
of NF-
B also influences gene activation. Only the p65, RelB, and
c-Rel subunits have strong transcriptional activation domains. NF-
B
forms without one of these subunits (for example, p50 or p52
homodimers) are weaker transcription activators and can even function
as repressors (5, 39, 40). In summary, cells are able to
modulate expression of NF-
B-regulated genes by regulating the I
Bs
expressed and the subunit composition of the NF-
B activated
(26).
NF-B has been implicated in regulating cell proliferation
and death in a number of cell types (6, 7, 29).
Intriguingly, NF-
B has been found to both promote and suppress cell
death. The role NF-
B assumes in regulating cell death appears to
depend on both cell type and apoptosis inducer (6, 7, 10, 12, 13,
34, 43, 56, 60, 63, 67). In addition to regulating genes that
directly influence cell proliferation and death, NF-
B regulates
expression of a number of cytokines and cell adhesion molecules
operative in immune and inflammatory responses (2, 5, 17, 23, 38,
39, 68). These molecules can also influence cell proliferation
and death in a tissue. Understanding how NF-
B regulates cell
proliferation and death in an integrated, functioning tissue is
therefore a major research challenge.
We are interested in better understanding the role of NF-B in
regulating cellular proliferation and apoptosis of colonic epithelial
cells. Colonic epithelium is a dynamic structure organized into
invaginations known as the crypts of Lieberkühn. These crypts open into the lumen of the intestinal tract. Located at the base of the
crypt are a number of stem cells that divide continuously and are
responsible for perpetual renewal of the epithelium (50). As cells migrate up the crypt axis (due to the mitotic pressure from
the cells below them), they stop dividing and differentiate. Differentiated cells continue their ascent toward the luminal surface,
where they undergo anoikis or apoptosis (24, 25). The lamina propria that underlies epithelial cells consists of connective tissue and a variety of wandering cells (lymphocytes, plasma
cells, eosinophils, and mast cells), which can interact with epithelial
cells. The regulation of cell turnover in the colon can therefore be
influenced by signals generated by other cells in the tissue.
In this study, we investigated the expression and activity of NF-B
in colonic epithelial cells. In addition, we determined the effect of
the NF-
B1 knockout on epithelial cell turnover in the mouse colon.
Our results indicate that NF-
B is active in epithelial cells under
normal physiological conditions and that it participates in the
regulation of epithelial cell turnover.
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MATERIALS AND METHODS |
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Immunohistochemistry.
Paraffin-embedded tissues were first deparaffinized by baking slides in
a 60°C oven for 20 min. Slides were then treated with Hemo-De (Fisher
Scientific, Pittsburgh, PA) before undergoing a series of ethanol
immersions. To neutralize endogenous peroxidase activity, the sections
were blocked for 20 min in 1% hydrogen peroxide in PBS. For the
microwave antigen retrieval procedure, slides were immersed in 10 mM
citrate buffer (pH 6.0) in a clean polyethylene chamber in a
full-powered microwave for 10 min and were then cooled for 10-20
min. After washing three times with PBS-Brij solution, the sections
were blocked in 10% normal horse or goat serum for 30-60 min to
suppress nonspecific binding of IgG. Each tissue section was then
incubated with primary antibody (anti-NF-B1 D-17, Santa Cruz
Biotechnologies, Santa Cruz, CA; anti-p65 AB1604, Chemicon). The
p65-nuclear localization signal (NLS) antibody that has been
used to detect active p65 in human cells (MAB3026; originally available
from Boehringer Mannheim and now sold by Chemicon) (52)
could not be used for this analysis because it does not recognize mouse
p65. Antibodies were incubated overnight at 4°C, except anti-p65,
which was incubated for 1 h at room temperature. Slides were then
washed three times with PBS-Brij and then incubated with anti-goat or
anti-rabbit biotinylated secondary antibodies (Vector Laboratories,
Burlingame, CA). For proliferating cell nuclear antigen (PNCA;
clone-10; Signet) and bromodeoxyuridine (BrdU; Clone BU33; Sigma)
staining, mouse primary antibodies were used with a Mouse-On-Mouse
detection kit (Vector) to suppress nonspecific binding. Briefly, after
citrate buffer retrieval, the tissues were incubated with blocking
reagent for 1 h and washed twice with PBS for 2 min each. The
sections were then incubated with Mouse-On-Mouse diluent for 5 min
before the primary antibody was applied. After 30 min of incubation
with primary antibodies, tissues were washed with PBS and incubated with biotinylated anti-mouse IgG for 10 min. Incubation with
ABC Reagent (Vectastain) was followed with three washes of
PBS-Brij, and then diaminobenzine reaction was applied. Slides
were then counterstained with hematoxylin for 20 s and washed in
graded ethanol and Histoclear. Finally, slides were mounted with
coverslips and analyzed by light microscopy. Staining specificity for
the NF-
B antibodies was confirmed by comparing their staining with staining obtained using normal serum (Fig. 1C). Competition
experiments were also performed with specific peptide inhibitors that
reduced the level of staining (data not shown).
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Colonic epithelial cell isolation. The isolation of colonic epithelial cells was based on a procedure described by Homaidan et al. (33). Distal colon was removed and washed three times with ~5 ml of washing buffer [150 mM NaCl and 1 mM freshly added dithiothreitol (DTT)] to remove adherent mucus. The colon was then placed in a 60-mm cell culture plate filled with ~5 ml of the dissociation buffer (130 mM NaCl, 1 mM Na2EDTA, 10 mM HEPES, 10% FCS, and 1 mM freshly added DTT, pH 7.4). The plate was placed on a shaker at 37°C and incubated for 15 min. Dissociated cells were collected into a 15-ml Falcon tube by removing the dissociation buffer from the colon. Incubation in dissociation buffer was repeated a total of six times. For the first and second collections, 1 mM EDTA was used, for the third and fourth, 5 mM was used, and for the final two, 10 mM EDTA was present. Isolated cells were processed immediately after isolation by centrifugation at 4°C (2,500 rpm) for 10 min. Proteins were extracted as described below.
Protein extraction.
Following centrifugation of the isolated cells, the resulting pellet
was washed twice with cold PBS. The cells were then lysed by incubating
them at 4°C for 8 min with 500 µl of buffer containing 0.1% NP-40
[10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 2 mM
MgCl2, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 M
sucrose, and 2 µg/ml leupeptin] and transferred to Eppendorf tubes.
The tubes were centrifuged at 4°C (14,000 rpm) for 10 min, and the
resulting supernatant was collected into a new tube. This is the
cytoplasmic extract. The resulting pellet was washed with 1 ml of the
above buffer without NP-40, resuspended in 30 µl of buffer (20 mM
HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA,
1 mM DTT, 5% glycerol, 0.5 mM PMSF, and 2 µg/ml leupeptin) and then
incubated for 40 min on ice. The nuclear suspension was centrifuged at
4°C (14,000 rpm) for 10 min. The resulting supernatant, the nuclear
extract, was transferred to new tube and diluted with 45 µl of a
buffer (20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 1 mM DTT, 20%
glycerol, 0.5 mM PMSF, and 2 µg/ml leupeptin). Protein concentrations
of nuclear and cytoplasmic extracts were measured by the Bradford
assay, and the tubes were placed in 80°C freezer for storage.
Immunoblotting. Thirty micrograms of proteins from each of the above-described samples were resolved on 12% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were blocked for 1 h with 5% nonfat dry milk. Specific proteins were detected with the appropriate antibodies using enhanced chemiluminescence for detection (Santa Cruz). The antibodies used were p65 (1:1,000 C-19; Santa Cruz), p50/p105 (1:1,000 D-17; Santa Cruz), PCNA (1:500 clone-10; Signet), and alkaline phosphatase (1:500; Chemicon).
Electrophoretic mobility shift assay.
Ten micrograms of nuclear extract, prepared as described
in Protein extraction, in 10 µl of buffer were mixed
with 2 µg poly-dIdC and 1 µg BSA to a final volume of 19 µl.
After 15-min incubation on ice, 1 µl of [-32P]ATP
end-labeled double-stranded NF-
B consensus oligonucleotide (TGAGGGGACTTTCCCAGGC) was added to each reaction and incubated at room
temperature for an additional 15 min. The reaction products were
separated on a 4% native polyacrylamide-0.5 × Tris-borate-EDTA gel and analyzed by autoradiography.
Supershift antibodies (1 µl) were included in the binding reaction as
indicated (all supershift antibodies were obtained from Santa Cruz).
Animals.
Six-week-old male NF-B1 knockout mice and their wild-type
counterparts (B6/129P) were purchased from the Jackson Laboratory (Bar
Harbor, ME) and housed in a controlled environment with a 12:12-h
light/dark cycle. After an acclimation period of 4 wk, mice were killed
and their colons were removed and processed for cell isolation or
paraffin imbedding as described in Colonic epithelial cell
isolation. For in vivo BrdU labeling, mice were injected intraperitoneally with BrdU (Zymed Laboratories, South San Francisco, CA) at a concentration of 50 mg/kg body wt. Two injections were performed, one at 24 h and the other at 12 h before death.
Colons were then processed for immunohistochemistry. BrdU labeling was estimated by determining the fraction of cells with positively stained
nuclei in each animal. Thirty crypts from the distal colon of each
animal were arbitrarily chosen and examined. Only crypts cut
longitudinally were considered in this analysis.
cDNA array analysis.
The Clontech Atlas Mouse 1.2 cDNA array was used (Clontech, Palo Alto,
CA). RNA was isolated from whole colon tissue immediately on removal
following the Clontech protocol, with the exception that the Qiagen
poly(A) mRNA isolation kit was used (Qiagen, Valencia, CA). cDNA
labeling was performed using Clontech reagents, and the resulting
probes were purified with a Centri-Spin column (Princeton Separations,
Adelphia, NJ). Clontech membranes were probed and washed as described
by Clontech, exposed to film, and then quantified with a Bio-Rad
PhosphorImager (Bio-Rad, Hercules, CA). Autoradiograms were compared
manually using a transparent alignment grid. Only hybridization signals
that were precisely positioned in the array were considered further (a
number of background "dots" appear on both arrays). True
hybridization signals with apparent differences in intensity were then
compared quantitatively on the PhosphorImager, with signal adjustments
made using the housekeeping genes present at the bottom of the
membrane. Nerve growth factor (NGF)- expression found to be enhanced
on the array was also analyzed by the RT-PCR assay, following a
published protocol (64). Primers for the NGF-
were
CACCCTGCCCACTGAGGAGCCCAA and CCATCCATCTCTCCTGCACACA, which generated a
195-bp fragment from the NGF-
mRNA. (These primers spanned an
intron, to prevent confusion with amplification products from trace
amounts of genomic DNA in the reaction). Primers to
hypoxanthine-guanine phosphoribosyltransferase, which were used as a
control, have been previously described (64).
RNase protection.
Two NF-B-regulated genes not found on the Clontech cDNA array are
TNF-
and Fas (3, 15). To assay the expression levels of
these genes, an RNase protection assay was performed using in vitro
transcription plasmids obtained from PharMingen (San Diego, CA). RNA
for this analysis was purified from whole mouse colon using TRIzol
reagent (Life Technologies, Grand Island, NY). Complementary RNA
labeling and RNase protection was performed as described by PharMingen.
Resulting sequencing gels were exposed to film and quantified by PhosphorImager.
In situ hybridization.
RNA probes for TNF- in situ hybridization were transcribed in vitro
from a pGEM-T Easy vector (Promega, Madison, WI). An RT-PCR-generated
TNF-
gene fragment (+292 to +578 relative to the translation start
site) was cloned into the pGEM-T Easy vector, and the orientation was
determined by PCR using TNF-
primers and universal priming sites
present in the plasmid. In vitro transcription of this clone with T7
RNA polymerase generated the antisense probe, whereas the Sp6 RNA
transcription produced the sense probe. The sense and antisense RNA
probes were labeled with psoralen-biotin using a BrightStar
psoralen-biotin labeling kit according to the manufacturer's
instructions (Ambion, Austin, TX). Hybridization was also
performed according to the manufacturer's instructions. Briefly,
6-µm sections were deparaffinized and rehydrated in nuclease-free alcohol. The tissue sections were treated with proteinase K, washed, prehybridized, and then probed with either the sense or antisense TNF-
probe in a humidifier chamber for 4 h at 55°C. After
hybridization, sections were washed and incubated with a
streptavidin-alkaline phosphatase conjugate. After several washes,
signal was detected by nitroblue tetrazolium salt and
5-bromo-4-chloro-3-indol phosphate. Slides were dehydrated by gradual
alcohol immersion, cleared by Histoclear, mounted with coverslips, and
analyzed by light microscopy.
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RESULTS |
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Immunohistochemical analysis of NF-B subunits in the mouse
colon.
If NF-
B is involved in regulating epithelial cell turnover, its
expression and activity may change as epithelial cells mature. We
therefore analyzed sections of mouse colon for the NF-
B subunits p50
and p65. These two subunits were chosen for analysis because NF-
B
isolated from the colon has been found to be composed primarily of
these subunits (see NF-
B activity in epithelial cell
isolates). As shown in Fig.
1A, p65 expression is
concentrated at the bottom of the crypt where proliferating cells are
present. Some of the staining is present in the nucleus, suggesting
that a portion of the p65 may be active. It is not clear, however,
whether this nuclear staining is an accurate indication of DNA binding
activity. [The antibody for active human p65 (52) does
not recognize mouse p65]. Figure 1B shows the
immunohistochemical staining for NF-
B1 proteins (p50 and p105). In
contrast to the results obtained for p65, NF-
B1 staining is more
even through the length of the crypt, with more cytoplasmic staining
present in cells closer to the luminal surface. Staining with normal
serum shows some background at the top of the crypts, but is
otherwise lower than that obtained with the NF-
B antibodies (Fig.
1C). Specific peptide inhibitors to these antibodies also
indicates that the observed staining patterns are specific (data not
shown). Immunoblot data described in Fig. 4 are also
consistent with these staining patterns.
NF-B activity in epithelial cell isolates.
To study the activity of NF-
B in colonic epithelial cells,
epithelial cells were nonenzymatically stripped from the mouse colon
(33). Sequential incubations in EDTA-containing buffers provided enriched populations of epithelial cells from different regions of the crypt. Three populations were isolated and characterized for markers of cell proliferation (PCNA) and differentiation (alkaline phosphatase). As shown in Fig.
2A, PCNA expression is highest in the last two isolated cell populations, whereas alkaline phosphatase is highest in the first isolated pool. These isolated cell populations were assayed for NF-
B activity by the electrophoretic mobility shift
assay (EMSA). As shown in Fig. 2B, NF-
B activity is
highest in proliferating cells. In addition, the size of the NF-
B
complex differs in the proliferating and mature cells, suggesting a
change in subunit composition. To further characterize the NF-
B from proliferating and mature cells, a supershift assay was performed. As
shown in Fig. 3, NF-
B complexes
isolated from proliferating cells include both the p65 and p50
subunits, whereas the mature cells have primarily p50 dimers (the low
intensity of the p65 supershift may result from antibody
destabilization of the protein-DNA complex). These results are
consistent with the immunohistochemical analysis shown in Fig. 1, in
which proliferating cells displayed nuclear staining for p65 and the
mature cells did not.
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NF-B activity in NF-
B1 knockout mice.
To further explore the role of NF-
B in regulating epithelial cell
turnover, we analyzed colonic epithelium of NF-
B1 knockout mice
(55). Unlike p65 knockout mice, NF-
B1 knockout mice are viable, although they have a number of immunological alterations, including impaired immunoglobin isotype switching (10,
55). An EMSA analysis was performed using nuclear extracts
prepared from colonic epithelial cells of NF-
B1 knockout animals and
their wild-type counterparts. As shown in Fig.
5, the expected NF-
B binding results
were obtained from the wild-type isolates, with both p65-p50 and
p50-p50 dimers present (isolated cell populations were combined for
this assay). However, no NF-
B binding activity was detected in the
colonic epithelial cells of the NF-
B1 knockout mice. The two faint
complexes that are observed in the knockout animals cannot be
supershifted with antibodies to any of the NF-
B subunits. The
NF-
B1 knockout therefore significantly reduces NF-
B activity in
colonic epithelial cells and therefore provides a useful model for
studying the role of NF-
B in epithelial cell turnover in the colon.
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Epithelial cell turnover NF-B1 knockout mice.
To determine the effect of NF-
B1 knockout on epithelial cell
proliferation, colonic crypts were analyzed for length and expression of the PCNA. Figure 6 shows
representative crypts from the distal colon of a wild-type (Fig.
6A) and an NF-
B1 knockout (Fig. 6B) animal.
Figure 7 shows quantitative data of crypt
length (in cell number) and proliferative zone (in cell number)
averaged from three animals. As summarized in Fig. 7, the average crypt
length in wild-type animals is 22 cells in length, whereas the length of a crypt in the knockout tissue is ~31 cells. In addition,
proliferating cells in the wild-type mice extend ~16 cells from the
base of the crypt, whereas in the knockout animals the region of
proliferation extends to ~25 cells from the crypt base. Also shown in
Fig. 6 are the results from an in vivo BrdU labeling of colonic crypts from wild-type (Fig. 6C) and NF-
B1 knockout mice (Fig.
6D). The BrdU labeling results confirm the PCNA staining and
show a cell labeling index ~2.3-fold higher in the knockout animal
(BrdU labeling was estimated as described in MATERIALS AND
METHODS). From these results, we conclude that NF-
B1 plays a
role in suppressing epithelial cell proliferation in the colon. Models
for how NF-
B1 may be performing this growth inhibitory role are
discussed below.
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Gene expression profiling in NF-B1 knockout mice.
To begin to address the mechanism by which the NF-
B1 disruption
leads to enhanced cell proliferation in the colon, gene expression profiling was performed. Two approaches were taken (using RNA isolated
from the entire tissue). First, a Clontech Atlas mouse 1.2 cDNA array
was probed (Fig. 8A).
Comparing the expression patterns on these arrays indicates that the
expression of most genes remains unchanged in the NF-
B1 knockout.
One exception was NGF-
, a subunit of NGF, which was found to be
upregulated ~2.5-fold in the knockout. This result was confirmed by
RT-PCR on an independent animal (Fig. 8B). Second, two
NF-
B-regulated genes of interest that are not on the cDNA array (Fas
and TNF-
) were analyzed by an RNase protection assay (Fig.
8C) (3, 15). In this assay, the NF-
B1
knockouts were found express TNF-
at levels more than five times
higher than their wild-type counterparts. Fas mRNA levels were similar
in the wild-type and NF-
B1 knockouts, indicating that altered
expression of Fas is probably not contributing to the increased
proliferation rate observed in the knockout mice. The increase in
TNF-
expression was confirmed by RT-PCR on two other animals (data
not shown). A number of cell types in the colon have been reported to
express TNF-
, including epithelial cells (45). To
determine whether epithelial cells were expressing TNF-
in the
NF-
B1 knockout, in situ hybridization was performed (Fig.
9). The results from the in situ
hybridization reveal that epithelial cells are at least partly
responsible for the elevated levels of TNF-
in the NF-
B1
knockout. The possible contribution of TNF-
and NGF-
to the
observed proliferation changes in the knockout are discussed below.
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DISCUSSION |
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The transcription factor NF-B participates in the regulation of
cell proliferation and death in many cell types. Its participation in
these events has been studied extensively in cell culture, where
NF-
B is typically found to promote cell proliferation and suppress
apoptosis (6, 7, 10, 12, 13, 29, 34, 43, 56, 60, 63, 67).
However, its role in regulating cell turnover in tissue where a variety
of cell types interact is less well understood. The complexity of
NF-
B function in an intact tissue is highlighted by the fact that
NF-
B is a positive regulator of a number of cytokines, which can
have a profound influence on cell viability (2, 5, 17, 23, 38,
39, 68).
Here we provide evidence that NF-B is active in normal mouse colonic
epithelial cells. Similar results have been reported by others in both
mouse and human tissue (although the level of NF-
B activation
increases in inflamed tissue) (49, 52). Other groups have
also reported expression of NF-
B-regulated genes by normal colonic
epithelial cells, suggesting a role for this transcription factor in
tissue homeostasis (45). Here we provide evidence that
NF-
B expression and activity change in mouse colonic epithelial
cells as they mature. In particular, both the expression and the
activity of the p65 subunit are lower in mature cells at the luminal
surface. Interestingly, the inhibitor I
B
is reciprocally expressed in colonic epithelium, being highly expressed in mature surface epithelial cells (66). Colonic epithelium
therefore appears to be designed to have a low NF-
B response at the
surface. The changes in p65 and I
B
expression during epithelial
cell maturation also suggest that NF-
B may participate in regulating epithelial cell turnover. The temporal and positional cues that are
influencing p65 and I
B
expression are not entirely clear but
could include growth factor availability, extracellular matrix composition, or exposure to luminal contents such as short-chain fatty
acids (9, 16, 44, 58).
To study the influence of NF-B on cell turnover in the colon, we
have analyzed NF-
B1 knockout mice. It was found that disruption of
the NF-
B1 gene leads to a loss in NF-
B activity in colonic epithelial cells and an increase in cell proliferation. In this regard,
colonic epithelium appears to be similar to stratified skin epithelium,
where NF-
B has been found to play a growth inhibitory role
(54). We were unable to detect any differences in
apoptosis in the NF-
B1 knockout by the TUNEL assay. Apoptosis can,
however, be difficult to analyze because apoptotic bodies may be
rapidly eliminated from the tissue by phagocytosis and shedding. For
these reasons it is premature to conclude that the rates of apoptosis have not been affected in the NF-
B1 knockouts. The precise mechanism of NF-
B in regulating cell proliferation in the colon may involve the activation and/or suppression of a battery of genes required in
tissue maintenance.
By gene profiling, two genes were found to be increased in NF-B1
knockout animals, NGF-
and TNF-
. Increased expression of these
genes may be contributing to the enhanced cell proliferation in the
colon of NF-
B1 knockout mice. NGF-
is a component of the mouse
NGF, and although it does not interact directly with cellular
receptors, it is involved in the processing and stabilization of the
receptor-binding NGF-
subunit (22). NGF is produced by
numerous cell types, including intestinal epithelial cells, and it can
promote proliferation of epithelial cells under some conditions
(18, 20, 61). Modest TNF-
levels have also been found
to enhance intestinal epithelial cell proliferation in cell culture
systems, and it has been suggested to play a role in tissue repair
(21, 35-37). The enhanced TNF-
expression may also
be partly responsible for the higher NGF-
expression (1,
46). Further study will be required to determine the
contribution of these proteins to the enhanced proliferation rates in
NF-
B1 knockout mice.
Given the central role of TNF- in intestinal inflammation, the
finding that this protein is upregulated in NF-
B1 knockouts is
particularly interesting (4, 37, 48, 65). This finding is
somewhat counterintuitive because the TNF-
promoter has a number of
functional NF-
B sites (3, 68). A possible explanation for the higher TNF-
expression level in NF-
B1 knockout mice comes
from the finding that the p50 dimer (generated from the NF-
B1 gene)
can act as a potent transcriptional repressor of the TNF-
promoter
through a high-affinity binding site specific for the p50 dimer
(3). Whether mouse intestinal epithelial cells use the p50
dimer to suppress TNF-
expression remains to be determined. The
finding that TNF-
can be produced in the NF-
B1 knockout mice also
implies that other transcription factors participate in the regulation
of TNF-
expression in epithelial cells. A number of other response
elements are present on the TNF-
promoter, and some of these
response elements may be activated in the knockout (3,
68).
In summary, our data show that NF-B is active and regulated in
colonic epithelial cells and that activation of this transcription factor is associated with a suppression of proliferation. Whether NF-
B plays a similar growth inhibitory role during different pathological situations is not clear. Changes in NF-
B activity have
also been implicated in carcinogenesis (8, 14, 51, 63). In
fact, the first NF-
B subunit identified was v-Rel, an oncogene from
the avian reticuloendotheliosis virus. The mechanism by which v-Rel
transforms cells appears to involve the ability of v-Rel to enhance
expression of a set of v-Rel-responsive genes and to interfere with the
ability of other NF-
B subunits to activate gene expression
(62). Other studies have revealed that activation of
NF-
B can enhance proliferation and reduce apoptosis in transformed cells (6, 28, 29, 31, 32, 41, 51, 63, 67). Whether
increased NF-
B activity is a general facilitator of tumor growth in
vivo is not clear. The influence of NF-
B on tumor growth is likely
to depend on a combination of effects, including the ability to promote
cell survival and the ability to stimulate immune and inflammatory
reactions to transformed cells.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Giardina, Dept. of Molecular and Cell Biology, Univ. of Connecticut, U-125, 75 North Eagleville Rd., Storrs, CT 06269-3125 (E-mail: giardina{at}uconnvm.uconn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 February 2000; accepted in final form 24 July 2000.
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