Departments of 1 Medicine and 2 Microbiology and Immunology, and the 3 Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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The ubiquitous
transcription factor NF-B is a central regulator of the
transcriptional activation of a number of genes involved in cell
adhesion, immune and proinflammatory responses, apoptosis, differentiation, and growth. Induction of these genes in intestinal epithelial cells (IECs) by activated NF-
B profoundly influences mucosal inflammation and repair. NF-
B activation requires the removal of I
B from NF-
B by inducible proteolysis, which liberates this transcription factor for migration to the nucleus, where it binds
to
B-regulatory elements and induces transcription. I
B
degradation is incomplete and delayed in IECs, resulting in buffered
responses to luminal stimuli. The stimulatory environment partially
determines whether the effect of NF-
B is protective or deleterious
for the host.
B-dependent proinflammatory gene expression,
particularly chemokines, major histocompatibility complex
class II antigens, and adhesion molecules may be extremely important in
early protective responses to mucosal pathogens but, when dysregulated,
could lead to the development of chronic inflammation, as seen in
inflammatory bowel diseases. The key role of NF-
B in regulating
expression of a number of proinflammatory genes makes this protein an
attractive target for selective therapeutic intervention.
cytokines; signal transduction; bacteria; gene manipulation; intestinal epithelial cells
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INTRODUCTION |
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AN EMERGING AREA OF RESEARCH in intestinal homeostasis
and inflammation is focused on the role of transcription factors in regulating intestinal epithelial cell (IEC) gene expression. This review discusses recent findings concerning the IB/NF-
B signaling pathway and the importance of this transcriptional system in IEC biology, mucosal inflammation, and infection.
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IMPORTANCE OF IECs IN THE MUCOSAL IMMUNE SYSTEM |
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IECs form a single layer of cells that isolate the host from the hostile gut luminal environment. Aside from their classical absorptive and physical barrier roles, an emerging concept views IECs as immunological sentinels of the intestinal mucosa (67). IECs are capable of responding to a wide array of biologically active agents commonly found in the lumen of the distal intestine, including bacterial products, adherent and invasive bacteria, cytokines, and short-chain fatty acids. IECs exert their immunological functions by processing and presenting antigens to T cells (45, 79), expressing cell adhesion molecules (30, 56, 68, 72), secreting various cytokines, particularly chemokines (32, 63, 66), releasing eicosanoids (28, 33, 60), producing nitric oxide (69, 70), and expressing costimulatory molecules (24, 79).
The strategic location of IECs allows them to interact both with
luminal antigens and with resident intraepithelial and lamina propria mononuclear cells to form a complex network of interrelated immunologically active cells that recognize and respond to mucosal infection, injury, and inflammation. Communication between IECs and
mucosal myeloid, lymphoid, and mesenchymal cells might be critical for
maintaining gut immune homeostasis (67). Because of the repertoire of
molecules they produce when activated by microbial agents or
proinflammatory cytokines, IECs can function as sensors of mucosal
injury and actively participate in the mucosal response to intestinal
inflammation and infection (67). In response to bacterial invasion
(66), bacterial products (57, 66), and proinflammatory cytokines (55),
IECs produce a wide variety of chemokines, adhesion molecules, major
histocompatibility complex (MHC) class II molecules, and inflammatory
mediators that influence the adjacent immune and mesenchymal mucosal
cells and recruit circulating inflammatory cells to the mucosa (15, 67,
71, 84). In turn, proinflammatory molecules such as interleukin-1 (IL-1
), tumor necrosis factor-
(TNF-
), and interferon-
(IFN-
), produced by recruited inflammatory and immune cells,
reciprocally stimulate adjacent IECs. Whereas lamina propria
mononuclear cells may be predominantly responsible for chronic,
immune-mediated inflammation, IECs likely are quite important in
maintaining mucosal homeostasis and responding to environmental
challenges that injure the intestine. Therefore, IECs not only
represent the gut's first line of defense but are also part of a
complex and well-orchestrated mucosal immune system.
Although these in vitro studies strongly suggest a role for IECs in intestinal inflammation, it is still unclear whether they participate in the initiating phase of inflammation. For example, transgenic mice overexpressing IL-8 in epithelial cells in the small intestine do not show signs of neutrophilic extravasation or tissue damage (110). However, genetic alteration of the keratin 8 or N-cadherin gene results in intestinal inflammation (9, 44). In addition, a human IEC xenograft model of Escherichia histolytica infection indicates that IECs participate in the initiating phase of inflammation (106). More recently, stimulation of the peroxisome proliferator-activated receptor or expression of the galanin-1 receptor of IECs has been shown to inhibit or potentiate intestinal inflammation, respectively (43, 112).
Most of the molecules synthesized during the course of IEC stimulation
are the result of a highly integrated complex cascade that includes
transmission of a membrane signal to the nucleus via activation of a
series of protein kinases and phosphatases. This sequence of events
ultimately leads to the upregulation of a characteristic set of genes
(66, 130). Most of the immune genes that are upregulated in stimulated
IECs are transcriptionally controlled, belonging to a class known as
immediate-early (IE) genes, for which induction occurs without new
protein synthesis. One of the transcription factors that binds to the
promoter/enhancer region of many IE genes is the nuclear factor B
(NF-
B).
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I![]() ![]() |
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NF-B is an inducible transcription factor comprised of subunits that
can include cRel, RelA, RelB, p50 and p52 (the latter two synthesized
as p105 and p100 precursors, respectively) (7, 10). In most cells the
NF-
B prototype is a heterodimer composed of the RelA (p65) and
NF-
B1 (p50) subunits. This variant is the most potent gene
transactivator among the NF-
B family (8, 97) and is the major
NF-
B protein found in the nucleus of cytokine-stimulated IECs (55,
59).
NF-B is activated by a wide variety of agents, including phorbol
esters, IL-1, TNF-
, lipopolysaccharide (LPS),
double-stranded RNA, cAMP, bacteria, and viral transactivators (7, 10)
(Table 1). Once activated, NF-
B
transcriptionally regulates many cellular genes implicated in early
immune, acute phase, and inflammatory responses, including IL-1
,
TNF-
, IL-2, IL-6, IL-8, IL-12, inducible nitric oxide synthase
(iNOS), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1
(ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), T cell
receptor-
(TCR-
), and MHC class II molecules (7, 10)
(Table 2). Thus the inducers and products
of NF-
B activation are highly relevant to intestinal inflammation
(37, 100).
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Endogenous cytoplasmic inhibitors, known as IBs, tightly regulate
NF-
B activation by complexing with the transcription factor and
trapping it in the cytoplasm. I
B molecules form a distinct family of
proteins that include I
B
, I
B
, I
B
, I
B
, Bcl3, p105, and p100 (41, 116). Proteins in this family are characterized by
an ankyrin repeat domain involved in protein/protein interaction (11,
41).
The most characterized and studied NF-B inhibitor is I
B
. This
protein binds avidly to the p65 (RelA) subunit of NF-
B through the
association of ankyrin repeat domains of I
B
with the nuclear localization signal and the Ig-like domain of p65 (52, 53). During
activation of NF-
B, numerous stimuli, including IL-1
and TNF-
, activate a complex of I
B kinases (IKK) that
phosphorylate I
B
on the amino terminus at serine residues 32 and
36 (Fig. 1) (21). Phosphorylation of these
serine residues is a necessary step for inducible I
B degradation;
replacement of these two amino acids by site-directed mutagenesis
prevents I
B degradation and NF-
B activation (16, 17, 21, 62,
118). Phosphorylated I
B
is then selectively ubiquinated and
rapidly degraded via a nonlysosomal, ATP-dependent 26S proteolytic
complex composed of a 700-kDa proteasome (73, 89, 103). Evidence for
the role of ubiquination comes from recent work showing that target
inactivation of a specific I
B-ubiquitin ligase inhibits NF-
B
activation (131, 132). In the final steps of the activation cascade,
phosphorylation and proteolytic degradation of I
B allows the release
and nuclear transmigration of NF-
B (36, 116). Degradation of I
B
exposes the NF-
B nuclear localization signal, resulting in
transportation of NF-
B into the nucleus (6).
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Together with IB
(37 kDa), the proteins I
B
and I
B
(each 45 kDa) are the most abundant and potent mammalian NF-
B
inhibitors. Although each of these three proteins acts to inhibit
NF-
B, there are differences among them with regard to their affinity
for NF-
B and their mechanism of action (6, 42 , 108, 109, 117, 119, 126). Regulation and activation of I
B
differs considerably from
that of I
B
and I
B
. In most mammalian cells I
B
is
rapidly and completely degraded (<10 min) following inducible
phosphorylation but is quickly resynthesized (60 min) in an
NF-
B-dependent manner (Fig. 1) (113). Newly synthesized I
B
complexes with nuclear NF-
B to terminate gene transcription (4, 6,
93, 120). Thus stimulation of NF-
B induces its own inhibitor to
regulate cellular activation. In contrast, I
B
transcription is
not regulated by NF-
B; therefore, the protein is slowly degraded (2 h) on IL-1 and LPS stimulation and leads to persistent activation of
NF-
B (>20 h) (65, 117, 124). The mechanism of persistent
activation on I
B
degradation may involve a chaperone-like role of
unphosphorylated, newly synthesized I
B
that allows transport of
NF-
B to the nucleus without being trapped by I
B
(114).
Preliminary reports show that degradation and resynthesis are slower
for I
B
than for I
B
and that the inhibitory effect of
I
B
is exerted in the cytoplasm (108, 109, 126). This pool of
variably responding I
B molecules, with different kinetics of
degradation, synthesis, and affinity, may allow cells to activate
NF-
B differentially and, consequently, to regulate downstream genes
differentially in response to the wide array of stimulating agents.
Another level of control for NF-B activation involves
cytokine-induced phosphorylation of RelA/p65, which modulates its
transactivation capacity (29, 123, 136). The kinase(s) responsible for
p65 phosphorylation remains to be formally identified, but
potential candidates include IKK and casein kinase II (12, 80).
Therefore, similar to c-jun regulation, NF-
B transactivational
activity could be induced by phosphorylation, independently of its
DNA-binding activity.
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CYTOKINE AND BACTERIAL PRODUCTS SIGNALING THROUGH THE
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Recent findings in cytokine signaling have provided a better
understanding of proximal events involved in NF-B activation, with
elucidation of the mechanisms by which cytokines transduce their
signals through the I
B/NF-
B system (Fig.
2). Most of the cytokine and microbial
product receptors have no intrinsic kinase activity and, therefore,
rely on scaffolding and adaptor proteins to transmit their
extracellular signal inside the cells. On ligand binding, these
receptors aggregate and form a multimer complex followed by the
recruitment of downstream scaffolding and adaptor proteins to the
cytoplasmic tail portion of the receptor. For example, following
TNF-
stimulation, the TNF receptor-1 (TNFR1) trimerizes and recruits
the TNF receptor-associated factor (TRAF)-2 and the receptor
interacting protein (RIP) to the cytoplasmic portion of
the TNFR1 via the intermediate action of TNF receptor 1-associated death domain (TRADD) (49, 50). In another
example, stimulation by the cytokine IL-1
initiates a
signaling cascade that requires the participation of the IL-1 receptor
accessory protein (IL-1RAcp), MyD88, and the IL-1 receptor-associated
kinase (IRAK). These proteins act together to associate with and
activate TRAF-6 (18-20, 125). In each case, the signal coming from
the respective TRAF/RIP protein is transmitted to the NF-
B-inducing kinase (NIK) (77), which in turn associates/activates the IKK complex
(Fig. 2) (75, 111, 121).
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The IKK complex is composed of at least IKK-, IKK-
, and
IKK-
/Nemo subunits and a scaffolding protein named IKK
complex-associated protein (IKAP) (102). Differences are seen in the
roles of the IKK complex proteins from in vitro studies compared with
in vivo studies. In vitro studies suggest that all of the IKK complex proteins are critical in mediating/controlling cytokine-induced I
B
phosphorylation (23, 81, 96, 128, 134). However, recent findings using
gene deletion technology provide a different picture regarding the
contribution of each kinase in NF-
B activation in vivo (78). From
these studies, it appears that IKK-
, but not IKK-
, mediates
cytokine-induced NF-
B activation (74), whereas the role of IKK-
in vivo seems to be restricted to transmitting signals involved in
skeletal development (51, 115). These results suggest that strategies
aimed at manipulating the cytokine inflammatory cascade during
inflammation should target IKK-
rather than IKK-
.
Another question concerns which upstream kinase mediates
cytokine-inducible NF-B activation in vivo. A puzzling observation is that IKK-
kinase activity is preferentially activated by the mitogen-activated protein kinase kinase kinase-1 (MEKK-1), whereas the
NF-
B-inducing kinase NIK has a higher affinity for IKK-
(83, 85).
Gene deletion experiments with MEKK-1 and NIK would help clarify the
physiological contribution of each kinase in cytokine-induced NF-
B
activation. Interestingly, adenoviral-mediated delivery of a
constitutively active NIK (Ad5NIK) strongly induces I
B
phosphorylation/degradation, NF-
B activation, and IL-8 secretion in
Caco-2 cells (64). This result suggests that NIK activates the critical
IKK
subunit responsible for NF-
B induction.
There are additional levels of regulation for NF-B induction by
cytokine-induced phosphorylation. Regulation of IKK
activity appears
to be mediated by a cluster of serine residues located next to the
helix-loop-helix (HLH) domain (27). The progressive phosphorylation of
these serine residues seems to weaken the interaction between the HLH
domain and the kinase domain, resulting in a decrease in kinase
activity and a consequent decrease in IKK
phosphorylation activity.
Bacterial products such as LPS and peptidoglycan-polysaccharide (PG-PS)
are found in high concentrations in the colon and are able to stimulate
IECs (67). Therefore, the elucidation of bacterial products signal
transduction through the IB/NF-
B system is highly relevant for
mucosal homeostasis. Recently, it was demonstrated that LPS signaling
involved the Toll-like receptors (TLR) (48, 129). Moreover, LPS was
shown to signal through the NF-
B system by utilizing components of
the IL-1 pathway such as MyD88, IRAK, and TRAF-6 in endothelial and
monocytic cell lines (135). Therefore, it would be important to
determine whether LPS and PG-PS signal through the NF-
B system in a
similar manner in IECs.
Because the signaling cascade leading to NF-B activation involves
the participation of multiple proteins, this complex network provides
many potential targets for therapeutic intervention. Recent studies
have focused on the role of NF-
B in the clinically important field
of intestinal inflammation.
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UNIQUE CYTOKINE SIGNAL TRANSDUCTION AND I![]() ![]() ![]() |
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In the past few years, there has been increased interest in how
cytokines, bacteria, and bacterial polymers induce IEC gene expression.
IEC gene expression must be tightly regulated to avoid overreaction to
normal microbial flora while at the same time remaining adequately
responsive to environmental pathogens. The IECs from the distal ileum
and colon are in constant contact with a rich microbial flora, with
luminal contents having as much as 108 aerobic and
1011-12 anaerobic enteric bacteria/g wet wt and 80 µg LPS/g feces, yet, under normal conditions, no
pathological inflammation is present. This observation, in conjunction
with our observation that mature IECs are relatively refractory to
IL-1, LPS, and PG-PS activation, suggests that IECs have developed
unique protective responses that allow them to remain quiescent in a
hostile environment (Table 3).
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The unique signaling pathways associated with cytokine-induced NF-B
activation in IECs has been investigated using various approaches.
Adenoviral gene delivery of a truncated TRAF-2 protein revealed that
TNF-
partially mediates NF-
B activation and IL-8 expression
through TRAF-2 in HT-29 and IEC-6 cells (59). In addition, in HT-29
cells, although not in several other cell types (26), TRAF-2 is
required for IL-1
-induced NF-
B and IL-8 gene expression (59).
These data suggest that interactions occur between the TRAF-2 protein
and several different cytokine receptors in IECs in contrast to signal
transduction in many bone marrow-derived cells, where TRAF-2 interacts
solely with the TNF receptor family.
Native colonic epithelial cells and most IEC lines have delayed and
incomplete IB
degradation following cytokine activation (55), in
addition to a strong decrease in IKK activity (64). Although viral
delivery of NIK induces a strong I
B
phosphorylation and
NF-
B-activated gene expression in HT-29 cells, only a marginal degradation of I
B
steady-state level is observed in these cells (64). By contrast, ectopic expression of NIK and IKK induces almost
complete I
B degradation in Caco-2 cells (54, 64). In addition, the
IL-1
signaling kinase IRAK is rapidly and completely degraded
in IL-1
-stimulated Caco-2 cells but not in HT-29 cells (14,
64). Because the proteasome pathway mediates degradation of both
I
B
and IRAK, it is tempting to speculate that HT-29 cells
have a defect in their proteasome pathway, although there is
currently insufficient evidence to support this hypothesis. The
physiological mechanisms of I
B resistance to degradation and its
relevance to intestinal homeostasis remain to be determined.
The intestinal mucosa is composed of a dynamic cell population in
perpetual change from a proliferative and undifferentiated stage
(crypt-base) to mature surface villus epithelial cells (1, 92, 127).
Migration of cells from the crypt to the surface of the colon is
accompanied by cellular differentiation that leads to important
morphological and functional changes. Although several studies have
shown that this process involves substantial changes of cellular
morphology, growth, proliferation, and expression of biochemical
markers (76, 137), little is known about the alteration of
immunological functions as IECs mature. Interestingly, the IL-1
signaling pathway leading to IKK and NF-
B activation is
downregulated in differentiated HT-29 cells (surfacelike cells) compared with undifferentiated cells (cryptlike cells) (14). In
addition, bacterial invasion is reduced in differentiated IECs (22).
These findings suggest that a gradient of NF-
B activation may exist
along the crypt-surface axis in response to stimulation by
proinflammatory cytokines and bacteria.
IEC apoptosis is an important phenomenon in mucosal homeostasis,
assuring a constant balance between cell production and cell loss.
Recently, NF-B was shown to play a protective role against apoptosis
mediated by some death signals such as TNF-
and radiation in many
cell types (5). Therefore, NF-
B activation status may have an impact
on intestinal hyperplasia through cell removal by apoptosis in a manner
similar to experimental rheumatoid arthritis (82). Of interest,
cellular differentiation dramatically sensitized HT-29 cells to
Fas-mediated apoptosis (M. P. Russo, R. B. Sartor, and C. Jobin,
unpublished observations). The combined effect of IEC differentiation
on downregulation of IL-1
signaling and increased susceptibility to
Fas-mediated apoptosis may represent a mechanism to maintain mucosal
homeostasis. However, the role of NF-
B in the IEC apoptotic process
remains to be established, since this transcription factor also seems
to play a proapoptotic role in HT-29 cells (40).
Although these data shed some light on the complex cytokine-induced
NF-B signal cascade in IECs, many questions remain unanswered. For example, the relative contribution and role of various
adaptor proteins and kinases in cytokine-induced NF-
B activation
remain unclear. The responsiveness of native IECs at various stages of differentiation to physiological stimuli needs to be determined to
address the hypothesis that IECs have dampened NF-
B-regulated responses that preserve homeostasis in an aggressive luminal environment.
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THE I![]() ![]() |
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NF-B regulates the transcription of a number of proinflammatory
molecules involved in acute responses to injury and in chronic intestinal inflammation, including IL-1
, TNF-
, IL-6, IL-8, IL-12, iNOS, ICAM-1, VCAM-1, TCR-
, and MHC class II molecules (Table 2) (7,
10, 87). In addition, activation of NF-
B in IECs has been
demonstrated in vivo (95). NF-
B activation as indicated by increased
DNA binding activity and p65 nuclear translocation has been documented
in the intestine of patients with Crohn's disease, ulcerative colitis,
and self-limited colitis (86, 95, 105), as well as in rodents with
experimental colitis. (86). The amount of activated NF-
B correlates
with the degree of mucosal inflammation. Immunohistochemistry performed
on tissue sections isolated from patients with inflammatory bowel
disease (IBD) demonstrates the presence of activated NF-
B in IECs
located at the crypts but not at the surface region (95).
Because IECs are surrounded by commensal bacteria and are the first
cells affected by pathogens, the effect of bacteria on NF-B
activation in IECs has been examined. Savkovic et al. (101) reported
that T84 cells infected with enteropathogenic Escherichia coli
secrete IL-8 through a NF-
B-dependent mechanism. However, IL-8 gene
expression was not induced in IECs infected with nonpathogenic E. coli, suggesting that IECs discriminate between pathogenic and
nonpathogenic bacteria with respect to NF-
B activation (101). This
discriminatory mechanism may ensure proper IEC regulation in response
to the natural enteric flora and its constituents. These data further
suggest that specific bacterial genes activate the IEC signaling
cascade leading to NF-
B activation and gene expression.
Hobbie and colleagues (47) have shown that Salmonella infection
of IECs triggers IB
degradation and IL-8 release. A number of
invasive bacteria as well as high doses of bacterial cell wall polymers
(LPS, endotoxins) and PG-PS induce NF-
B-dependent chemokines in a
wide variety of IEC lines (57, 67). Additional studies have shown that
bacterial invasion activates the IKK complex in IECs, thereby
triggering NF-
B activity (35). These findings provide a link between
bacterial invasion and induction of the I
B/NF-
B system, although
the exact mechanisms involved in signal transduction remain to be
eludicated. The requirement of bacterial invasion for NF-
B
activation is controversial, since noninvasive bacteria can also
trigger NF-
B activation (31). However, it is evident that a variety
of enteric microbial stimuli, including parasitic infection and
nonviable cell wall polymers, can also induce NF-
B-dependent
chemokine expression.
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MANIPULATION OF INTESTINAL INFLAMMATION BY TARGETING THE
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The critical role of NF-B in intestinal inflammation is eloquently
illustrated by the profound inhibition of inflammatory responses
following selective blockade of NF-
B activation. For example, local
or systemic administration of p65 antisense oligonucleotides reversed
chronic experimental colitis induced in mice by trinitrobenzene sulfonic acid (86). Likewise, administration of specific proteasome inhibitors markedly attenuated PG-PS-induced granulomatous colitis in
rats, presumably by inhibition of NF-
B (25). In both studies, however, it was unclear which cell populations responded to NF-
B inhibition in vivo.
Pharmacological agents and molecular approaches have been used in vitro
to investigate the role of NF-B in cytokine-stimulated gene
expression and signal transduction in IECs. Proteasome inhibitors effectively prevent cytokine-induced IL-8 and ICAM-1 gene expression by
suppressing I
B
degradation and NF-
B activation in transformed IECs (55, 56). Of considerable importance, several anti-inflammatory drugs used in treatment of IBD mediate their effects in part through inhibition of the I
B/NF-
B pathway. Dexamethasone at
pharmacologically relevant concentrations stimulates I
B
synthesis, stabilizes I
B mRNA, and apparently interferes with I
B
degradation in IEC-6 cells (58). Biopsy samples from IBD patients
treated in vivo with corticosteroids revealed a decrease of NF-
B
activity (3). Furthermore, the anti-inflammatory compound sulfasalazine
used for IBD treatment blocks degradation of I
B mediated by both
TNF-
and LPS and prevents NF-
B activation in transformed IECs
(122). Of interest, mesalamine, an aminosalicylate, inhibits
IL-1
-induced NF-
B activity by interfering with inducible p65
phosphorylation but not I
B
degradation (34). In addition, IL-10
has a dual mechanism of inhibiting NF-
B-mediated gene transcription
by transiently suppressing IKK activity and interfering with NF-
B
DNA binding by an undefined mechanism in monocytes (104).
To gain more specificity over the pharmacological approach, molecular
interventions using dominant- negative versions of individual components of the IB/NF-
B pathway delivered by adenoviral vectors were designed and tested in transformed and primary IECs. An adenoviral vector bearing a cytokine-resistant proteolysis I
B
mutated at serine-32 and serine-36 (Ad5I
B
AA) was effective in blocking gene
expression of IL-1, IL-8, iNOS, and COX-2 by mRNA and protein analysis
(60, 62). A dominant-negative TRAF-2 mutant was also effective
in blocking TNF-
-induced gene expression of IL-8 and NF-
B nuclear
transmigration, although to a much lesser extent than Ad5I
B
AA
(59, 62). This may suggest that targeting divergent adaptor proteins is
less effective in blocking NF-
B than targeting convergent proteins
(such as NIK and IKK). Strategies aimed at blocking NIK or IKK
using
adenoviral gene delivery should provide valuable answers regarding
their efficacy as therapeutic targets, as suggested by preliminary
results of NF-
B and IL-8 blockade by a NIK dominant-negative
molecule (61).
Although adenoviral molecular approaches have been successful in
inhibiting NF-B in vitro, there is no evidence that similar strategies would be effective in vivo. For example, the use of gene
therapy raises the concern of potential host immunological responses
against viral vector proteins (39, 133). In addition, a method for the
specific, efficient long-term delivery of an adenoviral vector into the
intestinal epithelium or lamina propria would be needed to sustain
exogene expression. Interestingly, natural dietary products have
recently been shown to be potent inhibitors of cytokine-mediated
NF-
B activation and IL-8 expression through IKK inhibition,
suggesting a potential therapeutic application in vivo (54) as a more
practical approach compared with antisense oligonucleotide (86) and
intranasal delivery systems (38).
The complexity of the NF-B activation pathway provides various
potential targets for selective therapeutic intervention in IECs. The
development of specific and safe NF-
B inhibitors delivered either
locally or systemically could create a useful arsenal to complement the
more globally acting drugs currently used to manage intestinal
inflammation. The biggest challenge in the therapeutic application of
this class of antagonists will be to inhibit the deleterious side of
NF-
B without impairing normal cell functions dependent on NF-
B.
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SUMMARY AND PERSPECTIVE |
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Many aspects of cellular function are regulated by the IB/NF-
B
system. Biological processes such as immune and inflammatory responses,
as well as cell growth and apoptosis, are in part regulated by NF-
B.
The multiple signals leading to NF-
B activation converge on a series
of adaptor proteins and kinases in a cascade that leads to I
B
phosphorylation/degradation, nuclear translocation of NF-
B dimers,
and transcriptional activation of NF-
B-dependent genes. Many
research groups have demonstrated the critical role of NF-
B in
proinflammatory gene expression by IECs after cytokine or microbial stimulation.
It appears that IB
is relatively resistant to degradation due to
decreased IKK complex activity in differentiated IEC lines and native
epithelial cells and that differentiated IECs respond less efficiently
to IL-1 stimulation and are more sensitive to Fas-mediated apoptosis.
We postulate that this relative unresponsiveness allows IECs to exist
in a quiescent state (Fig. 3A),
while in constant contact with ubiquitous endogenous luminal bacteria
and bacterial products, yet to be capable of mounting a rapid response to microbial pathogens, resulting in chemotactic signals and adhesion molecules that recruit phagocytic effector cells to the injured mucosa
(Fig. 3B).
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Despite these rapidly accumulating findings, many questions remain
unanswered. For example, the role that NF-B plays in growth, differentiation, and apoptosis in IECs is currently unknown. Recently, it has been show that NF-
B may control cell cycle regulation by
modulating cyclin D1 expression (46). These biological processes are
key determinants of intestinal development, repair, inflammation, and
carcinogenesis. Therefore, understanding the contribution of NF-
B could lead to novel therapeutic approaches to these
conditions. To design an efficient, nontoxic blocking strategy, more
data are needed to describe the precise mechanisms of bacterial and cytokine induction of the signaling cascade through the I
B/NF-
B system. Equally important is the relative input and possible
interaction of epithelial cells vs. lamina propria mononuclear cells in
the development and persistence of intestinal inflammation. The
generation of transgenic mice carrying an NF-
B inhibitor (such as
mutated, nondegradable I
B) or expressing a constitutively active
NF-
B (such as active IKK or NIK) regulated by an intestinal
cell-specific promoter should help address the role and contribution of
IECs in intestinal homeostasis and inflammation. Regulation of the I
B/NF-
B system in IECs represents a new and exciting era of research in intestinal inflammatory diseases and neoplasia that could
potentially give rise to new targets for therapeutic intervention.
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ACKNOWLEDGEMENTS |
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We thank Barbara J. Rutledge for editorial assistance. Grant support was provided by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47700 and by the Crohn's and Colitis Foundation of America.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Jobin, Division of Digestive Diseases and Nutrition, CB# 7038, Glaxo Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038 (E-mail: Job{at}med.unc.edu).
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