Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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Acute lung
injury occurs as a result of a cascade of cellular events initiated by
either infectious or noninfectious inflammatory stimuli. An elevated
level of proinflammatory mediators combined with a decreased expression
of anti-inflammatory molecules is a critical component of lung
inflammation. Expression of proinflammatory genes is regulated by
transcriptional mechanisms. Nuclear factor-B (NF-
B) is one
critical transcription factor required for maximal expression of many
cytokines involved in the pathogenesis of acute lung injury. Activation
and regulation of NF-
B are tightly controlled by a complicated
signaling cascade. In acute lung injury caused by infection of
bacteria, Toll-like receptors play a central role in initiating the
innate immune system and activating NF-
B. Anti-inflammatory cytokines such as interleukin-10 and interleukin-13 have been shown to
suppress inflammatory processes through inhibiting NF-
B activation.
NF-
B can interact with other transcription factors, and these
interactions thereby lead to greater transcriptional selectivity.
Modification of transcription is likely to be a logical therapeutic
target for acute lung injury.
nuclear factor-B; transcription factor; cytokine; pulmonary
inflammation
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INTRODUCTION |
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THE LUNG IS AN IMPORTANT TARGET ORGAN
for systemic inflammatory mediators released after major trauma
(6, 39, 44) and severe infection (113, 118).
Acute respiratory distress syndrome (ARDS), clinically manifested as
inflammatory lung injury of very rapid onset, is characterized by
intractable respiratory dysfunction. This syndrome is associated with
hypoxemia, reduced lung compliance, and diffuse infiltrations
(13). Neutrophils sequestered in the lung play a central
role in the pathogenesis of ARDS (50). Lung neutrophil
accumulation seen in ARDS is thought to occur as a result of a cascade
of cellular events initiated by either infectious or noninfectious
inflammatory stimuli. Local activation of resident cells in the lung
interstitium and alveolus, primarily macrophages, leads to elaboration
of several proinflammatory cytokines such as tumor necrosis factor
(TNF)- and interleukin (IL)-1
and chemokines including IL-8 and
macrophage inflammatory protein (MIP)-2
(35, 37, 47,
87). These mediators act in concert to promote neutrophil sequestration by activating endothelial cell adhesion molecule expression and to induce migration of neutrophils into the interstitium and alveolar space where they propagate inflammation and injury through
the release of reactive oxygen species (ROS) and proteolytic enzymes
(155). A counterinflammatory response in the lungs is also
initiated with the release of anti-inflammatory cytokines such as
IL-10, IL-4, IL-13, IL-1 receptor antagonist (IL-1RA), and transforming
growth factor (TGF)-
(36, 47, 109). Elevated levels of
proinflammatory mediators combined with decreased expression of
anti-inflammatory molecules correlate with the magnitude of lung injury
and its outcome (108). The imbalance between
proinflammatory and anti-inflammatory mechanisms is a critical
component of lung inflammation.
The signals that lead to increased gene expression and biosynthesis of
proinflammatory mediators by airspace inflammatory and epithelial cells
are thus of considerable interest. An understanding of the expression
of genes, in particular the transcriptional apparatus usually located
in the upstream region of the gene promoter, is required to explain the
regulation of expression of proinflammatory genes. Nuclear factor
(NF)-B is one such critical transcription factor required for
maximal expression of many cytokines involved in the pathogenesis of
ARDS. NF-
B, first identified by Sen and Baltimore
(124), functions to enhance the transcription of a variety
of genes, including cytokines and growth factors, adhesion molecules,
immunoreceptors, and acute-phase proteins (a list of genes containing
NF-
B sites in their promoters is shown in Table 1). Under resting condition, NF-
B
functions in regulating the expression of genes involved in normal
immunologic responses such as the generation of antibody light chains
and other immunoregulatory molecules (124, 152). NF-
B
activation is necessary for an intact host defense response, whereas
excessive activation of NF-
B results in overly exuberant
inflammatory injury of lungs and other organs (17, 18,
40).
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In this review, we focus on the recent progress in our understanding of
the role of NF-B and interactions between NF-
B and other
transcription factors in the pathogenesis of acute lung injury.
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MOLECULAR BIOLOGY OF NF-![]() |
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The NF-B family of transcription factors.
NF-
B belongs to the Rel family of proteins that are ubiquitous
transcription factors sharing a common structural motif for DNA binding
and dimerization (10). Although, for the sake of simplicity, NF-
B is often referred to as a single entity, it is, in
reality, a complex mixture of homodimers and heterodimers, all with
distinct characteristics and biological properties. The cloning of
NF-
B family members has revealed five distinct subunits homologous
to the protooncogene c-Rel and the Drosophila melanogaster morphogen dorsal: NF-
B1 (p50/105), NF-
B2 (p52/100),
Rel A (p65), Rel B, and c-Rel itself (10, 134). These
subunits are all highly related over an ~300-residue amino-terminal
DNA binding and dimerization domain termed the Rel homology domain.
Activation of NF-B.
NF-
B is normally sequestered in the cytoplasm through its
association with an inhibitor protein called inhibitory
B (I
B), which masks the nuclear translocation signal and thus prevents NF-
B
from entering the nucleus. NF-
B activation represents the terminal
step in a signal transduction pathway leading from the cell surface to
the nucleus. On exposure of the cell to activation signals such as the
binding of lipopolysaccharide (LPS), TNF-
, or IL-1
to cell
surface receptors, the I
B protein is phosphorylated on serine-32 and
serine-36, ubiquinated, and degraded in proteasomes. After being freed
from association with I
B, the NF-
B complex moves to the nucleus
where it binds to specific sequences in the promoter/enhancer regions
of genes.
NF-B inhibitors and their regulation.
The various forms of I
B include I
B-
, I
B-
, I
B-
, and
Bcl-3 (10, 45, 153). In addition, p105, which is the
precursor of p50, and p100, which is the precursor of p52, can bind Rel A and thus function as NF-
B inhibitors (31, 83, 97).
The COOH-terminal portions of p105 and p100 have been designated
I
B-
and I
B-
, respectively. These inhibitory units contain
multiple copies of ankyrin repeat domains that allow interaction with
NF-
B in a configuration that masks the nuclear localization signal domains of NF-
B, thereby preventing nuclear transport
(7). Signaling pathways leading to NF-
B activation
converge on phosphorylation, ubiquitination, and degradation of I
Bs
(except Bcl-3), which unmask the nuclear localization signal leading to
translocation of NF-
B/Rel dimers into the nucleus (4, 86,
125). I
Bs are also active in the nucleus. I
B-
and
I
B-
interact with p50/p65 NF-
B heterodimers in the nucleus as
in the cytoplasm, inhibiting transcriptional activity of NF-
B
(4, 144). In experiments examining NF-
B activation in
the lung, levels of I
B-
in the nucleus as well as in the
cytoplasm were elevated in a mixed population of intraparenchymal lung
cells after hemorrhagic shock in association with NF-
B nuclear
translocation (94). These findings suggest that
1) I
B-
is upregulated by NF-
B and 2)
other I
B proteins contribute to NF-
B activation in the lung after
blood loss.
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BACTERIA-INDUCED NF-![]() |
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Infection with microbial pathogens and septicemia are major causes
of acute lung injury. Major causative agents include gram-negative and/or gram-positive bacteria. Septic shock and acute lung injury follow a pattern in which one or more products of microbial pathogens such as the endotoxin of gram-negative bacteria, diverse membrane components of gram-positive bacteria, and nonmethylated CpG-rich sequences of bacterial DNA activate the innate immune system. This
system represents the immediate host defense mechanism that involves
the secretion of cytokines and other mediators with antimicrobial effects. It is well documented that phylogenetically conserved signaling mechanisms provide an immediate cellular reaction that utilizes NF-B at the center of its first line of defense
(89). The recognition of a pathogen is mediated by a set
of germ line-encoded receptors referred to as pattern-recognition
receptors, which directly recognize invariant molecular structures
(pathogen-associated molecular patterns). These are shared by large
groups of microorganisms (56, 88). The three functional
classes of pattern-recognition receptors are signaling receptors,
endocytic receptors, and secreted proteins. Studies over the past few
years have demonstrated that a family of signaling receptors, known as
the Toll-like receptors (TLRs), plays a crucial role in
Drosophila and mammalian host defense. In both organisms,
TLRs activate intracellular signaling, notably via NF-
B. At least
nine TLRs have been identified in humans, but only three of them have
been characterized as interacting with a ligand of bacterial origin
(89, 116, 142). TLR2 is a receptor for peptidoglycan and
lipopeptide from gram-positive bacteria and zymosan from yeast. TLR4
recognizes LPS from gram-negative bacteria (reviewed in Ref.
102). It was recently shown that TLR9 is a putative
receptor for bacterially derived CpG DNA (51). All members
of the Toll family are membrane proteins that span the membrane once
and share similar extracellular domains, which include 18-31
leucine-rich repeats and similar cytoplasmic domains of ~200 amino
acids. Interestingly, the latter domain is also similar to the
cytoplasmic domain of the IL-1 receptor [Toll/IL-1 receptor homologous
region (TIR)]. The cytoplasmic domains of the Toll family define a
subclass of TIR domains. The TIR domain of human TLR4, for example, is
32% identical to Drosophila Toll but less similar (20%
identical) to the TIR domain of the IL-1 receptor.
Specific components of microbial cell walls are strong activators of
innate immune responses. Human pathogen-associated molecular patterns
such as LPS of gram-negative bacteria and peptidoglycan and
lipoteichoic acid components of gram-positive bacteria trigger the
production of IL-1, TNF-
(103, 122, 141, 157), and
IL-6 (74) as well as of cyclooxygenase metabolites
(114). Recent genetic and biochemical experiments
have highlighted the critical role of TLRs in LPS-induced NF-
B
activation (27, 60, 100). Importantly, the responsiveness
of TLRs to LPS is dependent on LPS binding protein and CD14 (121,
139). On binding of LPS to TLRs via CD14, a number of molecules
are recruited to the receptor to mediate NF-
B activation (Fig.
2). First, the adaptor molecule myeloid
differentiation factor 88 (MyD88) binds to the receptor and interacts
through its death domain with the protein kinase IL-1
receptor-associated kinase (IRAK), which, in turn, recruits the adaptor
TRAF-6 to the receptor complex (49). The importance of
MyD88 in LPS signaling was confirmed by analysis of MyD88-deficient mice, which are normally highly resistant to LPS-induced shock (62). Evolutionarily conserved signaling intermediate in
Toll pathways (ECSIT) bridges TRAF-6 to MEKK1. This protein is specific for the Toll/IL-1 pathway and is a regulator of MEKK1 processing (64). Expression of wild-type ECSIT accelerates the
processing of MEKK1, whereas a dominant negative fragment of ECSIT
blocks MEKK1 processing and activation of NF-
B (64). A
recent study (3) indicated that stimulation of TLR2 by
Staphylococcus aureus induces NF-
B activation through the
small GTPases Rac1 and Cdc42.
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Resuscitated hemorrhagic shock is believed to promote the development
of lung injury by priming the immune system for an exaggerated inflammatory response to a second, often trivial, stimulus, the so-called "two-hit hypothesis" (41, 95). In recent
studies, a rodent model of LPS-induced lung injury after resuscitated
hemorrhagic shock was used to examine the regulation of TLR4 gene and
protein expression in vivo and to evaluate the impact of its regulation on the development of inflammation (Fan J and Rotstein OD,
unpublished data). The studies have demonstrated that intratracheal LPS
alone induces a rapid reduction of TLR4 mRNA in the whole lung and
recovered alveolar macrophages. This effect appeared to be due to a
lowering of TLR4 mRNA stability by ~69%. In contrast, although shock
and resuscitation alone have no effect on TLR4 mRNA levels, they
markedly alter the response to LPS. Specifically, the antecedent shock prevented the LPS-induced reduction in TLR4 mRNA levels in whole lung
tissue and alveolar macrophages. This reversal can be explained by the
ability of prior resuscitated shock to prevent the destabilization of
TLR4 mRNA by LPS as well as to augment LPS-stimulated TLR4 gene
transcription compared with LPS alone. Oxidant stress related to shock
or resuscitation appears to contribute to the regulation of TLR4 mRNA.
Supplementation of the resuscitation fluid with the antioxidant NAC
reversed the ability of shock or resuscitation to preserve TLR4 mRNA
levels after LPS. TLR4 protein levels in whole lung and surface
expression on alveolar macrophages mirrored the changes seen in TLR4
mRNA. Furthermore, preservation of surface TLR4 receptors in
LPS-treated alveolar macrophages recovered from shock or resuscitated
animals correlated with enhanced NIK activity, NF-B nuclear
translocation, and inflammatory cytokine gene transcription (Fan J and
Rotstein OD, personal communication). These data suggest that
Tlr4 expression is controlled both transcriptionally
as well as posttranscriptionally through altered mRNA stability and
that changes in the local cellular microenvironment profoundly
influence the net effect of these processes and, ultimately, cell responsiveness.
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ROLE OF NF-![]() |
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Increasing evidence shows an important role for NF-B in the
pathogenesis of acute lung inflammation. In vitro studies have shown that NF-
B regulates gene expression of cytokines (TNF-
, IL-1
), chemokines [MIP-2, cytokine-induced neutrophil
chemoattractant (CINC)], and adhesion molecules [intercellular
adhesion molecule (ICAM)-1, E-selectin] (see Ref. 17).
All of these factors play an important role in lung inflammatory injury
(30). These in vitro findings are supported by a study in
humans with ARDS (123) showing enhanced NF-
B activation
in alveolar macrophages recovered by bronchoalveolar lavage. In
addition, an in vivo study in animals (82) has shown an
association between NF-
B activation and the expression of cytokines,
chemokines, and vascular adhesion molecules (82). Recent
studies in vivo demonstrated that lung NF-
B activation is suppressed
by antioxidants (16, 76-78) or anti-inflammatory cytokines (70), resulting in decreased proinflammatory
mediator expression and reduced inflammatory injury. Thus it appears
that activation of NF-
B is central to the development of pulmonary inflammation and acute lung injury.
Lung neutrophil activation and apoptosis.
Acute lung injury is characterized by the accumulation of neutrophils
in the lungs, accompanied by the development of interstitial edema and
an intense inflammatory response. A study (2) has addressed the role of neutrophils as early immune effectors in hemorrhage- or endotoxemia-induced lung injury. Mice were made neutropenic with cyclophosphamide or anti-neutrophil antibodies, and it
was shown that endotoxemia- or hemorrhage-induced lung edema was
significantly reduced in those animals (2). Activation of
NF-B after hemorrhage or endotoxemia was also diminished in the
lungs of neutropenic mice compared with the lungs in nonneutropenic mice. These experiments show that neutrophils play a central role in
initiating acute inflammatory responses and inducing tissue injury in
lungs after hemorrhage or endotoxemia.
Alveolar macrophages.
Alveolar macrophages are strategically situated at the air-tissue
interface in the alveoli and alveolar ducts and are therefore the first
cells encountered by inhaled organisms and antigens in the lower
respiratory tract. Alveolar macrophages not only act in their
traditional role as phagocytes but also function as potent secretory
cells. Alveolar macrophages, on appropriate simulation, can release a
wide variety of biologically active products and thereby play an
important role in regulating inflammatory reactions within the lung.
Depletion of alveolar macrophages by intratracheal instillation of
liposomes containing the compound dichloromethylene diphosphonate has
been used to study alveolar macrophage functions in vivo (23, 30,
129). It has been shown with this technique that depletion of
alveolar macrophages reduces lung production of TNF- and neutrophil
recruitment in a model of LPS-induced lung inflammation (12,
48). In a model of bacterial pneumonia (23),
depletion of alveolar macrophages by dichloromethylene diphosphonate-liposomes unexpectedly caused increased lung production of TNF-
and increased lung neutrophil recruitment. Although the nature of the stimulus may dictate the function of alveolar
macrophages, it appears from the latter study that sources of TNF-
other than alveolar macrophages, under special circumstances, are
important to the development of lung inflammatory injury.
Endothelial activation.
Endothelial expression of molecules that mediate adhesion and signaling
of leukocytes is the key to understanding the transcriptional mechanisms of acute lung injury (28, 43, 79, 110). A
significant advance is the concept that only the activated endothelium
participates in the inflammatory response (150). In lung
injury, endothelial adhesion molecules have a role in recruiting
inflammatory cells such as neutrophils and lymphocytes from the
circulation to the site of inflammation. NF-B regulates the
expression of several genes that encode adhesion molecules such as
ICAM-1, vascular cell adhesion molecule-1, and E-selectin.
Cytokine-induced cell surface expression of E-selectin, vascular cell
adhesion molecule-1, and ICAM-1 and the secretion of IL-8 as well as of
other chemokines are regulated at the transcriptional level in
endothelial cells by the binding of NF-
B to its putative site in the
5'-flanking sequences (8). NF-
B-dependent expression of
these molecules is downregulated in endothelial cells by treatment with
antioxidants (25). This finding suggests a central role
for NF-
B in the activation of genes in endothelial cells, the
products of which promote the adhesion and extravasation of leukocytes
across the endothelial barrier.
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MODULATION OF NF-![]() |
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The regulation of inflammation by cytokines involves an intricate
balance of pro- and anti-inflammatory mediators. The principal anti-inflammatory cytokines released in lung tissue include IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13, and TGF-. The soluble cytokine receptors TNF receptor p55, TNF receptor p75, and IL-1 receptor type 2;
the membrane-bound IL-1 receptor type 2; and the IL-18 binding protein
also have anti-inflammatory activities. However, only a few of these
have been shown to involve in lung injury via interaction with NF-
B.
Intrapulmonary deposition of IgG immune complexes in rats causes
alveolar macrophage activation and results in neutrophil-dependent parenchymal cell injury as characterized by increased pulmonary vascular permeability and alveolar hemorrhage (61, 151).
Studies (32, 126-128) have identified IL-6, IL-10,
IL-1RA, and IL-13 as endogenous regulators in this model of
inflammatory lung injury. Lentsch et al. (70) showed that
exogenously administered IL-4, IL-10, or IL-13 greatly attenuated the
lung injury induced by IgG immune complexes and demonstrated that both
IL-10 and IL-13 inhibited nuclear localization of NF-B in alveolar
macrophages and lung tissues in a manner associated with preserved
expression of I
B-
protein. These findings suggest that IL-10 and
IL-13 reduce lung inflammation by preventing degradation of I
B-
,
thus inhibiting the activation of NF-
B.
Schottelius et al. (119) showed that IL-10 functions to
block NF-B activity at two levels, through 1) suppression
of IKK activity and 2) inhibition of NF-
B DNA binding
activity. To address the mechanism of action of IL-10, Wang et al.
(149) tested the effects of IL-10 on different
transcription factors including NF-
B, NF-IL-6, activator protein
(AP)-1, AP-2, glucocorticoid receptor, cAMP response element binding
protein (CREB), Oct-1, and Sp1. They found that IL-10 selectively
prevented NF-
B activation, with the effect occurring rapidly and in
a dose-dependent manner. Moreover, the effect was correlated with the
cytokine synthesis inhibitory activity of IL-10. IL-10 inhibited
chemokine expression (MIP-1
and MIP-2) by inducing both mRNA
destabilization and NF-
B inhibition (130). However,
IL-4, which also inhibited cytokine mRNA accumulation in monocytes,
showed little inhibitory effect on LPS-induced NF-
B activation
(149). Unlike IL-10, IL-4 enhanced mRNA degradation and
failed to suppress cytokine gene transcription. These data point to
distinct roles for IL-10 and IL-4 in inhibiting cytokine production by
different mechanisms.
TGF-, a pleiotropic cytokine/growth factor, is believed to play a
critical role in the modulation of inflammatory events. DiChiara et al.
(33) demonstrated that exogenous TGF-
1 inhibited the
expression of the proinflammatory adhesion molecule E-selectin in
vascular endothelium exposed to inflammatory stimuli. This inhibitory
effect occurred at the level of transcription of the E-selectin gene
and was dependent on the action of Smad proteins, a class of
intracellular signaling proteins mediating the cellular effects of
TGF-
1 (33). Furthermore, this work demonstrated that
Smad-mediated effects in endothelial cells resulted from a competitive
interaction between Smad proteins activated by TGF-
1 and NF-
B
activated by inflammatory stimuli (such as cytokines or bacterial LPS).
This interaction is mediated by the transcriptional coactivator
CREB-binding protein (CBP) (33). Augmentation of the
limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonized the ability of TGF-
1 to
block proinflammatory E-selectin expression (33). These
data demonstrate a potentially important interaction between
TGF
-1-regulated Smad proteins and NF-
B that is regulated by
inflammatory stimuli in endothelial cells.
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INTERACTIONS BETWEEN NF-![]() |
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NF-B can differentially regulate the expression of many genes
involved in inflammatory processes with different functions, in
different cell types, and at different times. This diversity arises
from a delicately balanced network of protein-protein interactions. NF-
B activity is determined not only through its regulated nuclear localization signal but also by the cellular context. NF-
B interacts with a large number of heterologous transcription factors, and these
interactions can select for specific NF-
B subunits and thereby lead
to greater transcriptional selectivity. Figure
3 is a hypothetical schema for possible
mechanisms by which NF-
B interacts with other transcription factors.
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AP-1 is one such transcription factor involved in the control of many
inflammatory mediators. AP-1 is composed of protooncogene products as
heterodimers of c-Fos and c-Jun or as c-Jun-c-Jun homodimers. Activated
AP-1 completely disrupts the structure of the nucleosome
(99) and is considered to be the important first step in
the chromatin remodeling process involved in the initial binding of
transcriptional factors to a nucleosomal template. Interestingly, the
interactions between NF-B and AP-1 sometimes do not involve a
precise promoter/enhancer organization or even require a
B element.
Fos and Jun can stimulate Rel A DNA binding and transactivation through
B elements in the absence of AP-1 sites, whereas Rel A can stimulate
AP-1 DNA binding and activation through AP-1 sites in the absence of a
B element (137). Armstead et al. (5)
reported that tissue factor mRNA level and protein activity in plasma
and lungs were markedly increased after trauma and the increase was
AP-1 and NF-
B activation dependent. However, other observations
suggest that AP-1 and NF-
B can function independently from each
other. Shenker and Abraham (131) studied the
transcriptional mechanisms involved in regulating pulmonary cytokine
expression after hemorrhage and showed activation of NF-
B and CREB
but not of AP-1, CCAAT/enhancer binding protein-
(C/EBP-
), or Sp1
in lung mononuclear cells isolated from mice. Liu et al.
(75) also observed that the expression of TNF-
by
NF-
B was independent of c-Jun in primary human macrophages. A 120-bp
TNF-
promoter-reporter possessing binding sites for NF-
B (
B3),
C/EBP-
, and c-Jun was activated by cotransfection of plasmids
expressing the wild-type version of each of these transcription
factors. Dominant negative versions of NF-
B p65 and c-Jun but not of
C/EBP-
suppressed LPS-induced TNF-
secretion in primary human
macrophages (75). NF-
B p50/p65 heterodimers were bound
to the
B3 site and c-Jun was bound to the
103 AP-1 site of the
TNF-
promoter after LPS stimulation. By transient transfection,
NF-
B p65 and p50 were shown to synergistically activate the TNF-
promoter. In contrast, such synergy was not observed between NF-
B
p65 or NF-
B p50 and c-Jun or C/EBP-
even in the presence of the
coactivator p300 (75). This result suggests that adjacent
B3 and AP-1 sites in the human TNF-
promoter contribute
independently to the LPS-induced activation response. More work is
needed to determine how AP-1 interacts with other transcriptional
factors such as NF-
B and thereby controls the inflammatory responses
resulting in acute lung injury.
The C/EBP family of transcription factors, together with AP-1 and
activating transcription factor/CREB, belong to a class of DNA binding
proteins termed bZIP proteins (146). These proteins are
characterized by their leucine zipper structure and the adjacent DNA
binding basic region, both located in the COOH-terminal portion of the
proteins. Four members of the C/EBP family, C/EBP-, C/EBP-
, C/EBP-
, and C/EBP-
(24), form homodimers and
heterodimers with each other and bind with a similar affinity to
various C/EBP binding sites (24, 66). Stein et al.
(138) demonstrated a functional and physical association
between NF-
B and C/EBP. These associations are characterized by
1) inhibition of NF-
B-induced expression of promoters
containing NF-
B binding sites by the expression of C/EBP family
members and 2) positive synergistic action of NF-
B family
members with C/EBP family members on promoters containing C/EBP binding sites.
Evidence indicates that NF-B subunits act in concert with members of
the C/EBP family of transcription factors in regulating gene expression
mediated by the multimerized c-Fos serum response element
(138). Several functional NF-
B subunits (p50, p65, and c-Rel) interacted with three different isoforms of C/EBP (
,
, and
) (138), indicating an interaction between these
families and not merely a limited interaction between individual
members. In another study (136), it was shown that C/EBP
repressed NF-
B transactivation through
B elements, whereas
NF-
B stimulated DNA binding and transactivation through C/EBP
binding sites. The IL-8 promoter contains a region with binding sites
for both NF-
B and C/EBP in close proximity that is required for
TNF-
- and IL-1
-mediated activation (136). NF-
B
and C/EBP cooperatively bind this element and thereby induce the
expression of IL-8 (136). Thus C/EBP specifically modulates NF-
B function and cooperatively stimulates the
transcription of genes.
Binding elements for NF-B and CREB are present in the
enhancer/promoter regions of the immunoregulatory cytokine genes
IL-1
and TNF-
and have important functions in modulating
transcription of these genes (9, 125, 145). NF-
B and
CREB were activated in the lungs after endotoxemia or blood loss
(16, 67, 131); however, precisely how these two
transcription factors cooperate to control inflammatory gene
transcription in acute lung injury remains unclear.
High-mobility group protein-1 (HMG-1) is a highly conserved protein
with >95% amino acid identity between rodents and humans (80,
105). HMG-1 was initially identified as a nonhistone nuclear protein that binds to the narrow minor groove of the AT sequence-rich B
form of DNA. HMG-1 has been implicated in the regulation of gene
transcription and in stabilizing nucleosome formation (15, 52,
58, 80, 105). A study (148) showed that HMG-1 is a key late mediator of endotoxin lethality. HMG-1 given intratracheally produced acute inflammatory injury in the lungs, with neutrophil accumulation, development of lung edema, and increased pulmonary production of IL-1, TNF-
, and MIP-2 (1). In
endotoxin-induced acute lung inflammation, administration of anti-HMG-1
antibody either before or after endotoxin challenge decreased the
migration of neutrophils into the lungs and prevented pulmonary edema.
These protective effects of the anti-HMG-1 antibody were specific
because pulmonary levels of IL-1
, TNF-
, or MIP-2 were not
decreased after therapy with the anti-HMG-1 antibody (1),
thus pointing to a role for HMG-1 as a distal mediator of acute
inflammatory lung injury.
The experiments of Brickman et al. (22) revealed the
interaction between HMG-1 protein and members of the NF-B family. They showed that the Drosophila protein DSP1, a HMG-1/2-like
protein, binds DNA cooperatively with NF-
B, the p50 subunit of
NF-
B, and the Rel domain of Dorsal (22). This
cooperativity is also apparent with other DNA molecules bearing
consensus Rel protein binding sites. Moreover, the cooperativity
observed in these DNA binding assays is paralleled by interactions
between protein pairs in the absence of DNA. It is apparent from the
work thus far that further studies addressing the significance of
interactions of HMG-1 with NF-
B in the mechanism of acute lung
injury will be important.
There is growing evidence that the regulation of gene expression is not
mediated solely by the presence or absence of a particular set of
transcription factors. The mechanisms regulating NF-B activation,
NF-
B by I
B, and the interactions of NF-
B with other transcription factors are complex. Additional studies that make functional sense of this complexity will not only enhance our understanding of the role played by transcription factors in mediating lung inflammation and acute lung injury but will also lead to novel
therapies directed against lung inflammation.
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SUMMARY |
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Transcriptional activities in which the NF-B family members
play a central position are key to understanding the pathobiology of
acute lung injury. Therefore, modification of transcription becomes a
logical therapeutic target for acute lung injury. Activation of NF-
B
and other transcription factors represents a complex network of finely
balanced protein-protein interactions. The balance of proinflammatory
to anti-inflammatory cytokines is regulated by a highly intricate
network of transcription factors. Under pathological conditions,
anti-inflammatory mediators provide insufficient control over
proinflammatory activities. Despite the complexities inherent in the
human immune response, therapeutic intervention with specific
inhibitors of transcription signaling may have significant benefits in
lung injury. Future studies in this area are likely to lead to novel
therapies for the wide range of NF-
B-mediated inflammatory diseases
including acute lung injury.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. Fan, Dept. of Pharmacology, Univ. of Illinois at Chicago College of Medicine, Chicago, IL 60612 (E-mail: jiefan{at}uic.edu).
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