1 Department of Environmental Health Sciences and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21205; and 2 Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405
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ABSTRACT |
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Aberrant cell proliferation and differentiation after toxic injury to airway epithelium can lead to the development of various lung diseases including cancer. The activator protein-1 (AP-1) transcription factor, composed of mainly Jun-Jun and Jun-Fos protein dimers, acts as an environmental biosensor to various external toxic stimuli and regulates gene expression involved in various biological processes. Gene disruption studies indicate that the AP-1 family members c-jun, junB, and fra1 are essential for embryonic development, whereas junD, c-fos, and fosB are required for normal postnatal growth. However, broad or target-specific transgenic overexpression of the some of these proteins gives very distinct phenotype(s), including tumor formation. This implies that, although they are required for normal cellular processes, their abnormal activation after toxic injury can lead to the pathogenesis of the lung disease. Consistent with this view, various environmental toxicants and carcinogens differentially regulate Jun and Fos expression in cells of the lung both in vivo and in vitro. Moreover, Jun and Fos proteins distinctly bind to the promoter regions of a wide variety of genes to differentially regulate their expression in epithelial injury, repair, and differentiation. Importantly, lung tumors induced by various carcinogens display a sustained expression of certain AP-1 family members. Therefore a better understanding of the mechanisms of regulation and functional role(s), as well as identification of target genes of members of the AP-1 family in airway epithelial cells, will provide additional insight into toxicant-induced lung diseases. These studies might offer a unique opportunity to use AP-1 family members and transactivation as potential diagnostic markers or drug targets for early detection and/or prevention of various lung diseases.
asbestos; tobacco smoke; silica; gene expression; carcinogenesis
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INTRODUCTION |
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AIRWAY EPITHELIUM is the primary target of various mixtures of environmental (habitual, chemical, and biological) toxicants and/or carcinogens. Some of these include tobacco or cigarette smoke (TS), asbestos, silica, ozone, particulates, cytotoxicants, viruses, and bacteria. It has been widely reported that exposure of airway epithelium to these toxins either alone and/or in combination can lead to the development of various respiratory diseases including lung cancer. For instance, exposure to TS causes chronic obstructive lung disease, bronchitis, emphysema, and cardiovascular disease as well as lung cancer (37, 123). Occupational exposure to asbestos has been linked to the development of pulmonary fibrosis, bronchogenic carcinoma, and mesothelioma (95). Exposure of animals to asbestos also causes lung inflammation and injury, which may play a role in asbestos-induced lung diseases (95). Similar to asbestos, exposure to silica also causes lung inflammation and fibrosis, as well as carcinogenesis, in rodents (28). Environmental toxicants may also act additively or synergistically. For example, TS exposure increases the incidence of asbestos-associated lung carcinogenesis in human populations (94). Prior exposure of mice to TS also impairs naphthalene (a cytotoxicant)-induced bronchiolar epithelial repair with the persistence of squamous cells in terminal bronchioles (141). Although there is considerable evidence documenting the effects of various environmental agents on lung injury and inflammation as well as their causative role in the development of respiratory pathogenesis, the cellular and molecular mechanisms governing these processes remain unclear.
Considerable experimental evidence generated in both tissue culture and animal models indicates that after toxic injury, airway epithelial cells, in a protective response, rapidly undergo changes in their structure and function to repair epithelium. This phenomenon is a very dynamic and multistep process by which epithelial cells rapidly migrate to the injured area, proliferate, and finally differentiate into a normal phenotype to restore regular airway functions (70, 113). However, aberrant cell proliferation and differentiation during this process can result in altered phenotype and tissue dysfunction. Although the molecular responses of the lung to toxic injury remain enigmatic, it has been documented that various toxicants or carcinogens, after interacting with epithelial cells, initiate a cascade of both cellular and molecular reactions that activates various transcription factors. This is accompanied by the induction of a plethora of proinflammatory cytokines, growth factors and their receptors, antioxidant enzymes, proliferation and differentiation markers, etc. (70, 113). However, abnormal expression and/or activation of these transcription factor(s) can deregulate the expression of their downstream target genes, thereby altering normal injury and repair processes, which may lead to the development of pathogenesis. Thus a better understanding of the mechanisms of regulation and role of the transcription factors governing toxicant-induced injury and the repair process is critical in order to develop an effective strategy to modulate respiratory pathogenesis.
It is widely documented that both cellular signaling mechanisms and
activation of transcription factors play a pivotal role in regulation
of gene expression. Among the transcription factors, activator protein
(AP)-1, comprising Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB,
Fra1, and Fra2) family members, plays a central role in regulating gene
transcription in various biological processes (125). AP-1
family members, also referred to as "immediate-early genes" and
"early response proto-oncogenes," directly couple intracellular signals initiated by various external mitogenic and toxic stimuli to
regulate gene expression involved in cell proliferation and differentiation, transformation, apoptosis, pulmonary defense, inflammation, immune responses, etc. (Fig.
1) (5). The
mitogen-activated protein kinase (MAPK) signal transduction pathway
uses AP-1 as a converging point not only to regulate expression of
various genes but also to autoregulate AP-1 gene transcription, thereby increasing their abundance to amplify the signals to various external stimuli. Most importantly, several genes, which play very important roles in injury, repair, and differentiation, contain a bona fide binding site(s) of AP-1 in their promoter and/or enhancer regions (5). Some of the genes include extracellular
matrix metalloproteinases (MMPs), antioxidant enzymes, surfactant
proteins, growth factors and their receptors, differentiation markers,
cytokines, chemokines, other transcription factors, etc.
(125).
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Several studies performed in both tissue culture and mouse models indicate an essential role for AP-1 proteins in normal cell growth and development (65). Paradoxically, overexpression of some of these proteins results in an overt phenotype(s), including tumor/cellular transformation both in vivo and in vitro, respectively (65). Thus altered AP-1 protein expression and/or activation by toxicants can deregulate gene expression, resulting in aberrant cell proliferation and differentiation, which may lead to the development of various diseases. Although much is known about their involvement in other systems, the mechanisms of activation and individual contribution of AP-1 family members both in lung homeostasis and in the development of toxicant-induced respiratory pathogenesis are unclear. The objective of this article is to provide a brief review on the biology and role of the members of the AP-1 family as well as their mechanisms of activation. In addition, their involvement in the regulation of gene expression involved in lung injury, repair, and transformation is briefly discussed.
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BIOLOGY OF AP-1 PROTEINS |
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AP-1 is a homo- or heterodimer mainly composed of Jun-Jun and
Jun-Fos transcription factors that belong to the basic region-leucine zipper (bZIP) group of DNA binding proteins. The basic region or DNA
binding domain (DBD) of bZIP proteins contains positively charged amino
acid residues required for DNA binding activity. The leucine-zipper
domain (LZD), located immediately downstream of DBD, contains a heptad
repeat of leucine residues (Fig.
2A). LZD mediates the
dimerization of proteins, bringing two DBDs into juxtaposition, thereby
facilitating the interaction of protein dimers with DNA (Fig.
2B). Unlike Jun proteins, Fos proteins cannot homodimerize
among themselves because of a subtle difference in amino acid
composition within their LZDs (65). However, they can
partner with Jun proteins to form Jun-Fos heterodimers, which are more
stable and therefore possess a higher DNA binding activity than the
Jun-Jun homodimers. Although LZD and DBD are highly conserved among
all AP-1 proteins, their amino (NH2)- and carboxy
(COOH)-terminal regions are quite divergent. The Jun proteins contain
the transactivation domain (TAD) at their NH2-terminal
region, whereas Fos members, except Fra1 and Fra2, possess TADs at
their NH2- and COOH-terminal regions (125).
Both homo- and heterodimers of AP-1 can bind to the 7-bp palindromic
DNA sequence 5'-TGAGTCA-3', also known as the
12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive
element (TRE or AP-1 site), in the promoter and/or enhancer regions of variety of genes to regulate their transcription (5). AP-1 can also bind to the variant TREs, such as 5'-TTAGTCA-3' and
5'-TGATTCA-3', which somewhat deviate from the perfect recognition
sequence by one or two bases (20). However, the AP-1
binding at these sites appears to be mostly influenced by the flanking
DNA sequences and cognate binding proteins. In addition, AP-1 proteins
can dimerize with other bZIP family of proteins, such as activating
transcription factors/cyclic AMP response element binding proteins
(ATFs/CREBs). These AP-1 heterodimers preferentially bind to the 8-bp
DNA sequence 5'-TGACGTCA-3', also known as cyclic AMP-responsive
element (CRE) (Fig. 2B). The CRE has an extra base insertion
into the AP-1 consensus recognition sequence TRE. Unlike TRE, the
Jun-Jun and Jun-Fos dimers have a lower affinity to the CRE. Although
both c-Jun and JunB contain a well-conserved LZD and DBD, unlike the
former, the homodimers of the latter bind less efficiently to the
promoters/enhancers that have a single TRE compared with the
promoters/enhancers that contain multiple TREs in tandem or in close
proximity (102). Therefore, it is envisioned that, by
virtue of both a selective dimerization and diverse binding
specificities, AP-1 proteins distinctly regulate both cell type- and
stimulus-specific gene expression involved in various biological
processes.
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FUNCTIONAL ROLE OF AP-1 PROTEINS |
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Cell culture studies. AP-1 proteins display a distinct expression pattern during cell cycle progression (71, 74). Upon mitogenic stimulation, the AP-1 complex is mainly composed of Jun-c-Fos heterodimers, whereas Jun-Fra1 and Jun-Fra2 dimers are predominant during exponential cell growth. Moreover, AP-1 proteins differentially regulate the expression of several key players of cell cycle machinery such as cyclin D1, p16, and p53 (91, 125). For example, c-Jun upregulates the promoter activity of cyclin D1, whose expression correlates well with cycle progression, whereas JunB has an opposite effect (7). Importantly, intracellular injection of neutralizing antibodies to AP-1 proteins alters cell cycle progression (71, 74), indicating their role in cell proliferation. Consistent with this view, mouse embryonic fibroblasts (MEFs) devoid of AP-1 protein(s) exhibit defects in proliferation and undergo senescence prematurely. Together, these studies underscore a pivotal role for AP-1 family members in cell proliferation.
Knockout studies.
Gene ablation (knockout or disruption) approaches have been used most
widely to examine the functional role of many protein factors involved
in cell growth and development and inflammation as well as
transformation. c-jun null (/
) embryos die at ~13 days
postcoital (dpc) from multiple defects in neural crest, heart, and
liver development (31). Similarly, embryos lacking
junB (120) die between 8.5 and 10 dpc due to
vascular defects in various extraembryonic tissues. In contrast to
c-jun
/
and
junB
/
,
junD
/
mice are viable and appear healthy.
Although male mice exhibit impaired spermotogenenesis with an altered
sperm structure, no fertility effects are noticeable in female mice
(133).
"Knockin" and transgenic complementation studies.
Intriguingly, deletion of specific AP-1 members does not result in
compensatory upregulation of other members, indicating some functional
redundancy among Jun and Fos proteins (65). To address
this issue, investigators have used knockin and/or transgenic
complementation approaches in which another gene is replaced or
ectopically overexpressed in mice lacking a particular AP-1 gene. The
knockin of junB cDNA in the c-jun locus rescues both liver and cardiac defects observed in
c-jun/
mice during development
(102). However, mice do not survive postnatally because of
certain defects in cardiac outflow. Interestingly, c-jun
/
mice overexpressing the
junB transgene develop normally and can survive up to 7 mo,
indicating that JunB, if present in sufficient amounts, can rescue main
defects that were noticed in the absence of c-Jun (102).
Although c-fos
/
mice develop osteopetrosis,
substitution of fra1 in the c-fos locus restores
the phenotypic defect. Consistent with this, ectopic expression of
fra1 also restores an abnormal phenotype in
c-fos
/
mice (64). These results
indicate an overlapping function between JunB and c-Jun, and Fra1 and
c-Fos. It is not clear why the presence of junB in
c-jun
/
mice and vice versa cannot restore
each other's functions during development. One possible
explanation for this paradox, as suggested by Passegue et al.
(102), is either the lack of sufficient amounts of
respective protein or incomplete activation of their target genes to
restore normal functions. For example, c-Jun and JunB differ
significantly in both their DNA binding and transactivation potential as well as their target gene regulation (26).
Transgenic overexpression studies. Overexpression of the individual members of Jun and Fos in transgenic mice under the control of a ubiquitous and/or targeted promoter displays specific phenotypes. Broad overexpression of c-jun (39) and junB (119) does not result in an abnormal phenotype, whereas junD overexpression studies have not been reported yet. Intriguingly, targeted expression of junB using a CD4 promoter interferes with T helper (Th) cell differentiation (78). In these studies, Th1 cells overexpressing JunB display a higher level of Th2 cytokines, such as IL-4 and IL-5. Consistent with this, junB expression levels are selectively induced in Th2, but not Th1, cells during differentiation. Moreover, JunB activates IL-4 transcription. Together, these observations strongly support a critical role for JunB in T cell differentiation.
The ectopic expression of c-fos results in mainly osteosarcomas, malignant bone tumors, due to transformation of osteoblasts (39). However, fosB overexpression has no effects (39). Overexpression of
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AP-1 FAMILY MEMBER EXPRESSION IN LUNG CELLS DURING DEVELOPMENT AND NEOPLASIA |
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The basal level of expression of members of the AP-1 family is quite different in various tissues and cell types during embryonic and postnatal development and adulthood. In general, the mRNA levels of c-jun, junB, and junD are high compared with fos family members in various tissues including the lung. c-jun transcripts are detectable during mouse lung development at 15.5-17.5 dpc (146), whereas expression in postnatal and adult mouse lungs is quite abundant (102). In contrast to jun family members, the expression of fos family members, with the exception of fra2, is very low during development and in adult lungs (97). During development, c-fos expression can be noticeable in developing bronchioles but not in mesenchyme of 14-dpc embryos. However, its expression decreases in 17-dpc bronchioles but is sporadically present in mesenchyme (96, 146). Immunohistochemical analysis of 17.5-dpc embryos reveals the localization of FosB in bronchial epithelial cells and endothelial cells of the mouse lung (41). However, its expression, with the exception of bone, is very low or undetectable in various adult mouse tissues including lung (154). Similar to fosB, the expression of fra1 is very low but can be detectable by RT-PCR in various adult mouse tissues, including the lung (13, 23, 64). The expression of fra2 is very low around midgestation but can be detected in the epithelial cells of the trachea and bronchi of 16.5-dpc embryos. In adult tissues, fra2 expression is somewhat similar to that of jun members with abundant localization in various differentiated epithelia, including the lung (13). It is noteworthy that the levels of JunB and JunD, but not c-Fos and c-Jun, are higher in the lungs of neonates (~12 h old) compared with adult rats, indicating certain differences in the expression pattern of Jun and Fos proteins during lung development (149). In another study, it was shown that at 14 dpc the expression of c-fos is low, whereas at birth its expression significantly higher compared with 14-dpc rat lungs (21). Conversely, compared with 17-dpc lung, c-fos expression in 14-dpc and neonatal hypoplastic lungs is very low (21). In summary, differential expression of jun and fos proto-oncogenes occurs during embryonic and postnatal development as well as in adult tissues.
Jun and fos expression is also variable between different normal and malignant cell lines. Consistent with in vivo observations, RNase protection analysis reveals high-level expression of c-jun, junB, junD, and fra2 in the nontransformed mouse type II alveolar epithelial cell line C10, whereas the expression of c-fos, fosB, and fra1 is very low or undetectable (126). However, detectable amounts of c-fos and fra1, in addition to c-jun, junB, and junD, were noticed in normal human bronchial epithelial (HBE) cells (76). Intriguingly, malignant HBE cells variably express AP-1 proto-oncogene expression. One study demonstrates significantly lower levels of junB, c-fos, and fra1 mRNA in malignant HBE cells compared with normal cells (11). In contrast, a different study shows a high but a variable expression of c-jun, junB, junD, and c-fos message levels in various human lung cancer cell lines (130). Immunohistochemical analysis of various neoplastic human lung tissues reveals a high level of expression of c-jun antigen in atypical, hyperplastic, and metaplastic epithelium, whereas its expression in surrounding normal bronchial and alveolar epithelia is very low or undetectable (130).
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TRANSCRIPTIONAL REGULATION OF AP-1 FAMILY MEMBERS |
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In response to various toxic and mitogenic stimuli, jun and fos mRNA expression is rapidly induced severalfold above the basal level in a wide variety of tissues and/or cell types. In general, the mRNA levels of c-jun, junB, junD, c-fos, and fosB peak within 15-30 min of stimulation and return to basal level within 1-2 h (5, 48). The induction of fra1 and fra2 mRNA expression mainly occurs between 30 and 60 min, peaking at 90-180 min. However, the mRNA expression remains elevated above basal level for 2-24 h, depending upon the stimuli (48).
Intriguingly, although all AP-1 members are rapidly induced, albeit
with somewhat different kinetics, the 5'-flanking regions of AP-1
family members are not well conserved. They contain both common and
distinct regulatory cis-acting elements, including their own
target sites (5, 48, 145). The proximal promoter of the
c-jun gene is highly conserved among species and contains a
TATA-like sequence, CAAT box, Sp1 site, and three CREs (Fig. 3A). Genomic footprinting
analysis in living cells reveals that these sites are constitutively
occupied in vivo (115) and respond rapidly to various
stimuli, such as TPA, insulin, and epidermal growth factor (EGF), to
induce c-jun transcription. Such induction is mediated
mainly by the c-Jun-ATF-2 heterodimer binding to the CRE
(68). This indicates that c-Jun positively regulates its own transcription. In addition to CRE, the protein binding at the
myocyte binding site located at 59 also regulates c-jun
transcription (44). Similar to c-jun,
the junB promoter is also well conserved and contains a TATA
box, CAAT box, Sp1 site, and a CRE (91). In addition, it
contains signal transducer and activator of transcription (STAT)
binding sites, multiple Ets transcription factor binding sites (EBSs),
and a serum-responsive element (SRE). The SRE mediates the
growth factor- and serum-inducible junB transcription
through recruitment of ternary complex factor (TCF). Recent studies
show that, in addition to the proximal CCAAT box (32),
Smad binding elements located at
2,980 to
2,611 regulate
transforming growth factor (TGF)-
-induced junB
transcription (66). The proximal promoter of mouse
junD also contains a TATA box, CAAT box, Sp1 site, and two
CREs (91). Unlike c-jun and junB,
the transcription of junD does not appear to be inducible by
various stimuli, including TPA and growth factors (91).
This is mainly attributed to the presence of a functional octomer
motif. This motif is uniquely present in junD promoter and
constitutively occupied by a ubiquitous protein, Oct-1
(91).
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Among the Fos family members, the transcriptional stimulation of
c-fos is most extensively studied. The promoter of
c-fos contains several cis-elements. The most
notable ones include a CRE, SRE, and the sis-inducible element/enhancer
(SIE) (24) (Fig. 3B). The SRE is generally
bound by the serum response factor (SRF). SRF plays a major role in the
recruitment of TCF after serum and growth factor stimulation. Upon
activation by various MAPKs, Elk1 and SAP-1, which belong to the family
of Ets transcription factors, are recruited at an SRE site to form TCF
with the bound SRF, thereby inducing c-fos transcription
(5, 24, 145). The SIE located distally to SRE also plays a
role in cytokine- and growth factor-inducible c-fos
transcription. In response to various cytokine stimuli, STAT1 and -3 bind at the SIE and act in concert with SRE to stimulate
c-fos transcription. The CRE located at 70 to
50
mediates cAMP and Ca2+-inducible c-fos
transcription (145). The most notable difference between c-jun and c-fos transcriptional
regulation is that the former is positively autoregulated by its own
product, whereas the expression of the latter is suppressed by Jun-Fos
dimers (5, 24, 145). The proximal promoter of
fosB contains a TATA box, SRE, and TRE, which are located at
nearly identical positions and in the same order as the
c-fos promoter. Similar to c-fos, fosB
transcription is also suppressed by its own protein and c-Fos (75).
The proximal promoter of human and mouse fra1,
although moderately conserved with that of c-fos (70%),
does not contain a TATA box and a consensus DNA sequence of SRE
(138). However, its expression can be strongly inducible
after serum stimulation in airway epithelial cells (S. P. M. Reddy, unpublished data). Unlike c-fos and fosB,
fra1 transcription can be upregulated by most of the AP-1
family members, including its own product (9, 121).
Moreover, some studies have shown that a 50-bp fragment of the first
intron containing a perfect TRE and two variant TREs can mediate the
transcriptional regulation of the mouse and rat fra1 gene
(9, 121). Although this is unique among fos
family members, examination of the human genomic sequence of
fra1 did not reveal a similar sequence in its first intron.
However, results from our laboratory indicate that TPA-, EGF-, and
serum-inducible fra1 transcription in A549 cells is mainly
mediated by the 379- to
283-bp DNA fragment containing multiple
motifs such as AP-1, EBS, and Sp1 (3). Unlike
fra1, the mouse and rat fra2 promoter contains a
TATA box, two TREs, CRE, SIE, and SRE (128). The TREs located immediately downstream of the start site are required for
serum-inducible fra2 transcription (128). In
unstimulated cells, the TREs are weakly occupied by the c-Jun-Fra2
dimers. Upon stimulation with serum, c-Jun-c-Fos dimers form a strong complex that is replaced by c-Jun-Fra2 dimers at later time periods (128). Similar to fra1, fra2
transcription is positively autoregulated by AP-1 proteins.
It is noteworthy that although Jun and Fos mRNA levels are rapidly induced, this is not reflected at the protein level (5, 145). Moreover, treatment of cells with cycloheximide also enhances AP-1 family member expression. Thus, in addition to transcription, both posttranscriptional and posttranslational modifications also regulate AP-1 expression (5, 145).
In summary, the presence of both common and distinct regulatory elements in the 5'-flanking regions of AP-1 family members, the fact that their transcription is differentially (positively or negatively) regulated by themselves and other proteins, and the existence of some functional redundancy among AP-1 proteins as demonstrated by in vivo genetic models strongly suggest that both spatial expression and regulation of AP-1 family members may play a central role in toxicant-induced injury, repair, and/or cellular transformation.
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AP-1 ACTIVATION BY MAPKS |
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It is well documented that different signaling cascades initiated by various extracellular stimuli converge at AP-1, which then regulates its own transcription as well as the transcription of other genes required for various biological processes (27). Among various MAPKs, the c-Jun NH2-terminal kinases (JNKs) and extracellular signal-regulated kinase (ERKs) mainly phosphorylate Jun and Fos proteins, respectively (27). Although Jun and Fos proteins are rapidly induced by various stimuli, most cells possess a certain amount of preexisting Jun and Fos proteins that are initial targets for JNK and ERK MAPKs (145). Upon their activation, ERKs and JNKs phosphorylate both preexisting and newly synthesized AP-1 proteins. The activated JNKs bind to the docking site located in the NH2-terminal region of c-Jun and phosphorylate Ser63 and -73 located within its transactivation domain (145). Due to its higher affinity to the docking site, JNK2 phosphorylates c-Jun more efficiently than JNK1 (42). JunB also contains the JNK docking site but lacks NH2-terminal acceptor serine residues and therefore appears to be poorly activated by JNKs. However, a recent study shows phosphorylation of Thr102 and -104 of JunB by JNKs in COS-7 cells and a B-lymphoma cell line, M12 (78). Although it contains a putative serine residue, JunD cannot be phosphorylated by JNKs due to the lack of a functional JNK docking site within its NH2-terminal region. However, after dimerization with c-Jun, JunD can be phosphorylated on its serine residue by the JNK (67). Because of these differences, although they contain similar DNA recognition properties and binding activities, Jun proteins apparently have different transactivating characteristics (42, 145).
In contrast to Jun proteins, which are mainly phosphorylated by JNKs within their NH2-terminal region, Fos proteins are mainly phosphorylated by ERKs on serine and/or threonine residues located within their COOH-terminal domain. Upon activation, ERK MAPKs translocate into the nuclei and phosphorylate c-Fos on Ser374 (19), whereas FosB appears to be phosphorylated on several serine residues: 284, 297, 299, 302, and 303 (127). In addition, Fos-regulating kinase and ribosomal S-6 kinase phosphorylate c-Fos on Ser133 and Thr232 (25) and Ser362 (19), respectively. ERK1 also phosphorylates Fra1 both in vitro and in vivo (40). Upon mitogenic stimulation, Fra1 displays different band sizes, as analyzed by Western analysis, indicating the existence of multiple phosphorylation sites. A recent study indicated the requirement of ERK-dependent phosphorylation of Thr231 of Fra1 for mitogen-activated epidermal transformation (150). Similar to Fra1, Fra2 contains putative serines and threonines (three each) that are extensively phosphorylated by the mitogen-activated protein/extracellular signal regulated-kinase (MEK)/ERK pathway both in vivo and in vitro (99).
Although p38 MAPKs do not activate AP-1 proteins directly, they can regulate jun and fos transcription by phosphorylating ATF-2, Elk1, SAP-1, and CCAAT enhancer binding proteins (C/EBPs), which then bind to the promoter elements of jun and fos and regulate their transcription. Regulation of c-jun transcription by ERK5 (also known as big mitogen-activated kinase 1) has been demonstrated (86). ERK5 plays a regulatory role in proliferation and differentiation and regulates c-jun expression through the activation of MEF-2D (86), which binds at the c-jun promoter to stimulate its transcription (44). In addition to various MAPKs, AP-1 protein phosphorylation by other kinases, including protein kinase (PK) C, PKA, casein kinase II, and Cdc2, has been documented, suggesting their potential roles in the activation of AP-1 proteins (145).
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REGULATION OF AP-1 GENE EXPRESSION AND/OR ACTIVATION BY TOXICANTS |
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TS contains a wide variety of compounds that are both volatile and nonvolatile. Some of the major volatile components include acrolein, napthalene, aldehydes, and hydrogen peroxide (H2O2), whereas the nonvolatile fraction contains tumor promoters such as phorbol ester analogs and carcinogens such as aflatoxin B1, benzopyrene, and 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (52). A variety of exposure protocols are being used to study the effects of smoke on the cells of the lung both in tissue culture and animal models. Some of these protocols include exposure of cells or animals directly to mainstream TS (MTS), TS bubbled through phosphate buffer solution [called TS extract (TSE)], or aged and diluted sidestream TS [referred to as environmental TS (ETS)]. Exposure of rats to MTS (1 h/day, 5 day/wk for 8 wk) enhanced c-Fos, but not c-Jun, expression in the terminal bronchioles. This was associated with the increased levels of MEK1 and ERK2 but no effect on MEK kinase 1 and p38 protein levels (18). However, a different study showed elevated levels of both c-Jun and c-Fos in the lung tissues of MTS-exposed (6 mo) ferrets (82). Moreover, elevated levels of c-Jun and c-Fos were positively correlated with the expression of proliferating cell nuclear antigen and squamous metaplasia in bronchial epithelium (82). TSE also enhanced c-fos mRNA levels in fibroblasts (98). Likewise, the exposure of A549 cells to TSE significantly enhanced AP-1 binding activity (110). The latter studies show that supplementation of cells with antioxidants or superoxide scavengers before TSE exposure attenuated c-fos induction (98) and AP-1 DNA binding activity (110), indicating a role of reactive oxygen species (ROS) in AP-1 induction. In contrast to A549 cells, TSE had no effect on AP-1 DNA binding activity in a human premonocytic cell line, U397 (34), indicating the existence of cell type-specific responses to TS.
Although studies above examined the effects of TS mainly on c-jun and c-fos expression, little is known with regard to other members of AP-1 family. Recently, we showed that Fra1 and Fra2 distinctly regulate transcription of the squamous differentiation marker SPRR1B in HBE cells (103). Because inhalation of TS causes squamous metaplasia in airways, the effects of MTS and ETS on AP-1 gene expression in HBE cells were investigated in tissue culture and animal models, respectively. Exposure of primary cultures of HBE or A549 cells to MTS markedly enhanced fra1 mRNA levels at 5 h, which remained elevated through 14 h, while fra2 message levels were unchanged and/or slightly reduced (112). Exposure of mice to ETS (6 h/day, 5 days) considerably induced fra1 mRNA levels in the lungs compared with the filtered air-exposed control group. In contrast, TS showed no effect on fra1 expression in the liver. Moreover, immunohistochemical analysis revealed an elevated level of Fra1 in both bronchial and alveolar type II epithelial cells of ETS-exposed mouse lungs, further corroborating mRNA levels. ETS did not alter junB or c-fos mRNA levels, whereas a slight reduction (< 30%) in c-jun message levels was noticeable after ETS exposure (112). Together, these results indicate that TS differentially regulates the induction of members of the AP-1 family in HBE cells. However, the discrepancy observed in the expression pattern of jun and fos members in lung cells after exposure to TS in the above studies may be related to differences in the exposure protocols or in the animal models as well as the detection methods. The individual contribution of AP-1 family members in smoke-induced injury and in the pathogenesis of the lung diseases, as well as downstream target genes, needs more thorough investigation.
Asbestos is a fibrogenic dust and potent inducer of pulmonary fibrosis as well as carcinogenesis. Asbestos fibers differentially upregulate jun and fos mRNA expression in the cells of the lung both in vivo and in vitro (85). Most notably, asbestos fibers cause a sustained induction of c-jun and c-fos mRNA expression as well as AP-1 DNA binding activity in pulmonary epithelial cells and mesothelial cells (53, 60, 135). Moreover, the increase in c-jun transcription positively correlates with cell proliferation and transformation of asbestos-exposed tracheal epithelial cells both in vivo and in vitro (108, 136). Recent studies have shown a protracted induction of fra1 mRNA both in asbestos-induced rat lung tumors (117) and in asbestos-exposed rat mesothelial cells (117), as well as in mouse pulmonary epithelial cells (126). The latter study revealed the mRNA expression of c-jun, junB, and c-fos to be somewhat less protracted than that of fra1, whereas no change in the expression of fra2 and junD was detected after the asbestos exposure (126). Studies performed by the same laboratory have also shown that the induction of jun and fos by asbestos is oxidant dependent (63) and mainly mediated through the EGF receptor (EGFR), a tyrosine receptor kinase, which activates the ERK/MAPK pathway (152, 153). In support of this notion, pretreatment of cells with EGFR antibodies, GSH, antioxidants, and/or pharmacological inhibitors of the MEK1/2 pathway significantly attenuates asbestos-inducible jun and fos expression (85). In contrast to the above studies, cDNA microarray analysis reveals an inducible (fourfold) expression of c-fos, but not c-jun and fra1, in asbestos-induced tumorigenic human bronchial cells, indicating certain species-specific differences in responses to asbestos (155). In a transgenic mouse model that expresses the luciferase reporter gene under the control of AP-1 binding sites or TREs, a strong AP-1 transactivation by asbestos was demonstrated in the lung and in bronchiolar and alveolar type II epithelial cells (29, 93). Together, these studies indicate that a protracted induction of certain AP-1 family members, such as Fra1, in epithelial cells of the lung after asbestos exposure can lead to deregulation of cell proliferation and differentiation and the development of pulmonary disease.
Similar to asbestos, silica selectively upregulates the expression of c-jun, junB, c-fos, and fra1 message levels in pulmonary epithelial cells, whereas the expression of junD and fra2 remained unchanged (126). Most notably, the junB and fra1 message levels persist over 24 h, indicating a potential role for these proteins in silica-induced pathogenesis. Furthermore, the induction of jun and fos expression is accompanied by an increase in AP-1 DNA binding activity and AP-1 transactivation associated with alterations in cell cycle progression (126). Indeed, the expression of fra1 correlates with cells entering into S phase (126). The increase in AP-1 expression and DNA binding activity correlates with sustained phosphorylation and activation of JNK but not p38 and ERK MAPKs (126). Pretreatment of cells with intracellular scavengers suppresses both silica-induced JNK activity and fra1 mRNA levels, suggesting a role for oxidative stress in this process (126). Silica-inducible AP-1 activation was also confirmed in a mouse model. Intratracheal instillation of freshly prepared crystalline silica to transgenic mice carrying a reporter luciferase gene under the control of TREs markedly elevated AP-1 transactivation in the lung (30). Although patterns of asbestos- and silica-induced AP-1 proto-oncogene expression are nearly identical and mediated by oxidative stress, how they activate upstream MAPK signaling cascades and their downstream target genes is unclear.
A growing body of evidence suggests an association between exposure to airborne particulate matter (PM) and increases in respiratory morbidity and mortality (2). Links to the development of lung cancers and increases in asthma, chronic bronchitis, and pneumonia in predisposed individuals suggest that injury to respiratory epithelium is an initiating factor in the development of these diseases. The composition of PM is complex, consisting of soluble agents, insoluble particles, metals, and contaminants such as endotoxin. In alveolar type II epithelial cells, endotoxin-free PM samples show a modest but significant transcriptional activation of AP-1-dependent genes compared with the known pathogenic fiber, asbestos, and earlier, transient increases (1 h) in JNK1 activity and phosphorylated Jun protein (134). These increases occur in the presence of increased incorporation of 5'-bromodeoxyuridine by epithelial cells, which later proved to be indicative of cell proliferation (137). In these recent studies, the development of dose-related proliferation and apoptosis occurs with unique patterns of jun and fos family member expression. For example, more protracted increases (24 h) in c-jun, junB, fra1, and fra2 are seen at lower, proliferative concentrations of PM, whereas transient induction (2 h) of all jun and fos family members occurs at apoptotic concentrations of PM. The ultrafine particulate component of PM appears to be important in eliciting both proliferation and increases in expression of AP-1 family members, whereas fine titanium dioxide and glass beads (nontoxic particles) have no effect.
ROS and reactive nitrogen species (RNS), such as peroxynitrite and nitric oxide (NO), are also components of air pollution and cause acute damage to respiratory epithelial cells. In rat alveolar epithelial cells, different species of RNS or ROS cause AP-1 transactivation, which correlates with their ability to induce membrane permeability or apoptosis (61). For example, NO generation or exposure to H2O2 causes increased c-jun and c-fos mRNA/protein levels and AP-1 DNA binding, leading to increased membrane permeability and apoptosis. In contrast, exogenously administered peroxynitrite does not induce toxicity or induction of early response genes despite increased nitration of tyrosine, a signature of exposure (61).
In conclusion, the results above suggest that increased jun and fos family member expression and AP-1 transactivation may be sensors for environmental stresses in the pulmonary epithelium, most importantly those that cause injury to and proliferation of epithelial cells. The lack of effect of nontoxic particles or inactive analogs in these models is especially encouraging.
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REDOX REGULATION OF AP-1 ACTIVATION |
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The redox state of the cysteine moieties of various proteins,
including transcription factors such as NF-B and AP-1, plays a
regulatory role in various biological processes, including gene regulation (72). The AP-1 proteins Jun and Fos contain
cysteine residues both in the DBD and in the flanking NH2-
and COOH-terminal regions (Fig. 4). Human
c-Jun contains three cysteine residues, located in the DBD, downstream
of the LZD, and in the NH2-terminal region.
Oxidation of the cysteine residue located in the DBD leads to
intermolecular disulfide formation or S-glutathionylation of c-Jun, thereby inhibiting its DNA binding activity (69,
72). Similar to c-Jun, the oxidation of Cys154 on human c-Fos
modulates its DNA binding activity (1). Supplementation of
cells with antioxidants such as GSH, thioredoxin (TRX), and redox
factor-1 (Ref-1), which promote a reducing environment, enhances AP-1
DNA binding activity (55); conversely, the oxidized form
of GSH, GSSG, has an opposite effect (69, 72). Whether
redox regulation modulates DNA binding activities of other AP-1
proteins is unknown. However, all of them contain several cysteine
residues located both within the DBD and downstream of the LZD (Fig.
4). The well-conserved nature of the two cysteine residues in both Jun
and Fos families of proteins suggests a potential a role for redox
regulation in the modulation of AP-1 DNA binding activity in various
cell types, including the lung.
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Paradoxically, exposure of cells of the lung to agents such as
H202, TNF-, TS, asbestos, and silica, which
are potent inducers of oxidative stress, stimulates both the DNA
binding activity as well as the transcription of AP-1 proto-oncogenes.
For example, TSE, which causes depletion of intracellular GSH levels,
increases AP-1 DNA binding activity, which correlates with the
transcription of
-glutamylcysteine synthetase (
-GCS), a regulator
of GSH synthesis, in A549 cells (110). Similar to GSH and
N-acetyl-L-cysteine (NAC), TRX acts as an
antioxidant. TRX is a ubiquitously expressed protein and contains two
functional redox-active cysteine residues that play an important role
in redox-dependent gene expression in various cell types. The
expression level of TRX is high in airway epithelia (46).
After translocation into the nucleus, TRX activates Ref-1, which in
turn reduces the cysteine residues of the Jun and Fos proteins thereby
stimulating AP-1 DNA binding activity (55). Recently, it
was shown that Ref-1 modulates granulocyte-machophage colony
stimulation factor-induced AP-1 DNA binding in human alveolar macrophages (35). Recent studies also indicate that TRX,
NAC, and GSH exhibit differential effects on the activation of
redox-dependent transcriptional activity. For instance, TRX inhibits
NF-
B activation in the cytoplasm (56, 131), whereas in
the nucleus it stimulates NF-
B transcriptional activity
(46). Thus it appears that compartmentalization and close proximity, as well as the availability of the GSH, TRX, Ref-1, and other reducing agents to oxidized cysteine residues probably
contribute the ultimate physiological response, depending on the toxic
stimuli and cellular context (69, 109).
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REGULATION OF GENE EXPRESSION BY AP-1 IN LUNG CELLS |
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After toxic injury, airway epithelial cells dedifferentiate, flatten, rapidly migrate, and proliferate to repair the injured area. During this process, extracellular matrix (ECM) protein deposition and degradation also play a key role in restoring normal cellular structures (70, 113). A plethora of genes coding for proinflammatory cytokines, growth factors and their receptors, antioxidant enzymes, and proliferation and differentiation markers, which participate during the injury and repair process, contain functional AP-1 binding site(s) or TREs in their promoter regions (5, 6). Although various studies demonstrate a correlation between the candidate gene expression and AP-1 DNA binding, relatively few studies have examined the role of individual members of the AP-1 family in detail. Here we discuss genes whose expression and regulation by AP-1 proteins have been investigated after toxicant-associated injury, repair, and cellular transformation.
Differentiation markers.
Upon exposure to toxicants and/or carcinogens, proximal and distal
alveolar epithelial cells can lose their normal secretory functions and
express squamous and keratinizing properties. Surfactant proteins (SP-A
to -D) and Clara cell secretory protein (CCSP) play important roles in
pulmonary function, host defense, and innate immunity of the lung.
Although Nkx2.1 (or TTF-1) and HNF-3 (Fox) family members mainly
regulate their expression, AP-1 proteins also distinctly regulate
SP-A, -B, -D, and CCSP transcription in alveolar
epithelial cells. The suppression of SP-A and -B
transcription by PMA and TNF-, potent inducers of AP-1 activation,
is attributed in part to the cytoplasmic trapping of Nkx2.1 as well as
to differentially enhanced binding of AP-1 proteins (104,
107). The suppression of SP-A transcription by TPA
correlates with an enhanced Jun binding to the functional TRE located
in the intron region (57). Intriguingly, overexpression of
junD upregulates mouse SP-B promoter activation, whereas c-jun and junB have opposite effects
(124). Likewise, c-Jun, ATF-2, and CREB binding to
81
CRE located adjacent to HNF-3 and Nkx2.1 sites suppresses rabbit
SP-B transcription (10). Unlike SP-C
transcription, which apparently is not regulated by AP-1
(81), mutation of
109 TRE totally abolishes human
SP-D promoter activity (51). Moreover, JunB,
JunD, and Fra1 bind to the TRE, and the overexpression of latter two
proteins upregulates SP-D promoter activity, whereas c-Jun
and c-Fos suppress it (51). The binding of JunB and Fra1
to a composite element, admixed with TRE, HNF-3, and octomer motifs,
correlates with rat CCSP expression in bronchiolar cells
(118). The fact that TRE/CRE is either embedded or located
in close proximity to other functional motifs, such as Nkx2.1, HNF-3,
and NF-1, strongly suggests that the distinct regulation of
SPs and CCSP transcription is probably mediated by complex, cooperative, and/or mutually exclusive interactions between
AP-1 proteins. Cell type and/or ubiquitous factors may also be important.
Antioxidant enzymes.
Exposure of pulmonary epithelial cells to TS, asbestos, silica, and
other toxicants induces the cellular stress arising from the generation
of ROS. The antioxidant enzymes (AOEs) play a pivotal role in the
detoxification of various ROS and are rapidly induced after toxic
injury. Among the AOEs, the regulation of -GCS,
NAD(P)H:quinone oxidoreductase 1 (NQO1), metallothionein-I (MT-I), heme
oxygenase 1 (HO-1), and glutathione S-transferases, which
play a key role in detoxification process, has been extensively
investigated. Because toxicant-induced AP-1 proto-oncogene expression
appears to precede AOE expression, and the regulatory
regions of several AOE contain functional TRE(s), a
potential role for AP-1 is implicated in antioxidant responses.
ECM proteins and growth factors. During the injury and repair process, deposition and degradation of ECM proteins (e.g., collagens, fibronectin, integrins) play a key role in restoration of normal cellular structures. AP-1 regulates the expression of these proteins, MMPs, tissue inhibitor of metalloproteinases, and growth factors (e.g., EGF) and their receptors (e.g., EGFR). Many of the above-mentioned genes also contain AP-1 binding sites in their promoter region. For example, recently, it was shown that downregulation of elastin gene expression by basic fibroblast growth factor-2 is probably mediated by a protracted induction of fra1 (14). TS exposure induces the expression of MMP-12, which degrades other ECM proteins in the mouse lungs (47), and also decreases the lung elastin content (17, 47). MMP-12 expression is also regulated by AP-1, and Fra1 is one of the AP-1 components that bind to its functional AP-1 site (148).
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INTERACTION OF AP-1 PROTEINS WITH OTHER TRANSCRIPTION FACTORS |
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In addition to self homo- or heterodimerization, Jun and Fos
proteins selectively partner with other related bZIP families of
proteins (Fig. 5), including ATFs,
C/EBPs, Nrf/Mafs, and helix-loop-helix ZIP proteins, such as upstream
stimulatory factors (USFs) (20). Furthermore, they can
physically interact with non-ZIP proteins, such as nuclear factor of
activated T cells, Ets, NF-B, glucocorticoid receptor, CREB-binding
protein, and TATA-binding protein (20). These interactions
not only increase the complexity and repertoire of the protein factors
present in a given cell type but also result in differential DNA
binding and transactivation activities, thereby tightly controlling
AP-1-dependent gene expression in various biological processes
(20). In addition, the involvement of a variety of MAPK
modules that differentially activate various partners can also
integrate signals elicited by diverse stimuli. The interactions between
AP-1 proteins and non-ZIP proteins and their role in inflammatory and
immune responses have been discussed elsewhere (33, 36, 54,
83). In this review, we briefly discuss some of the potential interactions between AP-1 and other bZIP proteins and their role in
regulation of gene expression in cells of the lung.
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Interactions with bZIP proteins.
Jun and Fos proteins can distinctly heterodimerize with ATF family
members, B-ATF, ATF-, ATFs 1-4 and -6, and CREB. These heterodimers more efficiently bind to CRE compared with TRE
(20). The specific interactions of AP-1 include c-Jun with
ATF-2, -3, and -4; JunB with ATF-3 and B-ATF; c-Fos with ATF-4; and
Fra1 with ATF-4 (20). Compared with c-Jun and c-Fos, Fra1
binds to ATF-4 with a stronger affinity (43). Several
genes expressed in the lung contain functional CRE in their regulatory
regions and are regulated by Jun-Fos-ATF heterodimers. Some of
them include c-jun, SP-B, TG-1,
HO-1, and interferon-
. Mice devoid of
ATF-2 display severe respiratory distress and die shortly after birth, suggesting a critical role for it in respiratory functions
(84). As described above, ATF-2-c-Jun binding at CRE
located at the proximal promoter plays a prominent role in regulation
of c-jun transcription (125). However,
c-jun transcription can be inducible in the cells that are
devoid of ATF-2, suggesting other factors might compensate for its
absence. A suppressive role for ATF-Jun dimers exists in regulation of
SP-B transcription (10), whereas the binding of
Jun and CREB positively correlates with TG-1 transcription (92). Disruption of ATF-4 results in severe
fetal anemia in mice (87). Because ATF-4, a ubiquitous
protein, positively upregulates Nrf2-induced HO-1
expression in other cell types (49) and because Nrf2 and HO-1 also play a role in pulmonary defense mechanisms (22, 100), it is likely that the interactions between
ATF-4 and AP-1 proteins have differential effects on gene expression in
cells of the lung.
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ROLE OF AP-1 FAMILY MEMBERS IN LUNG TUMORIGENESIS |
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Cells of the respiratory tract epithelium, a direct target of inhaled toxicants/carcinogens, are considered as the progenitor cell types for the majority of various lung tumors. Experimental evidence suggests that lung cancer development is a multistep process characterized by multiple but sequential morphological, molecular, and genetic changes involved in cell growth and differentiation (12, 45). In addition to deregulation of tumor suppressor gene expression (e.g., p53, Rb), the activation and/or overexpression of oncogenes (e.g., K-ras, c-myc) and growth factors and their receptors (e.g., EGFR) also play a role in cellular transformation (12, 45). Because various oncogenic and mitogenic signaling pathways converge at AP-1, which in turn regulates the expression of cell cycle machinery, it is likely that abnormal activation of members of the AP-1 family by toxicants amplifies the intracellular signals, resulting in unregulated epithelial cell growth and cellular transformation. Mutations or deletions in coding and/or noncoding regions of several genes have been correlated with the progression of several diseases including cancer. Intriguingly, none of these modifications have been documented for AP-1 family genes, alternatively suggesting that modulation of their abundance and/or activity by toxicants plays a more significant role in cellular transformation (65). In support of this notion, as discussed earlier, cells and mice overexpressing individual AP-1 components demonstrate cellular transformation and tumors, respectively (65). Although previous studies have mainly focused on the role of c-Jun and c-Fos in lung epithelial cell transformation (5, 11), relatively little is known about the involvement of other AP-1 components. Surprisingly, although thought to play a role in transformation in vitro, ectopic expression of either c-Jun or c-Fos does not result in an overt phenotype in the lung or any other organ, with the exception of bone tumors in the case of the latter (39). Recent studies also rule out a role for JunB (78), FosB (39), or Fra2 (89) in cellular transformation, as their overexpression produces no phenotype in mouse lungs. Although a transgenic overexpression of JunD has not been reported, it is unlikely to have a profound phenotypic effect in the lung, as, similar to c-Jun, JunB, and Fra2, the endogenous expression of JunD is very high in various tissues, including the lung, and is not significantly altered after mitogenic or toxic stimuli (133).
Does then Fra1 play a role in cellular transformation? As discussed earlier, several lines of recent evidence generated by various laboratories indicate a potential role for Fra1 in abnormal differentiation and transformation of lung cells. First, most importantly, the broad overexpression of fra1, but not other AP-1 components, induces some lung tumors in mice (64). Second, various toxicants and known carcinogens, such as TS (112), silica (126), and asbestos (53, 117, 135), persistently activate fra1 expression in lung cells both in vitro and in vivo. This appears to be selective, as exposure of bronchial epithelial cells to TS upregulates fra1, but not fra2, expression both in vitro and in vivo (112). Third, Fra1 positively upregulates gene expression associated with squamous cell metaplasia, a preneoplastic lesion (103, 143). Fourth, the transition from small cell to nonsmall cell lung cancer phenotype induced by H-Ras/c-Myc is associated with the specific induction of fra1 but not other AP-1 family members (114). Last, Fra1 is a predominant component of the AP-1 complex in asbestos-induced mesothelioma and proliferating rat mesothelioma cells, and overexpression of the dominant-negative Fra1 mutant inhibits growth of these cells in soft agar (110a). Fra1 mRNA is also highly induced in NNK-induced lung tumors compared with control lung (112). Together, these observations highlight a potential role for Fra1 in lung tumorigenesis.
In support of this hypothesis, a causal role for Fra1 in cellular transformation has been documented in other systems. For example, fra1 expression is high in stomach (88) and esophageal (59) squamous cell carcinomas, as well as in breast tumor cells (151). Ectopic expression of fra1 increases the motility and metastatic behavior of invasive mammary adenocarcinoma cells (73). A higher level of fra1 expression is essential for v-mos-induced transformation of thyroid cells (90). Consistent with this observation, human thyroid tumors also express a high levels of fra1 (140). Fra1 is also the predominant component of AP-1 complex formation induced by activated Ras, which promotes fibroblast transformation (8). Overexpression of fra1 in fibroblasts results in an anchorage-independent cell growth in vitro and tumor formation in nude mice (9, 147). Most recently, the requirement of Fra1, but not Fra2, for TPA- and EGF-induced neoplastic transformation of epidermal cells has been documented (150).
Somewhat puzzling is the mechanism by which Fra1 induces cellular transformation. Unlike c-Fos and FosB, Fra1 apparently lacks a transcriptional activation domain and therefore is thought to suppress or limit the transcriptional activity of AP-1 by forming stable heterodimers with Jun proteins (65). Although deletion of fra1 causes early embryonic lethality, and its expression is high during late G1 and asynchronous cell growth, MEFs devoid of fra1 surprisingly have no defects in cell proliferation (65). There are several possibilities to explain why Fra1 expression may not be required for MEF proliferation, including compensation of c-Fos, FosB, or Fra2 for Fra1. For example, MEFs devoid of both c-Fos and FosB, but not each individually, show proliferative defects, thus suggesting that Fos proteins indeed complement each other (65). This phenomenon is also confirmed by another study that showed that microinjection of antibodies specific for individual Fos proteins did not block cell proliferation, whereas their combined use did (65). If compensatory mechanisms exist, an unexplained question is why the disruption of fra1 causes embryonic lethality, whereas disruption of c-fos and fosB shows no effects. This paradox suggests that Fra1 might have functions distinct from those of other Fos proteins. Surprisingly, contrary to the previous belief that Fra1 lacks a transactivation domain, a recent study demonstrates that Fra1 possesses transactivation potential required for TPA- and EGF-induced neoplastic transformation of epidermal cells. This is mediated by the mitogen-activated ERK-dependent phosphorylation of Thr231 located in the COOH-terminal region of Fra1 (150).
Alternatively, it is possible that a protracted induction of Fra1 by
mitogens and/or toxicants alters the dynamics of AP-1 by changing dimer
composition (Fig. 5). This might either positively or negatively
influence the transcriptional activation of target genes. In addition,
Fra1 can distinctly interact with other proteins, such as USF-1
(105) and ATF-4 (43), thereby playing a
regulatory role in gene expression involved in pulmonary defense
mechanisms. For instance, USF-1 (105) regulates gene
expression of MT-I (80) and SP-A transcription
in alveolar type II cells (38), whereas ATF-4 interacts
with Nrf-2 (4, 49) and positively regulates gene
expression of NQO1, -GCS, and HO-1, which are involved in the detoxification of ROS (100). However, Fra1 suppresses
Nrf-2-inducible NQO1 (142) and possibly
-GCS expression (62). Thus one could speculate that the interaction of Fra1 with USF-1 and/or ATF-4 probably
modulates gene expression that plays a role in pulmonary defense mechanisms.
On the basis of the above observations, it is quite reasonable to assume that a protracted induction of fra1 in cells of the lung, which endogenously is very low or undetectable, could modulate downstream target gene expression by toxicants and carcinogens. This in turn may compromise normal cell growth and differentiation, thereby altering the injury and repair processes, and culminate in cellular transformation. The fact that Fra1 cannot bind to DNA by itself suggests that interactions of Fra1 with other transcription factors, as well as their posttranslational modifications, may play a central role in the pathogenesis.
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SUMMARY AND FUTURE DIRECTIONS |
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As the downstream transcription effector of a variety of signaling pathways activated by toxic stimuli, AP-1 can act as a "master switch" to regulate gene expression involved in lung injury, repair, and transformation (Fig. 1). Through heterodimerization of AP-1 family members and interactions with other transcription factors (Figs. 2 and 5), AP-1 proteins provide different layers of both multiplicity and diversity to control the regulation of gene expression tightly and distinctly. The lack of a well-conserved promoter structure, their distinct regulation by various stimuli (Fig. 3), and the existence of some functional redundancy (Table 1) strongly suggest that both spatial expression and regulation of AP-1 family members are important in various cellular responses. Intriguingly, the presence of TREs, which either overlap with or exist adjacently and/or in close proximity with other motifs recognized by cell type (e.g., Nkx2.1, HNF-3) and/or ubiquitous (e.g., Nrf2, Sp1, and Ets) transcription factors, indicates that activation of AP-1 family members by toxic agents may tilt the dynamics of transcription. These interactions may deregulate the expression of a particular set of target genes(s), which may lead to abnormal injury and repair process, culminating in lung disease. A daunting task, based on the fact that lung is a complex tissue consisting at least 40 different cell types, is the design of studies to gain a better understanding of the distinct regulatory mechanisms of individual AP-1 subunits in various lung cells. These investigations should provide additional insight into the molecular changes involved in abnormal differentiation and cellular transformation. The generation of in vitro and animal models that overexpress wild-type or mutant protein(s) in an "on/off" manner in the specific cell types may help identify the role of AP-1 family members in normal and abnormal lung biology. In addition, cDNA microarray and proteomic analysis will enable us to identify the critical players and the sequence of interactions of AP-1-inducible (both early- and late-responsive) gene expression. This information may offer unique opportunities to use AP-1 family members or target genes as potential diagnostic markers or drug targets for early detection and/or prevention of various lung diseases.
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ACKNOWLEDGEMENTS |
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We apologize to all colleagues whose work has not been referenced, due to page limit.
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FOOTNOTES |
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This work was supported by grants R29 HL-58122, R01 HL-66109, EPA-R826724, ES-09606, and RO1 ES-011863 to S. P. M. Reddy; and PO1 HL-67004 and R01 ES/HL-09213 to B. T. Mossman.
Address for reprint requests and other correspondence: S. P. M. Reddy, Johns Hopkins Univ., Dept. of Environmental Health Sciences, Div. of Physiology, Rm. W7006, 615 No. Wolfe St., Baltimore, MD 21205 (E-mail: sreddy{at}jhsph.edu).
10.1152/ajplung.00140.2002
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