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INTRODUCTION |
Cystic fibrosis (CF)1 is
the most common lethal, inherited pulmonary disorder and is caused by
mutations in the cystic fibrosis transmembrane conductance regulator
(CFTR) (1). More than 800 distinct mutations in the CFTR gene have been
associated with clinical diseases characteristic of cystic fibrosis
(2). Whereas many organs are affected in CF, morbidity and mortality in
the disease is primarily related to mucus accumulation, recurrent infections, and excessive inflammation in the lung. Whereas the pathogenesis of CF is not fully understood, abnormalities in cyclic AMP-dependent chloride secretion and excessive sodium
reuptake by epithelial cells related to CFTR deficiency are thought to alter fluid homeostasis at the airway surface liquid leading to its dehydration, impaired mucociliary clearance, and infection (see
Ref. 3 for review). Because the elucidation of the primary structure of
CFTR, a myriad of functions and numerous interactions with other
cellular proteins have been ascribed to CFTR. Thus, in addition to the
role of CFTR in the regulation of cAMP-dependent chloride
transport, this protein may play pleotropic roles in many cellular
processes by interacting with the cytoskeleton, membrane transport
proteins, as well as receptors, protein routing and degradation
machinery (2). A number of studies support the concept that the
excessive inflammatory responses occur in the CF lung, but the
mechanisms underlying these abnormalities have not been clarified.
Changes in levels of IL-8 and other proteins mediating inflammatory
signaling including NF
B and iNOS have been associated
with CF, in the presence or absence of infection, raising the
possibility that abnormalities in CFTR may constitutively alter
pathways mediating inflammation (4-6).
In the lung, CFTR is distributed primarily in apical regions of airway
and submucosal gland epithelial cells (7). Abundance and cellular sites
of expression of CFTR are strongly influenced by developmental,
spatial, and humoral factors, supporting the concept that the
expression and function of CFTR are regulated at both transcriptional
and post-transcriptional levels. Despite extensive study, the precise
role of CFTR in the pathogenesis of CF disease remains poorly
understood. At the clinical level, severity of CF disease is highly
variable even among individuals bearing identical mutations, supporting
the concept that environmental and hereditary factors may influence the
severity of the disorder (2). These clinical observations, and
observations demonstrating strain differences in the severity of CF
phenotype after CFTR gene targeting or mutation in mice (8), support
the concept that the expression of CFTR and its function in cellular
processes may be influenced by many genes or pathways intensifying or
mollifying CF disease in various organs. Morbidity and mortality in
patients with CF is strongly associated with pulmonary disease caused
by mucous accumulation, inflammation, and infection; however, deletion of CFTR mice does not cause significant pulmonary disease suggesting that expression of alternative channels or other complementary genes
maintains pulmonary homeostasis in the mouse. Whereas numerous in
vitro and in vivo models have been developed for study
of CFTR, analysis of genomic responses to the presence or absence of
CFTR are complicated by heterogeneity of cell models, and culture
conditions that may influence cell function and gene expression
independently of CFTR. Direct RNA analysis of pulmonary tissue from
humans with CF is complicated by the nearly ubiquitous, severe
pulmonary infections that may secondarily modify cellular responses and
gene expression, complicating identification of responses to CFTR
in vivo.
In the present study, we undertook experiments to identify RNAs
influenced by the presence and absence of CFTR in vivo,
seeking to identify genes and pathways that interact with or compensate for the CFTR to maintain pulmonary function. In this study, stereotypic genomic responses to the lack of CFTR were observed in pulmonary tissues in the absence of infection or disease.
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MATERIALS AND METHODS |
Transgenic mice bearing a null mutation in CFTR (CFTR(
)),
generally succumb to intestinal disease in the weanling period (9). To
generate healthy mice deficient in CFTR, the human CFTR cDNA was
expressed in the intestinal epithelium under control of the intestinal
fatty acid-binding protein gene promoter (iFABP), fully correcting
small intestinal pathology and supporting normal postnatal survival of
CFTR(
) mice (10). The iFABP-hCFTR, mCFTR(
) mice have been
maintained in a mixed FVB/N, C57BL/6 background without evidence of
gastrointestinal or pulmonary disease for nearly a decade in our
laboratory. Histological andbiochemical studies identified no overt
pathology in lung tissue from these mice compared with CFTR expressing
littermate controls (10, 11). Mice were maintained in filtered
microisolator cages. Sentinal mice were free of mouse pathogens. Lungs
of adult iFABP-hCFTR, mCFTR(
), and control mice were free of
bacterial pathogens or colonization as assessed by quantitative culture
of lung homogenates on blood agar plates.
RNA Preparation--
Matings of FABP-hCFTR(+/+)/mCFTR(
/
)
mice to wild type FVB/N-mCFTR(+/+) mice were used to produce F1
FABP-hCFTR(+/
)/mCFTR(
/+) mice. These mice were crossed to generate
F2 offspring littermates that were then genotyped, choosing
FABP-hCFTR(+)/mCFTR(
) and hCFTR(+)/mCFTR(+) mice for use in RNA
comparisons. To minimize the potential influence of strain differences
in this colony, lung RNA was isolated from sex-matched littermates at
3, 6, and 11 weeks of age. RNA was also isolated from lungs of
surviving CFTR(
)/hCFTR(
) and CFTR(+) littermates at 3 weeks of age
for comparison with those bearing the iFABP-hCFTR transgene. All
CFTR(+) mice were heterozygous for the targeted mCFTR gene. Lungs from sex-matched littermates were carefully dissected and the conducting airways and mediastinal structures removed. RNA, cDNA synthesis, and microarray analysis were performed in pairs to minimize technical variability related to RNA and isolation and hybridization conditions. Lungs were homogenized in TRIzol reagent (Invitrogen) using
methods recommended by the manufacturer. Genotyping was performed using the following primers: primers for mCFTR PCR: forward primer (intron 9), 5'-AGGGGCTCGCTCTTCTTTGTGAAC-3', and reverse primer (intron 10), 5'-TGGCTGTCTGCTTCCTGACTATGG-3'; neomycin resistance gene PCR:
forward primer, 5'-CACAACAGACAATCGGCTGCT-3', and reverse primer,
5'-ACAGTTCGGCTGGCGCGAG-3'; and hCFTR PCR: forward primer (exon 9),
5'-AAACTTCTAATGGTGATGACAG-3', and reverse primer (exon 11),
5'-AGAAATTCTTGCTCGTTGAC-3'.
Validation of RNAs by RT-PCR--
Changes in selected RNA levels
identified in the microarray analysis were validated by RT-PCR using a
Light CyclerTM or a regular thermocycler followed by gel
electrophoresis. For these studies, lung RNAs were isolated as
described above. cDNAs were generated by reverse transcription and
PCR analysis was performed using the following primers for: Kir 4.2 forward, 5'-CTTTGAGTTTGTGCCTGTGGTTTC-3', and reverse,
5'-GCTGTGTGATTTGGTAGTGCGG-3'; human CFTR, same as above; and mouse CFTR
forward, 5'-TGCTTCCCTACAGAGTCATCAACGG-3', and reverse,
5'-CACAGGATTTCCCACAACGCAGAG-3';
-actin for normalization, forward, 5'-TGGAATCCTGCGGCATCCATGAAC; reverse,
5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; glyceraldehyde-3-phosphate
dehydrogenase, forward, 5'-CTTCACCACCATGGAGAAGGC-3', and reverse,
5'-GGCATGGACTGTGGTCATGAG-3'; CCAAT enhancer-binding protein (CEBP)
, forward, 5'-CGCAACAACATCGCTGTG-3', and reverse, 5'-GGGCTGGGCAGTTTTTTG-3'; TNF-AIP-3, forward,
5'-GCACGAATACAAGAAATGGCAGG-3', and reverse,
5'-GGCATAAAGGCTGAGTGTTCACG-3'; and Grin 2d, forward, 5'-CCTTCTTTGCCGTCATCTTTCTTGC-3', and reverse,
5'-AAACTTCAGGGGTGGGTATTGCTCC-3'.
RNA Microarray and Data Analysis--
Total RNA was subjected to
reverse transcription using oligo(dT) with T7 promoter sequences
attached, followed by second strand cDNA synthesis. Antisera cRNA
was then amplified and biotinylated using T7 RNA polymerase, prior to
hybridization to the Affymetrix genechip mouse U74aV2 using the
Affymetrix recommended protocol (12, 13). Affymetrix MicroArray Suite
version 5.0 was used to scan and quantitate the genechips using default
scan settings. Intensity data were collected from each chip, scaled to
a target intensity of 1500, and the results were analyzed using both
MicroArray Suite and GeneSpring 5.0 (Silicon Genetics, Inc., Redwood
City, CA).
cDNAs were hybridized to U74aV2 chips (Affymetrix Inc.).
Hybridization data were normalized in a CCAAT enhancer-binding protein two-step process to remove or minimize systemic sources of variation at
both chip and gene level. Specifically, each chip was normalized to the
distribution of all genes on the chip to control for variation between
samples. Each RNA from mCFTR(
) mice was normalized to its specific
control (i.e. sex and age-matched mCFTR(+) littermates). Data were further transformed into log ratio for analysis and symmetry
of distribution. Changes in mRNAs were identified by the
combination of a distribution analysis (JMP4, SAS Institute, Inc.), and
the Welch analysis of variance. Outlier box and quartile box plots were
used to identify outliers with the definition of up-outlier > upper quartile + 1.5 (interquartile range), and the down-outlier < lower quartile
1.5 (interquartile range). Significant changes were calculated by the Welch t test at
p < 0.05. Adjusted p values were calculated
by Westfall and Young permutation for correction of false positives
(GeneSpring 4.2.1, Silicon Genetics). Comparisons between genotype and
age groups were performed using one-way analysis of variance. To
identify genes that were differentially expressed because of CFTR
genotype regardless of age, hierarchical, and k-means
clustering were used to identify consistent changes in gene expression
in response to the lack of CFTR at all three time points. Candidate
RNAs were further filtered on the basis of reproducibility and absolute
intensity. Mean, standard deviation, and coefficient variation were
calculated for each replicate. Replicates with coefficient
variations > 50% were deleted from analysis. Genes whose
expression was below the level of detection were eliminated as
experimental noise.
Pathway and Literature Analysis--
Selected genes were
subjected to intensive search to identify biological function and
associated regulatory pathways. A U74Av2 annotation data base with
system identifiers was constructed for all the array elements and their
associated GenBankTM accession numbers. Gene description,
functional categories, biological processes, molecular functions,
cellular components, protein domain, and literature information
were identified. Information resources included NetAffy
(www.affymetrix.com), Source Search
genome-www5.stanford.edu/cgi-bin/SMD/source/), BLAST
NCBI, Locus Link, mouse-human homolog search
(www.ncbi.nlm.nih.gov), and Gene Ontology data base
(www.godatabase.org/chi-bin/go.cgi). Differentially expressed genes
were classified into functional categories based on the gene ontology
definition. To determine which functional category is overrepresented
in the selected gene list, the binomial probability was calculated for
each category using the entire U74Av2 (contains 12488 mice genes) as a
reference data set. The binomial probability is defined by the following.
|
(Eq. 1)
|
It returns the probability of getting k successes of
n trials if the probability is p in the given
population (U74Av2). Potential protein/protein or protein/DNA
interactions were identified using the published literature information.
Lung Histology and Immunohistochemistry--
Lungs from
postnatal animals were inflation fixed with 4% paraformaldehyde at 25 cm H2O pressure via a tracheal cannula. Lung tissue was
processed according to standard methods and embedded in paraffin.
Procedures for immunostaining were previously described (14). Rabbit
monoclonal antibody against the 110-kDa Mac-3 antigen was used at
1:40,000 to identify alveolar macrophages (BD Pharmingen, San Diego, CA).
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RESULTS |
Histology and Immunohistochemistry--
Lung histology in adult
iFABP-hCFTR, CFTR(
); iFABP-hCFTR, CFTR(+), and CFTR(+) control mice
was not different (Fig. 1). There was no
evidence of pulmonary inflammation, infection, or remodeling. Mac-3
staining was used to identify alveolar macrophages. Numbers and
histology of alveolar macrophages were not altered by mCFTR.

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Fig. 1.
Normal lung histology in CFTR( )
mice. Lung tissue was obtained after inflation fixation
from iFABP-hCFTR, mCFTR( ) mice (B and D) and
iFABP-hCFTR, mCFTR(+) littermates (A and C) at 3 months of age. Lung histology after staining with hematoxylin-eosin
(A and B) or Mac-3 antibody (C and
D), a marker of alveolar macrophages, were not altered in
the CFTR( ) mice. Bar = 200 µm.
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Identification of Gene Responses to the Lung CFTR Deletion--
To
identify genes responsive to CFTR, lung RNAs from iFABP-hCFTR,
mCFTR(
), iFABP-hCFTR, mCFTR(+), mCFTR(
) and mCFTR(+) littermates at
the age of 3, 6, or 11 weeks of age were compared. Microarray analyses
were performed in duplicate from RNA isolated at 3 and 6 weeks of age.
Data from 10 Affymetrix Murine Genome U74Av2 chips were normalized and
statistical differences between CFTR-deficient (CFTR(
)) and control
(CFTR(+)) mice were identified. Differences related to age were
identified by outlier analysis and/or unpaired t test. After
normalization, normal distributions were observed in the intensity data
from lung tissue obtained at all ages. Lung RNA data from 3-week-old
mCFTR(+) and mCFTR(
) mice (lacking the iFABP-hCFTR transgene) were
similarly distributed to those bearing the FABP-hCFTR gene and were,
therefore, included in the analysis.
To identify RNAs that were differentially expressed in response to CFTR
regardless of age, mCFTR(
) and mCFTR(+) data were separated into two
groups. The log-ratio distribution and outlier plot of the combined
data set are represented by Fig. 2. A
total of 1977 outliers were identified from 12442 genes/expressed
sequence tags analyzed. The abundance of 848 RNAs was increased; 1129 were decreased. Welch t test together with the Westfall and
Young step-down permutation further narrowed the number of
differentially expressed RNAs to 315. Hierarchical clustering was used
to visualize and classify the data set (Fig.
3). Data are shown in a two-dimensional matrix to identify groups of genes with similar expression patterns and
show remarkably ordered gene expression profiles of 315 selected genes.
On the chip level (top dendrogram) RNAs influenced by CFTR formed two
distinct groups. Within each group, the samples collected from
age-matched pairs were more closely related than those from different
ages, suggesting that age also influenced gene expression. At the RNA
level (the dendrogram at the left side), genes were clearly separated
into two major groups: those mRNAs increased or decreased in
mCFTR(
) mice. Genes were further filtered for the consistency of
differences in expression levels across all time points (coefficient
variation < 50%) and for their absolute intensity above 243 (90% of genes called absent by Affymetrix software, <243 for this
data set). Additional filters reduced the number of RNAs to 54, of
which 29 were consistently increased and 25 were decreased in mCFTR(
)
compared with their mCFTR(+) littermates (Tables
I and II).
The expression profiles of these 54 genes are shown in Fig.
4, demonstrating consistent patterns of
expression of the CFTR-responsive RNAs regardless of age.

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Fig. 2.
Distribution of lung RNAs from mCFTR( ) and
mCFTR(+) mice. The left panel is a histogram of log
ratio and gene frequency. The right panel is the outlier box
plot. The ends of the dashed lines, denoted by an
x and y markers, are the outliers identified from
their respective quartiles.
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Fig. 3.
Two-dimensional hierarchial clustering of 315 genes/expressed sequence tag that were significantly altered in
response to the presence or absence of mCFTR. Intensity in the
red and green color range indicates up-regulated
and down-regulated RNAs, respectively. Each row represents a
single gene. Each column represents a particular
experimental condition. Each box represents the normalized
RNA intensity value. Clustering was performed by UPGMA. Similarity
measures were assessed utilizing Euclidean distance.
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Fig. 4.
Expression profile chart (A)
and hierarchical clustering (B) of 54 selected RNAs
that were consistently altered in response to the lack of CFTR
regardless of age. Hierarchical clustering was performed by UPGMA.
Data were normalized using Trimmed Mean and Z-score calculations. In
the profile chart, data were normalized by pairwise control. The
y axis is normalized intensity (log scale) and the x
axis represents experimental ages. Red lines represent
the profiles of lung RNAs increased in CFTR( ) mice. Green
lines represent the profiles of down-regulated RNAs.
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|
Differentially expressed genes were further classified according to
their known or predicted functions. Each gene was annotated and
assigned to a functional category. To simplify the calculation, we
assumed that genes in each category could be fit to a binomial distribution. The binomial probability was calculated for each category
using the entire U74Av2 as reference data set. "Inflammatory Response" was the most represented category of those RNAs increased in mCFTR(
) mice. Among RNAs whose abundance was increased by the lack
of CFTR, those influencing inflammation, transcription, and transport
were most highly represented and consisted of a group of functional
categories quite distinct from those whose expression was decreased in
mCFTR(
) mice (Tables I-III).
The potential influence of the FABP-hCFTR transgene on RNA expression
was also assessed using the Welch t test at the three ages.
Differentially regulated RNAs identified in analysis of gastrointestinal-corrected mice were similarly affected in mCFTR(
) mice, demonstrating a lack of effect of iFABP-hCFTR on this subset of
genes. Genes whose expression was independently altered by the
iFABP-hCFTR transgene included 7 RNAs decreased and 11 increased. Differences in their levels of expression were modest (less than 1.5-fold) (Table IV).
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Table IV
The effect of fabp-hCFTR(+) on lung gene expression
Lung RNA samples from iFABP-hCFTR,CFTR( ) (i.e. gut
corrected, GC) were compared with those from CFTR(+) (lacking the
iFARP-hCFTR transgene, i.e. gut uncorrected, NGC). Ratio is
defined as: R = GC/NGC, if (GC > NGC);
R = NGC/GC if (GC < NGC).
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Validation of Selected mRNAs--
To validate the responsive
RNAs identified by microarray analyses, real time RT-PCR was performed.
mRNA levels were normalized using
-actin or
glyceraldehyde-3-phosphate dehydrogenase. Kir 4.2 (Kcnj15), CEBP
,
TNF-AIP-3, and Grin 2d mRNA were significantly increased in
CFTR(
) mice compared with control littermates (Fig. 5). As expected, murine CFTR was not
detectable by RT-PCR in mCFTR(
) mice, nor was hCFTR mRNA detected
in lung from the iFABP-hCFTR bearing mice.

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Fig. 5.
Real time PCR analysis of Grin 2d
(A), Kir4.2 (B),
CEBP (C), and TNF-AIP-3
(D). Lung mRNA abundance was determined by
RT-PCR using a Light CyclerTM in tissue from mCFTR( )
(open) and mCFTR(+) mice (black). Values were
normalized to -actin or glyceraldehyde-3-phosphate dehydrogenase
RNAs and represent mean ± S.E., n = 56 determinations and differences were assessed by paired t
test.
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DISCUSSION |
The absence of CFTR caused stereotypic changes in gene
expression in the lungs of CFTR(
) mice in the absence of detectable infection or inflammation. Cellular responses to CFTR included enhanced
expression of transcription factors and signaling pathways known to
influence CFTR gene expression (IL-1
and CEBP
). Likewise, expression of RNAs modulating inflammation, ion transport, protein trafficking, and degradation were altered, indicating that a number of
cellular pathways may compensate for or mediate
CFTR-dependent functions that, in turn, may maintain normal
pulmonary homeostasis in the CFTR(
) mice or alternatively, influence
CF phenotype following response to pathogens. The absence of CFTR
initiates reproducible changes in lung gene expression that may modify
inflammation, host defense, and other cellular functions, that likely
contribute to or mollify the pathogenesis of pulmonary disease in
CF.
Because strain is known to influence pulmonary findings in CF mice (8),
and the transgenic mice presently studied were generated in a mixed
FVB/N, C57 Bl/6 background, lung RNA was compared from sex-matched
littermates at various ages. An extensive data set was utilized to
identify CFTR-dependent changes in gene expression that
were present throughout development and in the absence of inflammation,
seeking to identify pathways influenced primarily by CFTR, rather than
sex, strain, age, or other secondary phenomena. As expected, expression
of mCFTR was not detected in the gastrointestinal-corrected mCFTR(
)
mice, consistent with previous findings (10). Histologic analyses,
performed presently and previously, demonstrated no structural
abnormalities, infection or inflammation in the lungs of these mice as
maintained in our vivarium (10, 11), supporting the concept that
changes in gene expression were related to CFTR and not to age or lung
disease. Analysis of arrays prepared from pairs of mCFTR(
) mice and
mCFTR(+) littermates (those lacking the iFABP-hCFTR transgene),
confirmed the microarray findings, demonstrating both the lack of mCFTR mRNA in lungs of the mCFTR(
) and that RNA changes were
independent of the iFABP-hCFTR transgene.
Expression of Genes Modulating CFTR--
Expression of a number of
genes known to influence CFTR expression was enhanced in the lungs of
CFTR(
) mice, including CEBP
and IL-1
suggesting that CFTR cells
responded by enhancing levels of RNAs encoding transcription factors or
pathways that may compensate for the lack of CFTR. IL-1
increased
CFTR gene transcription in epithelial cells in vitro (15)
and cis-acting elements binding CEBP
(to CCAAT enhancer sites) and
c-Fos (to AP-1 elements) are present in the promoter-enhancer
regions of the mouse CFTR gene (16, 17). Previous studies demonstrated
that CEBP
directly enhanced CFTR gene transcription in
vitro (18), thus, the increased CEBP
expression may represent a
potential compensatory response to CFTR deficiency that, in turn, may
influence expression of genes unrelated to CFTR. Expression of
c-fos was also increased in the mCFTR(
) mice. Whereas AP-1
sites have been identified in the hCFTR promoter, the precise role of
c-fos in regulation of CFTR has not been clarified; although
treatment of various cell types with phorbol esters decreased CFTR gene
transcription in vitro (18). Because CEBP
and
c-fos activate or inhibit the expression of numerous genes
that share cis-active elements with the CFTR gene, changes in their
activity may broadly influence gene expression, perhaps inadvertently
linking CFTR deficiency to the expression of genes whose activities are
not directly related to CFTR protein function. RNA encoding TNF-AIP-3,
a zinc finger transcription factor, was also increased in the CFTR(
)
mice, further linking transcription responses to the lack of CFTR. Of considerable interest, TNF-AIP-3 RNA was induced by either IL-1
or
TNF. TNF-AIP-3 inhibited NF
B translocation in vitro, and
may represent a compensatory response to the increased expression of
IL-1
seen in the CFTR(
) mice (19). JAK-3, nuclear receptor subfamily 2, and interferon regulatory factor-1 RNAs were decreased in
CFTR(
) mice. These RNAs encode transcription factors that regulate
various pathways involved in inflammation and may, therefore, represent
responses to the proinflammatory proteins induced in the CFTR(
) mice,
in essence, being secondarily responsive to initial compensatory changes.
Enhanced Expression of Genes Modulating Inflammation--
Genes
involved in inflammation were overly represented among the RNAs induced
and were distinct from those that were decreased in the lungs from
CFTR(
) mice (Table III). Most prominent among genes whose expression
was increased were a family of calcium-binding proteins termed the
calgranulins (S100A8, S100A9, and calbindin D9K). S100A8 is expressed
primarily by macrophages and monocytes and its expression is enhanced
by various cytokines including TNF-
, IL-1
, and interferon
(20, 21). This family of peptides is expressed by various cell
types and share potent chemoattractant activities, stimulating
inflammatory cell trafficking. Increased expression of S100A8
(calgranulin A) was previously demonstrated in alveolar macrophages
from CFTR mutant mice, the authors suggesting that increased S100A8 may
contribute to the enhanced inflammatory responses seen in the absence
of CFTR (22). Expression of S100A8 by alveolar macrophages was induced
by TNF-
, interferon
and IL-1
, mediated at least in
part by AP-1-dependent pathways (20). Chitinase A mRNA
was also consistently increased in the lungs of CFTR(
) mice. The
acidic chitinase A is a small peptide containing a mammalian lectin
domain that binds complex carbohydrates on surfaces of microbial
pathogens including fungi. Like the S100 family of proteins, chitinase
family members are also expressed by alveolar macrophages in the lung
(23). Thus, decreased mCFTR initiated changes in expression of gene
expressed primarily in alveolar macrophages. It remains unclear whether
these responses are mediated by direct effects of CFTR in alveolar
macrophages or by altered cellular signaling initiated by the lack of
CFTR in epithelial or other pulmonary cells that secondarily alters macrophage activity.
Intense neutrophilic infiltration and an increased IL-8 production are
strongly associated with CF lung disease in humans (2). Increased IL-8
and neutrophilic infiltrates were observed in bronchoalveolar lavage
fluid from CF patients in the absence of documented pulmonary infection
(4). Although it remains possible that antecedent, but resolved,
infections may have contributed to the increased inflammation observed
in the CF, these observations support the concept that CF is associated
with increased susceptibility to pulmonary inflammation. In the present
study, IL-1
, IL-4, and CSF-1 receptor RNAs were increased and each
may contribute to the proinflammatory milieu. IL-1
enhances CFTR
gene transcription, induces inflammation, and is known to stimulate
production of the S100-calgranulins, perhaps indicating a network of
genes influenced by CFTR through IL-1
. IL-4 is a potent inflammatory
mediator that enhances inflammation and mucous production in airway
epithelia. Transgenic animals expressing IL-4 or animal models in which
IL-4 is induced developed severe goblet cell hyperplasia, increased mucous production, and inflammatory cell infiltrates (24), findings typically found in patients with cystic fibrosis. CSF-3r RNA encoding a
receptor that mediates monocytic cell migration, proliferation, and
activity in response to CSF-3 (G-CSF) was increased 3-4-fold in the
CFTR(
) mice (25). Thus, taken together, expression of a number of
genes; many influenced by IL-1
and mediating inflammation, were
induced in the lungs of CFTR(
) mice.
Despite the increased expression of proinflammatory molecules, there is
no evidence of inflammation in the lung of the CFTR(
) mice perhaps
indicating that normal homeostasis is maintained by the complex
responses of the lung to the lack of CFTR. The presence of stereotypic
changes in expression of many genes suggests that the presence of a
single ameliorating gene, for example, an alternative chloride channel,
does not fully explain the physiologic adjustment of the lung the
CFTR(
) mice. At present, it is unclear whether these proinflammatory
responses are secondary to changes in the expression and function of
CFTR in the epithelial cells that, in turn, modulate cell signaling and
cytokine production in the lung. Alternatively, the absence of CFTR in
the alveolar macrophages may alter expression of genes mediating
inflammation in those cells. It is of considerable interest that
changes in RNAs modifying inflammation were altered in the lungs of
mCFTR(
) mice in the absence of detectable bacterial infection or
inflammation, supporting the concept that the transcriptional
adjustment to CFTR deficiency suffices to maintain normal pulmonary
homeostasis in the mouse in vivo. Alternatively, the levels
of expression of the proinflammatory molecules may not be adequate to
cause histologically detectable inflammation. It remains unclear
whether these adjustments in gene expression may, in turn, render the CFTR(
) mice susceptible to inflammation following infection or injury.
Changes in NF
B- and TNF-
-dependent
Pathways--
RNAs encoding a number of proteins involved in TNF
signaling and NF
B activation were also induced in the CFTR(
) mice.
The abundance of TNF-AIP-3 mRNA, a zinc finger transcription
protein, a protein whose expression is induced by both TNF-
and
IL-1
, was increased in the CFTR(
) mice. TNF-AIP-3 inhibits NF
B
activity at target genes (19) and may represent a response to the
proinflammatory milieu established in the CFTR(
) lung. PEG-3, a
protein that regulates the induction of NF
B following TNF
stimulation, was also increased, providing for the support for
transcriptional relationships between CFTR deficiency and activity of
NF
B (26). Recent studies of Schroeder et al. (27) support
the concept that the CFTR is required for regulation of NF
B, serving
as a pattern-associated molecular recognition molecule,
following pulmonary exposure to Pseudomonas aeruginosa. In
that study, NF
B activation and its nuclear trafficking were
deficient in CF cells. Taken together with the observation that
IL-1
-mediated transcription of CFTR gene transcription is dependent
upon NF
B, this important pathway mediating inflammation appears to
be influenced by CFTR.
In the present microarray analysis, CFTR deficiency was associated with
increased claudin 8 and decreased gap junction connexin 37. Decreased
expression of gap junction proteins (Cx43, 40, -37, and -32) and
decreased gap junction communication were observed in various in
vitro cell systems after exposure to proinflammatory cytokines
(28). The observed changes in RNAs mediating cell adhesion and
increased expression of IL-1
seen in the CFTR(
) mice are
consistent with findings that in the absence of CFTR, IL-1
and
TNF-
failed to inhibit cell communication via gap junctions. Previous studies demonstrated that CFTR is required for the uncoupling of gap functions between epithelial cells during inflammation, a
process that may restrict the spread of pathogens or signaling among
adjacent cells. It will be of interest to test whether cell adhesion
mediated by the claudins is also linked to the pathogenesis of CF.
Changes in Protein Degradation Pathways--
Several genes
involved in protein degradation were altered in the absence of CFTR
compared with normal. Proteosome 26 S subunit (PSMC3) is the major
proteolytic component of the ubiquitin-dependent proteosome. Proteosome 26 S regulates degradation of proteins influencing cell cycle, oncogenesis, transcription, and immunity, including CFTR itself (29, 30). The proteosome is composed of two
subcomplexes, the 20 S proteosome and PA700. PSMC3 expression was
modestly, but significantly increased (1.5-fold) in the absence of
CFTR. In contrast, PA28
(PSME3), an activator of the 20 S proteosome, was decreased 1.6-fold. These observations are consistent with previous findings that proinflammatory cytokines, including TNF-
or interferon-
increased expression of the 26 S proteosome and its activators PA28
and
(31), whereas expression of PA28
was decreased by interferon-
(32). RNA encoding adaptor protein
complex AP-2,
1 subunit (Ap2a1), a protein involved in the formation
of intracellular transport vesicles was also decreased in the absence
of CFTR. CFTR co-precipitates with
-adaptin (33). Recent studies
demonstrated that a C-terminal domain of CFTR binds to the AP-2 adaptor
complex to form clathrin-coated vesicles that mediates CFTR
internalization (34). ADP-ribosylation factor 5 belongs to a family of
GTP-binding proteins that play important roles in the control of
membrane trafficking, including formation of secretory vesicles at the
trans-Golgi network, endosomal and vesicle-plasma membrane fusion (35).
Recent studies support the concept that CFTR regulates endosomal fusion
and vesicular trafficking (36, 37) indicating potential relationships
between CFTR and the actions of ADP-ribosylation factor 5. Another gene in this functional category is represented by kinesin3
, an mRNA that was decreased in the lungs of CFTR(
) mice.
Transport Proteins Influenced by CFTR--
RNAs encoding several
transmembrane transport proteins and receptors were also altered in the
lungs of CFTR(
) mice, including solute carrier 38 (member 4), the
potassium inwardly rectifying channel (Kir 4.2 or Kcnj 15), the
glutamate receptor (Grin 2d), the naturietic peptide receptor 3 (Npr-3), and the
3-adrenergic receptor (ADRB-3). Thus, expression of
a number of membrane transport proteins was influenced by CFTR, perhaps
representing compensatory responses to defects in CFTR-mediated
transport activity. Kir 4.2 and Grin 2d RNAs were increased 2-3-fold
in CFTR(
) mice. Kir 4.2 is expressed in respiratory epithelial cells
at sites similar to that of CFTR (38). Kir 4.2 regulates cation
transport upon which chloride transport via CFTR or other chloride
channels may be influenced. Surprisingly, there is evidence for
interactions between CFTR and Kir family members because they are both
known to bind via PDZ binding domains through interactions with channel interacting PDZ domain protein (39). Likewise, there is
precedence for PDZ-dependent interactions among glutamate
receptors, CFTR, and Kir family members (40). Thus, the lack of CFTR
enhanced the expression of a number of membrane proteins that may
interact with CFTR via PDZ domains, perhaps indicating that
CFTR-protein complexes may initiate changes in gene expression. These
findings support the concept that CFTR interacts with numerous membrane transport proteins and do not support a model in which activity of an
alternative Cl
transporter alone suffices to compensate
for the lack of CFTR in pulmonary cells in the CFTR(
) mice.
Regulation of Cell Receptors by CFTR--
Natriuretic peptide
receptor C (Npr-3 or NprC) was increased more than 2-fold in CFTR(
)
mice. Natriuretic peptides comprise a family of 3 structurally related
molecules: atrial (ANP), brain (BNP), and C-type (CNP) whose functions
are cGMP-dependent (41, 42). Among them, CNP increased
ciliary beat, mucociliary clearance in airway epithelial cells, and
activated CFTR-dependent chloride transport (42).
Natriuretic peptides regulate cytokine-stimulated NO production via the
binding of Npr-3 (43). Because deficient NO production was observed in
respiratory epithelial cells of the iFABP-hCFTR, CFTR(
) mice, the
increased expression of Npr-3 in CFTR(
) mice may represent a
compensatory response influencing airway clearance and nitric oxide
production via cGMP (44). In contrast to the transport/receptors that
were induced, ADRB3, a G protein-coupled transmembrane protein
mediating cAMP production was decreased in CFTR(
) mice. ADRB3 is
co-expressed with CFTR in airway epithelium (45) and may be
functionally coupled to CFTR via cAMP-independent pathways (46).
2-Adrenergic receptors directly interact with CFTR via the
Na(+)/H(+) exchanger regulatory factor to form a signal transduction
complex (47). Co-regulation of ADRB3 and CFTR may indicate that these
proteins interact closely at both structural and functional levels, a
finding that may be linked to the important role of
-adrenergic
stimulation and cAMP in the activation of chloride transport mediated
by CFTR.
Altered Expression of RNAs Encoding CFTR Interacting
Proteins--
Surprisingly, analysis of the RNA influenced by CFTR
identified a number of proteins that directly or indirectly interact with CFTR via protein-protein interactions. This list included proteins
involved in protein trafficking and degradation (proteosome 26 S and
PA28 subunits and
-adaptin), ion transport (Kcnj15 and Grin 2d), and
receptors (Npr-3 and ADRB3). Interaction of CFTR with many proteins
occurs via PDZ binding domains that mediate protein-protein complex
formation. The finding that expression of CFTR interacting proteins is
altered in the lungs of CFTR(
) mice, suggests that CFTR influences
networks of signaling and transport activities in the cell and that
cells respond to CFTR deficiency via transcriptional responses to
CFTR-protein complexes, rather than to CFTR per se.
Are Changes in Gene Expression Bystander Effects?--
Whereas
genomic responses may compensate for the lack of CFTR mRNA and
represent compensation for CFTR function, some responses may be
secondary and mediated by pathways not directly related to the action
of CFTR per se. Alterations in cytokine production or
changes in transcription factor activity caused by the absence of CFTR
may secondarily change the behavior of numerous cell types, that may,
in turn, contribute to the pathogenesis of CF lung disease by processes
that are not direct functions of CFTR. Because CFTR is known to form a
membrane-associated cyclic nucleotide-activated Cl
channel and interacts with numerous other cellular proteins, changes in
gene expression may indicate that cells sense the CFTR protein,
CFTR-dependent activity, or CFTR-protein complexes that initiate transcriptional responses related to or unrelated to CFTR
function. At present, it is unclear whether the "sensor" is
chloride ion, the presence of CFTR, or CFTR-protein complexes in
intracellular and membrane compartments or to other functions of CFTR.
Likewise, the hierarchy and interrelatedness of the networks of RNA
responses to CFTR deficiency remain to be unraveled.
Maintenance of pulmonary homeostasis in the mCFTR(
) mouse was
associated with complex adaptive responses in gene expression. CFTR
influenced RNAs encoding transcription factors, ion channels, membrane
receptors, cytokines, and intracellular trafficking proteins. Finally,
CFTR altered the expression of a number of proteins that interact with
CFTR via protein-protein interactions perhaps representing transcriptional responses to functions mediated by CFTR(
) protein complexes (Fig. 6). The diversity of
genes whose expression was altered by CFTR support the concept that, in
addition to regulation of Cl
transport, CFTR plays
diverse roles in multiple cellular functions. The present findings
support the hypothesis that pulmonary homeostasis in the CFTR(
) mouse
is maintained by complex genomic responses to the lack of CFTR rather
than by the action of a single alternative Cl
channel.
Finally, the genes and pathways identified in this study provide new
links between CFTR and cellular processes that may influence the
pathogenesis of CF lung disease.

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Fig. 6.
Molecular pathways and networks influenced by
CFTR. A model is proposed by which the lack of CFTR initiates
expression of genes that are known to enhance CFTR expression IL-1
and CEBP , secondarily modulating inflammation (1, 2). Increased
expression of cytokines, IL-4 and IL-1 , and the cytokine receptor,
CSF-3R, may influence inflammation (2) as well as expression of genes
modulating protein trafficking, degradation, and cell-cell
communication (3-5). Abundance of RNA of chemoattractant peptides of
the calgranulin family were enhanced, perhaps contributing to a
proinflammatory environment within the lung (2). CFTR altered the
expression of RNAs encoding transport proteins and membrane receptors
that likely interact with CFTR via PDZ domains (6, 7) supporting the
concept that pulmonary cells respond to the lack of CFTR or CFTR
complexes by regulating the expression of CFTR partners. Alterations in
pathways regulating second messengers, including cAMP, via the
3-adrenergic receptor (Adrb3), were observed (7). Absence
of CFTR also influenced expression of genes mediating endocytosis,
membrane recycling, and regulated secretion (4, 8, 9).
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