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INTRODUCTION |
Airway inflammation, characterized by intense influx of
neutrophils and elevated concentrations of proinflammatory mediators, is a prominent and early feature of cystic fibrosis
(CF).1 Despite an intense
inflammatory response, bacteria are not cleared efficiently from the
respiratory tract of CF patients, leading to recurrent infection and
progressive deterioration of lung function (1, 2). It has been proposed
that chronic inflammation is maintained by increased adherence (3),
decreased clearance (4, 5), or decreased killing (6, 7) of pathogens
associated with recurrent airway infections in CF. Other in
vitro and in vivo studies suggest that early
inflammation in CF airways is associated with abnormal production of
pro- and/or anti-inflammatory cytokines (8-13). Dysregulation of the
inflammatory response may represent an intrinsic component of the CF
phenotype because it is observed independently of the type of
infectious stimulus (14, 15). Consistent with this view, an increasing
number of reports have shown that CF airway epithelial cells exhibit
increased translocation of the nuclear factor-
B, a transcriptional
activator of immunomodulatory genes (11, 16-18). CF results from
mutations of the cystic fibrosis transmembrane regulator (CFTR) gene
(19). Thus, mutations in CFTR may cause alterations in intracellular
signal transduction pathways, resulting in an exaggerated recruitment
of neutrophils through constitutive and bacteria-induced release of
inflammatory mediators that is disproportionate to the infectious
stimulus (11, 20, 21). It is currently not known, however, how
mutations of CFTR lead to an abnormal inflammatory response.
It is now well established that the CFTR protein, in addition to
functioning as a Cl
channel, plays an important role in
regulating other ion channels and transporters of the plasma membrane.
Thus, CFTR influences the function and properties of Cl
,
Na+, and K+ channels in epithelial cells as
well as the control of electroneutral Na+ reabsorption,
Cl
/HCO
exchange and water permeability (22-24). We recently provided evidence for a role of CFTR in the control of gap junction channel connectivity (25, 26). Defects in
CFTR-dependent regulation of other transport mechanisms may contribute to some of the phenotypes that are observed in the CF
pathology (27). So far, the molecular mechanisms linking CFTR to other
channels and transporters are unknown.
Gap junctions, the only channels that allow direct exchange of ions and
small (<1 kDa) metabolites between cells, are composed of proteins
called connexins (Cx) in vertebrates. Gap junctions contribute to
tissue homeostasis and have been involved in the regulation of diverse
biological functions (28-31). Critical roles for gap junctions have
been elucidated by the discovery of disease-causing mutations in human
connexin genes and the observation that mice with targeted deletions of
connexins develop distinct phenotypes (32-34). These recent findings
confirm the view that perturbation of gap junction connectivity
contributes to disease initiation and/or progression. We recently
reported that the proinflammatory cytokine TNF-
differentially
regulates gap junctional communication in airway epithelial cell lines.
In non-CF airway cells, gap junction channels rapidly close in response
to TNF-
. In contrast, this mechanism is defective in CF airway cells
but can be rescued by expression of wild-type CFTR (26). These
observations, together with other reports (18, 35-38), suggest that
CFTR may interfere with some of the signal transduction pathways
initiated through receptor-ligand interactions. In this context,
differential regulation of channel activity may represent a model to
search for the signaling pathways that are defective in CF cells.
Elucidating the mechanisms linking genotype to disease may be of
critical importance to understand the pathogenesis of exaggerated
airway inflammation in the CF lung.
Here we have attempted to identify the signal transduction pathways
involved in the down-regulation of gap junction connectivity by TNF-
in non-CF airway cells. We provide evidence that TNF-
signaling
initiates the activation of the tyrosine kinase c-Src in airway
epithelial cells. We further show that inhibition of c-Src tyrosine
kinase activity abolished TNF-
-induced closure of gap junction
channels in non-CF airway cells. Moreover, we show that activation of
c-Src is defective in CF airway cells but rescued in CFTR-corrected CF
cells. These results indicate that c-Src signaling pathway links the
mediators of inflammation to gap junction channels. They also suggest
that mutations in the CFTR protein alter the c-Src tyrosine kinase
transduction pathway. The defective activity of c-Src in CF cells may
contribute to some dysfunction of the CF airway epithelium, including
ion channel activity, cytokine production, mucus secretion, and
epithelial cell differentiation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The normal human bronchial epithelial Beas2B
cell line was purchased from the American Type Culture Collection
(Manassas, VA); the human nasal epithelial CF15 cell line,
which was derived from a patient homozygous for the
Phe-508 mutation
of CFTR, was previously characterized by Jefferson and colleagues (39).
IB3-1 cells, a human bronchial epithelial cell line derived from a
patient with CF (
Phe-508/W1282X) and C38 cells, the rescued cell
line, which expresses a plasmid encoded copy of a functional CFTR (40, 41), were kindly provided by Dr. P. L. Zeitlin (The Johns Hopkins University School of Medicine, Baltimore, MD). The enhanced expression of CFTR in C38 cells was confirmed by Western blots (not shown). Beas2B
cells were maintained in Dulbecco's modified Eagle's medium; CF15 cells were cultured on surfaces coated with 50 µg/ml
of human placental collagen IV (Sigma) and maintained in 3:1 (v/v)
Dulbecco's modified Eagle's medium/F-12 supplemented with growth
factors (26). IB3-1 and C38 cells were cultured on surfaces coated with collagen IV and 10 µg/ml bovine plasma fibronectin (Invitrogen) and
maintained in bronchial epithelial cell growth medium (Promocell, Heidelberg, Germany). All media were supplemented with 10% fetal calf
serum (SeraTech, Griesbach, Switzerland), 30 units/ml penicillin, and
30 µg/ml streptomycin (Invitrogen). Beas2B cells are hereafter referred to as non-CF cells, CF15, and IB3-1 cells as CF
cells, and C38 cells as corrected cells.
Expression of Cx, c-Src, and Active c-Src--
For Western
blots, subconfluent monolayers of cells were rinsed with PBS and
scraped into an ice-cold solubilization buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride, and a mixture of
protease inhibitors (Roche Molecular Biochemicals). After a 30-min
incubation, the samples were centrifuged at 4 °C for 10 min at
50,000 × g. Supernatants were recovered, and total
amounts of protein were determined by a bicinchoninic acid
quantification assay (Sigma). Fifteen to 25 µg of protein were
electrophoresed on 10% SDS-PAGE and electrotransferred onto
Immobilon-P polyvinylidene difluoride membranes (Millipore AG,
Volketswill, Switzerland). Membranes were then soaked overnight at
4 °C in a 2% defatted milk saturation buffer containing 10 mM Tris-HCl (pH 7.4), 2 mM EDTA, 133 mM NaCl, 0.05% Triton X-100, and 0.2% sodium azide.
Blotted proteins were then incubated for 1 h at room temperature
with either a mouse monoclonal Cx43 (1:500 dilution) antibody (Chemicon
International Inc., Temecula, CA), a monoclonal v-Src (1:250 dilution)
antibody (Oncogene Research Products, La Jolla, CA), or a polyclonal
c-Src(Tyr(P)-418) (1:200 dilution) phosphospecific antibody (BioSource
International, Palo Alto, CA). This step was followed by a 1-h
incubation with goat anti-mouse or anti-rabbit IgG secondary antibodies
conjugated to peroxidase (The Jackson Laboratories, West Grove, PA).
Immunoreactivity was detected through the Super Signal West Pico kit (Pierce).
Localization of Cx43--
For immunofluorescence labeling,
non-CF and CF airway cell lines were cultured on glass coverslips and
fixed for 2-3 min with methanol at
20 °C. The coverslips were
rinsed and incubated successively with 0.2% Triton X-100 for
1 h, 0.5 M NH4Cl for 15 min, and PBS supplemented with 2% bovine serum albumin for another 30 min. Cells
were the rinsed and incubated overnight with a poylclonal antibody
(1:30 dilution) raised against Cx43 (Alpha Diagnostics, San
Antonio, TX). After washing in PBS, the coverslips were incubated with a secondary antibody conjugated to fluorescein isothiocyanate for
3 h, counter-stained with Evans Blue, and then examined using fluorescent microscopy. Images were acquired with a high sensitivity black and white CCD Visicam (Visitron systems GmbH, Puchheim, Germany)
camera connected to a personal computer. Images were captured using the
software Metafluor 4.01 (Universal Imaging Corp., Downington, PA) and
processed using Adobe Photoshop 5.5.
Expression Vectors--
pMT2-plasmids encoding either for a
kinase-dead version of c-Src (SrcK
), which acts as a
dominant negative (K295 M mutation), or a constitutively active c-Src
(SrcK+), in which the inhibitory intramolecular interaction
between phosphotyrosine 527 and the SH2 domain is disrupted (Y527A
mutation), were used (42, 43). A chimeric version of SrcK
was also constructed using PCR by destroying the stop codon and appending the cDNA for the enhanced green fluorescent protein (EGFP). Full-length cDNAs of wild-type human Cx43 (WT-Cx43) and of
Cx43 where tyrosines 247 and 265 were replaced by alanines (Y247A,Y265A-Cx43) were constructed using conventional two-step PCR mutagenesis and sub-cloned into pIRES2-EGFP
(Clontech, Palo Alto, CA). Point mutations were
verified by sequencing.
Cell Transfection--
About 50% confluent cells were
transfected either with the Effectene transfection reagent (Qiagen AG,
Basel, Switzerland) or the FuGENE transfection reagent (Roche
Diagnostics), according to manufacturers' instructions. The efficiency
of cell transfection when using pMT2 plasmids encoding for
SrcK
or SrcK+ was evaluated by
co-transfecting the cells with pMT2 encoding for EGFP. Thirty-48 h
later, small colonies of EGFP-positive cells among EGFP-negative cells
were observed and subjected to dye coupling.
Dye Coupling--
Dye coupling studies were performed on
subconfluent monolayers of cells incubated in a solution (external
solution) containing (in mM): 136 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, and 2.5 glucose and was
buffered to pH 7.4 with 10 mM HEPES-NaOH. Single cells were impaled with microelectrodes backfilled with a 4% Lucifer Yellow solution prepared in 150 mM LiCl (buffered to pH 7.2 with
10 mM HEPES). The fluorescent tracer was allowed to fill
the cells by simple diffusion for 3 min. After the injection period,
the electrode was removed, and the number of fluorescent cells was
counted. Cells were visualized using epifluorescent illumination
provided by a 100-watt mercury lamp and the appropriate set of filters. For dye coupling experiments performed in cell clusters expressing EGFP, a filter specific for fluorescein (excitation range 465-490 nm)
was used, and images of the fluorescent clusters were acquired before
the injection of Lucifer Yellow. The green fluorescent cells were then
injected with Lucifer Yellow as described above. The yellow
fluorescence of the dye could be distinguished from the green
fluorescence of EGFP using a filter with a broader excitation spectrum
(420-490 nm). Because of the brighter intensity of the Lucifer Yellow
signal, the image acquisition time was typically 10 times smaller than
that used for EGFP, so that the green fluorescence of the cells was not
detected by the camera. In Photoshop, the image with the EGFP signal
was ascribed a red color and the image with the Lucifer Yellow signal a
green color, allowing us to determine the extent of dye diffusion in
EGFP-positive cells. Phase-contrast views of each injected cell
clusters were also taken. Experiments on cells expressing
SrcK
or the chimeric versions of
SrcK
fused with EGFP yielding similar results, and
the data were pooled for quantitative analysis.
Electrical Coupling--
For electrical coupling studies, the
dual whole-cell patch clamp approach was applied on pairs of cells
incubated in the external solution. Both cells of a pair were
voltage-clamped at a common holding potential of 0 mV using the EPC-9
amplifier and a PC-501A amplifier (Warner Instruments, Hamden, CT). To
measure gap junctional currents (Ij), transjunctional potential
differences (Vj) were elicited by changing the
holding potential of one member of a cell pair. Ij was defined
as the current recorded in the cell kept at a 0 mV. Junctional
conductance (gj) was then calculated by
gj = Ij/Vj. Series
resistance was not compensated and was less than 2% of the combined
junctional and cell input resistance. To resolve the activity of single
gap junction channels, gj was pharmacologically
reduced with the gap junction blocker halothane. Digitized current
traces were filtered at 0.1-1.0 kHz for analysis and display of
single-channel activity using customized software (MacDAQ; kindly
provided by A.C.G. van Ginneken, University of Amsterdam, Amsterdam,
The Netherlands). To determine unitary gap junctional conductances
(
j), the amplitudes of single-channel transitions were
measured and divided by the applied Vj. Conductance
values were then converted into step-amplitude histograms with a bin
width of 4 pS. For these experiments, which were carried out under
strong buffering conditions for H+ and Ca2+,
patch electrodes were filled with a solution containing (in mM): 135 CsCl, 0.5 CaCl2, 5.5 EGTA, 3 MgATP,
and 0.1 GTP, buffered to pH 7.2 with 10 mM HEPES-CsOH. CsCl
was chosen to replace KCl in these experiments in order to improve
resolution of single gap junction channels by reducing activity of
non-junctional channels.
Cell Treatment--
Cells under experimental conditions were
incubated for 30 min in external solution supplemented with 100 units/ml TNF-
, 100-500 ng/ml Pseudomonas aeruginosa
lipopolysaccharide (LPS), or 10 nM IL-8. Ten percent fetal
calf serum was added to the external solution for experiments with LPS.
Lysophosphatidic acid (LPA) was used at a concentration of 1 µM and was applied for 5 min. For studies with
mitogen-activated protein kinase (MAPK) and tyrosine kinase inhibitors,
cells were pre-treated for 30-120 min with 20 µM
PD98059, 50 µM PP1, or 200 µM tyrphostin
47. All agents were from Sigma (Sigma) except TNF-
(Bachem AG,
Bubendorf, Germany). All data are shown as mean ± S.E. and
compared using unpaired t tests.
 |
RESULTS |
Effects of TNF-
on Gap Junction Channel Activity in Airway
Cells--
We have reported previously that 100 units/ml TNF-
, a
concentration that maximally stimulates the release of IL-8, decreased gap junction connectivity in non-CF but not CF airway cells within 15 min by a yet undefined mechanism (26). Plausible candidates for
transduction of cell uncoupling could include direct closure of the
channels by cytoplasmic factors like pH or Ca2+ or
decreased expression of Cx43, the gap junction protein that is
expressed in these airway cell lines (26). To address these possibilities, immunolabeling and Western blot analysis were performed to evaluate the expression of Cx43, and the dual patch clamp approach was used to monitor Cx43 channel activity. As shown in Fig.
1a (left panel),
Cx43 was detected in intracellular compartments as well as in cell-cell
contact areas as revealed by indirect immunofluorescence. No change in
the distribution of Cx43 was observed in cells exposed to TNF-
(right panel). Similar observations were made for Cx43 in
non-CF and rescued CF airway cells exposed either to TNF-
or the
proinflammatory endotoxin LPS for 30 min (not shown). The total amount
of Cx43 protein detected by Western blot did not change after exposure
of non-CF cells for 30 min with TNF-
or LPS. In addition, no changes
in the relative isoforms of Cx43 were detected for both mediators (Fig.
1b). Longer exposure (1, 3, 6, and 12 h) of the cells
to TNF-
yielded similar results (not shown).

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Fig. 1.
Localization and expression of Cx43 in
non-CF airway cells exposed to pro-inflammatory mediators.
a, indirect immunofluorescence of Cx43 in non-CF airway
cells. Positive labeling for Cx43 (green signal) was
detected at cell-cell contacts as well as in intracellular compartments
(left panel). Treatment of the cells with TNF- for 30 min
did not affect the basal localization of Cx43 (right panel).
The red fluorescence is from the counter staining with Evans Blue.
Bar, 10 µm. b, Western blot analysis was
performed on cellular lysates using either a Cx43 antibody (top
panel) or a -actin antibody (bottom panel) to
control protein loading. Cx43 was detected in its non-phosphorylated
(NP) and phosphorylated (P1 and P2)
isoforms, which were determined by treating cellular lysates with calf
intestinal phosphatase prior to Western blot analysis (not shown).
Treatment of the cells with TNF- or LPS for 30 min did not affect
basal expression or distribution of the Cx43 isoforms.
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Gap junction channel activity was studied under conditions of strong pH
and Ca2+ buffering. Under these conditions, exposure of
non-CF cells to TNF-
for 30 min decreased (p < 0.001) junctional conductance (gj) from 11.2 ± 3 (mean ± S.E., n = 6) to 2.8 ± 0.7 nS
(n = 8). To investigate the effects of TNF-
on
single gap junction channel activity, large driving forces were applied
to cell pairs in which gj was reduced with
halothane. The single channel conductance (
j) was measured
before and after 30 min of treatment with TNF-
, and frequency
histograms were constructed. As shown in Fig.
2, TNF-
did not change the distribution of
j values measured. These results suggest that one mechanism by which TNF-
modulates gap junction connectivity in non-CF cells is by decreasing the open probability of Cx43 channels,
likely via kinase activation of a signal transduction cascade.

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Fig. 2.
Unitary conductance values of the gap
junction channels expressed in non-CF airway cells. Frequency
distribution of single-channel transitions were measured in cell pairs
under basal conditions (top panel) or treated with TNF-
for 30 min (bottom panel). The distribution of unitary
conductance values did not differ under both conditions. Most
single-channel events occur at about 30 and 55 pS, which is typical for
Cx43. n indicates the number of transitions measured.
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Role of c-Src in Mediating TNF-
-induced Airway Cell
Uncoupling--
To identify other potent pro-inflammatory mediators on
gap junction connectivity, we have studied the effects of LPS, LPA, and
IL-8 on dye coupling of non-CF and CF cells. As shown in Table I, LPS and LPA markedly decreased dye
coupling within minutes in non-CF cells, whereas IL-8 had no effect.
LPS and LPA did not change the strength of gap junctional communication
in CF cells (Table I).
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Table I
Effects of proinflammatory mediators and phospholipids on dye coupling
of non-CF and CF airway epithelial cells
Values indicate the number of cells labeled with Lucifer Yellow. In
non-CF cells, TNF- , LPS, and LPA decreased (p < 0.01) the extent of dye coupling; IL-8 had no effects. LPA had
transient effect on dye coupling; cell uncoupling was observed after 5 min of treatment up to 15-20 min, likely because of desensitization of
G protein-coupled receptors (Postma et al. (46)). TNF- ,
LPS, or LPA did not affect the extensive dye coupling in CF cells. ND,
not determined.
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LPA has emerged as a ligand for G-coupled membrane receptors on various
cell types. MAPK and the tyrosine kinase c-Src have been proposed to
mediate the closure of Cx43 channels by LPA (44, 45). To investigate
whether these signaling pathways are involved in TNF-
-induced dye
uncoupling of non-CF cells, pharmacological inhibitors of MAPK
(PD98059) and of tyrosine kinases (PP1 and tyrphostin 47) were used. As
shown in Fig. 3, pretreatment of non-CF
cells for 30-120 min with MAPK or tyrosine kinase inhibitors did not
affect their normal extent of intercellular communication. However, the
uncoupling effect of TNF-
was abrogated in the presence of PP1 or
tyrphostin 47 but not PD98059 (Fig. 3). Similarly, the extent of dye
coupling reached by LPS in the presence of PP1 (5.1 ± 0.8 cells, n = 7) was not different from that observed under control conditions (Table I). Pervanadate, an inhibitor of
tyrosine phosphatases, is known to inhibit gap junctional communication by increased tyrosine phosphorylation activity (44-46). Indeed, treatment with pervanadate for 30 min markedly decreased dye coupling in both non-CF (2.7 ± 0.5 cells, n = 7) and
CF cells (1.5 ± 0.3 cells, n = 6).

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Fig. 3.
Quantitative evaluation of dye coupling in
non-CF airway cells exposed to inhibitors of MAPK and tyrosine
kinases. Pre-treatment of the cells with PD98059, PP1, or
tyrphostin 47 (Tyr47) did not change the basal extent of
intercellular communication, as revealed by intracellular
microinjection of Lucifer Yellow (LY). Exposure of the cells
for 30 min to TNF- decreased the extent of dye coupling. Although
inhibitors of p42-MAPK (PD98059) had no effect, tyrosine kinase
inhibitors abrogated TNF- -induced cell uncoupling.
Numbers indicate the dye injections performed.
Asterisks indicate significance at p < 0.01.
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To examine whether c-Src is involved in the regulation of gap junction
channel connectivity by TNF-
, non-CF cells were transiently transfected with a plasmid encoding a dominant negative
(SrcK
) version of the tyrosine kinase. Under these
conditions, the uncoupling effect of TNF-
and LPS was fully
prevented (Fig. 4). Conversely, the
expression of an active mutant of c-Src (SrcK+) abolished
gap junctional communication in CF cells. Under these conditions, the extent of dye coupling was reduced to 2.5 ± 0.3 cells (n = 14). These results suggest that tyrosine
kinase activity is stimulated by proinflammatory mediators and mediates gap junction closure in non-CF airway cells. To address further the
possible regulation of Cx43 channel activity by activated c-Src, non-CF
airway cells were transfected with a cDNA encoding for a mutant of
Cx43 in which the tyrosines at positions 247 and 265 have been replaced
with alanines (pIRES2-Y247A,Y265A-Cx43-EGFP), as these have been shown
previously (43, 47, 48) to be phosphorylation sites for c-Src.
Expression of Y247A,Y265A-Cx43, as visualized by the red color ascribed
to EGFP fluorescent cells in Fig.
5a, prevented the uncoupling
effect of TNF-
in EGFP-positive cells (6.9 ± 0.7 cells,
n = 20) but not in neighboring non-transfected cells
(2 ± 0.3 cells, n = 17). In control experiments
using pIRES2-WT-Cx43-EGFP, enhancing the expression of WT-Cx43
increased the average extent of dye coupling (9.1 ± 1.1 cells, n = 24) but did not alter (p < 0.01) the uncoupling effect of pro-inflammatory mediators (3.6 ± 0.6 cells, n = 26). Thus, the data
suggest that the double Y247A,Y265A mutation did interfere with the
effect of TNF-
on gap junctional communication in non-CF airway
cells, suggesting further that Cx43 is a target of the c-Src tyrosine kinase.

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Fig. 4.
Dye coupling in non-CF cells transfected with
a dominant negative version of c-Src. Under control conditions
(Control), TNF- and LPS markedly reduced basal dye
coupling in non-CF cells. In contrast, transient expression of a
dominant negative form of c-Src (SrcK )
prevented the uncoupling effect by both proinflammatory mediators.
Numbers indicate the dye injections performed.
Asterisks indicate significance at p < 0.01. The expression of SrcK was confirmed by
Western blot using an antibody directed against v-Src. As shown in the
inset, increased expression of the tyrosine kinase was
detected in cells transfected with SrcK
cDNA.
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Fig. 5.
Dye coupling in non-CF cells expressing a
mutant of Cx43 lacking Tyr-247 and Tyr-265 (Y247A,Y265A-Cx43) or
wild-type Cx43 (WT-Cx43). The expression vector pIRES2-EGFP was
used to visualize transfected cells by the fluorescence of EGFP. Small
EGFP-fluorescent clusters appear in red for cells expressing
Y247A,Y265A-Cx43 (a) or WT-Cx43 (d).
EGFP-positive clusters were subjected for dye coupling in the presence
or absence of pro-inflammatory mediators. In the presence of TNF- ,
Lucifer Yellow diffused from the injected cells to several neighboring
cells expressing Y247A,Y265A-Cx43; in contrast, microinjection of
Lucifer Yellow in an EGFP-negative cell (arrow in
a-c) showed no dye diffusion (b).
c, phase-contrast view of the injected field. Extensive
diffusion of Lucifer Yellow was also detected in EGFP-positive cells
expressing WT-Cx43 (e). In the presence of TNF- , however,
the diffusion of Lucifer Yellow was strongly reduced; red
and green images were merged to visualize Lucifer Yellow
labeling in EGFP-positive cells expressing WT-Cx43 (f).
Bar, 10 µm.
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c-Src Activity in Non-CF and CF Airway Cells--
The expression
of c-Src in non-CF airway cells was confirmed by Western blot
using antibodies against v-Src (Fig.
6a). To ensure that TNF-
activates c-Src, Western blots were performed using specific antibodies
against the phosphorylated Tyr-418, which is indicative of
tyrosine kinase activation (42, 49). As shown in Fig. 6a,
endogenous c-Src activity could be detected in these cells under
control conditions. The proportion of activated c-Src increased with
time of treatment with TNF-
. Quantitative analysis revealed that
TNF-
increased (p < 0.05) c-Src activity by 52.4 ± 6.7% (n = 4) after 10 min of treatment
(Fig. 6b). The increased activity of c-Src was transient and
reached basal levels within 30 min of treatment with TNF-
(not
shown).

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Fig. 6.
Expression and activity of c-Src in non-CF
airway cells exposed to TNF- . Western blot analysis
was performed on cellular lysates using either a v-Src antibody
(a, top panel) or an antibody directed against
the activated c-Src (a, bottom panel). Although
the expression of c-Src remained stable during TNF- treatment, the
amount of activated c-Src increased with time as compared with basal
conditions. Quantitation of the effect of TNF- was measured by
normalizing the activated c-Src signal with that of v-Src
(b). TNF- increased (p < 0.05) the
amount of activated c-Src by 52.4 ± 6.7% during 10 min of
treatment. Activated c-Src decreased with longer incubation times,
reaching basal levels within 30 min. The number of measurements, which
were performed in at least 4 independent experiments, is
indicated.
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The activity of c-Src was also found to be strongly stimulated by
refreshing non-CF cells with culture medium after 48 h of serum
starvation (Fig. 7, a and
c). By using similar experimental approaches, however, no
changes in c-Src activation could be detected in CF cells. This was
observed for both CF15 (Fig. 7, a and
c) and IB3-1 (Fig. 7, b and c) cells.
Interestingly, increased c-Src activity was detected in the
CFTR-corrected CF cell line C38, suggesting that expression of CFTR
rescued the activation of the tyrosine kinase signal transduction
cascade (Fig. 7, b and c). Additional experiments
using inhibitors of CFTR Cl
channels (200 µM diphenylamine carboxylic acid) or scavengers of
extracellular ATP (0.5 units/ml hexokinase) failed to affect the
activation of c-Src in non-CF cells (not shown). The expression of the
total c-Src protein remained stable during the stimulation of each cell
line, as revealed by Western blots using antibodies against v-Src (Fig.
7). These results indicate that activation of c-Src is defective in CF
airway epithelial cells.

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Fig. 7.
Comparison of the expression and activity of
c-Src in non-CF and CF cells. Western blot analysis was performed
on cellular lysates using a v-Src antibody (top panels) or
an antibody directed against the activated c-Src (bottom
panels). a, no marked difference in c-Src
expression was observed between Beas2B (non-CF) and CF15
cells (CF). Under basal conditions (B),
endogenously activated c-Src was detected in Beas2B cells. The amount
of activated c-Src markedly increased by refreshing the medium of
serum-starved cells for 48 h with complete culture medium
(S). The latter effect appeared specific because it could be
prevented in the presence of a tyrosine kinase inhibitor
(SI). In contrast, activated c-Src was not induced by this
procedure in CF15 cells. b, the
CFTR-corrected C38 cells showed higher amount of c-Src than their
parental IB3-1 CF cells. Endogenously activated c-Src was detected in
both cell lines (B). Although refreshing the medium markedly
increased the amount of activated c-Src in C38 cells (S), no
change was detected in IB3-1 cells subjected to the same procedure.
Quantitation of these effects was measured by normalizing the activated
c-Src signal with that of v-Src (c). The number of
measurements, which were performed in at least 3 independent
experiments, is indicated.
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|
 |
DISCUSSION |
Our results describe one possible mechanism that regulates gap
junction connectivity in non-CF and CF airway cell lines exposed to the
proinflammatory mediator TNF-
. We identified the c-Src tyrosine
kinase as a link between the mediators of inflammation and
Cx43-composed gap junction channels. Importantly, we provide evidence
that activation of c-Src is defective in CF cells.
It is well established that the products of the Rous sarcoma virus
oncogene pp60v-src or of the proto-oncogene
c-src can abolish gap junctional communication of
Cx43-expressing cells (44, 45, 50). There is increasing evidence that
v-Src or c-Src tyrosine kinases physically associate with Cx43 (43, 47,
51, 52), the SH3 and SH2 domains of Src being necessary for binding and
phosphorylation of Cx43. Thus, mutations introduced in the proline-rich
region (which binds to the SH3 region of Src) or on Tyr-265
(which binds to the SH2 region of Src) of Cx43 COOH terminus markedly
reduced the interaction between v-Src or c-Src and Cx43 in
vitro and in vivo (43, 51). Active Src phosphorylates
tyrosine residues in target proteins. Accordingly, mutation of Tyr-265
and Tyr-247 completely prevented Cx43 channel closure by Src,
suggesting that phosphorylation of both tyrosine residues is involved
in the disruption of gap junction connectivity (43, 47, 48). Recently,
a correlation between increased phosphotyrosine content of Cx43 and
c-Src activity was observed in the heart of Syrian BIO 14.6 hamsters,
which systematically develop heart dysfunction at a late stage in their
life (53).
Growth factors, tumor promoters, and mediators of inflammation have
been shown to inhibit gap junctional communication in various cell
types expressing Cx43 (54). Transduction mechanisms of cell uncoupling
have been difficult to identify because of the multiple intracellular
signaling pathways that are activated by these agents (44). In non-CF
airway cells, the uncoupling effect of TNF-
occurs within 15 min by
a mechanism that does not involve cytoplasmic factors like pH or
Ca2+ or decreased expression of Cx43. We present several
lines of evidence that indicate that TNF-
, as well as LPS, close
Cx43 channels in non-CF airway cells by activation of Src-like tyrosine kinases. First, the uncoupling effects of TNF-
and LPS were both abrogated by broad spectrum and Src family inhibitors of tyrosine kinases. Second, a dominant negative version of c-Src prevented the
uncoupling effects of TNF-
and LPS. Third, the uncoupling effect of
TNF-
was prevented in cells expressing a mutant of Cx43 lacking
Tyr-247 and Tyr-265. Finally, LPA, a lipid messenger that closes Cx43
gap junction channels by activation of c-Src tyrosine kinase (43, 46),
also abrogated intercellular communication in non-CF airway cells.
However, we could not detect Cx43 tyrosine phosphorylation in non-CF
cells where Cx43 and c-Src are endogenously expressed. The
stoichiometry of tyrosine phosphorylation on the 12 connexins that
constitute a gap junction channel to induce its closure is unknown.
Because most Cx43 detected by Western blots are not at the plasma
membrane, tyrosine phosphorylation of functional gap junction channels
may not be detectable biochemically in our cell model. Consistent with
previous observations (48, 55, 56), we did not detect changes in the
distribution of Cx43 isoforms and Cx43 single-channel unitary
conductances, suggesting that activation of c-Src alters the gating
properties of Cx43 channels in non-CF airway cells exposed to TNF-
.
In agreement with these results, TNF-
was found to activate c-Src in
non-CF cells with a time course that parallels the closure of gap
junction channels. Thus, activated TNF receptors may recruit adaptor
proteins that in turn bind and activate additional key pathway-specific tyrosine and serine/threonine kinases, including c-Src and MAPK (57-59). Similarly, c-Src has been involved in linking LPA and LPS
membrane receptors to MAPK signaling pathways (60-62).
TNF-
, LPS, and LPA did not affect the strength of intercellular
communication in the CF airway cell lines used in this study. It is
known that all these cell lines, irrespective of their CF or non-CF
origin, express Cx43 (26). The possibility that the association of
c-Src with Cx43 is altered in CF airway cells is unlikely. Indeed,
disruption of gap junction connectivity could be achieved in CF cells
treated with pervanadate, an inhibitor of tyrosine phosphatases, or
transiently transfected with a plasmid encoding for a constitutively
active version of c-Src. In contrast, we report that activation of
c-Src is defective in CF airway cells but can be rescued in
CFTR-corrected CF cells. Little is known about the involvement of c-Src
tyrosine kinase in CF pathogenesis. The stimulation of production of
mucin by TNF-
and growth factors has been shown to be prevented in
part by tyrosine kinase inhibitors (63, 64). Importantly for CF, the
induction of mucin production by normal airway epithelial cells
infected with P. aeruginosa involves an
Src-dependent Ras-MAPK-pp90rsk signaling
pathway that activates nuclear factor-
B (61). The Src-dependent Ras-MAPK-pp90rsk pathway is
the only downstream cascade known to activate mucin gene transcription
to date (65). Recently, increased expression of c-Src mRNA was
detected by differential display in the tracheobronchial epithelial
CFDE cells as compared with the same cells transfected with CFTR
(66). This increased expression was confirmed at the protein level and
was shown to be associated with increased endogenous tyrosine kinase
activity and mucin overproduction. An inverse relationship between the
presence of functional CFTR and c-Src expression was not observed in
our study. Instead, the expression level of c-Src appeared variable and
dependent on the cell line. CFDE is a non-
F508 CF cell line,
which was derived from a CF patient of unknown genotype. Possibly,
different genotypes may have diverse impacts on c-Src expression. On
the other hand, c-Src tyrosine kinase activity is extremely sensitive
to the extracellular environment and stimuli. In this context, we
observed that rapid activation of c-Src was altered in CF15
and IB3-1 cells, which express CFTR with class II mutations, whereas it
could be readily detected in non-CF cells and in CF cells corrected
with CFTR. Of note, invasion of human epithelial cells by P. aeruginosa has been shown to involve Src-like tyrosine kinases
(67). Possibly, the defective activation of c-Src may prevent the
internalization and thereby the clearance of P. aeruginosa
by CF airway cells, contributing to the recurrent airway infection (5).
Taken together, our results point to the existence of a link between
CFTR and c-Src-dependent signaling pathways. Furthermore,
activation of c-Src appears as a central element in the signaling
pathway connecting CFTR with gap junction channels in airway epithelial cells.
So far, the mechanisms that link CFTR to c-Src signaling are not known.
Blocking the Cl
activity of CFTR with
diphenylamine carboxylic acid or interfering with the
possible release of ATP by the addition of extracellular hexokinase did
not affect the activation of c-Src in non-CF airway cells. Recent data
demonstrated that the last amino acids at the COOH-terminal end of CFTR
form a PSD95/Dlg/ZO-1 protein (PDZ)-binding domain. PDZ-binding domains
are involved in the clustering of transmembrane ion channels and in
connecting intracellular signaling pathways. Interestingly, CFTR binds
to the PDZ1 domain of the ezrin-binding phosphoprotein 50 (EBP50) (38,
68). Thus, EBP50 and other scaffolding proteins have not only the
ability to target CFTR to membrane compartments via ezrin and the actin
cytoskeleton but may also mediate the regulation by CFTR of other ion
channels via protein-protein or signaling interactions (34, 38, 69, 70). Indeed, EBP50 has been involved in the interaction with tyrosine
kinases of the Src family, which are known to regulate ion channel
activity, such as the amiloride-sensitive Na+ channels. It
is tempting, therefore, to hypothesize that complexes containing CFTR
and c-Src tyrosine kinase may transduce signals from membrane receptors
to ion channels, including gap junction channels. In CF epithelial
cells, the absence of CFTR from the plasma membrane may compromise the
regulation and/or the stability of these complexes, resulting in
altered regulation of ion channels and gap junction connectivity.
The tyrosine kinase c-Src plays a central role in linking membrane
receptors to various signaling pathways. NF-
B signaling is an
essential component of inflammation by regulating the transcriptional activity of inflammatory and mucin genes. Thus, defects in upstream transduction signals that lead to nuclear factor-
B translocation may
contribute to the exaggerated inflammatory response that is characteristic of the CF phenotype. In addition, defects in
c-Src-dependent tyrosine phosphorylation of protein
substrates may have yet unsuspected consequences on the response of
airway epithelial cells to the environment. Interestingly, gap
junctions have been involved in most of the functions fulfilled by
c-Src, including membrane trafficking, cell proliferation, cell
adhesion, cell migration, and cell differentiation (28-31, 42). Future
studies will be necessary to unravel the contribution of gap junctional
communication in the CF pathogenesis.