Functional KCa3.1 Channels Regulate
Steroid Insensitivity in Bronchial Smooth
Muscle Cells
This information is current as
of June 18, 2017.
Latifa Chachi, Aarti Shikotra, S. Mark Duffy, Omar Tliba,
Christopher Brightling, Peter Bradding and Yassine Amrani
J Immunol 2013; 191:2624-2636; Prepublished online 31
July 2013;
doi: 10.4049/jimmunol.1300104
http://www.jimmunol.org/content/191/5/2624
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References
The Journal of Immunology
Functional KCa3.1 Channels Regulate Steroid Insensitivity in
Bronchial Smooth Muscle Cells
Latifa Chachi,* Aarti Shikotra,* S. Mark Duffy,* Omar Tliba,† Christopher Brightling,*
Peter Bradding,*,1 and Yassine Amrani*,1
A
lthough many patients with asthma are well controlled
with inhaled glucocorticoid (GC) therapy, ∼ 5–10% of
patients have difficult-to-control or severe disease that
is poorly responsive to this treatment. These patients account for
a disproportionate share of asthma-related health care costs and
morbidity (1). This situation represents a significant unmet clinical
need, and novel therapies are urgently required (1).
Despite significant progress in the field, the precise molecular
mechanisms mediating GC insensitivity in severe asthma are poorly
understood. Several potential mechanisms have been postulated,
*Department of Infection, Immunity and Inflammation, University of Leicester,
Leicester LE1 7RH, United Kingdom; and †Department of Pharmaceutical Sciences,
School of Pharmacy, Thomas Jefferson University, Philadelphia, PA 19107
1
P.B. and Y.A. contributed equally to this work.
Received for publication January 14, 2013. Accepted for publication June 24, 2013.
This work was supported by laboratories partially funded by European Regional
Development Fund Grant 05567, by the National Institute for Health Research
Leicester Respiratory Biomedical Research Unit, by the Department of Health, by
National Institutes of Health Grant R01 HL111541 (to O.T.) and, in part, by a Wellcome Senior Clinical Fellowship (to C.B.).
The views expressed are those of the author(s) and not necessarily those of the
National Health Service, the Department of Health, and the National Institute for
Health Research.
Address correspondence and reprint requests to Dr. Yassine Amrani at the current
address: Institute for Lung Health, Department of Respiratory Medicine, Clinical
Sciences Building, Glenfield Hospital, Groby Road, Leicester LE3 9QP, U.K.
E-mail address: [email protected]
Abbreviations used in this article: ADAM, a disintegrin and metalloprotease domain;
ASM, airway smooth muscle; COPD, chronic obstructive pulmonary disease; CX3CL,
chemokine (C-X3-C motif) ligand; CXCL10, chemokine (C-X-C motif) ligand 10; GC,
glucocorticoid; GILZ, glucocorticoid-induced leucine zipper; GR, glucocorticoid receptor; KCa, Ca2+-activated K+; PP5, protein phosphatase 5; shRNA, small hairpin RNA.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300104
identified mostly from studies in immune cells, including alveolar
macrophages and circulating PBMCs obtained from patients with
GC insensitivity (2–6). In addition to infiltrating inflammatory
cells, the airway smooth muscle (ASM) is another key player in
severe asthma pathophysiology, demonstrating heightened sensitivity to both direct and indirect contractile stimuli leading to
exaggerated airway narrowing and airflow obstruction (7). The
therapeutic benefit in those with severe asthma that is provided
by bronchial thermoplasty, a therapy that attenuates bronchoconstriction via reduction of ASM mass, has strongly supported the
idea that ASM plays a central part in the pathogenesis of the severe
disease (8–11). In addition to its central role in bronchoconstriction,
the role of ASM in the pathogenesis of severe asthma could derive
also from its immunomodulatory function, as it secretes a variety
of proinflammatory mediators (12). This characteristic is evident
both in vitro using cultured ASM cells and in vivo in the ASM
bundles of asthmatic patients (12).
Whether the pathogenesis of severe asthma is driven by the
steroid-resistant production of proteins from ASM cells represents
an interesting hypothesis. Indeed, previous reports convincingly
showed that despite high doses of inhaled or oral GC taken by
patients, there is ongoing expression of different “proasthmatic”
proteins in asthmatic ASM bundles, including the following chemokines: chemokine (C-X3-C motif) ligand (CX3CL) 1 (13), CCL11
(14), CCL15 (15), and CCL19 (16), and a disintegrin and metalloprotease domain (ADAM) 33 and ADAM8 (17, 18). These studies
provide indisputable in vivo evidence for the existence of steroidresistant pathways in ASM that are potentially driving inflammatory processes and ASM contractile dysfunction in asthmatic
airways. A better understanding of the underlying mechanisms
driving these steroid-resistant pathways in ASM could therefore
lead to novel therapeutic approaches.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma we see in vivo evidence of GC-resistant pathways in airway smooth
muscle (ASM) bundles that can be modeled in vitro by exposing cultured ASM cells to TNF-a/IFN-g. This action drives GC
insensitivity via protein phosphatase 5–dependent impairment of GC receptor phosphorylation. In this study, we investigated
whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and
nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and
immunofluorescence staining confirmed the nuclear localization of KCa3.1 channels in ASM cells. The functional importance of
KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNF-a/IFN-g was assessed using complementary
inhibitory strategies, including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific small hairpin RNA delivered by
adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5, and CCL11 gene and
protein expression. KCa3.1 channel blockade also restored fluticasone-induced GC receptor-a phosphorylation at Ser211 and
transactivation properties via the suppression of cytokine-induced protein phosphatase 5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary, KCa3.1 channels contribute to the
regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in
severe asthma. The Journal of Immunology, 2013, 191: 2624–2636.
The Journal of Immunology
Materials and Methods
Human subjects
Asthmatic subjects, patients with chronic obstructive pulmonary disease
(COPD), and healthy volunteers were recruited. Asthmatic and COPD
subjects had a consistent history and objective evidence of asthma or COPD,
as described previously (32). Asthma severity was defined by British
Guideline on the Management of Asthma treatment steps: mild = step 1,
b2-agonist only; moderate = steps 2 and 3, inhaled corticosteroid # 800 mg
beclomethasone equivalent per day 6 long-acting b2-agonist; severe =
steps 4 and 5 (33). Five of 7 patients at step 4/5 undergoing immunohistological staining met the American Thoracic Society criteria for refractory
asthma (1). All COPD patients used for the study were classified as GOLD
(Global Initiative for Chronic Obstructive Lung Disease) I and II. The
studies were approved by the Leicestershire, Northamptonshire, and Rutland Research Ethics Committee (references: 4977, 04/Q2502/74 and 08/
H0406/189). Written informed consent was gained from all participants
prior to their involvement. Tables I and II show the demographics of the
patients used in the immunohistochemistry studies and molecular/cellular
studies, respectively.
Fiberoptic bronchoscopy
Subjects underwent fiberoptic bronchoscopy, as described previously (32)
and according to British Thoracic Society guidelines (34). Bronchial
mucosal biopsy specimens were taken from the right middle lobe and
lower lobe carinae, fixed in acetone, and embedded in glycol methacrylate
(35). Additional biopsy specimens were dissected for the isolation and
culture of ASM (36).
KCa3.1 staining in endobronchial biopsies
Sequential 2-mm sections were cut from glycol methacrylate–embedded
bronchial biopsies and immunostained as described previously (35), using
a rabbit polyclonal anti-human KCa3.1 Ab (M20, 2.7 mg/ml; gift from
Dr. Chen, GlaxoSmithKline, Stevenage, U.K.; Ref. 37) and isotype control
rabbit IgG (2.7 mg/ml; R&D Systems, Minneapolis, MN). Cells staining
positively for KCa3.1 in the ASM compartment were quantified using a
semiquantitative intensity score: 0 = no staining, 0.5 = sparse and patchy,
1.0 = diffuse but weak, 2.0 = diffuse. The percentage of nuclei that were
stained positive within the ASM cells was also calculated. All sections
were counted by a blinded observer.
Culture of ASM cells
Primary human ASM cells were obtained from healthy subjects and isolated
from endobronchial biopsy specimens from COPD and asthmatic patients,
as previously described (36). Briefly, pure ASM bundles in airways were
dissected free of surrounding tissue. The small muscle bundles were cultured in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 100 U/
ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin.
ASM characteristics were determined by immunofluorescence and light
microscopy, with a-smooth muscle actin–FITC direct conjugate and myosin indirectly conjugated with FITC (Sigma-Aldrich, Dorset, U.K.). Cells
were used from passages 2–6.
Treatment of ASM cells
Cells were pretreated with the specific KCa3.1 channel blockers TRAM-34
(200 nM; a gift from Heike Wulff, University of California–Davis, Davis,
CA) (38) and ICA-17043 (60 nM; a gift from Icagen, Durham, NC) (39),
alone or in combination with fluticasone (100 nM), for 2 h and then
stimulated with TNF-a (10 ng/ml)/IFN-g (500 IU/ml) for an additional 24
h. Cell viability was assessed in cells from two asthmatic individuals (each
performed in triplicate), using the MTT assay, as described in our previous
report (40).
ELISA
ELISAs for assessing chemokine levels were performed on 100-ml cell
supernatants using the R&D Systems DuoSet kits according to the manufacturer’s instructions (41). DMSO controls were performed, with the
final concentration of DMSO 0.1% in all control and drug wells.
Quantitative PCR
Total RNA was extracted using the RNeasy Plus Mini Kit (#74134;
QIAGEN). The cDNAs were synthesized using a First Strand cDNA
Synthesis Kit (Fermentas Life Sciences). Real-time quantitative PCR was
performed using Brilliant SYBR Green QPCR Master Mix (Agilent), using
a Stratagene Mx3000P (Santa Clara, CA). Each sample from different
patients was assayed in duplicate. For relative quantification, the CT
method, ratio = 2 2 [(Ct (gene of interest) 2 Ct (b actin as internal control)] (22DDCt), was used as described in Ref. 42 to determine the fold
change caused by the different treatment conditions (fluticasone alone,
KCa3.1 blockers alone, and both drugs in combination). The primers were
as follows: b-actin forward: 59-CCCAAGGCCAACCGCGAGAAGAT-3
and reverse: 59-GTCCCGGCCAGCCAGGTCCAG-39; CX3CL1 forward:
59-GCTGAGGAACCCATCCAT-39 and reverse: 59-GAGGCTCTGGTAGGTGAACA-39; CCL5 forward: 59-TCTGCGCTCCTGCATCTG-39 and
reverse: 59-GGGCAATGTAGGCAAAGCA-39; CCL11 forward: 59-AAT
GTC CCC AGA AAG CTG TG, CCL11-39 and reverse: 59-TCC TGC
ACC CAC TTC TTC TT-39; and glucocorticoid-induced leucine zipper
(GILZ), forward: 59-TCTGCTTGGAGGGGATGTGG-39 and reverse:
59-ACTTGTGGGGATTCGGGAGC-39.
Immunofluorescence
ASM cells grown on the chamber slides were fixed with 10% neutral
buffered formalin for 15 min at room temperature, washed three times with
PBS, permeabilized using 90% ice-cold methanol for 20 min, and washed
again three times with PBS. The cells were then incubated with 3% BSA/
PBS for 1 h before anti-KCa3.1 (2.5 mg/ml; Biorbyt, Cambridge, U.K.),
anti-HDAC1 (1 mg/ml; Cell Signaling Technology, Danvers, MA), or appropriate isotype-matched control Abs (all prepared in 1% BSA/PBS) were
added overnight at 4˚C. After three washes, Alexa Fluor 488 goat antirabbit IgG Ab (Invitrogen, Paisley, U.K.) was added at 1:300 dilution for
40 min, then washed with PBS and mounted using VectaMount (Vector
Laboratories, Peterborough, U.K.). Images of the cells were viewed on
a confocal microscope (Leica TCS SP5 confocal) and captured using Leica
LAS AF software (Leica, Milton Keynes, U.K.).
Western blot
Immunoblots on total cell extracts from ASM cells were performed as
described previously (43), using anti–GR-S211 and anti–glucocorticoid
receptor (GR) Abs (Cell Signaling Technology). To ensure equal loading,
the membranes were stripped and reprobed with anti–b-actin Ab (Santa
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The intermediate-conductance Ca2+-activated K+ (KCa) channel
KCa3.1 channel (also known as IK1, SK4, or KCNN4) is closely
associated with the progression of number of human diseases, and
is expressed by human ASM cells (19, 20). In human ASM cells,
a KCa3.1 blocker attenuated mitogen-induced ASM cell proliferation (20). Inhibitors of KCa3.1 channels, such as TRAM-34,
are effective in treating various inflammatory diseases in preclinical models, including atherosclerosis (21), neurodegenerative
disorders (22), autoimmune encephalomyelitis (23), and coronary
vasculoproliferative diseases (24). With respect to asthma, we recently provided the first evidence, to our knowledge, that TRAM34 prevents the development of eosinophilic inflammation, airway hyperresponsiveness, and airway remodeling in a murine
model of allergic asthma (25). The underlying mechanisms by
which KCa3.1 channels contribute to the pathogenesis of allergic
asthma are yet to be defined, but we have shown that KCa3.1
channels regulate mast cell degranulation and migration (26–29),
as well as fibrocyte migration (30). Others have implicated
KCa3.1 channels in the migration of lung dendritic cells to CCL19
and CCL21 (31). These observations demonstrate that activation
of KCa3.1 channels on structural (smooth muscle/fibroblasts) airway cells may represent an important pathway driving key features of allergic asthma.
In the current study, we uncovered the surprising finding that
KCa3.1 channels are not only essential in driving the production of
GC-resistant chemokines by ASM cells but also contribute to the
reduced ASM sensitivity to GC therapy via the upregulation of
serine/threonine phosphatase protein phosphatase 5 (PP5). To our
knowledge, this is the first report to demonstrate a functional interaction between KCa3.1 channels and the impairment of GC
function. This study uncovers a novel molecular mechanism contributing to the development of GC insensitivity, a defining feature
of severe asthma.
2625
2626
KCa3.1 CHANNEL AND STEROID RESISTANCE
Cruz Biotechnology, Santa Cruz, CA). Immunoblots on nuclear fractions
used samples prepared using an extraction reagent kit (NE-PER) (Thermo
Fisher Scientific). A total of 30 mg nuclear extracts was analyzed by 10%
SDS-PAGE and assayed by immunoblot using anti-KCa3.1 Ab from
Sigma-Aldrich (1:500) and anti–b-actin (1:200 dilution). The anti-KCa3.1
Ab from Biorbyt (1:500 dilution) was used for assessing KCa3.1 expression
in total cell lysates (60 mg) in the silencing studies. Anti-mouse IgG-HRP
or anti-rabbit IgG-HRP (1:5000 dilution Santa Cruz Biotechnology) was
used as a secondary Ab. The bands were visualized by the ECL system
(Amersham Biosciences) and autoradiographed.
Small hairpin RNA gene knockdown of KCa3.1 channels
Flow cytometry
ASM cells were fixed and permeabilized in 4% paraformaldehyde/0.1%
saponin for 15 min on ice. Cells were then stained with either 2 mg/ml
rabbit anti-human PP5 Ab (Cell Signaling Technology) or isotype-matched
control (rabbit IgG; IMMUNOSTEP, Salamanca Spain) overnight, followed by secondary sheep anti-rabbit IgG:RPE Ab (Cell Signaling Technology) for 2 h. Staining was examined by flow cytometry using the
Becton Dickinson FACScan (Oxford, U.K.).
Patch-clamp electrophysiology
The whole-cell variant of the patch-clamp technique was used as previously
described (20). Briefly, ASM cells were cultured until confluent and then
maintained in insulin–transferrin–sodium selenite media as normal. Cells
were incubated overnight with TNF-a and IFN-g for 24 h, with control
cells being maintained under identical conditions without added cytokines.
Prior to electrophysiological recording, the cells were trypsinized and
resuspended in ASM culture media. Cells were pipetted into a heated (30˚C)
recording chamber ready for patching. Individual (nonadhered) cells were
voltage clamped using the whole-cell variant of the patch-clamp technique.
Statistical analysis
For cell culture studies across groups, comparisons were made using oneway ANOVA or repeated-measures ANOVA, as appropriate, with the Bonferroni post hoc test for comparisons between specific groups. Immunohistochemical staining was analyzed across groups using the Kruskal–Wallis test.
A p value ,0.05 was taken as statistically significant.
Results
Expression of KCa3.1 in ASM bundles
The clinical characteristics of the subjects assessed by immunohistochemistry are described in Table I. There was no immunostaining in isotype control sections (Fig. 1A), but KCa3.1 immunoreactivity was positive in ASM bundles (Fig. 1B–D), irrespective
of disease status (Fig. 1E). The overall intensity of staining of
KCa3.1 in the ASM was not different between healthy subjects
(Fig. 1B) and asthmatic patients with moderate (Fig. 1C) or severe
disease (Fig. 1D) (quantification not shown). Of interest, not only
was KCa3.1 localized to the plasma membrane and cytoplasm
(consistent with channel trafficking), but it was also localized to
the nuclear membrane (Fig. 1E). The immunostaining taken at a
higher magnification clearly shows a marked expression of KCa3.1
channel in the nucleus of three different cells in one asthmatic
patient (31000). The proportion of ASM cells expressing the
KCa3.1 channel in the nucleus did not differ between normal and
asthmatic conditions (Fig. 1F).
Nuclear expression of KCa3.1 in cultured ASM cells
The demographics of patients used for the immunostaining, chemokine expression assays, and immunoblot are shown in Table II.
To further investigate whether KCa3.1 channel was also present in
the nuclear compartment in cultured ASM cells, we conducted
immunofluorescence staining in permeabilized cells derived from
Table I. Demographics of patients used in the immunohistochemistry studies
Healthy Control
Subjects
Number
7
Age (mean 6 SEM)
35.71 6 6.98
Sex (M/F)
2/5
Asthma duration (mean 6 SEM)
N/A
N/A
Inhaled corticosteroid dose (beclomethasone
equivalentsb) (mg)
Number at BTS step 5
N/A
Number on long-acting b-agonist
N/A
Exacerbations in last year (median [range])
N/A
Mean daytime symptom score (median [range])
N/A
Mean daily night time symptom score (median [range])
N/A
Reliever use/week (median [range])
N/A
Sputum eosinophil count (%) (geometric mean [95% CI])
0.34 [0.20–0.56]
PEF amplitude % of the mean (mean 6 SEM)
N/A
FEV1 (% predicted)
102.4 6 3.45
FEV1/FVC (%)
84.0 6 3.90
.16
PC20 methacholine (mg/ml)(geometric mean [95% CI])
Serum IgE (kU/L) (geometric mean [95% CI]
31.59 [12.57–79.4]
Number with positive skin prick test
5
Number with positive skin prick test to Aspergillus fumigatus
0
a
Patients with Mild to Moderate
Asthma (BTS steps 1–3)
Patients with Severe
Asthma (BTS
steps 4 and 5)
5
36 6 7.86
2/3
17 6 5.38
512.5 6 234.9
7
42.29 6 4.18
3/4
33.86 6 5.63
1400 6 130.9
—
0.590
0.843
0.064
0.006
0
2
0 [0–1]
0.25 [0.18–0.54]
0 [0.0–0.0]
1.5 [0.0–5.25]
8.86 [2.08–37.75]
20.08 6 6.43
91.80 6 7.37
76.40 6 5.17
0.6841 [0.02–30.49]
501.5 [43.07–5840]
4
0
4
7
1 [0.0–3.0]
0.93 [0.14–1.39]
0.28 [0.0–1.07]
22.0 [6.0–37.0]
2.07 [0.43–9.98]
34.50 6 9.45
66.57 6 6.71
59.57 6 6.47
0.11 [0.02–0.72]
203.1 [28.98–1424]
5
1
—
—
0.238
0.176
0.109
0.037
0.006
0.272
0.006
0.018
0.123
0.021
0.368
0.368
Statistical analysis across asthma groups. Bold indicates significance.
Ratio for budesonide Turbuhaler calculated as 1.5.
BTS, British Thoracic Society; CI, confidence interval; FEV1, force expiratory volume in 1 s; FVC, force vital capacity; PEF, peak expiratory flow.
b
p Valuea
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KCa3.1 (V1 and V2), control (V5), and GFP–small hairpin RNA (shRNA)–
expressing adenoviruses (Ad5C20Att01) were purchased from BioFocus
DPI (Leden, the Netherlands). Preliminary transduction experiments using
this adenovirus expressing cDNA for GFP demonstrated high transduction
efficiency (∼80% of cells). Gene knockdown of KCa3.1 channels with
different shRNA adenoviruses was confirmed using 2 3 105 ASM cells
transduced using a multiplicity of infection of 30 and subsequent Western
blotting. For experiments, 24 h after transduction, the cells were serum
deprived for 3 h and then exposed to fluticasone (100 nM) for 2 h, followed
by addition of control media or TNF-a/IFN-g for an additional 22 h. After
24 h, protein supernatants were collected for ELISA assays (CCL5,
CX3CL1, CCL11), and cells were lysed for total cell extract preparation,
as described above.
Briefly, voltage commands were applied to potentials ranging from 2100
to +100mV from a holding potential of 220mV. Membrane currents were
recorded and subsequently plotted against command potential. Solutions
used were as follows: external (mM)—NaCl (140), KCl (5), MgCl2 (1),
and CaCl2 (2) (pH was adjusted to 7.4 using NaOH); internal (mM)—KCl
(140), MgCL2 (2), HEPES (10), ATP (2), and GTP (0.1) (pH was adjusted
to 7.3 using KOH).
The Journal of Immunology
2627
three patients. As shown in Fig. 2A, KCa3.1 channel was identified
within the nucleus in ASM cells. Staining for HDAC1 (Fig. 2B),
a protein exclusively found in the nucleus (44), was used as
a positive control for the immunostaining procedure. The intracellular expression of KCa3.1 in ASM cells is highly novel,
although one report identified KCa3.1 channels in mitochondrial
membranes in human colon carcinoma 116 cells (45). We also
performed Western blots directly on isolated nuclear extracts to
provide additional evidence for the nuclear expression of KCa3.1
channel in human ASM cells. Interestingly, the nuclear fraction
contained discreet bands recognized by an anti-KCa3.1 Ab of 47
and 53 kDa, as described previously in the cytoplasmic extracts of
several cell types (20, 37, 46) (Fig. 2C). The predicted size of
KCa3.1 is 48 kDa. The larger band is likely to represent alternative
glycosylation, although several smaller splice variants are also
described (46). The quantitative analysis of KCa3.1 expression in
the nuclear fractions by immunoblot assay revealed no significant
changes between healthy and asthmatic subjects (Fig. 2C, bottom
panel). This finding confirms the nuclear expression of intracellular KCa3.1 in human ASM cells.
Using patch-clamp electrophysiology, we also assessed whether
channel activity was affected in cells treated with TNF-a/IFN-g.
Fig. 2D compares the average current-voltage (I/V) curves performed
in 16 healthy cells from diluent or cytokine-treated conditions for
24 h. In agreement with our previous report (20), we found no
evidence of KCa3.1 currents in nonstimulated cells. Of note,
KCa3.1 currents were also not noticeable in cells treated with
TNF-a/IFN-g, suggesting that cytokines do not stimulate plasma
membrane KCa3.1 channel activity in ASM cells.
Production of steroid-resistant chemokines induced by TNFa/IFN-g in ASM cells from healthy controls and patients with
asthma and COPD
We first determined whether production of four different chemokines—CCL5, CCL11, CX3CL1, and chemokine (C-X-C motif)
ligand 10 (CXCL10)—in response to TNF-a/IFN-g was affected
by disease status (Fig. 3). We found that levels of CCL5 (Fig. 3A),
CCL11 (Fig. 3B), and CXCL10 (Fig. 3C) produced in response to
TNF-a/IFN-g at 24 h were similar in the different groups studied.
Thus net increases (induced minus basal levels) of CCL5 were
7269 6 1841, 5968 6 1388, and 7280 6 2297 pg/ml in healthy,
asthmatic, and COPD subjects, respectively (Fig. 3A). Net
increases of CCL11 were 417.2 6 93, 266.6 6 92, and 317 6 121
pg/ml in healthy, asthmatic, and COPD subjects, respectively (Fig.
3B). Net increases of CXCL10 were 72666 6 9740, 71478 6
11260, and 79401 6 11272 pg/ml in healthy, asthmatic, and
COPD subjects, respectively (Fig. 3C). Of interest, we found that
ASM cells from COPD patients produced significantly less CX3CL1
Table II. Demographics of patients used for the chemokine expression assays/immunoblot and
immunostaining studies
Number
Gender (M/F)
Age (mean 6 SEM)
FEV1 (6 SEM)
FEV1% predicted (6 SEM)
FEV1/FVC (%) (6 SEM)
a
Controls
Asthma
COPD
p valuea
12
5/7
42.25 6 4.15
3.7 6 0.27
88.75 6 4.08
88.7 6 8.3
10
5/5
49.4 6 2.98
2.48 6 0.22
70.92 6 9.97
64.4 6 3.32
6
6/0
66 6 4.31
1.9 6 0.31
61.5 6 7.04
53.82 6 5.16
—
Statistical analysis across subject groups. Bold indicates significance.
0.0046
0.0004
0.0116
0.0044
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FIGURE 1. In vivo KCa3.1 expression in human ASM bundles. Representative immunostaining in bronchial biopsies for isotype control Ab (3200) (A),
KCa3.1 in a healthy control (3400) (B), or in an asthmatic individual treated with inhaled corticosteroid (3400) (C) or with inhaled corticosteroid + longacting beta agonist (3400) (D). (E) Representative immunostaining at higher magnification (31000) showing the nuclear expression of KCa3.1 channel
in vivo in three different muscle areas. The upper left image is the tissue incubated with the isotype-matched control Ab. (F) The percentage of ASM cell
nuclei staining for KCa3.1 in normal subjects (n = 7) and in patients with moderate (n = 5) and severe asthma (n = 6). Two sections at least 10 mm apart were
analyzed for each subject, and the mean of the two sections taken as the value for each subject. A minimum of 15 cells per section were counted (range, 15–
100 cells; median, 49.5 cells). Data are expressed as means 6 SEM. Ep, Epithelium.
2628
KCa3.1 CHANNEL AND STEROID RESISTANCE
in comparison with healthy controls (774 6 142 versus 4763 6
1400 pg/ml, p , 0.05), whereas no difference was noticed when
compared with levels produced by asthmatic individuals (2927 6
501 pg/ml) (Fig. 3D).
KCa3.1 channel inhibition differentially suppresses the
production of TNF-a/IFN-g–induced steroid-resistant
chemokines
We next investigated the role of KCa3.1 inhibitors in our ASM cell
model of GC resistance (TNF-a/IFN-g–treated cells) by assessing
the expression of four different chemokines in ASM cells from
healthy (n = 4), asthmatic (n = 6), and COPD subjects (n = 4). As
reported for tracheal ASM cells (12), induction of CX3CL1,
CCL5, CCL11, and CXCL10 by TNF-a/IFN-g was completely
resistant to fluticasone treatment in ASM cells derived from the
bronchial tree (Figs. 4–7). Chemokine production was not affected
in cells treated with 0.1% DMSO, the final concentration of the
inhibitor solvent.
The specific KCa3.1 blockers TRAM-34 (200 nM) (38) and
ICA-17043 (60 nM) (39) alone did not affect TNF-a/IFN-g–induced production of CCL5 (Fig. 4) or CCL11 (Fig. 5), with the
exception of cells derived from asthmatic patients (Fig. 5B). The
inhibitory effect was further enhanced by . 90% in the presence
of fluticasone (Fig. 5B). TNF-a/IFN-g–induced production of
CCL5 in all tested individuals (Fig. 4) or CCL11 in healthy and
COPD subjects (Fig. 5A–C) was inhibited only when fluticasone
was combined with either KCa3.1 channel blocker. Fluticasone and
ICA-17043 in combination reduced TNF-a/IFN-g–induced CCL5
levels to 57 6 12%, 61 6 14.5%, and 52 6 14% in ASM cells
from healthy, asthmatic, and COPD subjects, respectively (Fig. 4).
Fluticasone and TRAM-34 reduced TNF-a/IFN-g–induced CCL5
levels to 57 6 11%, 51 6 21%, and 54 6 9.6% in cells from
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FIGURE 2. Nuclear expression of KCa3.1 channel in cultured human ASM cells. Permeabilized ASM cells were stained for KCa3.1 (original magnification 3200) (A), the nuclear marker HDAC1 (original magnification 3200) (B), or were incubated with the corresponding isotype-matched Abs used as
negative controls. DAPI counterstaining was performed on the same cell population to demonstrate the nuclear location of KCa3.1. Images are representative of experiments performed on cells from three different healthy subjects. (C) Top, KCa3.1 expression in the nucleus was also confirmed by assaying
ASM nuclear fractions for KCa3.1 by immunoblot analysis. Bottom, Scanning densitometric measurement of KCa3.1 expression normalized over the
corresponding b-actin showed no significant difference in KCa3.1 expression in ASM nuclear fractions between nonasthmatic and asthmatic subjects.
Results are representative of blots performed in six subjects (three healthy and three asthmatic subjects). (D) Current voltage curves showing the lack of
detectable KCa3.1 currents in ASM cells at baseline and after cytokine treatment. Data are expressed as means 6 SEM of measurement performed in n = 16
cells per condition taken from a healthy subject.
The Journal of Immunology
2629
healthy, asthmatic, and COPD subjects, respectively (Fig. 4). Production of CCL11 by TNF-a/IFN-g was reduced to 36 6 14%,
12 6 14%, and 28 6 12% of control by the fluticasone and ICA17043 combination in cells from healthy, asthmatic, and COPD
subjects, respectively. TNF-a/IFN-g–induced CCL11 production
was reduced to 38.5 6 14.2%, 9 6 9%, and 31 6 15% by the
fluticasone and TRAM-34 combination in cells from healthy,
asthmatic, and COPD subjects, respectively (Fig. 5).
Of note, induction of CX3CL1 by TNF-a/IFN-g in cells from
healthy, COPD, and asthmatic subjects was reduced to 31.4 6
12.1%, 8.8 6 3.4%, and 33.2 6 8.9% by ICA-17043 alone (60
nM) and to 28.9 6 10.8%, 21.1 6 10.9%, and 26.4 6 14.7% by
TRAM-34 alone (200 nM) (Fig. 6A–C). The degree of CX3CL1
inhibition by KCa3.1 blockade was not changed by the presence of
fluticasone (Fig. 6A–C, last two columns).
In contrast, production of CXCL10 by TNF-a/IFN-g was not
affected by either KCa3.1 inhibitors used alone or in combination
with fluticasone (Fig. 7), ruling out a toxic or nonspecific effect of
KCa3.1 inhibitors. Measuring cell viability using the MTT assay
did not show any signs of cytotoxicity in cells treated with the above
concentrations of KCa3.1 inhibitors. No differences were observed
between relative MTT-reducing activities in control cells (with 0.1%
DMSO) and in cells treated with either ICA-17043 or TRAM-34:
0.344 6 0.019, 0.317 6 0.022, and 0.346 6 0.063, respectively.
shRNA silencing of KCa3.1 channels
To confirm the previous findings with pharmacological blockers
of KCa3.1, we also used knockdown of KCa3.1 channels, using
shRNA delivered by adenoviruses. An shRNA control adenovirus
(V5) did not affect KCa3.1 channel protein expression (Fig. 8A),
but this was completely decreased by KCa3.1 shRNAs V1 and V2
(Fig. 8A, top panel). None of the shRNAs affected b-actin expression (Fig. 8A). TNF-a/IFN-g treatment alone or in the presence of fluticasone had no effect on KCa3.1 expression or on the
efficacy of shRNA adenoviruses to silence the channel (Fig. 8A,
bottom panel). The following functional assays were then per-
formed using shRNA-KCa3.1 adenoviruses V1 and V2, and shRNA
control adenovirus (V5) as a control.
KCa3.1 downregulation in ASM cells attenuates
TNF-a/IFN-g–induced steroid-resistant chemokine expression
As in untransduced ASM cells, expression of CX3CL1 (Fig. 8B)
and CCL5 (Fig. 8C) in V5 control transduced cells was increased
by TNF-a/IFN-g and unaffected by fluticasone. In contrast, CCL5
production was inhibited by ∼ 30% by KCa3.1 shRNAs V1 and
V2, with further inhibition of secretion with the addition of fluticasone to almost 50% (Fig. 8C). In agreement with the data
obtained with the soluble inhibitors, CX3CL1 expression was
inhibited by KCa3.1 shRNAs V1 and V2 irrespective of fluticasone
treatment (Fig. 8B). The percent inhibition of CX3CL1 induced
by shRNA V1 in the absence or presence of fluticasone when
normalized to CX3CL1 production in the presence of the control
shRNA was 68% or 49%, respectively. The percent inhibition of
CX3CL1 induced by shRNA V2 was 45% or 30% in the absence
or presence of fluticasone, respectively. These data confirm that
KCa3.1 channels are essential in the regulation of TNF-a/IFN-g–
inducible GC-resistant chemokine in ASM cells.
KCa3.1 channel inhibition suppresses TNF-a/IFN-g–induced
steroid-resistant chemokine mRNA expression
Quantitative PCR analyses were performed to determine whether
KCa3.1 channel blockers modulate TNF-a/IFN-g–induced chemokine expression at the transcriptional level. In cells exposed
to TNF-a/IFN-g for 6 h, CCL5, CX3CL1, and CCL11 mRNA
expression was increased significantly by 35.2- 6 19-fold, 96- 6
41-fold, and 1.8- 6 0.43-fold, respectively (p , 0.05). Fluticasone
did not alter the expression of CCL5 or CCL11 mRNA, but nonsignificant trends were noted for reduced expression of CCL5 and
CCL11 mRNA with KCa3.1 blockade (Fig. 9). Combining fluticasone with TRAM-34 or ICA-17043 led to a drastic inhibition of
both CCL5 and CCL11 expression (p , 0.05, n = 4 combined from
2 asthmatic patients, 1 control, and 1 COPD patient)(Fig. 9A–C).
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FIGURE 3. Chemokine production induced by TNF-a/IFN-g in ASM cells from healthy, asthmatic, and COPD subjects. Cells were stimulated with
TNF-a/IFN-g for 24 h. CCL5, CX3CL1, CCL11, and CXCL10 levels in the supernatants were assessed by ELISA assays, as described in Materials and
Methods. Data are expressed as means 6 SEM of the net TNF-a/IFN-g–induced increase of CCL5 (A), CCL11 (B), CXCL10 (C), and CX3CL1 (D) in n = 5
controls, n = 7 asthmatic patients, and n = 5 COPD patients, all performed in triplicate. All chemokines were significantly increased compared with those in
unstimulated control. ***p , 0.01 compared with healthy cells.
2630
KCa3.1 CHANNEL AND STEROID RESISTANCE
FIGURE 5. Inhibition of GC-resistant CCL11 by KCa3.1 blockers. Cells
pretreated with TRAM-34 (200 nM) or ICA-17043 (60 nM), with or
without fluticasone (100 nM, 2 h), were stimulated with TNF-a/IFN-g for
24 h. CCL11 levels in the supernatants were assessed by ELISA as described in Materials and Methods. Data are expressed as mean 6 SEM %
of TNF-a/IFN-g–induced CCL11 levels. Healthy controls (A), n = 4;
asthmatic patients (B), n = 6; and COPD patients (C), n = 4. *p , 0.05,
***p , 0.001 compared with TNF-a/IFN-g/DMSO control.
Fluticasone alone had no significant effect on TNF-a/IFN-g–
induced CX3CL1 expression in response to TNF-a/IFN-g, but this
expression was almost completely reduced in cells treated with
either KCa3.1 blocker alone, irrespective of fluticasone treatment
(Fig. 9B). These data demonstrate that KCa3.1 channel inhibition
regulates the expression of GC-resistant chemokines in ASM cells
at the transcriptional level.
GR phosphorylation induced by TNF-a/IFN-g was associated
with an impaired expression of the well-defined GRE-dependent
gene called GILZ (Fig. 10C). Although these data were generated
in cells from healthy subjects, similar findings were also observed
in cells derived from n = 2 persons with nonsevere asthma (data
not shown). More importantly, in the presence of the KCa3.1
channel blockers ICA-17043 or TRAM-34, the inhibitory effect of
TNF-a/IFN-g on both fluticasone-induced GRa phosphorylation
and GILZ expression was completely prevented (n = 3, p , 0.01)
(Fig. 10A–C). This finding demonstrates that KCa3.1 channel inhibition restores a key signaling component required for normal GRa
transcriptional function in this ASM model of steroid resistance.
Fluticasone-induced GRa phosphorylation at Ser211 and GILZ
expression are impaired in steroid-resistant states but restored
in the presence of KCa3.1 channel inhibitors
Cytokine-induced steroid resistance in human ASM cells involves
the inhibition of GRa transcriptional activity via multiple mechanisms (12). In this study, we confirmed that fluticasone-induced
GRa phosphorylation on Ser211, which is essential for its transcriptional activity, was almost completely inhibited in the presence of TNF-a/IFN-g (Fig. 10A, 10B). This reduced GC-induced
KCa3.1 channel inhibition suppresses cytokine-induced PP5
expression
We have recently uncovered that TNF-a/IFN-g–induced upregulation of PP5 is the key factor by which this cytokine combination
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FIGURE 4. Inhibition of GC-resistant CCL5 by KCa3.1 blockers.
Cells pretreated with TRAM-34 (200 nM) or ICA-17043 (60 nM), with
or without fluticasone (100 nM, 2 h), were stimulated with TNF-a/IFN-g
for 24 h. CCL5 levels in the supernatants were assessed by ELISA, as
described in Materials and Methods. Data are expressed as means 6 SEM %
of TNF-a/IFN-g–induced CCL5 levels. Healthy controls (A), n = 4;
asthmatic patients (B), n = 6; and COPD patients (C), n = 4. *p , 0.05,
**p , 0.01 compared with TNF-a/IFN-g/DMSO control.
The Journal of Immunology
impairs GC-induced GRa phosphorylation at Ser211 (43, 47). Flow
cytometry assays confirmed that PP5 was indeed upregulated
following treatment with TNF-a/IFN-g in nine subjects (four
asthmatic subjects and five healthy subjects), but interestingly, this
response was significantly inhibited by both KCa3.1 blockers (Fig.
11). This finding suggests that although KCa3.1 channel activity
directly regulates the expression of CX3CL1 (Fig. 6), it is also
involved in the induction of PP5, which in turn promotes GC
resistance by dephosphorylating GRa at Ser211 (Fig. 12).
Discussion
KCa3.1 channels may represent major players in the pathogenesis
of several lung diseases (19). The present article now reveals that
KCa3.1 channels may figure importantly in the regulation of GCinsensitive pathways. We found that TNF-a/IFN-g–induced expression of GC-resistant CX3CL1 was inhibited by KCa3.1 channel blockade or knockdown at both the mRNA and the protein
FIGURE 7. Lack of inhibition of GC-resistant CXCL10 by KCa3.1
blockers. Cells pretreated with TRAM-34 (200 nM) or ICA-17043 (60
nM), with or without fluticasone (100 nM, 2 h), were stimulated with TNFa/IFN-g for 24 h. CXCL10 levels in the supernatants were assessed by
ELISA, as described in Materials and Methods. Data are expressed as
mean 6 SEM % of TNF-a/IFN-g–induced CXCL10 levels. Healthy
controls (A), n = 4; asthmatic patients (B), n = 6; and COPD patients (C),
n = 4. None of the values were significantly different when compared with
TNF-a/IFN-g/DMSO control.
levels, irrespective of GC presence. In contrast, the inhibition of
GC-resistant CCL5 was visible only in cells cotreated with both
fluticasone and KCa3.1 channel inhibition. The failure of fluticasone to induce GRa phosphorylation at Ser211 or to promote the
expression of the GC-inducible gene GILZ in the GC-resistant state
was fully prevented by the presence of KCa3.1 inhibitors as a consequence of reduced PP5 expression. To our knowledge, this is the
first report in any given cell type or tissue that describes a functional interaction between KCa3.1 channels and GC signaling.
The mechanisms underlying GC resistance in asthma are not
clearly defined (48). However, combined in vitro and in vivo
studies have led to the important conclusion that GC-resistant
pathways are operative in ASM cells within asthmatic airways
(49). Indeed, many studies performed on bronchial biopsies from
asthmatic individuals showed marked expression of inflammatory
proteins such as CX3CL1 (13), CCL11 (14), CCL15 (15), CCL19
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FIGURE 6. Inhibition of GC-resistant CX3CL1 by KCa3.1 blockers.
Cells pretreated with TRAM-34 (200 nM) or ICA-17043 (60 nM), with
or without fluticasone (100 nM, 2 h), were stimulated with TNF-a/IFN-g
for 24 h. CX3CL1 levels in the supernatants were assessed by ELISA, as
described in Materials and Methods. Data are expressed as mean 6 SEM %
of TNF-a/IFN-g–induced CX3CL1 levels. Healthy controls (A), n = 4;
asthmatic patients (B), n = 6; and COPD patients (C), n = 4. *p , 0.05,
**p , 0.01, ***p , 0.01 compared with TNF-a/IFN-g/DMSO control.
2631
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KCa3.1 CHANNEL AND STEROID RESISTANCE
(16), ADAM33, and ADAM8 (17, 18) within ASM bundles, despite treatment of patients with either high-dose inhaled or oral
GC. We have previously modeled this GC-resistant state in vitro
by exposing cultured human tracheal ASM cells to a combination
of TNF-a/IFN-g, which results in the production of an array of
inflammatory proteins, including CD38, CCL5, CX3CL1, and IFN
regulatory factor 1, that are completely resistant to GC treatment
(12, 43, 47, 50, 51). In this study, we found that in addition to
CCL5 and CX3CL1, production of other chemokines, CCL11 and
CXCL10, is also insensitive to fluticasone treatment; more importantly, this GC-resistant state also occurs in primary bronchial
ASM cells in health, asthma, and COPD (as opposed to tracheal
ASM cells in our previous studies). Generally, TNF-a/IFN-g–
dependent steroid-resistant chemokine production was similar
irrespective of disease status (healthy status, COPD, asthma), although production of CX3CL1 was dramatically reduced by .
80% in COPD patients when compared with levels produced by
cells from healthy subjects. The reasons for this are unknown, and
additional studies are clearly required to confirm this observation.
Immunohistochemistry on bronchial biopsies demonstrated that
KCa3.1 channels are expressed in vivo in ASM bundles in asthmatic patients, and interestingly, this included a nuclear distribution. This observation was further confirmed by assessing KCa3.1
expression in cultured ASM cells, using immunoblot analysis directly on nuclear extracts, and immunofluorescence staining showing a nuclear distribution of KCa3.1. KCa3.1 expression in asthmatic
ASM bundles was not affected by disease severity and the associated intensity of antiasthma treatment, and no differences in immunostaining intensity were evident between healthy subjects and
asthmatic patients. These observations suggest that changes in
channel activity rather than protein expression within ASM bundles
could explain their contribution to the pathogenesis of asthma. In
contrast to the study showing that TGF-b increased both KCa3.1
expression and activity in ASM cells (20), we found that TNFa/IFN-g failed to significantly stimulate KCa3.1 protein levels or
plasma membrane KCa3.1 channel activity, suggesting that modulation of KCa3.1 expression or function in ASM is highly stimulus dependent (growth factors versus cytokines). The fact that no
change in KCa3.1 activity occurs when measured at the cell surface supports the concept that compartmental changes in Ca2+
levels rather than cytoplasmic changes could explain the activation
of KCa3.1 in GC sensitivity. Our data showing that KCa3.1 inhibition does not affect CXCL10 induction strongly suggest that the
inhibitory effect of KCa3.1 blockade is gene specific and not due to
generalized nonspecific effects linked to overall changes in Ca2+
levels inside the cells. In this study we also confirmed our previous
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FIGURE 8. shRNA KCa3.1 modulates TNF-a/IFN-g–induced chemokine expression. (A) Top, Representative immunoblot of KCa3.1 protein expression
in cells transduced with KCa3.1 shRNA (V1 and V2) and control (V5) adenoviruses in the presence or absence of cytokines and fluticasone (FP). Bottom,
Scanning densitometric measurement of KCa3.1 expression by immunoblot assays normalized over the corresponding b-actin showed complete knockdown
of KCa3.1 expression in ASM cells, using shKCa3.1 V1 and V2, when compared with control adenovirus. Note that the TNF-a/IFN-g combination had no
effect on KCa3.1 expression. Results are shown as means 6 SEM of blots performed in three healthy donors. (B and C) Effect of the same adenoviruses on
TNF-a/IFN-g–induced expression of CX3CL1 (B) and CCL5 (C) assessed by ELISA. Results are shown as means 6 SEM of experiments performed in
triplicate in three donors. **p , 0.01 compared with respective control shRNA.
The Journal of Immunology
reports (20) showing that KCa3.1 inhibitors had no cytotoxic action on ASM cells. Because KCa3.1 channels were found to be
expressed in the nuclear compartment, our study raises the possibility that nuclear modulation of KCa3.1 function by TNFa/IFN-g could explain their involvement in modulating GC signaling. Of interest, the expression of KCa3.1 channels has been
reported in cytoplasmic organelles such as mitochondria in a human colon tumor cell line (45). Additional studies are clearly
needed to define how intracellular KCa3.1 channels regulate cellular function in ASM cells.
We found that combining fluticasone with KCa3.1 blockers or
KCa3.1 downregulation was effective in inhibiting GC-resistant
CCL5, CCL11, and CX3CL1 in ASM cells. It is interesting that
although KCa3.1 blockade had a strong inhibitory effect on cytokine-induced CCL5 expression at the mRNA level, production
of CCL5 protein was reduced by only 50%. We believe that in
addition to transcriptional mechanisms, CCL5 induction by
cytokines is also regulated at the posttranscriptional level. Previous studies performed in A549 cells (52) and in human ASM cells
(53) support this hypothesis by showing that although IFN-g on its
own did not stimulate CCL5 expression, it does enhance TNF-a–
induced CCL5 expression via posttranscriptional mechanisms. We
also have some preliminary evidence, using a different pharmacological inhibitor, demonstrating that the degree of CCL5 inhibition seen at the mRNA level does not correlate with similar
changes at the protein level (Y. Amrani, unpublished observations).
The ability to reproduce the same results when channels were
downregulated by shRNA shows unequivocally that KCa3.1 regulates, at least in part, TNF-a/IFN-g–dependent GC-resistant
chemokine expression. In our study, we noted some expected
discrepancy between the two inhibitory strategies. For example,
cytokine-induced CCL5 expression was significantly affected by
KCa3.1 shRNA, but not by the pharmacological blockers, and the
degree of CX3CL1 inhibition was somewhat greater when combining fluticasone with pharmacological blockers and not with
shRNA vectors. In contrast to soluble inhibitors, channel knockdown could indirectly have an impact on other cellular signaling
pathways through the loss of interactions between the target
protein (in this case, the KCa3.1 channel) and other key binding
partners. In the case of KCa3.1, very little is known about the
nature of proteins that associate with the channel. The direct
binding of 59-AMP–activated protein kinase (54), mammalian
protein histidine phosphatase 1 (55), or nucleoside diphosphate
kinase B to KCa3.1 was found to be essential in regulating channel
activity (56). Considering the fact that these proteins have multifunctional properties, it is therefore plausible that reducing KCa3.1
levels could lead to downstream effects not evoked by channel
blockers alone. The apparent differences observed between the data
obtained with the soluble inhibitors and silencing adenoviruses
could also be due to the heterogeneity in patients’ responses because different subjects were used with the two inhibitory strategies.
Of note, GC-resistant CX3CL1 expression, at both protein and
mRNA levels, was inhibited by KCa3.1 inhibitors irrespective of
GC treatment, suggesting that KCa3.1 channel activity is a key
factor regulating the transcriptional expression of this chemokine
in response to TNF-a/IFN-g. The putative role of KCa3.1 in regulating expression of inflammatory mediators has been mostly
described in T lymphocytes, in which KCa3.1 blockade (either via
pharmacological inhibitors or the use of T cells deficient in the
channel) significantly reduces TCR-induced expression of IL-2,
TNF-a, and IFN-g (57–59). In T cells it is likely that this
results from the instrumental role of KCa3.1 in regulating Ca2+
entry through the plasma membrane, a vital signal for optimal
T cell activation and cytokine secretion. The fact that TNF-a/IFNg–dependent CX3CL1 expression in ASM cells is inhibited by
two selective blockers of the KCa3.1 pore demonstrates that the
ionic conductance of K+ is key in the mediation of this effect, and
not a regulatory channel domain. Interestingly, we and others (60,
61) have shown that TNF-a alters Ca2+ handling in ASM cells via
the induction of ectoenzyme CD38 or transient receptor potential
C3 channels (62). It is therefore plausible that KCa3.1 regulates
CX3CL1 via activation of Ca2+-dependent pathways. However, as
discussed above, it seems unlikely that this is due to channel activity located in the plasma membrane.
Another major observation in our study is the functional interaction between KCa3.1 channels and GC signaling pathways.
Specifically, KCa3.1 inhibitors restored the ability of fluticasone to
inhibit the production of CCL5 and CCL11 in TNF-a/IFN-g–induced GC-resistant conditions, irrespective of the KCa3.1 inhibition strategy used. In addition, this restoration of cell sensitivity to
fluticasone by KCa3.1 blockade was concomitantly associated with
a reinstatement of the GRa phosphorylation at Ser211 that was
impaired in TNF-a/IFN-g–treated cells. This finding indicates that
KCa3.1 activity drives TNF-a/IFNg–induced GC insensitivity in
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FIGURE 9. KCa3.1 blockers modulate the transcription of GC-resistant
chemokines. Cells pretreated with TRAM-34 (200 nM) or ICA-17043 (60
nM), with or without fluticasone (100 nM, 2 h), were stimulated with TNFa/IFN-g for an additional 4 h. Total RNA was extracted, and real-time
quantitative PCR was performed, as described in Materials and Methods.
Results are expressed as fold induction of chemokine expression by calculating the negative inverse of the 22DDCT value for each condition.
Expression levels of CCL5 (A), CX3CL1 (B), and CCL11 (C) were
expressed as % of TNF-a/IFN-g response. Means 6 SEM of experiments
performed in duplicate in four different subjects (two asthmatic patients,
one control, and one COPD patient). *p , 0.05, **p , 0.01 compared
with TNF-a/IFN-g/DMSO control.
2633
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KCa3.1 CHANNEL AND STEROID RESISTANCE
ASM cells, in part by via the modulation of GRa phosphorylation
status (Fig. 10A) and GRa transactivation activity (Fig. 10B). Our
data support the growing evidence showing the importance of
transactivation properties in the anti-inflammatory action of GC
(63, 64). Although GRa phosphorylation on three major residues
located on its N terminus (Ser203, Ser211, and Ser226) affects key
functions of the receptor, including turnover and subcellular trafficking, it is phosphorylation on Ser211 that is essential for optimal
GRa transcriptional activity (65). Indeed, in agreement with our
previous reports (12, 47, 50, 51), we confirmed that fluticasoneinduced GRa-dependent transactivation was significantly impaired
FIGURE 11. PP5 upregulation by TNF-a/IFN-g is
inhibited by KCa3.1 inhibitors. Cells pretreated with
TRAM-34 (200 nM) or ICA-17043 (60 nM) for 2 h
were stimulated with TNF-a/IFN-g for 22 h. PP5 levels
assessed by flow cytometry [representative histogram
of one cell line shown in (A)] were expressed as the
fold increase in mean fluorescence intensity (B) over
basal 6 SEM of experiments performed in 9 subjects
(n = 4 asthmatic patients and n = 5 healthy controls).
*p , 0.05, **p , 0.01.
in the TNF-a/IFN-g–induced GC-insensitive state (Fig. 10C). More
importantly, KCa3.1 blockade was also able to fully restore
fluticasone-induced GRa-dependent transactivation, suggesting
a role of KCa3.1 channel in impairing GC function in ASM cells.
Our previous report showed that the impaired GRa transactivation
was due to the upregulation of the serine/threonine phosphatase
PP5, which mediated cytokine-induced GRa dephosphorylation at
Ser211 (43). In this article, we confirm not only that PP5 levels are
increased by TNF-a/IFN-g in ASM cells independently of disease
status (healthy status and asthma) but also, more importantly, that
PP5 induction was dependent on functional KCa3.1 channels.
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FIGURE 10. Impaired fluticasone-induced GRa phosphorylation and GRE-dependent GILZ expression by cytokines is restored by KCa3.1 inhibitors.
Cells pretreated with TRAM-34 (200 nM) or ICA-17043 (60 nM), with or without fluticasone (100 nM, 2 h), were stimulated with TNF-a/IFN-g for 4 h.
(A) Total cell lysates were prepared and assayed for total GR, phosphoserine 211 GR Abs, and b-actin for loading by immunoblot analysis. (B) Means 6
SEM of scanning densitometric analyses of blots from n = 3 healthy patients, with each value normalized over the mean density of the corresponding total
GR bands. *p , 0.05 compared with fluticasone, *p , 0.05 compared with TNF-a/IFN-g/fluticasone as indicated. (C) RNA was extracted and purified
using the PureLink RNA Mini Kit according to the manufacturer’s instructions. Total mRNA (2 mg) was subjected to real-time RT-PCR with GILZ and
b-actin primers, and the relative quantification in each condition was performed using the standard curve method and expressed as fold increased over basal.
Each experiment was performed in duplicate and repeated in cells from three different healthy donors. **p , 0.01 compared with TNF-a/IFN-g/fluticasone, ***p , 0.001 compared with TNF-a/IFN-g/fluticasone.
The Journal of Immunology
2635
FIGURE 12. Role of KCa3.1 in mediating GC insensitivity in ASM cells. We uncovered two mechanisms by which KCa3.1 channels mediate TNF-a/IFNg–associated corticosteroid insensitivity. Transcription
of some proasthmatic genes involves KCa3.1-dependent
pathways that are insensitive to corticosteroids (right).
Other genes are resistant to corticosteroids via the induction of the KCa3.1-dependent serine/threonine
phosphatase PP5, which impairs GRa transcriptional
function through decreased GRa phosphorylation (left).
Disclosures
C.B. is on the advisory board for GlaxoSmithKline, AstraZeneca,
HoffmanLaRoche, Novartis, Genentech, and MedImmune and has received consultancy fees from Medimmune and Novartis and travel expenses
to attend scientific meetings from Boehringer-Ingelheim. P.B. has acted as
an advisor to Icagen (2007–2010). The other authors have no financial
conflicts of interest.
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Although a direct link between PP5 and steroid insensitivity has
been suggested by Goleva and colleagues (66), who found that
PP5 knockdown restored GC responsiveness in estrogen-treated
breast cancer cells, our report, to our knowledge, is the first to
show that this functional link between proasthmatic cytokines and
PP5 occurs via KCa3.1-dependent pathways. Our present study
supports the novel model that TNF-a/IFN-g impairs GC sensitivity in ASM cells by promoting GRa dephosphorylation via
KCa3.1-dependent upregulation of PP5 expression.
In summary, we have shown that KCa3.1 channels contribute to
TNF-a/IFN-g–associated GC insensitivity (Fig. 12). Transcription
of some proasthmatic genes such as CX3CL1 is driven by KCa3.1dependent pathways that are insensitive to corticosteroids. Other
genes such as CCL5 or CCL11 are indirectly rendered resistant to
GC via the induction of the KCa3.1-dependent serine/threonine
phosphatase PP5 which interferes with GRa receptor function
through decreased GRa phosphorylation and transactivation. Our
study reveals the potential therapeutic value of targeting the
KCa3.1–PP5 axis in the treatment of lung diseases such as severe
asthma, in which relative GC resistance is evident. The availability of a well-tolerated orally bioavailable KCa3.1 blocker that
has been used in phase III trials of sickle cell disease (ICA-17043
[Senicapoc]) (19) means that the potential exists for the rapid
translation of these findings to the clinic.
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KCa3.1 CHANNEL AND STEROID RESISTANCE