Article
The Rockefeller University Press $30.00
J. Exp. Med. 2017 Vol. 214 No. 1 143–163
https://doi.org/10.1084/jem.20160675
143
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is one of
the leading causes of morbidity and mortality in the world,
resulting in a growing social and economic burden (Mathers
and Loncar, 2006; Vestbo et al., 2013). It is anticipated that the
prevalence and burden of COPD will further rise over the
next decades, as a result of the aging population and the per-
sistent exposure of individuals to risk factors associated with
the disease (Mathers and Loncar, 2006). In accordance, aging
has recently been highlighted as a signicant risk factor for
chronic lung diseases (Meiners et al., 2015). Long-term cig-
arette smoke (CS) exposure is a primary causative risk factor
for COPD, although the disease can also develop in individ-
uals who never smoked (Salvi and Barnes, 2009; Vestbo et al.,
2013). COPD is characterized by progressive, irreversible air-
ow limitation and loss of functional parenchymal pulmonary
tissue, called emphysema. Emphysema comprises alveolar air-
space enlargement and impaired pulmonary regeneration; it
has a poor prognosis and there are currently no eective med-
ical treatments aside from lung transplantation. The molecular
mechanisms underlying the development and progression of
COPD/emphysema are not yet fully claried. Recent studies
from our laboratory and others have demonstrated that alter-
ations in the WNT microenvironment potentially contribute
to disease pathogenesis (Baarsma et al., 2011; Kneidinger et
al., 2011; Wang et al., 2011; Heijink et al., 2013).
WNT ligands (19 in human) are evolutionarily con-
served secreted glycoproteins that are indispensable for proper
organ, especially lung, development (Morrisey et al., 2013;
Kotton and Morrisey, 2014). Specic WNT ligands can either
activate the β-catenin–dependent (canonical) or β-catenin–
independent (noncanonical) pathways by acting on various
Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD
is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly de-
ned. Reduced WNT–β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for at-
tenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing
to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD
and increased posttranslationally modied WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung
broblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senes-
cence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdiffer-
entiation in vitro
.
Lung-specic WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in
vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored ex-
pression of β-catenin–driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models
of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal–epithelial cross talk in COPD patho-
genesis, which is amenable to therapy.
Noncanonical WNT-5A signaling impairs endogenous
lung repair in COPD
HoekeA.Baarsma,
1
WiolettaSkronska-Wasek,
1
KathrinMutze,
1
FlorianCiolek,
1
DarcyE.Wagner,
1
GerritJohn-Schuster,
1
KatharinaHeinzelmann,
1
AndreasGünther,
2
KenR.Bracke,
3
MaylisDagouassat,
4
JorgeBoczkowski,
4
GuyG.Brusselle,
3
RonSmits,
5
OliverEickelberg,
1
AliÖ.Yildirim,
1
and
MelanieKönigsho
1
1
Comprehensive Pneumology Center, Research Unit Lung Repair and Regeneration, Helmholtz Center Munich, Ludwig Maximilians University Munich,
University Hospital Grosshadern, 81377 Munich, Germany
2
University of Giessen Lung Center, 35392 Giessen, Germany
3
Department of Respiratory Medicine, Ghent University Hospital, 9000 Ghent, Belgium
4
Inserm U955, Equipe 4, 94000 Créteil, France
5
Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, 3000 Rotterdam, Netherlands
© 2017 Baarsma et al. This article is distributed under the terms of an Attribution–Noncommercial–Share
Alike–No Mirror Sites license for the rst six months after the publication date (see http ://www .rupress .org
/terms /). After six months it is available under a Creative Commons License(Attribution–Noncommercial–
Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /).
Correspondence to Melanie Königshoff: melanie.koenigshoff@ucdenver.edu
Abbreviations used: ABC, active β-catenin; ATI, alveolar epithelial type I; CM, condi-
tioned medium; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke;
CSE, CS extract; DOX, doxycycline; FA, ltered air; GOLD, Global Initiative for Chronic
Obstructive Lung Diseesae; IPF, idiopathic pulmonary brosis; phLF, primary human
lung broblast; PKC, protein kinase C; SFT PC, surfactant protein C; siRNA, small
interference RNA.
The Journal of Experimental Medicine
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WNT-5A impairs alveolar epithelial cell repair | Baarsma et al.144
transmembrane receptors (Baarsma et al., 2013). In emphy-
sematous COPD patients, nuclear expression of the tran-
scriptional coactivator β-catenin, a surrogate marker for active
canonical WNT signaling, is decreased in alveolar epithelial
type II (ATII) cells (Kneidinger et al., 2011; Jiang et al., 2016).
The cause of reduced canonical WNT–β-catenin signaling in
the alveolar epithelium and, consequently, limited lung repair
capacity in COPD patients remains to be elucidated.
The structural and cellular alterations observed in the
lungs of individuals with COPD phenotypically resemble ac-
celerated aging of the organ and WNT signal alterations have
been shown to impact cellular aging mechanisms, such as se-
nescence (Ito and Barnes, 2009; Muñoz-Espín et al., 2013;
Scheraga and Thannickal, 2014; Meiners et al., 2015). Recent
evidence indicates that noncanonical WNT signaling is able
to inhibit canonical WNT signaling, resulting in decreased
β-catenin stability and/or impaired downstream signaling
(Mikels and Nusse, 2006; Nemeth et al., 2007). Nevertheless,
this mechanism has not been linked to chronic lung disease
pathology. In the current study, we hypothesize that a tran-
sition of canonical to noncanonical WNT signaling contrib-
utes to COPD development.
We report for the rst time that WNT-5A expression,
a ligand known to trigger noncanonical WNT signaling, is
increased in experimental and human COPD. We provide
evidence of WNT signaling being crucially involved in im-
paired cellular crosstalk in which broblast-derived WNT-5A
negatively regulates canonical WNT–β-catenin signaling in
alveolar epithelial cells in vitro and in vivo, thereby impairing
the capacity of the lung for wound healing and regeneration.
RESULTS
Noncanonical WNT-5A is increased in murine models of
COPD and contributes to emphysema development in vivo
We rst examined the expression of the noncanonical
WNT ligands in well-established mouse models of COPD.
WNT-5A was the only noncanonical WNT ligand signi-
cantly increased in mice subjected to short-term (3 d) CS
(ΔCT:
Wnt-4
, −1.93 ± 0.04 vs. −1.98 ± 0.11; P > 0.05;
Wnt-5A
, −2.20 ± 0.18 vs. −1.61 ± 0.15; P < 0.01;
Wnt-5B
,
−4.50 ± 0.05 vs. −4.22 ± 0.19; P > 0.05; and
Wnt-11
, −2.45
± 0.10 vs. −2.00 ± 0.16; P > 0.05; ltered air [FA] versus
CS-exposed mice;
n
= 4). Increased WNT-5A protein ex-
pression, accompanied by reduced active β-catenin (ABC)
expression, was observed in whole-lung homogenate of mice
chronically exposed to CS (4 mo) in comparison to FA-ex-
posed mice (fold change, 3.5 ± 0.7; Fig.1A). Accordingly,
expression of the WNT/T–β-catenin target gene
Axin2
was
signicantly attenuated, whereas expression of the neutrophil
chemoattractant
KC
(
Cxcl1
) was increased by CS exposure
(Fig.1B). Similar results were obtained in our mouse model
of elastase-induced emphysema with WNT-5A transcript
and protein expression being increased (protein: 2.4 ± 0.3-
fold over vehicle control; Fig.1, C and D). Elastase-induced
emphysema development was also accompanied by a reduc-
tion in the canonical WNT target genes
Axin2
and
Naked1
(
Nkd1
) and by an up-regulation of elastin (
Eln
; Fig.1D), fur-
ther corroborating previously published results (Kneidinger
et al., 2011; Uhl et al., 2015).
Next, we analyzed whether WNT-5A contributes to
the development of experimental emphysema in vivo
.
To this
end, we used mice that conditionally overexpress WNT-5A
specically in the lung (SFT PC-rtTA TetO-WNT-5A mice).
Pulmonary overexpression of WNT-5A was initiated by
supplementation of in the drinking water with doxycycline
(DOX) 1 wk before the induction of emphysema and was
well tolerated by the mice in all the experimental groups
(Fig.1, E and F). Overexpression of the ligand aggravated the
development of elastase-induced emphysema, as determined
by an increase in tropoelastin expression (Fig.1F), histologi-
cal assessment, and quantitative morphometry (Fig.1, G and
H). A signicant increase in mean chord length was observed
in emphysematous mice that overexpressed WNT-5A com-
pared with respective control group (Fig.1H; 35.0 ± 3.0 ver-
sus 26.8 ± 1.9 µm, respectively; P < 0.05). Collectively, these
data indicate a potential pathophysiological role of increased
WNT-5A in the development and progression of emphysema.
Enhanced expression and posttranslationally modied
WNT-5A in biosamples of individuals with COPD
To determine the potential clinical relevance of WNT-5A in
human COPD, we investigated whether WNT-5A expres-
sion was altered in lung samples of COPD patients. Notably,
WNT-5A transcript was enhanced in lung tissue specimens
from individuals with COPD compared with tissue from
individuals without COPD (donor; Fig. 2 A). Additionally,
analysis of
WNT-5A
expression in induced sputum of COPD
patients revealed that expression of the ligand is dependent
on disease severity (i.e., Global Initiative for Chronic Ob-
structive Lung Disease [GOLD] status; Fig.2B; data derived
from Gene Expression Omnibus [GEO] microarray available
under accession no. GSE22148; Singh et al., 2011). The data
from this large cohort (GSE22148) with 140 COPD patients
(GOLD stage II,
n
= 71; stage III,
n
= 59; and stage IV,
n
= 13)
revealed a weak (r = 0.18), but signicant (P < 0.05), linear
correlation between age of the patient and
WNT-5A
expres-
sion, independent of disease severity (Fig.2C).
Monomeric WNT ligands are predicted to have a mo-
lecular mass of ∼40 kD; however, they are heavily subjected
to posttranslational modications, including palmitoylation
and glycosylation (Willert and Nusse, 2012; Baarsma et al.,
2013). In addition, (secreted) WNT ligands can form high
molecular weight homomers/oligomers, which may inu-
ence the signaling capacity of ligands (Cha et al., 2008; Mac-
Donald et al., 2014; Zhang et al., 2015). We rst veried if
the antibody that we used for detection of WNT-5A was also
able to detect the ligand when oligomerized (Fig. S1). Nota-
bly, mature (∼49 kD) and homomeric/oligomeric WNT-5A
protein (∼230 kD) was increased in lung tissue of individuals
with COPD, whereas the expression of WNT-5A with a mo-
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145JEM Vol. 214, No. 1
Figure 1. Noncanonical WNT-5A is increased in murine models of COPD and contributes to emphysema development in vivo. (A) Immunoblots
and quantication of WNT-5A and ABC in whole-lung homogenate of mice exposed to FA (
n
= 6) or CS (CS;
n
= 8) for 4 mo. (B) Expression
Axin2
and
KC
(CXCL1)
in whole-lung homogenate of mice exposed to FA (
n
= 6) or CS (
n
= 8) for 4 mo. (C) Immunoblots and quantication of WNT-5A expression in
whole lung homogenate of mice 14 d after exposure to vehicle (PBS,
n
= 3) or elastase (
n
= 3). (D) Expression of
Wnt-5A
,
Axin2
,
Nkd1
, and
Eln
in whole-lung
homogenate of mice 7 d after exposure to vehicle (PBS;
n
= 6) or elastase (
n
= 6–12). (A–D) *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s
t
test.
(E) Experimental setup to determine the impact of lung-specic WNT-5A overexpression on emphysema development. Animals had ad libitum access to
drinking water containing 5% sucrose (solid line) or DOX (2 mg/ml) in 5% sucrose (dashed line). Animals were treated on day 0 with either elastase (PPE; 40
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WNT-5A impairs alveolar epithelial cell repair | Baarsma et al.146
lecular weight of ∼45 kD tended to be increased, although
not signicantly (Fig.2D). In accordance, gene expression of
porcupine (
POR CN
), an O-acyltransferase that posttransla-
tionally modies WNT proteins, is increased in lung tissue of
COPD patients (Fig.2E).
Pulmonary broblasts are a cellular
source of noncanonical WNT-5A
We next aimed to identify the main cellular source of
WNT-5A in COPD. WNT-5A expression was analyzed
in 3D human lung tissue cultures (3D-LTCs), primary
human ATII (phATII) cells, and primary human lung -
broblasts (phLF; Fig.3A). The highest basal transcript level
of WNT-5A was detected in phLF compared with phATII
and 3D-LTCs. WNT-5A protein could be detected in su-
pernatants from phLF, but not in supernatants of ATII-like
A549 cells (Fig. 3 B). Notably, RNA sequencing of phLF
from individuals without (donor) and with COPD further
revealed aberrant gene expression of specic WNT ligands,
with increased noncanonical
WNT-5A
expression in COPD
broblasts (Fig. 3 C; 2.4-fold of mean donor; P < 0.001;
unpublished data). Moreover,
WNT-5A
expression was in-
creased in senescent phLF of smokers, as well as from indi-
viduals with COPD compared with respective nonsenescent
broblasts (Fig.3D). Next, we stimulated phLF with TGF-β
and/or CS extract (CSE). TGF-β treatment of phLF resulted
in increased expression of
WNT-5A,
whereas expression of
another noncanonical WNT ligand,
WNT-4
, was unaected
(Fig.3E). Stimulation with TGF-β led to increased WNT-5A
protein expression and secretion of the ligand (Fig.3, F and
G). Although CSE alone (up to 15%) only resulted in a slight
increase in intracellular WNT-5A protein expression, CSE
showed a synergistic eect on both TGF-β–induced WNT-5A
protein expression and secretion in phLF (Fig.3, F and G).
Given our initial nding that WNT-5A appears to be
posttranslationally modied and oligomerized dierently in
human COPD samples, we next investigated the role of gly-
cosylation on WNT-5A synthesis and secretion by phLF. The
phLF were stimulated without or with TGF-β in the absence
or presence of tunicamycin, a compound that prevents gly-
cosylation of proteins. Tunicamycin largely decreased cellular
expression and secretion of WNT-5A in cell lysates and su-
pernatants from phLF, respectively (Fig.3H). These ndings
highlight the requirement of WNT-5A glycosylation to be
produced and secreted by phLF, which is in line with a bio-
chemical study investigating the relevance of posttranslational
modications of WNT-5A (Kurayoshi et al., 2007).
Fibroblast-derived WNT-5A inhibits canonical WNT–β-
catenin signaling in alveolar epithelial cells
Next, we endeavored to elucidate the underlying mechanism
by which WNT-5A impairs lung repair. Our previous work
demonstrated that canonical WNT–β-catenin signaling is
attenuated in ATII cells of emphysematous COPD patients
(Kneidinger et al., 2011; Uhl et al., 2015). Given that canon-
ical and noncanonical WNT signaling can reciprocally regu-
late each other’s signaling activity, we investigated the direct
eect of WNT-5A on WNT–β-catenin signaling in alveolar
epithelial cells. Stimulation with WNT-3A resulted in in-
creased phosphorylation of LRP6 (p-ser1490 LRP6) and ac-
cumulation of ABC in human (A549) and murine (MLE12)
alveolar-like epithelial cells (Fig. 4, A and B). WNT-5A
cotreatment resulted in a concentration-dependent attenua-
tion of WNT-3A–induced LRP6 phosphorylation and ABC
accumulation. DVL2 phosphorylation, a positive control for
WNT signaling in general, was induced by either WNT-3A
or WNT-5A, and this response was enhanced by stimulation
with both ligands together (Fig.4, A and B). Moreover, in
murine alveolar epithelial cells (MLE12), WNT-3A enhanced
cytosolic and/or nuclear localization of β-catenin (indicated
by arrows), which was largely decreased by direct cotreatment
with WNT-5A (Fig.4 C). Accordingly, activation of β-cat-
enin–dependent gene transcription (determined by TOP/
FOP ash activity) by WNT-3A or pharmacological inhibi-
tion of GSK-3β by either LiCl (10mM) or SB216763 (5µM)
was attenuated by WNT-5A in lung epithelial cells (Fig. 4,
D–F). The intracellular eects of WNT-5A were at least in
part mediated by protein kinase C (PKC), but not JNK1/2
or TAK1/NLK (percentage inhibition of WNT-3A signaling
by WNT-5A in the absence or presence of the PKC inhibitor
GF109203X was 69.2 ± 3.3% and 43.3 + 9.4%, respectively;
P < 0.05; and not depicted). These ndings indicate that
WNT-5A impairs β-catenin activation and signaling not only
at the receptor level (i.e., regulating LRP6 activation) but also
downstream of canonical WNT receptors.
This eect was specic for WNT-5A, as WNT-4 was
not able to inhibit β-catenin activity in alveolar epithelial cells
(Fig.4G). In addition, the WNT-5A eect seems to be cell
specic, as WNT-5A was unable to attenuate WNT-3A–in-
duced activation of β-catenin–dependent gene transcription
in broblasts (Fig.4H). Collectively, these results suggest that
noncanonical WNT-5A is a specic negative regulator of
WNT–β-catenin signaling in alveolar epithelial cells.
We further investigated the importance of
N-
glycosyla-
tion on the signaling properties of WNT-5A. Deglycosylated
U/kg body weight) or vehicle control (PBS). Data are derived from two independent animal experiments. (F) Analysis of WNT-5A and tropoelastin at day 21 in
whole-lung homogenate of SFT PC rtTA TetO-WNT-5A mice exposed to DOX or 5% sucrose in the drinking water and treated with elastase or vehicle control
(
n
= 3–6 mice/group). *, P < 0.05; **, P < 0.01, determined by one-way ANO VA, followed by a Newman-Keuls multiple comparison test. (G) H&E-stained lung
tissue sections. Bars: (left) 1 mm; (right) 100 µm. (H) Mean chord length as determined by quantitative morphometry (
n
= 6–9 animals per group). **, P <
0.01; ***, P < 0.001, compared with vehicle treatment with sucrose;
###
, P < 0.001 compared with vehicle treatment with DOX;
$
, P < 0.05, compared with
elastase treatment with sucrose; determined by one-way ANO VA, followed by a Newman-Keuls multiple comparison test.
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147JEM Vol. 214, No. 1
WNT-5A was generated by treating WNT-5A conditioned
medium (WNT-5A CM) with Peptide-
N
-Glycosidase F
(PNGase F), which did not inuence protein stability of the
ligand (Fig.5A). Deglycosylated WNT-5A was not able to
signicantly attenuate canonical WNT signaling, whereas
untreated WNT-5A CM attenuated WNT-3A–induced ac-
tivation of β-catenin dependent gene transcription in A549
cells (Fig.5B, left). Furthermore, control CM treated with
PNGase F did not aect WNT-3A driven transcriptional
activity of β-catenin (Fig.5B, right). These results demon-
strate that glycosylation of the ligand is essential for the ob-
served negative regulation of WNT–β-catenin signaling by
WNT-5A in alveolar epithelial cells.
Next, we wondered if broblast-derived WNT-5A
was sucient to aect canonical WNT–β-catenin signaling
in alveolar epithelial cells. We experimentally addressed this
Figure 2. Enhanced expression and posttranslationally modied WNT-5A in biosamples of individuals with COPD. (A)
WNT-5A
in whole-lung
homogenate of individuals without (Donor;
n
= 19) and with COPD (
n
= 17). *, P < 0.05, unpaired Student’s
t
test with Welch’s correction. (B)
WNT-5A
expression in induced sputum of individuals with COPD (GOLD stage II,
n
= 71; III,
n
= 59; IV,
n
= 13). *, P < 0.05, determined by one-way ANO VA, followed
by a Newman-Keuls multiple comparison test. (C)
WNT-5A
expression in induced sputum of individuals with COPD correlated to age of the individual (COPD
stage II/III/IV;
n
= 143). Linear regression analysis; r = 0.18 and P < 0.05. Data presented in B and C are derived from microarray data available under GEO
accession no. GSE22148 (Singh et al., 2011). (D) Immunoblots and quantication of WNT-5A protein expression in whole-lung homogenate of individuals
without (Donor;
n
= 16) and with COPD (
n
= 22). *, P < 0.05, unpaired Student’s
t
test with Welch’s correction. (E) Expression of
porcupine
(
POR CN
) in whole-
lung homogenate of individuals without (Donor;
n
= 10) and with COPD (
n
= 14). *, P < 0.05, unpaired Student’s
t
test with Welch’s correction.
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