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WNT signaling in airway remodeling in asthmaKumawat, Kuldeep

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Citation for published version (APA):Kumawat, K. (2015). WNT signaling in airway remodeling in asthma: novel roles for WNT-5A in airwaysmooth muscle. [S.l.]: [S.n.].

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WNT signaling pathway is altered in

a mouse model of chronic allergic

airway inflammation

Kuldeep Kumawat

Mark H. Menzen

Nienke De Haas

Marieke Smit

Andrew J. Halayko

Reinoud Gosens

6

Chapter 6

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WNT pathway is altered in a mouse model of chronic allergic airway inflammation

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Abstract

Asthma is a chronic obstructive disease of the airways characterized by bronchial

hyperresponsiveness, airway inflammation and remodeling. The underlying mechanisms

that initiate, drive and maintain asthma are not completely understood. We have recently

reported increased abundance of the WNT ligand, WNT-5A, in asthmatic airway smooth

muscle (ASM) cells and have identified its novel role in TGF-β-induced extracellular matrix

(ECM) expression. The WNT pathway is composed of a multitude of signaling components

and participates in a myriad of biological functions in health and disease including

pulmonary disorders. We have undertaken a comprehensive analysis of expression of WNT

signaling members using an animal model of allergen-induced chronic inflammation to

identify potential mediators of asthma pathophysiology. We here show a wide ranging

modulation of most of the WNT ligands, Frizzled (FZD) receptors and various intracellular

and extracellular mediators and modulators in the lungs of mice after ovalbumin (OVA)

exposure. Notably, WNT-7A, -9A and -10B and FZD4 and FZD6 were significantly

downregulated along with a modest but significant reduction in β-catenin mRNA levels post-

OVA challenge. Of note, WIF1, a WNT signaling antagonist, showed strong downregulation

in OVA-challenged lungs as compared to saline-challenged controls. Moreover, TGF-β

reduced WIF1 mRNA in ASM cells. Owing to its antagonistic effect on WNT signaling, WIF1

downregulation may have key effects. In conclusion, allergen-induced modulation of the

WNT signaling pathway with suppression of WIF1 may indicate a significant pathological

event.

Introduction

Asthma is a heterogeneous chronic obstructive disease of the airways inflicting

approximately 300 million people worldwide and imposing a substantial burden on the

patients and healthcare system [1]. It is characterized by the presence of chronic

inflammation, bronchial hyperresponsiveness and extensive structural changes in the

airways, termed as airway remodeling [1,2].

Exposure to inhaled stimuli such as allergens or respiratory viruses triggers an exaggerated

response in asthmatic airways inducing airway constriction leading to episodes of

breathlessness and wheezing. Asthma can be effectively managed by β2 adrenoreceptor-

agonists and/ or corticosteroids in most patients providing substantial relief from the

episodic breathlessness. However, despite the most effective current therapies, a subset of

asthma patients remain poorly controlled even at the highest doses of the asthma medication

[3,4]. Asthma is a complex manifestation of (epi)genetic and environmental factors that

contribute to the evolution of the disease from childhood and often actively regulate the

course of disease and its management in later stages [1,5]. However, the factors and

mechanisms that govern the initiation, progression and maintenance of asthma remain

poorly understood hampering the efforts to develop new and more effective therapeutic

strategies targeting asthma.

Airway remodeling is a key pathological feature of individuals with asthma and is associated

with airway obstruction [6] and bronchial hyperresponsiveness [7]. It is characterized by

Chapter 6

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extensive structural changes in the airway wall which include airway smooth muscle (ASM)

cell hypertrophy and hyperplasia, subepithelial fibrosis, mucus hypersecretion,

neovascularization and increased and altered extracellular matrix (ECM) expression, leading

to airway wall thickening [8]. Airway remodeling is associated with the severity of disease.

For instance, in fatal asthma, the entire airway tree is massively remodeled whereas in non-

fatal asthma, remodeling is less prominent and afflicts mainly small airways [8]. Similarly,

the thickness of the remodeled airway wall also correlates with the severity of the disease [8-

11].

WNT signaling is an important developmental pathway which plays critical roles in

embryonic morphogenesis and regulates biological functions in post-natal life. The term

WNT is derived from a combination of two homologues genes integrase 1 (int1) and wingless

(wg) [12]. The Int1 gene was first identified as a locus for the integration of mouse mammary

tumor virus DNA leading to its activation and involvement in the development of virally-

induced breast tumors in mice [12,13]. Wg, which was identified for its role in development

of wing tissue in Drosophila and regulation of larval segment polarity, was later found to be

a homologue of Int1 [12]. The WNT signaling family has grown multifold since then both in

the number of its members and its complexity. In humans, the WNT family is comprised of

19 WNT ligands, 10 Frizzled (FZD) receptors, low-density lipoprotein receptor-related

protein (LRP) 5/6 coreceptors, several non-frizzled receptors such as RYK, ROR2, PTK7

along with intracellular mediators, several extracellular and intracellular antagonists and a

range of modulators [14]. These WNT ligands can function through signaling mechanisms

broadly categorized on the basis of the requirement of an intracellular mediator- β-catenin.

The β-catenin-dependent WNT signaling pathway is termed as canonical WNT signaling

whereas all the WNT ligands activated signaling cascades functioning independent of β-

catenin are collectively described as noncanonical WNT signaling.

Accumulating evidence point to a key role of WNT signaling pathway in asthma pathology.

A study has highlighted an indirect link between the expression of various WNT ligands such

as WNT-5A, -3A and 10A and the presence of Th2 inflammation in asthmatic subjects [15].

Moreover, we have recently identified significantly increased abundance of WNT-5A mRNA

and protein in the airway smooth muscle (ASM) cells of asthma patients as compared to the

healthy subjects (Chapter 3). We demonstrated that TGF-β, a growth factor implicated

extensively in airway remodeling and asthma pathobiology [16], induces WNT-5A

expression in ASM cells where it mediates ECM expression, thus, providing a possible link

between WNT signaling and features of asthma pathobiology (Chapter 3).

WNT is an inherently complex pathway comprised of large number of members. In view of

the crucial roles assigned to some of its members in lung development and diseases including

asthma, we evaluated WNT signaling pathway expression patterns in lungs during asthma

using a mouse model of allergen-induced chronic airway inflammation. Here, we present

evidence of extensive modulation of WNT signaling components in ovalbumin (OVA)-

challenged lungs and demonstrate that WNT inhibitory factor 1 (WIF1), a WNT signaling

antagonist, was significantly attenuated on OVA exposure.


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Material and Methods

Reagents- Recombinant human TGF-β1, recombinant human WIF1 and mouse anti-WIF1

antibody were from R&D systems (Abingdon, UK). Human WIF1 siRNA and mouse anti-

GAPDH, mouse anti-β-actin antibodies were purchased from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). Mouse anti-total β-catenin antibody was from BD Biosciences (San

Jose, CA, USA) and mouse anti-active β-catenin antibody (clone 8E7) was obtained from

Millipore (Amsterdam, the Netherlands). HRP-conjugated goat anti-mouse antibody and

human Interleukin-1β (IL-1β) was obtained from Sigma (St. Louis, MO, USA) and

Fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibody was procured

from Jackson Immunoresearch Europe (Suffolk, UK). Non-targeting siRNA was procured

from Qiagen (Venlo, The Netherlands) and rabbit- anti-α-smooth muscle-actin and rabbit

anti-WIF1 antibody were obtained from Abcam (Cambridge, UK). X-tremeGENE siRNA and

X-tremeGENE DNA HP transfection reagents were purchased from Roche Applied Science

(Mannheim, Germany). All other chemicals were of analytical grade.

Animals- Female BALB/c mice about 8-12 weeks old, were procured from Charles River

Laboratories (Leiden, the Netherlands). Animals were housed under a conventional 12-hour

light/dark cycle and received food and water ad libitum. All animal studies were conducted

according to the national guidelines and were approved by the University of Groningen

Committee for Animal Experimentation (no. 5912A).

Animal Model- Animals were sensitized to OVA (Sigma-Aldrich, Zwijndrecht, the

Netherlands) on Days 1, 14, and 21 by intraperitoneal administration of 10 µg OVA

emulsified in 1.5 mg aluminum hydroxide (Aluminject; Pierce Chemical, Etten-Leur, The

Netherlands) in a final volume of 200 µl in PBS. Subsequently, mice were divided into two

groups (n=10-12) and challenged with either saline or OVA aerosols (1% in PBS) for 20

minutes twice weekly on consecutive days for 4 weeks (Figure 1). The aerosol was delivered

to a Perspex exposure chamber (9 liter) by a De Vilbiss nebulizer (type 646; De Vilbiss,

Somerset, PA) driven by an airflow of 40 L/min, providing an aerosol with an output of 0.33

ml/min, as described previously (19).

Figure 1. Animal model of allergen-

induced chronic inflammation. Female

BALB/c mice (n=10 per group) were sensitized

to ovalbumin (OVA) on Day 1, 14 and 21.

Animals were subsequently challenged with

saline or OVA aerosols twice weekly for 4 weeks

as described in the material and methods

section. 24 hour following the last challenge,

animals were sacrificed and lungs were

harvested for further analysis.

Sensitization Challenge Sacrifice

1 14 21 26 33 40 47 49 27 34 41 48

Day

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Tissue collection- 24 hours following the last challenge, animals were anesthetized with

~2% (v/v) Isoflurane in oxygen and sacrificed by exsanguination. Right lung was inflated

with 50% Tissue-Tek in PBS (Sakura Finetek, Alphen aan de Rijn, the Netherlands) and

lungs were subsequently harvested. The smallest lower and the upper right lung lobes were

separately snap-frozen for mRNA analysis, the two middle right lung lobes were snap frozen

for immunohistochemistry. Uninflated left lung was snap frozen for protein analysis.

Immunohistochemistry- Morphometric analyses were performed on the transverse

tissue cryosections of 5µM thickness. Eosinophils were visualized by staining cryosections

with diaminobenzidine (Sigma-Aldrich, Zwijndrecht, the Netherlands) for cyanide-resistant

endogenous peroxidase activity. The number of eosinophils around the airways was counted

and expressed as number of cells per mm basement membrane.

Cryosections were stained for α-smooth muscle-actin using a rabbit anti-α-smooth muscle-

actin antibody (Abcam, Cambridge, UK) which was visualized by staining with horseradish-

peroxidase-linked secondary antibody and diaminobenzidine (Sigma-Aldrich, Zwijndrecht,

the Netherlands). Airways within sections were digitally photographed and α-smooth

muscle-actin around the airway was quantified using Image J (National Institute of Health).

The surface of positively stained tissue was expressed as mm2 per mm2 basement membrane.

Immunofluorescence- For immunofluorescence, cryosections were air-dried, fixed in

acetone for 10 minutes and blocked with a mix of 2% donkey serum and 1% BSA for 1 hour.

WIF1 was detected using rabbit anti-WIF1 antibody (Abcam, Cambridge, UK) and FITC-

conjugated donkey anti-rabbit antibody.

β-Catenin was visualized by mouse anti-β-catenin antibody using mouse-on-mouse (MOM)

elite peroxidase kit (Vector Laboratories, Burlingame, USA) as per manufacture’s instruction

with minor modifications. Briefly, following the blocking with a mix of 2% donkey serum and

1% BSA as mentioned above, cryosections were further incubated with the mouse Ig blocking

reagent for 1 hour and with MOM diluent for 5 minutes. Subsequently, cryosections were

incubated with anti-β-catenin antibody for 30 minutes and with a secondary rabbit anti-

mouse IgG for another 30 minutes. β-Catenin was visualized by using a tertiary FITC-

conjugated donkey anti-rabbit antibody.

Nuclei were stained with Hoechst diluted in ddH2O. After staining, cryosections were

mounted in ProLong Gold Antifade reagent (Life Technologies, Bleiswijk, the Netherlands).

Immunofluorescence was analyzed using Olympus AX70 microscope.

Cell culture- Immortalized human airway smooth muscle cell lines (ASM) cells and human

bronchial epithelial cell lines (HBE) were used for in vitro experiments. Cell lines were

maintained and used for experiments as described previously (Chapter 3 and [17]).

RNA isolation and real time PCR- Lung homogenates were prepared by pulverizing

tissues under liquid nitrogen. Total RNA was extracted from the lung homogenates and cell

lines using the Nucleospin RNAII kit (Macherey-Nagel, Duren, Germany) as per

manufacturer’s instructions. Equal amounts of total RNA were reverse transcribed using the


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Reverse Transcription System (Promega, Madison, USA). 1 µl of 1:2 diluted cDNA was

subjected to real-time PCR using FastStart Universal SYBR Green Master (Rox) from Roche

Applied Science (Mannheim, Germany) in an Illumina Eco Personal QPCR System

(Westburg, Leusden, the Netherlands). Real time PCR was performed with denaturation at

94°C for 30 seconds, annealing at the mentioned temperatures (Table 1) for 30 seconds and

extension at 72°C for 30 seconds for 40 cycles followed by 10 minutes at 72°C. Data was

analyzed using the comparative cycle threshold (Cq: amplification cycle number) method.

The amount of target gene was normalized to the endogenous reference gene 18S ribosomal

RNA (∆Cq). Where appropriate, relative differences were determined using the equation 2(-

∆∆Cq). Primers used for PCR amplification are listed in Table 1.

Western Blotting- Lung homogenates were prepared by pulverizing tissues under the

liquid nitrogen with subsequent sonication in SDS lysis buffer (62.5 mM Tris, 2% w/v SDS,

1 mM NaF, 1 mM Na3VO4, 1µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM

β-glycerophosphate, pH 6.8). The hTERT and HBE cells were also lysed in SDS lysis buffer

supplemented with protease inhibitors.

Protein concentration was determined using Pierce BCA protein assay kit (Thermo

Scientific, Rockford, IL, USA). Equal amounts of protein were separated by electrophoresis,

electro-transferred to nitrocellulose membranes and analyzed for proteins of interest using

specific primary and HRP-conjugated secondary antibodies. Westerns were subsequently

visualized using the G-box gel documentation system (Syngene, Cambridge, UK) using

enhanced chemiluminescence reagents and were quantified by densitometry using

Genetools software where necessary.

siRNA transfection- ASM cells were grown to ~90% confluence in 6-well cluster plates

and transfected with 200 pmol of WIF1-specific in serum and antibiotic free DMEM with X-

tremeGENE siRNA transfection reagent (Roche). Control transfections were performed

using a non-targeting control siRNA (Qiagen). After 6 hours of transfection, medium was

replaced with DMEM supplemented with antibiotics and ITS for a period of 42 hours before

TGF-β stimulation.

DNA transfection- ASM cells or HBE cells were grown to ~90% confluence in 6-well or

24-well cluster plates and were subsequently transfected with 1 µg of Myc-DDK-tagged

human WIF1 plasmid (Myc-WIF1) (Origene, Rockville, MD, USA) in serum and antibiotic

free medium using X-tremeGENE HP DNA transfection reagent. 1 µg of Green Fluorescent

Protein (GFP) expression vector was transfected as control. After 6 hours of transfection,

medium was replaced with DMEM or MEM supplemented with antibiotics and 10% (v/v)

fetal bovine serum (FBS) for 18 hours. Cells were then serum-deprived for 24 hours before

stimulation(s).

IL-8 ELISA- ASM cells were stimulated for 24 hours with IL-1β. Supernatants were

collected and IL-8 concentrations were determined by ELISA as per manufacturer's

instructions (Sanquin, the Netherlands).

Chapter 6

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Table 1: Primers used for WNT pathway gene expression analysis

Gene Forward 5’ ��3’ Reverse 5’ ��3’

WNT-1 ATTTTGCGCTGTGACCTCTT AGCAACCTCCTTTCCCACTT

WNT-2 GAAAGGAAGTGCCAAGGACA CCTTCCTTCCAGCTCTGTTG

WNT-2B CGAGGTGGCAAACATCCTAT CTTTGAAGGCTCCACTCCTG

WNT-3 CTCGAGGCTTACCTTTGCAC AGGCAGCTTGGACACAGAAT

WNT-3A GTGCACACCTGCAAGTAGGA TCTCCGCCCTCAAGTAAGAA

WNT-4 CTGGAGAAGTGTGGCTGTGA CAGCCTCGTTGTTGTGAAGA

WNT-5A CAAATAGGCAGCCGAGAGAC CTCTAGCGTCCACGAACTCC

WNT-5B GGTTCCACTGGTGTTGCTTT AGACTTTTGTGAGGCGGAGA

WNT-6 TTCGGGGATGAGAAGTCAAG AAAGCCCATGGCACTTACAC

WNT-7A GGTCAGATCACAGGCAGGAT TGCAGGAAACCCAGAATACC

WNT-7B TCCTTGCAGAACTCGAGGAT GCCTGACACAAGGGACATTT

WNT-8A AACGGTGGAATTGTCCTGAG GGTGACTGCGTACATGATGG

WNT-8B GTAGGCAGGTTGGCAATCAT TCCTTCCTATGCCCTTCCTT

WNT-9A TCGTGGGTGTGAAGGTGATA TGGCTTCATTGGTAGTGCTG

WNT-9B CTTCTCTGGGACGACTTTGC GGTTGCCAAGGAACACATCT

WNT-10A ATTGACATTCCTCCGCTCAC TGGGGAAGGGAAGAAGAGAT

WNT-10B CCACTGGTGCTGTTATGTGC AGCCCTCACACAGTGCTTCT

WNT-11 ACCCCTACACGGACAGAGTG AGGGAAGTCCACCAAGGTCT

WNT-16 GAGCTGTGCAAGAGGAAACC GCGACCATACAGTTCCACCT

FZD1 CAAGGTTTACGGGCTCATGT GTAACAGCCGGACAGGAAAA

FZD2 CCGTCTCTGCATCCTCACAT TAGCAGCCGGACAGAAAGAT

FZD3 GAAGCAAAGCAGGGAGTGTC ATGCTGCCGTGAGGTAGTCT

FZD4 CTGCAGCATGCCTAATGAGA CGTCTGCCTAGATGCAATCA

FZD5 AGCCAAATGAGGCACATACC TCTCCTTTTGCGAGCGTTAT

FZD6 TCCGACGCTTGAAGAAAACT CAACCCCAGGTCCTCAAGTA

FZD7 ATCATCTTCCTGTCGGGTTG AAGCACCATGAAGAGGATGG

FZD8 TCCGTTCAGTCATCAAGCAG CGGTTGTGCTGCTCATAGAA

FZD9 CCAGCTGTCAAGGTCAGACA CACTCCCTGCATGAGACAGA

FZD10 TGGTACGCATAGGGGTCTTC TCAGGCAGTCAGGTGTCTTG

SFRP1 ATCCCCCTCTTTCTGCCTTA GGCCAGTTCAAAAGCTGAAG

SRFP2 ACGACAACGACATCATGGAA ACGCCGTTCAGCTTGTAAAT

WIF1 GAGTGTCCGGATGGGTTCTA AGCAGGTGGTTGAGCAGTTT

DKK1 CAGCTCAATCCCAAGGATGT CAGGGGAGTTCCATCAAGAA

CTNNBIP1 (ICAT) TTGAACCCAAGCAGATACCC TCCCCCTTCTATGCTGACAC

β-catenin AGGGTGGGAATGGTTTTAGG GTGGCAAAAACATCAACGTG

Axin2 GTGGAGAGGATCGACTGAGC GCCCAGAGTGGTCATGTTTT

Wisp1 GGGTCTTCCCAGGTATCCAT TTCCTTGAACCAGGCCATA

18S AAACGGCTACCACATCCAAG CCTCCAATGGATCCTCGTTA


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Data Analysis- Values reported for all data are represented as mean ± SEM. The statistical

significance of differences between means was determined on log transformed data by

Student’s t-test or by 1-way ANOVA, followed by Student-Newman Keuls multiple

comparisons test. Differences were considered to be statistically significant when p<0.05.

Results

Inflammation and remodeling in the mouse model of allergen-induced chronic

airway inflammation. Persistent chronic inflammation and airway remodeling are

important characteristics of asthma. We assessed these parameters in our animal model to

ascertain the presence of asthma-like characteristics. OVA treatment induced eosinophilia

as observed by a massive increase in the number of eosinophils in the lungs of OVA-

challenged mice underlining the presence of inflammation in the animal model (Figure 2A).

Similarly, a profound increase in the α-smooth muscle-actin positive area surrounding the

airways was observed in OVA-challenged mice in comparison to saline-challenged mice

underlining increased ASM mass (Figure 2B). Taken together, our data shows the presence

of inflammation and airway remodeling post-OVA challenge validating our animal model

used in this study.

Figure 2. Morphometric analysis of the lungs. Animals were treated as described in Figure

1. Lungs were harvested 24 hours after the last challenge. (A) Eosinophils were analysed using

DAB, counted and expressed as number per mm of basal membrane (n=8 each group).

Quantification and representative image is shown here. (B) α-Smooth muscle-actin is stained using

specific antibody to determine airway smooth muscle mass. Positively stained area is quantified

and expressed as per mm2 of basement membrane (n=8 per group). Data represent mean ±SEM.

**p< 0.01, ***p<0.001 compared to saline-challenged control mice; 2-tailed Student’s t test for

unpaired observations. Magnification 200X.

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Extensive modulation of WNT signaling pathway by allergen challenge.

Having validated our animal model, we next screened the lung homogenates for mRNA

expression of various components of the WNT signaling pathway. OVA challenge led to a

wide ranging modulation in gene expression of WNT ligands. WNT-1, -3, -3A, -5A, -5B, -7B,

-9B, -10A, -11 and -16 showed varying degree of up or downregulation following OVA

challenge but the observed changes failed to achieve statistical significance. On the other

hand, WNT-7A, -9A and -10B were significantly downregulated upon OVA exposure in

comparison to the saline-challenged group (Figure 3A, 3B). Similarly, FZD4 and FZD6

expression were significantly downregulated by OVA exposure whereas other FZD receptors

remained either unaltered or showed minimum modulation (Figure 3C, 3D). The canonical

WNT signaling mediator-β-catenin, showed modest but significant reduction following OVA

challenge whereas Axin2, a member of the β-catenin destruction complex, remained

unchanged (Figure 3E). The expression of secreted FZD related protein (SFRPs) and

Dickkopf1 (DKK1) which function as extracellular inhibitors of WNT signaling. SFRP1

showed a trend of increased expression upon OVA challenge whereas SFRP2 and DKK1

didn’t change (Figure 3F). Similarly, the intracellular inhibitor of β-catenin-mediated

transcription- inhibitor of β-catenin and TCF (ICAT), also remained unaltered. One of the

most important observations came from the analysis of WIF1 which showed a significant

decline in response to OVA challenge (Figure 3F). WIF1 is an extracellular WNT inhibitor

which antagonizes WNT signaling by binding to various WNT ligands.

Since the reduction in WIF1 was among the most significant changes, we further expanded

the mRNA observations at protein level. We performed immunofluorescence microscopy on

lung cryosections using WIF1-specific antibody which indicated a reduction in WIF1

abundance in the OVA-challenged airways as compared to saline-challenged group (Figure

3G).

Thus, our data suggest a widespread modulation of WNT signaling pathway components in

the lungs of OVA-challenged animals with a significant decline in expression of the WNT

antagonist WIF1 among others.

Functional significance of WIF1 downregulation in asthma. We directed our

focus on WIF1 which was significantly downregulated by OVA-challenge. We hypothesized

that reduced abundance of WIF1 releases its target WNT ligands from inhibition and allows

enhanced intracellular WNT signaling, irrespective of their expression levels. To this aim,

we first analyzed the activation of β-catenin signaling. As revealed by immunofluorescence

staining, β-catenin shows a clear localization to the lateral membrane of airway epithelial

cells in the saline-challenged group, in line with its established role as a component of

adherens junctions (Figure 4A). Interestingly, OVA challenge completely altered the

membrane localization of β-catenin in the airway epithelial cells with the lateral staining

completely lost and replaced by a less clear apical staining pattern (Figure 4A). Of note, we

also failed to detect the nuclear localization of β-catenin, a marker of active β-catenin

signaling, post-OVA challenge. We also performed western blot analysis to detect any

changes in β-catenin signaling. As shown in Figure 4B, there was no difference in abundance


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Frizzled receptors

1 2 3 4 5 6 7 8 9 10

Re

lativ

e m

RN

A e

xpre

ssi

on

(fo

ld o

f re

spe

ctiv

e s

alin

e)

0

1

2

3

4

5 Saline

Ova

** *

WNT Ligand

1 2 2B 3 3A 4 5A 5B 7A 7B 8A 8B 9A 9B 10A 10B 11 16

Re

lative

mR

NA

exp

ressi

on

(fo

ld o

f re

sp

ective

sa

line

)

0

1

2

3

4

5

6 Saline Ova

* *** **

WNT Ligand

1 2 2B 3 3A 4 5A 5B 7A 7B 8A 8B 9A 9B 10A 10B 11 16

Re

lative

mR

NA

exp

ress

ion

(fold

of W

NT

-9A

)

0

2

4

6

8

10 Saline Ova

*

***

**

Frizzled receptors

1 2 3 4 5 6 7 8 9 10

Re

lative

mR

NA

exp

ressio

n(f

old

of F

ZD

5)

0

1

2

3

4

15

20

25 Saline

Ova

** *

A

B

C D

Chapter 6

184 | P a g e

of total β-catenin in lung homogenates obtained from saline- and OVA-challenged groups.

Interestingly, the nonphospho β-catenin which represents the active fraction of β-catenin

showed a modest increase in the lung homogenates of OVA-challenged group as revealed by

quantification of the westerns. Though the data indicate a possible activation of β-catenin

signaling, they remain inconclusive due to lack of any statistical significance in the effects.

Figure 3. Expression analysis of WNT family members. Animals were treated as described

in Figure 1. Lungs were harvested 24 hours after the last challenge. Total RNA was isolated from

whole lung homogenates and mRNA expression was determined using qRT-PCR. Expression of

genes is represented as Cq-values corrected for 18S rRNA. WNT ligands mRNA abundance as fold

of respective saline-challenged control (A) or relative to WNT-9A (B). FZD receptor mRNA

expression as fold of respective saline-challenged control mice (C) or relative to FDZ5 abundance

(D). (E, F) Expression of various WNT pathway components relative to saline-challenged control

mice as depicted in the respective panels. Data represent mean ±SEM; n=10 each group. *p<0.05,

**p<0.01, ***p<0.001 compared to respective saline-challenged control mice; 2-tailed Student’s t

test for unpaired observations. (G) Animals were treated as described in Figure 1. Lungs were

harvested 24 hours after the last challenge. Cryosections were prepared and immunofluorescence

microscopy was performed as described in material and methods. Representative image showing

WIF1 staining in saline- and OVA-challenged mice. Magnification 40X.

β-catenin Axin2

Re

lative

mR

NA

exp

ress

ion

(fo

ld o

f sa

line

)

saline Ova

0.0

0.5

1.0

1.5

2.0

*

E

SFRP1 SFRP2 WIF-1 WISP ICAT DKK-1

Re

lative

mR

NA

exp

ressio

n(f

old

of salin

e)

0

1

2

3saline

Ova

***

F


185 | P a g e

Figure 4. Effect of OVA exposure on β-catenin protein levels. (A) Animals were treated as

described in Figure 1. Lungs were harvested 24 hours after the last challenge. Cryosections were

prepared and immunofluorescence microscopy was performed as described in material and methods.

Representative image showing β-catenin staining in saline- and OVA-challenged mice. Magnification

40X. (B) Total and active β-catenin expression levels were analysed in whole lung extracts by western

blotting. Equal protein loading was verified by analysis of β-actin. Abundance of total and active β-

catenin bands is quantified and expressed as percentage of saline-challenged controls. Data represent

mean ±SEM; n=4 each group.

We expanded our investigation in vitro using ASM and HBE cell lines to identify which

compartments of lungs are contributing to the observed reduction in WIF1 expression in

OVA-challenged group. TGF-β is the most prominent cytokine which is increased in

asthmatic airways and regulates various aspects of asthma pathophysiology [18-21].

Interestingly, TGF-β significantly reduced WIF1 mRNA expression in ASM cells but not in

HBE cells underlining the cell-dependent effects (Figure 5A, 5B). Additionally, HBE cells

were treated with house dust mite extract (HDM), a common allergen, which also failed to

alter WIF1 mRNA levels (Figure 5B).

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186 | P a g e

Our previous study has shown that TGF-β induces expression of WNT-5A in ASM cells where

it mediates expression of TGF-β-induced ECM proteins (Chapter 3). As WIF1 can

antagonize WNT-5A and as observed, TGF-β decreases WIF1 expression in ASM cells (Figure

5A), we investigated the possible role of WIF1 in TGF-β-induced ECM expression. We

observed that the presence of recombinant WIF1 had no effect on TGF-β-induced collagen

1α1 and fibronectin expression in ASM cells (Figure 6A). Similarly, exogenous WIF1 failed to

affect other key TGF-β responses in ASM cells such as upregulation of WNT-5A and

reduction of SMAD3 expression (Figure 6B, 6C). An increase in WNT-5A message in ASM

cells appears only after 12 hours of TGF-β stimulation (Chapter 3), so we decided to

coincide WIF1 treatment with the time of TGF-β-induced WNT-5A expression. ASM cells

were first stimulated with TGF-β for 12 hours following which recombinant WIF1 was added

and cells were further incubated for 12 hours. However, we failed to observe any effect of

exogenous recombinant WIF1 on TGF-β-induced expression of collagen and fibronectin

(Figure 6D). Considering that complete absence of serum might have some effect on the

stability or function of WIF1 directly or indirectly, we decided to tweak our experimental

conditions. Instead of serum-free media, stimulations were carried out using a medium

supplemented with 0.5% serum and ASM cells treated with or without TGF-β and/ or

recombinant WIF1. However, presence of exogenous WIF1 didn’t change the expression of

TGF-β-induced ECM (Figure 6E).

Using a loss-of-function approach, we observed that knock-down of WIF1 using specific

siRNA had no effect on collagen Iα1 or fibronectin expression in response to TGF-β in ASM

cells (Figure 7A). In a reciprocal approach, we overexpressed WIF1 in ASM cells and

evaluated expression of TGF-β-induced ECM proteins. Following transfection with Myc-

Figure 5. WIF1 expression in ASM and HBE cells. (A) ASM cells were stimulated with TGF-

β (2 ng/ml) and (B) HBE cells were stimulated with TGF-β (2 ng/ml) or HDM extract (30 µg/ml)

for 24 hours. Expression of WIF1 mRNA was determined by qRT-PCR. Data represents mean ±

SEM of 4 independent experiments. *p<0.05 compared to untreated control; 2-tailed Student’s t-

test for paired observations.

A

0.0

0.5

1.0

1.5

2.0

Basal TGF-β

WIF

1 m

RN

A e

xp

ress

ion

(fo

ld o

f b

asa

l)

*

ASM cells

B

0.0

0.5

1.0

1.5

2.0

Basal TGF-β HDM

WIF

1 m

RN

A e

xpre

ssio

n (

fold

of

ba

sal)

HBE cells


187 | P a g e

WIF1, the WIF1 message showed ~26000 fold increase at basal level (Figure 7B). However,

similar to knock-down, WIF1 overexpression failed to alter collagen or fibronectin

expression in response to TGF-β (Figure 7C).

WNT signaling has recently been linked to Th2 inflammation in asthma subjects [15]. So, we

investigated whether WIF1 participates in the inflammatory responses in ASM cells. IL-1β

induces IL-8 secretion in ASM cells [22,23]. However, WIF1 overexpression had no effect on

IL-1β-induced IL-8 release in ASM cells (Figure 8).

Taken together, our comprehensive analysis revealed that WIF1 had no effect on TGF-β-

induced ECM expression and doesn’t participate in regulating IL-1β-induced inflammatory

responses in ASM cells.

Discussion

In the current study, we evaluated alterations in the expression of WNT pathway

components using an animal model of allergen-induced chronic airway inflammation. We

demonstrate a broad modulation of WNT family gene expression levels post-OVA challenge.

Most importantly, we identified a significant decrease in WIF1 in OVA-challenged lungs

compared to the saline-challenged controls. Moreover, functional studies using gain- and

loss-of-function approaches revealed that WIF1 doesn’t participate in TGF-β-induced ECM

production or IL-1β-induced IL-8 release in ASM cells.

Aberrant WNT signaling activation has been reported in several fibroproliferative disorders.

For instance, increased expression of WNT signaling pathway genes and enhanced nuclear

abundance of β-catenin is observed in fibroproliferative diseases of kidney, liver and bone

[24-27]. Similarly, expression of several WNT signaling pathway genes such as WNT-1,

WNT-7B, WNT-10B, FZD2, FZD3, β-catenin and LEF1 and nuclear localization of β-catenin

are augmented in idiopathic pulmonary fibrosis patients [28,29], supporting a

comprehensive role for WNT signaling in fibroproliferative disorders, including those of the

lung. We have recently shown that WNT-5A expression is increased in ASM cells derived

from asthmatic patients in comparison to healthy subjects. WNT-5A is also overexpressed in

fibroblasts of patients with usual interstitial pneumonia [30]. Here, we present evidence for

a wide range modulation of WNT signaling pathway in asthmatic lungs obtained from the

mouse model of allergen-induced chronic airway inflammation. WNT ligands exhibit

extensive up and downregulation in expression, with significant downregulation of WNT-

7A, WNT-9A and WNT-10B in OVA-challenged group in comparison to the saline-

challenged control group. Similarly, FZD receptors and other components of WNT pathway

examined also showed widespread modulation in expression post-allergen challenge.

Importantly, basal expression of WNT-5A was one of the highest in mice lungs with a trend

towards upregulation post-allergen challenge. WNT-5A along with WNT-5B and WNT-11

and FZD8 are targets of TGF-β in ASM cells as we reported previously. However, lack of a

specific TGF-β signature on WNT ligands and FZD receptor expression in the lungs obtained

from mouse model could be attributed to the use of whole lungs instead of isolated

compartments in this analysis. The effect of TGF-β on WNTs in ASM might be different from

its effect in epithelial cells or fibroblasts.

Chapter 6

188 | P a g e

Figure 6. Effects of exogenous WIF1 on TGF-β cellular responses. (A-C) ASM cells were

stimulated with TGF-β (2 ng/ml) in the presence or absence of recombinant WIF1 (3 µg/ml) for 24

hours. Expression of collagen 1α1, fibronectin and PAI1 mRNA (A), WNT-5A mRNA (B) and

SMAD3 mRNA (C) was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to

the untreated control. Data represent mean ±SEM of 3-7 independent expriments. *p<0.05,

**p<0.01, ***p<0.001 compared to untreated control; 1-way ANOVA followed by Newman-Keuls

multiple comparisons test. (D) ASM cells were stimulated with TGF-β (2 ng/ml) for 12 hours

following which recombinant WIF1 (3 µg/ml) was added and cells were further incubated for 12

hours. Expression of collagen 1α1, fibronectin mRNA was determined by qRT-PCR, corrected for

18S rRNA and expressed relative to the untreated control. Data represent mean ±SD of 2

Basal TGF-β Basal TGF-β Basal TGF-β

Extr

ace

llula

r m

atr

ix g

en

e e

xpre

ssi

on

(fo

ld o

f b

asa

l)

0

2

4

6

8

10

12 Vehicle

Rec. WIF1 (3 µg/ml)

Collagen 1α1 Fibronectin PAI-1

*** ***

*** ***

***

***

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Basal TGF-β

WN

T-5

A m

RN

A e

xpre

ssio

n (

fold

of

ba

sal)

Vehicle


**

**

0.0

0.5

1.0

1.5

2.0

Basal TGF-β

SM

AD

3 m

RN

A e

xpre

ssio

n (

fold

of

ba

sal)

Vehicle


* **

B

C D

E

Basal TGF-β Basal TGF-β

Extr

acellu

lar

matr

ix g

en

e e

xpre

ssio

n(f

old

of b

asal)

0

1

2

3

4

5

6 Vehicle

Rec. WIF1 (3 µg/ml; 12 hours)

Collagen 1α1 Fibronectin


Ext

race

llula

r m

atr

ix g

ene

expre

ssio

n(f

old

of

ba

sal)

0

1

2

3

4 Vehicle




189 | P a g e

independent experiments. (E) ASM cells were stimulated with TGF-β (2 ng/ml) in the presence or

absence of recombinant WIF1 (3 µg/ml) for 24 hours. DMEM supplemented with 0.5% FBS is used

for all treatment conditions. Expression of collagen 1α1, fibronectin mRNA was determined by

qRT-PCR, corrected for 18S rRNA and expressed relative to the untreated control. Data represent

mean ±SD of 2 independent experiments.

Figure 7. Effects of WIF1 loss- or gain-

of-function in ECM production in ASM

cells. (A) ASM cells were transfected with

WIF1-specific siRNA or a negative (control)

siRNA as control. Subsequently, cells were

stimulated with TGF-β (2 ng/ml) for 24

hours. Expression of collagen IαI and

fibronectin mRNA was analyzed by qRT-

PCR and expressed relative to negative

siRNA-transfected, untreated control. Data

represent mean ±SD of 2 independent

experiments. (B, C) ASM cells were

transfected with Myc-WIF or GFP as

control. Subsequently, cells were stimulated

with TGF-β (2 ng/ml) for 24 hours. Expression of WIF1 mRNA (B) and collagen IαI and fibronectin mRNA (C) was analyzed by qRT-

PCR and expressed relative to GFP-transfected, untreated control. Data represent mean ±SD of 2

independent experiments.

Earlier studies have provided some evidence about the possible involvement of WNT

signaling in asthma. Airway remodeling is a critical feature of the asthmatic airway which

involves extensive structural changes in the affected airway [1,2]. These changes include

ASM hypertrophy and hyperplasia, epithelial damage, basement membrane thickening and

altered and augmented ECM deposition [1,2]. A study from our lab showed that β-catenin

mediates TGF-β-induced expression of ECM expression in ASM cell, implicating cross-talk

between TGF-β and canonical WNT signaling mediator to regulate airway remodeling [31].

A


Ext

racellu

lar

ma

trix

ge

ne

exp

ress

ion

(fold

of

no

nta

rge

ting

siR

NA

)

0

1

2

3

4

5

6 Negative siRNA

WIF1 siRNA


GFP Myc-WIF1

WIF

1 m

RN

A e

xpre

ssio

n

(fold

of

GF

P-t

ransfe

cted

)

0

1

2

20000

30000

40000

B


Ext

racellu

lar

ma

trix

ge

ne e

xpre

ssio

n(f

old

of

GF

P-t

ran

sfe

cte

d b

asal)

0

2

4

6

8

10 GFP

Myc-WIF1


C

Chapter 6

190 | P a g e

Figure 8. Functional analysis of WIF1 in

inflammation in ASM cells. ASM cells were

transfected with Myc-WIF or GFP as control.

Subsequently, cells were stimulated with IL-1β

(1 ng/ml) for 24 hours. IL-8 abundance was

measured in cell supernatants by ELISA. Data

represent mean ±SEM of 3 independent

expriments. ***p<0.001 compared to GFP-

transfected, untreated control; 1-way ANOVA

followed by Newman-Keuls multiple

comparisons test.

TGF-β is a pleiotropic cytokine and highly increased in asthmatic lung tissue and broncho-

alveolar lavage (BAL) fluid and functions as master regulator of airway remodelling in

asthma [16,19-21]. While accumulating evidence have key roles for β-catenin in airway

remodeling (Chapter 2), a direct link between canonical or noncanonical WNT signaling

with asthma is not known. Increased expression of WNT signaling pathway members in

airway epithelium and endobronchial biopsies of asthma patients, including WNT-5A, have

been reported although the cellular localization and functional roles of these WNT ligands

were not determined [15,32]. Here, we demonstrate a direct link with WNT signaling family

modulation using our animal model.

Furthermore, we demonstrate a significant decrease in WIF1 mRNA levels in the lungs of

OVA-challenged mice as compared to saline-challenged mice. WIF1 is an evolutionary

conserved protein that can bind to and antagonize WNT signaling. WIF1 has been shown to

bind to WNT-3A, -4, -5A, -7A, -9B and -11 via its WIF domain with varying affinities (WNT-

5A>WNT-9B>WNT-11>WNT-4>WNT-7A>WNT-3A) [33]. WIF1 binds to WNT ligands via

its WIF domain and tethers this WIF1-WNT complex in the ECM by its interaction with the

heperan sulphate proteoglycan- glypican via epidermal growth factor-like domains [34].

Indeed, glypicans can have modulatory effects on WNT signaling [35] and formation of this

WNT-WIF1-glypican complex is required for complete WNT antagonizing activity of WIF1

[34]. Of note, the observed suppression of WIF1 is particularly important as single nucleotide

polymorphisms in WIF1 associate with lung function in two cohorts of asthma patients (14).

Moreover, WIF1 is a target of bone morphogenetic protein 4 (BMP4)-SMAD1 signaling in

developing lungs where it antagonizes WNT/β-catenin signaling and regulates lung

morphogenesis by fine tuning the WNT and BMP signaling. Abrogation of WIF1 expression

leads to severe fetal lung abnormalities, in part, due to hyperactivation of WNT/β-catenin

signaling [36]. In line with this, a study has demonstrated downregulation of SMAD1 and

WIF1 expression during the saccular stage of lung development in mouse model of

congenital diaphragmatic hernia, leading to the retardation of lung morphogenesis and

appearance of hypoplastic lung [37]. Owing to the crucial role of WIF1 in WNT signaling

Basal IL-1β

IL-8

secre

tion (

pg/m

l)

0

150

300

450

100000

150000

200000GFP

Myc-WIF1

******


191 | P a g e

regulation, suppression of WIF1 is often associated with malignancies such as prostate,

breast, lung, and bladder cancer [38]. Thus, WIF1 downregulation could lead to disruption

of lung homeostasis both during embryogenic and adult life.

Lungs are highly specialized organ composed of many different compartments. Epithelium

and ASM represent important structural and functional units of the lung. Epithelium

constitutes the barrier between airways and the external environment and also serves as the

first line of defense. ASM cells, on the other hand, provide mechanical properties to the

airways and maintain bronchial tone. Both epithelium and ASM cells can respond to a range

of growth factors and other substances present in the lung and as such actively participate,

not only in maintenance of lung homeostasis but also in respiratory disorders such as

asthma. In fact, anomalies in epithelial and ASM bundle are prominent features of a

remodeled airway in asthma [1,2,39]. We attempted to identify the lung compartment(s)

contributing to the reduction WIF1 abundance in OVA-challenged lung. We here

demonstrate that TGF-β suppresses WIF1 expression in ASM cells but not in HBE cells. Since

epithelial cells are exposed to external environmental allergens, we treated HBE cells with

HDM extract, a common aeroallergen. HDM extract alone or in combination with TGF-β

failed to convincingly reduce WIF1 mRNA levels in HBE cells (Figure 3B; data not shown)

suggesting a highly context dependent regulation of WIF1 expression. Moreover, basal

abundance of WIF1 transcripts is much higher in ASM cells as compared to HBE cells. In

view of these observations, reduced expression of WIF1, as seen in OVA-challenged lungs,

might be attributed to the ASM cells.

We have recently demonstrated that WNT-5A regulates TGF-β-induced ECM expression in

ASM cells (Chapter 3). We hypothesized that downregulation of WIF1 by TGF-β in ASM

cells could be an important mechanism for allowing maximum WNT-5A signaling to drive

ECM expression. However, the presence of exogenous WIF1 didn’t affect TGF-β-induced

ECM expression in ASM cells. Similarly, loss of WIF1 using siRNA or overexpression of WIF1

didn’t show substantial effects on TGF-β-induced ECM expression negating our preliminary

hypothesis about the role of WIF1 in TGF-β responses. However, the observations from WIF1

loss-of-function and gain-of-function investigation should be viewed with caution owing to

insufficient replicates for statistical analysis. Further experiments are warranted to

determine the functional significance of WIF1 downregulation in ASM cells.

Epigenetic mechanisms and posttranscriptional mechanisms can regulate WIF1 expression

levels. DNA methylation is an epigenetic modification that renders target gene inactive.

Promoter hypermethylation is associated with WIF1 suppression in lung [40], breast cancer

[41] and astrocytomas [42]. Further, a study demonstrated that miRNA-374a can target

WIF1 transcripts therby limiting its expression in breast cancer cells [43]. Interestingly TGF-

β has been shown to induce expression of DNA methyltransferases (DNMTs), enzymes

involved in DNA methylation, in prostate cancer cells to suppress its own signaling [44]. On

the contrary, TGF-β downregulates DNMTs in cardiac fibroblasts thereby reducing collagen

type I promoter methylation and allowing its subsequent expression [45] highlighting the

context-dependent regulation of DNA methylation by TGF-β. How TGF-β induces WIF1

Chapter 6

192 | P a g e

suppression in ASM cells and whether any epigenetic mechanisms are involved or not needs

further investigation.

Taken together, the data presented in current study establish that the WNT signaling

pathway is extensively modulated in response to allergen challenge. Of note, downregulation

of WIF1 by TGF-β may represent a detrimental event which may allow increased WNT

signaling in the lungs driving asthma pathological manifestations. The inference, however,

be taken with caution as more experiments are needed to substantiate the findings.

Acknowledgements

This study was supported by a Vidi Grant (grant nr. 016.126.307) from the Dutch

Organization for Scientific Research (NWO) to R. Gosens. The funders had no role in study

design, data collection and analysis, decision to publish, or preparation of the manuscript.


193 | P a g e

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