Osteoarthritis and Cartilage 24 (2016) 158e168

Intermittent cyclic mechanical tension promotes endplate cartilage

degeneration via canonical Wnt signaling pathway and E-cadherin/
b-catenin complex cross-talk
H.-g. Xu y *, Q. Zheng y, J.-x. Song z, J. Li x, H. Wang y, P. Liu y, J. Wang k, C.-d. Wang k,
X.-l. Zhang k **
y Department of Orthopedic Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, 241001, Anhui, China
z Department of Orthopedic Surgery, Chinese Medicine Hospitals of Wuhu, Wuhu, 241001, Anhui, China
x Department of Cell Biology and Genetics, Zunyi Medical College, Zunyi, 563003, Guizhou, China
k Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai, 200011, China

a r t i c l e i n f o s u m m a r y

Article history: Objective: This study aimed to investigate the role of the Wnt/b-catenin signaling pathway and E-cad-
Received 11 November 2014 herin/b-catenin complex in intermittent cyclic mechanical tension (ICMT)-induced endplate cartilage
Accepted 27 July 2015 degeneration.
Design: b-Catenin expression was measured in disc samples obtained from patients with disc degen-
Keywords: eration and those with cervical vertebrae fracture or dislocation. Histological staining was performed to
Mechanical tension
examine the disc tissue morphology and extracellular matrix after application of ICMT in vitro and in vivo.
Endplate cartilage
Multiple strategies were employed to examine activation of Wnt/b-catenin signaling after ICMT appli-
Degeneration
b-catenin cation in vivo and in vitro. Co-immunoprecipitation was performed to examine the interaction between
E-cadherin E-cadherin and b-catenin. Pathway-specific inhibitors and an E-cadherin expression plasmid were used
to regulate Wnt/b-catenin signaling and E-cadherin expression.
Results: b-Catenin protein expression was elevated significantly, whereas cartilaginous genes were
down-regulated in endplate cartilage samples obtained from patients with disc degeneration. ICMT
loading led to Wnt/b-catenin signaling activation and the loss of the chondrogenic phenotype of end-
plate chondrocytes in both an in vivo rabbit model and in vitro endplate chondrocyte culture system.
Inhibition of Wnt/b-catenin signaling suppressed the decrease in ICMT-induced cartilaginous gene
expression. Furthermore, E-cadherin expression was inhibited by ICMT stimulation, resulting in a
decrease in the interaction between E-cadherin and b-catenin proteins. Over-expression of E-cadherin
rescued the cartilaginous gene expression by enhancing the interaction between E-cadherin and b-
catenin proteins.
Conclusions: ICMT promotes endplate cartilage degeneration via activation of Wnt/b-catenin signaling
and suppression of physical proteineprotein interactions between E-cadherin and b-catenin.
© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Introduction

Back and neck pain is the most common chronic condition in

* Address correspondence and reprint requests to: H.-g. Xu, Department of Or-
thopedic Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, 241001,
Anhui, China. Tel: 86-0553-5739037.
** Address correspondence and reprint requests to: X.-l. Zhang, Shanghai Key
Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai
Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai,
200011, China. Tel: 86-021-54923338.
E-mail addresses: xuhg@medmail.com.cn (H.-g. Xu), xlzhang@sibs.ac.cn
(X.-l. Zhang).
adults. Intervertebral disc (IVD) degeneration is considered to be
the primary reason for back and neck pain1. Endplate cartilage is an
important structure of IVDs, which is a thin layer hyaline cartilage
between the vertebral body and IVD. It is the gateway of nutrient
transport between the vertebral marrow and IVD2. In addition,
endplate cartilage is a mechanical interface between vertebral and
resilient discs. It contributes to even distribution of the

http://dx.doi.org/10.1016/j.joca.2015.07.019
1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168
compressive load across the vertebral body3. Numerous studies
have demonstrated that cartilaginous endplate degeneration plays
a key role in the process of IVD degeneration4,5.
Biomechanical loading, which is transduced through cell adhe-
sion via intracellular signaling, is one of the major factors that
determine cartilage development and function6. A long duration of
mechanical loading promotes cartilage damage7,8. Some studies
indicate that dynamic mechanical loading may lead to IVD degen-
eration and morphological changes in endplate cartilage9. There-
fore, the role of mechanical force cannot be ignored in endplate
cartilage degeneration. Our previous study showed that intermit-
tent cyclic mechanical tension (ICMT) induces endplate chon-
drocyte calcification and decreases the expression chondrocyte
anabolic genes such as type II collagen (COL-2A), aggrecan (ACAN),
and Sox910e12. However, the detailed molecular signaling mecha-
nisms by which mechanical stimulation affects endplate cartilage
degeneration are still unclear.
Wnt signaling plays a central role in regulating the develop-
ment of many tissues and organs, and alterations in this pathway
are commonly associated with human disease. The Wnt/b-catenin
signaling pathway, often referred to as the canonical Wnt
pathway, is activated upon binding of the Wnt protein to a re-
ceptor complex including Frizzled receptors and low-density li-
poprotein receptor-related protein 5/6 on the cell surface13. In the
absence of Wnt ligands, b-catenin is phosphorylated by glycogen
synthase kinase 3 (GSK3) and targeted for ubiquitin-dependent
proteolysis14. However, in the presence of a Wnt ligand, which
inhibits GSK3 activity15, b-catenin accumulates and translocates to
the nucleus where it combines with T cell factor/lymphoid
enhancer factor (TCF/LEF) and transactivates target gene pro-
moters16,17. Therefore, b-catenin is a key molecule in the canonical
Wnt signaling pathway and plays a critical role in the pathogen-
esis of IVD degeneration. Activation if b-catenin signaling in
articular chondrocytes leads to differentiation of chondrocytes
with an osteoarthrosis-like phenotype18. In addition, activation of
Wnt/b-catenin promotes IVD degeneration by modulating the
expression of matrix metalloproteinases and transforming growth
factor-b1 in nucleus pulposus cells19.
Traditional cadherins, such as E-cadherin and N-cadherin, are
transmembrane proteins that mediate cellecell adhesion. Cadher-
ins bind to b-catenin and form a complex called the cadherin-b-
catenin complex20. This complex acts as a sensitive mechano-
transducer to maintain cell differentiation and the synthesis of
extracellular matrix proteins by connecting to the cytoskeleton
inside the cell and regulating intracellular signaling pathways21,22.
A previous study has suggested that the presence of E-cadherin in
the cytoplasm is required to inhibit Wnt/b-catenin-dependent gene
expression23. However, the relationship between the E-cadherin/b-
catenin complex and mechanical tension-induced endplate carti-
lage degeneration has not been explored yet.
To our knowledge, there are currently no studies that have
investigated the regulatory role of Wnt/b-catenin signaling or the
E-cadherin/b-catenin complex during ICMT-induced endplate
cartilage degeneration. In the present study, we investigated the
relationship between the canonical Wnt signaling pathway and
endplate cartilage degeneration during application of ICMT, and
further explored the regulatory role of the E-cadherin/b-catenin
complex in ICMT-induced endplate cartilage degeneration.
Methods
Reagents and E-cadherin expression plasmid transfection
In some experiments, endplate chondrocytes were treated with
10 mM XAV-939 (Wnt/b-catenin signaling inhibitor; Selleckchem,
159
USA) during ICMT application24. The pEGFPN1-E-cadherin expres-
sion plasmid, which contained the rat E-cadherin-coding sequence
with insertion of green fluorescent protein (GFP) at the C-terminal,
was transiently transfected into endplate chondrocytes using Lip-
ofectamine 2000 (Invitrogen, Carlsbad, CA). At 24 h post-
transfection, the cells were exposed to mechanical loading.
Patient samples
Endplate cartilage samples were obtained from 20 patients who
underwent open vertebral surgery at our department. The control
group included eight patients (mean age: 38 years; two females
and six males) who underwent vertebral surgery for cervical
vertebrae fracture or dislocation. Twelve patients were included in
the experimental group (mean age: 52 years; five females and
seven males) who underwent vertebral surgery for cervical spon-
dylosis. No subject had a tumor, tuberculosis, diabetes, infectious
disease, or bone metabolic disease. Magnetic resonance imaging
(MRI) was routinely performed before surgery. MRIs revealed that
all discs in the experimental group were classified as grades IIeIII
according to Miller's classification, whereas the control group had
no evident degeneration [Fig. 1(A)]. This study was approved by the
Ethics Committee of Yijishan Hospital of Wannan Medical College.
Informed consent was obtained from all participants.
Application of mechanical tensile strain in vivo
For in vivo experiments, 50 New Zealand white rabbits (skele-
tally mature, at least 6 months old, and weighing 3.2 ± 0.8 kg) were
subjected to general anesthesia and surgery as described previ-
ously25,26. The rabbits were randomly assigned to five groups: 0-
day control group (no treatment, n ¼ 10); 28-day (n ¼ 10) and
56-day (n ¼ 10) sham groups (animals underwent a sham operation
but no mechanical tensile strain application); 28-day (n ¼ 10) and
56-day (n ¼ 10) loaded groups [discs were subjected to 2.4 MPa
external tensile loading during daily activity of the animals using an
external loading device [Fig. 1(B)]]. Mechanical tensile strain was
applied for 8 h a day.
Chondrocyte isolation and mechanical tensile strain application
in vitro
Primary chondrocytes were isolated from the lumbar spine
endplate cartilage of SpragueeDawley rats (160e180 g) as reported
in our previous study11. Second passage cells were used in experi-
ments. Endplate chondrocytes were seeded at a density of 2  105
cells per well in 2 ml Dulbecco's modified Eagle's medium/F-12
medium (HyClone, USA) supplemented with 10% fetal bovine
serum (Gibco, USA) on six-well flexible silicone rubber BioFlex™
plates coated with collagen type I (Flexcell International, Hills-
borough, NC). The cells were cultured for 48 h to 80e90% conflu-
ence, and then cyclic mechanical strain with a 0.5 Hz sinusoidal
curve at 10% elongation was applied for 8 h a day using an FX-
5000T Flexercell Tension Plus unit (Flexcell International). The
cultures were incubated in a humidified atmosphere at 37 C with
5% CO2 while stretching.
Cell viability, proliferation and apoptosis assays
Endplate cartilage cell viability was detected by nitroblue
tetrazolium (NBT) and 40 ,6-diamidino-2-phenylindole (DAPI)
staining according to a previous study27. Cells stained with NBT
(Amresco, USA) were considered as live cells, while DAPI staining
indicated the total cells. The viable cell ratio ¼ live cells/total
cells  100%. For the proliferation assay, endplate chondrocytes
160 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168

Fig. 1. ICMT induces disc and endplate chondrocyte matrix degeneration. (A) Human endplate cartilage was obtained from patients with cervical vertebrae fracture (normal disc
group) or cervical disc degeneration (CDD group). MRIs of subjects and the vertebral surgery (green arrow) with the site of the obtained material (red arrow) are shown. (B) External
loading device and animal model. (C) COL-2A, ACAN, and Sox9 expression in normal disc and CDD groups was measured by real-time RT-PCR (normal disc group: n ¼ 8; CDD group
n ¼ 12). (D) Rabbit discs treated with (Loaded) or without (Sham) tensile loading for 0, 28, and 56 days, H&E and Safranin O-fast green staining showed changes in the disc structure
and matrix distribution. Original magnification: 40. Representative results from three samples are shown. (E) Real-time RT-PCR analysis of COL-2A, ACAN, and Sox9 expression in
endplate cartilage after tensile strain application in vitro. (F) Relative expression of COL-2A, ACAN, and Sox9 in endplates was measured by real-time RT-PCR after ICMT stimulation
in vitro. (G) Alcian Blue and Toluidine Blue staining showed decreases in the chondrocyte matrix after ICMT application for 7 days. Representative results from three donors are
shown. GAPDH was used as an internal control for the Real-time RT-PCR. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each
performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference.

were seeded on BioFlex™ plates and treated with 10% ICMT. Cell
proliferation was assessed by an Alamar Blue assay (Invitrogen)
according to the manufacturer's instructions. The absorbance at 0,
12, 24, 48 and 72 h were measured to reflect the proliferation of
endplate chondrocytes. For apoptosis analysis, chondrocytes were
treated with or without 10% ICMT for 3 days, incubated with 10 ml
propidium iodide and 10 ml annexin V (Invitrogen) at room tem-
perature while protected from light for 10 min, and then analyzed
by flow cytometry using a FACSCalibur (Becton Dickinson Bio-
sciences, San Diego, CA) with FACSDiva software. Propidium iodide-
 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168
positive and annexin V-positive cells were defined as apoptotic
cells.
Histological staining and immunohistochemical analysis
Human endplate cartilage and lumbar spines from rabbits
treated with or without mechanical tensile loading were fixed in
10% paraformaldehyde, decalcified, and embedded in paraffin. Se-
rial sections (7 mm thick) were staining with hematoxylin and eosin
(H&E) and Safranin O-fast green. For immunohistochemical anal-
ysis, paraffin-embedded sections (4 mm) were heated at 70 C for 2 h
and then treated with a dual endogenous enzyme blocking reagent
for 10 min. After blocking with normal goat serum (1:20 dilution)
for 30 min, the sections were incubated with a mouse anti-b-cat-
enin polyclonal antibody (1:50 dilution; SigmaeAldrich) and rabbit
anti-b-catenin monoclonal antibody (1:50 dilution; Cell Signaling
Technology) overnight at 4 C. The sections were then incubated
with a secondary biotinylated goat anti-rabbit antibody (1:200
dilution; Vector) for 30 min followed by streptavidin-peroxidase
(1:250 dilution; Pierce) for 30 min at room temperature. Peroxi-
dase activity was visualized by staining with Romulin AEC Chro-
mogen (RAEC810L; Bio Care Medical).
Real-time reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from chondrocytes using Trizol reagent
(Invitrogen) according to the manufacturer's instructions. After
reverse transcription, real-time PCR was performed by a Light-
Cycler480 System (Roche) using SYBR1 Premix Ex Taq™ (Takara,
Dalian, China) according to the manufacturer's instructions.
Dissociation curves revealed no nonspecific amplification.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
internal control. Data were analyzed using the Ct 2DDCt method
and expressed as the fold change compared with the control. Each
sample was analyzed in triplicate. The primer sequences, optimized
PCR conditions, and NCBI reference numbers are available from the
corresponding author.
Co-immunoprecipitation and western blotting
Total explant and cell proteins were collected in radio-
immunoprecipitation assay buffer (Cell Signaling Technology)
containing protease inhibitors (10 mg/ml leupeptin, 10 mg/ml
pepstatin A, and 10 mg/ml aprotinin). Nuclear-cytoplasmic protein
fractionation was conducted using a NE-PER Nuclear and Cyto-
plasmic Extraction Reagents kit (Thermo Fisher Scientific). Co-
immunoprecipitation was performed using a Dynabeads Co-
immunoprecipitation Kit (Invitrogen) according to the manufac-
turer's protocol. For western blot analysis, protein samples (20 mg)
were fractionated by 10% sodium dodecyl sulfate polyacrylamide
gel electrophoresis and then electrotransferred onto nitrocellulose
membranes (Whatman, Piscataway, NJ). The primary antibodies
included a goat anti-E-cadherin polyclonal antibody (Santa Cruz
Biotechnology), mouse anti-E-cadherin monoclonal antibody (BD
Biosciences), mouse anti-b-catenin polyclonal antibody (Sigma-
eAldrich) and rabbit anti-b-catenin monoclonal antibody Cell
Signaling Technology). Antibodies were diluted at 1:1000. Protein
loading was normalized to GAPDH using an anti-GAPDH antibody
(1:10,000 dilution; SigmaeAldrich). Horseradish peroxidase-
conjugated secondary antibodies were applied at a dilution of
1:5000. Antigen-antibody complexes were visualized using the
enhanced chemiluminescence detection system (Millipore, Bill-
erica, MA) as recommended by the manufacturer. Immunoreactive
bands were quantitatively analyzed in triplicate by normalizing the
band intensities to GAPDH using Alpha Image software.
161
Immunofluorescence
Endplate chondrocytes were seeded on six-well flexible silicone
rubber Bio-Flex™ plates and then treated with or without ICMT for
3 days. After treatment, the cells were fixed with 4% para-
formaldehyde for 15 min at room temperature, permeabilized in 3%
(v/v) Triton-X 100/PBS, and then blocked with 5% (v/v) goat serum
for 1 h. Chondrocytes were incubated with primary antibodies
overnight at 4 C in a humidified chamber. Primary antibodies
included a rabbit anti-b-catenin antibody (1:200 dilution; Cell
Signaling Technology) and mouse anti-E-cadherin antibody (1:100
dilution; BD Biosciences). After washing, the cells were incubated
with a goat anti-rabbit fluorescein isothiocyanate (FITC) secondary
antibody (1:500 dilution; Invitrogen) for 1 h. For F-actin staining,
the cells were washed and then incubated with Texas Red®-X
phalloidin (1:500 dilution; Invitrogen) for 1 h at room temperature.
Finally, the cells were incubated with 1.5 mg/ml DAPI (Shanghai Mai
Bio Co, Shanghai, China) for 15 min and then visualized under a
confocal microscope (TCSSP5; Leica, Wetzlar, Germany).
Statistical analysis
All quantitative data are expressed as the mean with 95% confi-
dence intervals (CI). Data were obtained from three independent ex-
periments and tested for normality using the ShapiroeWilk test prior
to parametric analyses. The means of two groups were compared by
the two-tailed Student's t-test. For multiple group comparisons, we
used one-way analysis of variance with Tukey's post-hoc test. All
statistical analyses were conducted with SPSS 18.0 (IBM, Chicago,
USA). P-values of less than 0.05 were considered significant.
Results
ICMT promotes endplate cartilage degeneration in vivo and in vitro
Because IVD degeneration is often correlated with endplate
cartilage degeneration in patients, we examined endplate
chondrocyte-related gene expression in patients with cervical
vertebrae fracture (normal disc) or cervical spondylosis (cervical
degeneration disc, CDD). We found decreases in the expression of
COL-2A, ACAN, and Sox9 in the endplate cartilage obtained from
patients with disc degeneration [Fig. 1(C)]. This result indicated that
IVD degeneration was accompanied by significant endplate carti-
lage degeneration.
After ICMT loading in vivo, H&E staining showed no significant
changes in 0 d control and sham control disc groups. However, in
the ICMT loading group, the annulus and pulposus were signifi-
cantly damaged as the load duration increased. Safranin O-fast
green staining revealed damage in the cartilage matrix following
ICMT loading as visualized by a loss of Safranine O staining
[Fig. 1(D)]. Real-time RT-PCR showed reductions in the expression
of COL-2A, ACAN, and Sox9 in the endplate cartilage after ICMT
loading [Fig. 1(E)]. Endplate chondrocytes with ICMT stimulation
in vitro showed similar changes [Fig. 1(F)]. Toluidine Blue and Alcian
Blue staining showed a decrease in the chondrocyte matrix after
application of ICMT [Fig. 1(G)]. These results indicated that ICMT
induced disc and endplate cartilage degeneration.
ICMT does not influence the cell viability or apoptosis of endplate
chondrocytes but alters the cell proliferation and cytoskeleton
To determine whether ICMT-induced endplate cartilage
degeneration was caused by cell death, we examined the cell
viability, proliferation and apoptosis of endplate chondrocytes after
exposure to ICMT. NBT/DAPI staining showed no obvious
162 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168

Fig. 2. ICMT does not induce endplate chondrocyte death but changes the cytoskeleton. (A and B) After mechanical loading in vivo, NBT/DAPI staining showed no obvious
differences in the viable cell ratio of endplate chondrocytes between 0 d, sham operation, and tensile loading groups. NBT staining indicates live cells, while DAPI staining shows the
total cells. Original magnification: 100. Representative results from three samples are shown. (C and D) Endplate chondrocytes were subjected to ICMT in vitro for 3 days, and then
cell apoptosis was examined by propidium iodide/annexin V staining (C) and cell proliferation was examined by an Alamar Blue assay (D). Data are presented as the means and 95%
CI from three independent experiments. (E) F-actin staining showed changes in the chondrocyte cytoskeleton after 3 days of ICMT treatment. Representative results from three
donors are shown, red indicates F-actin and blue indicates DAPI staining. Each experiment performed in triplicate. *Statistically significant difference.

differences in the viable cell ratio of 0 d control and sham operation
groups after ICMT loading for 28 and 56 days [Fig. 2(A) and (B)].
Moreover, annexin V-FITC and propidium iodide staining indicated
no significant differences in the percentages of apoptotic cells after
exposure to ICMT for 3 days compared with cells in the non-loading
group [Fig. 2(C)]. However, the Alamar Blue assay showed that
ICMT promoted cell proliferation at 24 and 48 h [Fig. 2(D)]. In
addition, Texas Red®-X phalloidin staining revealed obvious
changes in the F-actin cytoskeleton of endplate chondrocytes after
ICMT for 3 days [Fig. 2(E)]. These data suggested that ICMT did not
promote endplate chondrocyte death, but promoted chondrocyte
proliferation and re-orientation of the cytoskeleton.
Activation of the Wnt/b-catenin signaling pathway by ICMT in
endplate cartilage
To investigate the effect of Wnt/b-catenin signaling on endplate
cartilage degeneration, we first detected b-catenin expression in
human endplate cartilage isolated from normal disc and CDD
groups. The results showed that both the mRNA and protein levels
of b-catenin were up-regulated in the degenerated endplate carti-
lage [Fig. 3(A)]. Immunohistochemical analysis showed that b-
catenin levels were much higher and mainly located in the nuclei of
samples from patients with disc degeneration compared with the
normal disc group [Fig. 3(B)]. These data suggested activation of
Wnt/b-catenin signaling during human endplate cartilage
degeneration.
In contrast, b-catenin mRNA and protein expression was up-
regulated after ICMT stimulation of endplate cartilage in vivo and
in vitro-cultured chondrocytes [Fig. 3(C) and (D)]. We next exam-
ined the distribution of b-catenin in endplate chondrocytes stim-
ulated by ICMT in vivo and in vitro. Immunohistochemical and
immunofluorescence analyses showed significantly higher b-cat-
enin protein levels and accumulation in the nucleus after applica-
tion of ICMT [Fig. 3(E) and (F)]. Similar results were obtained in
western blot analyses of nuclear/cytoplasmic protein fractions
 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168 163

Fig. 3. ICMT promotes activation of Wnt/b-catenin signaling in endplate chondrocytes. (A) Real-time RT-PCR and western blot analyses of b-catenin mRNA and protein
expression in samples obtained from patients with cervical vertebrae fracture or cervical disc degeneration (normal disc group: n ¼ 8; CDD group n ¼ 12). For western blotting,
Representative results from three patients are shown. (B) Immunohistochemical analysis showed up-regulation of b-catenin protein in samples from patients with disc degen-
eration. Original magnification: 200. Representative results from three patients are shown. (C and D) b-Catenin mRNA and protein expression in endplate chondrocytes was
assessed by real-time RT-PCR and western blotting after ICMT application in vivo (C) and in vitro (D). For western blotting, representative results from three donors are shown. (E) b-
Catenin protein expression was examined by immunohistochemical analysis in rabbit endplate cartilage tissue after 28 days of tensile loading in vivo. Original magnification: 200.
Representative results from three samples are shown. (F) Endplate chondrocytes were subjected to ICMT for 3 days, and then the expression and distribution of b-catenin protein
were detected by immunofluorescence. Representative results from three donors are shown. (G) Nuclear-cytoplasmic protein fractionation was performed to measure nuclear and
cytoplasmic b-catenin protein expression after ICMT application. Representative results from three donors are shown. (H) Real-time RT-PCR showed changes in cyclinD1 and TCF-1
expression after endplate chondrocytes were subjected to ICMT for 3 days. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR
data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically
significant difference.

[Fig. 3(G)]. Because b-catenin translocation into the nucleus results significantly by ICMT for 3 days [Fig. 3(H)]. Taken together, these
in TCF/LEF transcription factor activation17, we assessed the data demonstrated that ICMT activated the Wnt/b-catenin signaling
expression level of TCF/LEF target genes cyclinD1 and TCF-128,29. pathway in endplate cartilage, which may be related to endplate
Expression of Both cyclinD1 and TCF-1 genes was increased cartilage degeneration.
164 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168

Inhibition of the Wnt/b-catenin signaling pathway relieves ICMT-
induced endplate chondrocyte dedifferentiation
Because Wnt/b-catenin signaling showed a correlation with
endplate cartilage degeneration after application of ICMT, we next
examined whether inhibition of the Wnt/b-catenin signaling
pathway could relieve the degeneration induced by ICMT in
endplate chondrocytes. Treatment with the Wnt inhibitor XAV-
939 deceased both the mRNA and protein levels of b-catenin
[Fig. 4(A) and (B)]. Immunofluorescence analysis showed similar
changes in b-catenin protein expression [Fig. 4(C)]. In addition,
compared with the ICMT group, the down-regulated expression of
COL-2A, ACAN and SOX9 was partially restored by XAV-939
treatment [Fig. 4(D)]. These results indicated that the Wnt/b-cat-
enin pathway was indispensable for ICMT-induced endplate
cartilage degeneration.
Cross-talk of E-cadherin/b-catenin and the Wnt/b-catenin signaling
pathway in endplate chondrocytes
E-cadherin is a sensitive mechanotransducer that binds to b-
catenin and actin. Because we found that ICMT altered the orien-
tation of the F-actin cytoskeleton in endplate chondrocytes, we
determined whether E-cadherin is involved in ICMT-induced
cartilage degeneration. E-cadherin mRNA and protein levels in
endplate chondrocytes were significantly down regulated by ICMT
both in vivo and in vitro [Fig. 5(A)e(D)]. Immunofluorescence
indicated an increase in b-catenin expression and translocation into
the nucleus, which were accompanied by down-regulation of E-
cadherin [Fig. 5(E)]. Co-immunoprecipitation showed that b-cat-
enin formed a complex with E-cadherin in endplate chondrocytes
under normal conditions, but the interaction between E-cadherin
and b-catenin was weakened after application of ICMT [Fig. 5(F)].
Taken together, these data suggested that ICMT suppressed the
direct interaction between E-cadherin and b-catenin proteins,
which was most likely caused by inhibition of E-cadherin
expression.
Over-expression of E-cadherin rescues ICMT-induced endplate
chondrocyte anabolic genes decreasing by interacting with b-
catenin
To further explore the role of E-cadherin in ICMT-induced
endplate chondrocyte dedifferentiation, we over-expressed GFP-
labeled E-cadherin in endplate chondrocytes during ICMT appli-
cation. E-cadherin expression was significantly increased at 3 days
after transfection [Fig. 6(A)e(C)]. Co-immunoprecipitation indi-
cated that the interaction between E-cadherin and b-catenin was
enhanced after E-cadherin overexpression during application of
ICMT [Fig. 6(D)]. Nuclear-cytoplasmic protein fractionation showed
that E-cadherin over-expression inhibited b-catenin translocation
into the nucleus of ICMT-stimulated endplate chondrocytes
[Fig. 6(E)]. In addition, E-cadherin over-expression suppressed
ICMT-induced expression of cyclinD1 and TCF-1 [Fig. 6(F)]. These
data suggested that over-expression of E-cadherin in endplate
chondrocytes suppressed the Wnt/b-catenin signaling pathway. To
test whether E-cadherin over-expression could rescue the effect of
ICMT on the endplate chondrogenic phenotype, chondrocytes were
pre-transfected with E-cadherin expression or control vectors
before ICMT stimulation. After 3 days of ICMT, cells transfected with
E-cadherin showed higher COL-2A, ACAN and Sox9 expression than
those transfected with the control vector [Fig. 6(G)]. Overall, these
results suggested that E-cadherin suppressed the ICMT-induced
loss of the endplate chondrogenic phenotype, most likely via in-
hibition of the Wnt/b-catenin pathway through the physical
interaction of b-catenin to block its nuclear translocation.

Fig. 4. Inhibition of Wnt/b-catenin signaling rescues ICMT-induced endplate chondrocyte anabolic genes decreasing. (A and B) Endplate chondrocytes were treated with or
without XAV-939 during ICMT application. Expression of b-catenin mRNA (A) and protein (B) indicated that XAV-939 suppressed the Wnt/b-catenin signaling activated by ICMT. For
western blotting, representative results from three donors are shown. (C) Immunofluorescence showed inhibition of b-catenin protein expression. Representative results from three
patients are shown. Representative results from three donors are shown. (D) Real-time RT-PCR showed alterations in the expression levels of COL-2A, ACAN, and Sox9 after ICMT
with or without XAV-939 treatment. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95%
CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference.
 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168 165

Fig. 5. ICMT suppresses the interaction between E-cadherin and b-catenin by down-regulation of E-cadherin expression. (A e D) Expression of E-cadherin mRNA and protein
was measured by real-time RT-PCR and western blotting after ICMT loaded in vivo (A and B) and in vitro (C and D). For western blotting, representative results from three donors are
shown. (E) Endplate chondrocytes were treated with ICMT for 3 days. Double immunofluorescence staining revealed colocalization of E-cadherin and b-catenin proteins (yellow) in
endplate chondrocytes. Representative results from three donors are shown. (F) Co-immunoprecipitation showed an interaction between E-cadherin and b-catenin proteins after
treatment with or without ICMT for 3 days. Representative results from three donors are shown. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots.
Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor.
*Statistically significant difference.

Discussion
IVDs bear and distribute the mechanical load during movements
of the spine. Endplate cartilage is an important component of IVDs
and the main pathway for nutritional supply to avascular IVDs. The
phenotype of endplate chondrocytes is mainly characterized by
expression of genes encoding cartilage-specific extracellular matrix
proteins or their regulators, such as COL-2A, ACAN, and Sox9, which
are responsible for the maintenance of cartilage anabolism.
Changes in the endplate cartilage structure through variations in
poromechanical properties affect fluid velocities and chondrocyte
mechanosensing30. Some studies have demonstrated that endplate
cartilage damage and loss of the chondrogenic phenotype increase
the risk of internal disruption in discs, which promotes disc
degeneration31,32.
Biomechanical stimulation is a major factor implicated in IVD
degeneration. Higher frequencies of mechanical stretching
(0.33e1.0 Hz) can differentially regulate anabolic and catabolic
gene expression in annulus fibrosus cells33. Excessive mechanical
strain or acute mechanical injury promote the secretion of
inflammatory factor from IVD cells and contribute to IVD degen-
eration, innervation, and pain34,35. Complex overloading of human
and animal spines has direct deleterious effects on IVD tissue,
resulting in endplate cartilage damage and IVD degeneration36,37.
Similarly, our data suggest that excessive intermittent mechanical
tensile strain (2.4 MP, 8 h/day) loading in vivo damages disc tissue
and negatively regulates metabolism of the endplate cartilage
matrix.
Alterations in the cartilage matrix in response to mechanical
loading have also been reported in vitro. Short-term moderate
mechanical loading protects chondrocytes from calcification and
increases proteoglycan release38,39, but high levels and long dura-
tions of mechanical loading induce phenotypic changes in chon-
drocytes7,8,40. Huang et al.41 suggested that cyclic tension strain
(0.5 Hz, 10% strain) for 3 h promotes expression of COL-2A and
ACAN, but their expression decreases to below control levels after
12 h of application. Our findings also demonstrated that 0.5 Hz, 10%
ICMT application induced endplate chondrocyte matrix damage
and decreased COL-2A, ACAN, and Sox9 expression in a time-
dependent manner.
166 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168

Fig. 6. E-cadherin relieves ICMT-induced down-regulation of endplate chondrocyte anabolic genes via inhibition of b-catenin signaling. (A and B) Overexpression of GFP-
labeled E-cadherin in rat endplate chondrocytes was confirmed by real-time RT-PCR (A) and western blotting (B). For western blotting, representative results from three do-
nors are shown. (C) GFP-labeled E-cadherin was observed by fluorescence microscopy at 3 days post-transfection. Original magnification: 100. Representative results from three
donors are shown. (D) Endplate chondrocytes were transfected with an E-cadherin expression plasmid (E-cad) or pEGFPN1 vector (Veh). At 1 day after transfection, ICMT was
applied for 3 days, and then the interaction between E-cadherin and b-catenin proteins was detected by co-immunoprecipitation. Representative results from three donors are
shown. (E) Nuclear and cytoplasmic b-catenin protein expression was examined in nuclear and cytoplasmic protein fractions. Representative results from three donors are shown.
(F) CyclinD1 and TCF-1 mRNA expression was detected by real-time RT-PCR. (G) Relative expression levels of COL-2A, ACAN, and Sox9 were measured by real-time RT-PCR. GAPDH
was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments,
each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. (H) Schematic diagram of ICMT-induced endplate cartilage
degeneration through E-cadherin/b-catenin complex and Wnt/b-catenin signaling cross-talk.

Previous studies have shown that intermittent mechanical
tensile strain induces endplate cartilage degeneration. Several
reports suggest that continuous compressed loading ex vivo and
long durations of mechanical compression induce chondrocyte
death42,43. In this study, we found that intermittent tensile
loading (8 h/day) did not affect the cell viability of endplate
chondrocytes both in vivo and in vitro. These results suggest that
ICMT does not promote endplate chondrocyte death, and other
factors are responsible for ICMT-induced endplate cartilage
degeneration.
b-Catenin is up-regulated in degenerated IVDs, and condi-
tional activation of b-catenin signaling promotes disc tissue
 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168
damage44,45. We also found up-regulation of b-catenin protein
expression in endplate cartilage from patients with disc degen-
eration. There were similar changes in endplate chondrocytes
induced by ICMT in vivo and in vitro. Moreover, b-catenin protein
accumulated and translocated to the nucleus, together with sig-
nificant increases in the expression of Wnt/b-catenin signaling
target genes such as cyclinD1 and TCF-1. These results indicate
that ICMT activated the Wnt/b-catenin signaling pathway in
endplate chondrocytes. Furthermore, during ICMT application,
inhibition of Wnt/b-catenin signaling by XAV-939 abolished the
inhibitive effect of ICMT on COL-2A, ACAN, and Sox9 expression.
These findings suggest that ICMT-induce endplate cartilage
degeneration is mediated by activation of the Wnt/b-catenin
signaling pathway.
E-cadherin mediates intercellular adhesion and transmits sig-
nals via mechanotransduction46,47. Engl et al. found that internally
generated tensile forces modulate E-cadherin recruitment at
cellecell contacts via actin dynamics48. Similarly, we found that
ICMT changed the F-actin cytoskeleton of endplate chondrocytes.
Therefore, we further examined the expression of E-cadherin in
endplate chondrocytes after ICMT treatment in vivo and in vitro. We
found that E-cadherin mRNA and protein expression was signifi-
cantly decreased during ICMT application.
E-cadherin inhibits Wnt signaling in human cancer cells
through an interaction b-catenin49. We therefore determined
whether ICMT altered the cross-talk between E-cadherin and Wnt/
b-catenin signaling in endplate chondrocytes. Remarkably, immu-
nofluorescence and co-immunoprecipitation demonstrated an
obvious reduction in the proteineprotein interactions between E-
cadherin and b-catenin after ICMT application. Wang et al.50 found
that E-cadherin promotes ACAN and COL2A expression through
induction of bone morphogenetic protein genes and Smad1/5
phosphorylation in IVD cells. Our data showed that the ICMT-
induced decreases in COL-2A, ACAN, and SOX9 expression in end-
plate chondrocytes were rescued by over-expression of E-cadherin.
This rescue effect might have been due to enhancing the interaction
between E-cadherin and b-catenin proteins, and suppression of the
Wnt/b-catenin signaling pathway after E-cadherin over-expression
in the endplate chondrocytes.
Based on these results, we speculate that ICMT probably acti-
vates the Wnt/b-catenin signaling pathway, resulting in down-
regulation of anabolic genes. Moreover, ICMT decreases the inter-
action between E-cadherin and b-catenin by suppressing E-cad-
herin expression, leading to promotion of Wnt/b-catenin signaling
activation, which might accelerate endplate cartilage degeneration
[Fig. 6(H)]. These findings will be useful as the basis for future
studies exploring the Wnt/b-catenin signaling pathway and E-
cadherin/b-catenin complex as potential therapeutic targets to
prevent IVD degeneration.
Author contributions
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors
approved the final version to be published. Dr. Xu had full access to
all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study conception and design: Hongguang Xu, Quan Zheng,
Junxing Song and Xiaoling Zhang.
Acquisition of data: Quan Zheng, Hongguang Xu, Junxing Song,
Jiao Li, Jing Wang, Chuang-dong Wang, Hong Wang, Ping Liu and
Xiaoling Zhang.
Analysis and interpretation of data: Quan Zheng, Hongguang
Xu, Junxing Song, Jiao Li, Jing Wang, Chuang-dong Wang, Hong
Wang, Ping Liu and Xiaoling Zhang.
167
Conflict of interest
The authors have declared that no competing interests exist.
Acknowledgments
This study was supported by grants from National Natural Sci-
ence Foundation of China (30973025, 81272048, 81311130314),
National Natural Science Foundation of Anhui province
(1308085MH152), Science and Technology Commission of
Shanghai (No. 12411951100), Shanghai Municipal Commission of
Health and Family Planning (No. 2013ZYJB0501), and key discipline
construction fund of Shanghai education commission (J50206).
References
1. Manek NJ, MacGregor AJ. Epidemiology of back disorders
prevalence, risk factors, and prognosis. Curr Opin Rheumatol
2005;17:134e40.
2. Benneker LM, Heini PF, Alini M, Anderson SE, Ito K. 2004
Young Investigator Award Winner vertebral endplate marrow
contact channel occlusions and intervertebral disc degenera-
tion. Spine (Phila Pa 1976) 2005;30:167e73.
3. Ferguson SJ, Steffen T. Biomechanics of the aging spine. Eur
Spine J 2003;(Suppl 2):S97eS103.
4. Haschtmann D, Stoyanov JV, Gedet P, Ferguson SJ. Vertebral
endplate trauma induces disc cell apoptosis and promotes
organ degeneration in vitro. Eur Spine J 2008;17:289e99.
5. Moore RJ. The vertebral endplate: disc degeneration, disc
regeneration. Eur Spine J 2006;(Suppl 3):S333e7.
6. O'Conor CJ, Case N, Guilak F. Mechanical regulation of chon-
drogenesis. Stem Cell Res Ther 2013;4:61.
7. Roemhildt ML, Beynnon BD, Gauthier AE, Gardner-Morse M,
Ertem F, Badger GJ. Chronic in vivo load alteration induces
degenerative changes in the rat tibiofemoral joint. Osteoar-
thritis Cartilage 2013;21:346e57.
8. Ko FC, Dragomir C, Plumb DA, Goldring SR, Wright TM,
Goldring MB, et al. In vivo cyclic compression causes cartilage
degeneration and subchondral bone changes in mouse tibiae.
Arthritis Rheum 2013;65:1569e78.
9. Ariga K, Yonenobu K, Nakase T, Hosono N, Okuda S, Meng W,
et al. Mechanical stress induced apoptosis of endplate chon-
drocytes in organ cultured mouse intervertebral discs an
ex vivo study. Spine (Phila Pa 1976) 2003;28:1528e33.
10. Xu HG, Yu YF, Zheng Q, Zhang W, Wang CD, Zhao XY, et al.
Autophagy protects end plate chondrocytes from intermittent
cyclic mechanical tension induced calcification. Bone 2014;66:
232e9.
11. Xu HG, Hu CJ, Wang H, Liu P, Yang XM, Zhang Y, et al. Effects of
mechanical strain on ANK, ENPP1 and TGF-beta1 expression in
rat endplate chondrocytes in vitro. Mol Med Rep 2011;4:
831e5.
12. Xu HG, Zhang XH, Wang H, Liu P, Wang LT, Zuo CJ, et al.
Intermittent cyclic mechanical tension-induced calcification
and downregulation of ankh gene expression of end plate
chondrocytes. Spine (Phila Pa 1976 2012;37:1192e7.
13. Joiner DM, Ke J, Zhong Z, Xu HE, Williams BO. LRP5 and LRP6 in
development and disease. Trends Endocrinol Metab 2013;24:
31e9.
14. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. beta-catenin
is a target for the ubiquitin-proteasome pathway. Embo J
1997;16:3797e804.
15. Nusse R. Wnt signaling in disease and in development. Cell Res
2005;15:28e32.
16. Cadigan KM, Nusse R. Wnt signaling: a common theme in
animal development. Genes Dev 1997;11:3286e305.
168 H.-g. Xu et al. / Osteoarthritis and Cartilage 24 (2016) 158e168
17. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling:
components, mechanisms, and diseases. Dev Cell 2009;17:
9e26.
18. Zhu M, Tang D, Wu Q, Hao S, Chen M, Xie C, et al. Activation of
b-catenin signaling in articular chondrocytes leads to
osteoarthritis-like phenotype in adult b-catenin conditional
activation mice. J Bone Miner Res 2009;24:12e21.
19. Hiyama A, Sakai D, Risbud MV, Tanaka M, Arai F, Abe K, et al.
Enhancement of intervertebral disc cell senescence by WNT/
beta-catenin signaling-induced matrix metalloproteinase
expression. Arthritis Rheum 2010;62:3036e47.
20. Gooding JM, Yap KL, Ikura M. The cadherin-catenin complex as
a focal point of cell adhesion and signalling: new insights from
three-dimensional structures. Bioessays 2004;26:497e511.
21. Stepniak E, Radice GL, Vasioukhin V. Adhesive and signaling
functions of cadherins and catenins in vertebrate develop-
ment. Cold Spring Harb Perspect Biol 2009;1:a002949.
22. Schwartz MA, DeSimone DW. Cell adhesion receptors in
mechanotransduction. Curr Opin Cell Biol 2008;20:551e6.
23. Gottardi CJ, Wong E, Gumbiner BM. E-cadherin suppresses
cellular transformation by inhibiting beta-catenin signaling in
an adhesion-independent manner. J Cell Biol 2001;153:
1049e60.
24. Zhang P, Li H, Yang B, Yang F, Zhang LL, Kong QY, et al. Bio-
logical significance and therapeutic implication of resveratrol-
inhibited Wnt, Notch and STAT3 signaling in cervical cancer
cells. Genes Cancer 2014;5:154e64.
25. Kroeber MW, Unglaub F, Wang H, Schmid C, Thomsen M,
Nerlich A, et al. New in vivo animal model to create interver-
tebral disc degeneration and to investigate the effects of
therapeutic strategies to stimulate disc regeneration. Spine
(Phila Pa 1976) 2002;27:2684e90.
26. Guehring T, Omlor GW, Lorenz H, Bertram H, Steck E, Richter W,
et al. Stimulation of gene expression and loss of anular archi-
tecture caused by experimental disc degenerationean in vivo
animal study. Spine (Phila Pa 1976) 2005;30:2510e5.
27. Lim TH, Ramakrishnan PS, Kurriger GL, Martin JA, Stevens JW,
Kim J, et al. Rat spinal motion segment in organ culture: a cell
viability study. Spine (Phila Pa 1976) 2006;31:1291e7.
28. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M,
Pestell R, et al. The cyclin D1 gene is a target of the beta-
catenin/LEF-1 pathway. Proc Natl Acad Sci U.S.A 1999;96:
5522e7.
29. Roose J, Huls G, van Beest M, Moerer P, van der Horn K,
Goldschmeding R, et al. Synergy between tumor suppressor
APC and the -catenin-Tcf4 target Tcf1. Science 1999;285:
1923e6.
30. Malandrino A, Lacroix D, Hellmich C, Ito K, Ferguson SJ,
Noailly J. The role of endplate poromechanical properties on
the nutrient availability in the intervertebral disc. Osteoar-
thritis Cartilage 2014;22:1053e60.
31. Dolan P, Luo J, Pollintine P, Landham PR, Stefanakis M,
Adams MA. Intervertebral disc decompression following end-
plate damage: implications for disc degeneration depend on
spinal level and age. Spine (Phila Pa 1976) 2013;38:1473e81.
32. Dudli S, Haschtmann D, Ferguson SJ. Fracture of the vertebral
endplates, but not equienergetic impact load, promotes disc
degeneration in vitro. J Orthop Res 2012;30:809e16.
33. Gilbert HT, Hoyland JA, Millward-Sadler SJ. The response of
human anulus fibrosus cells to cyclic tensile strain is
frequency-dependent and altered with disc degeneration.
Arthritis Rheum 2010;62:3385e94.
34. Gawri R, Rosenzweig DH, Krock E, Ouellet JA, Stone LS,
Quinn TM, et al. High mechanical strain of primary interver-
tebral disc cells promotes secretion of inflammatory factors
associated with disc degeneration and pain. Arthritis Res Ther
2014;16:R21.
35. Alkhatib B, Rosenzweig DH, Krock E, Roughley PJ, Beckman L,
Steffen T, et al. Acute mechanical injury of the human inter-
vertebral disc: link to degeneration and pain. Eur Cell Mater
2014;28:98e110.
36. Pollintine P, Dolan P, Tobias JH, Adams MA. Intervertebral disc
degeneration can lead to “stress-shielding” of the anterior
vertebral body: a cause of osteoporotic vertebral fracture?
Spine (Phila Pa 1976) 2004;29:774e82.
37. Walter BA, Korecki CL, Purmessur D, Roughley PJ, Michalek AJ,
Iatridis JC. Complex loading affects intervertebral disc me-
chanics and biology. Osteoarthritis Cartilage 2011;19:1011e8.
38. Xu H, Zhang X, Wang H, Zhang Y, Shi Y, Zhang X. Continuous
cyclic mechanical tension increases ank expression in endplate
chondrocytes through the TGF-beta1 and p38 pathway. Eur J
Histochem 2013;57:e28.
39. Quinn TM, Allen RG, Schalet BJ, Perumbuli P, Hunziker EB.
Matrix and cell injury due to sub-impact loading of adult
bovine articular cartilage explants: effects of strain rate and
peak stress. J Orthop Res 2001;19:242e9.
40. Rosenzweig DH, Quinn TM, Haglund L. Low-frequency high-
magnitude mechanical strain of articular chondrocytes acti-
vates p38 MAPK and induces phenotypic changes associated
with osteoarthritis and pain. Int J Mol Sci 2014;15:14427e41.
41. Huang J, Ballou LR, Hasty KA. Cyclic equibiaxial tensile strain
induces both anabolic and catabolic responses in articular
chondrocytes. Gene 2007;404:101e9.
42. Lucchinetti E, Adams CS, Horton Jr WE, Torzilli PA. Cartilage
viability after repetitive loading: a preliminary report. Osteo-
arthritis Cartilage 2002;10:71e81.
43. Yurube T, Hirata H, Kakutani K, Maeno K, Takada T, Zhang Z,
et al. Notochordal cell disappearance and modes of apoptotic
cell death in a rat tail static compression-induced disc
degeneration model. Arthritis Res Ther 2014;16:R31.
44. Smolders LA, Meij BP, Riemers FM, Licht R, Wubbolts R,
Heuvel D, et al. Canonical Wnt signaling in the notochordal cell
is upregulated in early intervertebral disk degeneration.
J Orthop Res 2012;30:950e7.
45. Wang M, Tang D, Shu B, Wang B, Jin H, Hao S, et al. Conditional
activation of beta-catenin signaling in mice leads to severe
defects in intervertebral disc tissue. Arthritis Rheum 2012;64:
2611e23.
46. Leckband DE, de Rooij J. Cadherin adhesion and mechano-
transduction. Annu Rev Cell Dev Biol 2014;30:291e315.
47. Bhatt T, Rizvi A, Batta SP, Kataria S, Jamora C. Signaling and
mechanical roles of E-cadherin. Cell Commun Adhes 2013;20:
189e99.
48. Engl W, Arasi B, Yap LL, Thiery JP, Viasnoff V. Actin dynamics
modulate mechanosensitive immobilization of E-cadherin at
adherens junctions. Nat Cell Biol 2014;16:587e94.
49. Klingelho€fer J, Troyanovsky RB, Laur OY, Troyanovsky S. Ex-
change of catenins in cadherin-catenin complex. Oncogene
2003;22:1181e8.
50. Wang Z, Kim SS, Hutton WC, Yoon ST. E-cadherin upregulates
expression of matrix macromolecules aggrecan and collagen II
in the intervertebral disc cells through activation of the
intracellular BMP-Smad1/5 pathway. J Orthop Res 2012;30:
1746e52.