Yeon Hee Lee1, Ashish Ranjan Sharma2, Supriya Jagga2, Sang Soo Lee2, Ju Suk Nam2. 1. Institute for Skeletal Aging and Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Korea. Electronic Address: 123sslee@gmail.com. 2. Institute for Skeletal Aging and Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Korea.
Degenerative osteoarthritis (OA) is hallmarked by synovial
joints that suffer from degeneration of articular cartilage,
causing alteration of cartilage structure and compositions
along with changes in subchondral bone architecture (1,
2). Initially, it was proposed that alterations in bone take
place secondary to cartilage degeneration, and they do
not participate in the process of disease augmentation.
Nevertheless, several animal studies have demonstrated that
alterations in subchondral bone takes place at the initial stages
of OA (3, 4), and that changes in subchondral bone can lead to
degeneration of cartilage (5). The intimate physical interface
amid cartilage and subchondral bone suggests biochemical
and molecular interaction throughout this interface in healthy
and osteoarthritic joints (6).Amplified vascular communicating channels,
microcracks and fissures throughout the interface and
the asymmetrical bone cartilage anatomy could provide
a transport conduit to assist molecular transport. During
the OA progress, hydraulic conductance between articular
cartilage and subchondral bone increases (7). Effector
molecules produced from bone matrix metalloproteinase
2 (MMP2), receptor activator of nuclear factor κ-B ligand
(RANKL), hepatocyte growth factor (HGF) or cartilage
(i.e. interleukin 1; also known as IL1), metalloproteinases
with thrombospondin motifs (ADAMTS) and MMP13
may crossover from one zone to another and they can alter
the homeostasis of each other (8). Studies have confirmed
that the nutrients from bone may nourish cartilage through
the channels that links them, apart from the blood vessels
(9, 10). In bovine explant cultures, chondrocyte survival
is significantly influenced by subchondral bone (11).
While, regulatory factors released from the chondrocytes
in degraded cartilage might contribute to induction of
osteoclastogenesis, and thus participate in the loss of
subchondral bone during OA (12). Taken together, it may
be proposed that interplay between the bone cartilage
complexes is a holistic system, whereby multiple factors
might contribute to OA pathogenesis.Among various molecular regulators that affect cartilage
and subchondral bone, WNT signaling pathway is crucial
for maintaining the biochemical unit of bone and cartilage.
Studies have demonstrated that both, inhibition or
activation of canonical WNT signaling can have adverse
effect on cartilage, including apoptosis of chondrocytes,
perturb phenotype of articular chondrocytes, OA-like
lesions, overexpression of markers related to hypertrophy
and terminal differentiation (13, 14). While, activation
of WNT signaling pathway, either by inhibiting WNT
antagonists or increasing the stability of β-catenin, can
have stimulatory effect on bone formation causing stiffer
and thicker bones (15, 16). Since, various agonists and
antagonists, which are often secretory in nature -like
secreted frizzled-related protein (sFRP), sclerostin
(SOST) and Dickkopf (DKK1)- tightly regulate WNT
signaling, it is possible that bone and cartilage modulate
each other via WNT signaling pathway and create
pathological environment like arthritis. Overexpression
of WNT signaling pathway agonists, WNT1-inducible
signaling pathway protein1 (WISP1) and WNT16, has
been described in human cartilage tissue samples after
initiation of cartilage damage and synovium of OA (17,
18). Release of agonists can directly induce secretion
of the aggrecanase and MMPs in chondrocytes, leading
to destruction of cartilage. While, remodeling process
in subchondral bone may be compelled toward bone
formation resulting in development of osteophytes (19).Rspondins (RSPOs) contain a thrombospondin type
1 domain/repeat-1 and they are cysteine-rich secretory
proteins (20). In mammals, four members (RSPO1,
RSPO2, RSPO3 and RSPO4) of RSPO protein family
have been identified, having overall resemblance index
of 40-60% in sequence homology and organization of
domain (21, 22). In a high throughput sequencing study
of human fetal brain cDNA library, RSPO3 was identified
as the first member of the RSPO family (23). Thereafter,
other members of RSPO family were identified from
different species (20, 24, 25). To activate WNT signaling
pathway, extracellular constituents of the WNT signaling
acts synergistically with RSPOs (25-27). It has been
observed that during developmental stages, expression
of Wnt and RSPO proteins are either close or overlaps
with each other, suggesting a probable relationship
between RSPOs and WNT signaling pathway (28). Due
to the capability of RSPOs to act as a regulator of WNT
signaling pathway, several possible roles of these proteins
have been suggested (27). Considering the functional
role of RSPOs as agonists of WNT signaling, we tried to
analyze expression pattern RSPOs along with its receptors
[leucine-rich repeat-containing G-protein coupled
receptors (LGRs)] during early and advanced stages of
OA. An insight into the expression pattern of RSPOs and
LGRs could be helpful in understanding the regulation
of WNT signaling, as a cross-talk signaling mechanism
between bone and cartilage during OA progression.
Materials and Methods
Histochemistry
In this experimental study, cartilage tissue samples from
human femoral condyles were acquired from healthy
patients (around 58- to 80-years old) undergoing surgery
for hip replacements. The Ethical Committee of Hallym
University-Sacred Heart Hospital, Chuncheon, South
Korea (2009-42) reviewed the experimental procedure and
granted permission. To examine the status of explanted
cartilage damage, histochemical staining was performed
on random samples of femoral condyles cartilage tissue
pertaining to early and advanced OA stages. Explanted
femoral heads were cleaned under sterilized conditions
and harvested cartilage soft tissue was fixed by immersing
in a solution of 2% paraformaldehyde (PFA, Merck,
USA) for 24 hours. The samples were decalcified in 10%
ethylenediaminetetraacetic acid solution (EDTA, SigmaAldrich, USA) before embedding in paraffin wax. Prior to
staining, the tissues were deparaffinized and rehydrated.
Hematoxylin and eosin staining
The paraffin-embedded samples were deparaffinized,
rehydrated and 5 μm thick sectioned samples were cut.
Representative sections from all cartilage subtype samples
were stained with hematoxylin and eosin (H&E) for
the descriptive analysis of morphological details. Light
microscope at ×10 magnification (Ziess AxioCam digital
camera, Carl Zeiss, Germany) was used to visualize and
photograph the stained sections.
Safranin-O staining
Safranin-O staining was carried out as follows. After
steeping in Weigert’s iron hematoxylin solution for about
10 minutes, the samples were rinsed with normal alkaline
tap water for 10 minutes. For 5 minutes, the samples were
stained in fast green solution and bathed with 1% acetic
acid for 10 seconds. Subsequently, 0.1% Safranin-O
solution (Sigma-Aldrich, USA) was used to stain the
samples for 5 minutes and they were dehydrated by using
a graded series of alcohol. Next, the samples were cleared
with xylene. Finally, each sample was mounted with
resinous mounting medium for observation and image
acquisition. The obtained results were visualized at ×10
magnification by a microscope and pictured by a Ziess
Axi℃am digital camera.
Immunohistochemistry
Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Immunohistochemistry
Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Biotechnology, USA), RSPO1 (Sigma-Aldrich, USA),
RSPO2 (Sigma-Aldrich, USA), LGR5 (Sigma-Aldrich,
USA), LGR6 (Abcam, England) and β-catenin (Santa
Cruz Biotechnology, USA) of 1:500 dilutions at 4˚C.
The slides were washed thrice with phosphate-buffered
saline (PBS, Wel Gene, Korea) and goat anti-rabbit
immunoglobulin G (IgG, Santa Cruz Biotechnology,
USA) was treated at room temperature for 30 minutes.
Western blot bands were developed for visualization with
3, 3′-diaminobenzidine as the chromogen. Each section
was photographed at ×10 magnification by a Zeiss Axio
Cam digital camera.
Preparation of primary human chondrocytes
Cartilage samples from human femoral condyles were
dissected in 100 mm dish under sterilize environment.
Samples were rinsed continually with Dulbecco’s
Modified Eagle Medium (DMEM, Gibco, USA)
including 10% fetal bovine serum (FBS, Gibco, USA)
supplemented with 1% Penicillin-Streptomycin (P/S,
Gibco). After digestion with Hyaluronidase in dish,
sterilized blade was used to dissect the cartilage samples
into small fragments. In serum free DMEM media,
minced pieces of cartilage were washed twice and
treated with protease buffer for one hour at 37˚C and
5% CO2. Again, in DMEM (serum free), the cartilage
fragments were rinsed twice followed by enzymatic
digestion with collagenase for nearly 2 hours and 30
minutes at similar condition as mentioned above. After
completion of the enzymatic degradation, solution
was filtered through cell strainer of 70 μm and stored
in a 50 ml tube. Then, the media was centrifuged at a
speed of 1500 rpm for 5 minutes and pellet so obtained
was rinsed while performing the procedure twice. At
the start, cell pellet was resuspended with complete
DMEM (20% FBS and 1% P/S). Three days later,
DMEM media (10% FBS and 1% P/S) was replaced to
maintain the cells.
Pellet culture of the primary human chondrocytes for
differentiation
To induce chondrocyte differentiation, aliquots of 5×105
cells were centrifuged at 250 g for 5 minutes (29).
Then, chondrocyte cells were treated with 1X insulintransferrin-selenium x supplement premix (ITS-X,
Gibco, USA). After 24 hours of incubation period,
spherical aggregate of the sedimented cells were
observed at the bottom of each tube. 1×105 cells were
grown in 60 mm dish for control. Primary chondrocytes
were differentiated for 1, 7 and 14 days. The cells were
cultured under optimal condition of 37˚C and 5% CO2.
Once attached, the cells were cultured and medium
was changed after every 3 days.
Cultivation and differentiation of osteoblasts
SaOS-2 cells (Human osteosarcoma cell line, ATCC,
HTB-85), were grown in complete DMEM (10% FBS and
1% P/S). To induce differentiation, osteoblasts were grown
in osteogenic medium, containing 50 μg/ml ascorbic acid
(Sigma-Aldrich, USA) and 10 mM β-glycerophosphate
(Sigma-Aldrich, USA). 1×105 cells per well were seeded
in a 60 mm petri dish and grown at 37˚C and 5% CO2.
After every 3 days of culturing in osteogenic medium, it
was replaced. Osteoblasts were then differentiated for 1,
7 and 14 days.
RNA isolation and quantitative reverse transcription
polymerase chain reaction
As per the manufacturer’s guidelines, TRIzol reagent
(Invitrogen, USA) was used to isolate entire RNA. First
strand of cDNA was synthesized by using SuperScript
ІІ (Invitrogen, USA) and 2 µg of total RNA. For each
PCR mixture one-tenth of the cDNA was used in each
quantitative reverse transcription PCR (qRT-PCR)
supermix (EXPRESS SYBR green, BioPrince, Korea).
qRT PCR was done by using a Rotor-Gene Q (Corbett
RG3000, Australia). PCR reaction was accomplished by
50 cycles amplification at 95˚C for 20 seconds, 60˚C for
20 seconds and 72˚C for 25 seconds. Relative mRNA
expression level of specific genes was standardized to
GAPDH and quantified by using ΔΔCt method. The
human PCR primer sequences utilized in the study are
listed in Table 1.Human primer sequences for quantitative reverse transcription polymerase chain reaction
Protein extraction and western blotting
The cells were instantly rinsed with PBS (ice-cold)
after removing the media and incubated for 15 minutes
with lysis cocktail buffer containing phosphatase and
protease inhibitor (Roche Diagnostics, Germany).
After centrifugation at 14,000 rpm for 15 minutes,
the entire cell lysates were collected (separated from
the cells debris). As per manufacturer’s protocol,
protein assay kit (BioRad, USA) was used to
determine protein amount in the samples. For each
sample, equal amount of protein was loaded into
10% sodium dodecyl sulfate (SDS)-polyacrylamide
gel followed by gel electrophoresis. Then, separated
proteins were transferred to polyvinylidene fluoride
(PVDF) membranes (Millipore, USA). The blots
were incubated with 1:1000 dilutions of primary
antibodies: RSPO1, RSPO2, LGR5 and LGR6, Col1α,
Col2, osterix, IkBα, β-catenin, β-Actin (Santa Cruz
Biotechnology, USA), Sox-9 (Abcam, USA) and Cox-
2 (Cell Signaling Technology, USA) in 1 % BSA.
Blots were washed three times with TBST (10 mM Tris
HCl, 50 mM NaCl, 0.25% Tween 20) and then treated
with a horseradish peroxidase-conjugated secondary
antibody (Jackson Immunoresearch, USA) followed
by two times washing with TBST. Finally, the obtained
bands were pictured by using chemiluminescence
(ECL) reagents (BioNote Inc., Korea). Antibody
against β-actin was considered as a loading control.
Densitometric analyses of the western blots were also
performed (Fusion FX, Vilver Lourmat, France).
Statistical analysis
All of the statistical data were evaluated by Graphpad
Prism 5.0 (GraphPad Software, USA) and assessed
by two-tailed Student t test. Value of P<0.05, P<0.01
and P<0.001 was considered to designate statistical
significance.
Results
Differential expression of RSPOs and LGRs at
early and advanced stages of human OA samples
To observe the pattern of expression of RSPO
proteins and its receptors (LGRs) in OA tissue sample
from human patients, tissue sections were categorized
as early or advanced stage samples and they were
immunostained for the proteins like lubricin, RSPOs,
LGRs and β-catenin, as described in material and
methods. H&E, lubricin and safranin-o staining
demonstrated intact cartilage structure in the case
of early stage OA samples, while a loss of articular
surface, reduced expression of lubricin and decrease
in width of cartilage was observed in advanced OA
samples (Fig.1A). In advanced stage OA samples,
spatial RSPO1 expression was observed close to
subchondral bone plate which includes lower area of
calcified cartilage and a thin cortical bone tissue layer
(Fig.1B). Though, no localized expression of RSPO1
was observed in early stage OA samples. Expression
of LGR6 appeared to overlap with the expression of
RSPO1 (i.e. near the subchondral bone plate). The
spatial expression of RSPO2 was very much localized
towards the middle and deep zone of articular cartilage
since upper layer of cartilage was found distorted in
the advanced stage tissue samples. LGR5 expression
was observed close to the area of expression of RSPO2
which is near to middle and deep zone of articular
cartilage area. No expression of RSPO2 or LGR5 was
visible in the early stage OA samples. Since, RSPOs
has the ability to activate WNT signaling pathway, we
evaluated expression level of β-catenin in early and
advanced stage OA samples. In the advanced stage OA
samples, expression level of β-catenin was increased
around the overlapped region of RSPO1 and RSPO2.
Fig.1
Expression of RSPOs, LGRs and β-catenin in human OA cartilage samples. A. H&E staining, safranin-O staining and IHC of lubricin in OA cartilage
tissue samples with early and advanced stages obtained from femoral condyles cartilage. B. IHC showed that the expression of RSPOs, LGRs and
β-catenin is significantly increased in advanced stage of OA cartilage compared to early stage. In these figures, above part of the dotted line represents
articular cartilage whereas the lower part represents subchondral bone (approximate estimation, magnification ×10, scale bar: 100 µm). AS; Articular
surface, SB; Subchondral bone, RSPOs; Rspondins, LGRs; Leucine-Rich Repeat-Containing G-Protein Coupled Receptors, OA; Osteoarthritis, and IHC;
Immunohistochemistry.
mRNA expression profile of RSPO proteins and
its receptors during differentiation process of
chondrocytes
Since RSPO2 showed increased expression level
along with its receptor (LGR5) in advanced stages
OA tissue samples, we tried to analyze the expression
pattern of RSPO family proteins in differentiating
chondrocytes. Initially, the primary chondrocytes were
cultured as pellet culture and treated with InsulinTransferrin-Selenium-X supplement 1X (ITS-X)
to induce differentiation for 14 days. mRNA was
collected at days 2, 7 and 14 of the differentiation
process of chondrocytes. qRT-PCR data displayed a
substantial increase in the expression of RSPO2 after
7 days (10 folds) of treatment which followed till day
14 (16 folds), while a small increase in expression
pattern of RSPO3 and RSPO4 was observed at day
14 (3 folds) of differentiation process. In the case of
RSPO receptors, LGR5 demonstrated a significant
increase in the mRNA expression level after 7 days
(10 folds) of differentiation process followed till day
14 (15 folds). mRNA expression levels of other two
receptors, namely LGR4 and LGR6, did not show any
significant change (Fig.2A). Findings obtained from
western blot confirmed the mRNA expression results
by demonstrating protein expression of RSPO2 in
human primary chondrocytes pellet culture after 2
days of differentiation process till day 14. While,
RSPO1 protein expression was not observed during
this time. Protein expression of LGR5 was also
observed to be enhanced during differentiation process
of the chondrocytes (Fig.2B). An increment in the
protein expression level of chondrogenic markers
-like collagen (Col) 2 and master transcription factor,
Sox-9 (sex-determining region Y-type high mobility group box protein; responsible for early chondrocyte
differentiation) confirmed induction of differentiation
process in the chondrocytes.
Fig.2
Expression of RSPOs and LGRs in human primary chondrocytes during differentiation. To induce differentiation, chondrocytes were treated with 1X
ITS-X supplement. A. mRNA levels were measured by qRT-PCR after 2, 7 and 14 days of differentiation. Results are represented as fold-increase relative
to GAPDH expression. B. Protein levels were measured by western blotting after 2, 7 and 14 days of differentiation. Quantitative densitometric analysis
of protein was performed by using Fusion FX software. The results were normalized with β-actin expression. Data are shown as the mean ± SD of three
independent experiments.
mRNA expression profile of RSPO proteins and its
receptors during differentiation process of osteoblasts
Osteoblasts are well known for differentiating into
osteocytes and contributing to bone formation. This
process is tightly regulated by several regulatory
molecules like RSPOs. To observe the expression
pattern of RSPOs during the process of osteoblast
differentiation process, SaOS-2 cells were induced
to differentiate by treating β-glycerophosphate (10
mM) and ascorbic acid (50 µg/ml). mRNA from
SaOS-2 cells was collected after 2, 7 and 14 days of
differentiation process. Expression level of RSPO1,
RSPO2, RSPO3 and RSPO4 as well the receptors for
RSPOs, LGR4, LGR5 and LGR6 were analyzed by
qRT-PCR. Among RSPOs, mRNA expression level of
RSPO1 was found to be significantly increased after
7 days (12 folds) of differentiation process till day 14
(20 folds). mRNA expression of the other RSPOs were
not found to be significantly affected during the day
14 of differentiation. Among the receptors for RSPOs,
LGR6 was found to be elevated after 7 days (3 folds) of
differentiation till day 14. LGR4 and LGR5 showed no
significant alteration in mRNA expression level during
differentiation process of osteoblasts (Fig.3A). In order
to confirm the expression of RSPOs and its receptor
at mRNA levels, we tried to analyze protein level of
significantly expressed genes by western blotting.
Western blot results demonstrated elevated protein
expression of RSPO1 after 2 days of differentiation
process in SaOS-2 cells, till day 14. However, protein
expression of RSPO2 was not in accordance with
the mRNA expression profile in osteoblasts during
differentiation. Similar to mRNA expression of LGR6,
western blot results also confirmed the expression of
LGR6 after 7 days of differentiation process, till day
14. The process of differentiation in osteoblasts was
confirmed with an increment in the protein level of
osteogenic marker like Col1α and osterix (OSX, an
osteoblast-specific transcription factor) from day 2 till
14 of differentiation process in osteoblasts (Fig.3B).
Fig.3
Expression of RSPOs and LGRs in osteoblasts during differentiation. To induce differentiation, osteoblasts were treated with 10 mM β-glycerophosphate
and 50 µg/ml ascorbic acid. A. mRNA levels were measured by qRT-PCR after 2, 7 and 14 days of differentiation. Results are represented as fold-increase
relative to GAPDH expression. B. Protein levels were measured by western blotting after 2, 7 and 14 days of differentiation. Quantitative densitometric
analysis of protein was carried out by using Fusion FX software. The results were normalized with β-actin expression. Data are shown as the mean ± SD of
three independent experiments. **; P≤0.01, ***; P≤0.001, RSPOs; Rspondins, LGRs; Leucine-Rich Repeat-Containing G-Protein Coupled Receptors, qRTPCR; Quantitative reverse transcription polymerase chain reaction, Cont; Control, and d; Day.
Expression of RSPOs, LGRs and β-catenin in human OA cartilage samples. A. H&E staining, safranin-O staining and IHC of lubricin in OA cartilage
tissue samples with early and advanced stages obtained from femoral condyles cartilage. B. IHC showed that the expression of RSPOs, LGRs and
β-catenin is significantly increased in advanced stage of OA cartilage compared to early stage. In these figures, above part of the dotted line represents
articular cartilage whereas the lower part represents subchondral bone (approximate estimation, magnification ×10, scale bar: 100 µm). AS; Articular
surface, SB; Subchondral bone, RSPOs; Rspondins, LGRs; Leucine-Rich Repeat-Containing G-Protein Coupled Receptors, OA; Osteoarthritis, and IHC;
Immunohistochemistry.Expression of RSPOs and LGRs in human primary chondrocytes during differentiation. To induce differentiation, chondrocytes were treated with 1X
ITS-X supplement. A. mRNA levels were measured by qRT-PCR after 2, 7 and 14 days of differentiation. Results are represented as fold-increase relative
to GAPDH expression. B. Protein levels were measured by western blotting after 2, 7 and 14 days of differentiation. Quantitative densitometric analysis
of protein was performed by using Fusion FX software. The results were normalized with β-actin expression. Data are shown as the mean ± SD of three
independent experiments.*; P≤0.05, **; P≤0.01, ***; P≤0.001, RSPOs; Rspondins, LGRs; Leucine-Rich Repeat-Containing G-Protein Coupled Receptors, 1X ITS-X; Insulin-TransferrinSelenium-Ethanolamine, qRT-PCR; Quantitative reverse transcription polymerase chain reaction, Cont; Control, and d; Day.Expression of RSPOs and LGRs in osteoblasts during differentiation. To induce differentiation, osteoblasts were treated with 10 mM β-glycerophosphate
and 50 µg/ml ascorbic acid. A. mRNA levels were measured by qRT-PCR after 2, 7 and 14 days of differentiation. Results are represented as fold-increase
relative to GAPDH expression. B. Protein levels were measured by western blotting after 2, 7 and 14 days of differentiation. Quantitative densitometric
analysis of protein was carried out by using Fusion FX software. The results were normalized with β-actin expression. Data are shown as the mean ± SD of
three independent experiments. **; P≤0.01, ***; P≤0.001, RSPOs; Rspondins, LGRs; Leucine-Rich Repeat-Containing G-Protein Coupled Receptors, qRTPCR; Quantitative reverse transcription polymerase chain reaction, Cont; Control, and d; Day.
Effect of RSPO2 during tumor necrosis factor alpha
stimulatory conditions in chondrocytes
Tumor necrosis factor alpha (TNFα) is a known
inflammatory marker and is a major cytokine released
during inflammatory pathological condition, like arthritis
(30). To depict an in vitro inflammatory condition, TNFα
(10 ng/ml) was induced to chondrocytes after 7 days
of differentiation and the protein expression level of
inflammatory marker like Cox-2 and stability of IκBα
(NFκB signaling activation) were measured by western
blotting (Fig.4). Moreover, to observe any effect by
RSPO2 on the inflammation induced by TNFα, RSPO2
(100 ng/ml) was treated along with TNFα and expression
levels of inflammatory marker were analyzed. Results
demonstrated an increased protein level of Cox-2 and
diminished IκBα stability in chondrocytes, indicating
activation of NFκB signaling by TNFα, as expected.
Interestingly, treatment of RSPO2 recovered the effect of
TNFα and suppressed the Cox-2 expression, in addition to
restoring the stability of IκBα. RSPO2 is known to induce
WNT signaling activity and it was evident by the increased
stability of β-catenin in chondrocytes. However, TNFα
inhibited the β-catenin stability which was restored by the
treatment of RSPO2. Effect of TNFα was also evident on
the expression level chondrogenic markers, like Col2 and
Sox-9, in differentiated chondrocytes which again was
recovered after the treatment of RSPO2.
Fig.4
Effect of RSPO2 during chondrocyte differentiation under inflammatory conditions. Primary human chondrocytes were differentiated till day 7
by treating with 1X ITS-X. Thereafter, the cells were treated either with TNFα (T: 10 ng/ml) or along with RSPO2 (R2: 100 ng/ml) recombinant proteins.
After 24 hours, protein expression levels of Cox-2, IkBα, Col2, Sox-9 and β-catenin were analyzed by western blotting. Protein expressions were detected
by western blot. A relative densitometry analysis of protein bands was performed using Fusion FX software. The results were normalized with β-actin
expression. Significant changes between the RSPO2 treated sample with TNF-α (alone) and TNFα with TNFα along with RSPO2 has been depicted. Data
are shown as the mean ± SD of three independent experiments. *; P≤0.05, **; P≤0.01, ***; P≤0.001, RSPOs; Rspondin, TNFα; Tumor necrosis factor-alpha,
Cont; Control, and d; Day.
Effect of RSPO2 during chondrocyte differentiation under inflammatory conditions. Primary human chondrocytes were differentiated till day 7
by treating with 1X ITS-X. Thereafter, the cells were treated either with TNFα (T: 10 ng/ml) or along with RSPO2 (R2: 100 ng/ml) recombinant proteins.
After 24 hours, protein expression levels of Cox-2, IkBα, Col2, Sox-9 and β-catenin were analyzed by western blotting. Protein expressions were detected
by western blot. A relative densitometry analysis of protein bands was performed using Fusion FX software. The results were normalized with β-actin
expression. Significant changes between the RSPO2 treated sample with TNF-α (alone) and TNFα with TNFα along with RSPO2 has been depicted. Data
are shown as the mean ± SD of three independent experiments. *; P≤0.05, **; P≤0.01, ***; P≤0.001, RSPOs; Rspondin, TNFα; Tumor necrosis factor-alpha,
Cont; Control, and d; Day.
Effect of RSPO1 during TNFα stimulatory conditions
in osteoblasts
Treatment of SaOS-2 cells with TNFα (10 ng/ml)
7 days after differentiation induced the expression of
Cox-2 and decreased the stability of IκBα, implicating
activation of NFκB signaling in SaOS-2 cells (Fig.5).
The ability of RSPO1 (100 ng/ml) to induce WNT
signaling activity was observed even in SaOS-2
cells as the protein level of β-catenin was found to
be increased after RSPO1 treatment. Stimulation of
RSPO1 to TNFα treated SaOS-2 cells decreased the
protein levels of Cox-2, while it restored the stability
of IκBα. Moreover, TNFα suppressed protein level of
β-catenin, while it was recovered after the stimulation
of RSPO1. As marker for differentiation process of
osteoblasts, the protein levels of Col1α and OSX was
increased in SaOS-2 cells after day 7 of differentiation
process. However, treating with TNFα was able to
suppress the protein levels of Col1α and OSX in 7 days
differentiated SaOS-2 cells. Stimulation of RSPO1 to
TNFα treated SaOS-2 cells restored the protein level
of both Col1α and OSX.
Fig.5
Effect of RSPO1 during osteoblast differentiation under inflammatory conditions. SaOS-2 cells were differentiated till day 7 by treating
with 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid. Then, SaOS-2 cells were treated either with TNFα (T: 10 ng/ml) or along with
RSPO1 (R1: 100 ng/ml) recombinant proteins. After 24 hours, the protein expression levels of Cox-2, IkBα, Col1α, OSX and β-catenin were
analyzed by western blotting. Protein expressions and a relative densitometry analysis were performed by using Fusion FX software. The
results were normalized with β-actin expression. Significant changes between the RSPO1 treated sample with TNFα (alone) and TNFα with
TNFα along with RSPO1 has been depicted. Data are shown as the mean ± SD of three independent experiments. *; P≤0.05, **; P≤0.01, ***;
P≤0.001, TNFα; Tumor necrosis factor-alpha, Cont; Control, and d; Day.
Effect of RSPO1 during osteoblast differentiation under inflammatory conditions. SaOS-2 cells were differentiated till day 7 by treating
with 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid. Then, SaOS-2 cells were treated either with TNFα (T: 10 ng/ml) or along with
RSPO1 (R1: 100 ng/ml) recombinant proteins. After 24 hours, the protein expression levels of Cox-2, IkBα, Col1α, OSX and β-catenin were
analyzed by western blotting. Protein expressions and a relative densitometry analysis were performed by using Fusion FX software. The
results were normalized with β-actin expression. Significant changes between the RSPO1 treated sample with TNFα (alone) and TNFα with
TNFα along with RSPO1 has been depicted. Data are shown as the mean ± SD of three independent experiments. *; P≤0.05, **; P≤0.01, ***;
P≤0.001, TNFα; Tumor necrosis factor-alpha, Cont; Control, and d; Day.
Discussion
OA is marked by a continual damage of articular cartilage
accompanied with gradual loss of extracellular matrix,
causing pain and functional disabilities in elder people
(2). Regardless of extensive research efforts on OA, there
is a massive need of effective therapies that can ultimately
alter the natural course of this painful disease. With due
efforts, recent researches have established that OA is not
just a disease of articular cartilage, but the subchondral
bone beneath. It also has a vital role in maintaining the
health of the osteochondral unit (9). Studies focused on
the molecular communications amid bone and cartilage
interfaces might provide an understanding into the various
mechanisms that control the vital molecular factors and
signaling pathways involved in pathophysiology of OA (6,
19). Among the various factors that affects both cartilage
and bone, WNT signaling pathway has been found to be
activated during OA and it is thought to play critical role
in tissue repair and fibrosis (31).RSPOs are secretory proteins that have an ability to
activate WNT signaling pathway and they are often
co-expressed with WNTs (21, 27). During mouse
development, RSPOs expression overlap with the
expression of WNT signaling proteins, suggesting a
likely association of RSPOs with the WNT signaling
pathway (28). Rspo genes are differentially expressed
during development of mouse limbs, implicating dynamic
role of RSPOs during skeletal development (24, 28, 32).
Recently, efforts were made to study the involvement of
RSPO proteins in inflammatory arthritis animal model
(TNFα transgenic mice) and it was demonstrated that
RSPO1 was able to prevent bone and cartilage from
inflammation-related damage (33). RSPO family proteins
are dynamically expressed with distinct patterns during
different mouse embryonic and fetal developmental
stages (28). Henceforth, in order to understand the
involvement of RSPO proteins in OA, we tried to analyze
the expression pattern of RSPOs along with their receptors
(LGRs) in early and advanced stage of human OA tissue
samples. A progression based dynamic expression of
RSPOs might explain its regulatory role during the
pathogenesis of OA. Moreover, we tried to understand the
pattern of expression of RSPO and LGR family during
differentiation process of chondrocytes and osteoblasts,
in vitro. In endochondral ossification, RSPO2 has been
implicated to facilitate differentiation of chondrocytes
by augmenting WNT signaling pathway (34). However,
in animal OA models, increased stimulation of WNT/β-
catenin signaling exerts hypertrophic differentiation in
articular chondrocytes, which, in turn, results in enhanced
expression of cartilage-degrading metalloproteinase and
subsequent aggravation of OA. RSPO2 exerts this effect
by binding to its receptor LGR5 (35). In agreement,
expression profile of our results demonstrated an overlap
between the expression of RSPO2 and LGR5 in advanced
stage OA samples. The expression of RSPO2 and LGR
was very much localized toward the deep region of
articular cartilage. Moreover, in vitro data showed the
expression of RSPO2 and LGR5 during differentiation
process of chondrocytes. Here, it appears that expression
of RSPO2 is critical for differentiation of chondrocytes
and it is enhanced under pathological conditions, like OA
the expression of RSPO2.Previously, we have shown that RSPO1 can promote
osteoblast differentiation process through WNT signaling
pathway (36). Increased expression of LGR6 has been
identified in the mesenchymal stem cells undergoing
osteogenic induction and LGR6 has been suggested as an
osteoblastic progenitor marker (37). In accordance to the
above studies, we also observed that LGR6 expression
overlapped with the expression of RSPO1 in advanced
stage OA samples. In addition, the expressions of RSPO1
and LGR6 were detected during differentiation process of
osteoblasts, implicating that LGR6 is possibly responsible
for recognizing RSPO1 and mediating its effect for WNT
signaling stimulation. However, further experiments are
needed to ascertain this fact.Numerous studies have indicated that TNFα plays
a critical role, not only during the pathogenesis of
inflammatory arthritis but also during degenerative
joint disease like OA (30, 38). TNFα is responsible for
maintaining the homoeostasis of matrix synthesis and
its degeneration in articular cartilage of tandem with
other cytokines like IL1, transforming growth factor β.
Moreover, TNFα role has been shown in induction of
bone loss during inflammatory conditions by affecting
WNT signaling pathway (39, 40). In order to mimic the
pathological conditions that might prevail during OA, we
simply stimulated the chondrocytes and osteoblasts with
TNFα and induced inflammatory response in these cells.
Interestingly, co-treatment of TNFα along with RSPO2
in chondrocytes and RSPO1 in osteoblasts not only
recovered the induction of inflammatory marker like Cox-
2, but also suppressed activated NFκB signaling in both of
the cell types. Moreover, TNFα, suppressed chondrogenic
markers (Col2 and Sox-9) and osteogenic markers (Col1α
and OSX), were found to be recovered after co-treatment
with RSPO2 and RSPO1, respectively. Additionally,
TNFα, suppressing β-catenin stability, was restored by
treatment of RSPO2 and RSPO1 in chondrocytes and
osteoblasts, respectively. These results point towards a
regulatory role of RSPOs in inflammation which might
be achieved by activating WNT signaling pathway. TNFα
has been shown to induce secretion of WNT antagonists,
like DDK1 and SOST, from differentiating osteoblasts
affecting their bone forming ability (39). Moreover,
the localized expression pattern of RSPO1 (near to
subchondral bone area) and RSPO2 (near to deep articular
cartilage area) in the OA samples raise a possibility of
interplay between chondrocytes and osteoblasts. Though,
it appears to be interesting, further studies would be
needed to delineate the mechanism by which WNT
signaling pathway might interact with the inflammatory
mechanism under the regulation of RSPOs. For example,
further studies focused on the release of WNT signaling
antagonists, in response to TNFα in chondrocytes and osteoblasts and finding any role of RSPOs in regulating
this process would be quite interesting. Limitation of our
study is that we have just considered TNFα as a stimulator
for inflammation in vitro, while inflammation during
OA pathogenesis is a multifactorial event, involving a
diverse kind of pro-inflammatory factors and cytokines.
For instance, other than TNFα, IL1β is the other cytokine
that affects both chondrocytes and osteoblasts in joints.
Hence, future studies should try to reveal the effect
and role of IL1β or a combination of other cytokines in
presence of RSPOs. A clear understanding of RSPOs antiinflammatory role under inflammatory conditions, like
OA, would be helpful to suggest novel therapeutic agents
in near future.
Conclusion
During pathogenesis of OA, both articular cartilage
and subchondral bone shows morphological and
biochemical changes. OA does not simply represent
an event of wear and tear process, but instead it is an
atypical remodeling process leading to joint failure. An
intermolecular interaction between articular cartilage
and subchondral bone interface is being regarded
as the contributing factor for altered structural and
functional characteristics of this unit. RSPO family of
proteins is known to stimulate WNT signaling pathway.
Chondrocytes and osteoblasts need functional role of
WNT signaling pathway during their developmental
process as well as in pathogenic state. Thus, as key
molecules for WNT signaling pathway, RSPOs might
play a crucial role during their cross-talk based on their
differential expression patterns. Our results in OA tissue
samples demonstrate spatial expression of RSPO1 and
RSPO2 along with their receptors, respectively LGR6
and LGR5, in early and advanced stage of OA samples.
In vitro differentiation analysis of chondrocytes and
osteoblasts also demonstrated correlation of expression
pattern of RSPOs along with its receptors. Interestingly,
the ability of RSPOs to recover adverse effect induced
by TNFα represents possible role of RSPOs in affecting
inflammatory pathways through WNT signaling.
However, more detailed studies would be required
to ascertain the functional role of RSPOs during
inflammation. In brief, RSPOs might be the regulatory
molecule and they may explain the relationship amongst
cartilage and subchondral bone under pathogenic
conditions like OA. A clear insight into the differential
expression of RSPOs and their functional role might
contribute to identify novel therapeutic targets for the
cure of OA in near future.
Table 1
Human primer sequences for quantitative reverse transcription polymerase chain reaction
Authors: Arjen B Blom; Sarah M Brockbank; Peter L van Lent; Henk M van Beuningen; Jeroen Geurts; Nozomi Takahashi; Peter M van der Kraan; Fons A van de Loo; B Wim Schreurs; Kristen Clements; Peter Newham; Wim B van den Berg Journal: Arthritis Rheum Date: 2009-02
Authors: Mei Zhu; Dezhi Tang; Qiuqian Wu; Suyang Hao; Mo Chen; Chao Xie; Randy N Rosier; Regis J O'Keefe; Michael Zuscik; Di Chen Journal: J Bone Miner Res Date: 2009-01 Impact factor: 6.741
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