Differential Expression Patterns of Rspondin Family and
Leucine-Rich Repeat-Containing G-Protein Coupled
Receptors in Chondrocytes and Osteoblasts
The first two authors equally contributed to this work.
Rspondins (RSPOs) are regarded as the significant modulators of WNT signaling pathway and they are expressed dynamically during developmental stages. Since in osteoarthritis (OA) both cartilage and subchondral bone suffer damages and WNT signaling pathway has a crucial role in their maintenance, the objective of the study was to analyze expression profile of RSPO family and its receptors [leucine-rich repeat-containing G-protein coupled receptors (LGRs)] in OA tissue samples as well as in differentiating chondrocytes and osteoblasts.
Materials and Methods:
In this experimental study, human early and advanced stage of OA tissue samples were analyzed for the morphological changes of articular cartilage by hematoxylin and eosin (H&E) staining, safranin-O staining and lubricin immunostaining. RSPOs and LGRs expression were confirmed by immunohistochemistry. Human primary chondrocytes and human osteoblast cell line, SaOS-2, were cultured in differentiation medium till day 14 and they were analyzed in terms of expression of RSPOs, LGRs and specific marker for chondrogenesis and osteogenesis by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Advanced stage OA tissue samples showed increased expression of RSPO1 and LGR6 in a region close to
subchondral bone. While RSPO2 and LGR5 expression were seen overlapping in the deep region of articular cartilage.
Differentiating chondrocytes demonstrated elevated expression of
Spatial expression of RSPOs during progression of OA might be dynamically controlled by cartilage and subchondral bone. Interplay amid chondrocytes and osteoblasts, via RSPOs, might provide probable mechanisms for treating inflammatory pathogenic conditions like OA.
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
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 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.
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
|Gene||Primer sequence (5ˊ-3ˊ)|
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).
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.
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.
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
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
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
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.
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,
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
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.
This research financially supported by Hallym University Research Fund and Basic Science Research Program through the National Research Foundation of Korea (NRF) established by the Ministry of Education (NRF- 2016R1D1A1B03931318 and NRF-2017R1A2B4012944). There is no conflict of interest in this study.
Y.-H.L., A.R.S.; Performed the experiments, collected and interpreted the data, drafted and edited the manuscript. S.J.; Participated in data analysis, evaluations and editing the manuscript. J.-S.N., S.-S.L.; Conceived the idea, participated in study design and supervised the project. All authors read and approved the final manuscript.