Tight regulation of wingless-type signaling in the articular cartilage - subchondral bone biomechanical unit: transcriptomics in Frzb-knockout mice

Introduction The aim of this research was to study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled-related protein (Frzb) compared to wild-type mice by transcriptome analysis. Methods Gene-expression analysis of the articular cartilage and subchondral bone of three wild-type and three Frzb-/- mice was performed by microarray. Data from three wild-type and two Frzb-/- samples could be used for pathway analysis of differentially expressed genes and were explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the wingless-type (WNT) pathway was analysed using Western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micro-masses and mouse ribcage chondrocytes. Results Extracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were up-regulated in Frzb-/- samples. Several WNT receptors, target genes and other antagonists were up-regulated, but no difference in active β-catenin was found. Analysis of ATDC5 cell micro-masses overexpressing FRZB indicated an up-regulation of aggrecan and Col2a1, and down-regulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in down-regulation of aggrecan and Col2a1. Pathways associated with cell cycle were down-regulated in this transcriptome analysis. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells. Conclusions Our analysis provides evidence for tight regulation of WNT signalling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint.


Introduction
Homeostasis of articular cartilage and subchondral bone is essential for maintenance of joint function which is critically dependent on the balance between anabolic and catabolic signaling pathways [1,2]. It requires maintenance of the stable phenotype that characterises the articular cartilage, sustained extracellular matrix (ECM) synthesis, efficient breakdown and clearance of damaged macromolecules and dead cells, as well as functional and molecular adaptations to mechanic loads. Loss of homeostasis results in gradual deterioration of cartilage quality and thickening of the subchondral bone, progressively leading to osteoarthritis (OA).
The wingless-type (WNT) signaling pathway plays an important role in cartilage, bone and joint development and has been associated with postnatal joint homeostasis and disease [3,4]. WNTs are a group of at least 19 structurally related secreted glycoproteins that activate different intracellular cascades [5]. Among these, canonical WNT signaling involving β-catenin has been studied best. In the absence of a WNT-Frizzled low density lipoprotein receptor-related protein-5/6 co-receptor interaction (WNT-FZD-LRP5/6), β-catenin is caught in a molecular destruction complex, phosphorylated and degraded by the proteasome. Upon WNT-receptor interaction, the destruction complex is disassembled, βcatenin accumulates in the cell, translocates to the nucleus and associates with transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Alternatively, non-canonical WNT signaling can alter calcium balances in the cell or activate protein kinases [5,6].
WNTs, their extracellular antagonists, such as the secreted frizzled-related proteins (SFRPs), co-receptor inhibitors, such as the dickkopfs (DKKs), and β-catenin have been studied in animal models of OA and OA patients [7][8][9][10][11][12][13]. Current data suggest that canonical WNT signaling plays an essential role in joint and bone formation [14,15] and in the maintenance of the articular cartilage phenotype, which is characterised by extended cell survival and absence of differentiation towards hypertrophy [16]. Cartilage-specific inhibition of β-catenin results in an OA-like phenotype with chondrocyte apoptosis [8]. Cartilage-specific overexpression of a constitutively active form of β-catenin also results in an OA-like phenotype, but here the disease is characterised by loss of the chondrocyte's differentiation status and expression of hypertrophic markers [9].
Frizzled-related protein (Frzb, also known as SFRP3) is a WNT antagonist originally identified from a chondrogenic extract of articular cartilage [17] and plays a role in skeletal development [17,18]. Polymorphisms in FRZB have been associated with OA [3]. We previously developed mice that are genetically deficient in Frzb. These mice do not develop spontaneous arthritis but are more susceptible to OA in induced models [7]. This observation has been linked to increased WNT signaling and Mmp3 expression in the articular cartilage. The cortical bone in these mice is thicker and the bones show an enhanced anabolic response upon mechanical loading compared to wild-type mice. In this study, we used Frzb -/mice to further evaluate how the absence of a WNT antagonist affects molecular homeostasis in the articular cartilage and subchondral bone.

Mice and tissue sampling
Frzb -/mice were generated in our research group [7] and back-crossed into the C57Bl/6J background for over 10 generations. Genotypes were determined as described [7]. Six-week-old male Frzb -/and wild-type mice were sacrificed by cervical dislocation. The articular cartilage and subchondral bone from the tibial plateau of the knee joint of the hind limb was carefully dissected in one piece at the growth plate region using micro-dissection forceps, a procedure easy to perform at this age when the growth plate is not yet closed. The tissues were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing or used for histology. Animal procedures were approved by the Ethical Committee for Animal Research, KULeuven.

Microarray hybridization and data acquisition
Per microarray, articular cartilage and subchondral bone from a single joint were used. Samples were homogenised using the Fastprep-24 tissue-homogeniser (MP Biomedicals, Solon, OH, USA) in lysing matrix A tubes and RLT lysis buffer (RNeasy Fibrous Tissue kit (Qiagen, Chatsworth, CA, USA)). Samples were kept under pre-cooled conditions using the CryoPrep Adaptor. RNA was isolated with the RNeasy Fibrous Tissue kit (Qiagen) with proteinase K and deoxyribonuclease (DNaseI) treatment. RNA concentration and purity were assessed with a NanoDrop Spectrophotometer (Nano-Drop Technologies, Centreville, DE, USA) and integrity was determined using RNA nanochips and the Agilent 2100 Bio-analyzer (Agilent Technologies, Diegem, Belgium). Only non-degraded RNA without impurities (RNA integrity number > 7.7), was considered for microarray analysis.
Transcriptional profiles of three Frzb -/and three wildtype samples were analyzed by the VIB Microarray Facility [19]. Per sample, 2 μg of total RNA spiked with bacterial RNA transcript positive controls (Affymetrix, Santa Clara, CA, USA) was converted to double stranded cDNA. Subsequently, the sample was converted and amplified to antisense cRNA and labeled with biotin. A mixture of purified and fragmented biotinylated cRNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Mouse Genome 430-2.0 arrays followed by staining and washing in a GeneChip fluidics station 450 (Affymetrix). To assess the raw probe signal intensities, chips were scanned using a GeneChip scanner 3000 (Affymetrix). Microarray data have been deposited in the Gene Expression Omnibus (GEO) [20] and are accessible through Gene Expression Omnibus accession number GSE33656.
In Frzb knock-down experiments, ATDC5 cells were transfected with control pGIPZ-non-silencing shRNAmir (Open Biosystems, Thermo Scientific IT IS Open Biosystems, Thermo Scientific, Lafayette, CO, USA) or with a pGIPZ-shRNAmir directed against Frzb (Open Biosystems) using lipo-polymeric agent Arrest-In (Open Biosystems). After 24 hours, selection with 0.5 μg/ml puromycin was initiated. Selection medium was renewed every day for seven days. Antibiotic resistant cells were dilution-cloned.
Stably-transfected ATDC5 cells were grown in micromasses to undergo chondrogenesis. Three drops cell suspension (2 × 10 5 cells) were placed in a single well of a standard 12-well culture plate. The cells were allowed to adhere for two hours at 37°C, then 1 ml maintenance medium was added to each well. Geneticin or puromycin pressure was maintained during chondrogenesis.
Protein extraction of the micro-masses stably overexpressing FRZB or controls after seven days was performed using cell extraction buffer supplemented with 1 mM phenylmethanesulfonyl and 5% protease inhibitor cocktail, followed by quantification using the Pierce BCA Protein Assay kit (Thermo Scientific).
Some ATDC5 micro-masses were fixed in 95% icecold methanol for staining. For Picrosirius Red, micromasses were stained for one hour in Picrosirius Red (0.1% Direct Red 80 (Sigma-Aldrich) in a saturated aqueous solution of picric acid), washed three times with 0.5% acetic acid in water and air-dried. For Safranin O, micro-masses were stained for one hour in Safranin O (1% alcoholic solution (Klinipath, Olen, Belgium)), washed three times with water and air-dried. Quantification of the staining was performed by dissolving the micro-masses with 1 M NaOH (for Picrosirius Red) or 6M Guanidine-HCl (for Safranin O) (both from Sigma-Aldrich) and by measuring the absorbance at 540 and 512 nm respectively with the Infinite M200 (Tecan, Männedorf, Switzerland).

Mouse rib chondrocyte isolation and proliferation analysis
Rib and sternum chondrocytes were isolated from three six-week-old wild-type and three Frzb -/mice, as described with minor modifications [23]. The sternum was longitudinally cut, followed by complete removal of the ventral part of the ribcage. The ribcage was washed three times in Dulbecco's phosphate buffered saline (DPBS) (Lonza, Verviers, Belgium) with 1% AB (Gibco). Soft tissues were digested in 3 mg/ml collagenase D (Roche Diagnostics) in medium (DMEM, 1% AB and 1% sodium pyruvate (Invitrogen)) for 1 h standing upright in a collection tube in humidified atmosphere of 5% CO 2 and 95% O 2 at 37°C, followed by rotation for a further 1.5 h. Soft tissues were carefully removed, followed by further digestion in fresh 3 mg/ml collagenase D in medium when the soft tissues kept adhering. After washing twice in DPBS with 1% AB, cartilage was digested using 1 mg/ml collagenase D in medium overnight in a petri dish in the incubator. The medium containing chondrocytes was transferred to a collection tube. The bones were rinsed with complete growth medium (10% FBS (Gibco)) and this was also transferred to the collection tube. After centrifugation, cells were resuspended in 4 ml complete growth medium, plated on a T25 plate (Greiner Bio-One, Frickenhausen, Germany) and grown until confluent. The medium was changed every two days. For the proliferation assay, chondrocytes from three Frzb -/and three wild-type mice were plated at different cell densities (500, 2,000 or 4,000 cells/well) in triplicate on fluorescence compatible 96-well flat bottom plates (μClear-plate, black, 98well, Greiner Bio-one). Fluorescence was measured 24 h and 1 week after plating using the CyQuant NF Cell proliferation kit (Molecular Probes, Invitrogen) and the Wallac Victor 1420 Multilabel counter (Perkin Elmer) at an excitation wavelength of 485 nm and emission of 535 nm. The difference in fluorescence between the two time points (24 h and 1 week) was calculated and considered the amount of proliferation in that time window. A different plate was used for each time point.

Bioinformatics analysis and statistics
The quality of hybridization and data acquisition was assessed by RNA-degradation plots, histograms of the perfect match values distribution and quality control graphs. Data were pre-processed by removal of the hybridisation, labeling control and absent probe sets, followed by a log2 transformation and normalisation of the results to obtain the Robust Multiarray Averaging (RMA) algorithm defined expression values and the Microarray Analysis Suite (MAS) 5.0 software detection calls. Significant differences in gene expression were defined using a modified t-test by the limma package from Bioconductor [24] followed by Benjamini-Hochberg multiple testing correction. For further analysis, we used the PANTHER [25], DAVID [26] and GSEA [27] tools [28][29][30][31][32][33].
PANTHER uses pathways compiled by experts and determines the representation of a specific pathway on the selected gene list by applying a binomial statistic to which we applied an additional false discovery rate (FDR) test. Only pathways that included at least 15 annotated genes were taken into consideration. With DAVID we interrogated representation in KEGG [34] and Biocarta pathways [35]. It uses a modified Fisher's exact test and applies a Benjamini-Hochberg multiple testing correction. The GSEA system uses all data in the microarray analysis in a ranked list and compares a maximal enrichment score to a series of 1,000 random permutations resulting in nominal P-values and FDR qvalues. For GSEA analysis, the KEGG curated pathway set, the miRNA motif and transcription factor motif gene sets were used applying 1,000 permutations defined by the gene set. A weighed enrichment statistic using log2-ratio of classes was applied. A stringent limit with a nominal P-value < 0.001 and a FDR q-value < 0.01 was applied. In addition, we compiled a list of WNT target genes based on the WNT homepage [36] (see Additional file 1) and used a Yates corrected Chi-square test to compare our selected gene lists with the reference list. Other datasets were analyzed using a Mann-Whitney test for unpaired samples.
In silico promoter analysis of the Col3a1, Col5a1 and Col5a3 genes was performed using the TFSearch [37] and ALIBABA [38] online software, based on the TRANSFAC algorithm. Stringent criteria were applied so that only the responsive elements with a high homology to the consensus sequence matched our search (> 90%). Additionally, TCF/LEF responsive elements, specific transcription factors associated with WNT signaling, were investigated using the different consensus sequences as previously identified [39].

Primary analysis of the microarrays
We were able to dissect the subchondral bone and articular cartilage in one piece ( Figure 1A). The heatmap of the RMA expression values from the microarray analysis showed clustering of the transcriptomes into groups formed by the three wild-type and two out of three Frzb -/mice, respectively ( Figure 1B). The third presumed Frzb -/mouse clustered with the wild-types and was subsequently identified by re-genotyping as a heterozygous animal. This sample was not used in the analysis. A total of 697 probe sets out of 30,590 that had a "present" detection call were significantly up-regulated in the Frzb -/samples and 1,524 were significantly down-regulated as compared to the wild-type mice (defined by a Pvalue < 0.01 after Benjamini-Hochberg correction and | log2|-ratio > 1). Cartilage-specific and bone-specific genes were found in the highest percentiles of expressed genes in the microarray analysis, whereas genes specifically related to T cells, B cells and platelets were found in lower percentiles; possibly from RNA originating from the subchondral bone marrow ( Figure 1C).

Pathway analysis
Different bioinformatics tools were used for analysis of the large dataset with emphasis on the identification of pathways differentially regulated between the Frzb -/and wild-type mice. The PANTHER pathway analysis is shown in Table 2. Among the up-regulated pathways the ECM-associated integrin pathway, the cadherin pathway, as well as WNT signaling, were most striking from a biological perspective. Down-regulated pathways pointed towards inflammation and immune cascades, the cell cycle, p53 activation and again integrins ( Table  2). Associations of the differentially regulated gene set using databases defining "biological processes" as analysed by PANTHER are shown in the additional materials (see Additional file 3).
We also applied the DAVID bioinformatics tools specifically interrogating gene representation in KEGG and Biocarta databases. Again, pathways associated with WNT signaling, cell adhesion and ECM interactions were most prominent among the up-regulated gene sets and appeared relevant from a biological perspective (see Additional file 4). Members of transforming growth factor-beta (TGFβ) superfamily signaling, including bone morphogenetic proteins (BMPs), were also up-regulated. Pathways among the down-regulated gene list were again linked to p53 signaling and the cell cycle, and to different systems associated with immunity and inflammation. The GSEA analysis further confirmed positive associations between Frzb -/mice and ECM interactions as well as negative associations with the cell cycle (see Additional file 5). No miRNAs were associated with the Frzb -/or wild-type phenotype using the stringent limit (nominal P-value < 0.001 and FDR q-value < 0.01). Only miRNA-147 had a nominal P-value < 0.001 and a FDR q-value < 0.25 (0.17). This miRNA has been associated with WNT and ECM pathways [40] (Table 3). In the transcription factor analysis, motifs associated with Foxd1, Znf238 and Pbx1 had nominal P-values < 0.001 and FDR q-values < 0.05. Foxd1 has been suggested as a WNT target gene in the developing chick retina [41] ( Table 3). In addition, two motifs without specific transcription factor association were also enriched with Pvalues < 0.001 and FDR q-values < 0.05 (Table 3). Genes overexpressed in the wild-type mice compared to the Frzb -/mice were associated with different members of the E2F family of transcription factors applying the stringent criteria. E2F1 has been negatively associated with WNT signaling [42].

Detailed pathway analysis
We focused on a detailed analysis of changes in the WNT, the integrin/cadherin/ECM and the cell cycle pathways. Many genes mapped in the down-regulated inflammation-associated signaling systems were specifically linked to immune cell populations present in the bone marrow and were not further taken into account for this study.
The WNT pathway gene set demonstrated up-regulation of different extracellullar WNT antagonists in the Frzb -/mice as compared to wild-types. These genes belonged to the SFRP/FRZB-family, to the DKK family and to a group of intracellular WNT pathway modulators (Table 4 -compiled from PANTHER and DAVID analysis). Different frizzled (FZD) receptors were upregulated and there was evidence for activation of both canonical and non-canonical signaling with increased expression of target genes, such as Rspo2, Wisp2, Sox17, Tbl1x and Acta2, and of intracellular messenger molecules Nfatc2 and 4 that are activated in the calciumdependent WNT pathway (Table 4). Confirmation experiments by RT-PCR showed lack of Frzb, significant up-regulation of Sfrp1, Sfrp2 and a similar trend for Dkk2 (Figure 2A). This up-regulation of other antagonists may represent a compensatory mechanism to minimise the effects of WNT pathway activation in Frzb -/mice. Western blot analysis showed only discrete amounts of these different antagonists in the dissected material and did not allow for reliable quantification of the individual proteins (data not shown). Baseline activation of the canonical signaling pathway was indeed not found different between Frzb -/and wild-type mice as demonstrated by Western blot and quantitative analysis by densitometry for the active form of β-catenin ( Figure 2B). Also, Western blot for intracellular messengers of the BMP pathway, P-Smad 1/5/8, showed no striking differences between wild-type and Frzb -/mice suggesting maintenance of WNT and BMP pathway balance at the tissue level in unchallenged mice ( Figure 2C). However, further comparison of the list with genes up-regulated in the Frzb -/mice with a user-compiled list of WNT target genes (see Additional file 1), did reveal consistent up-regulation of such targets indicating that more subtle changes at the molecular level are present (Yates corrected Chi-square test P < 0.0001).
Although we did not previously find structural abnormalities or spontaneous development of OA in Frzb -/mice, expression of ECM components and cell adhesion molecules showed a shift in this genetic model (Table 5). In particular, a number of collagens were differentially regulated and specific changes in integrins were found. Some of these link to the articular cartilage while others are more likely associated with the subchondral bone and with small vessels.
We performed complementary gain of function experiments to test the effect of FRZB on chondrogenesis and ECM composition in micro-masses from the mouse chondrogenic ATDC5 cell line. Expression of both Col2a1 and aggrecan was significantly increased in ATDC5 micro-masses overexpressing FRZB as compared to controls ( Figure 3A). Staining for collagen content (Picrosirius Red) and sulphated glycosaminoglycans (GAGs) (Safranin O) at Day 7 revealed some changes in the morphology of micro-masses overexpressing FRZB. Collagen fibers and sulphated GAG distribution in these micro-masses seemed to have spread out more from the center compared to the controls ( Figure  3B). Protein quantification of the micro-masses was, however, comparable between the two groups suggesting that the appearance reflects increased migration of ATDC5 cells overexpressing FRZB ( Figure 3C). Quantification of the stainings was not different between micro-masses overexpressing FRZB and controls for Picrosirius Red. For Safranin O staining intensity was mildly but significantly decreased in micro-masses overexpressing FRZB ( Figure 3D). Conversely silencing of Frzb resulted in down-regulation of these genes ( Figure  3E). RT-PCR analysis of other collagens, in particular Col3a1 and Col5a1, significantly up-regulated in the Frzb -/mice compared to wild-type mice in the microarray analysis, depicted a decreasing trend at Day 7 in FRZB overexpressing micro-masses compared to the control micro-masses; however, these comparisons did not reach statistical significance ( Figure 4A). A similar down-regulation compared to controls was seen during differentiation after silencing of Frzb ( Figure 4B), which can be explained by the lack of chondrogenesis. In silico promoter analysis of these collagens, including Col5a3, which was also significantly up-regulated in Frzb -/samples, indicated the presence of several TCF/LEF responsive elements known from literature [39] in each of the gene promoters matching at least 80% of the original sequence. Moreover, each promoter contained a unique 100% consensus sequence in the promoter region indicating a direct link by which FRZB could modulate transcription of these genes (Table 6). Further analysis also showed the presence of binding sites for other transcription factors linked to WNT signaling such as Oct-1, EP300, Gata and AP-1.
Among the down-regulated pathways and processes, effects on the cell cycle and partially overlapping p53 signaling were most striking (Table 7). Down-regulation of different cyclins and cyclin kinases as well as many other positive regulators of the cell cycle suggest inhibition of mitosis and cell proliferation. Ribcage chondrocytes derived from Frzb -/mice proliferated significantly less than those derived from the wild-type mice in vitro after one week, corroborating the effect of FRZB on chondrocyte proliferation ( Figure 4C).

Discussion
Our transcriptome analysis of the bone-cartilage biomechanical unit of Frzb -/and wild-type mice provides evidence for tight regulation of WNT signaling, shifts in ECM component synthesis and alterations in cell proliferation and differentiation. FRZB is a secreted WNT antagonist, originally identified from a chondrogenic  [17] and misexpression of FRZB in the chick limb inhibits chondrocyte hypertrophy [18]. Polymorphisms in the human FRZB gene have been associated with OA [3], although this link has been debated recently [43].
Here, absence of Frzb in the articular cartilage and subchondral bone induces a subtle increase in WNT signaling evident by up-regulation of several WNT target genes as demonstrated by pathway analysis and by comparison with a user-compiled list of WNT target genes.  Absence of Frzb also results in the up-regulation of other SFRP family members and different WNT modulators, suggesting that compensatory mechanisms exist in order to tightly control WNT signaling in these tissues. We previously demonstrated that Frzb -/mice show increased articular cartilage damage in different induced models of OA, although we did not see signs of spontaneous accelerated OA development in one-year old mice [7]. This contrasts with more direct and radical changes in the WNT canonical cascade as both tissuespecific gain and loss of function of β-catenin, result in premature OA [8,9]. FRZB can modulate both canonical and non-canonical WNT signaling. New insights into the differential activation of these pathways in articular chondrocytes may help to further explain why deletion of a single antagonist induces only subtle changes as compared to the dramatic effects of β-catenin modulation. Distinct SFRPs do not bind different WNTs with similar affinities and their effect may depend on the cell type and interactions with other pathways [44]. Nalesso et al. demonstrated that low amounts of WNT ligand can activate noncanonical signaling whereas higher amounts activate the β-catenin mediated pathway [45]. Moreover, inhibition of either pathway can de-repress the alternative one. In their system, Wnt3a induced articular chondrocyte dedifferentiation by activating the non-canonical Ca 2+ /CaM-KII pathway and stimulated proliferation by activating the canonical pathway.
The changes we detected are not limited to the articular cartilage. Increased WNT signaling in the subchondral bone can also contribute to OA development. In this context, local regulatory mechanisms may be different from tissue to tissue. Frzb -/mice appear to have normal subchondral bone but increased cortical bone thickness [7]. Also, anabolic responses in the cortical bone to cyclic loading are much greater in Frzb -/mice compared to wild-types [7].
Absence of FRZB resulted in shifts in collagens, integrins and cadherins. Among these, changes in type III and type V collagen are of interest. As articular cartilage matures and ages, collagen fibrils become thicker, the amount of types IX and XI collagens decreases relative to type II collagen [46], and these minor collagens are progressively replaced by type V collagen [47]. Type III collagen can be detected in small but significant amounts in articular cartilage of mature joints and is cross-linked to the surface of type II collagen [46]. Its presence is more prominent in OA [48,49]. The type III collagen content in articular cartilage tends to vary between individual joints, anatomical location and tissue microanatomy. It may also be dependent on the history of injuries and the wear and tear experienced by a normal joint [46]. Therefore, it seems likely that type III collagen is synthesised as a modifier of existing fibril networks in response to tissue and matrix damage [46]. Although no increased cartilage damage was found in unchallenged Frzb -/mice, the significant up-regulation of Col5a1, Col5a3 and Col3a1 in the articular cartilage and subchondral bone from Frzb -/mice, suggests increased damage and repair in the Frzb -/mice at the molecular level.
These observations were further corroborated by complementary experiments where FRZB was overexpressed in the ATDC5 in vitro chondrogenesis model. Under these conditions, expression of both Col3a1 and Col5a1 was decreased during chondrogenic differentiation, suggesting that either FRZB by itself, or by modulating WNT signaling, affects expression of these ECM molecules in different systems. The additional observation that silencing of Frzb also results in a decrease in these collagens can be explained by lack of chondrogenic differentiation in the latter system.
We also found that overexpression of FRZB appeared to stimulate chondrogenesis in this model, as shown by increased aggrecan and col2a1 expression. Matured aggrecan monomers in the cartilage are glycosylated macro-molecules in which the glycoconjugates are formed by sulphatation of GAG side chains on the core protein [50]. The amount of sulphated GAGs in the micro-masses, measured by Safranin O staining, was surprisingly decreased in FRZB overexpressing micromasses. Although the differences we observed were limited, these results might suggest that FRZB overexpression in this system impairs the maturation of these aggrecan monomers, for instance, by a relative excess in substrate due to the higher expression levels. Staining for collagens by Picrosirius Red indicated no major differences in total collagen content in FRZB overexpressing micro-masses and controls. The observed spreading of the fibers from the center, however, which was also noted in the Safranin O staining, suggests that overexpression of FRZB could modify matrix distribution, possibly by increasing ATDC5 migration. All these results are in line with earlier observations on FRZB and chondrogenesis [17,18].
Collagen type III and V are also found in the bone, co-distributed in much lower quantities next to the main collagen component type I collagen. Type V collagen expression is regulated by TGFβ in osteoblasts during osteogenesis [51]. Since members of the TGFβ pathway are up-regulated in our Frzb -/samples, this may affect expression in the subchondral bone. Collagen type V is increased in some patients with brittle bone disease and in patients with osteogenesis imperfecta, where collagen type V likely interferes with the normal process of mineralization [52]. Similar results were found for collagen type III, suggesting a role for collagen type III and V in defects in maturation of the bone [53][54][55][56][57].
The responsive elements for TCF/LEF but also other transcription factors, related to WNT signaling, in the Col3 and Col5 promoters suggest a direct link with WNT signaling by which FRZB can influence the composition of the cartilage and subchondral bone ECM. and collagen type 5α1 (Col5a1) expression in the ATDC5 micro-masses overexpressing frizzled-related protein (FRZB) compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to hypoxanthine guanine phosphoribosyl transferase (Hprt) (2 -ΔΔCt ) ± SEM (n = six samples/condition; Mann-Whitney test: for Col3a1 P = 0.24 and for Col5a1 P = 0.06). (B) RT-PCR analysis for Col3a1 and Col5a1 expression in the ATDC5 micro-masses where Frzb was knocked down using the pGIPZ-shRNAmir directed against Frzb compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to Hprt (2 -ΔΔCt ) ± SEM (n = six samples for pGIPZ-Ctrl and three samples for pGIPZ-FRZB; Mann-Whitney test: for Col3a1 P = 0.047 and for Col5a1 P = 0.54). (C) Proliferation assay of ribcage articular chondrocytes isolated from Frzb -/compared to wild-type (WT) mice. Data are shown as the difference in fluorescence after 24 h and one week. (n = nine conditions/group; Initial cell densities were 500 (black dots), 2,000 (grey dots) and 4,000 (black circles) cells per well; Mann-Whitney test: P = 0.0019, P = 0.0012 and P = 0.0008). On the other hand, considering the relatively mild effects on WNT signaling at the tissue level, our study also leaves open the possibility that FRZB has unexpected, more robust post-transcriptional or epigenomic effects in these tissues suggesting new directions for research [58]. Loss of Frzb resulted in a decrease of genes associated with cell cycle progression. Proliferation analysis of ribcage chondrocytes isolated from Frzb -/mice compared to those isolated from wild-type mice agreed with this observation. Canonical WNT signalling is known to promote cell cycle progression and proliferation through the up-regulation of target genes like c-myc and cyclin D, but also via regulation of the mitotic spindle apparatus [59]. This apparent discrepancy where Frzb -/chondrocytes proliferate slower instead of faster, may be dependent on the cell type, the differentiation state, the WNT ligand involved and antagonist interactions. Differences in activation of either canonical or alternative pathways may also play a role. The analysis presented here has a number of limitations. In particular, the number of samples used in the microarray experiment is small. Extraction of high quality RNA, required for microarray, from the articular cartilage is quite challenging due to a low cell content, the cross-linked extracellular matrix and considerably high levels of RNA degradation [60]. From this perspective, less than one-third of the extractions yielded RNA of sufficient quality and quantity for the analysis. In addition, transcriptome analysis does not convey information about proteins and posttranslational modifications.

Conclusions
These data further support an important role for FRZB in the homeostasis of the joint, in particular in the articular cartilage-bone biomechanical unit. The molecular up-regulation of other antagonists of the WNT signalling cascade in the absence of Frzb and the similar activation of the β-catenin mediated cascade also provide evidence for the important homeostatic potential of the joint. From the clinical perspective, this should encourage the search for compounds that stimulate tissue homeostasis. Further analyses and future studies should focus on fine mapping of the interactions between WNTs, their receptors and antagonists, as well as modulating effects of the inhibitors on their own. These investigations appear necessary to better understand the complex biology of WNTs and SFRPs in the joint, thereby, more precisely defining therapeutic targets and strategies. Again, from the clinical perspective, our study suggests that WNT pathway modulators should be carefully selected and linked to specific activation or inhibition of intracellular cascades in order to predict their potential effects and toxicity.