Wnt/β-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone
Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage.
Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells.
Wnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4.
We demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.
KeywordsOsteoarthritis Chondrocyte Superficial zone Wnt signaling
Glycogen synthase kinase 3 beta
T cell factor/lymphoid enhancer factor
Red fluorescence protein
Osteoarthritis Research Society International
Constitutively active Lef1
Fluid flow shear stress
Protein kinase A
Osteoarthritis (OA), which involves articular cartilage degradation, is the most prevalent joint disorder. A large number of studies using genetically modified mice have revealed signaling pathways or molecules responsible for OA, such as a disintegrin-like metallopeptidase with a thrombospondin type 1 motif 5, hedgehog signaling, syndecan-4, matrix metalloproteinase-13, hypoxia-inducible factor 2-alpha, nuclear factor kappa-light-chain-enhancer of activated B cells, Notch signaling, and Wnt signaling [1, 2, 3, 4, 5, 6, 7, 8, 9]. However, the entirety of OA pathophysiology is not yet understood despite all of these findings.
Wnt is one of the most investigated signaling pathways in developmental biology and pathophysiology of various diseases. Wnt genes encode a family of about 20 small secreted proteins, which are highly conserved across various organisms . Wnt receptor activation leads to signal transduction via three different pathways: a canonical Wnt/β-catenin cascade, noncanonical planar cell polarity pathway, and the Wnt/Ca2+ pathway . Notably, the Wnt/β-catenin pathway is the best understood of these signaling cascades. When Wnt proteins are not bound to a receptor, a serine/threonine kinase complex that includes Axin, casein kinase 1, and glycogen synthase kinase 3 beta (GSK-3β) phosphorylates β-catenin, which can then be degraded by E3 ubiquitin ligases . However, once Wnt proteins bind their receptors, the kinase complex is recruited to the receptor, which leads to the stabilization of β-catenin . Subsequently, β-catenin translocates into the nucleus, binds to T cell factor/lymphoid enhancer factor (Tcf/Lef), and activates the transcription of target genes .
Regulation of chondrocyte generation and differentiation by the canonical Wnt/β-catenin pathway has been extensively investigated using transgenic and tissue-specific knockout mice. Skeletal formation is severely impaired in mouse embryos by chondrocyte-specific constitutive activation of Wnt/β-catenin signaling [11, 12, 13]. Interestingly, inactivation or inhibition of Wnt/β-catenin signaling also leads to severe impairment of skeletal growth [14, 15, 16]. These findings indicate that Wnt/β-catenin signaling activity is restrictively regulated in chondrocyte generation and differentiation during development . Similarly, regulation of articular cartilage by Wnt/β-catenin signaling is complicated and somewhat contradictory. Transgenic mice expressing an inhibitor of β-catenin and Tcf in chondrocytes displayed enhanced articular cartilage destruction . However, stabilization of β-catenin in adult chondrocytes also enhanced OA progression . Reasons underlying these contradictory findings have not yet been reported.
Recently, roles of articular cartilage superficial zone (SFZ) cells have been the focus of several studies. SFZ cells are thought to have different properties from chondrocytes in the deeper zone (DZ). SFZ cells produce lubricin (encoded by Prg4), a protein responsible for the lubrication of articular joints that contributes to the protection of articular cartilage [18, 19]. In the newborn period, joints of Prg4-knockout mice appear normal . However, as the mice aged, their joints exhibited early wear and increased friction compared with WT mice . Previous studies have shown that Wnt/β-catenin signaling is constitutively activated in the SFZ and required for SFZ formation during skeletal development [20, 21]. A recent cell tracking study using Prg4-CreERT2 mice further revealed that Prg4-expressing cells located at the joint surface in embryos or young mice include a progenitor population for mature DZ chondrocytes . Based on these findings, we hypothesized that Wnt/β-catenin signaling activity may contribute to the maintenance of the SFZ and further homeostasis of articular joints in adulthood similarly to the developmental period.
Herein, we describe roles of Wnt/β-catenin signaling in the SFZ of adult articular cartilage. We examined Wnt/β-catenin signaling activity in the SFZ and its in vivo roles in OA development, using SFZ-specific β-catenin knockout or stabilization. We further investigated expression levels of Wnt ligands and the involvement of mechanical loading as upstream triggers.
All animal experiments were authorized by the Animal Care and Use Committee of The University of Tokyo. We also complied with all relevant ethical regulations. In each experiment, we compared the genotypes of littermates maintained in a C57BL/6J background. TOPGAL, Prg4-CreERT2, Rosa26-tdTomato (Ai14), and Ctnnb1-flox mice were obtained from The Jackson Laboratory (Bar Harbor, ME) [22, 23, 24]. Ctnnb1-ex3-flox mice were generated as previously described .
Tissue samples were fixed in 4% paraformaldehyde buffered with phosphate-buffered saline (PBS, pH 7.4) at 4 °C for 1 day. Specimens were decalcified with 10% EDTA (pH 7.4) at 4 °C for 2 weeks, embedded in paraffin, and 5-μm-thick sagittal sections were cut from specimens. Safranin O staining was performed in accordance with standard protocols. For immunohistochemistry, sections were incubated with antibodies against red fluorescence protein (RFP; 1:2000; 600-401-379, Rockland, Limerick, PA), Ctnnb1 (1:1000, Ab2365, Abcam, Cambridge, UK), and Prg4 (1:500, Ab28484, Abcam). For visualization, simple stain mouse-MAX-PO(R) (Nichirei Bioscience, Tokyo, Japan) was used.
Osteoarthritis (OA) experiments
For OA experiments, Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice were generated by mating Prg4-CreERT2 with Ctnnb1-flox and Ctnnb1-ex3-flox mice, respectively. Tamoxifen (Sigma Aldrich, St. Louis, MO; 100 μg per g of body weight) was intraperitoneally injected into 7-week-old Prg4-CreERT2;Ctnnb1fl/fl, Ctnnb1fl/fl, Prg4-CreERT2;Ctnnb1-ex3fl/wt, and Ctnnb1-ex3fl/wt mice daily for 5 days. A surgical procedure was then performed to establish an experimental OA model in 8-week-old male mice . Under general anesthesia, resection of the medial collateral ligament and medial meniscus was performed under a surgical microscope. For sham surgery, only the skin incision was performed. Mice were analyzed 8 weeks after surgery. For the aging model, Prg4-CreERT2;Ctnnb1fl/fl and Ctnnb1fl/fl mice were analyzed at 18 months of age. All mice were maintained under the same conditions (three mice per cage). OA severity was quantified using the Osteoarthritis Research Society International (OARSI) system , which was assessed by two observers blinded to the experimental groups.
Primary SFZ cells were isolated as previously described . Briefly, the primary end of the femur and the distal end of the tibia were dissected from P5 mice, and ligaments and tendons were excised. Cartilage tissues were incubated with 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA) for 1 h, followed by 1.5-h digestion with 173 U/mL of type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ). DZ cells were isolated by additional digestion of residual epiphyseal cartilage tissue with 43 U/ml collagenase type I for 5 h. Dissociated cells were seeded on fibronectin-coated culture dishes. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS). The cells were cultured as a monolayer in all experiments. We used 2 μM 4-hydroxytamoxifen (4OHT; Sigma Aldrich, St Louis, MO, USA) to induce Cre recombination in the cultured SFZ cells. Recombinant human (rh) WNT5A (R&D Systems, Minneapolis, MN), rhWNT5B (R&D Systems), rhWNT9A (R&D Systems), CHIR99021 (Cayman, Ann Arbor, MI), FH535 (Cayman), and 666-15(Tocris Bioscience, Bristol, UK) were used at the concentrations described in the figures and figure legends. For the luciferase assay, ATDC5 cells (Riken BRC, Tsukuba, Japan) were cultured in DMEM/F-12 (Wako) containing 5% FBS.
Cyclic tensile strain loading of mouse primary SFZ cells
Mouse primary SFZ cells were seeded on silicon stretch chambers at a density of 1 × 105 cells/chamber (chamber area = 2 × 2 cm). The cells were cultured in DMEM containing 10% FBS. Cyclic tensile strain (0.5 Hz, 15% elongation) was applied by an STB-140 mechanical stretch system (STREX, Osaka, Japan) in a CO2 incubator. Control cells were cultured without cyclic tensile strain.
Fluid flow shear stress (FFSS) conditions of mouse primary SFZ cells
Mouse primary SFZ cells were seeded on tissue culture plates (culture plate diameter = 3.5 cm) at a density of 2 × 105 cells/cm2. The cells were cultured in DMEM containing 10% FBS. FFSS (180 rotations per minute) was applied by a WB-101SRC orbital shaker (WAKEN, Kyoto, Japan) in a CO2 incubator. Control cells were cultured under a static condition.
Cells were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific). Cell lysates were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). After blocking with 6% skim milk, membranes were incubated with primary antibodies against Ctnnb1 (1:250; #9652, Cell Signaling Technology, Danvers, MA), CREB1 (1:250; 9197S, Cell Signaling Technology), and actin (1:250; AC-74, Sigma Aldrich). Membranes were incubated with a horseradish peroxidase-conjugated antibody (Promega, Madison, WI), and immunoreactive proteins were visualized with ECL prime (GE Healthcare, Chicago, IL) and iBright CL 1000 (Thermo Fisher Scientific). The signal intensity of three independent experiments was quantified using the iBright CL 1000 and normalized to actin.
Total RNA was purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse transcribed using ReverTraAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Each PCR reaction contained 1× THUNDERBIRD SYBR qPCR Mix (Toyobo), 0.3 mM specific primers, and 20 ng of cDNA. The full-length or partial cDNAs of target genes, including PCR amplicon sequences, were amplified by PCR, cloned into pCR-TOPO Zero II or pCR-TOPO II vectors (Invitrogen), and used as standard templates after linearization. Copy numbers of target mRNAs in each total RNA were calculated by reference to standard curves and adjusted to mouse standard total RNA (Thermo Fisher Scientific) with rodent actin as an internal control. All reactions were run in triplicate. Primer sequences for real-time RT-PCR are shown in Additional file 1: Table S1.
Proximal regions around the transcription start site of Prg4 (from − 3460, − 1470, or − 500 to + 592 bp relative to the transcription start site) were amplified by PCR using mouse genomic DNA as the template and cloned into the PGL4.10[luc2] vector (Promega). We performed the luciferase assay using ATDC5 cells and a Dual-Luciferase Reporter Assay System (Promega) and reported the data as the ratio of firefly to Renilla activities.
To assess the statistical significance of experimental data, we used a two-tailed unpaired Student’s t test for comparison of two groups, one-way ANOVA followed by Tukey’s correction for comparison of multiple groups, and the Mann-Whitney U test for the OARSI score, as appropriate. P values less than 0.05 were considered significant.
Wnt/β-catenin signaling activity in the SFZ
Enhancement of OA development by β-catenin knockout in the SFZ
Suppression of OA development by β-catenin stabilization in the SFZ
Regulation of Prg4 expression by Wnt/β-catenin signaling in the SFZ
Activation of Wnt/β-catenin signaling by mechanical loading
CREB1 induction by Wnt/β-catenin signaling
The present study demonstrated that Wnt/β-catenin signaling plays essential roles in the SFZ of adult articular cartilage. Indeed, Wnt/β-catenin signaling is activated only in the SFZ of adult articular cartilage. Loss-of-function in Prg4-positive cells leads to enhanced OA development with degenerated SFZ, while gain-of-function results in resistance to OA. Prg4 expression is dependent on Wnt/β-catenin signaling, and mechanical stress loading increases expression of Wnt ligands and Prg4 in SFZ cells. Wnt/β-catenin signaling induces CREB1, a representative transcription factor of Prg4. Similar to previous findings that Wnt/β-catenin signaling contributes to characterization and formation of the SFZ during skeletal development , it is also necessary for the maintenance of adult articular cartilage.
Previous studies have shown that activation of Wnt/β-catenin signaling in chondrocytes enhances endochondral ossification and replacement by bone [14, 30]. These findings are consistent with a previous report of enhanced OA progression in Col2a1-CreERT2;Ctnnb1-ex3fllwt mice . In this previous study, Safranin O and Alcian blue staining was reduced, and the articular cartilage area was reduced significantly in Col2a1-CreERT2;Ctnnb1-ex3fllwt mice without surgical OA induction at 2 months after tamoxifen injection . However, exacerbation of OA was also observed in transgenic mice expressing an inhibitor of β-catenin and Lef1 (ICAT) under the Col2a1 promoter . The cartilage degeneration in transgenic mice was rather slow and mild, which may be partially because the binding of ICAT to β-catenin was not irreversible . Notably, in Col2a1-ICAT mice, mild cartilage degeneration was observed at the articular surface, particularly in the weight-bearing area of knee joints . Considering that Col2a1 is expressed both in the SFZ and DZ, exacerbation of OA in Col2a1-ICAT mice may be associated with decreased activity of Wnt/β-catenin signaling in the SFZ, although Prg4 expression was not examined. In the present study, Prg4-Cre-driven β-catenin stabilization led to resistance against OA accompanied by enhanced expression of Prg4, whereas Prg4-Cre-driven β-catenin knockout resulted in OA exacerbation, indicating that Wnt/β-catenin signaling in the SFZ contributes to cartilage homeostasis at least by Prg4 induction. A recent study showed that SFZ cells include a population of progenitors for articular chondrocytes . Considering that Wnt is a major regulator of mesodermal cells , Wnt/β-catenin signaling may be involved not only in Prg4 induction, but in maintenance of chondrocyte progenitors in the SFZ of adult articular cartilage.
The mechanisms underlying the high expression level of β-catenin protein in the SFZ are still unknown. We found that Wnt5a, Wnt5b, Wnt9a, and Wnt16 were highly expressed in SFZ cells (Fig. 5a). Among them, mRNA expression of Wnt5a, Wnt5b, and Wnt9a was induced by mechanical stress loading (Fig. 5b). However, we could not obtain further findings to explain the upregulation of Wnt/β-catenin signaling in the SFZ. Notably, a recent study showed that Wnt16 protects articular cartilage by antagonization of excessive Wnt/β-catenin signaling . Wnt16 expression is increased in the SFZ during the early stage of mouse experimental knee OA . Treatment with recombinant Wnt16 induces Prg4 expression, although the molecular pathway is unknown . It is widely known that the potent Wnt antagonist FRZB is expressed in DZ chondrocytes and exerts cartilage-protective effects [33, 34]. Various molecules are likely to be involved in the tight regulation of Wnt/β-catenin signaling in the SFZ.
CREB, a member of the leucine zipper family of DNA binding proteins, was identified as a protein that binds to the cAMP-responsive element . Generally, transcriptional activity of CREB is regulated by phosphorylation . Notably, in the present study, Creb1 mRNA expression was induced by Wnt/β-catenin signaling. Although the interaction of Wnt/β-catenin signaling and CREB-binding protein has been well described , induction of CREB by Wnt/β-catenin signaling has not been revealed. However, as both CREB and Wnt/β-catenin are widely expressed, the Wnt/β-catenin-CREB pathway may be involved in other cell activities. We could not reveal the precise molecular mechanisms underlying transcriptional induction of CREB by Wnt/β-catenin, i.e., whether the induction is direct or indirect. Furthermore, molecules or signaling pathways other than CREB may be also associated with Prg4 induction by Wnt/β-catenin signaling.
Ogawa et al. previously reported that mechanical loading enhances Prg4 expression both in vivo and in vitro . Their results indicated that FFSS induces Prg4 mainly via protein kinase A (PKA)/CREB signaling . Enhanced prostaglandin E2 production and parathyroid hormone-related protein expression elicited CREB phosphorylation through PKA, resulting in increased Prg4 expression . In their study, FFSS enhanced the activity of a luciferase reporter containing TCF-binding sites (TOP-luc), as well as CRE-luc . Considering the present data, both enhanced CREB transcription induced by Wnt/β-catenin and CREB phosphorylation induced by the PKA pathway are likely involved in Prg4 induction by mechanical loading.
In conclusion, we demonstrated that Wnt/β-catenin signaling is activated specifically in the SFZ of adult mouse articular cartilage, whereby it contributes to Prg4 expression, which plays essential roles in the homeostasis of articular cartilage.
We thank J. Sugita and K. Kaneko for the technical assistance. We also thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
FX, FY, and TS conceived and designed the study. FX, FY, DM, RC, YM, YMaenohara, YMakii, and TO performed the experiment. FX, FY, MMT, HH, SO, UC, ST, and TS analyzed and interpreted the data. TS wrote the paper. All authors have read and approved the final manuscript.
This study was supported by JSPS KAKENHI Grant Numbers JP16K10888, JP19109007, JP17H04310, JP17K10996, and JP17K10998.
Ethics approval and consent to participate
All animal experiments were authorized by the Animal Care and Use Committee of The University of Tokyo (M-P17-091).
Consent for publication
The authors declare that they have no competing interests.
- 6.Little CB, Barai A, Burkhardt D, Smith SM, Fosang AJ, Werb Z, Shah M, Thompson EW. Matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development. Arthritis Rheum. 2009;60(12):3723–33.PubMedPubMedCentralCrossRefGoogle Scholar
- 13.Tamamura Y, Otani T, Kanatani N, Koyama E, Kitagaki J, Komori T, Yamada Y, Costantini F, Wakisaka S, Pacifici M, et al. Developmental regulation of Wnt/beta-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification. J Biol Chem. 2005;280(19):19185–95.PubMedCrossRefGoogle Scholar
- 20.Koyama E, Shibukawa Y, Nagayama M, Sugito H, Young B, Yuasa T, Okabe T, Ochiai T, Kamiya N, Rountree RB, et al. A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Dev Biol. 2008;316(1):62–73.PubMedPubMedCentralCrossRefGoogle Scholar
- 21.Yasuhara R, Ohta Y, Yuasa T, Kondo N, Hoang T, Addya S, Fortina P, Pacifici M, Iwamoto M, Enomoto-Iwamoto M. Roles of beta-catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells. Lab Invest. 2011;91(12):1739–52.PubMedPubMedCentralCrossRefGoogle Scholar
- 23.Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128(8):1253–64.PubMedGoogle Scholar
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