Dataset Information

Wnt/?-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

ABSTRACT: BACKGROUND:Both loss- and gain-of-function of Wnt/?-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/?-catenin signaling in the superficial zone (SFZ) of articular cartilage. METHODS:Wnt/?-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/?-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/?-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/?-catenin signaling in SFZ cells. RESULTS:Wnt/?-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 ?-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 ?-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by ?-catenin knockout, while it was upregulated by ?-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/?-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. CONCLUSIONS:We demonstrated that Wnt/?-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

SUBMITTER: Xuan F

PROVIDER: S-EPMC6880374 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Wnt/β-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

Xuan Fengjun F Yano Fumiko F Mori Daisuke D Chijimatsu Ryota R Maenohara Yuji Y Nakamoto Hideki H Mori Yoshifumi Y Makii Yuma Y Oichi Takeshi T Taketo Makoto Mark MM Hojo Hironori H Ohba Shinsuke S Chung Ung-Il UI Tanaka Sakae S Saito Taku T

Arthritis research & therapy 20191127 1

<h4>Background</h4>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.<h4>Methods</h4>Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-Cre<sup>ERT2</sup>;Ctnnb1<sup>fl/fl</sup> and Prg4-Cre<sup>ERT2</sup>;Ctnnb1-ex3<sup>fl/wt</sup> mice for loss- and gain-of-function, respe ...[more]

PMID: 31771658

Similar Datasets

Project description:The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, is thought to promote self-renewal of articular cartilage, and is thus important for joint long-term function, but the mechanisms regulating its properties remain unclear. Previous studies revealed that Wnt/β-catenin signaling is continuously active in SFZ, indicating that it may be essential for SFZ function. Thus, we examined whether Wnt/β-catenin signaling regulates proliferation and phenotypic expression in SFZ cells. Using transgenic mice, we found that acute activation of Wnt/β-catenin signaling increases SFZ thickness, Proteoglycan 4 (Prg4, also called lubricin) expression and the number of slow-cell cycle cells, whereas conditional ablation of β-catenin causes the opposite. We developed a novel method to isolate SFZ cell-rich populations from the epiphyseal articular cartilage of neonatal mice, and found that the SFZ cells in culture exhibit a fibroblastic cytoarchitecture and higher Prg4 and Ets-related gene (Erg) expression and lower aggrecan expression compared with chondrocyte cultures. Gene array analyses indicated that SFZ cells have distinct gene expression profiles compared with underlying articular chondrocytes. Treatment of Wnt3a strongly stimulated SFZ cell proliferation and maintained strong expression of Prg4 and Erg, whereas ablation of β-catenin strongly impaired proliferation and phenotypic expression. When the cells were transplanted into athymic mice, they formed Prg4- and aggrecan-expressing cartilaginous masses attesting to their autonomous phenotypic capacity. Ablation of β-catenin caused a rapid loss of Prg4 gene expression and strong increases in expression of aggrecan and collagen 10, the latter being a trait of hypertrophic chondrocytes. Together, the data reveal that Wnt/β-catenin signaling is a key regulator of SFZ cell phenotype and proliferation, and may be as important for articular cartilage long-term function.

Project description:BackgroundLubricin, a mucinous glycoprotein secreted by synoviocytes and chondrocytes plays an important role in reducing the coefficient of friction in mammalian joints. Elevated cartilage surface friction is thought to cause chondrocyte loss; however, its quantification and methodological approaches have not been reported. We adapted a stereological method and incorporated vital cell staining to assess cellular loss in superficial and upper intermediate zones in lubricin deficient mouse cartilage.MethodsThe femoral condyle cartilage of the intact knees from lubricin wild type (Prg4 (+/+)), heterozygote (Prg4 (+/-)), and knockout (Prg4 (-/-)) mice was imaged using fluorescein diacetate (FDA), propidium iodide (PI), and Hoechst staining, and confocal microscopy. Three dimensional reconstructions of confocal images to a depth of 14 ?m were analyzed using Matlab to determine the volume fraction occupied by chondrocytes in cartilage of both medial and lateral femoral condyles. Living chondrocyte volume fraction was defined as FDA stained chondrocyte volume/total volume of superficial?+?upper intermediate zone. Living and dead (total) chondrocyte volume fraction was defined as FDA?+?PI stained chondrocyte volume/total volume of superficial?+?upper intermediate zone. MicroCT provided an orthogonal measure of cartilage thickness. Immunohistology for activated caspase-3 and TUNEL staining were performed to evaluate the presence of apoptotic chondrocytes in Prg4 mutant mice.ResultsLiving chondrocyte volume fraction of the medial femoral condyle was significantly lower in Prg4 (-/-) mice compared to Prg4 (+/+) (p?=?0.002) and Prg4 (+/-) (p?=?0.002) littermates. There was no significant difference in medial condyle chondrocyte volume fraction between Prg4 (+/+) and Prg4 (+/-) mice (p?=?0.82). No significant differences were observed for the chondrocyte volume fraction for the lateral condyle (p?>?0.26). Cartilage thickness increased in the medial condyle for Prg4 (-/-) mice compared to Prg4 (+/+) (p?=?0.02) and Prg4 (+/-) (p?=?0.03) littermates, and the lateral condyle for Prg4 (-/-) mice compared to Prg4 (+/+) (p?<?0.0001) and Prg4 (+/-) (p?<?0.0001) littermates, indicating that a multi-dimensional increase in cartilage volume did not artifactually lower the chondrocyte volume fraction in the medial condyle. Significantly higher number of caspase-3 positive cells were observed in the superficial and upper intermediate zone cartilage of the medial femoral condyle of Prg4 (-/-) mice compared to Prg4 (+/+) (p?=?0.01) and Prg4 (+/-) (p?=?0.04) littermates, and the lateral femoral condyle of Prg4 (-/-) mice compared to Prg4 (+/+) (p?=?0.02) and Prg4 (+/-) (p?=?0.02) littermates. There were no significant differences in TUNEL staining among different Prg4 genotypes in both condyles (p?>?0.05 for all comparisons).ConclusionsIncreased Caspase-3 activation is observed in Prg4 deficient mice compared to Prg4 sufficient littermates. Absence of Prg4 induces loss of chondrocytes in the superficial and upper intermediate zone of mouse cartilage that is quantifiable by a novel image processing technique.

Dataset Information

Wnt/?-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

Publications

Wnt/β-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets