Loss of Wnt9a and Wnt4 causes degenerative joint alterations

Abstract

Purpose: Both induced and reduced Wnt-signaling has been shown in literature to contribute to osteoarthritis. The Wnt-ligands Wnt9a and Wnt4 are of special interest since they are expressed in cells of the joint forming region and are positive regulators of chondrocyte maturation. More specifically, Wnt9a regulates the pace of chondrocyte proliferation and maturation, while Wnt4 regulates chondrocyte maturation at the transition from prehypertrophic to hypertrophic chondrocytes and misexpression accelerates chondrocyte maturation. Wnt9a downregulation has been shown in OA patients, and knockout or inhibition of β-catenin leads to the destruction of articular cartilage in mice. In addition, Wnt-antagonists Dkk-1 and Frzb-1 are expressed by articular chondrocytes, suggesting that canonical Wnt-signaling in articular chondrocytes has to be tightly controlled. Methods: Here, we investigate the effects of loss of Wnt9a and the combined loss of Wnt9a and Wnt4 with respect to joint maintenance in aged mice. To circumvent the postnatal lethality of loss of Wnt9a and Wnt4, the genes for both Wnt ligands were either conditionally deleted in the limb mesenchyme (Prx1-Cre), or in the case of Wnt9a in the superficial zone of the articular cartilage (Prg4-Cre). The resulting skeletal phenotype was analyzed at the ages of 6, 9 and 12 months by μCT and histology. To investigate expression changes in chondrocytes and synovial fibroblasts upon deletion of Wnt9a and Wnt4, we isolated superficial zone cells and deeper layered chondrocytes from femoral condyles and tibial plateaus of the knee and synovial fibroblasts from paws of 8-day-old Wnt9afl/fl and Wnt4fl/fl mice. Cultured cells were either infected with a lentivirus expressing or non-expressing Cre and alterations in gene expression levels were measured via qPCR. Results: The limb specific Wnt9a knockout mice show a joint phenotype at the age of 9 months. The onset of the phenotypic changes in the joints is accelerated by the additional loss of Wnt4. Phenotypically, the articular cartilage shows loss of proteoglycans, erosion, reduction of subchondral cortical thickness, and formation of ectopic calcifications and osteophytes at the knee joint. These alterations are progressively worsening with the age of the mice. Concomitant with the reduction of subchondral cortical thickness, an enriched TRAP-positive cell population can be found in the subchondral compartment. Mice with a conditional loss of Wnt9a in the superficial articular zone show degenerative changes of the articular cartilage at an age of 12 months. In the in vitro chondrocyte culture experiments a downregulation of Prg4 and an upregulation of Tnfsf11 (encoding RANKL) was observed upon the combined knockdown of Wnt9a and Wnt4 while an upregulation of Il-6 was noted upon deletion of Wnt9a alone. In fibroblast cultures an upregulation of Cd34 and metalloproteases Mmp9, Adamts-4 and Adamts-5 and can be detected in Wnt9a single and combined Wnt9a and Wnt4 knowdowns. Conclusions: Taken together, our results further support the notion that imbalanced Wnt-signaling can cause degenerative joint alterations. Furthermore, we show that Wnt9a and Wnt4 signaling are critical for maintenance of the joint. Yet, the underlying mechanisms remain to be elucidated in the future.