Cartilage β-catenin signaling plays a key role in the development of spondyloarthritis

Abstract

Purpose: Spondyloarthritis (SpA) is a group of autoimmune disease affecting spine and joint. Cartilage is the major tissue involved in this disease. Up-regulation of Wnt/β-catenin signaling has been reported in patients with SpA. In this study, we will determine if activation of β-catenin signaling in chondrocytes is the key event leading to bony overgrowth phenotype of the spine during the development of SpA. Methods: 1) Chondrocyte-specific β-catenin conditional activation mice, β-catenin(ex3)Col2ER, were generated by breeding β-catenin(ex3)flox/flox mice with Col2-CreERT2 transgenic mice. Deletion of exon3 of the β-catenin gene leads to the production of truncated β-catenin protein which is resistant to the GSK-3β-induced phosphorylation and subsequent ubiquitination and proteaome degradation. 2) Histology and histomorphometric analyses were performed in 3- and 6-month-old mice and changes in disc tissue were analyzed. Histological changes in facet joints were also analyzed in 4- and 9-month-old mice. 3) μCT analysis was performed to determine osteophyte formation in the spine. 4) Immortalized annular fibrosus (AF) and nucleus pulposus (NP) cells were generated. The cells maintain proliferation and differentiation properties and respond to growth factors even after 50 passages. These cells were used for β-catenin signaling studies. 5) Pain-related behavior tests were performed in 4–9 month old β-catenin(ex3)Col2ER and Cre-negative control mice. Results: 1) We found that severe destruction of disc tissues in 3- and 6-month-old β-catenin(ex3)Col2ER mice, including severe loss of cartilage tissues in endplate, disorganized AF and NP tissues and large amounts of osteophyte formation in the entire spine. 2) Using immortalized rat AF and NP cell lines, we found that treatment with BIO, a GSK-3β inhibitor (mimic β-catenin signaling activation), stimulated Top-flash reporter activity (6-15 folds) in both AF and NP cells after 24 h treatment. BIO induced Mmp13 (8 and 11 folds) and Ccl2 (6 and 11 folds) expression in AF and NP cells. 3) To determine if Mmp13 is a key downstream target of β-catenin signaling, we generated β-catenin(ex3)/Mmp13Col2ER double mutant mice and found that deletion of Mmp13 significantly reversed defects observed in disc tissue of β-catenin(ex3)Col2ER mice. 4) In addition to the disc tissues, we also observed severe damage and loss of proteoglycan and defects in cartilage structure in the facet joint in 4- and 9-month-old β-catenin(ex3)Col2ERmice. 5) CCL2 has been demonstrated as a key mediator of osteoarthritis (OA) pain. In the present studies we found that Ccl2 was significantly up-regulated by the activation of β-catenin signaling in both AF and NP cells. We then determined changes in pain-related behavior in 4–9 month old β-catenin(ex3)Col2ER mice. The β-catenin(ex3)Col2ER mice had significantly increased pain sensitivity as evidenced by lower von Frey thresholds, and this pain behavior sustained throughout the entire experimental period (9 months). The rearing as well as ambulation counts were also reduced in 4–9 month old β-catenin(ex3)Col2ER mice compared to Cre-negative controls. Conclusions: Taking consideration of our previous observations from β-catenin(ex3)Col2ER mice with OA-like phenotype in knee joint and temporomandibular joint (TMJ), the findings observed in disc and facet joint tissues of β-catenin(ex3)Col2ER mice largely resemble key features found in patients with SpA. Our findings suggest that activation of β-catenin signaling in cartilage tissue may be the key event leading to the bony overgrowth of the spine in patients with SpA.