The role of smoothened for the osteophyte formation in mouse osteoarthritis

Abstract

Objective: Osteoarthritis (OA) shows a progressive degeneration of articular cartilage and osteophyte formation. Endochondral ossification is considered an important pathophysiological process for the osteophyte formation. Chondrocyte hypertrophy is also an essential process required for endochondral bone formation. It is reported that the expression of Indian hedgehog (Ihh) in pre- and early hypertrophic chondrocytes of growth plate and osteophytes, and a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy. Therefore, in this study, we investigated whether the deletion of smoothened (Smo), an essential transducer of Hedgehog signaling, affected osteophyte formation in mouse osteoarthritis. Methods: To investigate the role of Smo in osteophyte formation, we crossed Smo/flox mice, which express a null allele of Smo upon exposure to Cre recombinase, with RosaCreER(T) mice to generate Smo conditional knockout (CKO) mice upon administration of Tamoxifen. CKO mice and Cre-negative control mice were administrated Tamoxifen on 3, 5 weeks after birth and 3days, 1day before surgery, resulting in downregulated Hedgehog signaling. OA was surgically induced in 8-week-old CKO mice and Cre-negative control mice using the anterior cruciate ligament transection model. 1week after surgery, mice were caged in climbing cage to accelerate degenerative change. Mice were sacrificed at 4, 6, 8 weeks after surgery. Histomorphometric and immunohistochemical (IHC) assessment was performed. Result: The immature osteophyte-like lesion composed of chondrocytes and cartilage was observed at the posterior margins of tibia in both CKO mice and Cre-negative control mice in 4 weeks post-surgery. However, the maturity of osteophytes was suppressed in CKO mice compared to Cre-negative control mice at 6 and 8 weeks post-surgery. In contrast, Cre-negative control mice showed mature ossifying lesion and hypertrophic chondrocytes in osteophytes, and had a larger and more mature osteophytes at 6 and 8 weeks post-surgery by histomorphometric analysis. Furthermore, it is revealed that lesser expression of type X collagen in osteophytes of CKO mice. These data suggests that the deletion of Smo delayed the formation of osteophytes and the suppression of the endochondral ossification process with blocking the hypertrophy of chondrocytes and type X collagen expression. Conclusions: Smo plays a crucial role of osteophyte formation in mouse osteoarthritis, especially, the maturity of osteophytes through endochondral ossification process.