Developmental Regulation of Wnt/β-catenin Signals Is Required for Growth Plate Assembly, Cartilage Integrity, and Endochondral Ossification

Yoshihiro, Tamamura, Tomohiro Otani, Naoko Kanatani, Eiki Koyama, Jirota Kitagaki, Toshihisa Komori, Yoshihiko Yamada, Frank Costantini, Satoshi Wakisaka, Maurizio Pacifici,Masahiro Iwamoto and Motomi Enomoto-Iwamoto From theDepartment of Orthopaedic Surgery, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107; Department of Oral Cytology and Cell Biology, School of Dentistry, Nagasaki University, Nagasaki 852-8501, Japan; Osaka University Faculty of Dentistry, Suita, Osaka 565-0871, Japan; Molecular Biology Section NIH/NIDCR, Bethesda MD, 20892; and Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY 10032. Running title: Wnt/β-catenin signals and limb cartilage development Address Correspondence to: Motomi Enomoto-Iwamoto, Department of Orthopedic Surgery Jefferson Medical College, Thomas Jefferson University, 1015 Walnut Street, Curtis Bldg. Suite 501, Philadelphia, PA 19107, Tel. 215-955-7624; Fax, 215-955-9159; E-mail: [email protected] Studies have suggested that continuous Wnt/β-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, while signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/β-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of β-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated β-catenin mutant (Δ−β-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/β-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization and expression of terminal markers such as MMP-13 and VEGF. Since proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Δ−β-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP and ADAMTS5. In conclusion, Wnt/β-catenin signaling regulates chondrocyte phenotype, maturation and function in a developmentally-regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.