Developmental Regulation of Wnt/β-Catenin Signals Is Required for Growth Plate Assembly, Cartilage Integrity, and Endochondral Ossification
Studies have suggested that continuous Wnt/β-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas 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 metalloprotease (MMP)-13 and vascular endothelial growth factor. Because 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. Studies have suggested that continuous Wnt/β-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas 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 metalloprotease (MMP)-13 and vascular endothelial growth factor. Because 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. Skeletogenesis continues to attract much research interest because of its fundamental roles in embryonic development and growth and its susceptibility to pathologies. The process initiates with formation of mesenchymal or ectomesenchymal cell condensations at specific sites and times in the early embryo. As exemplified by the limb, the mesenchymal condensations then undergo chondrogenesis and give rise to cartilaginous long bone anlagen (1Horton W.A. Royce P.M. Steinmann B. Connective Tissue and Its Heritable Disorders. Wiley-Liss, Inc., New York1993: 73-84Google Scholar, 2Karsenty G. Nature. 2003; 423: 316-318Crossref PubMed Scopus (348) Google Scholar). The chondrocytes become organized in growth plates, proliferate, and mature into pre-hypertrophic and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes express genes such as Indian hedgehog, and hypertrophic chondrocytes express a number of characteristic genes, including collagen X, alkaline phosphatase, osteopontin, and metalloprotease (MMP) 1The abbreviations used are: MMP, metalloprotease; IHH, Indian hedgehog; HA, hemagglutinin; RT, reverse transcriptase; CA-LEF, constitutive active form of mouse LEF-1.-13. Eventually, the hypertrophic chondrocytes mineralize their matrix by deposition of apatitic crystals and are replaced by marrow, vascular, and bone cells via an endochondral ossification process. Whereas these stepwise endochondral events proceed, diarthrodial synovial joints form at each epiphyseal end. Joint formation begins with appearance of a mesenchymal interzone that comes to separate adjacent cartilaginous anlagen (3Pacifici M. Koyama E. Iwamoto M. Gentili C. Connect. Tissue Res. 2000; 41: 17-784Crossref Scopus (59) Google Scholar). Although the above principles and general mechanisms are clear and well established, much remains to be understood about skeletogenesis, particularly at the molecular signaling level. Wnt proteins are powerful secreted signaling factors that regulate a number of developmental processes (4Nusse R. Varmus H.E. Cell. 1992; 69: 1073-1087Abstract Full Text PDF PubMed Scopus (843) Google Scholar, 5Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (495) Google Scholar). The vertebrate Wnt family currently comprises 20 members. Wnt proteins act by binding to Frizzled and low density lipoprotein receptor-related protein cell surface receptors (4Nusse R. Varmus H.E. 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