Axin2 regulates chondrocyte maturation and axial skeletal development.

During embryonic development, skeletal elements are formed via two distinct processes: intramembranous and endochondral ossification. Several molecular signals coordinating endochondral bone formation have been discovered, including Wnt/β-catenin signaling and TGF-β signaling. Recent studies have demonstrated that Wnt/β-catenin signaling is required to drive proper osteoblast and chondrocyte differentiation during endochondral ossification.1–3 Additionally, TGF-β has been shown to stimulate early stages of chondrogenesis and chondrocyte proliferation while inhibiting the terminal differentiation of chondrocytes.4–6 Thus, Wnt and TGF-β signaling appear to oppose one another during select periods of endochondral bone development. Axins regulate both the Wnt/β-catenin and TGF-β signaling pathways. In the absence of Wnt signal, Axins bind GSK-3β and β-catenin, facilitating GSK-3β-mediated phosphorylation of β-catenin, marking the protein for ubiquitination and proteasomal degradation.7 Axins are stabilized when phosphorylated by GSK-3β.8 In the presence of Wnt signal, Axins are recruited to the Wnt co-receptor LRP5 or 6, and are dephosphorylated. 9,10 The protein complex formed by Axin is therefore destabilized, allowing β-catenin to accumulate and translocate to the nucleus where it regulates gene transcription through TCF and LEF transcription factors.11,12 In a negative feedback loop, Wnt/β-catenin/TCF signaling induces Axin2 expression, which in turn inhibits canonical Wnt signaling.13,14 While Axins inhibit Wnt signaling, they enhance TGF-β signaling in two ways. First, Axins facilitate the phosphorylation and activation of Smad3.15 Activated Smad3 associates with Smad4, translocates to the nucleus, and initiates gene transcription. Second, Axins facilitate phosphorylation of the inhibitory Smad7, which competitively inhibits Smad3 activity. Phosphorylation of Smad7 marks it for ubiquitination and proteasomal degradation.16 Axin1 and Axin2 are master scaffolding proteins originally identified as negative regulators of canonical Wnt/β-catenin signaling (see review17). Although these proteins are similar in function, Axin1 appears to be ubiquitously expressed, while Axin2 has a more restricted expression pattern.18 Further, Axin2 expression is directly induced by canonical Wnt signaling and therefore acts in a negative feedback loop.8,13 In addition to interactions with several canonical Wnt signaling proteins,9,19 the Axin proteins have been shown to interact with MEKK1 and Smad3 establishing their importance in other key signaling pathways.15,20 Given the role of Axins in at least two pathways relevant to endochondral ossification and embryogenesis in general, it is likely that Axins specifically contribute to normal skeletogenesis. Therefore, we examined Axin2 expression in the cartilage of Axin2LacZ/LacZ mice and examined the skeletal phenotype of Axin2-deficient mice to ascertain the effects of disrupted Wnt/β-catenin signaling on endochondral ossification. Intramembranous bone formation has already been shown to involve Axin2 as Axin2-deficient mice are characterized by craniofacial defects.21 Interestingly, mice deficient in the functional homolog Axin1 die in utero and are characterized by the presence of axis determination defects22–24; however, mice heterozygous for a mutation in Axin1 survive with no abnormalities. Hence, we also explored the skeletal phenotype of Axin2-deficient mice on a genetic background of Axin1 heterozygosity to determine if the presence of Axin2 in Axin1 heterozygotes is compensates for the absence of Axin1. We hypothesized that Axin2 would be expressed in cartilage cells, that Axin2-deficient mice would have significant changes in endochondral skeletal development when compared to heterozygous or wild-type littermate controls, and that these alterations would be even more profound in the Axin1+/−; Axin2−/− animals.