Emerging evidence suggests that the canonical Wnt pathway is crucial for bone and cartilage formation (Akiyama et al., 2004
; Bennett et al., 2005
; Day et al., 2005
; Glass et al., 2005
; Gong et al., 2001
; Hill et al., 2005
; Kolpakova and Olsen, 2005
; Li et al., 2005
; Little et al., 2002
). A growing list of molecules, regulating β-catenin signaling, has been linked to various skeletal diseases (Hartmann, 2006
). However, the exact role of β-catenin in sequential steps of bone development remains elusive. As a result of accelerated intramembranous ossification, targeted disruption of Axin2 induces premature suture closure (Yu et al., 2005a
). Axin2 apparently regulates expansion of osteoprogenitors and maturation of osteoblasts through its modulation on the canonical Wnt pathway (Yu et al., 2005a
). Using the Axin2-null mouse model, we investigated the exact functions and downstream signaling effects of β-catenin in osteoblast proliferation and differentiation. The results lead us to the following theory (). The enhanced proliferation is mediated by stimulation of a Wnt target gene cyclin D1 to promote cell divisions in osteoprogenitors. BMP signaling, elevated by Axin2 deficiency, is intimately involved in the Axin2/β-catenin-dependent osteogenesis. Inhibition of BMP by Noggin has no effects on nuclear accumulation of β-catenin as well as expansion of osteoprogenitors in the Axin2 mutants. However, Noggin strongly affects osteoblast maturation by regulating adherens junctions. During osteoblast differentiation, a BMP-dependent event, initially activated by Wnt, is required to promote cell–cell interaction by stimulating membrane accumulations of β-catenin and OBcad. Thus, it results in accelerated intramembranous ossification occurring in the Axin2−/− skulls. Disturbance of cell–cell interaction interferes with osteoblast differentiation, suggesting that adherens junctions play a significant role in the maturation processes. The presence of Noggin not only disrupts membrane distribution and stimulation of β-catenin and OBcad, but also represses osteoblast differentiation. These findings strongly support our model for distinct roles of β-catenin at different stages of calvarial osteoblast development (). β-catenin regulates cell cycle progression and cell–cell adhesion to control expansion of osteoprogenitors and maturation of osteoblasts, respectively.
Fig. 9 Model for calvarial osteoblast development mediated by Wnt signaling. β-catenin is located to the cytoplasm of resting osteoprogenitors. An active Wnt signal promotes cell cycle entry by inducing nuclear localization of β-catenin (β-Cat) (more ...)
Haploid deficiency of β-catenin alleviates the skull defects caused by Axin2 deficiency. The data reveal an absolute requirement of β-catenin in the Axin2 mediated skull morphogenesis. Haploid deficiency of β-catenin prevents premature fusion of the Axin2-null sutures by interfering with the calvarial osteoblast development. In the Axin2−/−; β-catenin+/− mutants, BMP signaling is reduced that places BMP downstream of β-catenin in osteoblast development. This is consistent with stimulation of phosphorylated Smad1/5/8 by the Axin2 deletion. On the other hand, Noggin was only able to inhibit the differentiation but not proliferation abnormalities, caused by Axin2 deficiency. Noggin interferes with membrane accumulations of β-catenin, suggesting that it is regulated by BMP during osteoblast maturation. Therefore, these two pathways appear to work in a feedback regulatory mechanism (). Initial β-catenin activation is responsible for stimulating the β-catenin/cyclin D1 pathway to enhance proliferation in a BMP-independent fashion. Nuclear localization of β-catenin then turns on BMP signaling that induces a second event of β-catenin activation upon osteoblast differentiation. This BMP-dependent signal facilitates the maturation processes by promoting cell–cell interaction mediated by β-catenin/OBcad.
The functions of β-catenin can also be molecularly distinct. In C. elegans
, there are three different β-catenin gene products for adhesion and signaling functions (Korswagen et al., 2000
). BAR-1 mediates Wnt signaling by forming a transcription complex with POP-1 (TCF/LEF). HMP-2 interacts exclusively with cadherin. WRM-1 is involved in a divergent Wnt pathway where it regulates POP-1 indirectly. Nevertheless, there is no evidence for multiple β-catenin gene products in vertebrates. This raises the possibility that β-catenin utilizes its distinct functions in regulating osteoblast development. Previous genetic analyses demonstrated that β-catenin is essential for different stages of skeletal development (Day et al., 2005
; Hill et al., 2005
). However, the nature of these studies could not distinguish which function of β-catenin is critically involved. The exact roles of β-catenin remain unclear because it is a multifunctional protein, which is involved in cellular responses including Wnt signaling (Logan and Nusse, 2004
; Moon et al., 2004
) and cell adhesion (Bienz, 2005
; Gumbiner, 2005
; Harris and Peifer, 2005
). Only the binding of β-catenin to the degradation complex is disrupted in the Axin2-null mice. These mutants provide an excellent model to investigate the functional involvement of β-catenin in the adhesion and transcription complexes. Our results demonstrated that β-catenin utilizes its disparate roles to control expansion of osteoprogenitors and maturation of osteoblasts in different contexts. They also provide evidence for how one molecule with multiple cellular functions can regulate the proliferation and differentiation processes of single lineage-specific development. Although prior studies suggested that distinct molecular forms of β-catenin possess preferential binding affinity to adhesion, transcription and degradation complexes to ensure proper tissue architecture and cell-fate decisions (Brembeck et al., 2004
; Gottardi and Gumbiner, 2004
), details of such mechanisms regulated in these microenvironments remain to be elucidated. Further investigations of our system might gain new insights into these mechanistic regulations.
The cadherin–catenin mediated cell adhesion has been shown to promote osteogenesis (Ferrari et al., 2000
; Stains and Civitelli, 2005
). Our data strongly support the importance of cell–cell interaction mediated by adherens junctions in osteoblast differentiation. OBcad, but not Ncad or Ecad, is specifically stimulated in the Axin2-null mutants. Similar to the Axin2-null mutants, the loss of OBcad caused defects in skeletal development that occurred preferentially in the mouse skull (Kawaguchi et al., 2001
). It has been suggested that Ncad, activated by BMP-2, is a direct target of Sox9 in chondrogenesis (Panda et al., 2001
). In human craniosynostosis with FGFR2 mutation, Ncad overexpression plays a role in premature suture closure (Muenke and Schell, 1995
). Therefore, promoting cell adhesion by stimulating the cadherin–catenin complex formation might be a key part of the skull morphogenetic signaling network, as well as other processes in musculoskeletal development.
FGF signaling plays a key role in skull morphogenesis and craniosynostosis (Ornitz and Marie, 2002
) although the mechanism by which FGF interacts with Wnt and BMP remains to be illustrated. Expression of two potential Wnt downstream targets FGF4 and FGF18 is elevated by Axin2 deficiency during osteoblast differentiation (Yu et al., 2005a
). In the Axin2-null sutures, increased numbers of the FGFR1-expressing cells are evident (Yu et al., 2005a
). This process depends on β-catenin as shown in the Axin2−/−; β-catenin+/− mice. Previous studies suggested that FGF might induce BMP signaling by inhibiting the BMP-dependent activation of Noggin (Warren et al., 2003
). It is possible that FGF acts in between Wnt and BMP to orchestrate skull morphogenesis. The exact role of FGF signaling in the hierarchy of skull signaling cascade and in the calvarial osteoblast developmental processes remains to be determined. Future studies focused on delineating the skull morphogenetic circuitry that regulates tissue architecture and cell-fate decisions promise important insights into the molecular and cellular bases of craniosynostosis.