JCI - Regulation of bone mass by Wnt signaling

As described above, Wnt signaling increases bone mass through diverse mechanisms. While effects on osteoblastogenesis and apoptosis have been studied in some mechanistic detail and will be elaborated upon here, this is not to diminish the potential importance of other mechanisms mentioned earlier that are less well studied, including renewal of stem cells (35), stimulation of preosteoblast replication (13), and enhancement of osteoblast activity (Figure 2) (13, 17).

Osteoblastogenesis versus adipogenesis. There is considerable evidence for the existence of a mesenchymal stem cell that gives rise to both osteogenic and adipogenic cells, and in vitro and in vivo experimental models have provided compelling evidence for a reciprocal relationship between these cell lineages (41–43). For example, cultures of bone marrow stromal cells as well as immortalized clonal lines (e.g., ST2) are capable of both osteogenic and adipogenic differentiation, depending upon culture conditions. Furthermore, single cell clones from bone marrow can differentiate in vitro into either adipocytes or osteoblasts (44). In addition to signaling by Wnt/β-catenin, a number of factors influence the fate of these marrow-derived mesenchymal stem cells, including retinoic acid, BMPs, vitamin D3, glucocorticoids, notch, sonic hedgehog, parathyroid hormone, parathyroid hormone–related peptide, and PPARγ ligands (24, 43, 45–47). Indeed, Wnt signaling may be required for or even mediate a subset of effects of BMP, parathyroid hormone, and hedgehog on cell fate decisions toward osteoblastogenesis (25, 48).

Pharmacological and genetic treatments that activate Wnt/β-catenin signaling in mesenchymal precursors repress adipogenesis and stimulate osteoblastogenesis (Figure 2). In preadipocyte models expression of Wnt does not influence induction of the transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPδ, but Wnt signaling blocks induction of master adipogenic transcription factors C/EBPα and PPARγ (30). Suppression of Wnt/β-catenin signaling with dominant-negative TCFs or sFRPs stimulates spontaneous adipogenesis, indicating that endogenous Wnts inhibit preadipocyte differentiation (30, 31). Wnt signaling is initiated in part by Wnt10b. Its expression is high in dividing and confluent preadipocytes, and Wnt10b is rapidly suppressed upon induction of differentiation (30, 31). In addition, ectopic expression of Wnt10b stabilizes free cytosolic β-catenin and is a potent inhibitor of adipogenesis. Most conclusively, Wnt10b antisera promotes adipogenesis when added to media of 3T3-L1 preadipocytes. Interestingly, expression of Wnt5b is transiently induced during differentiation of 3T3-L1 cells, and adenoviral expression of Wnt5b causes a slight increase in adipogenesis, presumably due to destabilization of β-catenin (49, 50). Wnt5b may activate noncanonical Wnt signaling, which has been reported to antagonize Wnt/β-catenin signaling (51), or Wnt5b may compete with other Wnts for binding to frizzled receptors. Further work is required to assess whether Wnt5b inhibits osteoblastogenesis.

Mesenchymal precursors such as ST2 cells express low but biologically relevant levels of adipogenic transcription factors C/EBPα and PPARγ and osteoblast transcription factors such as runt-related transcription factor 2 (Runx2), msh homeobox homolog 2 (Msx2), distal-less homeobox 5 (Dlx5), and osterix (24). Expression of these 2 classes of transcription factors is maintained at low levels due to negative feedback, and imbalance leads to differentiation. For example, Msx2 binds to C/EBPα and inhibits its ability to transactivate the PPARγ promoter, and Msx2 represses adipogenesis (52, 53). Similarly, PPARγ binds to Runx2 and inhibits transactivation of the osteocalcin promoter, and activation of PPARγ represses osteoblastogenesis (54). Constitutive Wnt/β-catenin signaling favors expression of osteoblast genes at the expense of adipocyte genes (24). Wnt signaling could regulate the fate of mesenchymal precursors by repressing adipocyte transcription factors, stimulating osteoblast transcription factors, or both (Figure 2). Increased bone mass in Pparg^+/– mice, increased osteogenesis in precursor cells from PPARγ-null mice, and decreased bone density following treatment of mice with a PPARγ agonist make this factor an attractive target (55–57). Indeed, suppression of PPARγ is required for Wnt10b to stimulate osteoblastogenesis (24). A recent report indicated that a transcriptional regulator, transcription coactivator with PDZ domain (TAZ), mediates the effects of BMP-2 on mesenchymal cell fate by inhibiting PPARγ activity while stimulating that of Runx2; however, the potential role of TAZ-mediated effects of Wnt/β-catenin signaling has not been reported (58).

Apoptosis. Induction of bone accrual in mouse models with increased Wnt signaling is due in part to reduced apoptosis of osteoblasts and osteocytes (14, 17, 59). Wnt signaling inhibits apoptosis in response to a wide variety of cellular insults, including chemotherapeutic agents and serum deprivation (60–62). Prevention of apoptosis occurs in a wide variety of cell models, including mesenchymal precursors, preosteoblasts, and osteoblasts. While signaling by canonical Wnts appears to universally protect against apoptosis through mechanisms involving β-catenin and activation of PI3K/Akt, other mechanistic aspects are dependent on cell type. For example, in rat intestinal epithelial cells, induction of cyclooxygenase-2 and Wnt-induced secreted protein 1 (WISP-1), but not Bcl-2, are critical for repression of apoptosis caused by c-myc (60). In preadipocytes, increased production of insulin-like growth factors feeds back through an autocrine/paracrine mechanism to block apoptosis due to serum deprivation (61). Finally, in preosteoblasts, activation of Src, ERK, and Akt by Wnt3a is required for prevention of apoptosis. In this cell model, Wnt signaling induces expression of Bcl-2 through a process requiring active ERK (62). The mechanism or mechanisms by which Wnt/β-catenin signaling brings about an increase in the number of osteoblasts and osteocytes in vivo have yet to be determined.