Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin

Although Wnt signaling is considered a key regulatory pathway for bone formation, inactivation of β-catenin in osteoblasts does not affect their activity but rather causes increased osteoclastogenesis due to insufficient production of osteoprotegerin (Opg). By monitoring the expression pattern of all known genes encoding Wnt receptors in mouse tissues and bone cells we identified Frizzled 8 (Fzd8) as a candidate regulator of bone remodeling. Fzd8-deficient mice displayed osteopenia with normal bone formation and increased osteoclastogenesis, but this phenotype was not associated with impaired Wnt signaling or Opg production by osteoblasts. The deduced direct negative influence of canonical Wnt signaling on osteoclastogenesis was confirmed in vitro and through the generation of mice lacking β-catenin in the osteoclast lineage. Here, we observed increased bone resorption despite normal Opg production and a resistance to the anti-osteoclastogenic effect of Wnt3a. These results demonstrate that Fzd8 and β-catenin negatively regulate osteoclast differentiation independent of osteoblasts and that canonical Wnt signaling controls bone resorption by two different mechanisms.     

Frizzled-4 is required for normal bone acquisition despite compensation by Frizzled-8

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.     

The Wnt Serpentine Receptor Frizzled-9 Regulates New Bone Formation in Fracture Healing

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9 ^−/− mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures.     

Essential role of beta-catenin in postnatal bone acquisition

Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the beta-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of beta-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of beta-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the beta-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-kappaB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/beta-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.     

Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation

Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.     

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.     

Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits transformed growth of NSCLC and promotes epithelial differentiation of these cells. The goal of this study was to determine whether the effects of Wnt 7a/Fzd 9 were mediated through PPARgamma. We found that Wnt 7a and Fzd 9 expression led to increased PPARgamma activity. This effect was not mediated by altered expression of the protein. Wnt 7a and Fzd 9 expression resulted in activation of ERK5, which was required for PPARgamma activation in NSCLC. SR 202, a known PPARgamma inhibitor, blocked the increase in PPARgamma activity and restored anchorage-independent growth in NSCLC expressing Wnt 7a and Fzd 9. SR 202 also reversed the increase in E-cadherin expression mediated by Wnt 7a and Fzd 9. These data suggest that ERK5-dependent activation of PPARgamma represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.     

Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of beta-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes.     

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.     

Insulin receptor substrate-2 maintains predominance of anabolic function over catabolic function of osteoblasts

Insulin receptor substrates (IRS-1 and IRS-2) are essential for intracellular signaling by insulin and insulin-like growth factor-I (IGF-I), anabolic regulators of bone metabolism. Although mice lacking the IRS-2 gene (IRS-2-/- mice) developed normally, they exhibited osteopenia with decreased bone formation and increased bone resorption. Cultured IRS-2-/- osteoblasts showed reduced differentiation and matrix synthesis compared with wild-type osteoblasts. However, they showed increased receptor activator of nuclear factor kappaB ligand (RANKL) expression and osteoclastogenesis in the coculture with bone marrow cells, which were restored by reintroduction of IRS-2 using an adenovirus vector. Although IRS-2 was expressed and phosphorylated by insulin and IGF-I in both osteoblasts and osteoclastic cells, cultures in the absence of osteoblasts revealed that intrinsic IRS-2 signaling in osteoclastic cells was not important for their differentiation, function, or survival. It is concluded that IRS-2 deficiency in osteoblasts causes osteopenia through impaired anabolic function and enhanced supporting ability of osteoclastogenesis. We propose that IRS-2 is needed to maintain the predominance of bone formation over bone resorption, whereas IRS-1 maintains bone turnover, as we previously reported; the integration of these two signalings causes a potent bone anabolic action by insulin and IGF-I.     

Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK

Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.     

Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5

Wnt7b is a Wnt ligand that has been demonstrated to play critical roles in several developmental processes, including lung airway and vascular development and chorion-allantois fusion during placental development. Wnt signaling involves the binding of Wnt ligands to cell surface receptors of the frizzled family and coreceptors of the LRP5/6 family. However, little is known of the ligand-receptor specificity exhibited by different Wnts, Fzds, and LRPs in Wnt signaling. Expression analysis of Fzds and LRP5/6 in the developing lung and vasculature showed that Fzd1, -4, -7, and -10 and LRP5/6 are expressed in tissue-specific patterns during lung development. Fzd1, -4, and -7 are expressed primarily in the developing lung mesenchyme, and Fzd10 is expressed in airway epithelium. LRP5 and LRP6 are expressed in airway epithelium during lung development, whereas LRP5 but not LRP6 expression is observed in the muscular component of large blood vessels, including the aorta. Cell transfection studies demonstrate that Wnt7b can activate the canonical Wnt pathway but not the noncanonical Wnt pathway in a cell-specific manner. Biochemical analysis demonstrates that Wnt7b can bind to Fzd1 and -10 on the cell surface and cooperatively activate canonical Wnt signaling with these receptors in the presence of LRP5. Together, these data demonstrate that Wnt7b signals through Fzd1 and -10 and LRP5 and implicate these Wnt coreceptors in the regulation of lung airway and vascular development.