Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development

Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling.     

Wnt pathway,an essential role in bone regeneration

Fracture repair is a complex regenerative process initiated in response to injury, resulting in optimal restoration of skeletal function. Although histology characteristics at various phases of fracture repair are clear and well established, much remains to be understood about the process of bone healing, particularly at the molecular signaling level. During the past decade, secreted signaling molecules of the Wnt family have been widely investigated and found to play a central role in controlling embryonic development processes. Wnt signaling pathway also plays a pivotal role in the regulation of bone mass. Recent published data reveal that Wnt signaling pathway is activated during postnatal bone regenerative events, such as ectopic endochondral bone formation and fracture repair. Dysregulation of this pathway greatly inhibits bone formation and healing process. Interestingly, activation of Wnt pathway has potential to improve bone healing, but only utilized after mesenchymal cells have become committed to the osteoblast lineage. These advances suggest an essential role of Wnt pathway in bone regeneration. J. Cell. Biochem. 106: 353–362, 2009. © 2009 Wiley‐Liss, Inc.     

Ecto-5′-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice

Ecto-5′-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46–52-week-old wild type (WT) and CD73 knock-out mice (CD73^?/?) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73^?/? mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.     

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.     

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.     

Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors

Hedgehog and canonical Wnt/beta-catenin signaling are implicated in development of the osteoblast, the bone matrix-secreting cell of the vertebrate skeleton. We have used genetic approaches to dissect the roles of these pathways in specification of the osteoblast lineage. Previous studies indicate that Ihh signaling in the long bones is essential for initial specification of an osteoblast progenitor to a Runx2+ osteoblast precursor. We show here that this is a transient requirement, as removal of Hh responsiveness in later Runx2+, Osx1+ osteoblast precursors does not disrupt the formation of mature osteoblasts. By contrast, the removal of canonical Wnt signaling by conditional removal of the beta-catenin gene in early osteoblast progenitors or in Runx2+, Osx1+ osteoblast precursors results in a similar phenotype: osteoblasts fail to progress to a terminal osteocalcin+ fate and instead convert to a chondrocyte fate. By contrast, stabilization of beta-catenin signaling in Runx2+, Osx1+ osteoblast precursors leads to the premature differentiation of bone matrix secreting osteoblasts. These data demonstrate that commitment within the osteoblast lineage requires sequential, stage-specific, Ihh and canonical Wnt/beta-catenin signaling to promote osteogenic, and block chondrogenic, programs of cell fate specification.     

Combined deficiency of the Cnr1 and Cnr2 receptors protects against age‐related bone loss by osteoclast inhibition

The endocannabinoid system plays a role in regulating bone mass and bone cell activity and inactivation of the type 1 (Cnr1) or type 2 (Cnr2) cannabinoid receptors influences peak bone mass and age‐related bone loss. As the Cnr1 and Cnr2 receptors have limited homology and are activated by different ligands, we have evaluated the effects of combined deficiency of Cnr1 and 2 receptors (Cnr1/2^?/?) on bone development from birth to old age and studied ovariectomy induced bone loss in female mice. The Cnr1/2^?/? mice had accelerated bone accrual at birth when compared with wild type littermates, and by 3 months of age, they had higher trabecular bone mass. They were also significantly protected against ovariectomy‐induced bone loss due to a reduction in osteoclast number. The Cnr1/2^?/? mice had reduced age‐related bone loss when compared with wild‐type due to a reduction in osteoclast number. Although bone formation was reduced and bone marrow adiposity increased in Cnr1/2^?/? mice, the osteoclast defect outweighed the reduction in bone formation causing preservation of bone mass with aging. This contrasts with the situation previously reported in mice with inactivation of the Cnr1 or Cnr2 receptors individually where aged‐related bone loss was greater than in wild‐type. We conclude that the Cnr1 and Cnr2 receptors have overlapping but nonredundant roles in regulating osteoclast and osteoblast activities. These observations indicate that combined inhibition of Cnr1 and Cnr2 receptors may be beneficial in preventing age‐related bone loss, whereas blockade of individual receptors may be detrimental.     

sFRP3 inhibition improves age‐related cellular changes in BubR1 progeroid mice

Wnt signaling is a well‐known molecular pathway in age‐related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1^H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1^H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1^H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.     

sFRP2 Suppression of Bone Morphogenic Protein (BMP) and Wnt Signaling Mediates Mesenchymal Stem Cell (MSC) Self-renewal Promoting Engraftment and Myocardial Repair

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.     

Wnt signaling control of bone cell apoptosis

Wnts are a large family of growth factors that mediate essential biological processes like embryogenesis, morpho- genesis and organogenesis. These proteins also play a role in oncogenesis, and they regulate apoptosis in many tissues. Wnts bind to a membrane receptor complex comprised of a frizzled （FZD） G-protein-coupled receptor and a low-density lipoprotein （LDL） receptor-related protein （LRP）. The formation of this ligand-receptor complex initiates a number of signaling cascades that include the canonical/beta-catenin pathway as well as several noncanonical pathways. In recent years, canonical Wnt signaling has been reported to play a significant role in the control of bone formation. Clinical studies have found that mutations in LRP-5 are associated with reduced bone mineral density （BMD） and fractures. Investigations of knockout and transgenic mouse models of Wnt pathway components have shown that canonical Wnt signaling modulates most aspects ofosteoblast physiology including proliferation, differentiation, function and apoptosis. Transgenic mice expressing a gain of function mutant of LRP-5 in bone, or mice lacking the Wnt antagonist secreted frizzled-related protein-l, exhibit elevated BMD and suppressed osteoblast apoptosis. In addition, preclinical studies with pharmacologic compounds such as those that inhibit glycogen synthase kinase-3β support the importance of the canonical Wnt pathway in modulation of bone formation and osteoblast apoptosis.     

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.     

Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice

The canonical Wnt/β-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5^−/−) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5^−/− mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5^−/− mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active β-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.     

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC^Bone) or adipogenic (mMSC^Adipo) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC^Bone versus mMSC^Adipo using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC^Bone while genes related to lipid metabolism and immune response were highly expressed in mMSC^Adipo. In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC^Bone compared to mMSC^Adipo (p < 0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC^Bone compared to mMSC^Adipo. Reduced Wnt activity in mMSC^Adipo was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC^Adipo. Interestingly, conditioned medium (CM) collected from mMSC^Adipo (mMSC-CM^Adipo) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM^Adipo abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.     

Sequential roles of Hedgehog and Wnt signaling in osteoblast development

Signals that govern development of the osteoblast lineage are not well understood. Indian hedgehog (Ihh), a member of the hedgehog (Hh) family of proteins, is essential for osteogenesis in the endochondral skeleton during embryogenesis. The canonical pathway of Wnt signaling has been implicated by studies of Lrp5, a co-receptor for Wnt proteins, in postnatal bone mass homeostasis. In the present study we demonstrate that beta-catenin, a central player in the canonical Wnt pathway, is indispensable for osteoblast differentiation in the mouse embryo. Moreover, we present evidence that Wnt signaling functions downstream of Ihh in development of the osteoblast lineage. Finally Wnt7b is identified as a potential endogenous ligand regulating osteogenesis. These data support a model that integrates Hh and Wnt signaling in the regulation of osteoblast development.