Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both μCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL–OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL–OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.     

Protection From Glucocorticoid‐Induced Osteoporosis by Anti‐Catabolic Signaling in the Absence of Sost/Sclerostin

Excess of glucocorticoids, either due to disease or iatrogenic, increases bone resorption and decreases bone formation and is a leading cause of osteoporosis and bone fractures worldwide. Improved therapeutic strategies are sorely needed. We investigated whether activating Wnt/β‐catenin signaling protects against the skeletal actions of glucocorticoids, using female mice lacking the Wnt/β‐catenin antagonist and bone formation inhibitor Sost. Glucocorticoids decreased the mass, deteriorated the microarchitecture, and reduced the structural and material strength of bone in wild‐type (WT), but not in Sost^–/– mice. The high bone mass exhibited by Sost^–/– mice is due to increased bone formation with unchanged resorption. However, unexpectedly, preservation of bone mass and strength in Sost^–/– mice was due to prevention of glucocorticoid‐induced bone resorption and not to restoration of bone formation. In WT mice, glucocorticoids increased the expression of Sost and the number of sclerostin‐positive osteocytes, and altered the molecular signature of the Wnt/β‐catenin pathway by decreasing the expression of genes associated with both anti‐catabolism, including osteoprotegerin (OPG), and anabolism/survival, such as cyclin D1. In contrast in Sost^–/– mice, glucocorticoids did not decrease OPG but still reduced cyclin D1. Thus, in the context of glucocorticoid excess, activation of Wnt/β‐catenin signaling by Sost/sclerostin deficiency sustains bone integrity by opposing bone catabolism despite markedly reduced bone formation and increased apoptosis. This crosstalk between glucocorticoids and Wnt/β‐catenin signaling could be exploited therapeutically to halt resorption and bone loss induced by glucocorticoids and to inhibit the exaggerated bone formation in diseases of unwanted hyperactivation of Wnt/β‐catenin signaling. © 2016 American Society for Bone and Mineral Research.     

Conditional deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation,increasing volume of remodeling bone in mice

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast‐specific manner using a knock‐in Cre mouse line to the cathepsin K locus (Ctsk^Cre/+;Bmpr1a^flox/flox, designated as Bmpr1a^ΔOc/ΔOc). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre‐dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1a^ΔOc/ΔOc with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1a^ΔOc/ΔOc mice. Expression of bone‐resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5‐, 8‐, and 12‐week‐old Bmpr1a^ΔOc/ΔOc mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone‐formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1a^ΔOc/ΔOc tibias at 8 weeks of age. For comparison, we also induced an osteoblast‐specific deletion of Bmpr1a using Col1a1‐Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone‐formation activity during bone remodeling. © 2011 American Society for Bone and Mineral Research     

Sclerostin Mediates Bone Response to Mechanical Unloading Through Antagonizing Wnt/β‐Catenin Signaling

Reduced mechanical stress leads to bone loss, as evidenced by disuse osteoporosis in bedridden patients and astronauts. Osteocytes have been identified as major cells responsible for mechanotransduction; however, the mechanism underlying the response of bone to mechanical unloading remains poorly understood. In this study, we found that mechanical unloading of wildtype mice caused decrease of Wnt/β‐catenin signaling activity accompanied by upregulation of Sost. To further analyze the causal relationship among these events, Sost gene targeting mice were generated. We showed that sclerostin selectively inhibited Wnt/β‐catenin in vivo, and sclerostin suppressed the activity of osteoblast and viability of osteoblasts and osteocytes. Interestingly, Sost^?/? mice were resistant to mechanical unloading‐induced bone loss. Reduction in bone formation in response to unloading was also abrogated in the mutant mice. Moreover, in contrast to wildtype mice, Wnt/β‐catenin signaling was not altered by unloading in Sost^?/? mice. Those data implied that sclerostin played an essential role in mediating bone response to mechanical unloading, likely through Wnt/β‐catenin signaling. Our findings also indicated sclerostin is a promising target for preventing disuse osteoporosis.     

N‐cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage–Specific

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N‐cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N‐cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin‐insensitive Lrp5^A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2‐deleted mice, suggesting N‐cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β‐catenin accumulation after administration of an anti‐Dkk1 antibody are enhanced in N‐cadherin–deficient mice. Thus, although lack of N‐cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N‐cadherin in osteolineage cells favors bone formation. Hence, N‐cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.     

The role of Dkk1 in bone mass regulation: Correlating serum Dkk1 expression with bone mineral density

The Wnt/β‐catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signaling resulting in disturbances in bone mass. The objectives of our study were to correlate serum Dkk1 expression with bone mineral density (BMD) and assess the potential role of Dkk1 as a serological marker of bone mass. Serum was collected from a cohort of patients (n = 36), 18 patients with a reduced BMD and 18 control patients. Serum Dkk1 expression as quantified by ELISA was correlated with lumbar and femoral t‐ and z‐scores. Serum Dkk1 concentration in the osteoporosis group was significantly higher than control group (941 ± 116 vs. 558 ± 47 pg/ml, p < 0.01). Serum Dkk1 expression was highly correlated with bone mass variables with inverse associations found between serum Dkk1 expression and lumbar t‐score (r = ?0.34, p = 0.00433), lumbar z‐score (r = ?0.22, p = 0.1907), femur t‐score (r = ?0.42, p = 0.0101), and femur z‐score (r = ?0.43, p = 0.0089). Our data further emphasizes the pivotal role played by Wnt/β‐catenin signaling in bone mass regulation. Dkk1, a powerful antagonist of canonical Wnt signaling, may have a role to play as a serological marker for disorders of bone mass, warranting further evaluation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:414–418, 2011     

Different bone remodeling levels of trabecular and cortical bone in response to changes in Wnt/β‐catenin signaling in mice

Trabecular bone and cortical bone have different bone remodeling levels, and the underlying mechanisms are not fully understood. In the present study, the expression of Wnt/β‐catenin signaling and its downstream molecules along with bone mass in trabecular and cortical bone were compared in wild‐type mice, constitutive activation of β‐catenin (CA‐β‐catenin) mice and β‐catenin deletion mice. It was found that the expression level of most of the examined genes such as Wnt3a, β‐catenin, osteocalcin and RANKL/OPG ratio were significantly higher in trabecular bone than in cortical bone in wild‐type mice. CA‐β‐catenin resulted in up‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio, which were down‐regulated. Also, CA‐β‐catenin led to increased number of osteoblasts, decreased number of osteoclasts and increased bone mass in both the trabecular bone and cortical bone compared with wild‐type mice; however, the extent of changes was much greater in the trabecular bone than in the cortical bone. By contrast, null β‐catenin led to down‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio. Furthermore, β‐catenin deletion led to decreased number of osteoblasts, increased number of osteoclasts and decreased bone mass when compared with wild‐type mice. Again, the extent of these changes was more significant in trabecular bone than cortical bone. Taken together, we found that the expression level of Wnt/β‐catenin signaling and bone remodeling‐related molecules were different in cortical bone and trabecular bone, and the trabecular bone was more readily affected by changes in the Wnt/β‐catenin signaling pathway. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:812–819, 2017.

     

Reversing LRP5‐Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low‐density lipoprotein receptor‐related protein (LRP) 5/6 Wnt co‐receptors, thereby inhibiting Wnt/β‐catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis‐pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost^?/?), Lrp5 (Lrp5^?/?), or both (Sost^?/?;Lrp5^?/?) to elucidate the mechanism of action of Sost in vivo. Sost deficiency–induced bone gain was significantly blunted in Sost^?/?;Lrp5^?/? mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost^?/?;Lrp5^?/? mice and most bone parameters were elevated relative to wild‐type. To test whether the remaining bone increases in Sost^?/?;Lrp5^?/? animals depend on Lrp6, we treated wild‐type, Sost^?/?, and Sost^?/?;Lrp5^?/? mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class–mediated Lrp6 signaling reduced cancellous bone mass and density in wild‐type mice. Surprisingly, it reversed the abnormal bone gain in Sost^?/? and Sost^?/?;Lrp5^?/? mice to wild‐type levels irrespective of enhancement or blockage of Wnt3a class‐mediated Lrp6 activity. Thus, whereas Sost deficiency–induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class–induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss‐of‐function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options. © 2014 American Society for Bone and Mineral Research.     

Bone morphogenetic protein‐2 promotes osteosarcoma growth by promoting epithelial‐mesenchymal transition (EMT) through the Wnt/β‐catenin signaling pathway

The correlation between BMP‐2 and osteosarcoma growth has gained increased interest in the recent years, however, there is still no consensus. In this study, we tested the effects of BMP‐2 on osteosarcoma cells through both in vitro and in vivo experiments. The effect of BMP‐2 on the proliferation, migration and invasion of osteosarcoma cells was tested in vitro. Subcutaneous and intratibial tumor models were used for the in vivo experiments in nude mice. The effects of BMP‐2 on EMT of osteosarcoma cells and the Wnt/β‐catenin signaling pathway were also tested using a variety of biochemical methods. In vitro tests did not show a significant effect of BMP‐2 on tumor cell proliferation. However, BMP‐2 increased the mobility of tumor cells and the invasion assay demonstrated that BMP‐2 promoted invasion of osteosarcoma cells in vitro. In vivo animal study showed that BMP‐2 dramatically enhanced tumor growth. We also found that BMP‐2 induced EMT of osteosarcoma cells. The expression levels of Axin2 and Dkk‐1 were both down regulated by BMP‐2 treatment, while β‐catenin, c‐myc and Cyclin‐D1 were all upregulated. The expression of Wnt3α and p‐GSK‐3β were also significantly upregulated indicating that the Wnt/β‐catenin signaling pathway was activated during the EMT of osteosarcoma driven by BMP‐2. From this study, we can conclude that BMP‐2 significantly promotes growth of osteosarcoma cells (143B, MG63), and enhances mobility and invasiveness of tumor cells as demonstrated in vitro. The underlying mechanism might be that BMP‐2 promotes EMT of osteosarcoma through the Wnt/β‐catenin signaling pathway. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1638–1648, 2019.     

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1^+/−) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2^c/c;Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1^+/− mice were crossed with Bmp2^c/c;Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.     

Deletion of a Single β‐Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β‐catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β‐catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18‐ to 24‐week‐old osteocyte β‐catenin haploinsufficient mice (Dmp1‐Cre × β‐catenin fl/ + ; HET cKO) were compared with their β‐catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µ?, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro‐computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone‐formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β‐catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β‐catenin is required for bone formation in response to mechanical loading. © 2014 American Society for Bone and Mineral Research     

Adverse Effects of Osteocytic Constitutive Activation of ß‐Catenin on Bone Strength and Bone Growth

The activation of the canonical Wnt/β‐catenin signaling pathway in both mesenchymal stem cells and osteoblasts has been demonstrated to increase bone mass, showing promise for the treatment of low bone volume conditions such as osteoporosis. However, the possible side effects of manipulating this pathway have not been fully addressed. Previously, we reported that the constitutive activation of ß‐catenin in osteoblasts impaired vertebral linear growth. In the present study, β‐catenin was constitutively activated in osteocytes by crossing Catnb+/lox(exon 3) mice with dentin matrix protein 1(DMP1)‐Cre transgenic mice, and the effects of this activation on bone mass, bone growth and bone strength were then observed. DMP1‐Cre was found to be predominantly expressed in osteocytes, with weak expression in a small portion of osteoblasts and growth plate chondrocytes. After the activation, the cancellous bone mass was dramatically increased, almost filling the entire bone marrow cavity in long bones. However, bone strength decreased significantly. Thinner and more porous cortical bone along with impaired mineralization were responsible for the decrease in bone strength. Furthermore, the mice showed shorter stature with impaired linear growth of the long bones. Moreover, the concentration of serum phosphate decreased significantly after the activation of ß‐catenin, and a high inorganic phosphate (Pi) diet could partially rescue the phenotype of decreased mineralization level and impaired linear growth. Taken together, the constitutive activation of β‐catenin in osteocytes may increase cancellous bone mass; however, the activation also had adverse effects on bone strength and bone growth. These adverse effects should be addressed before the adoption of any therapeutic clinical application involving adjustment of the Wnt/β‐catenin signaling pathway. © 2015 American Society for Bone and Mineral Research.     

Thyroid hormone‐mediated growth and differentiation of growth plate chondrocytes involves IGF‐1 modulation of β‐catenin signaling

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β‐catenin signaling pathway. Insulin‐like growth factor 1 (IGF‐1) has been described as a stabilizer of β‐catenin, and thyroid hormone is a known stimulator of IGF‐1 receptor expression. The purpose of this study was to test the hypothesis that IGF‐1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β‐catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF‐1 and the IGF‐ receptor (IGF1R) stimulate Wnt‐4 expression and β‐catenin activation in growth plate chondrocytes. The positive effects of IGF‐1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T₃ activates IGF‐1/IGF1R signaling and IGF‐1‐dependent PI3K/Akt/GSK‐3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T₃‐mediated Wnt‐4 expression, β‐catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β‐catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF‐1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF‐1/IGF1R‐mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β‐catenin signaling, which is at least in part initiated by IGF‐1 signaling or the IGF‐1‐activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research     

Loss of wnt/β‐catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β‐catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β‐catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β‐catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow–derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β‐catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell‐fate shift of preosteoblasts that had to express osterix (lineage‐committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of β‐catenin from preosteoblasts caused a cell‐fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β‐catenin signaling exerts control over the fate of lineage‐committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity. © 2012 American Society for Bone and Mineral Research.     

PTH receptor signaling in osteocytes governs periosteal bone formation and intracortical remodeling

The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling. We report herein that transgenic mice expressing a constitutive active PTH receptor in osteocytes (DMP1‐caPTHR1 mice) exhibit increased cortical bone area and an elevated rate of periosteal and endocortical bone formation. In addition, DMP1‐caPTHR1 mice display a marked increase in intracortical remodeling and cortical porosity. Crossing DMP1‐caPTHR1 mice with mice lacking the Wnt coreceptor, LDL‐related receptor 5 (LRP5), or with mice overexpressing the Wnt antagonist Sost in osteocytes (DMP1‐Sost mice) reduced or abolished, respectively, the increased cortical bone area, periosteal bone formation rate, and expression of osteoblast markers and Wnt target genes exhibited by the DMP1‐caPTHR1 mice. In addition, DMP1‐caPTHR1 lacking LRP5 or double transgenic DMP1‐caPTHR1;DMP1‐Sost mice exhibit exacerbated intracortical remodeling and increased osteoclast numbers, and markedly decreased expression of the RANK decoy receptor osteoprotegerin. Thus, whereas Sost downregulation and the consequent Wnt activation is required for the stimulatory effect of PTH receptor signaling on periosteal bone formation, the Wnt‐independent increase in osteoclastogenesis induced by PTH receptor activation in osteocytes overrides the effect on Sost. These findings demonstrate that PTH receptor signaling influences cortical bone through actions on osteocytes and defines the role of Wnt signaling in PTH receptor action. © 2011 American Society for Bone and Mineral Research.     

Stretch‐induced inhibition of Wnt/β‐catenin signaling in mineralizing osteoblasts

Wnt signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of Wnt signaling in bone mechanotransduction. However, negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre‐osteoblasts to study short‐ and long‐term effects of mechanical loading on Wnt/β‐catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in Wnt/β‐catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear β‐catenin, but ultimately resulted in significant decrease at 12 and 40 h after stretch. Downregulation of Wnt‐responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0126 did not alter the stretch‐induced decrease of β‐catenin levels. Our data indicate a biphasic effect of mechanical loading on β‐catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of Wnt/β‐catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:390–396, 2010     

Elevated Dickkopf‐2 levels contribute to the abnormal phenotype of human osteoarthritic osteoblasts

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/β‐catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf‐1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a‐dependent Wnt/β‐catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/β‐catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor β1 (TGF‐β1) in OA osteoblasts, and exogenous TGF‐β1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF‐β1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF‐β1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF‐β1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype. © 2011 American Society for Bone and Mineral Research.