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     

Indirubin‐3′‐Oxime Reverses Bone Loss in Ovariectomized and Hindlimb‐Unloaded Mice Via Activation of the Wnt/β‐Catenin Signaling

Osteoporosis is a major global health issue in elderly people. Because Wnt/β‐catenin signaling plays a key role in bone homeostasis, we screened activators of this pathway through cell‐based screening, and investigated indirubin‐3′‐oxime (I3O), one of the positive compounds known to inhibit GSK3β, as a potential anti‐osteoporotic agent. Here, we show that I3O activated Wnt/β‐catenin signaling via inhibition of the interaction of GSK3β with β‐catenin, and induced osteoblast differentiation in vitro and increased calvarial bone thickness ex vivo. Intraperitoneal injection of I3O increased bone mass and improved microarchitecture in normal mice and reversed bone loss in an ovariectomized mouse model of age‐related osteoporosis. I3O also increased thickness and area of cortical bone, indicating improved bone strength. Enhanced bone mass and strength correlated with activated Wnt/β‐catenin signaling, as shown by histological analyses of both trabecular and cortical bones. I3O also restored mass and density of bone in hindlimb‐unloaded mice compared with control, suspended mice, demonstrating bone‐restoration effects of I3O in non‐aged–related osteoporosis as well. Overall, I3O, a pharmacologically active small molecule, could be a potential therapeutic agent for the treatment and prevention of osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Diet‐Derived Phenolic Acids Regulate Osteoblast and Adipocyte Lineage Commitment and Differentiation in Young Mice

A blueberry (BB)‐supplemented diet has been previously shown to significantly stimulate bone formation in rapidly growing male and female rodents. Phenolic acids (PAs) are metabolites derived from polyphenols found in fruits and vegetables as a result of the actions of gut bacteria, and they were found in the serum of rats fed BB‐containing diet. We conducted in vitro studies with PAs and demonstrated stimulation of osteoblast differentiation and proliferation. On the other hand, adipogenesis was inhibited. To more fully understand the mechanistic actions of PAs on bone formation, we administered hippuric acid, one of the major metabolites found in animal circulation after BB consumption, to prepubertal female mice for 2 weeks. We found that hippuric acid was able to stimulate bone‐forming gene expression but suppress PPARγ expression, leading to increased bone mass dose‐dependently. Cellular signaling studies further suggested that the skeletal effects of PAs appeared to be mediated through activation of G‐protein‐coupled receptor 109A and downstream p38 MAP kinase and osterix. In conclusion, PAs are capable of altering the mesenchymal stem cell differentiation program and merit investigation as potential dietary therapeutic alternatives to drugs for degenerative bone disorders. © 2014 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.     

Osteoblast‐Targeted Expression of Sfrp4 in Mice Results in Low Bone Mass

Transgenic mice overexpressing Sfrp4 in osteoblasts were established. These mice exhibited low bone mass caused by a decrease in bone formation. Introduction: We recently reported that single nucleotide polymorphisms in the secreted frizzled‐related protein 4 (Sfrp4) gene are responsible for low peak BMD in senescence‐accelerated mouse (SAM) P6. In vitro studies revealed inhibition of osteoblast proliferation by Sfrp4, which is supposed to be mediated by canonical Wnt signaling. Materials and Methods: We examined the expression of Sfrp4 in neonate long bones by in situ hybridization and generated transgenic mice in which Sfrp4 was specifically overexpressed in osteoblasts under the control of a 2.3‐kb Col1a1 osteoblast‐specific promoter. Next, we compared the phenotype of Sfrp4 transgenic (Sfrp4 TG) mice with that of mice in which one allele of β‐catenin was conditionally disrupted in osteoblasts (βChet), and administered lithium chloride (LiCl) to Sfrp4 TG mice. Results: Hemizygous Sfrp4 TG mice exhibited a 30% reduction of trabecular bone mass compared with that in wildtype littermates at 8 wk of age, and histomorphometrical analysis showed decreases in both osteoblast numbers and bone formation rate. βChet mice exhibited a 17% reduction of trabecular bone mass in distal femora caused by an increase in the osteoclast number and a decrease in bone formation rate. Furthermore, LiCl administration rescued the bone phenotype of Sfrp4 TG mice. Conclusions: Expression of Sfrp4 in periosteum and bone tissues suggested the role of Sfrp4 in osteoblasts, and we identified that overexpression of Sfrp4 in osteoblasts suppressed osteoblast proliferation, resulting in a decrease in bone formation in vivo. Partial suppression of β‐catenin/canonical Wnt signaling also impaired bone formation, and activation of the signaling restored low bone mass of Sfrp4 TG mice. Thus, these results indicate that Sfrp4 decreases bone formation at least in part by attenuating canonical Wnt signaling in vivo.     

β‐catenin promotes bone formation and suppresses bone resorption in postnatal growing mice

Genetic studies in the mouse have demonstrated multiple roles for β‐catenin in the skeleton. In the embryo, β‐catenin is critical for the early stages of osteoblast differentiation. Postnatally, β‐catenin in mature osteoblasts and osteocytes indirectly suppresses osteoclast differentiation. However, a direct role for β‐catenin in regulating osteoblast number and/or function specifically in the postnatal life has not been demonstrated. Addressing this knowledge gap is important because low‐density lipoprotein receptor‐related protein 5 (LRP5), a coreceptor for WNT signaling proposed to function through β‐catenin, controls osteoblast number and function in postnatal mice or humans. To overcome the neonatal lethality caused by embryonic deletion of β‐catenin in early‐stage osteoblast‐lineage cells, we use the Osx‐CreER^T2 mouse strain to remove β‐catenin in Osterix (Osx)‐expressing cells by administering tamoxifen (TM) temporarily to postnatal mice. Lineage‐tracing experiments in the long bones demonstrate that Osx‐CreER^T2 targets predominantly osteoblast‐lineage cells on the bone surface, but also transient progenitors that contribute to bone marrow stromal cells and adipocytes. Deletion of β‐catenin by this strategy greatly reduces the bone formation activity of the targeted osteoblasts. However, the targeted osteoblasts rapidly turn over and are replaced by an excessive number of non‐targeted osteoblasts, causing an unexpected increase in bone formation, but an even greater increase in osteoclast number and activity produces a net effect of severe osteopenia. With time, the mutant mice also exhibit a marked increase in bone marrow adiposity. Thus, β‐catenin in postnatal Osx‐lineage cells critically regulates bone homeostasis by promoting osteoblast activity and suppressing osteoblast turnover, while restraining osteoclast and marrow fat formation. © 2013 American Society for Bone and Mineral Research.     

High levels of β‐catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway

Periodontal ligament stem cells (PDLSCs), a new population of mesenchymal stem cells (MSCs), have been isolated from the periodontal ligament (PDL). The capacity of multipotency and self‐renewal makes them an excellent cell source for bone regeneration and repair. However, their bone‐regeneration ability could be awakened in inflammatory microenvironments, which may be the result of changes in their differentiation potential. Recently, genetic evidences has shown that the Wnt pathway plays an important role in bone homeostasis. In this study we have determined the specific role of β‐catenin in osteogenic differentiation of PDLSCs obtained from inflammatory microenvironments (P‐PDLSCs). The inflammatory microenvironment, while inhibiting osteogenic differentiation potential, promotes proliferation of MSCs. A higher the level of β‐catenin in P‐PDLSCs than in H‐PDLSCs (PDLSCs obtained from a healthy microenvironment) resulted in the same disparity in canonical Wnt signaling pathway activation between each cell type. Here we show that activation of β‐catenin suppresses the noncanonical Wnt/Ca²⁺ pathway, leading to increased proliferation but reduced osteogenic differentiation of P‐PDLSCs. Downregulation of the levels of β‐catenin by treatment with dickkopf‐1 (DKK‐1) leads to activation of the noncanonical Wnt/Ca²⁺ pathway, which, in turn, results in the promotion of osteogenic differentiation in P‐PDLSCs. Interestingly, β‐catenin can affect both the canonical Wnt/β‐catenin pathway and the noncanonical Wnt/Ca²⁺ pathway. Our data indicate that β‐catenin plays a central role in regulating osteogenic differentiation of MSCs in inflammatory microenvironments. Given the important role of Wnt signaling in osteogenic differentiation, it is possible that agents that can modify this pathway may be of value in bone regeneration by MSCs in chronic inflammatory microenvironments. © 2011 American Society for Bone and Mineral Research     

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.

     

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     

Plasticizer di(2‐ethylhexyl)phthalate interferes with osteoblastogenesis and adipogenesis in a mouse model

Plasticizer di(2‐ethylhexyl)phthalate (DEHP) can leach from medical devices such as blood storage bags and the tubing. Recently, epidemiological studies showed that phthalate metabolites levels in the urine are associated with low bone mineral density (BMD) in older women. The detailed effect and mechanism of DEHP on osteoblastogenesis and adipogenesis, and bone loss remain to be clarified. Here, we investigated the effect and mechanism of DEHP and its active metabolite mono(2‐ethylhexyl)phthalate (MEHP) on osteoblastogenesis and adipogenesis. The in vitro study showed that osteoblast differentiation of bone marrow stromal cells (BMSCs) was significantly and dose‐dependently decreased by DEHP and MEHP (10–100 µM) without cytotoxicity to BMSCs. The mRNA expressions of alkaline phosphatase, Runx2, osteocalcin (OCN), Wnt1, and β‐catenin were significantly decreased in DEHP‐ and MEHP‐treated BMSCs during differentiation. MEHP, but not DEHP, significantly increased the adipocyte differentiation of BMSCs and PPARγ mRNA expression. Both DEHP and MEHP significantly increased the ratios of phosphorylated β‐catenin/β‐catenin and inhibited osteoblastogenesis, which could be reversed by Wnt activator lithium chloride and PPARγ inhibitor T0070907. Moreover, exposure of mice to DEHP (1, 10, and 100 mg/kg) for 8 weeks altered BMD and microstructure. In BMSCs isolated from DEHP‐treated mice, osteoblastogenesis and Runx2, Wnt1, and β‐catenin expression were decreased, but adipogenesis and PPARγ expression were increased. These findings suggest that DEHP and its metabolite MEHP exposure may inhibit osteoblastogenesis and promote adipogenesis of BMSCs through the Wnt/β‐catenin‐regulated and thus triggering bone loss. PPARγ signaling may play an important role in MEHP‐ and DEHP‐induced suppression of osteogenesis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1124–1134, 2018.

     

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.     

β‐Adrenergic Blockade and Leptin Replacement Effectively Mitigate Disuse Bone Loss

Our objective was to test effects of β‐adrenergic blockade on hindlimb unloading (HU)‐induced bone loss and serum leptin and to compare these responses with those observed with leptin replacement. Adult male rats were randomized into six groups (n = 10 each): HU rats treated with vehicle (VEHHU), leptin analog (LEPHU), or β‐blocker (BBHU) during a 28‐day HU and cage activity controls (CC) treated with the same three agents and pair‐fed to HU rats. On days 0 and 28, pQCT scans of proximal tibia and serum collections for leptin assays were performed, and histomorphometric measures of proximal tibia cancellous bone were assessed. The 20% decrease in cancellous vBMD observed in the VEHHU group was halved in BBHU rats and LEPHU rats. Bone formation rate (BFR) in BBHU rats, but not in LEPHU rats, was preserved. The 3‐fold increase in resorption surface with HU was abolished by BB and LEP treatments. The decrease in serum leptin after a 28‐day HU was attenuated in BBHU and LEPHU rats and was predictive of the decrease in BFR with HU. Blocking sympathetic adrenergic signaling by peripheral administration of a β‐blocker during HU mitigates disuse‐induced decreases in cancellous bone mass through stimulation of osteoblastic activity and suppression of osteoclastic activity. A direct effect of β‐adrenergic blockade on bone cells during HU may be enhanced by an indirect effect mitigating reductions in circulating leptin, possibly through disinhibition of leptin release from adipocytes.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.