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.     

Regulation of RANKL‐induced osteoclastogenesis by TGF‐β through molecular interaction between Smad3 and Traf6

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling. © 2011 American Society for Bone and Mineral Research.     

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     

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.     

Mechanical induction of PGE2 in osteocytes blocks glucocorticoid‐induced apoptosis through both the β‐catenin and PKA pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid‐flow shear stress blocks dexamethasone‐induced apoptosis of osteocyte‐like cells (MLO‐Y4). Prostaglandin E₂ (PGE₂), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone‐induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3‐kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β‐catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid‐induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase‐2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β‐catenin protein expression. β‐catenin siRNA knockdown experiments abrogated the protective effects of PGE₂, confirming the central role of β‐catenin in mediating the protection against dexamethasone‐induced cell death. Our data support a central role for PGE₂ acting through the cAMP/PKA and β‐catenin signaling pathways in the protection of osteocyte apoptosis by fluid‐flow shear stress. © 2010 American Society for Bone and Mineral Research.     

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.     

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     

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.

     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Distribution of slow‐cycling cells in epiphyseal cartilage and requirement of β‐catenin signaling for their maintenance in growth plate

Slow proliferation is one of the characteristics of stem cells. We examined the presence, distribution, and regulation of slow‐cycling cells in the developing and growing skeleton using a pulse‐chase method with a new nucleoside derivative, 5‐ethynyl‐2′‐deoxyuridine (EdU). C57BL/6 mice received daily intraperitoneal injections of EdU from postnatal day 4 to day 7. One day after the last EdU injection, a large population of cells in articular cartilage and growth plate was labeled. Six weeks after the last injection, the number of EdU‐labeled cells dramatically decreased, but a small number of them were dominantly present in the articular surface, and the labeling index was significantly higher in the surface than that in the rest of articular cartilage. In the growth plate, most EdU‐positive cells were found in the top layer that lies immediately below the secondary ossification center. Interestingly, postnatal conditional ablation of β‐catenin in cartilage caused a complete loss of the EdU‐labeled cells in growth plate that displayed disorganization and dysfunction. Together, our data demonstrate that slow‐cycling cells do reside in specific locations and numbers in both articular cartilage and growth plate. The β‐catenin signaling pathway appears to play a previously unsuspected role in maintenance of the slow‐cycling cells. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:661–668, 2014.     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

β‐catenin expression and claudin expression pattern as prognostic factors of prostatic cancer progression

OBJECTIVE

To investigate the patterns of expression of the junctional proteins β‐catenin and claudins in different prognostic groups of patients with prostatic cancer, to determine their value as prognostic markers.

PATIENTS AND METHODS

We evaluated the samples of 30 patients who had a radical prostatectomy for organ‐confined cancer (pT2N0M0), men with clinically advanced cancer, and a control group with benign prostatic hyperplasia. Using immunohistochemistry applied to tissue microarrays, each group was evaluated for claudin‐1, ‐2, ‐3, ‐4, ‐5, ‐7, ‐8 and ‐10, and β‐catenin expression.

RESULTS

There were differences among the three groups in the expression of claudin‐1 (P = 0.001), ‐2 (P = 0.014), ‐3 (P = 0.027), ‐4 (P = 0.001), ‐8 (P = 0.001) and β‐catenin (P = 0.002), regardless of Gleason score. By contrast, claudin‐5, ‐7 and ‐10 patterns were not significantly different among the groups. Furthermore, claudin‐1 (P = 0.014) and ‐4 (P = 0.004) could be used to distinguish between those patients who had metastases and those who did not.

CONCLUSIONS

The pattern of claudin expression could be a novel diagnostic marker in re‐classifying adenocarcinomas, and an additional sensitive predictive factor for a clinically poor prognosis. Our results suggest that patients with organ‐confined and advanced cancer are subsets with distinct claudin expression profiles, and that claudin‐4 is related to cellular differentiation in prostate cancer, which is not only the receptor molecule for the Clostridium perfringens enterotoxin, and thus a theoretical future therapeutic target for prostate cancer, but also a marker of progression.