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.     

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.

     

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.     

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.     

Response to tumor necrosis factor‐α mediated inflammation involving activation of prostaglandin E2 and Wnt signaling in nucleus pulposus cells

The cyclooxygenase 2 (COX‐2) product, prostaglandin E₂ (PGE₂), acts through a family of G protein‐coupled receptors designated E‐prostanoid (EP) receptors that mediate intracellular signaling by multiple pathways. However, it is not known whether crosstalk between tumor necrosis factor‐α(TNF‐α)–PGE₂‐mediated signaling and Wnt signaling plays a role in the regulation of intervertebral disc (IVD) cells. In this study, we investigated the relationship between TNF‐α–PGE₂ signaling and Wnt signaling in IVD cells. TNF‐α increased the expression of COX‐2 in IVD cells. The EP receptors EP1, EP3, and EP4 were expressed in IVD cells, and TNF‐α significantly increased PGE₂ production. Stimulation with TNF‐α also upregulated EP3 and EP4 mRNA and protein expression in IVD cells. The inductive effect of the EP3 and EP4 receptors on Topflash promoter activity was confirmed through gain‐ and loss‐of‐function studies using selective EP agonists and antagonists. PGE₂ treatment activated Wnt–β‐catenin signaling through activation of EP3. We conclude that TNF‐α‐induced COX‐2 and PGE₂ stimulate Wnt signaling and activate Wnt target genes. Suppression of the EP3 receptor via TNF‐α–PGE₂ signaling seems to suppress IVD degeneration by controlling the activation of Wnt signaling. These findings may help identify the underlying mechanism and role of Wnt signaling in IVD degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1756–1768, 2015.     

Immunomodulator ‘mushroom beta glucan’ induces Wnt/β catenin signalling and improves wound recovery in tilapia and rat skin: a histopathological study

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β‐catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery.     

Wnt/β‐catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin‐embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine‐specific Wnt signaling molecule mRNA expression levels were evaluated by two‐step real‐time qPCR. Spatial tissue protein expression of β‐catenin, Wnt‐11, Wnt‐4, and Dkk‐1 was determined by immunohistochemistry. There was significantly decreased Wnt‐11 and increased β‐catenin, Wnt‐5b, Dkk‐1, Lrp6, Wif‐1, Axin1, and SC‐PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β‐catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1433–1438, 2015.     

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet‐derived growth factor‐BB (PDGF‐BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF‐BB, and HBO combined with PDGF‐BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF‐α receptor (PDGFR‐α) and β receptor (PDGFR‐β) was detected by RT‐PCR. Protein expression of PDGFR‐β was detected by Western blotting. In vivo, chondrocytes and PDGF‐BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF‐BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up‐regulated mRNA expression of PDGFR‐α and increased cell accumulation in the S‐phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR‐β than HBO or PDGF‐BB. In vivo results suggested that PDGF‐BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin‐O and type II collagen staining confirmed the hyaline‐like cartilage regeneration in the repaired tissues. In situ up‐regulation of PDGFR‐β expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1439–1446, 2009     

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.     

p130Cas,E‐cadherin and β‐catenin in human transitional cell carcinoma of the bladder: Expression and clinicopathological significance

Objectives: To investigate the effects of junction protein, p130 Crk‐associated substance (p130Cas), and adhesion molecules, E‐cadherin and β‐catenin, on the biological behavior of transitional cell carcinoma of the bladder. Methods: In 72 paraffin embedded specimens of transitional cell carcinoma of the bladder and 20 normal controls, the expression of p130Cas, E‐cadherin and β‐catenin was examined by quantum dot‐based immunofluorescence histochemistry (QD‐IHC) and conventional immunohistochemistry (IHC). Results: QD‐IHC was consistent with IHC in detecting the expression of the three molecules (P > 0.05 for all comparisons). The positive expression rate of p130Cas in bladder cancer tissues increased more significantly than that in normal bladder tissues (P < 0.001). Similarly, the aberrant expression rates of E‐cadherin and β‐catenin in bladder cancer tissues were significantly higher than those in normal bladder tissues (P < 0.001 for both comparisons). The expression of each molecule was correlated with tumor pathological grade and clinical stage (P < 0.05 for all comparisons), but not with tumor number and size (P > 0.05 for all comparisons). Furthermore, negative correlations were found between the expression intensities of p130Cas and E‐cadherin or β‐catenin in transitional cell carcinoma of the bladder (P < 0.05 for both comparisons). Conclusions: p130Cas, E‐cadherin and β‐catenin might represent useful predictors of malignant degree of transitional cell carcinoma of the bladder.