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1.  Chondrocyte-Specific Inhibition of β-Catenin Signaling Leads to Dysplasia of the Caudal Vertebrae in Mice 
Spine  2013;38(24):2079-2084.
Study Design
To inhibit β-catenin specifically signaling in chondrocytes Col2-ICAT transgenic mice were generated. Anomalies in caudal vertebrae were detected during embryonic and postnatal stages of Col2-ICAT transgenic mice.
Objective
To determine the role of canonical β-catenin signaling in caudal vertebral development.
Summary of Background Data
β-catenin signaling plays a critical role in skeletal development. Col2-ICAT transgenic mice were generated to selectively block β-catenin signaling by overexpression of the ICAT gene in chondrocytes.
Methods
Tails of E16.5 transgenic embryos and adult Col2-ICAT transgenic mice and their wild-type littermates were collected and analyzed. Skeletal preparation, 3-dimensional micro-computed tomographic and histological analyses were performed to evaluate changes in the structure of caudal vertebrae. Bromodeoxyuridine labeling was performed to evaluate changes in chondrocyte proliferation in caudal vertebrae.
Results
Skeletal preparation and 3-dimensional micro-computed tomographic analyses revealed bone deformation and angulated deformities in tail tissue in Col2-ICAT transgenic mice. Histological studies revealed abnormal bone development and dysplastic caudal vertebrae in Col2-ICAT transgenic mice. Inhibition of β-catenin signaling in cartilage resulted in vertebral dysplasia leading to aberrant resegmenting process. Thus, 2 poorly developed sclerotomes failed to fuse to form a complete vertebrae. BrdU labeling revealed a decreased chondrocyte proliferation in both cartilageous templates of transgenic embryos and the growth plate of adult Col2-ICAT transgenic mice.
Conclusion
Wnt/β-catenin signaling plays an important role in vertebral development. Inhibition of β-catenin signaling in chondrocytes results in caudal vertebra deformity in mice, which may occur as early as in the stage of sclerotome formation.
doi:10.1097/01.brs.0000435024.57940.8d
PMCID: PMC3928445  PMID: 24026150
β-catenin; ICAT; chondrocyte; caudal vertebral dysplasia
2.  Inhibition of the Wnt-β-catenin and Notch signaling pathways sensitizes osteosarcoma cells to chemotherapy 
Osteosarcoma (OS) is one of the most common malignant bone tumors in early adolescence. Multi-drug chemotherapy has greatly increased the five year survival rate from 20% to 70%. However, the rate has been staggering for 30 years and the prognosis is particularly poor for patients with recurrence and metastasis. Our study aimed to investigate the role of Wnt-β-catenin, Notch and Hedgehog pathway in OS development because all these pathways are involved in skeletal development, tumorigenesis and chemoresistance. Our results showed that the major components in Wnt-β-catenin pathway, e.g. Wnt3a, β-catenin and Lef1, were consistently upregulated in human osteosarcoma cell line Saos2 cells compared to human fetal osteoblasts (hFOB), whereas the changes in the expression levels of Notch and Hh signaling molecules were not consistent. Knocking down β-catenin increased the Saos2 sensitivity to methotrexate (MTX) induced cell death. Consistently, the expression level of β-catenin protein correlated with the invasiveness of OS, as evidenced by more intensive β-catenin immunoreactivity in higher grade OS samples. Chemical inhibition of the Wnt-β-catenin signaling enhanced MTX mediated death of Saos2 cells. A synergistic effect with MTX was observed when both inhibitors for Wnt-β-catenin and Notch pathways were simultaneously used, while the addition of the Hh inhibitor did not further improve the efficacy. Our findings provide some novel insight to OS pathogenesis and lay a foundation for future application of Wnt-β-catenin and Notch inhibitors together with the currently used chemotherapeutic drugs to improve the outcome of OS treatment.
doi:10.1016/j.bbrc.2012.12.118
PMCID: PMC3710725  PMID: 23291185
Osteosarcoma; Catenin; Notch; Pathway; Methotrexate; Apoptosis
3.  Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges 
Recent years have witnessed much progress in both basic research and clinical trials regarding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells. The unique structure of CAR endows T cell tumor specific cytotoxicity and resistance to immunosuppressive microenvironment in cancers, which helps patients to better tackle the issue of immunological tolerance. Adoptive immunotherapy (AIT) using this supernatural T cell have gained momentum after decades of intense debates because of the promising results obtained from preclinical models and clinical trials. However, it is very important for us to evaluate thoroughly the challenges/obstacles before widespread clinical application, which clearly warrants more studies to improve our understanding of the mechanism underlying AIT. In this review, we focus on the critical issues related to the clinical outcomes of CAR-based adoptive immunotherapy and discuss the rationales to refine this new cancer therapeutic modality.
doi:10.1186/1756-8722-6-47
PMCID: PMC3706354  PMID: 23829929
Chimeric antigen receptor; Adoptive immunotherapy; Single chain variable fragment; T cell receptor
4.  Aberrant hypertrophy in Smad3-deficient chondrocytes is rescued by restoring TAK1-ATF-2 signaling: a potential clinical implication for osteoarthritis 
Arthritis and rheumatism  2010;62(8):2359-2369.
Objective
To investigate the biological significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and ATF-2 in the TGF-β signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.
Methods
Joint disease in Smad3 knockout (Smad3−/−) mice was examined by micro-CT and histology. Numerous in vitro methods including immunostaining, real-time PCR, Western blotting, an ATF-2 DNA-binding assay and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3−/− mice.
Results
Smad3−/− mice gradually developed an end-stage OA phenotype. TGF-β-induced TAK1-ATF-2 signaling was disrupted in Smad3−/− chondrocytes at the level of p38 MAP kinase activation resulting in reduced ATF-2 phosphorylation and transcriptional activity. Re-introduction of Smad3 into the Smad3−/− cells restored the normal p38 response to TGF-β. Phospho-p38 formed a complex with Smad3 by binding to the Smad3 MH1-linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAP kinase phosphatase-1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild type and Smad3−/− chondrocytes. p38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms α, β and γ, but not δ.
Conclusions
Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1-ATF-2 signaling, most likely by disrupting the Smad3-phospho-p38 complex and, thereby, promoting p38 dephosphorylation and inactivation by MKP-1. p38 and ATF-2 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.
doi:10.1002/art.27537
PMCID: PMC2921996  PMID: 20506210
5.  Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of β-Catenin–BMP Signaling 
Journal of Bone and Mineral Research  2010;25(6):1234-1245.
Osteoporosis is defined as reduced bone mineral density with a high risk of fragile fracture. Current available treatment regimens include antiresorptive drugs such as estrogen receptor analogues and bisphosphates and anabolic agents such as parathyroid hormone (PTH). However, neither option is completely satisfactory because of adverse effects. It is thus highly desirable to identify novel anabolic agents to improve future osteoporosis treatment. Osthole, a coumarin-like derivative extracted from Chinese herbs, has been shown to stimulate osteoblast proliferation and differentiation, but its effect on bone formation in vivo and underlying mechanism remain unknown. In this study, we found that local injection of Osthole significantly increased new bone formation on the surface of mouse calvaria. Ovariectomy caused evident bone loss in rats, whereas Osthole largely prevented such loss, as shown by improved bone microarchitecture, histomorphometric parameters, and biomechanical properties. In vitro studies demonstrated that Osthole activated Wnt/β-catenin signaling, increased Bmp2 expression, and stimulated osteoblast differentiation. Targeted deletion of the β-catenin and Bmp2 genes abolished the stimulatory effect of Osthole on osteoblast differentiation. Since deletion of the Bmp2 gene did not affect Osthole-induced β-catenin expression and the deletion of the β-catenin gene inhibited Osthole-regulated Bmp2 expression in osteoblasts, we propose that Osthole acts through β-catenin–BMP signaling to promote osteoblast differentiation. Our findings demonstrate that Osthole could be a potential anabolic agent to stimulate bone formation and prevent estrogen deficiency–induced bone loss. © 2010 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.21
PMCID: PMC3153131  PMID: 20200936
osthole; β-catenin; BMP-2; osteoblast; osteoporosis; ovariectomy
6.  Prostaglandin E2 Inhibits BMP Signaling and Delays Chondrocyte Maturation 
While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the BMP signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2 treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the Protein Kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-β to activate the TGF-β-responsive reporter, 4XSBE. Finally, PGE2 down regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation.
doi:10.1002/jor.20805
PMCID: PMC2737521  PMID: 19023895
7.  Transforming Growth Factor-β Stimulates Cyclin D1 Expression through Activation of β-Catenin Signaling in Chondrocytes* 
The Journal of biological chemistry  2006;281(30):21296-21304.
Transforming growth factor-β (TGF-β) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-β rapidly induced β-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-β-increased β-catenin induction was reproduced by over-expression of SMAD3 and was absent in Smad3-/- chondrocytes treated with TGF-β. SMAD3 inhibited β-transducin repeat-containing protein-mediated degradation of β-catenin and immunoprecipitated with β-catenin following TGF-β treatment. Both SMAD3 and β-catenin co-localized to the nucleus after TGF-β treatment. Although both TGF-β and β-catenin stimulated cyclin D1 expression in chondrocytes, the effect of TGF-β was inhibited with β-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-β stimulates cyclin D1 expression at least in part through activation of β-catenin signaling.
doi:10.1074/jbc.M600514200
PMCID: PMC2649822  PMID: 16690606
8.  Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation 
Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-β signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3−/− chondrocytes. BMP signaling is increased in TGF-β deficiency and is required for accelerated chondrocyte maturation.
Introduction
Disruption of TGF-β signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.
Materials and Methods
Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3−/− mice. Expressions of maturational markers, as well as genes involved in TGF-β and BMP signaling were examined. Chondrocytes were treated with TGF-β and BMP-2, and effects on maturation-related genes and BMP/TGF-β responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.
Results
Expression of colX and other maturational markers was markedly increased in Smad3−/− chondrocytes. Smad3−/− chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-β 1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-β toward BMP signaling. In Smad3−/− chondrocytes, alternative TGF-β signaling pathways remained responsive, as shown by luciferase assays. These non–Smad3-dependent TGF-β pathways reduced colX expression and alkaline phosphatase activity in TGF-β–treated Smad3−/− cultures, but only partially. In contrast, Smad3−/− chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.
Conclusions
These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-β signaling with important implications for osteoarthritis and cartilage diseases.
doi:10.1359/JBMR.050911
PMCID: PMC2649698  PMID: 16355269
Smad3; TGF-β; BMP; chondrocyte maturation
9.  TGF-β SIGNALING IN CHONDROCYTES 
Transforming growth factor-beta (TGF-β) regulates a large variety of cellular activities. Binding of TGF-β to its cell surface receptor triggers several signaling cascades, among which the TGF-β-Smad pathway is the most extensively studied. TGF-β also activates protein kinases, including MAPK, PKA and PKC, and modulates gene expression via its delicate interaction with other signaling pathways. During endochondral bone formation, TGF-β acts as a potent inhibitor of the terminal differentiation of epiphyseal growth plate chondrocytes. This effect appears to be primarily mediated by Smad molecules, although MAPK-ATF2 signaling is also involved. The rate of chondrocyte maturation is tightly regulated through the interactions of Smad-mediated signaling, the Wnt signaling pathway, and the transcription factor Runx2. Improving our understanding of the exact mechanisms underlying TGF-β-mediated signaling pathways and their effects may greatly impact the diagnosis and treatment of many common orthopaedic diseases.
PMCID: PMC2647990  PMID: 15569609
TGF-β; Receptor; Smad; Chondrocyte; Maturation; Review; Gene
10.  Inhibition of β-catenin signaling causes defects in postnatal cartilage development 
Journal of cell science  2008;121(Pt 9):1455-1465.
Summary
The Wnt/β-catenin signaling pathway is essential for normal skeletal development because conditional gain or loss of function of β-catenin in cartilage results in embryonic or early postnatal death. To address the role of β-catenin in postnatal skeletal growth and development, Col2a1-ICAT transgenic mice were generated. Mice were viable and had normal size at birth, but became progressively runted. Transgene expression was limited to the chondrocytes in the growth plate and articular cartilages and was associated with decreased β-catenin signaling. Col2a1-ICAT transgenic mice showed reduced chondrocyte proliferation and differentiation, and an increase in chondrocyte apoptosis, leading to decreased widths of the proliferating and hypertrophic zones, delayed formation of the secondary ossification center, and reduced skeletal growth. Isolated primary Col2a1-ICAT transgenic chondrocytes showed reduced expression of chondrocyte genes associated with maturation, and demonstrated that VEGF gene expression requires cooperative interactions between BMP2 and β-catenin signaling. Altogether the findings confirm a crucial role for Wnt/β-catenin in postnatal growth.
doi:10.1242/jcs.020362
PMCID: PMC2636704  PMID: 18397998
Chondrocyte; Endochondral bone formation; Inhibitor of β-catenin and TCF (ICAT); Vascular endothelial growth factor (VEGF); β-catenin
11.  Fibroblast biology: Signals targeting the synovial fibroblast in arthritis 
Arthritis Research  2000;2(5):348-355.
Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.
doi:10.1186/ar111
PMCID: PMC130135  PMID: 11094447
fibroblast; rheumatoid arthritis; synovial membrane
12.  Chondrocyte-Specific Inhibition of β-Catenin Signaling Leads to Dysplasia of the Caudal Vertebrae in Mice 
Spine  2013;38(24):2079-2084.
Inhibition of β-catenin signaling with overexpression of ICAT driven by the Col2a1 romoter resulted in dysplastic caudal vertebrae and angulated deformities in mouse tails. The poorly developed sclerotomes failed to resegment and to form complete caudal vertebrae.
Study Design.
To inhibit β-catenin specifically signaling in chondrocytes Col2-ICAT transgenic mice were generated. Anomalies in caudal vertebrae were detected during embryonic and postnatal stages of Col2-ICAT transgenic mice.
Objective.
To determine the role of canonical β-catenin signaling in caudal vertebral development.
Summary of Background Data.
β-catenin signaling plays a critical role in skeletal development. Col2-ICAT transgenic mice were generated to selectively block β-catenin signaling by overexpression of the ICAT gene in chondrocytes.
Methods.
Tails of E16.5 transgenic embryos and adult Col2-ICAT transgenic mice and their wild-type littermates were collected and analyzed. Skeletal preparation, 3-dimensional micro-computed tomographic and histological analyses were performed to evaluate changes in the structure of caudal vertebrae. Bromodeoxyuridine labeling was performed to evaluate changes in chondrocyte proliferation in caudal vertebrae.
Results.
Skeletal preparation and 3-dimensional micro-computed tomographic analyses revealed bone deformation and angulated deformities in tail tissue in Col2-ICAT transgenic mice. Histological studies revealed abnormal bone development and dysplastic caudal vertebrae in Col2-ICAT transgenic mice. Inhibition of β-catenin signaling in cartilage resulted in vertebral dysplasia leading to aberrant resegmenting process. Thus, 2 poorly developed sclerotomes failed to fuse to form a complete vertebrae. BrdU labeling revealed a decreased chondrocyte proliferation in both cartilageous templates of transgenic embryos and the growth plate of adult Col2-ICAT transgenic mice.
Conclusion.
Wnt/β-catenin signaling plays an important role in vertebral development. Inhibition of β-catenin signaling in chondrocytes results in caudal vertebra deformity in mice, which may occur as early as in the stage of sclerotome formation.
Level of Evidence: N/A
doi:10.1097/01.brs.0000435024.57940.8d
PMCID: PMC3928445  PMID: 24026150
β-catenin; ICAT; chondrocyte; caudal vertebral dysplasia

Results 1-12 (12)