As a component of both the adherens junctions and desmosomes, plakoglobin plays a pivotal role in the regulation of cell-cell adhesion. Furthermore, similar to β-catenin, plakoglobin is capable of participating in cell signaling. However, unlike β-catenin that has well-documented oncogenic potential through its involvement in the Wnt signaling pathway, plakoglobin generally acts as a tumor/metastasis suppressor. The exact roles that plakoglobin plays during tumorigenesis and metastasis are not clear; however, recent evidence suggests that it may regulate gene expression, cell proliferation, apoptosis, invasion, and migration. In this paper, we describe plakoglobin, its discovery and characterization, its role in regulating cell-cell adhesion, and its signaling capabilities in regulation of tumorigenesis and metastasis.Plakoglobin ( These catenin proteins have two major roles in the cell: the mediation of cell-cell adhesion and cell signaling. As adhesive proteins, both β-catenin and plakoglobin interact with the cytoplasmic domain of cadherins, thereby tethering the cadherin proteins to the cytoskeleton. In addition to their cell-cell adhesive functions, both β-catenin and plakoglobin interact with a number of intracellular partners including signaling proteins and transcription factors, which accounts for their involvement in cellular signaling [β-catenin and plakoglobin emerges when considering their signaling functions. While β-catenin has a well-defined oncogenic potential as the terminal component of the Wnt signaling pathway [Plakoglobin  pool of plakoglobin was serine phosphorylated, the soluble pool was serine, threonine, and tyrosine phosphorylated, suggesting that these different pools of plakoglobin are differentially regulated and perform varying functions . Collectβ-catenin or plakoglobin, which then interacts with α-catenin, which is an actin-binding protein. A fourth catenin protein, p120-catenin, interacts with the juxtamembrane domain of E-cadherin and is important for E-cadherin dimerization and stability at the membrane  were unable to form desmosomes upon exogenous expression of E- or P-cadherin, despite the formation of the adherens junction in these cells . InteresFollowing this study, our laboratory demonstrated the role of plakoglobin in adhesive junction formation by expressing low/physiological levels of plakoglobin in SCC9 cells, a squamous cell carcinoma cell line that lacks the expression of both plakoglobin and E-cadherin . Followiβ-catenin levels, Wnt-1 can modulate plakoglobin levels and localization [Xenopus embryos, resulting in dorsalized gastrulation and anterior axis duplication. In this study, the exogenously expressed plakoglobin localized both at the plasma membrane and in punctate nuclear aggregates. Furthermore, the coinjection of mRNAs-encoding plakoglobin as well as the cytoplasmic domain of desmoglein suppressed both dorsalized gastrulation and anterior axis duplication. In these embryos, plakoglobin was localized primarily to the plasma membrane with some perinuclear distribution. These results suggested that plakoglobin has signaling ability similar to β-catenin, but when it is sequestered at the plasma membrane (as part of desmosomes), plakoglobin is unable to participate in cell signaling.The first clue that plakoglobin might participate in cell signaling came from studies of the exogenous expression of Wnt-1 in PC12 cells. In these cells, plakoglobin levels were increased, and it underwent membrane redistribution, suggesting that, in addition to lization . Subsequlization  demonstrβ-catenin and the Drosophila Armadillo protein. However, subsequent studies from various groups have demonstrated that while plakoglobin does indeed have signaling capabilities, it appears to function as a tumor suppressor rather than a tumor promoter. The first demonstration of this phenomenon occurred when Simcha et al. [α-catenin, β-catenin, plakoglobin, and desmoplakin and induces tumor formation in mice. Plakoglobin expression in KTCTL 60 cells also inhibited the tumorigenicity of these cells in syngeneic mice. Notably, the authors showed that the majority of the plakoglobin in these cells was Triton X-100 soluble, suggesting that it was not junction associated. This result was of significance because it demonstrated that plakoglobin could suppress tumor formation independent of its role in mediating cell-cell adhesion.This initial finding suggested that plakoglobin may have signaling functions similar to its homologs a et al.  found thβ-catenin, plakoglobin may have oncogenic activity. In the following sections, we will present the experimental evidence for both the tumorigenic and tumor suppressive activities of plakoglobin and propose possible explanations for these observed discrepancies.These studies made it clear that plakoglobin was capable of cell signaling and able to act as a tumor suppressor. Numerous subsequent studies have described the signaling function of plakoglobin as primarily one of tumor suppression, although a few reports have suggested that similar to β-catenin, could also regulate plakoglobin protein levels. In this study, the authors also showed that exogenous expression of plakoglobin in rat RK3E cells, which express considerable amounts of endogenous plakoglobin and β-catenin [β-catenin protein that cannot be degraded [Kolligs et al.  have sho-catenin , 58, res-catenin  have shodegraded , resulteβ-catenin [β-catenin interact with some of the same proteins and display high sequence homology , which would result in increased levels of cytoplasmic and nuclear β-catenin and in turn enhanced signaling. Indeed, following the observation that plakoglobin expression resulted in Xenopus axis duplication [β-catenin from, and the increased association of plakoglobin with, the N-cadherin-containing adherens junctions. Furthermore, excess cytoplasmic β-catenin was able to translocate into the nucleus. This was supported by other work, which showed that overexpression of plakoglobin in NIH3T3 cells resulted in the nuclear accumulation of β-catenin and that overexpression of the Wnt coactivator Lef-1 in MDCK cells resulted in its preferential interaction with β-catenin (instead of plakoglobin). Subsequently, the β-catenin-Lef complexes were localized to the nucleus [β-catenin were present within the cell, β-catenin-Lef complexes were more readily formed and transcriptionally active. Further examination of the ability of plakoglobin to signal via interactions with the Tcf/Lef family of transcription factors showed that although plakoglobin interacted with Lef-1, this complex was inefficient in binding to DNA, whereas β-catenin-Lef-1 complexes more readily bound DNA [β-catenin-Lef-1 complex formation and its association with DNA. Further analysis of the transactivation potential of β-catenin and plakoglobin demonstrated that β-catenin was a much stronger activator of Tcf/Lef target genes than plakoglobin [Both of these studies concluded that plakoglobin possessed oncogenic activity. However, it must be noted that several lines of evidence suggest that the oncogenic activity of plakoglobin may be indirect and achieved through modulation of the protein levels and signaling ability of -catenin –67. Sinclication , the samlication . This delication  showed t nucleus , suggestound DNA . This stkoglobin .β-catenin-N-cadherin interactions were decreased, and β-catenin accumulated in the nucleus, interacted with Tcf, and its signaling was increased [β-catenin.As mentioned earlier, we have previously shown that the expression of low/physiological levels of plakoglobin in plakoglobin-deficient SCC9 cells induced a mesenchymal to epidermoid change in phenotype, whereas its overexpression resulted in foci formation and decreased apoptosis, which was concurrent with the upregulation of the prosurvival protein Bcl-2 . Using cncreased , confirmβ-catenin. In Kolligs's study [β-catenin and plakoglobin [β-catenin or whether either of these catenins was detected in the nucleus in association with the c-Myc promoter. In addition, in Pan's study [β-catenin was a mutant protein that was unable to be phosphorylated and subsequently degraded [β-catenin localized to the membrane in these cells [β-catenin-cadherin interactions and increased β-catenin signaling. In support of this prediction, HCT116 cells overexpressing plakoglobin showed increased expression of the oncogenes securin and c-Myc and decreased expression of E-cadherin, all of which are documented β-catenin target genes [β-catenin signaling rather than oncogenic activity due directly to plakoglobin.The above studies describing the oncogenic potential of plakoglobin may also be as a result of 's study  where plkoglobin ), it was's study  in whichdegraded . While mse cells , plakoglet genes –81. Takeβ-catenin rather than plakoglobin itself, this cannot account for all of the observations regarding plakoglobin signaling. Recent studies attempting to discern the signaling activity of plakoglobin independent of β-catenin have used tissue culture cell lines that lack the endogenous expression of β-catenin [β-catenin, plakoglobin does indeed have Tcf/Lef-mediated transcriptional activity, although this activity is less than that of β-catenin-Tcf complexes. Interestingly, although these studies have demonstrated that plakoglobin can signal through forming transcriptional complexes with Tcf/Lef transcription factors, they did not assess the tumor-forming properties of these cells, so it remains unclear as to whether these cells possessed transformed or nontransformed properties. To that end, it has been demonstrated that plakoglobin or β-catenin expression in renal carcinomas lacking endogenous β-catenin and plakoglobin resulted in the upregulation of Nr-CAM, a neuronal cell adhesion molecule that can be regulated by both β-catenin and plakoglobin [β-catenin-target genes in the absence of β-catenin, it still may suppress tumorigenesis in the same cells. The homology between plakoglobin and β-catenin explains the ability of plakoglobin to signal through Tcf/Lef in the absence of β-catenin. Taken together, indeed, it is not surprising that if β-catenin is completely absent from a cell line, plakoglobin cannot only replace it in junctional complexes, but may also be able to regulate some β-catenin target genes  cell lines and lung primary tumors, plakoglobin expression was very low or completely absent [β-catenin-Tcf signaling, which was concurrent with decreased cell and anchorage-independent growth. This result further supported the idea that plakoglobin can act as a tumor suppressor by inhibiting the oncogenic activity of β-catenin.Further evidence suggesting a growth suppressive activity for plakoglobin was provided in lung cancer, when it was shown that while y absent . The autInterestingly, when the authors treated these NSCLC cell lines with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (AZA) or the histone deacetylase inhibitor trichostatin A (TSA), plakoglobin levels were increased. Previous analysis of the plakoglobin promoter had described CpG islands within the promoter , and whiThe occurrence of methylated CpG islands within the plakoglobin promoter as well as histone deacetylation has not been limited to NSCLC cell lines. Various groups have shown that the plakoglobin promoter is methylated in prostate, bladder, trophoblastic, and mammary carcinomas –92, whicL were higher in the plakoglobin null keratinocytes, which could potentially have prevented the translocation of cytochrome c from the mitochondria. Finally, the expression of plakoglobin in the null keratinocytes resulted in decreased Bcl-XL levels, caspase 3 activation, and apoptosis induction following etoposide treatment. Taken together, these studies have demonstrated that plakoglobin does have some role in apoptosis signaling and potentially may exert part of its tumor suppressor activity through the modulation of apoptosis.Several lines of evidence suggest that plakoglobin plays a role in regulating apoptosis, in addition to acting as a growth suppressor. In their work describing the effects of plakoglobin on hair growth in transgenic mice, Charpentier et al.  showed tAs the tumor suppressor activity of plakoglobin began to be revealed, it soon became evident that in addition to inhibiting the growth properties of carcinoma cell lines, plakoglobin also plays a role in regulating the invasive and migratory properties of cancer cells. The initial observation of plakoglobin metastasis suppressor activity was documented in human umbilical vascular endothelial cells (HUVEC), where plakoglobin was typically associated with sites of cell-cell contact . PlakoglMukhina et al.  further β-catenin signaling in these bladder carcinomas, which suggested that plakoglobin possessed tumor and metastasis suppressor activities independent of β-catenin.The metastasis suppressor activity of plakoglobin has also been described in bladder carcinomas, where the expression of plakoglobin in plakoglobin null cell lines resulted not only in decreased growth and tumorigenicity , but also in decreased invasive and migratory capabilities of the transfectants . Similarα-catenin binding and transactivation domain, resp.). The expression of either of these mutant proteins resulted in increased keratinocyte adhesiveness when compared to the plakoglobin null cells, demonstrating that these domains were dispensable for the adhesive function of plakoglobin. Importantly, the authors showed that whereas individual keratinocytes expressing the N-terminal-deleted plakoglobin were not migratory, those that expressed the C-terminal-deleted plakoglobin were migratory. This showed that plakoglobin could indeed suppress migration independent of its adhesive function . Subsequent work using these plakoglobin null keratinocytes has suggested that plakoglobin affected individual cell motility by regulating the deposition of the extracellular matrix (ECM) protein fibronectin, actin cytoskeleton organization , and RhoGTPases [The ability of plakoglobin to act as a metastasis suppressor independent of its role in cell-cell adhesion has been demonstrated using plakoglobin null keratinocytes , which woGTPases . CollectWhen discussing roles for plakoglobin during tumorigenesis and metastasis, it is important to consider that while plakoglobin may function as both a regulator of cell-cell adhesion and an intracellular signaling molecule, it may also play a more active role in these processes through the regulation of gene expression. Evidence supporting the plakoglobin-mediated regulation of gene expression has started to emerge, and work from several groups, including ours, has suggested that plakoglobin can regulate the expression of genes involved in cell-cycle control, apoptosis, cell proliferation, and invasion.β-catenin null keratinocytes, demonstrating that plakoglobin could regulate gene expression independent of β-catenin. Finally, using chromatin immunoprecipitation with plakoglobin antibodies, the authors demonstrated that plakoglobin and Lef-1 associated with the c-Myc promoter in keratinocytes undergoing growth arrest, which implicated the downregulation of c-Myc gene expression as a possible reason for the suppression of cell growth by plakoglobin.Williamson et al.  have shoβ-catenin and plakoglobin resulted in the increased expression of the tumor suppressor gene PML, a nuclear protein that forms nuclear bodies and is involved in the regulation of p53 activity. Importantly, the increased PML levels due to plakoglobin expression were independent of β-catenin and Tcf, since β-catenin was not detected in the plakoglobin-expressing cells and the deletion of Tcf/Lef sites in the PML promoter did not affect the ability of plakoglobin to increase PML gene expression. Together, these observations suggest that plakoglobin may regulate gene expression independent of Tcf/Lef.Plakoglobin-mediated regulation of gene expression has also been shown in renal carcinoma cells. Shtutman et al.  found thβ-catenin null mesothelioma and colon carcinoma cells, Wnt3a stimulation led to the nuclear accumulation of plakoglobin and induced the expression of the antiapoptotic gene Survivin [β-catenin target genes in a β-catenin null background, it is again of importance to emphasize that β-catenin null tumors are very rare and that the plakoglobin-mediated regulation of gene expression occurs mainly in the presence of cellular β-catenin.In Survivin . CoimmunSurvivin . While tAs previously discussed, Todorović et al.  have shoJUP, the gene encoding plakoglobin, mapped the gene to chromosome 17q21, proximal to the BRCA1 gene [α-catenin, β-catenin, desmoglein, or desmoplakin resulted in increased tumor formation and size and was correlated with increased tumor stage, poor patient survival, and increased metastasis in bladder, pituitary, oral, pharyngeal, skin, prostate, and NSCLC tumors [The initial characterization of CA1 gene . In thisC tumors –111. HowThe loss of plakoglobin expression has been observed in melanocytic and thyroid tumors , 113. Ceβ-catenin accumulated in the nuclei of cells of primary and metastatic adenocarcinoma and adenoma lesions, while the levels of nuclear plakoglobin were decreased in these tumors, suggesting that nuclear plakoglobin did not promote tumorigenesis in the colon. In esophageal cancers, while decreased levels of E-cadherin and plakoglobin were associated with poor differentiation and decreased patient survival, reduced plakoglobin levels alone correlated with lymph node metastasis [In colon carcinomas, Lifschitz-Mercer et al.  showed ttastasis . The fintastasis . Aberrantastasis , 119. Intastasis , whereastastasis . Finallytastasis , 123. Coin vitro migration and invasiveness. These analyses led to the identification of several growth regulatory genes that were differentially expressed in plakoglobin-expressing transfectants compared to their plakoglobin-deficient parental cells.We have developed two experimental model systems using squamous and breast carcinoma cell lines with no or very low plakoglobin expression and various degrees of transformation/invasiveness to specifically assess the growth/metastasis inhibitory activities of plakoglobin. Using a combination of molecular and cell biological approaches, including proteomics and transcriptome analysis, we compared the protein and mRNA profiles of plakoglobin-deficient and plakoglobin-expressing cell lines and their Comparison of the proteomic profiles of plakoglobin null SCC9 cells and their plakoglobin-expressing transfectants allowed us to identify several tumor/metastasis regulating proteins, which were differentially expressed in plakoglobin-expressing transfectants (SCC9-PG-WT) relative to parental SCC9 cells. We performed RNA microarray experiments to determine whether changes in gene expression upon plakoglobin expression accompanied these changes in protein levels and compared the transcriptome profiles of SCC9 cells and SCC9-PG-WT transfectants. Furthermore, to determine whether the subcellular distribution of plakoglobin had an effect on gene expression, we also compared the RNA profiles of SCC9 and SCC9-PG-WT cells with those of SCC9 cells transfected either with cDNAs-encoding plakoglobin fused with a nuclear localization signal (NLS) to express plakoglobin exclusively in the nucleus (SCC9-PG-NLS), or cDNAs-encoding plakoglobin fused with a nuclear export signal (NES) to express plakoglobin exclusively in the cytoplasm (SCC9-PG-NES). From these experiments, we identified three subsets of genes that were differentially expressed based on plakoglobin expression and its subcellular distribution: those whose differential expression required exclusively cytoplasmic plakoglobin, those whose differential expression required nuclear plakoglobin, and those whose differential expression required the ability of plakoglobin to shuttle between the nucleus and the cytoplasm. Based on the results of these experiments and analysis of the expression patterns of plakoglobin-target genes in relation to plakoglobin subcellular distribution, we propose that plakoglobin can regulate gene expression by three concurrent mechanisms .The first of these mechanisms involves the action of plakoglobin in the cytoplasm, where it would sequester a protein involved in the regulation of gene expression. In this case, plakoglobin would prevent an inhibitor of a tumor suppressor gene or a promoter of an oncogenic gene from entering the nucleus and affecting gene expression. Plakoglobin target genes whose expression patterns were similar in SCC9-PG-WT and SCC9-PG-NES cells and were opposite to SCC9-PG-NLS cells would be considered part of this group.The second mechanism involves nuclear localized plakoglobin, which would directly associate with a nuclear factor and regulate gene expression. In this case, plakoglobin would interact with a transcriptional activator and promote gene expression, or, conversely, it would interact with a transcriptional repressor and silence gene expression. Plakoglobin target genes whose expression patterns were similar in SCC9-PG-WT and SCC9-PG-NLS cells and were opposite to SCC9-PG-NES cells would be considered part of this group.The vast majority of plakoglobin target genes, however, belonged to the third group of genes: those whose differential expression depended on the ability of plakoglobin to shuttle between the nucleus and the cytoplasm. In this case, plakoglobin would interact with some cytoplasmic cofactor, translocate into the nucleus, and regulate gene expression. Plakoglobin target genes whose expression patterns were similar in SCC9-PG-NES and SCC9-PG-NLS cells and were opposite to SCC9-PG-WT cells would be considered part of this group.β-catenin/Wnt signaling is not activated in SCC9 cells [β-catenin. We are currently characterizing whether plakoglobin directly regulates Nm23 expression. Furthermore, we have also shown that plakoglobin interacts with the transcription factor p53 and regulates the expression of a number of p53 target genes (manuscript in preparation).Following these proteomics and microarray analyses, we began our initial characterization of the regulation of potential target genes by plakoglobin. We have recently shown that plakoglobin expression in SCC9 cells resulted in the increased expression of the metastasis suppressors Nm23-H1 and -H2, both at the mRNA and protein levels . Nm23 waC9 cells , these rRecent work has demonstrated that plakoglobin has novel roles in intracellular signaling and the regulation of gene expression, in addition to its previously well-established roles in cell-cell adhesion. Plakoglobin has emerged as a tumor/metastasis suppressor protein based on evidence from the great majority of the studies that have examined its signaling function. As more work focuses on the role of plakoglobin in tumorigenesis and metastasis, it is becoming clear that plakoglobin is a key, important player in these processes and consequently may be a useful therapeutic target in the treatment of cancer.


Events normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.(TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.Low-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.LRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on Our findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis. Osteoarthritis (OA) is a progressively degenerative joint disorder characterized by extracellular matrix degradation, articular cartilage loss, and osteophyte formation . It is cOsteoarthritis involves mainly the dysfunction of articular chondrocytes, which leads to cartilage degradation through chondrocyte maturation and MMPs production ,4. In grc-myc and cyclin D1 [FRZB, which encode frizzled-related protein, a soluble antagonist of the Wnt canonic signaling pathway [frzd knockout mice are more susceptible to chemically induced OA [-catenin gene in articular chondrocytes in adult mice leads to premature chondrocytes differentiation and the development of an OA-like phenotype [The function of articular chondrocytes is regulated by different growth factors, including Wnt ligands and BMPs, which have been shown to play a critical role in chondrocyte proliferation, differentiation, and apoptosis -13. The yclin D1 . The rol pathway -17. Addiduced OA . Besidesduced OA . A recenduced OA . Recentlduced OA . Moreoveduced OA , and thehenotype . Increashenotype -26.et al. [et al. [in vitro chondrogenic differentiation of human mesenchymal stem cells. Moreover, in preosteoblastic cells, BMP-2 was found to increase nuclear β-catenin protein levels and induce the expression of different Wnts, suggesting the interaction between Wnt and BMP-2 signaling during osteoblastic differentiation [Besides the Wnt/β-catenin pathway, BMPs also play a significant role in chondrocyte differentiation. BMP signaling is initiated by BMPs binding to their receptor (BMPR-I and II), resulting in increased phosphorylation of downstream signaling molecules, including Smad1, Smad5, and Smad8 (R-Smads). The R-Smads form complexes with Smad-4 and translocate into the nucleus, where they bind to regulatory regions of target genes regulating their expression . Althouget al.  reported [et al.  showed tntiation .As activation of Wnt/β-catenin and BMP-2 signaling pathways may signify a causal relation to chondrocyte differentiation and matrix degradation, we sought to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in the catabolic action and hypertrophy of osteoarthritic chondrocytes.MMP-7, 9, 13, 14, ADAMTS-5, 4, and COL10A1 genes were obtained from CHIP Bioinformatics Tools [LRP-5 promoter was tested for Smad binding sites .Promoter sequences of cs Tools . For eaca priori approval by the Local Ethical Committee of the University Hospital of Larissa.Articular cartilage samples were obtained from femoral condyles and tibial plateaus of patients with primary osteoarthritis undergoing knee-replacement surgery at the Orthopaedics Department of University Hospital of Larissa. In total, 18 patients were included in this study . All osteoarthritic specimens had Mankin scores of 10 to 14. Radiographs were obtained before surgery and graded according to the Kellgren-Lawrence system. All patients had K/L scores ≥ 2. The radiographs were assessed by two independent observers who were blinded to all data of the individuals. Normal articular cartilage was obtained from nine individuals  with 0 Mankin score, undergoing fracture-repair surgery, with no history of joint disease. Patients with rheumatoid arthritis and other autoimmune diseases, as well as chondrodysplasias, infection-induced OA, and posttraumatic OA, were excluded from the study. Written informed consent was obtained from all individuals in the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in οC under a humidified 5% CO2 atmosphere until reaching confluence for 4 to 6 days.Articular cartilage was dissected and subjected to sequential digestion with 1 mg/ml proteinase mixture  and 1 mg/ml collagenase P . Chondrocytes were counted and checked for viability by using trypan blue staining. More than 95% of the cells were viable after isolation. Isolated chondrocytes from individual specimens were separately cultured with Dulbecco Modified Eagle Medium/Ham F-12 (DMEM/F-12)  plus 5% fetal bovine serum  at 37-ΔΔCT method. Normalized gene-expression values for each gene based on cycle threshold (CT) values for each of the genes and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were generated.Total cellular RNA was extracted from cultured chondrocytes by using Trizol reagent . Preservation of 28S and 18S ribosomal RNA (rRNA) species was used to assess RNA integrity. All the samples included the study had prominent 28S and 18S rRNA components. The yield was quantified spectrophotometrically. Transcription of 0.1 μg RNA to cDNA was performed by using SuperScript III reverse transcriptase  and random primers . Quantification of COL2A1, COL10A1, aggrecan, MMP-13, LRP-5, LRP-6, BMP-2, BMP-4, BMP-7, BMPR-IA, BMPR-IB, LEF-1, and TCF-4 mRNA expression was performed with real-time PCR . Reactions were done in triplicate by using 2 μl of cDNA per reaction. All primers used are shown in Table Chondrocytes were lysed by using RIPA buffer and a cocktail of protease and phosphatase inhibitors. Protein concentration was quantified by using the Bio-Rad Bradford protein assay  with bovine serum albumin as standard. Cell lysates from normal and OA chondrocytes were electrophoresed and separated on a 4% to 20% Tris-HCl gel  and transferred to a Hybond-ECL nitrocellulose membrane . The membrane was probed with anti-LRP-5 , BMP-2 , BMP-4  BMPR-IA , LEF-1 antibody , and phospho-β-catenin , and signals were detected by using immunoglobulin (IgG) conjugated with horseradish peroxidase . The results were normalized by using anti-β-actin polyclonal antibody . The nitrocellulose membranes were then exposed to photographic film, which was scanned, and the intensities of the protein bands were determined with computerized densitometry.2O2 to block endogenous peroxidase. Nonspecific staining was blocked with TBS containing 2% bovine serum albumin  for 1 hour at room temperature, followed by incubation with primary antibody BMP-2  overnight at room temperature. Normal serum of the same species as the primary antibody was used as a control for the primary antibody. After extensive washing with TBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 hour at room temperature in a humid chamber. Finally, color reaction was performed by using the substrate reagent 3,3'-diaminobenzidine tetrahydrochloride . After being washed, the sections were incubated with DAB and coverslipped with mounting medium.Cartilage specimens were fixed in 10% formalin overnight and decalcified in 13% EDTA  for 3 weeks. Paraffin-embedded sections were deparaffinized in xylene, dehydrated through graded alcohols, and placed in 3% H5 cells/well, and 3 days after seeding, cells were treated with 50 ng/ml of BMP-2 or BMP-4 for 12, 24, and 48 hours; each experiment was conducted in triplicate wells. RNA was extracted, and real-time PCR analysis for osteocalcin, LRP-5, and LRP-6 was performed, as described earlier. All primers used are shown in Table Normal and osteoarthritic chondrocytes were seeded on six-well plates at 3 × 105 cells/well, and 3 days after seeding, cells were treated with 20 mM LiCl  for 12, 24, and 48 hours; each experiment was conducted in triplicate wells. RNA was extracted, and real-time PCR analysis for MMP-7, 9, 13, 14, ADAMTS-4, 5, and collagen X was performed as described earlier. All primers used are shown in Table Normal and osteoarthritic chondrocytes were seeded on six-well plates at 3 × 10ChIP was performed by using a ChIP assay kit  on BMP-2-treated, LiCl-treated, and untreated normal chondrocytes, as previously described . The celM LiCl (cultures with siRNA against LEF-1) for 48 hours. In each experiment, the results from three of the six-well plates were averaged and considered as n = 1. No significant variance was observed among the individual wells in each averaged group. RNA was extracted, and real-time PCR analysis for MMP-7, 9, 13, 14, ADAMTS-4, 5, and collagen X was performed, as described earlier. Moreover, cell lysates were extracted, and phospho- as well as total β-catenin protein levels were evaluated in BMP-2-treated chondrocytes after LRP-5 siRNA transfection by using Western blot analysis, as described earlier.Normal and osteoarthritic chondrocytes were seeded in six-well plates in DMEM/F-12 containing 5% FBS. After overnight incubation, cells were transfected with specific siRNA against LRP-5 , LEF-1 , and scrambled siRNA  by using LipofectAMINE 2000 , as described by the manufacturer. No cell toxicity was detected from the transfection agent. Four hours after siRNA transfection, the medium was changed, and cells were treated with 50 ng/ml BMP-2 (cultures with siRNA against LRP-5) or 20 mt test with a confidence level of 95% (P < 0.05).Statistical significance was determined by using the Student P < 0.05; Figure P < 0.05; Figure To evaluate differences in chondrocyte differentiation-related genes, we evaluated COL2A1, COL10A1, aggrecan, and MMP-13 mRNA expression levels in normal and osteoarthritic chondrocytes. We found that COL2A1 and aggrecan were significantly upregulated in normal chondrocytes , ADAMTSs (ADAMTS-4 and 5), and collagen X expression levels. siRNA against LRP-5 effectively inhibited LRP-5 expression, as we observed a downregulation of LRP-5 mRNA and protein expression in si-RNA-transfected chondrocytes compared with the untransfected and the scrambled siRNA-transfected chondrocytes 24 hours after siRNA transfection  and hypertrophic markers (collagen X). We found that normal and osteoarthritic chondrocyte treatment with LiCl reduced phospho-β-catenin levels , whereas MMP-14, ADAMTS-5, and collagen X expression was significantly upregulated in normal chondrocytes (P < 0.05) and showed a trend to increase in osteoarthritic chondrocytes , whereas ADAMTS-5 and collagen X mRNA expression was significantly downregulated in LiCl-treated osteoarthritic chondrocytes (P < 0.05) and showed a trend to decrease in LiCl-treated normal chondrocytes , MMP-13 (from -1142 to -1138), MMP-14 (from -575 to -571), ADAMTS-5 (from -755 to -751), and COL10A1 (from -784 to -781) promoters, and the binding was stronger in treated-LiCl chondrocytes, as LiCl stabilizes β-catenin active form and increases nuclear β-catenin protein levels, enhancing thus the complex between β-catenin and LEF-1 and the subsequent binding on gene promoters, suggesting that these genes are Wnt targets in adult articular cartilage (Figure MMP-7 and ADAMTS-4 promoter in the region from 1,500 base pairs upstream of the transcript start to 100 base pairs downstream of the coding-sequence start (Figure AXIN-2 promoter served as positive control.To obtain direct evidence that the Wnt/β-catenin signaling pathway can trigger extracellular matrix degradation and hypertrophic chondrocyte differentiation in osteoarthritis, we activated the pathway s Figure . siRNA as Figure . Howevers Figure . To invee Figure . No LEF-t Figure . LEF-1 bArticular chondrocyte proliferation, expression of hypertrophy markers, and remodeling of the cartilage matrix by proteases are among the main characteristics of osteoarthritis . Recent In the present study, we provide for the first time, to our knowledge, evidence for a cross-talk between BMP-2 and Wnt/β-catenin signaling pathways in osteoarthritic chondrocytes. Although BMPs are involved in all phases of chondrogenesis affecting chondrocyte differentiation and cartilage anabolism -30, receIt has been suggested that BMP-2 modulates β-catenin signaling through stimulation of Wnts, LRPs, and Frizzled receptors expression in mesenchymal cells and osteoblasts ,40. To iLRP-5 promoter activity in chondrocytes. Chromatin immunoprecipitation indicated that BMP-2 directly modulates LRP-5 expression, as we found, for the first time, that Smads complexes bind to SBEs on the LRP-5 promoter, suggesting the BMP-2 induced-Wnt/β-catenin signaling-pathway activation through direct modulation of LRP-5 expression. Previous studies have shown that osteoblast differentiation and new-bone formation require the interaction of BMP-2 and the Wnt/β-catenin signaling pathway [To investigate further the molecular mechanism involved in the BMP-2-induced LRP-5 upregulation, we examined the effect of BMP-2 through Smads binding on  pathway ,42. BMP-The Wnt/β-catenin signaling pathway participates in normal adult bone and cartilage biology and seems to be involved in cartilage degeneration and subsequent OA progression ,44. To dMMP-9, 13, 14, ADAMTS-5, and COL10A1 promoters, responsible for their promoter activity and is associated directly with the β-catenin/LEF-1 complex. Moreover, LEF-1 downregulation using siRNA reduced MMPs, ADAMTS-5, and collagen X mRNA expression, whose levels increased after treatment with LiCl, providing strong evidence of gene-expression regulation of catabolic factors by LEF-1. No upregulation was observed in MMP-7 and ADAMTS-4 levels, as no conserved LEF-binding sites were found on their promoters. It has been shown that Lef-1 binding to the 3' region of mmp-13 is involved in the transcriptional regulation of the mmp-13 gene in mouse chondrocytes [ex vivo and decreased the severity of osteoarthritis [To test for a possible association between the LEF-1 transcription factor of the Wnt/β-catenin signaling pathway and catabolic action, as well as hypertrophy in osteoarthritic chondrocytes, we activated Wnt/β-catenin signaling by using LiCl in cultured chondrocytes. LiCl is often used to mimic canonic Wnt signaling, as it inhibits GSK-3β, therefore stimulating downstream components of the Wnt-signaling pathway in an LRP-5-independent manner . The actdrocytes . It can drocytes ,48, wherdrocytes . In addirthritis . We alsoIn conclusion, we demonstrated, for the first time to our knowledge, that the BMP-2-induced Wnt/β-catenin signaling pathway activation through LRP-5 induces chondrocyte catabolic action and hypertrophy, providing thus novel and direct evidence on the role of BMP-2 mediated by Wnt/β-catenin signaling in osteoarthritis progression.BMPs: bone morphogenetic proteins; BMPR-IA: bone morphogenetic protein receptor-IA; BSA: bovine serum albumin; ChΙP: chromatin immunoprecipitation; COL10A1: collagen X; Dkk-1: Dickkopf-related protein 1; DMEM/F-12: Dulbecco Modified Eagles Medium/Ham F-12; FBS: fetal bovine serum; FRZB: Frizzled-related protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LEF-1: lymphoid enhancer factor-1; LRP-5: low-density-lipoprotein receptor-related protein 5; MMPs: matrix metalloproteinases; OA: osteoarthritis; SBEs: Smad-binding elements.The authors declare that they have no competing interests.IP performed the experiments and drafted the manuscript, KNM provided cartilage samples, clinical evaluation of patients, and helped in data interpretation, and AT conducted data analysis, results interpretation, and finalized the manuscript. All authors read and approved the final manuscript.


B. mallei as a biological weapon and its high mortality in inhalation animal studies has affirmed B. mallei as a biodefense concern. This threat requires the development of new glanders medical countermeasures (MCMs), as there is a lack of an effective vaccine and lengthy courses of multiple antibiotics needed to eradicate B. mallei. Here, we present a literature review of human glanders in which we discuss the clinical epidemiology and risk factors, potential routes of exposure, symptoms, the incubation period, and specific diagnostics. This review focuses on pulmonary glanders, as this is the most likely outcome of a biological weapons attack. Additionally, we outline current treatment regimens and propose a clinical definition of human pulmonary glanders infection.Glanders is a highly contagious and often fatal zoonotic disease, primarily of solipds. In the developed world, glanders has been eradicated. However, prior use of  Burkholderia mallei. Donkeys are prone to develop acute forms of glanders while horses are more likely to develop chronic and latent diseases. Mules are susceptible to acute and chronic forms of the disease as well as latent infections[B. mallei, the etiological agent of glanders, is a Gram-negative, non-motile, facultative intracellular pathogen. At one time, B. mallei infections occurred worldwide, but over the last 100 years the occurrence of glanders has decreased with the reduced economic reliance on solipeds as the primary mode of transportation, the implementation of testing all solipeds for glanders, and euthanizing those that are confirmed positive. The last naturally occurring human case in the United States was reported in 1934[B. mallei as a biological weapon have been documented[Glanders is a highly contagious and often fatal zoonotic disease primarily of solipeds such as horses, mules, and donkeys. It was first described by the Greeks in 450–425 BC and again by the Romans in 400–500 AD. Throughout history glanders has been known by other names including equinia, malleus, droes, and farcy-5. Glandfections-8. B. ma in 1934. However in 1934-14. Althcumented,16.B. mallei being used as a weapon is very real. In fact, B. mallei was one of the first biological warfare agents used in the 20th century, specifically during World War I[B. mallei against opposition forces in Afghanistan between 1982 and 1984[B. mallei, it did not weaponize B. mallei. However, during this work, seven individuals working in the US labs became infected. B. mallei has been considered a potential threat since the 1940’s because of its high infectivity, degree of incapacitation, and agent availability[Burkholderia workshop in order to develop consensus recommendations for post-exposure prophylaxis against and treatment for B. mallei[Although glanders has been eradicated from many parts of the world, the threat of ld War I. These mlability. As furtB. mallei. GlanderB. mallei from solipeds to humans appears to be uncommon, even in cases of frequent and close contact with infected animals. However, rigorous examination regarding the rarity of transmission has not been conducted. Hypotheses to explain this low incidence include low organism concentrations at the infection sites or a high infectious dose required to cause disease in humans[Zoonotic transmission of n humans. Despiten humans. Subclinn humans-20.Human-to-human transmission is also rare. However, it may occur during occupational exposure in medical practice or at autopsies,9,19,21.B. mallei cultures. In contrast to zoonotic transmission, aerosols of B. mallei are highly infectious[While laboratory workers have rarely been infected, their close contact with high concentrations of virulent organism puts them at a higher risk for infection. Eight cases of laboratory acquired glanders have been reported between 1943 and the present at US research centers, specifically Fort Detrick, Maryland. Seven of the eight Fort Detrick laboratory-acquired infections occurred prior to modern biological containment practices. The firfectious. Unfortufectious.B. mallei is not believed to penetrate normal intact skin, although wounds or penetrations during the likely exposure interval were not identified in many cases[Glanders is transmitted by direct invasion of abraded or lacerated skin; inhalation with deep lung deposition; and by bacterial invasion of the nasal, oral, and conjunctival mucous membranes. The occupational exposures described above most often occur through exposed skin, particularly the hands, arms, neck, and face. B. mallny cases. Indeed,ny cases. In the ny cases. In lighB. mallei , including two notable cases[B. mallei was dropped and broken thus potentially creating aerosol droplets. Both patients were admitted to the hospital on the same day, approximately two weeks after the incident and were the only personnel present in the lab when the flask was dropped. The other four patients (patients three to six) were actively engaged in washing B. mallei growth from agar plates for the preparation of vaccines approximately 10–14 days prior to the onset of their symptoms. While all workers wore the required protective clothing at the time and used caution while performing their duties, the possibility that they inhaled an aerosol could not be eliminated. Patient four also failed to heat kill the B. mallei sample they were working with prior to making dilutions for turbidity measurements two weeks before symptom onset. Approximately two weeks before their admission to the hospital, patients five and six had engaged in procedures involving the aeration of cultures. They recalled that on at least two occasions, the containers had been opened immediately after the air current had been turned off, rather than after a period of delay long enough to avoid the escape of aerosolized organisms into the room. Additionally, these two patients were working with a strain of organism of higher virulence than the other four. These events strongly suggest aerosol route of infection by B. mallei can result in human glanders cases, especially in the laboratory setting[Six of the eight cases above were described in Howe and Miller’s report which detailed glanders cases occurring within one year among Fort Detrick laboratory personnel working with le cases. Two wor setting.The acute form of disease has a typical incubation period of 1–14 days, while tin vitro diagnostic test for glanders, experimental serological tests, such as agglutination and complement fixation tests[Burkholderia antigens to coat sheep red blood cells prior to their incubation with serum from suspected melioidosis (or glanders) patients. In the resulting reaction the antigen-coated sheep RBCs agglutinate or form a pellet if the patient is seropositive for B. pseudomallei or B. mallei [Burkholderia species, the IHA assay is not a specific diagnostic test and other glanders-specific diagnostic tests are needed[The definitive diagnosis of glanders requires isolation of the organism and positive identification. Radiology may reveal abcesses in multiple organs including lungs, liver, and spleen, but these abcesses are indistinguishable from those caused by other disease and specific diagnosis is required. While ton tests-29, and on tests have beeon tests. Brieflythogens),32. Since needed,33. The e needed. HoweverMany forms of glanders have been described, including chronic, disseminated, pulmonary, and septicemic. The variety of infections is largely explained by various routes of infection. Localized infections are generally regionally confined and typically characterized by foci of suppuration. The abscesses can ulcerate and drain for long periods of time. However, localized infections may disseminate, leading to pulmonary, septicemic, or multi-tissue infection. The mosB. mallei infection presents with excessive lacrimation and photophobia. Nasal involvement is characterized by inflammation and swelling of the nose, which is common following inhalation of B. mallei. This may be followed by copious nasal discharge. Additionally, infection may invade the nasal septum and bony tissues, causing fistulae and tissue destruction[Involvement of the eye and conjunctiva in a truction. The factruction.B. mallei entry through an abrasion is typically followed by an inflammatory response, including pain and swelling. In these cases, a glanders node may appear as a single blister, gradually developing into an ulcer that may become hemorrhagic[B. mallei strains affect the smooth muscle of the lymphatics[Cutaneous manifestations include papular lesions that may erupt anywhere on the body with a more chronic, indolent course of infection. orrhagic,35. A lomphatics by enhanA pulmonary infection typically results in pneumonia, pulmonary abscess, pleuritis, and plural effusion. Signs and symptoms of pulmonary infection can include cough, dyspnea, chest pain, and mucoplurent sputum. Nonspecific signs and symptoms such as fatigue, fever (often exceeding 102°F), chills, headache, myalgias, lymphangitis, sore throat, pleuritic chest pain, cough, tachypnea, dyspnea, discharge, and gastrointestinal signs often accompany respiratory infections. Many syDissemination from local cutaneous or mucosal infection result in septicemia and the colonization of internal organs such as the spleen, liver, and lungs with the development of abscesses. UltrasoB. mallei infections have responded to antibiotic therapy despite slow recovery times after delayed diagnosis or disseminated disease. B. mallei has been reported to be susceptible to many antibiotics in vitro[B. mallei is a facultative intracellular pathogen, aminoglycosides and other antibiotics that are incapable of penetrating host cells are most likely not useful for the treatment of infection[B. mallei strains exhibit resistance to several antibiotics  aerosol challenge[3 CFUs or 160 LD50s) of B. mallei delivered by aerosol[Antibiotic efficacy has been tested against glanders in solipeds, hamsters, guinea pigs, and monkeys,43,45-47hallenge,45. All The majority of human glanders cases occurred before antibiotic treatment was available, and over 90% of infected people succumbed to disease. Of the B. mallei or patient intolerance consist of doxycycline, macrolides, chloramphenicol, quinolones, or amoxicillin-clavulanate as noted in the Occupational Health Manual from the United States Army Medical Research Institute of Infectious Diseases (USAMRIID)[B. mallei and B. pseudomallei are similar, it is postulated that drug therapy experiences should be similar as well. Thus, doxycycline, amoxicillin-clavulante and quinolones monotherapies are at a greater risk of relapse than TMP-SMX and chloramphenicol.Given the lack of historical data, current treatment regimens for human glanders are not well understood at this point in time, and no FDA approved treatment or treatment regimen exists. An exhaustive review of current and experimental treatments for both glanders and melioidosis has been published, but this review also describes the lack of clinical information for glanders treatment. HoweverSAMRIID). Since BDosing regimen recommendations are shown in Table The mortality rate for the pulmonary form of glanders has been reported to be 90-95% without treatment and up to 40% with treatment. The reported mortality rate for the septicemic form of glanders is greater than 95% without treatment and as high as 50% with treatment. The mortality rate for the untreated cutaneous form of glanders is 90-95% if it becomes systemic and as high as 50% with treatment. In the case of chronic glanders, the mortality rate may be as high as 50% despite treatment. It shouThe following first two criteria and at least one of last two criteria are proposed for diagnostic confirmation of acute pulmonary glanders infection in humans.1. Development of constitutional symptoms such as fever, rigors, myalgias, fatigue, headache, severe malaise, and pleuritic chest pain.2. Chest X-Ray exhibiting new onset of infiltrates either segmental or lobar which may appear as nodular opacities. May also exhibit cavitary lesions or abscesses not only in the lungs but virtually any remote sites such as spleen, liver, subcutaneous tissue and muscle.B. mallei organism from sites such as cavitary lesions or abscesses not only in the lungs but virtually any remote site such as spleen, liver, subcutaneous tissue, and muscle as well as secretions, sputum, blood, and urine[3. Positive isolation of nd urine.Burkholderia species non-specific IHA titer and if positive a follow-up four-fold titer increase[4. Positive increase,54.MCMs: Medical countermeasures; IHA: Indirect hemagglutination assay; IV: Intravenous; TMP-SMX: Trimethoprim-sulfamethoxazole.The authors declare that they have no competing interest.KVZ conducted the literature review and helped draft the manuscript. MTG developed the clinical definition of glanders and helped draft the manuscript. HCG helped draft the manuscript. All authors read and approved the final manuscript.


Antechinus flavipes) in 60 000 ha of extant river red gum  forests in south-eastern Australia in 2004, 2005 and 2011. Stand condition was assessed remotely using models built from ground assessments of stand condition and satellite-derived reflectance. Other covariates, such as volumes of fallen timber, distances to floods, rainfall and life stages were included in the model. Trapping of animals was conducted at 272 plots (0.25 ha) across the region. Densities of second-year females  and of second-year females with suckled teats (i.e. inferred to have been successful mothers) were higher in stands with the highest condition. There was no evidence of a relationship with stand condition for males or all females. These outcomes show that remotely-sensed estimates of stand condition (here floodplain forests) are relatable to some demographic characteristics of a small mammal species, and may provide useful information about the capacity of ecosystems to support animal populations. Over-regulation of large, lowland rivers has led to declines in many facets of floodplain function. If management of water resources continues as it has in recent decades, then our results suggest that there will be further deterioration in stand condition and a decreased capacity for female yellow-footed antechinuses to breed multiple times.Many ecological systems around the world are changing rapidly in response to direct (land-use change) and indirect (climate change) human actions. We need tools to assess dynamically, and over appropriate management scales, condition of ecosystems and their responses to potential mitigation of pressures. Using a validated model, we determined whether stand condition of floodplain forests is related to densities of a small mammal (a carnivorous marsupial,  Fast, extensive change now is the dominant characteristic of ecological systems across the world. This is due to climate change, many direct actions of humans, and myriad indirect effects arising from those actions Setophaga ceruleanRemote sensing offers the capacity to represent dynamically, and at appropriate spatial scales, the condition of surrogates (e.g. land use or vegetation condition) and then to project changes in biodiversity as the surrogate itself responds to anthropogenic pressures (e.g. land-use change) or natural processes . For example, in the Cumberland Mountains in the USA, 200 000 ha were assessed remotely as a potential habitat for cerulean warblers Remotely sensed data have been used to estimate forest stand condition (an indicator of dieback) over >200 000 ha in the southern Murray-Darling Basin in south-eastern Australia Antechinus flavipes Waterhouse is the only native, terrestrial, carnivorous mammal  on these floodplains. This species is most abundant in floodplain forests et al.The yellow-footed antechinus If there are relationships between modeled stand condition and population characteristics of antechinus , these could provide a tool for rapid, remote assessment of antechinus across vast landscapes.In this paper we explored the relative importance of modeled stand condition and of in-stand variables in explaining population characteristics of antechinus.Eucalyptus camaldulensis Dehnh.) in south-eastern Australia  −1 and mean monthly maximum temperatures range from 12.9°C for the coldest month to 31.8°C for the hottest month during the year http://spatialecology.com) using an existing map of stand condition Antechinuses were captured in the austral summer, autumn and winter in 2004, 2005 This work was carried out under permits BSCI/2011/03, BSCI/2003/02 . Traps contained lure and bedding, were protected by plastic bags and covered with bark. Animals were released at the point of capture.E. camaldulensis stands (see Methods S in N  =  140 stands) in that year and had high predictive power in an independent, follow-up validation survey in 2007 . The stand condition model for 2010 was built from on-ground surveys in that year (N  =  175 stands).Stand-condition scores see  were obtNephila sp. were counted within 2 m of each trap line. We considered the number of webs as a proxy for abundances of spiders and possibly of other macroinvertebrates, which are food for antechinus et al.Volume of fallen timber  was estiWe included two indicator variables to determine whether capture rates were affected by a life stage: juvenile dispersal variable, which was used to compare capture rates in January  to those in June and July (breeding season); and post-juvenile dispersal variable (capture rates in March to May vs during breeding season).We used logistic regression in WinBUGS v1.4 iy is the total number of individuals  captured over in trap-nights  at site i, and ip is the corresponding capture rate to be estimated . The αs are year-specific intercepts, logit-transformed mean capture rates for 2004, 2005 and 2011, and jβ is the regression coefficient for the tjh predictor variable. The εs are random effects for forest and site. Forest random effects were assigned exchangeable, normal prior distributions (Gelman 2005) with means 0 and variances, forestσ2. The site random effects were modeled with a spatial disc function such that the correlation between sites declined nearly linearly with distances up to 500 m . We assigned uniform priors  to the standard deviations of the random effects terms, siteσ, and forestσHere, β are non-zero (see Methods S1 in jβ ≠ 0) is a measure of the evidence that variable j is a predictor of the response. We considered Pr(jβ ≠ 0) > 0.75, which is equivalent to a threefold increase in the prior odds (which were unity), to be evidence that a predictor has a strong effect on the response variable jβ ∼ N; βσ ∼ Uniform. We re-fitted models with a range of plausible upper limits on βσ  and obtained similar results.We used Bayesian model selection, implemented with the reversible jump MCMC add-on to WinBUGS Models were estimated with three Markov chain Monte Carlo (MCMC) chains run for 100000 iterations following burns-in of 50000 iterations.2-discrepancy statistic. We also calculated 2pseudo-R values (proportion of deviance explained divided by maximum) as a measure of model fit. We performed 10-fold cross-validation on models to check that estimated relationships were not spurious and to evaluate likely predictive capacity. The data were split into 10 sets of sites (folds), and each fold, comprising all surveys at each site, served as test data for models built with the remaining sites. Sites were semi-randomly allocated to folds, but sites within 500 m of each other were allocated to the same fold so that predictions were not informed by spatial autocorrelation. We calculated cross-validation 2pseudo-R2pseudo-R values by chance.We checked the adequacy of the model structure by posterior predictive checks using the χTo track changes in stand condition across these floodplains, a temporal series  of condition maps for the region was compiled from previous work . There were 557 captures of antechinus (same-site recaptures discounted) with the average of 1.1 ± 1.4 SD individuals per site. There was strong evidence that capture rates of second-year females and of second-year females with suckled teats increased with increasing stand condition  > 0.75 in the full model, Pr(jβ ≠ 0) values exceeded 0.75 in at least 8 of 10 cross-validation iterations and were never < 0.6 . Predictive capacity of models was low (2pseudo-R ≤ 10%) but better than random for all response variables [Pr(observed or lower 2pseudo-R | random) < 0.001].Variable-selection results in cross-validation tests were consistent with values obtained for the full data . For allThe best estimate of the percentage of the floodplain forest stands in ‘good’ condition  and fallen timber volumes (which were not correlated with stand condition) that will cause local variation in antechinus densities independently of stand condition.There is a lack of information on how changes in land-use and management actions, such as management floods, affect (meta) population dynamics Our findings have relevance to management of floodplains in semi-arid areas. River regulation and water diversions are associated with similar tree population declines on floodplains in southwest of the USA Our results suggest that remote sensing offers a means to assess rapidly habitat quality for one mammal species over large areas. We linked a stand condition model, which originally was developed to assess health of river red gum forests, to population dynamics of antechinus and birds Figure S1Examples of stand condition states: good, poor and severe.(TIFF)Click here for additional data file.File S1Methods S1. Model selection with reversible jump MCMC. Stand-condition scores. Estimates of temporal trends in fractions of stand condition. References S1. Table S1 Study area and site information. Table S2 Mean trapping rates of the yellow-footed antechinus in 2004, 2005 and 2011 over all sampled sites in river red gum forests.Contains the following files: (DOC)Click here for additional data file.


We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of β-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of β-catenin/Tcf4. Moreover, TRα1 physically interacted with β-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased β-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis  THRa and THRb loci 0/0vil-TRα1 mice) allows increased cell proliferation and adenoma development; this overexpression also enhances the intestinal tumorigenic process in an Apc mutated genetic background (vil-TRα1/Apc+/1638N mice) vil-TRα1/Apc mice is the increased activity of the WNT pathway compared with that of the Apc mutants via the transcriptional regulation of the genes Ctnnb1 and Sfrp2The intestinal epithelium is a dynamic tissue that is continuously renewed through stem cells and committed progenitors, located in the crypts of Liberkün Ctnnb1, was originally identified as a structural component of cell adhesion complexes β-Catenin, the protein encoded by in vivo. We show that TRα1 interacts with the β-catenin/Tcf4 complex in the nuclei of Caco2 cells and that this interaction strongly reduces TRα1 functionality, but has a positive effect on the WNT downstream response in vitro. Finally, we observed the recruitment of TRα1 on the WRE regions in pre-cancerous and cancerous intestinal lesions.We established that the increased levels of the β-catenin/Tcf4 complex affects the transcriptional activity of TRα1 vil-TRα1 +/1638Nvil-TRα1/Apc vil-TRα1 mice are characterized by hyperproliferation in the intestinal crypts and a low rate of adenoma development in the small intestine and colon +/1638N develop a small number of adenocarcinomas, starting at approximately six months of age; these tumors occur primarily in the small intestine vil-TRα1/Apc mice develop adenocarcinomas in the small intestine and colon at a higher frequency than do the Apc mutants. Moreover, the tumor appearance and progression to invasiveness are also accelerated in these mice. Finally, WNT activity in the tumors of these animals is increased compared with Apc mutants We analyzed the expression profile of TRα1 target genes in normal mucosae or tumors from mice of different genotypes to gain additional insights on the cross-regulation between TRα1 and WNT pathways in gut tumorigenesis. Six-month-old Ctnnb1 and Sfrp2, TRα1-direct target genes Ccnb1 and Cdc2a, TRα1-indirect target genes Ctnnb1 and Sfrp2, we observed an increased mRNA levels in the vil-TRα1 mice compared with the WT mice, while in the normal mucosa or tumors of the Apc mice, the mRNA levels slightly decreased  We also measured the DR4-luc activity in cells maintained in culture medium containing physiological concentrations of T3 in vivo. To define the mechanism involved, we evaluated whether TRα1 could physically interact with β-catenin and/or Tcf4, as has been shown for other nuclear receptors Our findings strongly indicated that the increased level of the β-catenin/Tcf4 complex significantly reduced the TRα1 transcriptional activity both in cell lines and in vivo chromatin immunoprecipitation (ChIP) approach to analyze the TREs and WREs of the specific target genes of TRα1 and WNT, respectively. The ChIP assay was performed on WT intestine and the normal mucosa or tumors from vil-TRα1/Apc mice using anti-TRα1, anti-Tcf4, anti-β-catenin antibodies or rabbit IgG (as negative control). As shown in Sfrp2 gene, which contains the TRE in vitro EMS assay in which TRα1 binds to a DR4 element without forming a complex with β-catenin and/or Tcf4  to search for the presence of putative nuclear receptor binding sites in silico. No TREs were found within the Axin2 or c-Myc genomic regions containing the WREs analyzed (data not shown).Next, we analyzed the specific DNA binding of Tcf4 and β-catenin on their target genes and the eventual presence of TRα1 in the same regions. A WRE has been described within the Apc mice  and S3C.romoters ,F. GivenThe process of intestinal maturation and its homeostatic control by TH-TRα1 in mice results from a complex modulation of genes and signaling pathways, including the WNT, Notch and BMP Previously, we described several levels of complex relationships between WNT and the TRα1 pathways vil-TRα1/Apc mice is evident and might play a critical role during the early steps of gut tumorigenesis. With regard to the direct targets of TRα1, the overexpression of β-catenin/Tcf4 clearly inhibits their TRα1-dependent upregulation, but it does not affect the expression of the TRα1 indirect targets Ccnb1 and Cdc2a. It is probable that other transcription factors and compensatory mechanism(s) occurring in vivo may also be responsible for their expression levels.It has been shown that the functional interactions between signaling pathways involved in gut homeostasis can have a primary importance in the process of cell transformation. This is the case, for instance, of the synergy between WNT and Notch Apc, Ctnnb1 and Axin2) vil-TRα1/Apc mice. Interestingly, we also observed an increased expression of Tcf4 in the normal mucosa of the vil-TRα1/Apc mice but not in the normal mucosa of the Apc animals. Taken together, our previous and present data strongly suggest that even if we refer to “vil-TRα1/Apc mice normal mucosa”, this may only be indicative of the morphological appearance. Indeed, from a molecular standpoint, it differs in several ways from the WT mucosa Our results suggest that the binding of TRα1 to its DNA targets is shifted to the WNT targets when the β-catenin/Tcf4 protein levels increase . This co0/0/Apc mice sustain this assumption (our unpublished observations); however, it is necessary to further confirm this result to account for the delay in or absence of tumor formation in these animals. A future challenge will be to define how complex cross-regulation and integrated networks, such as those involving the NHR and WNT pathways, can affect the cellular phenotype when signals are altered. These data will surely be of importance for translational research and could help explain the contradictory results concerning NHRs and carcinogenesis.In conclusion, we have shown that in the context of intestinal lesions, TRα1 reshapes its function and induces specific genomic and non-genomic events. TRα1 transcriptional activity was negatively modulated by increased levels of β-catenin/Tcf4 and the binding to its target genes was also impaired. Interestingly, TRα1 was recruited to WNT target genes specifically within the lesions. This dual mechanism can have important repercussions and opens a new perspective in studying the sequential events of tumor development. An obvious speculation is that β-catenin/Tcf4 activity on their target genes could be reduced in the absence of TRα1. Our preliminary results from TRαvil-TRα1, vil-TRα1/Apc, Apc+/1638N and wild-type animals We used −7 M T3 and/or 10 ng/µl of recombinant Wnt3a (R&D system) was added to the culture medium for 24 hours.Intestinal epithelial primary cultures were derived from 4–6 day old neonatal mice according to Evans and colleagues Immunolabeling was performed on 2% paraformaldehyde-fixed cultures, as previously described by Rezza and colleagues Western blot (WB) was performed on whole-protein extracts obtained by homogenizing the samples in RIPA buffer, as described in −6 M) or vehicle alone was added to the culture medium 24 hours before the end of the culture. Luciferase activity was measured 48 hours after transfection using the luciferase dual system (Promega).This study was performed on the human Caco2 colorectal cancer cell line (from the American Type Culture Collection). Caco2 cells  were cultured in DMEM supplemented with 10% heat- inactivated fetal calf serum. We used the following vectors: pGl2-DR4-Luc and pGl2-mutDR4-Luc (200 ng/well) and pGS5-TRα1 Coimmunoprecipitation (CoIP) studies were conducted on Caco2 cells using a slightly modified procedure described by Guigon and colleagues RNA was extracted from tissue samples using the Qiagen RNeasy Kit (Qiagen). To avoid the presence of contaminating DNA, DNase digestion was performed on all preparations. Reverse transcription was performed using MuMLV reverse transcriptase (Promega) on 1 µg of total RNA using random hexanucleotide priming (Promega) according to the manufacturer's instructions. For the primary cultures, RNA was extracted using the Absolutely RNA Nanoprep Kit (Stratagene). Reverse transcription was performed using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) on 300 ng of total RNA. All of the cDNA samples were purified using the Qiagen PCR Purification Kit (Qiagen) before use for the qPCR experiments. The qPCR was performed with SYBR green PCR master mix (Qiagen) in an MxP3000 apparatus (Stratagene). The data from the qPCR were normalized to 36B4 levels for each sample. The primers are listed in Tcf4, β-catenin, TRα1 and RXRα full-length cDNAs were transcribed/translated in vitro using the quick TNT kit (Promega) according to the manufacturer's protocol. We performed the EMSA assay on the DR4-radiolabeled probe, as previously described vil-TRα1/Apc  and WT mice. The samples were minced with a razor blade and crosslinked with 1% formaldehyde at room temperature for 15 minutes. The crosslinking reaction was stopped by the addition of glycine to a final concentration of 0.125 M, and the samples were incubated at room temperature for 5 minutes. The samples were then centrifuged at 200 g to pellet the pieces. The fragments were washed once in cold PBS containing protease inhibitor cocktail (Roche) and then disaggregated by 10 strokes in a Dounce homogenizer. The resulting homogenate was centrifuged at 2000 g, the pellet recovered was then incubated in SDS buffer  on ice for 10 minutes. After 15 minutes of sonication , the samples were centrifuged at maximum speed at 4°C, and the supernatant was recovered and quantified by Nanodrop . The same amount of each sample was diluted 1∶10 in dilution buffer . After pre-clearing with Protein A magnetic beads (Invitrogen), each sample was divided into four samples, and the precipitation was performed using 9 µg of each antibody  promoters, as negative controls Ppia gene was used in all reactions as internal control.The ChIP study was performed on 10 mg of samples collected from the intestine of Figure S1RT-qPCR analysis in mice.  Analysis of TRα1-direct target genes in 1 month-old WT, vil-TRα1 and vil-TRα1/Apc mice. RT-qPCR analysis was performed to evaluate the mRNA levels of Ctnnb1 and Sfrp2. Values represent fold change ± sd after normalization to the wild-type (WT) animals. *: P<0.05, **: P<0.01 compared with the WT by two-tailed Student's t-test (n = 4). (C) Analysis of iodothyronine deiodinase selenoenzyme type 1 (Dio1) mRNA expression in WT, vil-TRα1, Apc and vil-TRα1/Apc mice. RT-qPCR experiments were performed in the intestine of 6-month-old mice. Values represent specific mRNA expression ± sd after normalization to 36B4 (n = 4). N, normal mucosa; T, tumor.(PDF)Click here for additional data file.Figure S2Physical interaction between TRα1 and the β-catenin in nuclei. Cellular lysates from Caco2 cells, maintained in T3-depleted (−) or T3-supplemented (+) serum, were fractionated into nuclear (N) and cytoplasmic (C) extracts. Lysates were immunoprecipitated with antibodies directed against endogenous β-catenin (upper panel) or TRα1 (lower panel), and the WB analysis of the indicated proteins was performed. Rabbit IgG was used as negative control. Ponceau red was used as whole-protein (50 µg/lane) loading control. Histone H1 was used to check the enrichment and was the loading control for the nuclear proteins in the inputs. The pictures are representative of at least three independent experiments.(PDF)Click here for additional data file.Figure S3Chromatin occupancy of TRα1, β-catenin and Tcf4 on genomic regions of target genes. ChIP analysis was performed with chromatin isolated from the intestine of WT or vil-TRα1/Apc mice  and immunoprecipitated with anti-TRα1, anti-β-catenin, anti-Tcf4 antibodies or rabbit IgG (negative control). qPCR was performed using specific primers covering the TRE of Sfrp2 (A) and Ctnnb1 (B), the WRE of Axin2 (C) and c-Myc (D) or the promoters of Villin (E) and 36B4 (F); the Ppia gene was used as internal control. Histograms represent the specific-DNA enrichment in each sample immunoprecipitated with the indicated antibody. The black bar in A–D delineates the threshold of binding specificity determined by the IgG non-specific binding. N, normal mucosa; T, tumor.(PDF)Click here for additional data file.Figure S4Electromobility Shift Assay on DR4 response element. (A) EMSA analysis was performed to examine the TRα1/β-catenin/Tcf4 complex formation on a DR4-radiolabeled probe. In vitrotranscribed/translated proteins were added to the mixture as indicated, and the DNA-protein complexes were separated by native gel electrophoresis. Specific antibodies were added to assess the protein[s] involved in the complex. In all of the conditions, only TRα1 alone binds to the labeled probe. (B) EMSA performed in the presence of the TRα1 transcriptional partner RXRα. It is worth noting that even in the presence of RXRα, there is no β-catenin/Tcf4 binding to TRα1 on a TRE.(PDF)Click here for additional data file.Table S1(A) Oligonucleotides used for the RT-qPCR study. (B) Oligonucleotides used for qPCR study after ChIP assay.(PDF)Click here for additional data file.


Results. Regardless of the level of induced β-catenin expression in the stable transfectants, there were no marked differences induced in their phenotype or invasion and migration capacity. Perhaps more importantly, none of them formed tumors when injected into immunocompromised mice. Moreover, the resulting transformed cells could be induced to osteogenic and chondrogenic differentiation but not to adipogenic differentiation. Conclusions. β-catenin, although fostering osteogenic differentiation, does not induce the malignant features and tumorigenicity conveyed by oncogenic H-RAS when introduced into partly transformed mesenchymal stem cells. This may have implications for the role of β-catenin in osteosarcoma pathogenesis. It also may suggest that adipogenesis is an earlier branch point than osteogenesis and chondrogenesis in normal mesenchymal differentiation.Although osteosarcoma is the most common primary malignant bone tumor in children and adolescents, its cell of origin and the genetic alterations are unclear. Previous studies have shown that serially introducing hTERT, SV40 large TAg, and H-Ras transforms human mesenchymal stem cells into two distinct sarcomas cell populations, but they do not form osteoid. In this study,  Osteosarcoma is the most common primary malignant bone tumor in children and young adults , 2. The β-catenin, the key downstream mediator of Wnt-signaling pathway, allowing the translocation of β-catenin to the nucleus resulting in transcriptional activation of downstream targets, many of which are involved in embryonic development and oncogenesis. The activation of Wnt pathway and β-catenin has been implicated in the pathogenesis and progression of an increasingly number of human malignancies, including colorectal cancer, melanoma, myeloma, and lung cancer [β-catenin mutation and elevated levels of nuclear β-catenin have been also noted in osteosarcoma and associated with lung metastasis [β-catenin signaling in the development and progression of osteosarcoma.The extremely complex Wnt pathway encodes highly conserved genes and secreted proteins, which modulate cell fate and cell proliferation during embryonic development and carcinogenesis through activation of receptor-mediated signaling pathways –10. In tg cancer –13. It hg cancer , 15 as wg cancer , 17. In tastasis –21, highβ-catenin in both tumor development and osteogenic differentiation, β-catenin was introduced into hMSC already transformed by hTERT and SV40 TAg. In this paper, the creation of these cells along with their characterization is reported, providing insights into the potential role of β-catenin in osteosarcoma pathogenesis.In order to better understand osteosarcomas genetic complexity, our efforts have been directed towards developing a tumor which recapitulates osteosarcomas phenotype by introducing defined genetic elements into human mesenchymal stem cells (hMSCs). Initially, hMSCs were transformed by the serial introduction of hTERT, SV40 TAg and H-Ras as had been described previously for transformation of other normal cell types , 23. The2. Cell morphologies were observed and pictures were taken using a Nikon Inverted Microscope ECLIPSE TE200 attached to a cooled charge coupled device . HT1080, HOS, and NIH 3T3 cells were purchased from American Tissue Type Culture Collection  and cultured as per their instructions.hMSCs and their transformed derivatives, transfected with hTERT and SV40 TAg, named, MSC-TS, were obtained, produced, and cultured as previously described  in MSC mβ-catenin constructed in pCMV6-XL5 vector was purchased from OriGene . The lentiviral plasmid pLenti-β-catenin-blast was constructed by subcloning the above β-catenin entry clone using pLenti6.2/V5-DEST Gateway Vector Kit and ViralPower Lentiviral Expression Systems . PLenti-β-catenin-blast was transiently transfected into the 293FT packaging cell line . Viral stock was harvested at 30 hours and MSC-TS cells were infected with 6 μg/mL polybrene when they were at 50% confluence. Following 24 hours of coculture, drug selection of infected cells was performed using 3 μg per mL blasticidin. Ten drug resistant colonies were picked up after 6 weeks and were separately subcultured. Expression of β-catenin was measured by immunoblotting 15 μg total protein with β-catenin monoclonal antibody, 6B3 , according to the manufacturer's instructions. Clones which had the highest, lowest, and intermediate β-catenin level were selected for further analysis.A full length cDNA clone of β-catenin in cultured cells was carried out using immunofluorescence staining methods. Cells were plated and cultured to 30–40% confluence on four-well chamber slides. Cells were then washed with PBS, fixed in ice-cold methanol for 5 min, washed, permeabilized with 0.25% Triton X-100 in PBS, and washed again. Nonspecific sites were blocked with 10% normal serum in 1% BSA in PBS for 1 h at RT. Cells were then incubated with 2 ug/mL rabbit polyclonal anti-β-catenin antibody  or purified IgG for isotype controls overnight at 4′. The next day, cells were incubated with a 1 : 250 dilution of goat anti-rabbit IgG secondary antibody conjugated to Alexa-488 green fluorescence  for 1 h at RT. Cells were then washed and counterstained with prolong gold antifade reagent with DAPI . Immunostaining was visualized by Zeiss Axio Observer inverted microscope at 63X magnification.Immunocytochemical staining of β-catenin, which were named MSC-TSB, as described previously [Six-to-eight-week-old CB-17 SCID mice  were injected with the MSC-TS and now also transfected with eviously . ParalleμL of extraction buffer. Absorbance at 562 nm was measured using a microplate reader .Motility (random migration) was measured by the wound healing assay as previously described . Cells wμm pore size polycarbonate membrane, over which a thin layer of extracellular matrix (ECM) is applied. The invasive cells which migrate through the matrix layer become attached to the bottom of the polycarbonate membrane, which is then stained and extracted.Invasion was measured using the Chemicon Cell Invasion Assay . Serum-starved cells were plated in the invasion chambers which have 8 β3) , respectively. Cells were cultured in differentiation induction medium for 4–8 weeks. Differentiated cells were stained with Alizarin Red, Oil Red O, and immunohistochemical staining of type II collagen using antibody collagen type II 003-02 , which can stain calcium, fat, and type II collagen, respectively, to verify formation of osteocytes, adipocytes, and chondrocytes, respectively. Chondrogenesis was also performed using a StemPro chondrogenesis differentiation kit  and was subsequently stained with alcian blue  for proteoglycans produced by chondrocytes. Pictures were taken using a Nikon Inverted Microscope ECLIPSE TE200 attached to a CCD .The osteogenic, adipogenic and chondrogenic differentiation capacity were measured according to manufacturer's instructions using an MSC osteogenesis kit, mesenchymal adipogenesis kit, and chondrogenic differentiation media with transforming growth factor .Student's β-catenin was introduced into the MSC-TS cells using blasticidin as the selectable marker. This new stably transfected MSC derived line was named MSC-TSB with T representing hTERT, S representing SV40 Tag, and B representing β-catenin, respectively. The levels of β-catenin expression were confirmed by western blot (β-catenin was expressed at varying levels in the ten clones that were selected after transfection, and also to some extent, in the parental cell lines, MSC, and MSC-TS. Clones that had the highest level of β-catenin, MSC-TSB-H1 and MSC-TSB-H2, lowest or null β-catenin level, MSC-TSB-L3 and MSC-TSB-L7, and intermediate level, MSC-TSB-I9 and MSC-TSB-I10, were selected for further analysis of the impact of β-catenin on their behavior.Stably transformed MSC-TS cell lines had been developed previously using neomycin and puromycin as the selectable markers . β-catenern blot . β-catenβ-catenin (β-catenin transfection, compared to MSC-TS with a population doubling time of less than 24 hours. Consistent with the behavior of the MSC-TSR cell line, the MSC-TSB cell lines also showed immortalization with the cells proliferating beyond 50 passages in culture.No distinguishable changes in cellular localization of -catenin , morpholβ-catenin levels and the parental MSC-TS with P values > 0.05, statistically insignificant.Two different kinds of motility were measured: random migration by wound healing assay  and haptβ-catenin levels with the degree of invasion similar to that of MSC-TS, P values > 0.05. Their invasion capacity was far less than that of HT1080 which was used as the positive control. The noninvasive cell line 3T3 was used as the negative control (Upon measuring the cellular invasiveness into the ECM, again, MSC-TSB clones did not show much variability between clones with differing  control . MSC-TSBIn tumorigenicity assays, no palpable tumor was found with any of the MSC-TSB clones in both subcutaneous and orthotopic-injected mice 6 weeks after the implantation. As a positive control, we used MSC-TSR6 which formed tumors in 7 out of 8 mice subcutaneously, and 4 out of 4 orthotopically. The parental MSC-TS also did not form tumors.β-catenin expression level, as compared to MSC-TS. Strong intensity staining with Alizarin Red could be detected as early as 3 weeks in differentiation media (β-catenin, the cells lost the capacity for adipogenic differentiation as demonstrated by the lack of Oil Red O staining after being in differentiation media 8 weeks (Osteogenic differentiation capacity was more rapid in MSC-TSB cell lines, once again independent of on media . With th 8 weeks . For the 8 weeks , whereasβ-catenin was successfully introduced into MSC-TS cells with the resulting cells being proliferative and immortalized, but without marked changes in motility or invasion and unable to produce orthotopic or heterotopic tumors in immunodeficient mice. Additionally these cells more rapidly underwent osteogenic differentiation but were unable to undergo adipogenic differentiation. Although these experiments did not produce an experimental model of osteosarcoma, some insights into the role of β-catenin in mesenchymal stem cell differentiation may be inferred. In addition, some implications may exist for intermediate stages of MSC differentiation.In summary, β-catenin instead of H-Ras into the MSC-TS cells. Some of the resulting clones showed lower level of β-catenin than the parental MSC-TS on the western blot. In this context, it is unclear why these clones have lower protein levels; however, possible explanations include either posttranslational modification or negative feedback regulation of β-catenin/Tcf pathway in which increased β-catenin signalings induce Tcf-dependent βTrCP protein expression resulting in a rapid degradation of wild-type β-catenin [There have been several reports indicating that murine mesenchymal stem cells could be transformed into malignant phenotype due to spontaneous mutations after many passages in culture, and therefore MSC could be a potential cell of origin of many sarcomas –26. In a-catenin . The preβ-catenin to transform these cells has been discussed previously but the involvement of the Wnt pathway in osteosarcoma pathogenesis and progression is somewhat controversial [β-catenin to the cell surface to promote cell-cell adhesion [β-catenin localization has been detected in the majority of osteosarcomas and may correlate with metastasis [β-catenin pathway activity in osteosarcoma cells by measuring the nuclear β-catenin level, and using a GSK3β inhibitor [β-catenin into MSC-TS, clones with different β-catenin level were deliberately picked to detect the effects of β-catenin on pathogenesis. No significant changes were detected among clones both phenotypically and functionally. Moreover, no tumor formation was observed in mice. These findings support the view that the expression of β-catenin alone is not involved in osteosarcoma pathogenesis regardless of the activity of the Wnt-signaling pathway. Frequent β-catenin accumulation in osteosarcoma may relate to the stage at which most cases become clinically apparent rather than reflecting events early in pathogenesis [β-catenin or other selected Wnt pathway components [The rationale for the use of oversial . Numerouoversial , 29, 30.adhesion . This isadhesion , 32. Furtastasis , 33. Thitastasis . Interestastasis . That sanhibitor . Althougogenesis . The othmponents , 32.β-catenin transfection into MSC-TS cell lines, the full multilineage differentiation capacity of the parental MSCs was lost. When MSC-TSBs were induced to osteoblasts, adipocytes, and chondrocytes via the respective induction media, osteogenesis was favored. No adipogenic differentiation was observed and chondrogenesis was delayed. This finding is also supported by previous studies in which inactivating mutations in LRP-5, Wnt coreceptor, are associated with decreased bone mass, as a consequence of reduced osteoblast differentiation and vice versa [A potentially important observation is that after ce versa –40. Lossce versa , 4. Osteβ-catenin to MSC-TS cells did not result in cells with tumorigenic properties. This suggests that β-catenin may not have a role in osteosarcoma pathogenesis; however, this does not rule out its potential involvement in osteosarcoma progression. The generation of cells with the capacity to differentiate into selected but not all mesenchymal stem cell lineages suggests that intermediate stages may exist. Further studies will be necessary to clarify the role of other components of the Wnt-signaling pathway in osteosarcoma pathogenesis and progression.In conclusion, this study demonstrated the addition of