These results suggest that, by down-regulating β-catenin, Gsk3β preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term β-catenin up-regulation in cartilage secondary to Gsk3β inhibition may be sufficient to induce osteoarthritis-like features in vivo.

In adult articular cartilage, the extracellular matrix is maintained by a balance between the degradation and the synthesis of matrix components. Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. There are indications for the involvement of the WNT signaling pathway in maintaining articular cartilage. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration. However, most of the studies on elucidating the role of WNT signaling in these processes were conducted using in vitro or in vivo animal models. Discrepancies have been found in the role of WNT signaling between chondrocytes of mouse and human origin, and extrapolation of results from mouse models to the human situation remains a challenge. Elucidation of detailed WNT signaling functions will provide knowledge to improve cartilage regeneration.

Background: During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin.

Co-culture models have been increasingly used in tissue engineering applications to understand cell-cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human expanded chondrocytes (HECs), human dermal fibroblasts (HDFs), mouse embryonic stem cells (MESCs), or mouse-3T3 feeder cells (M3T3s) in micromasses. BPCs were either co-cultured (1 : 5 ratio) with all cell types allowing direct cell-cell contacts or as separate micromasses in the same well with HECs. In co-culture groups with direct cell-cell contacts cartilaginous tissue was formed in all experimental groups. In situ hybridization showed that only 16-27% of the cells expressed type II collagen mRNA. Corresponding with the fact that micromasses consisted for approximately 20% only of BPCs, the amount of GAG was similar between 100% BPC micromass and the co-culture groups with HECs and HDFs. Therefore, co-culture micromasses support cartilage tissue formation predominantly originating from primary chondrocytes in direct contact with a variety of cell types. These findings potentially could be applied to optimize cell-therapy treatments for cartilage regeneration.

We have performed microarray analysis to identify PTHrP target genes in chondrocytes. ATDC5 cells were cultured as micromasses to induce chondrocyte differentiation. On d 8 of culture, the cells had a prehypertrophic appearance. This time point was chosen for isolation of RNA at 0, 1, 2, and 4 h after a challenge with 10(-7) M PTHrP. Samples were subjected to a cDNA microarray using competition hybridization. A list of 12 genes (P < 10(-3)), the expression regulation of which by PTHrP was confirmed by quantitative PCR analysis, was generated. This included seven up-regulated and five down-regulated genes. Three genes were known to be involved in PTHrP regulation, and six were previously found in growth plate chondrocytes. Most of the genes (10 of 12) were implicated in signal transduction and regulation. PTHrP also induced expression of the up-regulated genes in KS483 osteoblasts, suggesting involvement in a more generalized response to PTHrP. The vast majority of the up-regulated genes (six of seven) contained cAMP response element-binding protein- and/or activating protein-1 transcription factor-binding sites in their promoter regions. Remarkably, a number of PTHrP-regulated genes contained signal transducer and activator of transcription factor (Stat)-binding sites in their promoters. In transient transfection assays, we show that PTHrP is able to positively regulate the activity of Stat3-specific and negatively regulate the activity of Stat5-specific promoter-reporter constructs in ATDC5 and UMR106 cells. In combination with the expression regulation of genes involved in Janus kinase/Stat signaling, this data suggest a previously unrecognized interaction between PTHrP and Janus kinase/Stat signaling.

Correct diagnosis of the liver tumor phenotype is crucial for treatment planning, especially the distinction between malignant and benign lesions. Clinical practice includes manual scoring of the tumors on Magnetic Resonance (MR) images by a radiologist. As this is challenging and subjective, it is often followed by a biopsy. In this study, we propose a radiomics approach as an objective and non-invasive alternative for distinguishing between malignant and benign phenotypes. T2-weighted (T2w) MR sequences of 119 patients from multiple centers were collected. We developed an efficient semi-automatic segmentation method, which was used by a radiologist to delineate the tumors. Within these regions, features quantifying tumor shape, intensity, texture, heterogeneity and orientation were extracted. Patient characteristics and semantic features were added for a total of 424 features. Classification was performed using Support Vector Machines (SVMs). The performance was evaluated using internal random-split cross-validation. On the training set within each iteration, feature selection and hyperparameter optimization were performed. To this end, another cross validation was performed by splitting the training sets in training and validation parts. The optimal settings were evaluated on the independent test sets. Manual scoring by a radiologist was also performed. The radiomics approach resulted in 95% confidence intervals of the AUC of [0.75, 0.92], specificity [0.76, 0.96] and sensitivity [0.52, 0.82]. These approach the performance of the radiologist, which were an AUC of 0.93, specificity 0.70 and sensitivity 0.93. Hence, radiomics has the potential to predict the liver tumor benignity in an objective and non-invasive manner.

IntroductionIn this study, we tested the ability of small molecule inhibitors of WNT/β-catenin signaling to block interleukin 1β (IL-1β)- and tumor necrosis factor α (TNFα)-induced cartilage degradation. Proinflammatory cytokines such as IL-1β and TNFα are potent inducers of cartilage degradation by upregulating matrix metalloproteinase (MMP) expression and activity. Because WNT/β-catenin signaling was found to be involved in IL-1β- and TNFα-induced upregulation of MMP activity, we hypothesized that inhibition of WNT/β-catenin signaling might block IL-1β- and TNFα-induced cartilage degradation. We tested the effect of small molecules that block the interaction between β-catenin and TCF/Lef transcription factors on IL-1β- and TNFα-induced cartilage degradation in mouse fetal metatarsals.MethodsWe used mouse fetal metatarsals treated with IL-1β and TNFα as an ex vivo model for cytokine-induced cartilage degradation. Metatarsals were treated with IL-1β and TNFα in combination with the small molecules PKF115-584, PKF118-310 and CGP049090 at different concentrations and then harvested them for histological and gene expression analysis.ResultsWe found that IL-1β- and TNFα-induced cartilage degradation in mouse fetal metatarsals was blocked by inhibiting WNT/β-catenin signaling using small molecule PKF115-584 and partially using CGP049090 dose-dependently. In addition, we found that PKF115-584 blocked IL-1β- and TNFα-induced MMP mRNA expression, but did not reverse the inhibitory effect of IL-1β on the expression of cartilage anabolic genes.ConclusionIn this study, we show that inhibition of WNT/β-catenin signaling by small molecules can effectively prevent IL-1β- and TNFα-induced cartilage degradation by blocking MMP expression and activity. Furthermore, we elucidate the involvement of WNT/β-catenin signaling in IL-1β- and TNFα-induced cartilage degradation.

The canonical Wnt pathway plays a key regulatory role in osteoblastogenesis and bone mass acquisition through its main effector, bcatenin. Adenomatous polyposis coli (APC) represents the key intracellular gatekeeper of b-catenin turnover, and heterozygous germline mutations in the APC gene cause familial adenomatous polyposis (FAP). Whether APC mutations affect bone mass has not been previously investigated. We conducted a cross-sectional study evaluating skeletal status in FAP patients with a documented APC mutation. Twenty-two FAP patients with a mean age of 42 years (54.5% women) were included in this study. Mean bone mineral density (BMD) Z-scores were significantly increased above normal at all measured sites: lumbar spine (p < .01), total hip (p < .01), femoral neck (p < .05), and trochanter (p < .01). Z-scores were þ1 or greater in 14 patients (63.6%) and þ2 or greater in 5 (22.7%). Mean values of bone turnover markers were within normal ranges. There was a significant positive correlation between procollagen type I N-terminal propeptide (P1NP) and b-crosslaps (b-CTX) (r ¼ 0.70, p < .001) and between these markers and sclerostin and BMD measurements. We demonstrate that FAP patients display a significantly higher than normal mean BMD compared with age-and sex-matched healthy controls in the presence of a balanced bone turnover. Our data suggest a state of ''controlled'' activation of the Wnt signaling pathway in heterozygous carriers of APC mutations, most likely owing to upregulation of cytoplasmic b-catenin levels. ß