Supplementary Data 1 from Syndecan-2 Affects the Basal and Chemotherapy-Induced Apoptosis in Osteosarcoma

Metastases of osteosarcomas are heterogeneous. They may grow simultaneously with the primary tumor, during treatment or shortly after, or a long time after the end of the treatment. They occur mainly in lungs but also in bone and various soft tissues. They can have the same histology as the primary tumor or show a shift towards a different differentiation path. However, the metastatic capacities of osteosarcoma cells can be predicted by gene and microRNA signatures. Despite the identification of numerous metastasis-promoting/predicting factors, there is no efficient therapeutic strategy to reduce the number of patients developing a metastatic disease or to cure these metastatic patients, except surgery. Indeed, these patients are generally resistant to the classical chemo- and to immuno-therapy. Hence, the knowledge of specific mechanisms should be extended to reveal novel therapeutic approaches. Recent studies that used DNA and RNA sequencing technologies highlighted complex relations between primary and secondary tumors. The reported results also supported a hierarchical organization of the tumor cell clones, suggesting that cancer stem cells are involved. Because of their chemoresistance, their plasticity, and their ability to modulate the immune environment, the osteosarcoma stem cells could be important players in the metastatic process.

Sarcomas include cancer stem cells, but how these cells contribute to local and metastatic relapse is largely unknown. We previously showed the pro-tumor functions of calpain-6 in sarcoma stem cells. Here, we use an osteosarcoma cell model, osteosarcoma tissues and transcriptomic data from human tumors to study gene patterns associated with calpain-6 expression or suppression. Calpain-6 modulates the expression of Hippo pathway genes and stabilizes the hippo effector YAP. It also modulates the vesicular trafficking of β-catenin degradation complexes. Calpain-6 expression is associated with genes of the G2M phase of the cell cycle, supports G2M-related YAP activities and up-regulated genes controlling mitosis in sarcoma stem cells and tissues. In mouse models of bone sarcoma, most tumor cells expressed calpain-6 during the early steps of tumor out-growth. YAP inhibition prevented the neoformation of primary tumors and metastases but had no effect on already developed tumors. It could even accelerate lung metastasis associated with large bone tumors by affecting tumor-associated inflammation in the host tissues. Our results highlight a specific mechanism involving YAP transcriptional activity in cancer stem cells that is crucial during the early steps of tumor and metastasis outgrowth and that could be targeted to prevent sarcoma relapse.

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Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6-expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.

Stimulating bone formation is an important challenge for bone anabolism in osteoporotic patients or to repair bone defects. The osteogenic properties of matrix glycosaminoglycans (GAGs) have been explored; however, the functions of GAGs at the surface of bone-forming cells are less documented. Syndecan-2 is a membrane heparan sulfate proteoglycan that is associated with osteoblastic differentiation. We used a transgenic mouse model with high syndecan-2 expression in osteoblasts to enrich the bone surface with cellular GAGs. Bone mass was increased in these transgenic mice. Syndecan-2 overexpression reduced the expression of receptor activator of NF- k B ligand (RANKL) in bone marrow cells and strongly inhibited bone resorption. Osteoblast activity was not modified in the transgenic mice, but bone formation was decreased in 4-month-old transgenic mice because of reduced osteoblast number. Increased proteoglycan expression at the bone surface resulted in decreased osteoblastic and osteoclastic precursors in bone marrow. Indeed, syndecan-2 overexpression increased apoptosis of mesenchymal precursors within the bone marrow. However, syndecan-2 specifically promoted the vasculature characterized by high expression of CD31 and Endomucin in 6-week-old transgenic mice, but this was reduced in 12-week-old transgenic mice. Finally, syndecan-2 functions as an inhibitor of Wnt-β-catenin–T-cell factor signaling pathway, activating glycogen synthase kinase 3 and then decreasing the Wnt-dependent production of Wnt ligands and R-spondin. In conclusion, our results show that GAG supply may improve osteogenesis, but also interfere with the crosstalk between the bone surface and marrow cells, altering the supporting function of osteoblasts.

Purpose: Articular cartilage is an avascular and highly hypoxic tissue. HIF1α is a crucial hypoxia factor involved in chondrocyte activity and survival. The role of hypoxia in the regulation of cartilage remodeling and maintenance in osteoarthritis (OA) remains unknown. We and others have demonstrated the important role of Wnt/β-catenin pathway as a major chondrocyte regulator, the down-regulation of which preserving cartilage from damage. Here we investigated the level of hypoxia during OA and how this interferes with the Wnt/β-catenin signaling to modulate cartilage loss. Methods: To determine the role of hypoxia in OA, fl/fl HIF1α mice underwent destabilization of the medial meniscus (DMM). Hypoxyprobe assay was used to quantify the level of hypoxia in OA cartilage. Conditional Col2CreERT;fl/fl HIF1α (ΔHIF1αchon) mice were used to investigate the role of HIF1α. Knees were fixed and embedded in paraffin to assess cartilage loss using safranin-O staining and the expression of MMp-13 using immunohistochemistry. Primary chondrocyte cultures were used for understanding cellular mechanisms using RT-qPCR, Western Blot analysis and co-immunoprecipitation as well chip assay. Results: In WT mice (fl/fl), DMM downregulates hypoxia levels in the articular cartilage along with decreased HIF1α expression. Conditional deletion of HIF1α promoted cartilage breakdown and MMP-13 expression compared to WT. Hypoxia inhibits the expression of catabolic genes (Mmp-13, Mmp3) induced by Wnt/β-catenin activation in primary chondrocytes. Co-immunoprecipitations and Chip assays demonstrated the formation of β-catenin/HIF1α complexes in the nucleus. Moreover, this complex reduced β-catenin/TCF4 transcriptional activity resulting to an inhibition of Mmp13 expression. This effect was totally abolished when HIF1α was knocked-down by SiRNA strategy. Finally, blockade of β-catenin/TCF4 interaction by PKF 118-310, a specific inhibitor of this binding, alleviated the OA phenotype and downregulated Mmp13 expression in ΔHIF1αchon mice. Conclusions: Loss of hypoxia reduces HIF1α/β-catenin interaction in favor of β-catenin/TCF4 in OA. This complex acts as a positive regulator of MMP13 thus increasing cartilage degradation. Moreover, loss of HIF1α is rescued by inhibiting the β-catenin/TCF4 complex. Our study sheds light on a new role of HIF1α/β-catenin interaction in cartilage and brings new insights into the impact of hypoxia in articular cartilage and OA.

Significance Hypoxia-inducible factor 1α (HIF1α) is important for cell growth and survival. It modulates Wnt signaling, regulating cell differentiation and fate. Osteoarthritis (OA) is an increasingly frequent joint disorder characterized by progressive cartilage breakdown in which Wnt/β-catenin signaling triggers matrix metalloproteinase 13 (MMP13) expression and chondrocyte catabolism. Here we demonstrate HIF1α inhibits β-catenin signaling by blocking transcription factor 4 (TCF4)–β-catenin interaction and down-regulates MMP13 expression, thereby alleviating cartilage lesions, whereas the TCF4–β-catenin signaling induces an OA phenotype in mice. In OA joints, PKF118-310, a small molecule that blocked TCF4–β-catenin interaction, significantly reduced the progression of OA cartilage lesions. Thus, blockade of TCF4–β-catenin signaling by HIF1α represents a promising strategy to prevent articular cartilage loss in OA.

Contribution of Osteocytes to Cancer-Associated Bone Pain via Connexin43-Mediated Communications with Sensory Neurons Under the Acidic Microenvironment in Bone Metastasis Toshiyuki Yoneda1,2, Masahiro Hiasa1, Yohance Allette1, Matthew Ripsch1, Jesús Delgado-Calle1, Teresita Bellido1, David Roodman1, Lilian Plotkin1, Fletcher White1 1Indiana University School of Medicine, Indianapolis, IN, USA, 2Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

Syndecans 1-4 are a family of transmembrane proteins composed of a core protein and glycosaminoglycan chains. Although the four syndecans have common functions, they appear to be connected to different signaling pathways, and their expression occurs in a cell-and development-specific pattern. In contrast to other syndecans, syndecan-2 expression increases during osteoblast differentiation. Mechanistically, syndecan-2 exerts multiple functions in cells of the osteoblast lineage as it serves as a co-receptor for fibroblast growth factors and Wnt proteins and controls cell adhesion, proliferation, differentiation and apoptosis. Recent studies indicate that syndecan-2 also contributes to osteosarcoma cell response to cytotoxic agents through interactions with Wnt/beta-catenin signaling. Here we summarize our current understanding of the role of syndecan-2 in the control of osteoblast biology and pathology and discuss how syndecan-2 acts as a modulator of the bone cell microenvironment.

Osteosarcoma, chondrosarcoma, and Ewing sarcomas are the most common primary bone tumors in children and young adults. Significant progress has been made in our understanding of the etiology of these tumors. These tumors are now considered as a disease of cells of the osteoblast lineage characterized by uncontrolled proliferating cells maintained in an undifferentiated state, halted osteoblast differentiation and decreased cell apoptosis. These features have been shown to result from alterations in the expression or activity of oncogenes, tumor repressors and transcription factors controlling cell growth and differentiation. Additionally, we and others have shown the implication of abnormal Wnt signaling, aberrant expression or downregulation of tyrosine receptor kinase receptors, as well as a role of the bone marrow microenvironment. Here we review the mechanisms implicated in the deregulated cell growth and differentiation in primary bone tumors, and we discuss potential therapeutic approaches targeting these mechanisms which may offer novel strategies in conjunction with chemotherapy to reduce bone tumorigenesis.

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.

Background Activation of Wnt/β-catenin pathway triggers chondrocyte catabolism and MMP13 activation that contribute to osteoarthritis (OA). The mechanism of down-regulation of Wnt/β-catenin pathway in cartilage homeostasis is unknown. Because chondrocytes are in a hypoxic environment that is lost in OA, we speculated that Hypoxia Inducible Factor 1α (HIF1α) inhibits Wnt signaling and chondrocyte catabolism. Objectives We here investigated the interaction of β-catenin/TCF4 and the molecular regulation of Mmp-13 in articular chondrocytes and in OA mice. Methods Primary murine chondrocytes from WT and ΔHIF1α were cultured with Wnt3a in normoxic (21% O2) and hypoxic (1% O2) conditions and analyzed the expression of catabolic markers. The binding of TCF4 to Mmp-13 regulatory regions was assessed in Chip assay in both conditions. To determine the impact of the interaction of Wnt and HIF1α in healthy cartilage and in OA, ΔHIF1αchon and fl/fl HIF1α underwent DMM and received articular injection of PKF 118-310, an inhibitor of β-catenin and TCF4 interaction. Results Hypoxia prevented the increase in Mmp-13 and the decrease in Col2 and ACAN expression induced by Wnt. Blocking HIF1α by siRNA or cre-recombinase enhanced the expression of Mmp-13, but not Col2 expression suggesting that HIF1α regulated catabolic rather than anabolic genes. In hypoxic chondrocytes, Chip assay revealed that HIF1α lowered β-catenin/TCF4 transcriptional activity and the expression of Mmp13. Indeed, HIF1α interacted with β-catenin which displaced TCF4 from Mmp13 regulatory sequences. Moreover, DMM resulted in decreased HIF1α expression in articular cartilage of control mice. Furthermore, cartilage lesions were higher in ΔHIF1αchon mice submitted to DMM along with higher expression of β-catenin and Mmp13. Local administration of PKF 118-310 in ΔHIF1αchon reduced Mmp13 expression and prevented cartilage lesions. These results showed that HIF1α limited cartilage breakdown by blocking the binding of β-catenin to TCF4 subsequently leading to a lower Mmp-13 activation. Conclusions We show here that the HIF1α/β-catenin complex acts as a negative regulator of Wnt signaling responsible for Mmp13 transcription in chondrocytes and in mice osteoarthritic joints. Therefore, targeting the HIF1α/β-catenin interaction is a new approach to reduce chondrocyte catabolism and prevent from osteoarthritis. Disclosure of Interest None declared

Purpose: Activation of Wnt/β-catenin pathway triggers chondrocyte catabolism and MMP13 activation that contribute to osteoarthritis (OA). The mechanism of down-regulation of Wnt/β-catenin pathway in cartilage homeostasis is unknown. Because chondrocytes are in a hypoxic environment that is lost in OA, we speculated that Hypoxia Inducible Factor 1α (HIF1α) inhibits Wnt signaling and chondrocyte catabolism. Therefore, we here investigated the interaction of β-catenin/TCF4 and the molecular regulation of Mmp-13 in articular chondrocytes and in OA mice Methods: Primary murine chondrocytes from WT and ΔHIF1α were cultured with Wnt3a in normoxic (21% O2) and hypoxic (1% O2) conditions and analyzed the expression of catabolic markers. The binding of TCF4 to Mmp-13 regulatory regions was assessed in Chip assay in both conditions. To determine the impact of the interaction of Wnt and HIF1α in healthy cartilage and in OA, ΔHIF1αchon and fl/fl HIF1α underwent DMM and received articular injection of PKF 118-310, an inhibitor of β-catenin and TCF4 interaction. Results: Hypoxia prevented the increase in Mmp-13 and the decrease in Col2 and ACAN expression induced by Wnt. Blocking HIF1α by siRNA or cre-recombinase enhanced the expression of Mmp-13, but not Col2 expression suggesting that HIF1α regulated catabolic rather than anabolic genes. In hypoxic chondrocytes, Chip assay revealed that HIF1α lowered β-catenin/TCF4 transcriptional activity and the expression of Mmp13. Indeed, HIF1α interacted with β-catenin which displaced TCF4 from Mmp13 regulatory sequences. Moreover, DMM resulted in decreased HIF1α expression in articular cartilage of control mice. Furthermore, cartilage lesions were higher in ΔHIF1αchon mice submitted to DMM along with higher expression of β-catenin and Mmp13. Local administration of PKF 118-310 in ΔHIF1αchon reduced Mmp13 expression and prevented cartilage lesions. These results showed that HIF1α limited cartilage breakdown by blocking the binding of β-catenin to TCF4 subsequently leading to a lower Mmp-13 activation. Conclusions: We show here that HIF1α forms a complex with β-catenin that acts as a negative regulator of Wnt signaling and Mmp13 transcription. Therefore, targeting the interaction is a new approach to reduce chondrocyte catabolism and prevent from osteoarthritis.

The prevalent human F508-mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the F508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analysed the osteoblast phenotype in F508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the F508-CFTR mutation negatively impacts the differentiation of bone marrow osteoprogenitor cells (BMSCs) into osteoblasts and the activity of osteoblasts, demonstrating that the F508CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in F508-CFTR osteoblasts. Mechanistic analysis revealed that NF-B signaling and transcriptional activity are increased in mutant osteoblasts. Functional studies showed that the activation of NF-B transcriptional activity in mutant osteoblasts results in increased -catenin phosphorylation, reduced osteoblast catenin expression, and altered expression of Wnt--catenin target genes. Pharmacological inhibition of NF-B activity, or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in BMSCs and osteoblasts from F508-CFTR mice. Overall, the results show that the F508CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-B and reduced Wnt-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-B or activation of Wnt--catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent F508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. ______________________________________ http://www.jbc.org/cgi/doi/10.1074/jbc.M115.646208 The latest version is at JBC Papers in Press. Published on June 9, 2015 as Manuscript M115.646208 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at U N IV O F N E W O R L E A N S on Jne 2, 2015 hp://w w w .jb.org/ D ow nladed from Altered NF-B/Wnt signaling in F508-CFTR osteoblasts 2 Cystic fibrosis is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). The main function of the CFTR protein is a chloride channel in epithelia, and the most common mutation in humans, the F508-CFTR, is responsible for a channelopathy in epithelial cells (1). Several studies indicate that cystic fibrosis mutations may impact the skeleton in children and adults. Most patients with cystic fibrosis display low bone mass associated with fractures (2-5). The mechanisms underlying this bone pathology are complex and may involve inflammation and altered physical activity and nutritional status (6). In addition to these general mechanisms, the bone disease in cystic fibrosis may result from abnormal bone cell activity. Bone homeostasis is ensured by a balance between the resorption of the bone matrix by osteoclasts and its replacement by new bone formed by osteoblasts (7). Although CFTR was found to be expressed in human osteoclasts and osteoblasts and in mouse osteoblasts (8,9,10), the impact of CFTR mutations on bone cells remains largely unknown. Genetic studies in Cftr -/mice have shown that Cftr invalidation causes low bone mass and altered bone microarchitecture, a phenotype associated with decreased bone formation and increased bone resorption (1115). Others have reported altered osteoblast differentiation in cultured calvaria cells from Cftr -/mice (10). However, the relevance of the global Cftr invalidation to cystic fibrosis disease resulting from CFTR mutations is not known. Recent studies showed that the prevalent F508-CFTR mutation causes reduced bone mass as a result of decreased osteoblast activity and bone formation in mice (16) which may be partially corrected by treatment with a CFTR corrector (17). While these studies revealed that the F508-CFTR mutation impacts osteogenesis, the molecular mechanisms underlying the defective bone formation induced by the mutation have not been depicted yet. Previous studies in epithelial cells suggest that CFTR levels may control NF-B signaling (18-20) albeit the underlying mechanisms are not fully established. Notably, the F508CFTR mutation is associated with activated NF-B signaling in lung epithelial cells (21). In bone, exacerbated NF-B signaling is known to cause inflammation (22) and to promote osteoclastogenesis (23). In addition, recent studies indicate that NF-B signaling negatively controls bone formation (23,24,25,26 ). Mechanistically, NF-B activation in osteoblastic cells reduces the expression of the key osteogenic transcription factor Runx2 (27) and increases the expression of the E3 ubiquitin ligase Smurf1 (28), resulting in increased proteasomal degradation of RUNX2 (29,30,31,32). The potential implication of NF-B signaling in the abnormal bone formation in cystic fibrosis has not been investigated. In this study, we analysed the impact of the prevalent F508-CFTR mutation on the osteoblast phenotype in mice, and determined the mechanisms underlying this phenotype. We show here that the F508-CFTR mutation induces defective osteoblast differentiation and function in a cell-autonomous manner as a consequence of increased NF-B activity and reduced Wnt--catenin signaling, and that targeting these pathways leads to correct the osteoblast dysfunctions induced by the F508CFTR mutation in mice. EXPERIMENTAL PROCEDURES Mice-Rotterdam homozygous F508-CFTR mice (F508-Cftr tm1Eur ) which express the clinically common F508 mutation in Cftr gene at wild type protein level and their normal Cftr +/+ homozygous littermates (WT mice in the FVB background) were obtained from the Centre de Distribution, Typage et Archivage Animal (CDTA), Centre National de la Recherche Scientifique (CNRS, Orléans, France). We used adult 10 week-old F508CFTR male mice that exhibit decreased bone mass and bone formation related to their normal littermates (16). Cell Cultures and Treatments-Bone marrow stromal cells (BMSCs) were harvested from left tibiae in F508-CFTR and WT mice and cultured as described (33). In addition, osteoblasts were obtained by migration from trabecular bone fragments from long bone metaphysis as described previously (34). Cells at passage 2 were used in the different assays. In some experiments, cells were treated with the CFTR corrector miglustat (10 M, Actelion, Allschwil, Switzerland) (17) which acts by improving CFTR processing (35). In other experiments, cells were treated with the at U N IV O F N E W O R L E A N S on Jne 2, 2015 hp://w w w .jb.org/ D ow nladed from Altered NF-B/Wnt signaling in F508-CFTR osteoblasts 3 specific IκB kinase (IKK) inhibitor, a specific inhibitor of NF-κB activation (36) used at a dose (20 nM, SantaCruz, CA) that inhibits the upstream kinase that activates NF-κB (27), or with WNT3A conditioned medium (CM, 30%) prepared as described (37). Proliferation Assay-BMSCs and trabecular osteoblasts isolated from F508-CFTR and WT mice were cultured in DMEM supplemented with 10 % FCS. Cell replication was determined using the BrdU ELISA assay (Roche, Mannheim, France) according to the manufacturer's instructions. Osteoblast Differentiation Assays-Alkaline phosphatase (ALP) activity was assayed using an Alkaline Phosphatase kit (Bio-Rad, Hercules, USA). For osteogenic differentiation, cell culture medium was supplemented with 50 μmol/L ascorbic acid and 3 mM inorganic phosphate (NaH2PO4) to allow matrix synthesis and mineralization. At 21 and 28 days of culture, BMSC cultures were fixed in 4% paraformaldehyde, and matrix mineralization was evaluated by alizarin red staining and calcium deposition as described (38). Reporter Assay-Cells were seeded in 24well plates then co-transfected with 0.5 μg/well of the reporter plasmid, 10 ng/well of phRLSV40, a Renilla expression plasmid as internal transfection control (Clontech, Mountain View, CA). Empty pGL3-BASIC served as control for reporter activity. Firefly and Renilla luciferase activities were measured sequentially using Luciferase Reporter Assay System (Promega, Charbonnières-les-Bains, France) 48 hours after transfection. Luciferase activity was normalized both to Renilla activity, as transfection control, and to values obtained with cells transfected with an empty pGL3-BASIC, as control of the variations of the phRL-SV40 induced by treatment. Results are expressed as relative luciferase unit (RLU). Quantitative PCR Analysis-Total RNA was extracted using Trizol reagent (InVitrogen, Cergy Pontoise, France). One μg of total RNA from each sample was reverse-transcribed (Applied Biosystems kit, Courtaboeuf, France. The relative mRNA levels were evaluated by quantitative PCR analysis (LightCycler; Roche Applied Science, Indianapolis, OH, USA) using a SYBR Green PCR kit (ABGen, Courtabœuf, France) and specific primers (33). Signals were normalized to HPRT as internal control. Western Blot Analysis-Trabecular osteoblasts isolated from F508-CFTR and WT mice were cultured at preconfluence, then treated with WNT3A CM (37) for 1 or 24 hours. In other experiments, the cells were serum starved overnight and treated with recombinant mouse TNF (10 ng/ml, Apotech, Epalinges, Switzerland) and cell lysates were prepared as described (39). Protein concentrations were measured using the DC Protein assay (Bio-Rad, Marnes-la-Coquette, France). Equal aliquots (40-60 μg) of protein extracts were resolved on 10% SDS-PAGE. Western blot was performed using specific primary antibodies raised against -catenin (SantaCruz, 1/100), phospho--catenin (SantaCruz, 1/100), p65 (a gift from N. Rice, National Cancer Institute Frederick Cancer Research and Development Center, Frederick,