By targeting all fibroblasts or selectively targeting Dlk1+ lower dermal fibroblasts, we found that β-catenin stabilization between developmental stages E16.5 and P2 resulted in a reduction in the dermal adipocyte layer with a corresponding increase in dermal fibrosis and an altered hair cycle. The fibrotic phenotype correlated with a reduction in the potential of Sca1+ fibroblasts to undergo adipogenic differentiation ex vivo. Our findings indicate that Wnt/β-catenin signaling controls adipogenic cell fate within the lower dermis, which potentially contributes to the pathogenesis of fibrotic skin diseases.The Wnt/β-catenin pathway plays a central role in epidermal homeostasis and regeneration, but how it affects fibroblast fate decisions is unknown. We investigated the effect of targeted β-catenin stabilization in dermal fibroblasts. Comparative gene expression profiling of stem cell antigen 1 The epidermis is maintained by distinct subpopulations of stem cells whose fate is regulated by intrinsic mechanisms and external signals from the niche . SignaliThe dermis contains a variety of subpopulations of mesenchymal cells with different locations and functions . At the Epidermal Wnt/β-catenin signaling causes profound changes in the underlying dermis, leading to expansion of both the upper and lower dermal lineages, de novo formation of DPs, and an increase in adipocyte differentiation . In addiIn this study, we performed comparative gene expression profiling of distinct populations of neonatal fibroblasts, which showed differential expression of Wnt pathway genes. Using a conditional targeting approach, we examined the effects of β-catenin stabilization in all fibroblasts or selectively in reticular fibroblasts. Our findings indicate a key role of Wnt/β-catenin signaling in regulating the differentiation of reticular fibroblasts into adipocytes.+ fibroblasts and mature adipocytes present in P2 and adult skin, although Dlk1 is no longer expressed in adult skin  was highly enriched in the Pdgfrα+/Sca1+ fraction  analysis of mRNA isolated from the different dermal subpopulations showed that mRNAs corresponding to α- cells f–h, wherfraction i, confir reports   (To explore the differences between Pdgfrα < 0.05)  online,  < 0.05)  and regu < 0.05) b and c.+/Sca1+ and Pdgfrα+/Sca1- fibroblasts, which we confirmed by QPCR in independent biological samples , whereas Lef1 stained the upper papillary dermis (Pdgfrα+/Sca1-)  produces a variant of β-catenin that is resistant to phosphorylation by glycogen synthase kinase 3β and degradation by the adenomatous polyposis complex. Littermates were treated topically with 4-hydroxy-tamoxifen (4-OHT) on the day of birth and analyzed at different time points thereafter , and Ctnnb1 Exon3Flox/+ mice, we could show equal recombination efficiency whether or not β-catenin was stabilized. Fifty percent to 60% of all Pdgfrα+ fibroblasts were tdTomato+ at P4 in control and mutant (cΔex3) littermates , CD31 , CD45 or other markers of hematopoietic lineages  and lower (Sca1+) dermis. Interestingly, immunostaining showed that β-catenin and Lef1 are more abundant in Sca1- cells. β-Catenin stabilization in all fibroblast populations in neonatal skin resulted in a decrease in mature adipocytes and the appearance of fibrotic regions in the adipose layer, accompanied by stimulation of fibroblast proliferation throughout the dermis. The replacement of adipocytes by ectopic reticular fibroblasts also occurred when the lower dermal lineage was selectively targeted at E16.5, before adipocyte differentiation.Here we have examined the effect of dermal Wnt/β-catenin signaling in the context of fibroblast heterogeneity. We show that expression of Wnt pathway genes such as + cells rather than to promote expansion of preadipocytes or selective proliferation of reticular fibroblasts. This is similar to the effect of β-catenin stabilization on cultured human keratinocytes, which is to expand the stem cell compartment without stimulating proliferation , PdgfrαEGFP (PdgfrαH2B-eGFP) , Ctnnb1lox(ex3)/+ (T2 (The following mouse strains were maintained on a C57 Bl6/CBA background: Dlk1CreERx(ex3)/+ . PdgfrαC3)/+ (T2  and RosaSkin samples were harvested and processed to generate paraffin (5 μm) or thick cryopreserved whole-mount tissue (50–100 μm) sections as previously described . ImmunosImmunostained tissue sections were imaged using a Nikon A1R confocal microscope. Adobe Photoshop CS6  was used to adjust images and correct background. Bright field images were collected using either a Hamamatsu NanoZoomer slide scanner  or a Zeiss Axiophot microscope  with a ×10 objective and an AxioCam HRc camera. Image measurements were performed on at least six microscopic fields per biological sample.-/LIN-/CD31-). Data analysis and visualization were performed using FlowJo software version 7.6.5 .Dermis was separated from back skin of embryos (E18.5–E19.5) or postnatal pups (P2–P4) by incubation with thermolysin (0.25 mg/ml) (Sigma T7902) overnight at 4°C, digested in Dulbecco’s Modified Eagle’s Medium + 10% fetal bovine serum containing 2.5 mg/mL collagenase I , and further processed as previously described . Cells w5/ml in μClear 96-well plates (Corning). Cultures were maintained for 10 days at 37°C/5% CO2 in standard medium  or Adipogenic medium , and the medium was changed every 2–3 days.Sorted fibroblasts were collected and encapsulated in Extracel (Glycosan Biosystems) as previously described , at a deCultures were fixed with 4% paraformaldehyde for 10 minutes, washed twice in phosphate buffered saline, permeabilized with 0.1% Triton X-100 for 5 minutes and stained with LipidTox, DAPI, and rabbit anti-Rfp . Multiple image stacks of each colony were recorded using an Operetta High Content Imaging system . Spheres were identified by staining for tdTomato, and the number of nuclei per sphere was determined using quantification algorithms in the Columbus analysis software . The fluorescence intensity of LipidTox staining was measured for each sphere.t test. Error bars represent standard error of the mean.GraphPad Prism 6 software  was used for all analyses of numerical data and generation of graphs and statistical tests, including one-way analysis of variance or Student GSE76751. Complementary DNA was hybridized to Affymetrix MG430.2A arrays . Array images produced by the Affymetrix PICR 3000 scanner were imported as CEL files into Genespring GX11  for analysis. Robust Multi-array Average normalization  was used. Analyses were performed on genes selected for expression above the bottom 20th percentile in all three samples within at least one of two experimental groups. To identify differentially expressed genes, we compared the two groups using the Student t test . Entities regulated by greater than 2-fold were selected for further analysis.Genome-wide expression profiling was carried out by the Paterson Institute Microarray Core Facility, as described previously . The dathttp://orcid.org/0000-0001-6882-0257Beate M. Lichtenberger: http://orcid.org/0000-0003-4978-2237Maria M. Mastrogiannaki: http://orcid.org/0000-0001-9151-5154Fiona M. Watt: The authors state no conflict of interest.This work was supported by grants to FMW from the Medical Research Council and the Wellcome Trust. MM and BML were supported by long-term postdoctoral fellowships from the European Molecular Biology Organization (MM) and Federation of European Biochemical Societies (BML). We gratefully acknowledge excellent support from staff at King’s College London and Wellcome Trust Centre for Stem Cell Research, particularly J. Harris, Head of the Nikon Microscopy Centre at King’s. We gratefully acknowledge financial support in the form of access to the Biomedical Research Centre flow cytometry core of Guy’s & St. Thomas’ National Health Service (NHS) Foundation Trust.MM and BML equally contributed to performing and analyzing the experiments shown in


Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs.This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs , and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and  Titanium (Ti) substrates are commonly used as biomaterials in dental implantology because they provide excellent biocompatibility for peri-implant bone formation. Many clinical and experimental studies have demonstrated that surface properties, such as topography, roughness, surface energy, and hydrophilicity, are pivotal factors in enhancing osseointegration , 2. AlthPreliminary assessments of potential biomaterials are often made using osteoblasts, osteoblast-like cells, or bone marrow-derived mesenchymal stem cells , 6. Peri in vitro osteogenic differentiation of an immortalized human PDL cell line cultivated on two Ti scaffolds with different topographies were analyzed; the responses of these cells differed from those of osteoblasts, suggesting the cell-type specificity of responses to different surface structures , blocked with 5% skim milk for 1 h, and incubated with the appropriate primary antibodies  at the dilutions recommended by the supplier. The membranes were then washed and the primary antibodies were detected with goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase. The blots were developed with enhanced chemiluminescence (Santa Cruz Biotechnology) and exposed to X-ray film .Western blot analysis was conducted as previously reported . ProteinCells were fixed and treated with mouse anti-COLIA or anti-OPN antibody  for 1 h at room temperature. Fluorescein isothiocyanate-conjugated anti-mouse IgG (1 : 100) was then added for 1 h at room temperature. as previously reported . Images β-catenin  or an unrelated control siRNA targeting the green fluorescent protein (5′-CCA CTA CCT GAG CAC CCA GTT-3′), using the Lipofectamine 2000 according to the manufacturer's instructions. as previously described [Cells were transfected for 24 h with a Stealth small interfering RNA (siRNA) specific to escribed .P value <0.05 was considered to indicate statistical significance.All data are expressed as means ± standard deviations. One-way analysis of variance was used for multiple comparisons (Duncan's multiple range test). Analyses were performed with the SPSS software . A The PT and SLA surfaces showed water contact angles of 82.23° and 79.22°, respectively, whereas the contact angles of pmodPT and modSLA surfaces were close to 0°, indicating that the PT and SLA surfaces were hydrophobic, while the pmodPT and modSLA substrates were hydrophilic . SEM imaTo confirm the effects of surface topography on the biological responses of PDLSCs, the cells were cultured on tissue culture polystyrene (TCPS), PT, pmodPT, SLA, and modSLA surfaces, and alkaline phosphatase (ALP) activity was then assessed 4 and 7 days after induction to identify surface-specific osteogenic differentiation of PDLSCs. ALP activity was significantly higher in cells cultured on all Ti surfaces compared with the control TCPS Figures . In partTo further support the effect of surface properties on PDLSC behavior, we determined the mRNA expression of known osteogenic target genes  using real-time RT-PCR. According to ALP activity, mRNA expression of each osteogenic factor was increased on all Ti surfaces compared with the control TCPS. The expression of all genes was highest on SLA surfaces Figures . We alsoβ-catenin, were higher on SLA and modSLA surfaces than on TCPS and smooth surfaces. In contrast, gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT surfaces compared with TCPS, but it was downregulated on SLA and modSLA surfaces in comparison with TCPS and PT surfaces . Gene expression of the transcription factor for Shh, Gli1, was also increased significantly on smooth substrates , but it was decreased on SLA and modSLA surfaces in comparison with PT , but it was unchanged on hydrophilic PT and SLA surfaces compared with TCPS  inhibitor treatment decreased ALP activity of cells on PT and pmodPT surfaces but had no effect on SLA and modSLA surfaces, indicating that calcium-dependent Wnt signaling played a prominent role in regulating PDLSC osteogenesis on smooth surfaces . Howeverbstrates . in vitro and in vivo [ versus rough surfaces [ in vivo and in vitro environments need further confirmation.In the present study, we provide experimental evidence that implant roughness and hydrophilicity can affect differential signaling molecules targeting the early stages of osteogenic differentiation of PDLSCs. Moreover, the PDLSC osteogenic response was regulated in a roughness-dependent manner, in which osteogenesis-related factors were increased on SLA surfaces compared with PT surfaces. However, increased hydrophilicity contributed to cell response in a different way; the osteogenic properties of PDLSCs were hydrophilicity dependent on PT, but not on SLA, surfaces. This unexpected response is not consistent with previous reports of increased osteogenic activity of cells on hydrophilic compared with conventional SLA surfaces in vivo , 13. The in vivo , 15. How in vivo . Moreovesurfaces . In a stsurfaces . Thus, tβ-catenin, and the noncanonical Wnt pathways, which activates downstream signaling independent of β-catenin [β-catenin, whereas smooth PT surfaces affected one noncanonical Wnt pathway, the calcium-dependent molecules Wnt5a, calmodulin, and NFATc1. Consistently, previous studies reported that different Wnt pathways were activated in response to individual implant properties [Cell fate depends on mutual extracellular signaling and the activation or repression of specific transcription factors that affect common intracellular signaling cascades. The gene expression analysis conducted in this study indicated that differential, substrate-dependent signaling activation may be responsible for the increased osteogenic activity of PDLSCs. We first identified the dependence of Wnt factor regulation on implant surfaces. The role of Wnt signaling in bone formation has been examined recently, and it is considered to be a fundamental signaling cascade for osteoblast differentiation . Moreove-catenin , 20. Intoperties –23. Thusα2 and β1 signaling is known to regulate the osteogenic factor osteoprotegerin and the integrin α2/β1 pair is required for osteoblast differentiation on microstructured Ti [α2/β1 on SLA and modSLA substrates, consistent with the pattern of canonical Wnt signaling molecules, and decreased integrin expression with β-catenin siRNA, suggesting that the canonical pathways are involved in the regulation of integrins.In addition to Wnt signaling pathways, the present study classified several other molecules supporting the osteogenic response of PDLSCs to Ti surfaces. Among them, integrin tured Ti , 25. Simβ-catenin siRNA and noncanonical Wnt pathway with NFAT inhibitor decreased mRNA expression of Shh and Notch signaling molecules. Thus, our findings demonstrated that these signaling molecules involved in osteogenesis were differentially expressed according to implant properties and that Wnt signaling may act as an upstream regulator of the Shh and Notch pathways. Finally, ALP activity on smooth and rough substrates was inhibited by an NFAT inhibitor  and a β-catenin knockdown using siRNA , respectively. These results suggest that differentially activated Wnt pathways, depending on Ra and hydrophilicity, play important roles in the osteoinductive activity of PDLSCs.We have also demonstrated the different involvement of Shh/Gli and Notch signal transduction pathways with the different substrates. Each pathway has been suggested to play an important role in various cell types by regulating cell fate determination and differentiation –28. SeveThese findings show that implant properties exert complex modulation of PDLSC differentiation through these various pathways. In future studies, the interactions of these pathways will be explored in detail. In conclusion, the present study showed that Ti implant surfaces can increase the osteogenic capacity of PDLSCs with no added osteoinductive factor and suggest what kinds of surface topography and chemistry may be optimal for PDLSCs. Moreover, we suggest that the many signaling molecules may play roles in surface-induced osteogenic differentiation of PDLSCs, and they may represent useful therapeutic targets for improving clinical performance and future cell-based implant engineering.


Ghrelin (10−10M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys3-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression.Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/ Accumulating evidence, including in vitro and in vivo studies in animal models as well as observational studies in humans, indicate a role for ghrelin in the control of bone cell activities –4.Ghrelin, an endogenous ligand of the GH secretagogue receptor (GHS-R), and its receptor GHS-R1a are expressed in several osteoblasts and osteoblastic cell lines. Ghrelin directly promotes both osteoblast proliferation and differentiation –8 and inIn addition to a well characterized anabolic action, the data regarding the effects of ghrelin on osteoclastogenesis are contradictory. Costa et al.  have shoIn vivo studies showed that intraperitoneal  as well β-catenin pathway is critically associated with the entire osteoblast life cycle, that is, commitment, proliferation, apoptosis, and function, we hypothesized that ghrelin exerts its anabolic action in bone through canonical Wnt signaling and β-catenin stabilization. This hypothesis is supported by the observation that in rat cortical neuron cells ghrelin exerts specific, receptor mediated antiapoptotic effects by activating ERK1/2 pathway and by stabilizing β-catenin via PI3K/Akt-mediated inactivation of glycogen synthase kinase (GSK-3β) [Despite convincing evidence that ghrelin has positive effects on bone metabolism, the signaling mechanism and the specific involvement of GHS-R1a in osteoblast are still controversial  and rema(GSK-3β) .β-catenin, which is a 130 amino acid protein that, in the absence of Wnt proteins, is phosphorylated by GSK-3β and degraded through the proteosomal machinery. When not degraded, stable β-catenin is accumulated in the cytoplasm and upon reaching a certain concentration level is translocated into the nucleus to stimulate gene transcription [Wnt canonical signaling pathway relies on the cytosolic stabilization of cription .β-catenin intracellular levels in primary rat osteoblasts. Then, we characterized the receptor involved in the stimulatory action of ghrelin on β-catenin and examined the effects of ghrelin on the expression of osteoprotegerin (OPG) a target gene of β-catenin that plays an important role in osteoblasts and osteoclasts cross talk. Furthermore, we evaluated whether or not ghrelin, by modulating the OPG/RANKL/RANK system, could influence osteoclastogenesis in cultured rat osteoclast precursors cells.To test our hypothesis, we first examined the effects of increasing concentrations of ghrelin on 3-GHRP-6 was purchased from Bachem AG . H89 was from Sigma, Milan, Italy. Rat macrophage colony-stimulatory factor (M-CSF) and rat RANKL were from Peprotech, Rocky Hill, NJ.Ghrelin was synthesized by conventional solid phase synthesis and purified to at least 98% purity by HPLC by Neosystem . D-Lys2 flask and allowed to grow to confluence in DMEM containing 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B; the culture medium was changed every 2-3 days. Cells reached confluence after approximately one week. The cell population was tested for alkaline phosphatase production after PTH 10−8 M treatment for 48 h to ensure that the cells were endowed with osteoblast characteristics. Cells were all used at first passage to reduce the possibility of phenotype changes.Parietal and frontal calvariae  were explanted aseptically from 3-day-old Sprague Dawley rats. After removing the periosteum, the calvariae were cut in small pieces and placed in Joklik's modified MEM  serum-free medium containing 0.5 mg/mL type IV collagenase  for 20 minutes at 37°C with rotation. Digestion was stopped by the addition of Dulbecco's medium  containing 10% foetal bovine serum . Calvariae obtained from three animals were plated in 75 cm3 cells/well in a 96-well culture plate. After treatment, cells were washed with phosphate buffered saline  and incubated at 37°C with 0.5 mg/mL 3--2,5-diphenyltetrazoliumbromide  for 3 h. The conversion of the tetrazolium salt MTT to a colored formazan was used to assess cell viability. After the supernatant was removed, dimethyl sulfoxide was added to each well and the absorbance was recorded by a microplate spectrophotometer (WALLAC) at 550 nm.Cells were plated at the density of 3 × 10Cell phenotypic observations were made using Olympus TH4-200 inverted phase-contrast microscope, fitted with a digital camera Olympus C-4040 zoom to record any change during treatment.−11–10−7 M for 15, 30, 60, 90, and 120 min; the GHSR1a receptor antagonist, D-Lys3-GHRP-6 (10−7 M), was added 30 min before ghrelin 10−10 M; incubation with 2 × 10−6 M H89, an inhibitor of PKA activity, was performed 30 min before ghrelin 10−10 M.Confluent cells plated in 6-well multiwell plates were treated with ghrelin 10μL RIPA buffer  per well. Two wells were collected for each treatment. Total protein concentration was determined by BCA assay . Thirty micrograms of total protein extract was mixed with the appropriate volume of Laemmli's sample loading buffer, heated at 100°C for 5 min and loaded onto 10% SDS polyacrylamide gels. Western blots were performed using a specific antibody against rat β-catenin diluted 1 : 5000 in 5% milk in Tris–HCl with 0.1% Tween-20 (TBST); rat P-GSK3β (1 : 2000 5% milk in TBST); rat total GSK3β (1 : 2000 5% milk in TBST)  or against rat β-actin (1 : 2000 5% milk in TBST) . Nuclear fractions were isolated from untreated cells or cells treated with ghrelin (10−10 M) alone or in association with D-Lys3-GHRP-6 (10−7 M) by the Qproteome Cell Compartment Assay  that allows selectively isolating proteins in different cell compartments by sequential addition of different extraction buffers to a cell pellet. Scion Image software  was used to quantify the Western blots. β-Actin or Tot-GSK3β was used as endogenous controls to avoid incorrect estimations of the signal from the protein of interest. The intensity of the treated samples was normalized to that of the untreated samples (set as 1) and presented as a ratio of untreated controls.After removing the medium, adherent cells were gently scraped into 75 β-catenin, receptor activator of NFkB ligand (RANKL), and Dickkopf 1 (DKK1) mRNA relative expression were evaluated by real-time PCR after exposing rOB at confluence to ghrelin 10−11 M–10−7 M for 1, 4, and 24 h in serum-free medium. In order to assess if the effect of ghrelin on OPG gene expression was receptor mediated, cells were pretreated with D-Lys3-GHRP-6 10−7 M 30 min before adding ghrelin. Total RNA was extracted using TRIzol according to the manufacturer's instructions . RNA pellet concentrations were assessed spectrophotometrically using Epoch Spectrophotometer System (Biotek) (OD260/280). Two micrograms of total RNA were retrotranscribed in a total volume of 25 μL using an oligodT primer (0.5 mM), 200 U of M-MLV reverse transcriptase, deoxynucleotides (0.5 mM), M-MLV reaction Buffer 1x, and rRNasin ribonuclease inhibitor 1 U/mL . Relative quantification of OPG β-catenin, RANKL, and DKK1 was performed on an ABI PRISM 7900 sequence detector  using 20 ng cDNA of the RT-PCR solution in a final volume of 25 μL. The primer-probe sets were purchased as assay-on-demand for gene expression from Applied Biosystems. Real-time PCR was performed with FAM-labelled specific probes for OPG, β-catenin, RANKL, and DKK1 detection and VIC-labelled probes for the detection of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as an endogenous control. Real-time PCR was run according to the following protocol: an initial step of 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. mRNA levels were quantified using the comparative threshold-cycle (Ct) method.Osteoprotegerin (OPG), 4 cells/well) with M-CSF (25 ng/mL) and RANK-L (50 ng/mL) for osteoclast generation in presence of 50% of conditioning medium derived from rOB treated with ghrelin 10−10 M for 24 h, 48 h, or untreated. Half of the media was changed every day and cytokines were added freshly. After 5 days of culture cells were fixed with 4% paraformaldehyde in PBS for 10 min and osteoclast formation was assessed by tartrate resistant acid phosphatase (TRAcP) staining. Briefly, fixed cells were incubated with naphthol-ASBI-phosphate, pararosanilin, and tartrate in acetate buffer (30 μM) at 37°C for 45 min. TRAcP positive cells with three or more nuclei were considered to be osteoclasts.Osteoclast precursors cells were obtained from 3-day-old rat pups long bones. Bone marrow was flushed out with DMEM and the cell suspension was pushed through needles of decreasing size (19 G–25 G) until homogenously resuspended. Cells were then centrifuged and plated in petri dishes in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B in the presence of M-CSF (100 ng/mL) for 48 h. Nonadherent erythrocytes were removed while adherent bone marrow cells were washed with PBS resuspended in culture medium and plated on 96-well plates . All data are represented as the mean ± SEM. Differences between groups were assessed by one-way analysis of variance (ANOVA) for nonparametric data  followed by a multiple comparison test (Dunn's test) and by Mann-Whitney β-catenin levels, primary rat osteoblast-like cells were treated for 60 min with increasing ghrelin concentrations. Using Western blot analysis, we have shown that ghrelin significantly increased β-catenin levels starting from 10−10 M to 10−8 M (To examine the effects of ghrelin on o 10−8 M . Althougβ-catenin involves GSK-3β phosphorylation and inactivation as shown by the significant increase of phosphorylated GSK-3β induced by ghrelin (10−10 M) starting from 30 min of incubation , and then challenged with ghrelin (10−10 M). β-catenin levels were measured in both the cytosolic and nuclear compartments after fractioning of the whole cell lysate. Pretreatment with D-Lys3-GHRP-6 abolished both ghrelin-induced cytoplasm β-catenin stabilization and β-catenin nuclear migration  showed a significant increase in the expression of OPG. This ghrelin action involves an interaction with GHS-R1a since it was removed by pretreatment with D-Lys3-GHRP-6  was found (data not shown).Considering that OPG is a target gene of  Figures . We nextes −10 M . No sign−10 M for 24 and 48 h, in the presence of M-CSF (25 ng/mL) and of a subeffective dose of RANKL (50 ng/mL). The presence of osteoclast-like cells was evaluated by counting the number of multinucleated TRAcP positive cells per well. The number of osteoclasts was significantly reduced as compared with that detected incubating osteoclast precursors with medium from untreated-control osteoblasts, when bone marrow precursors were exposed to conditioned media from 48 h ghrelin- (10−10 M) treated rOB,  and constitutively phosphorylated via interaction with casein kinase I and GSK3-β. The presence of Wnt ligands inhibits the kinase activity of GSK-3β leading to β-catenin accumulation in the cytoplasm followed by its translocation into the nucleus where it interacts with T cell factor/lymphoid enhancer family members to induce the transcription of key osteoblastic genes [β-catenin is associated with a decrease or an increase of bone mass, respectively [β-catenin cytoplasmic levels leading to the nuclear translocation of the protein. The effect was obtained under physiological dosages of ghrelin to show its fine regulation of bone cells; its magnitude was in agreement with data recently observed in other systems [β: a critical element of β-catenin activation.Conversely, our study was based on the hypothesis that ghrelin, acting on osteoblasts, could exert a direct anabolic action through an autocrine/paracrine way that involves Wnt/function . In the ic genes . Canonicectively . Here we systems ; it was β phosphorylation  exposure of the cultures. It remains to be clarified whether ghrelin-induced β-catenin stabilization via cAMP-dependent protein kinase activation involves only the inhibition of GSK-3β.Kim and colleagues  have shoβ-catenin on ser552 by PKA enhances the transcriptional ability of β-catenin independent of the phosphorylation of β-catenin in the N-terminal domain at canonical sites regulated by Wnt pathway [β-catenin stabilization could be involved in ghrelin-induced β-catenin cytoplasmatic accumulation. Our data show that ghrelin enhances rOB viability through GHSR1a and PKA activation and, by acting on the same pathways, promotes stabilization of intracellular β-catenin levels. Therefore, we can speculate that the anabolic action of ghrelin in rOB is exerted through β-catenin stabilization. Further studies will be required to examine the direct involvement of β-catenin stabilization in ghrelin enhancement of rOB viability.A number of recent reports showed that the phosphorylation of  pathway , 33. Thiβ-catenin in osteoblasts affect not only bone formation but also bone resorption and osteoclast differentiation due to deregulation of the OPG gene. Activation of the Wnt/β-catenin pathway in osteoblasts suppresses bone resorption through upregulation of OPG expression and downregulation of RANKL expression [Loss- and gain-of-function mutations of pression , 35.3-GHRP-6, abolished the stimulatory action of ghrelin on OPG, thus indicating that ghrelin effects on OPG are specific and receptor mediated. The evidence that ghrelin induces OPG gene expression and reduces RANKL/OPG ratio suggests for the peptide an inhibitory role on osteoclastogenesis. This assumption is in line with the results obtained by exposing bone marrow precursor cells to the conditioned medium after 24 hr and 48 hr treatment with ghrelin. As expected, from the understanding of the OPG/RANK/RANKL system [Our results show that ghrelin stimulates OPG expression and that pretreatment with the GHS-R1a antagonist, D-LysL system , we founIt remains to be clarified whether ghrelin could have a direct action on osteoclastogenesis since the peptide is present in conditioned medium (CM) from rOB. This hypothesis is unlikely because CM from 24 h treated cells had no effect on multinucleated TRAcP positive cell formation. Consistent with this view is the here observed presence of the effect at 48 hours  that fol3-GHRP-6 abolished ghrelin effect on OPG expression in rOB, we can speculate that ghrelin affects indirectly osteoclastogenesis by binding GHS-R1a in primary rat osteoblasts. Our findings are in line with previous in vivo studies with hexarelin, a synthetic GH secretagogue that recognizes GHS-R1a. It has been reported, in fact, that long-term hexarelin administration inhibits bone resorption induced by gonadectomy in the rat [−9 M increased the bone resorption activity of mature rat osteoclasts but did not modify osteoclast differentiation in mouse bone marrow cultures. However, the conditioned medium used in our experimental condition mimics the inhibitory effect of OPG addition in the osteoclastogenesis assay applied by Costa et al. [−8 M) induces a global hypermethylation of primary rat osteoblasts DNA (data not shown). This effect might counteract the typical global hypomethylation of DNA present in postmenopausal women with osteoporosis [Our data show a stimulatory action of ghrelin on OPG expression and consequently an inhibition of osteoblast mediated osteoclastogenesis and corroborate the results of Delhanty and colleagues  that dem the rat . Contras the rat  have shoa et al. . More rea et al.  reportedoporosis .More research on this complex but fascinating area of gerontology and future in vitro and in vivo studies will be required to clarify the role of ghrelin in the regulation of bone turnover and to suggest the potential utility of GHS-R1a-agonists to stimulate bone formation and to inhibit osteoclastogenesis, thus improving bone structure in conditions such as osteoporosis.


The full-length cDNA of β-catenin was 3,353 bp, including a 2,511 bp open reading frame that encoded a predicted 836 amino acid protein. Level of the β-catenin mRNA increased significantly (P<0.05) with C. farreri gonadal development and presented a sexually dimorphic expression pattern in the gonads, which was significantly high in ovaries detected by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis revealed that the β-catenin was mainly located in germ cells of the gonads, with obvious positive immune signals in the oogonia and oocytes of ovaries as well as in the spermatogonia and spermatocytes of testes, implying β-catenin might be involved in the gametogenesis of C. farreri. Furthermore, when 0.1 µg/mL and 0.2 µg/mL DKK-1  were added in vitro to culture medium containing testis cells of C. farreri, the expression of β-catenin decreased significantly detected by qRT-PCR (P<0.05), suggesting the canonical Wnt signal pathway exists in the scallop testis. Similarly, when 50 µM and 100 µM quercetin (an inhibitor of β-catenin) were added in vitro to the culture system, Dax1 expression was significantly down-regulated compared with controls (P<0.05), implying the β-catenin is an upstream gene of Dax1 and is involved in the regulation of C. farreri spermatogenesis.β-catenin is a key signaling molecule in the canonical Wnt pathway, which is involved in animal development. However, little information has been reported for β-catenin in bivalves. In the present study, we cloned a homolog of  Drosophila melanogaster, Caenorhabditis elegans, and Hemicentrotus pulcherrimus, and vertebrates such as Danio rerio, Xenopus laevis, Mus musculus, and Homo sapiens have provided abundant information regarding Wnt signaling in signaling transduction and function The Wnt signaling transduction pathway plays an important role in many developmental processes, including vertebrate limb regeneration, nervous system development, body axis formation, and adrenocortical development To date, at least three Wnt intracellular signaling pathways have been identified: the Wnt/β-catenin pathway, the Wnt/planar polarity pathway, and the Wnt/calcium pathway β-catenin activates the expression of Dax1 through the canonical Wnt pathway. Therefore, the Wnt-β-catenin-Dax1 signaling pathway exists in these cells Dax1, implying that this signal pathway may commonly exist in mammals β-catenin is a key transcriptional effector of the canonical Wnt signal transduction pathway but also functions as a cell adhesion molecule at the plasma membrane by linking cadherins to α-catenin Chlamys farreri (Jones and Preston 1904) is an important commercial shellfish in China and is characterized by its relatively stable sex composition C. farreri Dax1 is suggested to involve in both oogenesis and spermatogenesis while DAX1 expressed much higher in testis than in ovary Dax1 during C. farreri spermatogenesis, we cloned β-catenin cDNA in the present study using the Rapid Amplification of cDNA Ends (RACE) technique, and determined its expression pattern in gonads during the reproductive cycle of C. farreri. Furthermore, the type of Wnt pathway and correlations between the β-catenin and Dax1 in C. farreri spermatogenesis were investigated using an in vitro cultured testis cells treated with DKK-1 and quercetin. Our aim was to reveal the β-catenin expression characteristics in the developing gonads of C. farreri, and provide evidence for the existence of the Wnt-β-catenin-Dax1 pathway in the C. farreri spermatogenesis.Shellfish comprise a group of bivalves with rich diversity in species and biological characteristics. Sex is an important characteristic, and sexual variability induced usually by interactions between environmental factors and genes is common in bivalves. However, the molecular mechanisms involved in sex variability are not clear because of a deficiency of data regarding sex-related genes. The scallop The collection and handling of these animals was approved by the Animal Care and Use Committee at the Ocean University of China.C. farreri with mean shell height 6.27±0.32 cm were purchased from the Aquatic Product Market . Gonads were dissected and weighed. Parts of the gonads were immediately frozen in liquid nitrogen, then stored at −80°C until RNA extraction. Parts of the gonads were fixed with Bouin's solution  for 24 h, embedded in paraffin wax, sliced at a thickness of 5 µm, and stained by hematoxylin-eosin (H&E) for histological analysis to determine developmental stages of the gonads. Parts of the gonads were fixed in 4% paraformaldehyde (pH 7.4) at 4°C for 24 h, and then dehydrated through a methanol series  and stored in 100% methanol at −20°C for immunohistochemistry analysis.Healthy male and female scallops According to the morphologic characteristics described by Liao et al. C. farreri ovaries at the proliferative stage with Trizol reagents  according to the manufacturer's instructions. The total RNA was then treated with DNase (Takara Bio Inc.) and purified using the RNeasy mini kit . RACE-Ready First-strand cDNA was synthesized, and 5′ and 3′ RACE PCR was conducted using the SMART-RACE cDNA Amplification kit . The specific primers (5′-CATTCAGCGTGTAGCAGCAGGAGTCCTC-3′ for 3′ RACE and 5′- CTTCGGACATGCGGAAAAGGACTGCTGC-3′ for 5′ RACE) were designed according to a 325 bp expressed sequence tag (EST) sequence (GenBank accession no. DT718886) with BLAST search from the C. farreri EST collection (http://www.ncbi.nlm.nih.gov/nucest/DT718886.1). RACE cDNAs were denatured at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min, ending with a 5-min extension at 72°C. PCR products were separated on an agarose gel (1.2%) and purified with a DNA purification kit (Takara Bio Inc.). Then, the purified 5′ and 3′ RACE products were subcloned into a PMD-18T vector (Takara Bio Inc.) and sequenced.Total RNA was isolated from β-catenin. Sequence identity and similarity of the C. farreri β-catenin with other known β-catenins were analyzed using the online BLAST suite of programs at the National Center for Biotechnology Information. Phylogenetic analysis was conducted using MEGA software (version 4.0) with the neighbor-joining method.5′ and 3′ RACE fragments were assembled using DNAstar software  to get the full-length cDNA of β-catenin in gonads of C. farreri during the reproductive cycle was conducted as described previously C. farreri β-catenin; forward primer: 5′-CAATCAGCAGCAAGGGTGGA-3′ and reverse primer: 5′-TCTGGGAACATGGCGTCTCG-3′. The β-actin gene was used as reference gene following previous studies in C. farreriβ-actin (GenBank accession no. AY335441) was amplified using the forward primer: 5′-TTCTTGGGAATGGAATCTGC-3′ and reverse primer: 5′-ATTGTGCTACCACCGGAAAG-3′. Primer specificity during the qRT-PCR was verified by a single distinct peak obtained by melting curve analysis. Quantification of target and reference genes was conducted simultaneously using the ABI 7500 detection system (Applied Biosystems) with SYBR Green Master Mix (Takara Bio Inc.). The qRT-PCR reaction consisted of 5 min at 94°C, followed by 40 cycles of 94°C for 15 s, and 60°C for 1 min. Gonads from six individuals at the same stage were sampled, and duplicate assays for each gonad sample were conducted. Data were analyzed using the ABI 7500 system SDS software version 1.4 (Applied Biosystems) with automatically set baseline and cycle threshold values. The 2−ΔΔct method was used to analyze the relative expression levels of the β-cateninqRT-PCR for analyzing expression levels of the P<0.05. All assays in the qRT-PCR were validated in compliance with “the MIQE guidelines” All data were presented as means ± SEM of six samples with two parallel repetitions. Differences between means were tested using one-way analysis of variance (ANOVA) followed by Duncan's post-hoc test , and the significant level was set at β-catenin was predicated to have a relative high antigenicity, and was amplified using the forward primer: GGATCCTCCAGTAACAAGCCAGCTGT-3′5′- (BamHI site underlined) and reverse primer: CTCGAGCAGATCAGTGTCATACCAGTTG-3′5′- (XhoI site underlined). After subcloning into a PMD-18T vector (Takara Bio Inc.), the recombinant plasmid was extracted and digested by BamHI and XhoI. The digested fragment was then ligated into a BamHI/XhoI site of the bacterial expression vector pet28a (Invitrogen) and verified by sequencing. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) (TransGen Bio Inc.). Glutathione-S-transferase (GST)-β-catenin fusion protein was expressed and affinity purified on an anti-GST Ni-NTA His Bind Resins (Invitrogen), and was injected into New Zealand white rabbits for the production of polyclonal antibodies. Antibody specificity was detected by Western blotting using the gonads of C. farreri at the growth stage, as previously described Based on the analysis of antigen clusters (DNAstar software), the partial ORF fragment (encoded from 378 amino acids to 836 amino acids) of the C. farreri with mean shell height 5.67±0.24 cm and mean GSI of 3.95±0.016 from the growth stage were purchased from the Aquatic Product Market . The scallops were maintained in seawater for 1 week at room temperature before sampling. The testis tissues were dissected and cut into pieces (mean size  = 1 mm3) in PBS (pH 7.4) containing penicillin  and streptomycin (800 µg/mL). These tissue pieces were cultivated in vitro at 23°C in primary medium that consisted of L15 medium (pH 7.2–7.4) plus 5% fetal bovine serum, 20.2 g/L NaCl, 0.54 g/L KCl, 0.60 g/L CaCl2, 1 g/L MgSO4, 3.9 g/L MgCl2, 10 ng/mL EGF, 2 ng/mL bFGF, 10 ng/mL LIF, 100 IU/mL penicillin, and 100 µg/mL streptomycin. After approximately 5 h of adherent culture, most somatic cells and germ cells migrated out from these tissue pieces. According to the differential adherent ability, we made the germ cells apart from bottom of the culture dish and suspend in the medium by shaking slightly the dish, meanwhile the somatic cells and tissue pieces were still attached to the bottom. Then the culture medium containing germ cells was collected carefully and transferred to six-well plates in which new primary medium was added. After cultivating at 23°C for 10 h when the germ cells were completely attached to the bottom of the plate, the culture medium was replaced by new medium containing the new primary medium plus human recombinant DKK-1  at the concentrations of 0.1 µg/mL or 0.2 µg/mL, and quercetin  at the concentrations of 50 µM or 100 µM for the treated groups, respectively; for the control, it was added only with new primary medium. The germ cells of the treatment group and control were cultivated for 48 h, and then harvested by centrifugation at 100 g for 10 min. After washing with PBS twice, the germ cells were stored in liquid nitrogen until RNA extraction. Three replicates were conducted for each group. Total RNAs from the cells of each sample were extracted with the MicroElute Total RNA kit . The RNAs were digested with DNase (Takara Bio Inc.), and transcribed to cDNA as described before. The expressions of β-catenin and Dax1 were detected using qRT-PCR. The primers for C. farreri β-catenin were the same as the gonadal expression described above by qRT-PCR detection, and the primers for C. farreri Dax1 (GenBank accession no. JQ071986) were as follows: forward primer 5′-TCTTCCTCGCCTCATTGTCG-3′ and reverse primer 5′-CGTCGGTATTGGAGCCTTTG-3′. The qRT-PCR data of both treated groups and controls were analyzed by the methods described above.Healthy male scallop C. farreri ovary at the proliferative stage were 1,241 bp and 2,279 bp in length, respectively. A full-length sequence of 3,353 bp was assembled  in C. farreri gonads during the reproductive cycle. Then, the level markedly decreased to a minimal level at the resting stage (P<0.05). Significant differences at expression levels (P<0.05) were also found between testes and ovaries at the same stage except the resting stage, which was approximately two times higher in the ovary than testis at the proliferative stage, 0.5 times higher at the growth stage, and two times higher at the mature stage.qRT-PCR results  indicateThe titer of β-catenin polyclonal antibody was detected to be 1∶512,000, and a single band with a molecular mass of approximately 90 kDa in gonads at the growth stage was detected by Western blotting , indicatβ-catenin and Dax1 decreased significantly (P<0.05) in C. farreri testis cells cultured in vitro, by a concentration-dependent mechanism when DKK-1 was added for 48 h. The expression levels of β-catenin decreased about 15% in the 0.1 µg/mL DKK-1 group and 26% in the 0.2 µg/mL DKK-1 group, compared with the control group  and its expression levels increased significantly (P<0.05) in both testes and ovaries during the development, implying the β-catenin might be involved in gametogenesis and gonadal development. Furthermore, we observed the expression of C. farreri β-catenin presented a sexually dimorphic expression pattern in gonads, with significantly (P<0.05) higher levels in ovaries than in testes during the reproductive cycle, except for gonads of the resting stage. Immunohistochemical analysis demonstrated strong β-catenin staining in ovaries while a relatively weak signal was observed in testes, indicating its sex dimorphic expression at the protein level. A similar result has also been observed in mice, where β-catenin is predominately expressed in ovaries Rspo1 knockout mice shows masculinized gonads, suggesting β-catenin is required for the development of ovaries C. farreri. Further study is required to reveal the precise function of β-catenin in bivalve gonads.Generally, the structure and developmental status of the gonads are non-permanent and change as an annual reproductive cycle in most marine animals. In the adult scallop, gametogenesis is initiated annually through mitosis and the differentiation of spermatogonia/oogonia at the beginning of the proliferative stage. This progress is gone on through meiosis and differentiation of germ cells to form mature gametes in the germinal scini of the growth and mature gonads β-catenin, which is a downstream and key mediator molecule in the canonical Wnt pathway C. farreri significantly (P<0.05) down-regulated the expression of β-catenin compared with the control. This result is coincident with that of adult human mesenchymal stem cells from bone marrow stroma (hMSCs). In hMSCs, the canonical Wnt signaling pathway has been confirmed to exist, and DKK-1 at a concentration of 0.1 µg/mL can down-regulate the expression of β-catenin both in the cytoplasm and nucleus of cultured hMSCs C. farreri gonads.Wnt regulates a variety of cellular events via canonical or noncanonical pathways Dax1 by interacting with Ad4BP/SF-1 in vitro, thus β-catenin is upstream of Dax1in vivo study in mice, where mutation of Wnt4, an upstream activator of β-catenin, causes the down-regulated expression of Dax1, suggesting the conservation of this signaling pathway in mammals C. farreri, we examined the expressions of β-catenin and Dax1 in cultured testis cells in vitro treated with quercetin, an inhibitor against β-catenin/Tcf signaling that down-regulates the expression of β-catenin or its downstream elements in SW480 colon cancer cells and HEK293 cells C. farreri testis cells cultured in vitro that the transcription of β-catenin was significantly down-regulated after treatment with quercetin for 48 h, particularly at concentrations of 100 µM Click here for additional data file.S2 TableExpression of β-catenin and Dax1 in in vitro cultured testis cells treated with DKK-1 and quercetin. A: Relative mRNA expression of β-catenin in cultured testis cells in vitro treated with DKK-1 for 48 h; B: Relative mRNA expression of Dax1 in cultured testis cells in vitro treated with DKK-1 for 48 h; C: Expression of β-catenin in cultured testis cells in vitro treated with quercetin for 48 h; D: Expression of Dax1 in cultured testis cells in vitro treated with quercetin for 48 h.(XLSX)Click here for additional data file.


Mc4r KO mice. Consequently, we hypothesized that other GPCRs involved in the control of energy homeostasis are likely to be regulated by MRAP2. In this study we identified PKR1 as the first non-melanocortin GPCR to be regulated by MRAP2. We show that MRAP2 significantly and specifically inhibits PKR1 signaling. We also demonstrate that PKR1 and MRAP2 co-localize in neurons and that Mrap2 KO mice are hypersensitive to PKR1 stimulation. This study not only identifies new partners of MRAP2 but also a new pathway through which MRAP2 regulates energy homeostasis.The Melanocortin Receptor Accessory Protein 2 (MRAP2) is an important regulator of energy homeostasis and its loss causes severe obesity in rodents. MRAP2 mediates its action in part through the potentiation of the MC4R, however, it is clear that MRAP2 is expressed in tissues that do not express MC4R, and that the deletion of MRAP2 does not recapitulate the phenotype of DOI:http://dx.doi.org/10.7554/eLife.12397.001 The brain plays a major role in controlling how much food animals eat. The nerve cells (neurons) involved in this process contain “receptors” that respond to cues from various parts of the body. For example, a receptor called PKR1 acts to limit food intake. The activities of PKR1 and other receptors are tightly regulated in cells, but it is not clear how this works.A protein called MRAP2 is known to regulate the activity of a receptor that regulates food intake and energy use in the brain. However, MRAP2 may also interact with other receptors to control food intake. Here, Chaly, Srisai et al. investigated whether MRAP2 can regulate the activity of PKR1 in animal cells and rodents.The experiments show that MRAP2 can interact with and inhibit the activity of PKR1. Furthermore, both MRAP2 and PKR1 can be found in the same neurons. Mutant mice that lack the gene that encodes MRAP2 have higher levels of PKR1 activity and eat less than normal mice when PKR1 is stimulated. Together the experiments suggest that MRAP2 can increase food intake by preventing PKR1 from being activated in the brain. The next steps are to find out if this protein regulates other receptors involved in the control of food intake, and to test whether PKR1 and MRAP2 also play a role in regulating energy usage.DOI:http://dx.doi.org/10.7554/eLife.12397.002 G-Protein Coupled Receptors (GPCRs) are responsible for a wide variety of physiological functions, and their activity is precisely regulated, not only by their ligands (agonists and in some cases antagonists) but also by other factors including G-proteins, β-arrestins, kinases, lipids, ions and accessory proteins . GPCR acout/Cin and Nin/Cout orientation, with MRAPs of opposite orientation forming anti-parallel homodimers . Activation of PKR1 in vivo, through central or peripheral injection of its ligand prokineticin 2 (PK2), was shown to significantly decrease food intake . In addiIn this study we identify PKR1 as the first non-melanocortin receptor to be regulated by MRAP2 and discover a novel mechanism of regulation of energy homeostasis by MRAP2 through the modulation of PKR1 signaling.Mrap2 KO mouse because of the way this mouse model was generated. Indeed, to produce this mouse, the cassette was inserted in the intron between exon 3 and 4 before the region coding for the transmembrane domain of MRAP2, thus allowing the N-terminal region to be produced (details can be found on the EUCOMM website). This region by itself is soluble and non-functional but can be recognized by the polyclonal MRAP2 antibody. Due to the lack of specific antibody to detect PKR1 in-vivo, we generated a mouse expressing a tagged PKR1. In order for PKR1 expression level, localization and activity to remain unchanged in this mouse model, we used the CRISPR / Cas9 technology to insert 2XHA tag immediately after the start codon of the endogenous PKR1 gene in chromosome 6 , the adrenal glands, the lungs, the spleen and the kidneys, but also, at lower level, in the heart and the pancreas . Both PKmosome 6 . The insmosome 6 . Using ikr1 mice . In contstaining . Finallystaining .10.7554/To determine if MRAP2 is a regulatory protein of PKR1, we first assessed the ability of those two proteins to form a complex. To this end, we transfected CHO cells with 2HA-PKR1 and MRAP2-3Flag and pulled down either PKR1 with a monoclonal anti-HA antibody or MRAP2 with a monoclonal anti-Flag antibody from the cell lysates. The precipitated proteins were then identified by western blot using an anti-HA antibody to detect the receptor and an anti-flag antibody to detect MRAP2. We observed that a significant fraction of PKR1 co-immunoprecipitates with MRAP2  and thatWe then assessed the effect of MRAP2 on PKR1 signaling. Prokineticin receptors are coupled to both the Gαs and the Gαq signaling pathways. In the former they activate adenylyl cyclase, leading to the production of cAMP. In the latter they activate phospholipases, leading to the production of IP3 and to the release of calcium from the ER. In order to measure the effects of MRAP2 on PKRs signaling through the Gαs pathway, CHO cells were transfected with the CRE-luciferase reporter and either PKR1 or PKR2 in the presence or absence of MRAP2. We find that MRAP2 dose-dependently inhibits PKR1 efficacy and decreases its potency four to five fold with a maximum effect at a receptor to MRAP2 DNA ratio between 1:7 and 1:10 . For thiBecause PKR1 can also couple to Gαq, we tested the effect of MRAP2 on PKR1-mediated production of IP3. For those experiments, we used CHO-M1 cells, which stably express the Gαq-coupled muscarinic M1 receptor; this allowed us to use the response to the M1R agonist carbachol to normalize the results and correct for possible differences in growth rates in the different transfection conditions. The assay used measures the accumulation of IP1, a degradation product of IP3, in the presence of lithium, an inhibitor of the inositol monophosphatase. We found that, as with the cAMP pathway, MRAP2 significantly inhibits PKR1-mediated IP3 production in response to both PK1  and PK2 Since several GPCR accessory proteins have been shown to modulate GPCR trafficking , we testWe also investigated the ability of MRAP2 to regulate PKR2, a GPCR closely related to PKR1. We transfected CHO cells with 2HA- PKR2 and MRAP2-3Flag and performed a co-immunoprecipitation as described earlier. A significant fraction of PKR2 was pulled down with MRAP2  and convIt is clear that MRAP2 can regulate multiple GPCRs, including PKRs, MC4R  and MC5RTo further validate our findings, we assessed the regulatory action of MRAP2 on PKR1 and PKR2 signaling  and trafMrap2 KO sibling mice were cannulated in the lateral ventricle of the brain. After recovering from the surgery, mice were fasted overnight and injected ICV with vehicle or the indicated dose of PK2 10 min prior to being given access to food. Food intake was then measured at 30 min, 1, 2, 4 and 6 hr. As expected the highest dose of PK2 significantly inhibited food intake in both WT and Mrap2 KO mice, however, the anorexigenic effect was more pronounced in Mrap2 KO mice  , rabbit anti-HA , mouse monoclonal anti-V5 , M2 anti-Flag , horseradish peroxidase (HRP)-conjugated antibodies against mouse and rabbit immunoglobulin  and anti-MRAP2 rabbit polyclonal antibody .HEK293T and CHO-K1 were purchased from ATCC, CHO-M1 cells (stably expressing the M1 muscarinic receptor) were obtained from Molecular Devices, GT1-1 cells were kindly provided by Dr. Richard Weiner, University of California San Francisco School of Medicine. All cell lines are mycoplasma free.2 at 37°C. For transfection, cells were grown to 70% confluency. Transfections were performed using LipoD293 in vitro transfection reagent  for all cells except GT1-1 cell that were transfected with Lipofectamine 3000. Total plasmid concentration was kept identical for all transfections by the addition of empty vector.HEK293T, CHO-K1, CHO-M1 and GT1-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin, in a humidified atmosphere consisting of 5% COn-dodecyl-β-maltoside in PBS with protease inhibitors. Lysates were centrifuged and supernatants were incubated with the indicated antibody (mouse anti-HA or mouse anti-Flag M2 ) at 1/5000 dilution overnight at 4°C. Immune complexes were collected with protein-G Dynabeads  at 4°C for 1 hr. Beads were washed three times and resuspended in LDS loading buffer with 5% β-mercaptoethanol and boiled 5 min. Proteins were resolved by SDS/PAGE and detected by western blot using rabbit anti-HA for PKR1 and PKR2 coimmunoprecipitation experiments, mouse anti-HA or anti-Flag for all other experiments. For MRAP2 deglycosylation, samples were incubated with PNGaseF for 1h at 37°C after the immunoprecipitation step.HEK293T cells transfected with the indicated plasmids were lysed in 0.1% Hercules, CA). Prokr1, Prokr2, Mrap2 and Gapdh were then amplified using the prepared cDNA as template and the following primers: mProkr1 Forward: 5’ CAC CAA CTT GCT TAT CGC CAA CC 3’; mProkr1 Reverse: 5’ GGC CAG ATC TGA CCA CAG AAG AT 3’; mProkr2 Forward: 5’ TGG CCA TCT CTG ACT TCC TGG T 3’; mProkr2 reverse:: 5’ TAG GAT TTG TAG TAG AGC TGC TGG T 3’; mMrap2 Forward: 5’TGT AAA GCC TGC GGT AAC CC 3’; mMrap2 Reverse: 5’ AGG ACT CCG CGT TGT CTT G 3’; Gapdh Forward: 5’ GGA GAG TGT TTG CTC GTC CC 3’ and Gapdh Reverse: 5’ ACT GTG CCG TTG AAT TTG CC 3’.Tissues were harvested from mice and mRNA was extracted using Trizol. Reverse transcription was carried out using 1 μg of mRNA per sample using the iScript kit , 12.2 mM Acetyl Coenzyme A; 30 μg/ml luciferin , was added. Samples were incubated at room temperature for 5 min and luminescence was then measured using a Spectramax I3 plate reader . Because CRE drives the expression of luciferase, the level of expression of the enzyme is proportional to the cAMP produced in the cell and luminescence serves as a reporter of cAMP production. In addition to the CRE-luciferase reporter, cells were transfected with a constant amount of PKR1 and increasing concentrations of MRAP2 plus enough empty vector to maintain a constant total DNA concentration. Cells were then treated with vehicle or with the indicated concentration of PK2 prior to measuring the cAMP production. In order to correct for possible variations in transfection efficiencies, the results were normalized to the signal obtained in response to 20 μM forskolin in each condition. The results were then normalized to the highest signal obtained in cells expressing PKR1 and CRE-luc in the absence of MRAP2.CHO-K1 cells were plated in 10 cm dishes. The next day, they were transiently transfected with a plasmid encoding the CRE-luciferase reporter , along with PKR1 or PKR2 and either empty vector or MRAP2 (1:10 ratio receptor to MRAP2). 24 hr following transfection, the cells were transferred to a white 96-well plate and left to adhere overnight. Cells were then incubated with vehicle or varying concentrations of peptide agonist (PK1 or PK2) or 20 μM forskolin, in DMEM/F-12 supplemented with 0.1% bovine serum albumin (BSA) for 4 hr at 37°C. The medium was removed and 100 μl of luciferin in lysis buffer, 200 mM Tris-HCl, 10 mM MgClWaltham, MA) following manufacturer’s manual. Plate was read with a Spectramax i3 plate reader equipped with a HTRF (homogeneous time resolved fluorescence) module.CHO cells in 10 cm dishes were transfected with indicated plasmids. The next day, cells were plated in a white 96 well plate and allowed to adhere overnight. Cells were then incubated with agonist at the indicated concentration in the presence of 0.1 mM 3-isobutyl-1-methylxanthine for 30 min at 37°C. Cells were then processed using the LANCE cAMP assay kit , with 2HA-PKR1 or 2HA-PKR2 and empty vector, or with 2HA-PKR1 or 2HA-PKR2 and MRAP2. 24 hr after transfection, the cells were washed with PBS, fixed for 10 min with 4% paraformaldehyde in PBS, washed, blocked in either 5% milk in PBS (surface-expression assay) or 5% milk in RIPA buffer  . Cells were then incubated with 1/5000 anti-HA antibody in blocking buffer for 2 hr at RT, washed three times for 5 min in PBS, incubated with 1/5000 anti-mouse-HRP antibody for 1 hr RT, and washed three times with PBS for 5 min. ELISA substrate  was then added until blue color was visible, and the reaction was stopped with 10% sulfuric acid. Absorbance was measured at 450 nm using a Spectramax I3 plate reader.CHO-K1 cells were seeded on sterilized coverslips and transfected with MRAP2-Y1, PKR1-Y2 or PKR2-Y2 plus empty vector, or PKR1-Y2 or PKR2-Y2 plus MRAP2-Y1. The next day, cells were incubated at room temperature for 1 hr and nuclei were stained with 1 μg/mL Hoechst 33,342 (Life Technologies) for 15 min. Coverslips were then mounted in imaging chambers, and imaging was performed using a 60X objective with an Olympus IX83 inverted fluorescence microscope.Inositol phosphate assay was performed using the IP-One-Tb HTRF kit  following the manufacturer’s instructions. CHO-M1 cells were transfected with PKR1 or PKR2 plus either empty vector or MRAP2 at a ratio of 1:10 (receptor to MRAP2). The day before the experiment, the transfected cells were plated in a white 384-well plate at a density of 20,000 cells/well. The next day the cells were treated with different concentrations of PK1, PK2 or 1 μM carbachol in stimulation buffer containing lithium for 1 hr at 37°C, and then incubated with IP1-d2 conjugate and Anti-IP1 cryptate Tb in lysis buffer for 1 hr at room temperature. HTRF signal was read at 615 nm and 665 nm using a Spectramax I3 plate reader equipped with the HTRF Cisbio cassette. For each condition, signal obtained with 1 μM carbachol, an agonist of the M1 muscarinic receptor, was used for normalization. Results were then normalized to the highest signal from the control (receptor + empty vector).Prokr1 mice and wild type littermates were deeply anesthetized with isoflurane and perfused transcardially with PBS and subsequently with 4% PFA in PBS. The whole brains were dissected out and post-fixed in 4% PFA overnight at 4°C. The brains were put in 30% sucrose in PBS until sunk. 40 μm frozen brain sections were cut by a cryostat and treated with 0.4% Triton X-100 in PBS for 1 hr, subsequently incubated in 3% hydrogen peroxide in PBS for 30 min and rinsed two times with PBS before incubation in 10 mM sodium citrate at 80°C for 30 min. The sections were blocked in 5% normal goat serum in 0.4% Triton X-100 in PBS and then in Mouse on mouse (MOM) Ig block reagent at room temperature for 1 hr according to the manufacturing protocol , rinsed with PBS twice, further incubated in MOM diluent for 5 min and subsequently incubated in 1:300 mouse anti-HA antibody in MOM diluent for 30 min. The free floating sections were washed twice with PBS and incubated in 1:250 biotinylated anti mouse IgG antibody for 10 min, washed with PBS three times and incubated in 1:1000 MRAP2 rabbit antibody in 0.4% Triton X-100 in PBS supplemented with 5% normal goat serum overnight at 4°C. The sections were washed with 0.4% Triton X-100 in PBS three times and incubated in 1:250 Pierce high sensitivity streptavidin-HRP  mixed with 1:250 Alex Fluor 546 goat anti rabbit antibody for 1 hr in 5% normal goat serum, 0.4% triton X-100 in PBS. The sections were washed four times and incubated in 1:100 Alexa Fluor 647 tyramide in amplification buffer for 10 min according to the manufacturer protocol  and washed with 0.4% Triton X-100 in PBS four times. The sections were mounted on a glass slide and air-dried. Prolong diamond antifade mountant with DAPI was applied on the sections before images were acquired with a Leica SP8 STED confocal microscope.2HA-For immunofluorescence in cells, GT1-1-GFP and GT1-1-MRAP2 cells were plated on sterile coverslips, fixed in 4% PFA, blocked in 5% goat serum in PBS supplemented with 0.3% Triton X-100 for 1 hr, then incubated with anti-MRAP2 antibody at 1/1000 dilution in blocking buffer for 2 hr at room temperature, washed with PBS, incubated with anti-rabbit-alexa 546 sary antibody for 1 hr, washed with PBS. The coverslips were then mounted on slides with prolong diamond with dapi and imaged.Mc4r KO and sibling WT at age 6–7 week-old were anesthetized with 17.5 mg/ml ketamine/ 2.5 mg/ml xylazine mix at dose 0.1 ml per 20 g body weight. A stainless steel guide cannula  was stereotaxically placed into the lateral ventricle . Perioperatively, mice were administered subcutaneously 0.1 mg/kg buprenorphine. Animals were kept in a temperature and humidity controlled room under a 12 hr light/dark cycle and allowed to recover for seven days after surgery and were handled for five days before the start of the experiment. Animals were fasted overnight and injected ICV with vehicle, 0.25 μg or 0.65 μg PK2 in 3 µl saline 10 min before food was returned to the cages (only vehicle or 0.65 μg PK2 were used for the Mc4r KO experiments). Food in each cage was weighed at 30 min, 1, 2, 4 and 6 hr following re-feeding. A minimum of three days drug-free period was maintained between infusions. During this period, the animals were handled but not tested. The mice that had received PK2 at the first injection received saline at the second and the mice that received saline the first time received PK2.Individually housed male and female MRAP2 knockout mice and sibling WT or loxTB The cannula placement was verified using histological methods. Animals were euthanized with carbon dioxide and received an ICV injection with 2 µl of ink. Brains were removed and the lateral ventricles opened to check for ink staining. The minimal number of number of animals to be used was determined by power analysis.Mrap2 KO mice, statistics were measured using a one way ANOVA within the result for the different doses of PK2 at a single time point and for one genotype and one gender. Lines connect the results compared for which the statistical significance is noted. For food intake studies in WT and Mc4r KO mice, statistics were measured a T-test between the result for vehicle or PK2 injected within the same gender and same time point, asterisk were color coded for clarity. *p<0.05, **p<0.01 and ***p<0.001. Results are shown as mean ± SEM.All experiments were repeated separately a minimum of three times. Statistics were calculated as follow. For ELISA histograms, one-way ANOVA with Tukey post-test was used and significance was measured between the control (no MRAP2) and the test (with MRAP2). For food intake studies in WT and  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your work entitled "The Melanocortin Receptor Accessory Protein 2 Promotes Food Intake Through Inhibition of the Prokineticin Receptor-1" for consideration by Summary:Chaly and colleagues describe results from a series of studies investigating the functional interactions of melanocortin receptor accessory protein 2 (MRAP2) and the prokineticin receptor 1 (PKR1). The manuscript provides a strong series of in vitro experiments, all properly controlled, that MRAP2 directly interacts with PKR1 and PKR2 and MRAP2 inhibits the activity of the prokineticin receptors through both Gs and Gq signaling pathways. There was little evidence for altered trafficking of PKR1 to the cell surface, while MRAP2 decreased the cell surface expression of PKR2. These are novel and potentially important findings as they demonstrate interaction of MRAP2 with a non-melanocortin receptor, and they show inhibition of PKR signaling instead of activation, as with the melanocortin receptors. The studies are well described and performed and provide novel information that may be of wide interest. A few issues need to be addressed.Essential revisions:1) The majority of the signaling and interaction data is done using heterologous cell systems. While this is clearly useful and understandable the in vivo data is less developed. They report extensive co-expression of the PKRs and MRAP2 at the tissue level, and in individual cells in the arcuate nucleus of the hypothalamus. On the basis of the latter finding, the authors then tested whether icv injection of PK2 had differential effects on short-term food intake in wild-type and MRAP2 KO mice. PK2 appeared to be more potent in the MRAP2 KOs. Although somewhat supportive of the in vitro work performed with transfected CHO cells, clearly much more work beyond the scope of this manuscript will need to be done to further explore the interactions of PKRs and MRAP2 in vivo.2) The authors use a MRAP2 antisera to demonstrate that neurons co-express PRK1 mRNA. The photos provided are somewhat difficult to discern double labeling. More importantly, the authors demonstrate specificity of the antisera by using transfected cells. It is unclear why knockout brain tissue was not used to validate the antisera.3) Some more information is needed regarded the food intake paradigms. Specifically, is PK2 known to cause non-specific effects such as malaise or sickness like behavior when given at higher doses? If this has been described in the literature (or not) should be discussed at a minimum.4) Another concern with the Introduction to the paper is why the authors focused on PKRs in the first place. There are many GPCRs to test, but no specific rationale was provided to go after PKRs in the first place. It might make more sense to first present the expression data in 5) Another concern is the immunoprecipitation studies of 6) In 7) In 8)  1) The majority of the signaling and interaction data is done using heterologous cell systems. While this is clearly useful and understandable the in vivo data is less developed. They report extensive co-expression of the PKRs and MRAP2 at the tissue level, and in individual cells in the arcuate nucleus of the hypothalamus. On the basis of the latter finding, the authors then tested whether icv injection of PK2 had differential effects on short-term food intake in wild-type and MRAP2 KO mice. PK2 appeared to be more potent in the MRAP2 KOs. Although somewhat supportive of the in vitro work performed with transfected CHO cells, clearly much more work beyond the scope of this manuscript will need to be done to further explore the interactions of PKRs and MRAP2 in vivo.We appreciate that most of the in-vitro work is done in CHO cells and in order to confirm our findings in a more relevant system we tested the effect of MRAP2 on PKR1 trafficking and signaling in the GT1-1 hypothalamic neuronal cell line. Additionally, because another group suggested that PKR1 might be downstream of MC4R, we tested the requirement of MC4R signaling for the PKR1-mediated decrease in food intake by using MC4R KO mice. Finally, we agree with the reviewers that more work needs to be done on the role of PKR1 and MRAP2 in- vivo and that these studies are indeed out of the scope of this manuscript, however, it will be addressed in a following study on the physiology of the PKR1/MRAP2 complex.2) The authors use a MRAP2 antisera to demonstrate that neurons co-express PRK1 mRNA. The photos provided are somewhat difficult to discern double labeling. More importantly, the authors demonstrate specificity of the antisera by using transfected cells. It is unclear why knockout brain tissue was not used to validate the antisera.We agree with the reviewers that being able to use the MRAP2 KO as a negative control for the immunofluorescence would have been ideal, however, like we now clarify in the body of the manuscript, the MRAP2 KO still produces a non-functional but immunogenic fragment of MRAP2. In order to further prove the specificity of the MRAP2 antisera (in addition to the western blot previously included) we validated it for immunofluorescence using control GT1-cells (not expressing MRAP2) and GT1-1 line stably expressing MRAP2.We also understand why the reviewers found the PKR1 staining too weak and not convincing but we were limited to in-situ hybridization due to the lack of a validated antibody raised against PKR1. In order to definitively establish the colocalization of PKR1 and MRAP2 in the brain we took advantage of a new mouse model we recently generated using CRISPR/Cas9 in which we inserted 2HA tags after the start codon of the endogenous gene, thus allowing us to detect PKR1 with the highly specific anti-HA antibody without perturbing PKR1 expression level, localization or function We hope the new microscopy results will be satisfactory.3) Some more information is needed regarded the food intake paradigms. Specifically, is PK2 known to cause non-specific effects such as malaise or sickness like behavior when given at higher doses? If this has been described in the literature (or not) should be discussed at a minimum.We now discuss the fact that in our hands and in published work, PK2 injection has not been observed to cause malaise or sickness like behavior. It was an important point, thank you for bringing it up.4) Another concern with the Introduction to the paper is why the authors focused on PKRs in the first place. There are many GPCRs to test, but no specific rationale was provided to go after PKRs in the first place. It might make more sense to first present the expression data in This is a good point and even though we mention that we did test numerous GPCRs involved in energy homeostasis and found that several were not regulated by MRAP2  we can see how this may feel random. For this reason, we went ahead and followed your advice to move 5) Another concern is the immunoprecipitation studies of It is true that even though most GPCRs run as several bands and that the high molecular weight smear has been shown to be a glycosylated form for many of them, we did not provide proof that it was the case for PKR1 and PKR2. In response to your comment we tried different approaches. First, we mutated the asparagine of the 2 predicted glycosylation sites to glutamine in order to prevent glycosylation of the receptors but those mutations caused the receptors to not be expressed. We then attempted to deglycosylate the receptors with PNGase F and with an Endo F1, F2, F3 mix but the receptors were mostly resistant to enzymatic deglycosylation. Because the glycosylation state of the receptors is irrelevant to our finding that MRAP2 interacts and regulate PKR1 in-vitro and in-vivo, and because we could not prove that the high molecular weight smears were glycosylated forms of the receptor, we opted to remove the label “glycosylated PKRs” from the figures. We were, however, successful in deglycosylating MRAP2 and showing that all bands collapse to the lowest molecular weight band. This result is now included in 6) In The statistics are now more detailed in the Methods section and the values that are compared are now linked by solid lines for clarity. We now discuss the effect of MRAP2 on PKR1 and PKR2 potency in the Results section. The “PKR2” label in the former 7) In In the former 8) The actual amount of PK2 is now indicated and the statistical analysis has been clarified in the Methods. Compared values are linked with lines in the figure.


Here we show that exposure to youthful circulation by heterochronic parabiosis reverses the aged fracture repair phenotype and the diminished osteoblastic differentiation capacity of old animals. This rejuvenation effect is recapitulated by engraftment of young haematopoietic cells into old animals. During rejuvenation, β-catenin signalling, a pathway important in osteoblast differentiation, is modulated in the early repair process and required for rejuvenation of the aged phenotype. Temporal reduction of β-catenin signalling during early fracture repair improves bone healing in old mice. Our data indicate that young haematopoietic cells have the capacity to rejuvenate bone repair and this is mediated at least in part through β-catenin, raising the possibility that agents that modulate β-catenin can improve the pace or quality of fracture repair in the ageing population.The capacity for tissues to repair and regenerate diminishes with age. We sought to determine the age-dependent contribution of native mesenchymal cells and circulating factors on  The reparative capacity of tissues decreases with age. Here, Baht et al. perform parabiosis experiments and provide evidence for the existence of a soluble factor produced by young, but not old, haematopoietic cells that is capable of improving fracture repair in old mice. Accordingly, bone repair and related processes, such as osseous integration of implants, occurs at a slower pace in older patients than in younger patients. This results in the need for additional surgical procedures to attain proper bone healing and is associated with increased rates of morbidity and mortality in the elderly1in vivo bone-fracture repair and in vitro osteoblast differentiation. This rejuvenation was independent of young osteoblastic cells and relied on a signal to the endogenous, aged osteoblasts to increase bone matrix deposition and mineralization. A similar rejuvenation was observed upon engraftment of young bone marrow (young haematopoietic cells) into aged mice. In cell culture conditions, media conditioned by young BMSCs were able to rejuvenate osteoblastogenesis of old cells. Modulation of the β-catenin protein level was found to be required for this rejuvenation. Decreasing levels of β-catenin early during fracture repair was able to rescue the aged fracture-repair phenotype.We sought to determine the effect of mesenchymal cell age and the age of the circulation on bone regeneration and osteoblast differentiation to understand the mechanism by which repair rate is slowed. Exposure to a young circulation rejuvenated This work demonstrates that diminished tissue-regeneration capacity is at least partly dependent on the regulation of β-catenin levels in the cell. With age, signalling of the canonical β-catenin pathway increases11The capacity for bone-fracture repair and osteoblastic differentiation potential diminish with age . Heterocin vitro osteogenic potential, but these changes were not significant  relative to control  rather than haematopoietic differentiation. During this process, tissue culture plastic-adherent haematopoietic cells may remain attached to the culture surface. To determine the proportion of haematopoietic lineage cells in our BMSC ‘conditioning cultures', we used flow cytometry to identify the percentage of CD45+ cells. We found that as much as 50% of cells adhered to the tissue culture surface are CD45+ at the initiation of differentiation. Thus, in our Temporal regulation of β-catenin is important for robust differentiation of mesenchyaml precursors to osteoblasts. Indeed, β-catenin levels are elevated during the early phases of bone fracture repair and dysregulation either to higher or lower levels inhibits normal healing. Dysregulation of β-catenin has been implicated in age-dependent changes in tissue regeneration9Axin2 transcript, a β-catenin target genein vitro), differentiating osteoblasts were lower  and is associated with decreased amounts of fibrotic tissue being deposited in the fracture callus. Mice in which the aged fracture repair was rejuvenated though heterochronic parabiosis showed a reduced level of total β-catenin, activated (unphosphorylated) β-catenin protein and To confirm the importance of β-catenin modulation during rejuvenation, conditional β-catenin-stabilized and β-catenin-null mice were engrafted with young, EYFP+ bone marrow. The protein produced by the stabilized or null alleles of β-catenin lack the ability to be normally regulated by canonical Wnt signalling. In recipient mice expressing the stabilized or null β-catenin alleles, engraftment with young bone narrow cells did not alter the observed fracture repair phenotype, despite rejuvenation of the control animals . These dAge-dependent differences in β-catenin levels between old and young fracture calluses were greatest during early fracture repair . β-caten10Here we demonstrated that aged fracture repair was rejuvenated through the modulation of β-catenin by exposure to a youthful circulation. Heterochronic parabiosis or engraftment of young bone marrow was able to rejuvenate bone repair and osteoblast differentiation. Within the population of bone marrow cells, CD45+ haematopoietic cells play an important role in this reversal of age. This rejuvenation was driven by a factor that was able to modulate Wnt/β-catenin signalling. Having demonstrated the necessity of β-catenin modulation, we also show that decreasing levels of β-catenin in old fracture calluses early in fracture repair can rejuvenate the fracture repair process.Interestingly, these findings indicate a potential role for inflammation in rejuvenation of fracture repair. With age, this inflammatory response due to injury changes leads to increased fibrosis and inefficient tissue regeneration. In bone marrow-derived rejuvenation, only CD45+ haematopoietic cells were engrafted into the marrow space. These cells are responsible for the inflammatory response during injury and, as such, these cells likely result in a rejuvenated inflammatory response. Several studies, including work from our group, have confirmed the importance of an appropriate inflammatory response for effective fracture repair3738Tg(CAC-cre/Esr1*)5AMC/J mice, which express Cre-recombinase in a ubiquitous chicken β-actin promoter. Cre-recombinase is restricted to the cytoplasm; however, treatment with tamoxifen allows localization to the nucleus, leading to recombination of the target gene. Tamoxifen was administered using two intraperitoneal injections per week (100 mg kg−1) beginning 1 week before and for the duration after fracture. Recombination of the conditional alleles was observed to be ∼80% at the fracture sites, confirming our previous observationsAll mice were housed at the Toronto Centre for Phenogenomics and all animal use protocols were approved by the Animal Care Committee at the Toronto Centre for Phenogenomics. EYFP+ mice [129-Tg(Actb-EYFP)2Nagy] express enhanced yellow fluorescent protein under control of the ubiquitous chicken β-actin promoter8 p.f.u. of adenovirus harbouring Dickkopf-1 cDNA tagged with a C-terminal hexa-His tag or adenovirus harbouring GFP was injected at the fracture site 1-day pre-fractureFor temporal decrease in β-catenin, 2 × 10−g) in saline was delivered using peritoneal injections at 7 and 2 days before harvest of the fracture callus. Specimens were fixed in 70% ethanol and embedded in methylmethacrylate. Sections were then cut to 5 μm, and fluorescent microscopy imaging  was used to observe the dye and the distance between the dye fronts was measured.Fluorochrome labelling was used to investigate active bone deposition. Calcein green  and heterochronic (young–old or old–young) pairs were established. A shared blood supply was ensured 3 weeks after surgery using FLOW cytometry6 cells in 200 μl PBS). Engraftment into the bone marrow was allowed to occur for 2 months and verified using FLOW cytometry.Two- or sixteen-month-old mice were irradiated (900cG) and tail vein-injected with bone marrow isolated from 2- or 16-month-old mice . EDTA  was used to decalcify bone for 4 weeks with two solution changes per week. Calluses were then embedded in paraffin and sectioned at 5 μm in thickness. Samples were stained using Safranin-O and Fast Green. Here red staining confirms the presence of proteoglycans that indicates the deposition of cartilagenous tissue. Representative sections from a minimum of five fractured limbs were analysed using computer-assisted histomorphometry analysis and results were presented as percent of bone, fibrous or cartilagenous tissue deposited relative to fracture callus.−l Penn/Strep) for 7 days. Colony-forming units (CFUs) were defined as individual clusters of stained cells or matrix. To determine CFU-F, wells were washed with PBS, fixed with 10% formalin and stained with 0.25% crystal violet. For osteogenic differentiation, the media was changed to osteogenic media . The first day in osteogenic media was considered Day 1, and media were changed every 2 days thereafter. In conditioned media experiments, media was transferred directly from well to well after two days of conditioning. For heat denaturation, media was heated to 95 °C for 10 min and then cooled to 37 °C. After 15 days in differentiation media, cells were stained with ALP (indicate osteoblasts), Alizaren Red  or VK .BMSCs were aspirated from tibiae of unfractured mice, and progenitor cells were isolated on the basis of their adherence to tissue culture plastic. Cells were plated in plating media , BSP (Mm00492555_m1) and Col1 (Mm00801666_g1) were purchased from Applied Biosystems. For western blot analysis, indicated antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL). Relative band intensities were quantified using the ImageJ software ( http://imagej.nih.gov/ij/). Anti-β-catenin antibody (working concentration of 1.0 μg ml−l—06-734) was purchased from Millipore, anti-active β-catenin antibody (working concentration of 1.5 μg ml−1—05-665) was purchased from Millipore, anti-β-actin antibody (working concentration of 0.5 ng ml−1—CP01) was purchased from Calbiochem and anti-His antibody (working concentration of 0.2 ng ml−1—s-804) was purchased from Santa Cruz. Full western blot images are shown in Total RNA or protein was extracted from fracture calluses after dissection. The proximal and distal borders of the fracture callus are roughly outlined by dashed lines in the low-magnification histological images. For real-time PCR, cDNA template was generated using random hexamers and data were related to the transcript of ribosomal protein 18S as a housekeeping control. Primers for P<0.05).Data are expressed as mean±95% confidence interval. One-way analysis of variance was performed followed by Dunnett's test when significant differences were detected. The data were considered to be statistically significant at a confidence level of 95% .Supplementary Figures 1-12