A dozen years ago the identification of causal mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene involved in two rare bone disorders propelled research in the bone field ...in totally new directions. Since then, there have been an explosion in the number of reports that highlight the role of the Wnt/β-catenin pathway in the regulation of bone homeostasis. In this review we discuss some of the most recent reports (in the past 2 years) highlighting the involvement of the members of the LRP family (LRP5, LRP6, LRP4, and more recently LRP8) in the maintenance of bone and their implications in bone diseases. These reports include records of new single nucleotides polymorphisms (SNPs) and haplotypes that suggest variants in these genes can contribute to subtle variation in bone traits to mutations that give rise to extreme bone phenotypes. All of these serve to further support and reinforce the importance of this tightly regulated pathway in bone. Furthermore, we discuss provocative reports suggesting novel approaches through inhibitors of this pathway to treat rarer diseases such as Osteoporosis-Pseudoglioma Syndrome (OPPG), Osteogenesis Imperfecta (OI), and Sclerosteosis/Van Buchem disease. It is hoped that by understanding the role of each component of the pathway and their involvement in bone diseases that this knowledge will allow us to develop new, more effective therapeutic approaches for more common diseases such as post-menopausal osteoporosis, osteoarthritis, and rheumatoid arthritis as well as these rarer bone diseases.
Abstract The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary ...mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo , we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1 h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24 h. This early activation at 1 h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro , PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24 h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo.
Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton. Recently considerable focus has been placed on signaling ...factors/molecules produced by these two tissues that may act to modulate the function of the other tissue. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5–6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 με. At 24 h post-load we observed a 2.5-fold increase in β-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active β-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based β-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1–2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6–8 fold increase in pAkt compared to a 3–4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of β-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.
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•Muscle paraylysis prevents load-induced osteocyte β-catenin signaling.•Contracted EDL muscle produces factor(s) that induce β-catenin signalning.•C2C12 myotubes factor(s) synergize with Wnt3a to induce β-catenin signaling.•A luciferase based MLO-Y4 β-catenin reporter cell line was developed.
It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have ...positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.
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This study presents a criterion for predicting the martensitic variants (MVs) that appear during the stress-induced martensitic transformation (SIMT) in a polycrystalline sample of ...Cu–11.5% wt. Al–0.5% wt. Be under simple tension. Our criterion is based on crystallographic parameters, such as the crystal orientation and Schmid factor (SF). The displacement vector fields (DVFs) were obtained in the observation system by a mathematical model and were used to distort the boundary of a set of grains. From the DVF, the strain tensor for each grain was obtained, and the strain ratio (SR) in the observation system was calculated. Electron backscattering diffraction (EBSD) measurements were performed to determine the crystal orientation of the grains. The inverse SF was used to determine the in-plane stress transformation diagrams (STDs) for each studied grain. The combination of a balance criterion (BC) and STD provided a criterion that allowed us to predict the possible order of stress-induced MVs formed as a function of the crystal orientation and thermomechanical parameters of the shape memory alloy (SMA) with higher accuracy than when using the criteria separately. To validate our criteria, we tested other researchers’ published results. Our results were in agreement and were capable of predicting the stress-induced MVs in a polycrystalline SMA.
It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have ...positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from
ex vivo
electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.
Genetic factors involved in the susceptibility to drug addiction still remain largely unknown. MiRNAs seem to play key roles in the drug-induced plasticity of the brain that likely drives the ...emergence of addiction. In this work we explored the role of miRNAs in drug addiction. With this aim, we selected 62 SNPs located in the 3'UTR of target genes that are predicted to alter the binding of miRNA molecules and performed a case-control association study in a Spanish sample of 735 cases (mainly cocaine-dependent subjects with multiple drug dependencies) and 739 controls. We found an association between rs1047383 in the PLCB1 gene and drug dependence that was replicated in an independent sample (663 cases and 667 controls). Then we selected 9 miRNAs predicted to bind the rs1047383 region, but none of them showed any effect on PLCB1 expression. We also assessed two miRNAs binding a region that contains a SNP in linkage disequilibrium with rs1047383, but although one of them, hsa-miR-582, was found to downregulate PLCB1, no differences were observed between alleles. Finally, we explored the possibility that PLCB1 expression is altered by cocaine and we observed a significant upregulation of the gene in the nucleus accumbens of cocaine abusers and in human dopaminergic-like neurons after cocaine treatment. Our results, together with previous studies, suggest that PLCB1 participates in the susceptibility to drug dependence.
Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal ...abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated network-generated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models.
Little is known regarding the mechanisms underlying the loss of tolerance in the early and preclinical stages of autoimmune diseases. The aim of this work was to identify the transcriptional profile ...and signaling pathways associated to non-treated early rheumatoid arthritis (RA) and subjects at high risk. Several biomarker candidates for early RA are proposed.
Whole blood total RNA was obtained from non-treated early RA patients with <1 year of evolution as well as from healthy first-degree relatives of patients with RA (FDR) classified as ACCP+ and ACCP- according to their antibodies serum levels against cyclic citrullinated peptides. Complementary RNA (cRNA) was synthetized and hybridized to high-density microarrays. Data was analyzed in Genespring Software and functional categories were assigned to a specific transcriptome identified in subjects with RA and FDR ACCP positive. Specific signaling pathways for genes associated to RA were identified. Gene expression was evaluated by qPCR. Receiver operating characteristic (ROC) analysis was used to evaluate these genes as biomarkers.
A characteristic transcriptome of 551 induced genes and 4,402 repressed genes were identified in early RA patients. Bioinformatics analysis of the data identified a specific transcriptome in RA patients. Moreover, some overlapped transcriptional profiles between patients with RA and ACCP+ were identified, suggesting an up-regulated distinctive transcriptome from the preclinical stages up to progression to an early RA state. A total of 203 pathways have up-regulated genes that are shared between RA and ACCP+. Some of these genes show potential to be used as progression biomarkers for early RA with area under the curve of ROC > 0.92. These genes come from several functional categories associated to inflammation, Wnt signaling and type I interferon pathways.
The presence of a specific transcriptome in whole blood of RA patients suggests the activation of a specific inflammatory transcriptional signature in early RA development. The set of overexpressed genes in early RA patients that are shared with ACCP+ subjects but not with ACCP- subjects, can represent a transcriptional signature involved with the transition of a preclinical to a clinical RA stage. Some of these particular up-regulated and down-regulated genes are related to inflammatory processes and could be considered as biomarker candidates for disease progression in subjects at risk to develop RA.
We present the first catalog of TeV gamma-ray sources realized with data from the newly completed High Altitude Water Cherenkov Observatory (HAWC). It is the most sensitive wide field-of-view TeV ...telescope currently in operation, with a one-year survey sensitivity of ∼5%-10% of the flux of the Crab Nebula. With an instantaneous field of view >1.5 sr and >90% duty cycle, it continuously surveys and monitors the sky for gamma-ray energies between hundreds of GeV and tens of TeV. HAWC is located in Mexico, at a latitude of 19° N, and was completed in 2015 March. Here, we present the 2HWC catalog, which is the result of the first source search performed with the complete HAWC detector. Realized with 507 days of data, it represents the most sensitive TeV survey to date for such a large fraction of the sky. A total of 39 sources were detected, with an expected number of false detections of 0.5 due to background fluctuation. Out of these sources, 19 are new sources that are not associated with previously known TeV sources (association criteria: <0 5 away). The source list, including the position measurement, spectrum measurement, and uncertainties, is reported, then each source is briefly discussed. Of the 2HWC associated sources, 10 are reported in TeVCat as PWN or SNR: 2 as blazars and the remaining eight as unidentified.
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