TRAF-6 Dependent Signaling Pathway Is Essential for TNF-Related Apoptosis-Inducing Ligand (TRAIL) Induces Osteoclast Differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017) includes a comprehensive assessment of incidence, prevalence, and years lived with disability (YLDs) for 354 causes in 195 countries and territories from 1990 to 2017. Previous GBD studies have shown how the decline of mortality rates from 1990 to 2016 has led to an increase in life expectancy, an ageing global population, and an expansion of the non-fatal burden of disease and injury. These studies have also shown how a substantial portion of the world's population experiences non-fatal health loss with considerable heterogeneity among different causes, locations, ages, and sexes. Ongoing objectives of the GBD study include increasing the level of estimation detail, improving analytical strategies, and increasing the amount of high-quality data. Methods: We estimated incidence and prevalence for 354 diseases and injuries and 3484 sequelae. We used an updated and extensive body of literature studies, survey data, surveillance data, inpatient admission records, outpatient visit records, and health insurance claims, and additionally used results from cause of death models to inform estimates using a total of 68 781 data sources. Newly available clinical data from India, Iran, Japan, Jordan, Nepal, China, Brazil, Norway, and Italy were incorporated, as well as updated claims data from the USA and new claims data from Taiwan (province of China) and Singapore. We used DisMod-MR 2.1, a Bayesian meta-regression tool, as the main method of estimation, ensuring consistency between rates of incidence, prevalence, remission, and cause of death for each condition. YLDs were estimated as the product of a prevalence estimate and a disability weight for health states of each mutually exclusive sequela, adjusted for comorbidity. We updated the Socio-demographic Index (SDI), a summary development indicator of income per capita, years of schooling, and total fertility rate. Additionally, we calculated differences between male and female YLDs to identify divergent trends across sexes. GBD 2017 complies with the Guidelines for Accurate and Transparent Health Estimates Reporting. Findings: Globally, for females, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and haemoglobinopathies and haemolytic anaemias in both 1990 and 2017. For males, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and tuberculosis including latent tuberculosis infection in both 1990 and 2017. In terms of YLDs, low back pain, headache disorders, and dietary iron deficiency were the leading Level 3 causes of YLD counts in 1990, whereas low back pain, headache disorders, and depressive disorders were the leading causes in 2017 for both sexes combined. All-cause age-standardised YLD rates decreased by 3·9% (95% uncertainty interval [UI] 3·1-4·6) from 1990 to 2017; however, the all-age YLD rate increased by 7·2% (6·0-8·4) while the total sum of global YLDs increased from 562 million (421-723) to 853 million (642-1100). The increases for males and females were similar, with increases in all-age YLD rates of 7·9% (6·6-9·2) for males and 6·5% (5·4-7·7) for females. We found significant differences between males and females in terms of age-standardised prevalence estimates for multiple causes. The causes with the greatest relative differences between sexes in 2017 included substance use disorders (3018 cases [95% UI 2782-3252] per 100 000 in males vs 1400 [1279-1524] per 100 000 in females), transport injuries (3322 [3082-3583] vs 2336 [2154-2535]), and self-harm and interpersonal violence (3265 [2943-3630] vs 5643 [5057-6302]). Interpretation: Global all-cause age-standardised YLD rates have improved only slightly over a period spanning nearly three decades. However, the magnitude of the non-fatal disease burden has expanded globally, with increasing numbers of people who have a wide spectrum of conditions. A subset of conditions has remained globally pervasive since 1990, whereas other conditions have displayed more dynamic trends, with different ages, sexes, and geographies across the globe experiencing varying burdens and trends of health loss. This study emphasises how global improvements in premature mortality for select conditions have led to older populations with complex and potentially expensive diseases, yet also highlights global achievements in certain domains of disease and injury

Global, regional, and national age-sex-specific mortality and life expectancy, 1950-2017: a systematic analysis for the Global Burden of Disease Study 2017

Background: Assessments of age-specific mortality and life expectancy have been done by the UN Population Division, Department of Economics and Social Affairs (UNPOP), the United States Census Bureau, WHO, and as part of previous iterations of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD). Previous iterations of the GBD used population estimates from UNPOP, which were not derived in a way that was internally consistent with the estimates of the numbers of deaths in the GBD. The present iteration of the GBD, GBD 2017, improves on previous assessments and provides timely estimates of the mortality experience of populations globally. Methods: The GBD uses all available data to produce estimates of mortality rates between 1950 and 2017 for 23 age groups, both sexes, and 918 locations, including 195 countries and territories and subnational locations for 16 countries. Data used include vital registration systems, sample registration systems, household surveys (complete birth histories, summary birth histories, sibling histories), censuses (summary birth histories, household deaths), and Demographic Surveillance Sites. In total, this analysis used 8259 data sources. Estimates of the probability of death between birth and the age of 5 years and between ages 15 and 60 years are generated and then input into a model life table system to produce complete life tables for all locations and years. Fatal discontinuities and mortality due to HIV/AIDS are analysed separately and then incorporated into the estimation. We analyse the relationship between age-specific mortality and development status using the Socio-demographic Index, a composite measure based on fertility under the age of 25 years, education, and income. There are four main methodological improvements in GBD 2017 compared with GBD 2016: 622 additional data sources have been incorporated; new estimates of population, generated by the GBD study, are used; statistical methods used in different components of the analysis have been further standardised and improved; and the analysis has been extended backwards in time by two decades to start in 1950. Findings: Globally, 18·7% (95% uncertainty interval 18·4–19·0) of deaths were registered in 1950 and that proportion has been steadily increasing since, with 58·8% (58·2–59·3) of all deaths being registered in 2015. At the global level, between 1950 and 2017, life expectancy increased from 48·1 years (46·5–49·6) to 70·5 years (70·1–70·8) for men and from 52·9 years (51·7–54·0) to 75·6 years (75·3–75·9) for women. Despite this overall progress, there remains substantial variation in life expectancy at birth in 2017, which ranges from 49·1 years (46·5–51·7) for men in the Central African Republic to 87·6 years (86·9–88·1) among women in Singapore. The greatest progress across age groups was for children younger than 5 years; under-5 mortality dropped from 216·0 deaths (196·3–238·1) per 1000 livebirths in 1950 to 38·9 deaths (35·6–42·83) per 1000 livebirths in 2017, with huge reductions across countries. Nevertheless, there were still 5·4 million (5·2–5·6) deaths among children younger than 5 years in the world in 2017. Progress has been less pronounced and more variable for adults, especially for adult males, who had stagnant or increasing mortality rates in several countries. The gap between male and female life expectancy between 1950 and 2017, while relatively stable at the global level, shows distinctive patterns across super-regions and has consistently been the largest in central Europe, eastern Europe, and central Asia, and smallest in south Asia. Performance was also variable across countries and time in observed mortality rates compared with those expected on the basis of development. Interpretation: This analysis of age-sex-specific mortality shows that there are remarkably complex patterns in population mortality across countries. The findings of this study highlight global successes, such as the large decline in under-5 mortality, which reflects significant local, national, and global commitment and investment over several decades. However, they also bring attention to mortality patterns that are a cause for concern, particularly among adult men and, to a lesser extent, women, whose mortality rates have stagnated in many countries over the time period of this study, and in some cases are increasing

A model for TRAIL-induced activation of the TRAF6 associated signaling pathway in osteolcast differentiation.

<p>Upon receptor engagement, in addition to transduce apoptosis signaling pathway, TRAIL induces activation of TRAF6, at least in part through the E3 ubiquitin ligase activity of the TRAF6 RING finger domain via a distinct signaling pathway. In osteoclast precursors, TRAIL is not able to induce sufficient amount of signals to activate the downstream apoptosis signaling to induce apoptosis; instead, TRAIL induces signaling to activate TRAF6 after receptor engagement. TRAF6 is the key molecule linking cytoplasmic signaling to the nuclear transcriptional program in osteoclast differentiation and bone resorption. These signals include activation of NF-κB by a process of ubiquitination in which TRAF6 functions as an E3 ligase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038048#pone.0038048-Deng1" target="_blank">[36]</a> and recruitment of the TAB1-TAB2-TAK1 complex, which lead to the osteoclast-specific event, that is, autoamplification of NFATc1, the master transcription factor for osteoclast differentiation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038048#pone.0038048-NegishiKoga1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038048#pone.0038048-Takayanagi2" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038048#pone.0038048-Takayanagi3" target="_blank">[31]</a>.</p

TRAIL-induced activation of NFATc1 in osteoclast differentiation is dependent on TRAF6.

<p>(A). RAW264.7 cells were treated with TRAIL, M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA, TRAIL receptor (TRAIL-R) siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. After stimulation, cells lysates of the nuclear fraction were prepared, and immunoblotted with anti-NFATc1 and anti-HDAC antibodies. The results are representative of three separate experiments. (B).TRAF6 is ubiquitinated after TRAIL stimulation. Ubiquitin transfected RAW 264.7 cells were treated with TRAIL in the presence or absence of TRAF6 siRNA, TRAIL-R siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. The cell lyses were immunoprecipitated with anti-TRAF6 mAb. Bound proteins were subjected to SDS–PAGE and immunoblotted with anti-Ubiquitin mAb, and then the membrane was stripped and reprobed with anti-TRAF6 mAb. The results are representative of three separate experiments.</p

TRAIL-induced osteoclast differentiation was abolished in TRAF6 knock out bone marrow-derived macrophages.

<p>(A) Bone marrow-derived macrophages isolated from TRAF6 knock out (TRAF6 KO) mice were treated with TRAIL (500 ng/ml) or RANKL (100 ng/ml) with M-CSF (200 ng/ml) for 7 days. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from bone marrow-derived macrophages isolated from wild type and TRAF6 KO mice. Data in (B) represent the mean±SD from three independent experiments. *<i>p</i> < 0.005.</p

TRAIL induced osteoclast differentiation is dependent on TRAF6.

<p>(A) Human peripheral blood mononuclear cells (PBMCs) were plated in 96-well plates at 1.5 × 10⁵ cells/well, and the next day the adherent monocytes were treated with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated in the figure for 30 days. The transfection of siRNAs into human macrophages cells was performed by electroporation. The TRAF6 inhibitory peptide, T6PD and its control peptide were used to examine its effects on TRAIL-mediated osteoclast differentiation. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. The bar in each figure represents 100 µM. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from human monocytes. Data in (B) represent the mean±SD from three to six independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.005.</p

TRAIL-induced osteoclast bone resorption activity is TRAF6-dependent.

<p>(A). Human monocytes were plated on an artificial bone matrix slide (calcium phosphate apatite-coated plate; OAAS) and were cultured with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated. Cells were detached after 30 days of culture. The number of resorption pits and resorptive area in each well was observed and counted using a microscope. (B). The resorption pits were photographed and the resorptive area in each well was counted. Data in (B) represent the mean±SD from five independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.005.</p