The adaptor protein p62/SQSTM1 in osteoclast signaling pathways

Paget's disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover and overactive osteoclasts. The discovery of mutations in the SQSTM1/p62 gene in numerous patients has identified protein p62 as an important modulator of bone turnover. In both precursors and mature osteoclasts, the interaction between receptor activator of NF-κB ligand (RANKL) and its receptor RANK results in signaling cascades that ultimately activate transcription factors, particularly NF-κB and NFATc1, promoting and regulating the osteoclast differentiation, activity, and survival. As a scaffold with multiple protein-protein interaction motifs, p62 is involved in virtually all the RANKL-activated osteoclast signaling pathways, along with being implicated in numerous other cellular processes. The p62 adaptor protein is one of the functional links reported between RANKL and TRAF6-mediated NF-κB activation, and also plays a major role as a shuttling factor that targets polyubiquitinated proteins for degradation by either the autophagy or proteasome pathways. The dysregulated expression and/or activity of p62 in bone disease up-regulates osteoclast functions. This review aims to outline and summarize the role of p62 in RANKL-induced signaling pathways and in ubiquitin-mediated signaling in osteoclasts, and the impact of PDB-associated p62 mutations on these processes

Regulation of osteoclast activation and autophagy through altered protein kinase pathways in Paget's disease of bone

Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique.Abstract : Paget’s disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover. In PDB, osteoclasts are larger, more active, more numerous, and resistant to apoptotic stimuli. While no single root cause has been identified, mutations to the gene encoding the p62 protein, SQSTM1, have been described in a significant population of patients with PDB. Among these mutations, the P392L substitution is the most prevalent, and overexpression of p62P392L in osteoclasts generates at least a partial pagetic phenotype in vitro. Normally this protein mediates a number of cell functions, from control of NF-κB signaling to autophagy. In human osteoclasts, a multiprotein complex containing p62 and protein kinases PKCζ and PDK1 (the principal kinase of Akt), form in response to stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL), the principal osteoclastogenic-signaling cytokine. We found that PKCζ is involved in RANKL-induced activation of NF-κB, and that it contributed to a basal activation of NF-κB observed in p62P392L mutants. This may be regulated in part by a PKCζ dependent increase in p65 phosphorylation at Ser536 which we characterized, independent of IκB. This could represent one alternative pathway by which mutant p62 leads to increased NF-κB activation. We observed increased basal phosphorylation of survival regulators ERK and Akt in PDB that was reduced upon PDK1 inhibition. The activity of 4EBP1 and Raptor, associated with mTOR activity, were also altered in pagetic osteoclasts and regulated by PDK1 inhibition. We then identified autophagic defects common to pagetic osteoclasts; with higher basal levels of LC3II (associated with autophagic structures), regardless of p62 mutation, and reduced sensitivity to autophagy induction in PDB. These results suggest an accumulation of non-degradative autophagosomes. Inhibition of PDK1 not only induced apoptosis in PDB and controls, but significantly reduced resorption in PDB, and with regards to autophagy, PDK1 inhibition was more potent in PDB than in controls. Therefore PDK1/Akt signaling represents an important checkpoint to PDB osteoclast activation. In sum, these results demonstrate the importance of several p62-associated kinases in the over-activation of pagetic osteoclasts, through increased survival and altered signaling. As p62 mutations alone do not account for most cases of PDB, the characterization of these pathways may identify a common factor linking pagetic osteoclasts. Therefore these studies represent a novel approach to osteoclast apoptosis, activation, and autophagy associated with PDB

Modulation of tumor necrosis factor related apoptosis-inducing ligand (trail) receptors in a human osteoclast model in vitro

Il a été démontré dans notre laboratoire que les ostéoclastes ( OCLs) provenant de patients atteints de myélome multiple (MM) présentaient une variation de l'expression des récepteurs de TRAIL en comparaison avec des ostéoclastes normaux. Il a également été montré que TRAIL (TNF-Related Apoptosis-Inducing Ligand), un membre de la famille TNF, était capable d'induire l'apoptose des cellules exprimant ses récepteurs, en liant les récepteurs DR4 et DRS, mais pas DcRl et DcR2, qui sont des récepteurs "decoy", dont le domaine intracellulaire de mort est absent. La modulation observée de ces récepteurs pourrait créer une résistance à l'apoptose dans le microenvironment du MM et pourrait être associée au profil cytokinique de cette maladie, caractérisé par une forte expression du « Receptor Activator of NF-KB Ligand" (RANKL) et de "Macrophage Inflammatory Protein lei' (MIP-la), cytokines majeures de la résorption osseuse. Le but de notre étude a été de déterminer quelles cytokines présentes dans cette pathologie pourraient en être responsables. Lors de la maturation cellulaire, les précurseurs d'OCL provenant de sang cordon et mis en présence de M-CSF et RANKL se différencient en cellules multinucléées (MNCs) qui expriment des marqueurs OCL et sont capables de résorber l'os. Par immunocytochimie, il avait été montré que ces cellules OCL exprimaient les 4 récepteurs de TRAIL. En stimulant de telles cultures d'OCLs avec différentes cytokines (RANKL, MIP-la, Transforming Factor (3 (TGF-{3), osteoprotegerin (OPG), TRAIL) et la parathormone (PTH), nous avons observé une modulation de l'expression de ces récepteurs de TRAIL, au niveau mARN par RT-PCR, au niveau protéique par immunobuvardage de type western et au niveau de la membrane cellulaire par immunocytochimie. Afin de voir si ces changements conduisaient à une modulation du niveau de résistance à l'apoptose, des MNCs, cultivées en présence de ces différentes cytokines pendant 5 jours, ont été stimulées avec TRAIL pendant 24h. L'apoptose des OCLs a été évaluée par la méthode TUNEL. Aucune corrélation n'a pu être établie entre la modification de l'expression de ces récepteurs et le niveau d'apoptose, mais le nombre d'expériences est encore trop faible pour conclure, et de nouvelles expériences doivent être réalisées afin de pouvoir analyser cette corrélation de manière adéquate. Nos résultats suggèrent néanmoins la possibilité qu'une modulation des récepteurs de TRAIL à la surface des OCLs soit induite par certaines cytokines fortement exprimées dans les maladies osseuses avec hyperrésorption osseuse, et puisse ainsi induire une résistance à l'apoptose de ces cellules. En prolongeant la vie des OCLs, ces influences régulatrices pourraient rendre compte en partie de l'augmentation de la résorption dans le micro environnement osseux des maladies comme l'ostéoporose, la maladie de Paget, ou le myélome multiple.Abstract: We have previously shown that osteoclasts (OCLs) from multiple myeloma (MM) specimens vary from healthy OCLs in their expression of the TRAIL receptors. TRAIL (TNF-Related Apoptosis-Inducing Ligand), a member of the TNF superfamily, has been shown to induce apoptosis in cells by binding receptors DR4 and DR5, but not DcR1 and DcR2, its decoy receptors, which lack the necessary internal death domain. The observed modulation of these receptors may confer a resistance to apoptosis in the MM environment, and could be related to the cytokine pattern that primarily involves the resorption promoting Receptor Activator of NF-[kappa]B Ligand (RANKL) and Macrophage Inflammatory Protein 1 (MIP-1[alpha]). The aim of our study was to determine which cytokines present in the disease might be responsible for this modulation. In long term cultures of OCL precursors from cord blood in the presence of M-CSF and RANKL, multinucleated cells (MNCs) that express OCL markers form, and can resorb bone. Through immunocytochemistry we showed that these MNCs can express all four TRAIL receptors. By stimulating with various cytokines (RANKL, MIP-1[alpha], Transforming Factor [bêta] (TGF[bêta]), osteoprotegerin (OPG), TRAIL), and parathyroid hormone (PTH) in OCL cultures, we were able to observe receptor modulation at the mRNA level using real time PCR, the protein level using Western blot analysis, and cell surface expression via immunocytochemistry. To determine if these changes translated to a difference in resistance to apoptosis, cells treated with [with] apoptosis-inducing levels of TRAIL after 5 days of stimulation with the selected cytokines were evaluated via TUNEL to quantify apoptosis. While no correlation has yet been established between the observed receptor modification and apoptosis induction, sample size is a factor, and further tests will be performed. Our results suggest the possibility that TRAIL receptor modification is induced by multiple cytokines present in bone diseases, capable of altering both the susceptibility and resistance pathways in osteoclasts. By potentially prolonging the lifespan of the OCL, these regulatory influences may ultimately be contributory factors to the augmentation of resorption in the micro-environment of bone resorptive diseases like multiple myeloma, Paget's disease of bone, or osteoporosis

Common mental disorders and ethnicity in England : the EMPIRIC Study

Background. There is little population-based evidence on ethnic variation in the most common mental disorders (CMD), anxiety and depression. We compared the prevalence of CMD among representative samples of White, Irish, Black Caribbean, Bangladeshi, Indian and Pakistani individuals living in England using a standardized clinical interview. Method. Cross-sectional survey of 4281 adults aged 16–74 years living in private households in England. CMD were assessed using the Revised Clinical Interview Schedule (CIS-R), a standardized clinical interview. Results. Ethnic differences in the prevalence of CMD were modest, and some variation with age and sex was noted. Compared to White counterparts, the prevalence of CMD was higher to a statistically significant degree among Irish [adjusted rate ratios (RR) 2.09, 95% CI 1.16–2.95, p=0.02] and Pakistani (adjusted RR 2.38, 95% CI 1.25–3.53, p=0.02) men aged 35–54 years, even after adjusting for differences in socio-economic status. Higher rates of CMD were also observed among Indian and Pakistani women aged 55–74 years, compared to White women of similar age. The prevalence of CMD among Bangladeshi women was lower than among White women, although this was restricted to those not interviewed in English. There were no differences in rates between Black Caribbean and White samples. Conclusions. Middle-aged Irish and Pakistani men, and older Indian and Pakistani women, had significantly higher rates of CMD than their White counterparts. The very low prevalence of CMD among Bangladeshi women contrasted with high levels of socio-economic deprivation among this group. Further study is needed to explore reasons for this variation

Argonaute2 Is Essential for Mammalian Gastrulation and Proper Mesoderm Formation

Mammalian Argonaute proteins (EIF2C1−4) play an essential role in RNA-induced silencing. Here, we show that the loss of eIF2C2 (Argonaute2 or Ago2) results in gastrulation arrest, ectopic expression of Brachyury (T), and mesoderm expansion. We identify a genetic interaction between Ago2 and T, as Ago2 haploinsufficiency partially rescues the classic T/+ short-tail phenotype. Finally, we demonstrate that the ectopic T expression and concomitant mesoderm expansion result from disrupted fibroblast growth factor signaling, likely due to aberrant expression of Eomesodermin. Together, these data indicate that a factor best known as a key component of the RNA-induced silencing complex is required for proper fibroblast growth factor signaling during gastrulation, suggesting a possible micro-RNA function in the formation of a mammalian germ layer

Grazing of dairy cows on pasture versus indoor feeding on total mixed ration: Effects on low-moisture part-skim Mozzarella cheese yield and quality characteristics in mid and late lactation

peer-reviewedThis study investigated the effects of 3 dairy cow feeding systems on the composition, yield, and biochemical and physical properties of low-moisture part-skim Mozzarella cheese in mid (ML; May–June) and late (LL; October–November) lactation. Sixty spring-calving cows were assigned to 3 herds, each consisting of 20 cows, and balanced on parity, calving date, and pre-experimental milk yield and milk solids yield. Each herd was allocated to 1 of the following feeding systems: grazing on perennial ryegrass (Lolium perenne L.) pasture (GRO), grazing on perennial ryegrass and white clover (Trifolium repens L.) pasture (GRC), or housed indoors and offered total mixed ration (TMR). Mozzarella cheese was manufactured on 3 separate occasions in ML and 4 in LL in 2016. Feeding system had significant effects on milk composition, cheese yield, the elemental composition of cheese, cheese color (green to red and blue to yellow color coordinates), the extent of flow on heating, and the fluidity of the melted cheese. Compared with TMR milk, GRO and GRC milks had higher concentrations of protein and casein and lower concentrations of I, Cu, and Se, higher cheese-yielding capacity, and produced cheese with lower concentrations of the trace elements I, Cu, and Se and higher yellowness value. Cheese from GRO milk had higher heat-induced flow and fluidity than cheese from TMR milk. These effects were observed over the entire lactation period (ML + LL), but varied somewhat in ML and LL. Feeding system had little, or no, effect on gross composition of the cheese, the proportions of milk protein or fat lost to cheese whey, the texture of the unheated cheese, or the energy required to extend the molten cheese. The differences in color and melt characteristics of cheeses obtained from milks with the different feeding systems may provide a basis for creating points of differentiation suited to different markets.This study was funded by the Department of Agriculture, Food and the Marine (Dublin, Ireland) Stimulus fund (11/sf/309), and additionally supported by funding from the Dairy Levy Trust Co-Operative Society Limited (Dublin, Ireland)

Antiretroviral treatment use, co-morbidities and clinical outcomes among Aboriginal participants in the Australian HIV Observational Database (AHOD)

Background: There are few data regarding clinical care and outcomes of Indigenous Australians living with HIV and it is unknown if these differ from non-Indigenous HIV-positive Australians. Methods: AHOD commenced enrolment in 1999 and is a prospective cohort of HIV-positive participants attending HIV outpatient services throughout Australia, of which 20 (74 %) sites report Indigenous status. Data were collected up until March 2013 and compared between Indigenous and non-Indigenous participants. Person-year methods were used to compare death rates, rates of loss to follow-up and rates of laboratory testing during follow-up between Indigenous and non-Indigenous participants. Factors associated with time to first combination antiretroviral therapy (cART) regimen change were assessed using Kaplan Meier and Cox Proportional hazards methods. Results: Forty-two of 2197 (1.9 %) participants were Indigenous. Follow-up amongst Indigenous and non-Indigenous participants was 332 & 16270 person-years, respectively. HIV virological suppression was achieved in similar proportions of Indigenous and non-Indigenous participants 2 years after initiation of cART (81.0 % vs 76.5 %, p = 0.635). Indigenous status was not independently associated with shorter time to change from first- to second-line cART (aHR 0.95, 95 % CI 0.51-1.76, p = 0.957). Compared with non-Indigenous participants, Indigenous participants had significantly less frequent laboratory monitoring of CD4 count (rate:2.76 tests/year vs 2.97 tests/year, p = 0.025) and HIV viral load (rate:2.53 tests/year vs 2.93 tests/year, p < 0.001), while testing rates for lipids and blood glucose were almost half that of non-indigenous participants (rate:0.43/year vs 0.71 tests/year, p < 0.001). Loss to follow-up (23.8 % vs 29.8 %, p = 0.496) and death (2.4 % vs 7.1 %, p = 0.361) occurred in similar proportions of indigenous and non-Indigenous participants, respectively, although causes of death in both groups were mostly non-HIV-related. Conclusions: As far as we are aware, these are the first data comparing clinical outcomes between Indigenous and non-Indigenous HIV-positive Australians. The forty-two Indigenous participants represent over 10 % of all Indigenous Australians ever diagnosed with HIV. Although outcomes were not significantly different, Indigenous patients had lower rates of laboratory testing for HIV and lipid/glucose parameters. Given the elevated risk of cardiovascular disease in the general Indigenous community, the additional risk factor of HIV infection warrants further focus on modifiable risk factors to maximise life expectancy in this population

Comorbidity in mental and physical illness

Comorbidity refers to the presence of two or more conditions at the same time. In the 2007 report in this series, comorbidity between mental disorders was examined. In this chapter comorbidity across mental disorders, chronic physical conditions, psychological wellbeing and intellectual impairment is profiled. • Physical health conditions were measured by showing participants a list of health conditions and asking whether a health professional had diagnosed them. Five chronic conditions were considered. Mental wellbeing was assessed using the Warwick Edinburgh Mental Wellbeing Scale (WEMWBS), where a higher score indicates greater psychological wellbeing. Intellectual impairment was also included, assessed using the Natonal Adult Reading Test (NART). • Overall, just over a quarter of adults (27.7%) reported having at least one of the five chronic physical conditions considered in this chapter diagnosed, and present in the last 12 months. High blood pressure was the most common, followed by asthma, diabetes, and cancer. A relatively small number of participants (52; 0.7% of adults) reported epilepsy; analysis by this group should therefore be treated with caution. • There was an association between common mental disorder (CMD) and chronic physical conditions. In people with severe CMD symptoms (revised Clinical Interview Schedule (CIS-R) score 18 or more) over a third (37.6%) reported a chronic physical condition, compared with a quarter (25.3%) of those with no or few symptoms of CMD (CIS-R score 0 to 5). • This pattern held for each of the chronic conditions examined. For xample, people with severe symptoms of CMD (CIS-R score 18+) were twice as likely to have asthma as people with no or few symptoms (CIS-R score 0–5): 14.5% compared with 7.2%. • Having a chronic physical condition was associated with lower levels of mental wellbeing. Overall, the mean WEMWBS score was 51.0 in people with at least one of the five chronic conditions considered, compared with 53.2 in people without a chronic physical condition. • Both the presence of self-reported diagnosed asthma and high blood pressure were associated with a wide range of different mental disorders, including depression, anxiety disorders (such as generalised anxiety disorder (GAD) and phobias), CMD Not Otherwise Specified (NOS), and posttraumatic stress disorder (PTSD). Asthma and high blood pressure were the most common chronic physical conditions examined; the larger sample of people with these conditions meant that statistically significant differences were more likely to be detectable. • Cancer and diabetes were also strongly associated with CMD-NOS, but higher rates of most other mental disorders were not statistically significant for these chronic physical conditions. • Adults with low wellbeing (with the lowest 15% of WEMWBS scores) experienced extremely high levels of psychiatric morbidity, including 21.0% screening positive for PTSD, 25.9% for attention-deficit/hyperactivity disorder (ADHD) and 6.0% for drug dependence. 20.6% of this group had made a suicide attempt. These rates were between 8 and 30 times higher than those for people with the highest mental wellbeing scores. • People with lower intellectual ability were more likely to have poorer mental health than those with average or above average intellectual functioning. • The results indicate that people with one condition tend to be more likely to have another, and that even subthreshold symptoms of common mental disorder are associated with having a chronic physical condition. These findings provide evidence to support the bringing of physical and mental health care provision closer together

Design rules for the self-assembly of a protein crystal

Theories of protein crystallization based on spheres that form close-packed crystals predict optimal assembly within a `slot' of second virial coefficients and enhanced assembly near the metastable liquid-vapor critical point. However, most protein crystals are open structures stabilized by anisotropic interactions. Here, we use theory and simulation to show that assembly of one such structure is not predicted by the second virial coefficient or enhanced by the critical point. Instead, good assembly requires that the thermodynamic driving force be on the order of the thermal energy and that interactions be made as nonspecific as possible without promoting liquid-vapor phase separation.Comment: 5 pages, 4 figure