Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5–hyaluronan complexes in articular cartilages

SummaryObjectiveHuman osteoarthritis (OA) is characterized by aggrecanase-mediated depletion of cartilage aggrecan. We have examined the abundance, location and some biochemical properties of the six known aggrecanases (A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) 4, 5, 8, 9 and 15) in normal and OA human cartilages.MethodsFormalin-fixed, ethylenediamine tetraacetic acid (EDTA)-decalcified sections of full-depth cartilage from human OA tibial plateaus and normal control samples were studied by confocal imaging. Probes included specific antibodies to aggrecanases and two aggrecan epitopes, as well as biotinylated hyaluronan binding protein (HABP) for hyaluronan (HA) visualization. Cartilage extracts were analyzed by Western blot for the individual proteinases and aggrecan fragments.ResultsADAMTS5 was present in association with cells throughout normal cartilage and was markedly increased in OA, particularly in clonal groups in the superficial and transitional zones, where it was predominantly co-localized with HA. Consistent with the confocal analysis, a high molecular weight complex of ADAMTS5 and HA was isolated from human OA cartilage by isotonic salt extraction and chromatography on Superose 6. The complex eluted with an apparent molecular size of about 2×106 and contained major ADAMTS5 forms of 150, 60, 40 and 30kDa. The yield of most forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was markedly enhanced by prior digestion of the complex with either Streptomyces hyaluronidase or chondroitinase ABC.ConclusionADAMTS5 abundance and distribution in human OA cartilages is consistent with a central role for this enzyme in destructive aggrecanolysis. HA-dependent sequestration of ADAMTS5 in the pericellular matrix may be a mechanism for regulating the activity of this proteinase in human OA cartilage

In Caenorhabditis elegans Nanoparticle-Bio-Interactions Become Transparent: Silica-Nanoparticles Induce Reproductive Senescence

While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the ‘bag of worms’ phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production

TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.Funding Agencies|German Research Foundation (DFG)|SFB670-NG01|Swedish Society of Medicine||Regional Research Council of South-East Sweden (FORSS)||Swedish Research Council division of Medicine||Gustav V 90th anniversary foundation||Italian Telethon Foundation||DFG|SE 1122/2-1|</p

A Genome-Wide Scan of Ashkenazi Jewish Crohn's Disease Suggests Novel Susceptibility Loci

Crohn's disease (CD) is a complex disorder resulting from the interaction of intestinal microbiota with the host immune system in genetically susceptible individuals. The largest meta-analysis of genome-wide association to date identified 71 CD–susceptibility loci in individuals of European ancestry. An important epidemiological feature of CD is that it is 2–4 times more prevalent among individuals of Ashkenazi Jewish (AJ) descent compared to non-Jewish Europeans (NJ). To explore genetic variation associated with CD in AJs, we conducted a genome-wide association study (GWAS) by combining raw genotype data across 10 AJ cohorts consisting of 907 cases and 2,345 controls in the discovery stage, followed up by a replication study in 971 cases and 2,124 controls. We confirmed genome-wide significant associations of 9 known CD loci in AJs and replicated 3 additional loci with strong signal (p<5×10−6). Novel signals detected among AJs were mapped to chromosomes 5q21.1 (rs7705924, combined p = 2×10−8; combined odds ratio OR = 1.48), 2p15 (rs6545946, p = 7×10−9; OR = 1.16), 8q21.11 (rs12677663, p = 2×10−8; OR = 1.15), 10q26.3 (rs10734105, p = 3×10−8; OR = 1.27), and 11q12.1 (rs11229030, p = 8×10−9; OR = 1.15), implicating biologically plausible candidate genes, including RPL7, CPAMD8, PRG2, and PRG3. In all, the 16 replicated and newly discovered loci, in addition to the three coding NOD2 variants, accounted for 11.2% of the total genetic variance for CD risk in the AJ population. This study demonstrates the complementary value of genetic studies in the Ashkenazim

The application of ‘elite interviewing’ methodology in transdisciplinary research: A record of process and lessons learned during a 3-year pilot in urban planetary health research

This paper sets out the rationale and process for the interviewing methodology utilized during a 3-year research pilot, ‘Moving Health Upstream in Urban Development’ (UPSTREAM). The project had two primary aims: firstly, to attempt to value economically the health cost benefits associated with the quality of urban environments and secondly, to engage with those in control of urban development in the UK in order to determine what are the barriers to and opportunities for creating healthy urban environments, including those identified through the utilisation of economic valuation. Engagement at senior level with those who have most control over key facets of planning and development implementation—such as land disposal, investment, development delivery and planning permission—was central to the approach, which encompassed the adoption of ‘elite interviewing’, a method developed in the USA in the 1950s and used in the political sciences but relatively unutilized in the health and environmental sciences [1]. Two rounds of semi-structured interviews were undertaken with 15 senior decision-makers from the UK’s main urban development delivery agencies, both public and private. The ‘elite interviewing’ approach successfully enabled the UPSTREAM project to capture and analyse the information received from the interviewees, all of whom held influential or leadership posts in organisations that are important actors in the process of planning, developing and constructing the built environment in the UK. Having academic and practitioner research leads on an equal footing created some minor tensions, but it also appeared to strengthen the rigor of the approach through a broad knowledge of context ‘in-house’. This form of co-production at times challenged academic traditions in qualitative analysis, but it also appeared to build trust with interviewees and provided greater clarity of the real-world context under investigation. Findings from this study are written up in a separate paper

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

The Role of Proteasome Beta Subunits in Gastrin-Mediated Transcription of Plasminogen Activator Inhibitor-2 and Regenerating Protein1

The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1(Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role