The integrins of the urochordate Ciona intestinalis provide novel insights into the molecular evolution of the vertebrate integrin family

BACKGROUND: Integrins are a functionally significant family of metazoan cell surface adhesion receptors. The receptors are dimers composed of an alpha and a beta chain. Vertebrate genomes encode an expanded set of integrin alpha and beta chains in comparison with protostomes such as drosophila or the nematode worm. The publication of the genome of a basal chordate, Ciona intestinalis, provides a unique opportunity to gain further insight into how and when the expanded integrin supergene family found in vertebrates evolved. RESULTS: The Ciona genome encodes eleven α and five β chain genes that are highly homologous to their vertebrate homologues. Eight of the α chains contain an A-domain that lacks the short alpha helical region present in the collagen-binding vertebrate alpha chains. Phylogenetic analyses indicate the eight A-domain containing α chains cluster to form an ascidian-specific clade that is related to but, distinct from, the vertebrate A-domain clade. Two Ciona α chains cluster in laminin-binding clade and the remaining chain clusters in the clade that binds the RGD tripeptide sequence. Of the five Ciona β chains, three form an ascidian-specific clade, one clusters in the vertebrate β1 clade and the remaining Ciona chain is the orthologue of the vertebrate β4 chain. CONCLUSION: The Ciona repertoire of integrin genes provides new insight into the basic set of these receptors available at the beginning of vertebrate evolution. The ascidian and vertebrate α chain A-domain clades originated from a common precursor but radiated separately in each lineage. It would appear that the acquisition of collagen binding capabilities occurred in the chordate lineage after the divergence of ascidians

Superoxide dismutase downregulation in osteoarthritis progression and end-stage disease

Oxidative stress is proposed as an important factor in osteoarthritis (OA). To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin–Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin–Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function

A Novel Form of Chondrocyte Stress is Triggered by a COMP Mutation Causing Pseudoachondroplasia

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012. © 2011 Wiley Periodicals, Inc

Reduced cell proliferation and increased apoptosis are significant pathological mechanisms in a murine model of mild pseudoachondroplasia resulting from a mutation in the C-terminal domain of COMP

Pseudoachondroplasia (PSACH) is one of the more common skeletal dysplasias and results from mutations in cartilage oligomeric matrix protein (COMP). Most COMP mutations identified to date cluster in the TSP3 repeat region of COMP and the mutant protein is retained in the rough endoplasmic reticulum (rER) of chondrocytes and may result in increased cell death. In contrast, the pathomolecular mechanism of PSACH resulting from C-terminal domain COMP mutations remain largely unknown. This study describes the generation and analysis of a murine model of mild PSACH resulting from a p.Thr583Met mutation in the C-terminal globular domain (CTD) of COMP. Mutant animals are normal at birth, but grow slower than their wild-type littermates and by 9 weeks of age they have mild short-limb dwarfism. Furthermore, by 16 months of age mutant animals exhibit severe degeneration of articular cartilage, which is consistent with early onset osteoarthritis seen in PSACH patients. In the growth plates of mutant mice the chondrocyte columns are sparser and poorly organized. Mutant COMP is secreted into the extracellular matrix, but its localization is disrupted along with the distribution of several COMP-binding proteins. Although mutant COMP is not retained within the rER there is an unfolded protein/cell stress response and chondrocyte proliferation is significantly reduced, while apoptosis is both increased and spatially dysregulated. Overall, these data suggests a mutation in the CTD of COMP exerts a dominant-negative effect on both intra- and extracellular processes. This ultimately affects the morphology and proliferation of growth plate chondrocytes, eventually leading to chondrodysplasia and reduced long bone growth

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration

ObjectivesThe circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration.MethodsClock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containingPer2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock geneBmal1.ResultsHere we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout ofBmal1in their disc cells demonstrated age-related degeneration of IVDs.ConclusionsWe have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.</jats:sec

Targeted Induction of Endoplasmic Reticulum Stress Induces Cartilage Pathology

Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS–causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF–mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress–inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix proteins

XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease

Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-β activity in MCDS chondrocytes may offer therapeutic opportunities

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2

SummaryTranscriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5–7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock

DEVELOPMENT OF HIGH STRENGTH COMPOSITE WIRES BASED ON CU-MG

Разработаны технологии по получению высокопрочных композитных проводов на основе Cu–Mg. Методом гидроэкструзии сформирован композитный провод с большим количеством волокон магния. Проведены эксперименты по получению композитного провода Cu–Mg путем введения смеси из порошков меди и магния. Исследована микроструктура полученных композитов. Проведены измерения электрических и механических свойств.The techniques to obtain high-strength Cu–Mg composite wires were developed. Hydrostatic extrusion method was used to obtain the Cu–Mg composite wire with lots of magnesium fibers. Also the experiments to acquire a composite wire from the mixture of copper and magnesium powders were conducted. The microstructure of obtained composites was investigated. The electrical and mechanical properties were measured.Работа выполнена в рамках государственного задания ФАНО России (тема «Деформация», № 01201463327)

Identification of multiple integrin β1 homologs in zebrafish (Danio rerio)

BACKGROUND: Integrins comprise a large family of α,β heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single β1 gene, and the β1 subunit associates with a large number of α subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about β1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish β1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two β1 paralogs (β1–1 and β1–2) that have a high degree of identity to other vertebrate β1 subunits. In addition, a third, more divergent, β1 paralog is present (β1–3), which may have altered ligand-binding properties. Zebrafish also have other divergent β1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with β1–3 these truncated forms comprise a novel group of β1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to β1–1 and β1–2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of β1–2 may have given rise to β1–3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated β1 paralogs appears to have taken place. The different zebrafish β1 paralogs have varied patterns of temporal expression during development. β1–1 and β1–2 are ubiquitously expressed in adult tissues, whereas the other β1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin β1 paralogs. β1–1 and β1–2 may share the roles of the solitary β1 subunit found in other vertebrates, whereas β1–3 and the truncated β1 paralogs may have acquired novel functions