A Microarray study of Carpet-Shell Clam (Ruditapes decussatus) shows common and organ-specific growth-related gene expression Differences in gills and digestive gland

Growth rate is one of the most important traits from the point of view of individual fitness and commercial production in mollusks, but its molecular and physiological basis is poorly known. We have studied differential gene expression related to differences in growth rate in adult individuals of the commercial marine clam Ruditapes decussatus. Gene expression in the gills and the digestive gland was analyzed in 5 fast-growing and five slow-growing animals by means of an oligonucleotide microarray containing 14,003 probes. A total of 356 differentially expressed genes (DEG) were found. We tested the hypothesis that differential expression might be concentrated at the growth control gene core (GCGC), i. e., the set of genes that underlie the molecular mechanisms of genetic control of tissue and organ growth and body size, as demonstrated in model organisms. The GCGC includes the genes coding for enzymes of the insulin/ insulin-like growth factor signaling pathway (IIS), enzymes of four additional signaling pathways (Raf/ Ras/ Mapk, Jnk, TOR, and Hippo), and transcription factors acting at the end of those pathways. Only two out of 97 GCGC genes present in themicroarray showed differential expression, indicating a very little contribution of GCGC genes to growth-related differential gene expression. Forty eight DEGs were shared by both organs, with gene ontology (GO) annotations corresponding to transcription regulation, RNA splicing, sugar metabolism, protein catabolism, immunity, defense against pathogens, and fatty acid biosynthesis. GO termenrichment tests indicated that genes related to growth regulation, development and morphogenesis, extracellular matrix proteins, and proteolysis were overrepresented in the gills. In the digestive gland overrepresented GO terms referred to gene expression control through chromatin rearrangement, RAS-related small GTPases, glucolysis, and energy metabolism. These analyses suggest a relevant role of, among others, some genes related to the IIS, such as the ParaHox gene Xlox, CCAR and the CCN family of secreted proteins, in the regulation of growth in bivalves.Direccion General de Investigacion Cientifica y Tecnica of the Spanish Government [AGL2010-16743, AGL2013-49144-C3-3-R]; COMPETE Program; Portuguese National Funds [PEst-255 C/MAR/LA0015/2011]; Portuguese FCT [UID/Multi/04326/2013]; Generalitat Valenciana; Ministry of Education, Culture, and Sports of the Spanish Government; Association of European Marine Biology Laboratoriesinfo:eu-repo/semantics/publishedVersio

Fish models of induced osteoporosis

Osteopenia and osteoporosis are bone disorders characterized by reduced bone mineral density (BMD), altered bone microarchitecture and increased bone fragility. Because of global aging, their incidence is rapidly increasing worldwide and novel treatments that would be more efficient at preventing disease progression and at reducing the risk of bone fractures are needed. Preclinical studies are today a major bottleneck to the collection of new data and the discovery of new drugs, since they are commonly based on rodent in vivo systems that are time consuming and expensive, or in vitro systems that do not exactly recapitulate the complexity of low BMD disorders. In this regard, teleost fish, in particular zebrafish and medaka, have recently emerged as suitable alternatives to study bone formation and mineralization and to model human bone disorders. In addition to the many technical advantages that allow faster and larger studies, the availability of several fish models that efficiently mimic human osteopenia and osteoporosis phenotypes has stimulated the interest of the academia and industry toward a better understanding of the mechanisms of pathogenesis but also toward the discovery of new bone anabolic or antiresorptive compounds. This mini review recapitulates the in vivo teleost fish systems available to study low BMD disorders and highlights their applications and the recent advances in the field.UIDB/04326/2020, EAPA_151/2016/BLUEHUMANinfo:eu-repo/semantics/publishedVersio

Temporal and spatial expression patterns of pregnane X receptor and vitamin K epoxide reductase genes, two core molecular players on fish vitamin K homeostasis and skeletal development.

Vitamin K (VK) is a liposoluble vitamin known to be essential for bone metabolism by two different pathways: (i) by its role as a coenzyme in the gamma-carboxylation of some skeletal proteins (e.g. osteocalcin (OC) and matrix Gla protein (MGP); Price et al., 1998); and (ii) through its role in skeletal gene transcription via binding to the pregnane X receptor (PXR; Azuma et al., 2010)

Transcriptional regulation of gilthead seabream bone morphogenetic protein (BMP) 2 gene by bone- and cartilage-related transcription factors

Bone morphogenetic protein (BMP) 2 belongs to the transforming growth factor (3 (TGF(3) superfamily of cytokines and growth factors. While it plays important roles in embryo morphogenesis and organogenesis, BMP2 is also critical to bone and cartilage formation. Protein structure and function have been remarkably conserved throughout evolution and BMP2 transcription has been proposed to be tightly regulated, although few data is available. In this work we report the cloning and functional analysis of gilthead seabream BMP2 promoter. As in other vertebrates, seabream BMP2 gene has a 5' non-coding exon, a feature already present in DPP gene, the fruit fly ortholog of vertebrate BMP2 gene, and maintained throughout evolution. In silico analysis of seabream BMP2 promoter revealed several binding sites for bone and cartilage related transcription factors (TFs) and their functionality was evaluated using promoter-luciferase constructions and TF-expressing vectors. Runt -related transcription factor 3 (RUNX3) was shown to negatively regulate BMP2 transcription and combination with the core binding factor beta (CBF(3) further reduced transcriptional activity of the promoter. Although to a lesser extent, myocyte enhancer factor 2C (MEF2C) had also a negative effect on the regulation of BMP2 gene transcription, when associated with SRY (sex determining region Y)-box 9 (SOX9b). Finally, v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) was able to slightly enhance BMP2 transcription. Data reported here provides new insights toward the better understanding of the transcriptional regulation of BMP2 gene in a bone and cartilage context. (C) 2015 Elsevier B.V. All rights reserved

Molecular effect of an OPTN common variant associated to Paget's disease of bone

Paget's disease of bone (PDB) is a chronic bone disorder and although genetic factors appear to play an important role in its pathogenesis, to date PDB causing mutations were identified only in the Sequestosome 1 (SQSTM1) gene at the PDB3 locus. PDB6 locus, also previously linked to PDB, contains several candidate genes for metabolic bone diseases. We focused our analysis in the most significantly associated variant with PDB, within the Optineurin (OPTN) gene, i.e. the common variant rs1561570. Although it was previously shown to be strongly associated with PDB in several populations, its contribution to PDB pathogenesis remains unclear. In this study we have shown that rs1561570 may contribute to PDB since its Tallele results in the loss of a methylation site in patients' DNA, leading to higher levels of OPTN gene expression and a corresponding increase in protein levels in patients' osteoclasts. This increase in OPTN expression leads to higher levels of NF-KB translocation into the nucleus and increasing expression of its target genes, which may contribute to the overactivity of osteoclasts observed in PDB. We also reported a tendency for a more severe clinical phenotype in the presence of a haplotype containing the rs1561570 T allele, which appear to be re-enforced with the presence of the SQSTM1/P392L mutation. In conclusion, our work provides novel insight towards understanding the functional effects of this variant, located in OPTN intron 7, and its implication in the contribution to PDB pathogenesis.national funds from Foundation for Science and Technology (FCT) [UID/Multi/04326/2013]; Canadian Institutes for Health Research, Canada [MOP130457]; CHU de Quebec Foundation; Canadian Foundation for Innovation; Fonds de recherche du Quebec-sante; Laval University; CHU de Quebec-Universite Laval Research Centre; FCT [SFRH/BD/77227/2011, SFRH/BPD/111898/2015]; Fonds de recherche Quebec-Sante (FRQ-S), Quebec, Canad

Vanadate and bone metabolism: effect on proliferation and mineralization of fish bone-derived cells

Vanadate is known for mimicking insulin action through activation of insulin and/or insulin like growth factor 1 (IGF 1) receptors. Vanadate insulin- like effect on bone-related metabolism has been previously investigated using mammalian in vitro cell systems but other vertebrate systems have rarely been used. We have recently demonstrated the suitability of a fish bone derived cell line (VSa13) to study anti-mineralogenic effects of vanadate. Here, we propose that vanadate stimulation of cell proliferation involves MAPK signalling pathway and IGF 1 receptor activation, while impairment of extracellular matrix (ECM) mineralization is likely to involve both MAPK and PI 3K pathways and insulin receptor activation

Gene expression during regeneration of zebrafish (danio rerio) fins: relative expression levels of mineralization – related gla proteins

Most animals have the ability to regenerate epidermal injuries yet only a few can regenerate largely severed appendages that comprise several different tissues. Nowadays zebrafish is one of the most used metazoan models in regeneration studies in particular for investigation of molecular events during fin regeneration process. Fin regeneration starts through the formation of a blastema, a set of heterogeneous mesenchyma-like cells located between stump tissues and the wounded epidermis. This event, denominated epimorphic regeneration, comprises strict growth control and cell reprogramming leading to faithful restoration of the lost parts

Vanadate effects on bone metabolism: fish cell lines as an alternative to mammalian in vitro systems

Vanadate, one of the most relevant forms of vanadium in solution, has been associated with the regulation of various enzyme activities (e.g. phosphatases, ribonucleases, ATPases, etc.) and shown to exhibit important biological effects. Several in vivo and in vitro studies have clearly demonstrated that any deficiency or excess of vanadium can seriously affect bone formation and its metabolism. Bone-related effects result largely from vanadium insulino-mimetic capabilities mediated by specific inhibition of protein tyrosine phosphatases (PTPases) and consequent activation of tyrosine kinase receptors (e.g. insulin receptor). Although mammals have been repetitively shown to be appropriate models to study vanadate mechanisms of action, fish have recently emerged as alternative models. Fish has been recognized as suitable model to study vertebrate bone formation and the natural presence of high quantities of vanadium in water makes it even more suitable to investigate vanadium effect on bone formation. Recent data obtained using fish bone-derived cells revealed that micromolar concentrations (5 mM) of monomeric and decameric vanadate slightly stimulate growth performances while strongly inhibiting extracellular matrix mineralization through mechanisms involving both alkaline phosphatase and MAPK pathways. Recent data obtained in fish cells will be discussed here and further compared to results obtained in mammalian systems

Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line

Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85±0.40 versus 3.95±0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems