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- Vanadate effects on bone metabolism: fish cell lines as an alternative to mammalian in vitro systemsPublication . Tiago, Daniel; Laizé, Vincent; Aureliano, M.; Cancela, LeonorVanadate, 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.
- Sturgeon osteocalcin shares structural features with matrix gla protein evolutionary relationship and functional implicationsPublication . S B Viegas, Carla; Simes, Dina; Williamson, Matthew K.; Cavaco, Sofia; Laizé, Vincent; Price, Paul A.; Leonor Cancela, M.Osteocalcin (OC) and matrix Gla protein (MGP) are considered evolutionarily related because they share key structural features, although they have been described to exert different functions. In this work, we report the identification and characterization of both OC and MGP from the Adriatic sturgeon, a ray-finned fish characterized by a slow evolution and the retention of many ancestral features. Sturgeon MGP shows a primary structure, post-translation modifications, and patterns of mRNA/protein distribution and accumulation typical of known MGPs, and it contains seven possible Gla residues that would make the sturgeon protein the most gamma-carboxylated among known MGPs. In contrast, sturgeon OC was found to present a hybrid structure. Indeed, although exhibiting protein domains typical of known OCs, it also contains structural features usually found in MGPs (e. g. a putative phosphorylated propeptide). Moreover, patterns of OC gene expression and protein accumulation overlap with those reported for MGP; OC was detected in bone cells and mineralized structures but also in soft and cartilaginous tissues. We propose that, in a context of a reduced rate of evolution, sturgeon OC has retained structural features of the ancestral protein that emerged millions of years ago from the duplication of an ancient MGP gene and may exhibit intermediate functional features.
- ZFBONE: an ImageJ toolset for semi-automatic analysis of zebrafish bone structuresPublication . Tarasco, Marco; Cordelieres, Fabrice P.; Cancela, M. Leonor; Laizé, VincentThe last decade has seen an increased interest in the discovery of compounds with bone anabolic activity to treat skeletal disorders such as osteoporosis and increase the well-being of patients. Due to the many technical advantages over classical rodent systems, zebrafish (Danio rerio) has been increasingly used in screening pipelines, in particular those aiming at identifying osteoactive compounds with pharmacological potential. Because compound osteoactivity is mostly determined in zebrafish through the morphometric analysis of bone structures, image analysis, rather than screening assay implementation, molecule availability and image acquisition, represents a bottleneck to the screening throughput. The absence of auto/semi-automatic tools for image analysis of fish bone structures is also a limitation to a broader usage of zebrafish screening pipelines. We present here ZFBONE (for ZebraFish BONE), an open-source, freely available, user-friendly, rapid and reliable toolset, aiming at accelerating image analysis by automating the morphometric assessment of zebrafish bone structures, but also at increasing data accuracy by reducing operator bias. Tools included in ZFBONE allow users to assess, from 2D images, morphometric parameters of several bone structures (e.g. operculum, caudal fin rays and scales) but also the extent and the intensity of bone-specific colorations. ZFBONE has been developed using the open-source ImageJ software, to make it available to the whole zebrafish research community, but also to have it easily modifiable according to user demands. ZFBONE can also be used toward the standardization of zebrafish screening protocols in academia and industry.
- The zebrafish operculum: a powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevancePublication . Tarasco, Marco; Laizé, Vincent; Cardeira, João; Leonor Cancela, M.; Gavaia, PauloBone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.
- Vanadate and bone metabolism: effect on proliferation and mineralization of fish bone-derived cellsPublication . Tiago, Daniel; Laizé, Vincent; Cancela, Leonor; Aureliano, M.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.