Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

<p>Abstract</p> <p>Background</p> <p>Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (<it>Sparus aurata</it>), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, <it>e.g. </it>mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of <it>in vitro </it>mineralization and genes involved in anti-mineralogenic action of vanadate.</p> <p>Results</p> <p>Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during <it>in vitro </it>mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization.</p> <p>Conclusions</p> <p>Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of <it>in vitro </it>mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation.</p

Proteínas gla do osso e cartilagem: importância dos anfíbios e peixes como modelos biológicos para elucidação da sua função e evolução - gla proteins in bone and cartilage: the importance of fish and amphibian models to understand their function and evolution

Gla proteins, as the name indicates, undergo a post-translation modification where specific glutamic acid residues are γ-carboxylated through the action of the ubiquitous enzyme γ-carboxylase and using vitamin K as cofactor. Therefore, these proteins are also called vitamin K dependent proteins or VKD [1, 2]. We can assign VKDs to essentially four different groups: 1) those involved in blood coagulation (such as prothrombin and various coagulation factors; the first group to be discovered), 2) those involved in tissue mineralization (bone and matrix Gla proteins), 3) a nerve growth factor (gas6), and 4) those of unknown function (the latest group to be discovered). The carboxylase enzyme is present in fly and worm but not in yeast, indicating that γ-carboxylation is likely a feature appearing in multicellular eukaryotes

Marine green macroalgae: a source of natural compounds with mineralogenic and antioxidant activities

Marine macroalgae represent a valuable natural resource for bioactive phytochemicals with promising applications in therapeutics, although they remain largely under-exploited. In this work, the potential of two marine green macroalgae (Cladophora rupestris and Codium fragile) as a source of bioactive phenolic compounds was explored, and antioxidant, mineralogenic, and osteogenic activities were evaluated. For each species, a crude hydroalcoholic extract (CE) was prepared by solid/liquid extraction and fractionated by liquid/liquid purification into an ethyl acetate fraction (EAF) enriched in phenolic compounds and an aqueous fraction (AF). Antioxidant activity, assessed through radical scavenging activity and reducing power assay, was increased in EAF fraction of both species and closely related to the phenolic content in each fraction. Mineralogenic activity, assessed through extracellular matrix mineralization of a fish bone-derived cell line, was induced by EAF fractions (up to 600 % for C. rupestris EAF). Quantitative analysis of operculum formation in zebrafish larvae stained with alizarin red S further confirmed the osteogenic potential of EAF fractions in vivo, with an increase of more than 1.5-fold for both C. fragile and C. rupestris fractions, similar to vitamin D (control). Our results demonstrated a positive correlation between phenolic fractions and biological activity, suggesting that phenolic compounds extracted from marine green macroalgae may represent promising molecules toward therapeutic applications in the field of bone biology.European Regional Development Fund (ERDF)-Atlantic Area Programme through MARMED project [2011-1/164]European Era-Net, Seas-Era program through the project INVASIVES [ANR-12-SEAS-0002-01]Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04326/2013