Abstract
BackgroundFish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), 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, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate.ResultsGlobal analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro 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.ConclusionsComparative 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 in vitro 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.
Highlights
Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), 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
To investigate mechanisms of tissue mineralization, various bonederived cell lines of fish origin are available [18] and gilthead seabream VSa13 and VSa16 cell lines are of particular interest due to their pre-chondrocyte and pre-osteoblast phenotypes. They are both capable of mineralizing their extracellular matrix, VSa13 and VSa16 cell lines behave differently regarding their degree of mineral deposition [19], levels of alkaline phosphatase activity [19], expression of mineralogenic genes, e.g. matrix gla protein (MGP), osteocalcin (OC), osteopontin (SPP1), bone morphogenetic protein-2 (BMP-2) [19,20,21], and susceptibility to mineralogenic or anti-mineralogenic molecules, e.g. insulin, IGF-1, vanadate [22,23] and retinoic acid
Genes differentially expressed during in vitro mineralization of gilthead seabream vertebra-derived cell lines Confluent cultures of VSa13 and VSa16 cells were cultivated during 4 weeks under control or mineralizing conditions
Summary
Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), 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. To investigate mechanisms of tissue mineralization, various bonederived cell lines of fish origin are available [18] and gilthead seabream VSa13 and VSa16 cell lines (derived from vertebra) are of particular interest due to their pre-chondrocyte and pre-osteoblast phenotypes They are both capable of mineralizing their extracellular matrix, VSa13 and VSa16 cell lines behave differently regarding their degree of mineral deposition [19], levels of alkaline phosphatase activity [19], expression of mineralogenic genes, e.g. matrix gla protein (MGP), osteocalcin (OC), osteopontin (SPP1), bone morphogenetic protein-2 (BMP-2) [19,20,21], and susceptibility to mineralogenic or anti-mineralogenic molecules, e.g. insulin, IGF-1, vanadate [22,23] and retinoic acid [unpublished results]. We have identified in VSa13 cell line the presence of mineralogenic genes whose expression was altered upon cell exposure to vanadate, a molecule with anti-mineralogenic activity [23]
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