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- Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fishPublication . Rosa, Joana; Tiago, Daniel; Marques, Cátia L.; Vijayakumar, Parameswaran; Fonseca, Luís; Cancela, Leonor; Laizé, VincentBackground: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.Methods: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences.Results: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.Conclusions: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.General significance: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
- Proliferative and mineralogenic effects of insulin, IGF-1, and vanadate in fish osteoblast-like cellsPublication . Tiago, Daniel; Cancela, Leonor; Laizé, VincentFish have recently been recognized as a suitable model and a promising alternative to mammalian systems to study skeletogenesis. In this regard, several fish bone-derived cell lines have been developed and are being used to investigate mechanisms associated with insulin-like action of vanadium on extracellular matrix (ECM) mineralization. Although proliferative and mineralogenic effects of vanadate, insulin-like growth factor 1 (IGF-1), and insulin have recently been evaluated in a fish prechondrocyte cell line, no data are available in fish bone-forming cells, the osteoblasts. Using fish preosteoblast cells, we showed that IGF-1, but not insulin or vanadate, stimulated cell proliferation through the mitogen-activated protein kinase (MAPK) pathway, while both IGF-1 and vanadate inhibited cell differentiation/ECM mineralization through the same mechanism. Our data also indicated that the phosphatidyl inositol-3 kinase (PI-3K) pathway stimulates differentiation/ECM mineralization in osteoblasts and could represent a way to balance MAPK pathway action. The comparison of these new data obtained in fish with those available in mammals clearly evidenced a conservation of regulatory mechanisms among vertebrate bone-derived systems, although different players are involved.
- 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.
- Desenvolvimento de sistemas celulares de peixe adequados ao estudo da mineralização in vitroPublication . Marques, C. L.; Rafael, Marta S.; Tiago, Daniel; Cancela, Leonor; Laizé, VincentOs peixes foram recentemente reconhecidos como organismos modelo apropriados para o estudo da biologia de vertebrados, particularmente de processos relacionados com a mineralização tecidular e o desenvolvimento do esqueleto. Apesar de existirem alguns estudos in vivo, a identificação de mecanismos associados à calcificação em peixes tem sido prejudicada pelo facto de não existirem sistemas celulares para estudos in vitro. Este artigo descreve um protocolo simples e de baixo custo adequado ao desenvolvimento de culturas celulares mineralogénicas, derivadas de tecidos calcificados de peixes.
- Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell linePublication . Tiago, Daniel; Laizé, Vincent; Cancela, Leonor; Aureliano, M.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.
- Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: Different gene expression of nuclear receptors and ECM proteinsPublication . Fernández, Ignacio; Tiago, Daniel; Laizé, Vincent; Cancela, Leonor; Gisbert, EnricRetinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36–59% in VSa13 and 17–46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50–62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11–57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner.
- Alternatively spliced transcripts of Sparus aurata insulin-like growth factor 1 are differentially expressed in adult tissues and during early developmentPublication . Tiago, Daniel; Laizé, Vincent; Cancela, LeonorSpliced variants of insulin-like growth factor 1 (IGF-1), a small peptide with a critical role in metabolism and growth, have been identified in various vertebrate species. However, despite recent functional data in mammalian systems suggesting specific roles (e.g. in muscle formation) for their pro-peptides and/or E domains, their function remains unclear. In this study, three alternatively spliced variants of Sparus aurata proIGF-1 (1a, 1b, and 1c) were identified and their expression analyzed. In adult fish, IGF-1 gene expression was observed in various soft tissues (highest levels in liver) and calcified tissues, with IGF-1c being always the most expressed isoform. In developing larvae, each isoform presented a specific pattern of expression, characterized by different onset and extent and consistent with a possible role of IGF-1a and 1b during early post-hatching events (e.g. bone or muscle formation), while IGF-1c would be rather involved in early larvae formation but probably acts in concerted action with other isoforms at later stages. We also propose that, in adults, IGF-1a and 1b isoforms may have a local action, while isoform 1c would assume a systemic action, as its mammalian counterpart. This hypothesis was further supported by in silico analysis of isoform distribution, revealing that only IGF-1c/Ea isoform has been conserved throughout evolution and that other fish isoforms (i.e. 1a and 1b) may be associated with mechanisms of osmoregulation. We finally propose that IGF-1 variants may exhibit different modes of action (systemic or local) and may be involved in different developmental and adaptive mechanisms.
- Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadatePublication . Tiago, Daniel; Laizé, Vincent; Bargelloni, Luca; Ferraresso, Serena; Romualdi, Chiara; Cancela, LeonorAbstract Background 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. 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. Results Global 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. Conclusions 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 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.
- Serum-specific stimulation of proliferation and mineralization of fish bone-derived cellsPublication . Rosa, Joana; Tiago, Daniel; Dias, J.; Cancela, Leonor; Laizé, VincentTeleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone-derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose-dependent and cell type-specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast-like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization-related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone-derived cells.
- New insights into mineralogenic effects of vanadatePublication . Laizé, Vincent; Tiago, Daniel; Aureliano, M.; Cancela, LeonorVanadium is a transition metal that occurs naturally in a variety of minerals and exhibits an exceptional complex chemistry in solution, e.g., several oxidation states ranging from ?2 to ?5, and formation of vanadium oligomers such as decameric vanadate (?5) species [1–4]. Besides its metallurgical role in steel alloys, vanadium is also an ultra trace element known to participate in many biological processes and considered to be essential for living organisms [5, 6]. It accumulates in a variety of organisms ranging from microbes to vertebrates, where it modulates the activity of an array of key enzymes or participates as a cofactor in the active centre of others [1, 2, 5–9]. In mammals, vanadium compounds can mimic insulin action and may prevent chemical carcinogenesis, most probably through the inhibition of cellular tyrosine phosphatases and subsequent activation of signalling pathways, suggesting their use as pharmacological tools to treat human diabetes mellitus and cancer, respectively [10–14]. Anti-tumoral action of vanadium is, however, controversial as several studies have proposed that vanadium could act as a mitogen, tumor promoter and co-carcinogen (see [15] and references therein). Other studies have reported an osteogenic role for vanadium compounds and suggest that vanadium could also have a therapeutic application in bone-related diseases, such as osteoporosis [16–18]. Decades of research have thus provided evidence for vanadium’s physiological and pharmacological properties, supporting the claim that it may represent a promising therapeutic agent for diseases targeting billions of human beings and affecting a wide range of pathological conditions. However, the development of vanadium-based pharmaceuticals will probably take some time since various issues related to vanadium toxicity, speciation and multiple targeting will need to be solved before advancing to clinical trials. Despite being used for decades by researchers as an inhibitor of protein tyrosine phosphatases, it is still not totally clear which vanadium species induce or which signalling pathways transduce physiological and pharmacological effects. Vanadium chemistry is complex, and different species or complexes may induce different pathways [5], affecting different biological processes. This work intends to review what is presently known about the bone-related role of vanadium in mammals and present recent in vitro data on the mineralogenic effect of vanadate in fish, which have become promising model organisms for vertebrate bone-related studies.