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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.
- 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.
- 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.
- 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.
- 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.
- Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadiumPublication . Tiago, Daniel; Cancela, Leonor; Aureliano, M.; Laizé, VincentInsulina e IGF-1 são pequenos péptidos circulantes conhecidos por regular a densidade mineral e formação óssea em vertebrados através da activação vias de sinalização celular PI-3K e MAPK. Por sua vez, demonstrou-se recentemente que o vanádio/vanadato regula a proliferação e diferenciação de linhas celulares derivadas do osso de mamíferos através mesmas vias. Neste estudo, os efeitos proliferativos e mineralogénicos da insulina, IGF-1 e vanadato foram investigados em linhas derivadas do osso de peixe: VSa13 (tipo condrócito) e VSa16 (tipo osteoblasto). Tratamentos agudos com metavanadato (espécies n-méricas 1 a 5) nas células VSa13 revelaram uma acumulação mais rápida e mais elevada de vanádio, bem como uma toxicidade mais baixa do que decavanadato (n = 1 ou 10). Tratamentos crónicos com ambas as soluções estimularam a proliferação das VSa13, tal como a IGF-1, e preveniram a mineralização, tal como a insulina. Tratamentos com wortmanina e PD98959 demonstraram o envolvimento das vias MAPK e PI-3K\Ras\ERK nos efeitos proliferativos e mineralogénicos, respectivamente. Os dados obtidos são consistentes com observações recentes nas células de mamífero ATDC5 (condrócitos) sugerindo uma conservação de mecanismos ao longo da evolução. Aparentemente o vanadato não só terá interferido com as vias de sinalização mas também com a actividade da fosfatase alcalina, uma enzima muito importante na formação óssea. Nas células VSa16 foram observados efeitos semelhantes na proliferação através de tratamentos com IGF-1 (estimulação), e na mineralização através de tratamentos com vanadato e IGF-1 (inibição), sugerindo uma conservação de mecanismos através dos tipos celulares de osso. Em paralelo, neste trabalho foram identificadas em seabream 3 variantes da proIGF-1 (IGF-1a, 1b e 1c) e associadas a diferentes modos de acção (local e sistémico) em tecidos de adultos e durante o desenvolvimento, tal como previamente demonstrado em mamíferos. vi Propôs-se também que em peixes as variantes 1a e 1b estariam relacionadas com uma adaptação a diferentes salinidades. Em geral, os resultados aqui apresentados indicam uma conservação de mecanismos em vertebrados, demonstrando a importância dos sistemas derivados de peixes no estudo dos efeitos semelhantes à insulina no osso.
- Vanadate proliferative and anti-mineralogic effects are mediated by MAPK and PI-3K/Ras/Erk pathways in a fish chondrocyte cell linePublication . Tiago, Daniel; Cancela, Leonor; Aureliano, M.; Laizé, VincentWe recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/ Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.
- 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.