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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.
- Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concernsPublication . Granadeiro, Luis; Dirks, Ron P.; Ortiz-Delgado, Juan B.; J. Gavaia, Paulo; Sarasquete, Carmen; Laizé, Vincent; Leonor Cancela, M.; I, FernándezWarfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly over-represented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.
- Functional expression of the human CHIP28 water channel in a yeast secretory mutantPublication . Laizé, Vincent; Rousselet, G.; Verbavatz, J. M.; Berthonaud, V.; Gobin, R.; Roudier, N.; Abrami, L.; Ripoche, P.; Tacnet, F.The temperature-sensitive Saccharomyces cerevisiae mutant strain NY17, deficient in the secretory pathway (sec6-4 mutation), is used for the heterologous expression of the human CHIP28 water channel. After a heat-shock, the protein is present in partially purified post-golgi secretory vesicles, lmmunodetection and water transport studies, directly made on the vesicles, showed that CHIP28 is highly expressed and active in the yeast membranes.
- Overexpression of four and a half LIM domains protein 2 promotes epithelial-mesenchymal transition-like phenotype in fish pre-osteoblastsPublication . Rafael, Marta S.; Laizé, Vincent; Florindo, C.; Ferraresso, Serena; Bargelloni, Luca; Cancela, LeonorFHL2 is a multifunctional protein involved in gene transcription regulation and cytoarchitecture modulation that has been recently associated with epithelial-mesenchymal transition (EMT) in colon cancer. Overexpression of FHL2 in a fish pre-osteoblastic cell line promoted cell dedifferentiation and impaired its extracellular matrix mineralization capacity. Cell cultures also acquired a novel threedimensional structure organization, their proliferation rate was enhanced and gene expression profile was altered in agreement with an EMT-like phenotype upon overexpression of FHL2. Altogether, our results provide additional support to the relevance of FHL2 for cell differentiation and its association with hallmarks of cancer phenotype.
- 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.
- Existence of a tightly regulated water channel in saccharomyces cerevisiaePublication . Meyrial, V.; Laizé, Vincent; Gobin, R.; Ripoche, P.; Hohmann, S.; Tacnet, F.The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.
- Cells isolated from regenerating caudal fin of Sparus aurata can differentiate into distinct bone cell lineagesPublication . Vijayakumar, Parameswaran; Cardeira, João; Laizé, Vincent; J. Gavaia, Paulo; Cancela, M. LeonorTeleosts have the ability to regenerate their caudal fin upon amputation. A highly proliferative mass of undifferentiated cells called blastema forms beneath wound epidermis and differentiates to regenerate all missing parts of the fin. To date, the origin and fate of the blastema is not completely understood. However, current hypotheses suggest that the blastema is comprised of lineage-restricted dedifferentiated cells. To investigate the differentiation capacity of regenerating fin-derived cells, primary cultures were initiated from the explants of 2-days post-amputation (dpa) regenerates of juvenile gilthead seabream (Sparus aurata). These cells were subcultured for over 30 passages and were named as BSa2. After 10 passages they were characterized for their ability to differentiate towards different bone cell lineages and mineralize their extracellular matrix, through immunocytochemistry, histology, and RT-PCR. Exogenous DNA was efficiently delivered into these cells by nucleofection. Assessment of lineage-specific markers revealed that BSa2 cells were capable of osteo/chondroblastic differentiation. BSa2 cells were also found to be capable of osteoclastic differentiation, as demonstrated through TRAP-specific staining and pit resorption assay. Here, we describe the development of the first successful cell line viz., BSa2, from S. aurata 2-dpa regenerating caudal fins, which has the ability of multilineage differentiation and is capable of in vitro mineralization. The availability of such in vitro cell systems has the potential to stimulate research on the mechanisms of cell differentiation during fin regeneration and provide new insights into the mechanisms of bone formation.
- Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebratesPublication . S B Viegas, Carla; Simes, D; Laizé, Vincent; Williamson, M. K.; Price, P. A.; Cancela, LeonorWe report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 -carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative 16 positive 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a -carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment.